Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Sharafianardakani, Zohreh
Doctor of Philosophy - PhD
University of British Columbia
Infants are susceptible to mucocutaneous candidiasis, which manifests via oral thrush and diaper rashes. Th17 cells are responsible for limiting mucosal invasion by commensal Candida spp. Dysregulated Th17 cells are also involved in necrotizing enterocolitis (NEC) pathogenesis, a surgical complication of the gut affecting about 5% of pre-term neonates. It was previously shown that neonatal peripheral blood naïve T cells produce low levels of IL-17, which might be protective against NEC while increasing susceptibility to mucocutaneous candidiasis. However, neonatal naïve CD4 T cells inherently produced high levels of IL-22 upon stimulation by Th17 cells. We sought the molecular mechanism underpinning the developmental lack of IL-17 responses in circulating blood T cells from neonates. A genome-wide transcriptome analysis comparing neonatal and adult naïve CD4 T cells revealed major developmental differences in the expression profile of genes associated with SMAD and STAT3 signaling. Functional assays confirmed that the requirement for TGF-β in human Th17 cell differentiation increased with age. Moreover, expression of STAT3 was relatively lower in neonatal naïve CD4 T cells; accordingly, overexpression of the STAT3 gene in naïve CD4 T cells restored IL-17 production in neonatal Th17 cells. Moreover, I generated infant (<6 months old)- and pediatric (6-14 years old)-derived enteroids and co-cultured them with recombinant human IL-22 and supernatant from Th17 cells, to investigate the effect of neonatal Th17 cells, a frequent type of T cell in the intestine, on the function of intestinal epithelial cells. I hypothesized that infantsâ gut epithelium is developmentally different when compared to older children and that neonatal Th17 cell-produced IL-22 plays an important protective role in the gut. Indeed, I show that IL-22-treated enteroids upregulate antimicrobial genes such as Reg3ðâ§¸ð¸. Infant-derived enteroids also showed exaggerated proliferation and mucus production compared to pediatric enteroids, after exposure to Th17 cell supernatants. The infant gut epithelium demonstrated increased proliferation and epithelial cells differentiation as compared to pediatric enteroids, as evidenced by higher expression of Ki67 and MUC2, respectively. These data suggest developmental cross-talk between Th17 cells and gut epithelium, as a means to maintain a healthy gut in the context of an undeveloped immune system.
Understanding T cell and gut interactions in early post-natal life by Zohreh Sharafianardakani M.S., Simon Fraser University, 2015 M.S., Tarbiat Modarres University, 2012 B.Sc., Zanjan University, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Zohreh Sharafianardakani, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled Understanding T cell and gut interactions in early post-natal life submitted by Zohreh Sharafianardakani in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Examining Committee Dr. Pascal Lavoie, Professor, Pediatrics, UBC Supervisor Dr. Bruce Vallance, Professor, Pediatrics, UBC Supervisory Committee Member Dr. Martin Hirst, Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Stefan Taubert, Associate Professor, Medical Genetics, UBC University Examiner Dr. Manon Ranger, Assistant Professor, Nursing, UBC University Examiner Additional Supervisory Committee Members: Dr. Gregor Reid, Associate Professor, Pediatrics, UBC Supervisory Committee Member iii Abstract Infants are susceptible to mucocutaneous candidiasis, which manifests via oral thrush and diaper rashes. Th17 cells are responsible for limiting mucosal invasion by commensal Candida spp. Dysregulated Th17 cells are also involved in necrotizing enterocolitis (NEC) pathogenesis, a surgical complication of the gut affecting about 5% of pre-term neonates. It was previously shown that neonatal peripheral blood naïve T cells produce low levels of IL-17, which might be protective against NEC while increasing susceptibility to mucocutaneous candidiasis. However, neonatal naïve CD4 T cells inherently produced high levels of IL-22 upon stimulation by Th17 cells. We sought the molecular mechanism underpinning the developmental lack of IL-17 responses in circulating blood T cells from neonates. A genome-wide transcriptome analysis comparing neonatal and adult naïve CD4 T cells revealed major developmental differences in the expression profile of genes associated with SMAD and STAT3 signaling. Functional assays confirmed that the requirement for TGF-β in human Th17 cell differentiation increased with age. Moreover, expression of STAT3 was relatively lower in neonatal naïve CD4 T cells; accordingly, overexpression of the STAT3 gene in naïve CD4 T cells restored IL-17 production in neonatal Th17 cells. Moreover, I generated infant (<6 months old)- and pediatric (6-14 years old)-derived enteroids and co-cultured them with recombinant human IL-22 and supernatant from Th17 cells, to investigate the effect of neonatal Th17 cells, a frequent type of T cell in the intestine, on the function of intestinal epithelial cells. I hypothesized that infantsâ gut epithelium is developmentally different when compared to older children and that neonatal Th17 cell-produced IL-22 plays an important protective role in the gut. Indeed, I show that IL-22-treated enteroids upregulate antimicrobial genes such as Reg3a/g. Infant-derived enteroids also showed exaggerated proliferation and mucus production compared to pediatric enteroids, after exposure to Th17 cell iv supernatants. The infant gut epithelium demonstrated increased proliferation and epithelial cells differentiation as compared to pediatric enteroids, as evidenced by higher expression of Ki67 and MUC2, respectively. These data suggest developmental cross-talk between Th17 cells and gut epithelium, as a means to maintain a healthy gut in the context of an undeveloped immune system. v Lay Summary I studied the role of immune cells to understand why babies are more susceptible to fungal infections, and necrotizing enterocolitis, a surgical complication causing serious damage to the gut. I showed that a babyâs immune cells called âT cellsâ produce a beneficial protein called IL-22 rather than its more pathological counterpart IL-17. Furthermore, I created âmini-gutâ organoids using stem cells obtained from gut biopsies obtained from babies under the age of 6 months, to compare to similarly created mini-guts from older children. babies gut cells grew faster compared older children, and the IL-22 produced by babiesâ T cells helps their gut make more protective mucus and antimicrobial molecules. overall, the T cells of babies respond very differently than the T cells of adults and this developmental difference may be important to help ensure that the gut of babies remains healthy, even if it makes them more susceptible to diaper rashes. vi Preface Ethics approval for using human cord blood and adult peripheral blood samples was obtained from the UBC Childrenâs & Womenâs and Fraser Health Research Ethics Boards (REBs) (#H19-02989). The acquisition of human infant intestinal biopsies for Chapters 3 & 4 was approved by REBs at the UBC Childrenâs & Womenâs and Fraser Health Research Ethics Boards (#H14-03199). Chapter 1: Figure 1.1 was reprinted with permission from Elsevier (license number 5426060640142) from Joannie M. Allaire, Shauna M. Crowley, Hong T. Law, Sun-Young Chang, Hyun-Jeong Ko, Bruce A. Vallance (2018). The Intestinal Epithelium: Central Coordinator of Mucosal Immunity. Trends in Immunology. DOI: 10.1016/j.it.2018.04.002 Figure 1.2 was reprinted with permission from John Wiley and Sons (license number 5426070016885) from Natalia Torow, Mathias W. Hornef (2019). âLayered immunityâ and the âneonatal window of opportunityâ â timed succession of nonâredundant phases to establish mucosal hostâmicrobial homeostasis after birth. https://doi.org/10.1111/imm.13149 Chapter 2: A version of this chapter has been published as Razzaghian HR & Sharafian Z, Sharma AA, Boyce GK, Lee K, Da Silva R, Orban PC, Sekaly R-P, Ross CJ and Lavoie PM (2021) Neonatal T Helper 17 Responses Are Skewed Towards an Immunoregulatory Interleukin-22 Phenotype. Front. Immunol. 12:655027. doi: 10.3389/fimmu.2021.655027, licensed under a Creative Commons Attributer 4.0 International License. The introduction, results, and discussion sections have been rephrased but the figures, figure captions, and part of the method section are still the same as in the published document. vii Experiments were designed by Z. Sharafianardakani, H. R. Razzaghian, A. A. Sharma, and P. C. Orban under the supervision of P. M. Lavoie. Cord blood samples were collected by Z. Sharafianardakani, H. R. Razzaghian, A. A. Sharma, K. Lee, and R. Da Silva. Naïve CD4 T cells were isolated for figures 2.1 and 2.2, and further experiments were conducted by Z. Sharafianardakani, H. R. Razzaghian, and A. A. Sharma. Multiplex ELISA experiment and analysis from figure 2.4 were analyzed and performed by A. A. Sharma and H. R. Razzaghian. Microarray experiments (Figure 2.5) were designed and analyzed by H. R. Razzaghian and AA Sharma in collaboration with Dr. Rossâs lab at BCCHRI. For figure 2.6, western blot and flow cytometry experiments were designed, performed, and analyzed by Z. Sharafianardakani and H. R. Razzaghian. Heatmaps in figure 2.6 were part of the microarray data analysis. For figure 2.7, Z. Sharafianardakani and H. R. Razzaghian designed the experiments and performed the Meso scale assay. Z. Sharafianardakani performed flow cytometry analysis for figure 2.7 C&D. For figure 2.8A, qPCR analysis was generated by H. R. Razzaghian. IL-6 receptor experiment and phospho-STAT3 assay were designed and performed by Z. Sharafianardakani (Figure 2.8B-D). For figure 2.9, Z. Sharafianardakani designed and run the experiments including the STAT3 cloning, transfection of the STAT3 gene into naïve CD4 T cells, and differentiation of STAT3-transfected naïve T cells into Th17 cells with assistance from R. Da Silva. Figure 2.10 is part of the Meso scale array that was performed by Z. Sharafianardakani and H. R. Razzaghian. viii Chapter 3: This chapter includes experiments designed by J. Allaire and Z. Sharafianardakani under the supervision of P. M. Lavoie and B. Vallance as detailed below. The human ethics application was prepared by Z. Sharafianardakani under the supervision of P. M. Lavoie. Subject recruitment for Chapters 3 & 4 was done by Z. Sharafianardakani and J. Allaire. The biopsy preparation, isolation, and organoid culturing were performed by Z. Sharafianardakani and J. Allaire with the assistance of M. Hill and B. Poon. Chapter 4: This chapter includes experiments designed by J. Allaire and Z. Sharafianardakani under the supervision of B. Vallance and P. M. Lavoie as detailed below. Patient recruitment was done by Z. Sharafianardakani and J. Allaire. Data were analyzed and interpreted by J. Allaire and Z. Sharafianardakani. Culturing and harvesting of infant and pediatric enteroids were done by Z. Sharafianardakani, J. Allaire, and B. Poon. Isolation and differentiation of naïve T cells into Th17 cells were performed by Z. Sharafianardakani with assistance from B. Poon. RNA-sequencing samples were prepared by Z. Sharafianardakani, J. Allaire, and B. Poon. RNA-sequencing data were analyzed by C. Michalski. Immunofluorescence staining was completed by Z. Sharafianardakani, J. Allaire, and B. Poon. ix Table of Contents Abstract .......................................................................................................................................... iii Lay Summary .................................................................................................................................. v Preface ........................................................................................................................................... vi Table of Contents .......................................................................................................................... ix List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................. xiii List of Abbreviations ..................................................................................................................... xv Acknowledgments ....................................................................................................................... xix Dedication .................................................................................................................................... xxi Chapter 1: Introduction ................................................................................................................. 1 1.1 The intestinal immune niche ..................................................................................................... 1 1.1.1 Intestinal epithelial cells (IECs) ..............................................................................................................1 1.1.1.1 Cell type specification of the intestinal epithelium .......................................................................2 1.2 Development of the intestinal niche ......................................................................................... 5 1.2.1 Prenatal intestinal mucosal development ................................................................................................5 1.2.1.1 Fetal Intestinal mucosal development in human ...........................................................................7 1.2.2 Prenatal intestinal immune system development ....................................................................................8 1.2.3 Intestinal mucosal maturation during the postnatal age ..........................................................................8 1.2.4 Postnatal development of the intestinal immune system ........................................................................9 1.2.4.1 Maturation of the intestinal innate immune system during postnatal age .....................................9 1.2.4.1.1 Innate lymphoid cells .............................................................................................................11 1.2.4.1.2 Dendritic cells .........................................................................................................................12 1.2.4.2 Maturation of intestinal adaptive immune system during postnatal age .....................................13 1.2.4.2.1 Regulatory T cells ...................................................................................................................14 1.2.4.2.2 Th17 cells ...............................................................................................................................16 1.2.4.3 IL-17 ............................................................................................................................................17 1.2.4.3.1 Members of the IL-17 family .................................................................................................17 1.2.4.3.2 IL-17 functional roles in the intestine ....................................................................................18 1.2.4.4 IL-22 ............................................................................................................................................19 1.2.4.4.1 Functional roles of IL-22 in the intestine ...............................................................................19 1.2.5 Role of the microbiome ........................................................................................................................20 1.2.5.1 Role of microbiota during fetal life .............................................................................................22 1.2.6 Breast milk as a modulator of postnatal intestinal maturation ..............................................................23 1.2.6.1 Breast milk Microbiota ................................................................................................................24 1.2.6.2 Human milk oligosaccharides .....................................................................................................24 1.2.6.3 Protein and peptides in breast milk .............................................................................................25 1.2.6.4 Immune cells and other immunological compounds ...................................................................25 1.3 Necrotizing enterocolitis: intestinal disease of prematurity ................................................ 26 1.3.1 NEC clinical manifestations .................................................................................................................27 1.3.2 Affected age group ................................................................................................................................27 1.3.3 Contributing factors in developing NEC ..............................................................................................27 1.3.3.1 Prematurity ..................................................................................................................................28 1.3.3.2 Intestinal dysbiosis ......................................................................................................................28 1.3.3.3 Antibiotic therapy ........................................................................................................................28 x 1.3.4 The molecular mechanism involved in NEC development ..................................................................29 1.3.5 Current treatment strategies ..................................................................................................................29 1.4 Thesis rationale and objectives ............................................................................................... 30 Chapter 2: Neonatal T-helper 17 responses are skewed towards an immunoregulatory interleukin-22 phenotype ............................................................................................................. 31 2.1 Introduction ............................................................................................................................. 31 2.2 Material and Methods ............................................................................................................. 33 2.2.1 Blood sample collection ........................................................................................................................33 2.2.2 Naïve T cell purification .......................................................................................................................34 2.2.3 RNA extraction and gene expression profiling .....................................................................................35 2.2.4 Real-time qPCR ....................................................................................................................................37 2.2.5 Stimulation of naïve T cells ..................................................................................................................37 2.2.6 STAT3 phosphorylation ........................................................................................................................38 2.2.7 STAT3 gene cloning .............................................................................................................................38 2.2.8 STAT3 Transfection .............................................................................................................................39 2.2.9 SMAD2/3 phosphorylation ...................................................................................................................40 2.2.10 Western Blot ....................................................................................................................................41 2.2.11 Data analysis ....................................................................................................................................41 2.3 Results ....................................................................................................................................... 42 2.3.1 Neonatal T cells are inherently biased towards high IL-22 cytokine production .................................42 2.3.2 Unbiased transcriptome analyses reveal altered TGF-β signaling in neonatal T cells .........................45 2.3.3 The requirement for TGF-β during the differentiation of human Th17 cells is age-dependent ...........48 2.3.4 Reduced STAT3 limits Th17 cell differentiation of neonatal T cells ...................................................50 2.4 Discussion ................................................................................................................................. 54 Chapter 3: Establishment of human infant intestinal organoid model ..................................... 58 3.1 Introduction ............................................................................................................................. 58 3.2 Material and Methods ............................................................................................................. 60 3.2.1 Ethics statement ....................................................................................................................................60 3.2.2 Inclusion criteria ...................................................................................................................................61 3.2.3 Exclusion criteria ..................................................................................................................................61 3.2.4 Patient recruitment ................................................................................................................................61 3.2.5 Sample collection ..................................................................................................................................62 3.2.6 Clinical Data Collection ........................................................................................................................62 3.2.7 Isolation and growth of infant intestinal organoids ..............................................................................63 3.2.8 Optimization of infant intestinal organoids ..........................................................................................63 3.2.8.1 Enzymatical splitting ...................................................................................................................64 3.2.8.2 Mechanical splitting ....................................................................................................................64 3.2.9 Growth media and passaging ................................................................................................................64 3.2.10 Harvesting and fixation of human enteroids for immunohistochemistry .........................................65 3.2.11 Immunofluorescence and staining of enteroid cultures ...................................................................65 3.3 Results ....................................................................................................................................... 65 3.3.1 Collecting infant intestinal biopsies and establishing a biobank ..........................................................65 3.3.2 Human infant intestinal organoid cultures ............................................................................................66 3.3.3 Optimization of human infant intestinal organoid cultures ..................................................................67 3.4 Discussion ................................................................................................................................. 70 Chapter 4: Cross-talk between Th17 cells and intestinal epithelium promotes gut health in infants through the actions of IL-22 ........................................................................................... 74 xi 4.1 Introduction ............................................................................................................................. 74 4.2 Material and Methods ............................................................................................................. 76 4.2.1 Ethics statement ....................................................................................................................................76 4.2.2 Naïve CD4 T cell isolation and differentiation .....................................................................................76 4.2.3 Collection of infant and pediatric biopsies ...........................................................................................77 4.2.4 Generation and maintenance of human infant and pediatric-derived enteroids ...................................77 4.2.5 Stimulation of human enteroids ............................................................................................................79 4.2.6 Harvesting and fixation of human enteroids for immunohistochemistry .............................................79 4.2.7 Immunofluorescence and staining of enteroid cultures ........................................................................79 4.2.8 RNA Extraction and qPCR analysis of enteroid cultures .....................................................................80 4.2.9 RNA Seq analysis .................................................................................................................................80 4.2.10 Statistical analysis ............................................................................................................................81 4.3 Results ....................................................................................................................................... 81 4.3.1 Neonatal Th17 cell-derived cytokines support the growth of infant enteroids .....................................81 4.3.2 IL-22 is the key cytokine underlying the effects of the human infant Th17 cell supernatant ..............84 4.3.3 IL-22 upregulates anti-microbial pathways in both infant and pediatric enteroids ..............................87 ................................................................................................................................................................ 88 4.3.4 Il-22 induces the JAK-STAT-signaling pathway and attenuates stemness ..........................................89 4.4 Discussion ................................................................................................................................. 92 Chapter 5: Conclusion ................................................................................................................. 97 5.1 Summary of findings ............................................................................................................... 97 5.2 Future directions .................................................................................................................... 105 Bibliography ............................................................................................................................... 109 xii List of Tables Table 1.1 The innate lymphoid cell family ................................................................................... 13 Table 2.1 List of primers used for STAT3 gene analysis ............................................................. 39 Table 3.1 Data set of human infant enteroids ............................................................................... 66 Table 4.1 List of genes differentially expressed in IEC subtypes in infant enteroids .................. 89 xiii List of Figures Figure 1.1. Representative small intestinal epithelium cell types ................................................... 4 Figure 1.2. Intestinal mucosal immune system development ....................................................... 15 Figure 2.1. Representative flow cytometry analysis of naïve CD4 T cells ................................... 35 Figure 2.2. Representative of gating strategy and flow cytometry analysis of naïve adult and neonatal CD4 T cells ..................................................................................................................... 36 Figure 2.3. Genetic map of cloning human STAT3 gene in pIRES2EGFP vector ...................... 39 Figure 2.4. Neonatal T cells are inherently biased against Th17 responses independent of antigen-presenting cell signals. ..................................................................................................... 44 Figure 2.5. Genome-wide gene expression comparison identifies a unique developmental Th22 signature at baseline in naïve neonatal CD4 T cells. .................................................................... 47 Figure 2.6. Expression and activity of SMAD2 and SMAD3. ..................................................... 49 Figure 2.7. Neonatal Th17 cells differ from adult T cells in their cytokine responses and are skewed toward high IL-22 production .......................................................................................... 50 Figure 2.8. STAT3 expression is down-regulated in neonatal Th17 cells. .................................... 51 Figure 2.9. Reduced STAT3 expression limits neonatal Th17 cell differentiation. ...................... 53 Figure 2.10. Effect of IL-10 blocking on IL-17 production in neonatal and adult naïve CD4 T cells ............................................................................................................................................... 54 Figure 3.1. Organoid culture of epithelial cells derived from infantsâ intestines ......................... 68 Figure 3.2. Surface area and the number of buds of infant enteroids ........................................... 68 Figure 3.3. Representative immunohistochemistry staining of infant enteroids .......................... 69 Figure 4.1. Neonatal Th17 cell-derived cytokines support infant organoids growth ................... 84 xiv Figure 4.2. IL-22 is the key cytokine underlying the effects of the human infant Th17 cell supernatant .................................................................................................................................... 86 Figure 4.3. IL-22 induces differential transcriptomic changes in infant and pediatric enteroids . 88 Figure 4.4. Il-22 attenuates stemness-associated pathways and induces the JAK-STAT-signaling pathway ......................................................................................................................................... 92 Figure 5.1 Schematic model explaining the cross-talk between Th17 cells and IEC in the human infant intestine ............................................................................................................................. 105 xv List of Abbreviations AhR Activate aryl hydrocarbon AMPs anti-microbial peptides APCs antigen-presenting cells ASC adult stem cell BATF Basic Leucine Zipper ATF-Like Transcription Factor BMP Bone Morphogenic protein BC British Columbia BF bright field CBS Canadian Blood Service CBMC cord blood mononuclear cell CCR7 CC-chemokine receptor CCS cold chelating solution CD cluster of differentiation ChIP-Seq chromatin immunoprecipitation sequencing CLP common lymphoid progenitor CRAMP cathelicidin-related antimicrobial peptide CFSE carboxyfluorescein succinimidyl ester CXCR C-X-C motif chemokine receptor DC dendritic cell dsRNA double-stranded RNA ECM extracellular matrix xvi EGF epidermal growth factor ESC embryonic stem cells EV Empty Vector FACS fluorescence activated cell sorting Fcgbp Fcg-binding protein FAE follicle-associated epithelium FDR False discovery rate FMO Fluorescence minus one FOXP3 forkhead box protein 3 FUT2 fucosyltransferase 2 GA gestational age GATA3 GATA binding protein 3 GALT gut-associated lymphoid tissues G-CSF granulocyte- colony stimulating factor GM-CSF granulocyte macrophage-colony stimulating factor GI gastrointestinal GF germ free HMO human milk oligosaccharides Id2 inhibitor of DNA binding 2 IECs intestinal epithelial cells IHC immunohistochemistry ILCs innate immune lymphocytes IFN interferon xvii IL Interleukin iPSC induced pluripotent stem cells IRAK1 Interleukin 1 receptor associated kinase 1 IRF Interferon Regulatory Factor ISC intestinal stem cells LPS Lipopolysaccharide LP Lamina Propria MC mucocutaneous candidiasis MLN Mesenteric lymph nodes miR146a MicroRNA 146a moDC monocyte-derived dendritic cells MUC1 mucin-1 NEC Necrotizing enterocolitis NF-kB Nuclease factor kappa B NK cell Natural killer cell NOD Nucleotide-binding oligomerization domain PPs Peyerâs patches PSC pluripotent stem cell qPCR Quantitative polymerase chain reaction RELMb resistin-like molecule RegIIIg regenerating islet-derived protein IIIg RIN RNA Integrity Numbers RORgt retinoic acid receptor-related orphan receptor gt xviii scRNA-seq single cell RNA-sequencing SILT solitary isolated lymphoid tissue sIgA secretory immunoglobulin A SFB Segmented filamentous bacteria SLEA Sample level enrichment analysis SLO secondary lymphoid tissue SMAD Small Mothers Against Decapentaplegic STAT Signal Transducer and Activator of Transcription TGF- β Transforming Growth Factor beta Th T helper TLR toll-like Receptor TFF3 trefoil factor 3 Treg regulatory T cells TSLP Thymic stromal lymphopoietin Zg16 Zymogen granule protein16 xix Acknowledgments I would like to acknowledge that my Ph.D. thesis research at BC Childrenâs Hospital was performed on the traditional, ancestral, and unceded territory of Coast Salish peoples â xÊ·mÉÎ¸kÊ·ÉyÌÉm (Musqueam), Sḵwx̱wú7mesh (Squamish), and SÉlÌílwÉtaÊ/Selilwitulh (Tsleil-Waututh) Nations. Many thanks to the funding provided by the Canadian Institute of Health Research (CIHR), BC Childrenâs Hospital, and Healthy Start research team through personal scholarships or grants to Dr. Lavoie. I would like to thank my senior supervisor, Dr. Pascal Lavoie for his constant support and dedication to my educational goals. I valued all the optimism, courage, knowledge, and patience that Pascal gave me during my Ph.D. journey. I would also like to express gratitude to my committee member Dr. Bruce Vallance, who gave me the opportunity to conduct infant organoid research in his lab and allowed me to be trained by his friendly and supportive lab members. I would also like to acknowledge committee members Dr. Martin Hirst and Dr. Gregor Reid for providing constructive feedback on my project and reviewing this thesis. My special thanks go to the present and past members of Lavoie lab. Ashish, who walked me through the transition from the Biochemistry field to the world of Immunology. Christina, who was a constant source of support and read the early drafts of my thesis. Elizabeth, who thought me many techniques and the foundations of flow cytometry. I also was fortunate to mentor many talented undergraduate and summer students; I had a wonderful time with them including Rachel, Jolie, and Bethany. Bethany, thanks for the past two years of help, support, and editing my thesis! You were the best student (and of course a sweet friend) that I could ask for and never forget all those moments that we spent on the Infant organoids project together! All those happy and sad moments! xx My special acknowledgment is dedicated to my wonderful mentor, Dr. Joannie Allaire. Joannie, thank you for always supporting me and being an amazing source of positive energy from the first day that we met. Thank you for showing me how to change gears and dive into the gastroenterology world! Thank you for always being on my side! You are not only a wonderful mentor but also a trustworthy friend and a great listener. I am very grateful to have a friend like you in my life. Last but most definitely not least, I would like to thank my family and friends who have supported me throughout this journey. My parents and in-laws who made themselves available, traveling so far to take care of my baby Nick and provided a supportive environment for me to write my thesis. Finally, a very special thanks to my husband Amir. Amir, thank you for your unconditional love and support, for never being disappointed in me, for the sense of humour that kept me laughing after every time crying, and for all the time you watch our baby so that I could have extra time to work on my thesis. xxi Dedication To Amir, who has been my rock and my source of unwavering support throughout my academic journey and Nick for his unconditional love. 1 Chapter 1: Introduction 1.1 The intestinal immune niche The primary function of the gut is to digest and absorb nutrients. It also plays an effective role as a barrier against invading pathogens and prevents the escape of harmful macromolecules from the gut lumen into the body1. However, the establishment of an efficient immune response against invasive pathogens must be balanced with maintaining tolerance against the microbiota. In the adult intestine, the presence of diverse and competitive microbiota, a protein-enriched mucus layer, highly proliferative epithelium, and a developed adaptive immune system support this dual role. This differs entirely in the neonatal intestine, due to the nutritious gut environment and lack of microbial competitors, facilitating the establishment of incoming pathogens2. In neonates, both innate and adaptive immune systems are functionally different from their adult counterparts3. This also applies to the neonatal gut and intestinal immune system, which must adapt to the rapid colonization by various microbes, including potential pathogens. In the following sections, the human gut-immune axis will be described before the discussion of the pre-and postnatal development of mucosal immunity. 1.1.1 Intestinal epithelial cells (IECs) The intestinal epithelium represents a single layer of intestinal epithelial cells (IECs) with tight junctions that physically separate the luminal bacteria and immune cells in the mucosa4. To achieve the functions of digestion and absorption, IECs require specialization and high metabolic activity, with a relatively rapid turnover throughout the life of the organism5. 2 IECs play an important role in orchestrating a variety of immune functions such as establishing barrier integrity and maintaining homeostasis of the intestine. The structure and cellular composition of IECs differ significantly in the small and large intestines. In the small intestine, structures called âvilliâ project into the lumen, increasing mucosal surface area and nutrition absorption. In contrast, the colon lacks villi, resulting in a flat mucosal surface; presumably due to the impact of stool, since this structure minimizes potential damage in the large bowel6. The gut epithelium contains a large variety of IECs with specific roles (Figure 1.1) and as detailed below. 1.1.1.1 Cell type specification of the intestinal epithelium Enterocytes: Specific enterocytes known as columnar cells are the most predominant type of IECs in the gut epithelium (small and large intestine), responsible for absorbing nutrients and water7. Enterocytes contain specialized apical membranes with particular transporters that uptake sodium ions from the lumen to their cytoplasm8. Paneth cells: Paneth cells are secretory cells located at the bottom of small intestinal crypts and are responsible for two main functions. First, they are responsible for the secretion of anti-microbial peptides (AMPs). These include a-defensin, RegIII-b, and RegIII-g, which regulate interactions between microbiota and their hosts through antibacterial activity against both Gram-positive and Gram-negative microbes4,7. Recent studies show exogenous bacteria and bacterial antigens induce Paneth cells to produce a-defensin. Commensal microbiota enhances the production of a-defensin in Paneth cells by TLR-MyD88 signaling, presumably as a protective function against pathogen invasion9. 3 Second, Paneth cells play an important role in establishing the intestinal stem cell (ISC) niche by providing essential signals required for stem cell regeneration and survival such as epidermal growth factor (EGF), Wnt3, and Notch10. It has been shown that the produced Wnt by Paneth cells, the ligand of b-catenin signaling pathway, is essential for the maintenance of ISCs11. Goblet cells: Intestinal goblet cells can be characterized by the production and secretion of the glycosylated mucin protein Muc2, which forms a protective mucus barrier on top of the intestinal epithelium12. Goblet cells also secrete trefoil factor 3 (Tff3), a tissue-protective factor, that stimulates epithelial restitution and mucosal repair through inhibition of apoptosis, supporting cell migration, and angiogenesis13. Proinflammatory Resistin-like molecule (RELMb) is another factor produced by goblet cells. Following infection by various enteric pathogens, RELMb strongly regulates the expression of inflammatory cytokines14,15. RELMb has been shown to have antimicrobial activity by killing selective Gram-negative bacteria, such as Pseudomonas aeruginosa and C. rodentium 16. To form the mucus layer, goblet cells secret Muc2 and other substances including Clca1, zymogen granule protein16 (Zg16), Fcg-binding protein (Fcgbp), and Agr28. Fcgbp binds directly to MUC2 to support the MUC2 network; Agr2 is a disulfide isomerase-like protein in the endoplasmic reticulum of goblet cells and is released in the gut lumen. Agr2 is also involved in the differentiation of goblet cells. Zg16, a lectin-like protein, helps the aggregation of Gram-positive bacteria by binding to peptidoglycan7,17. 4 Enteroendocrine cells: Enteroendocrine cells consist of eight cellular subtypes, producing diverse types of peptide hormones, and are found in both the colon and small intestine7. M cells: M cells are small intestine-specific cells located in the follicle-associated epithelium (FAE) overlying Peyerâs patches, contributing to the uptake of antigens and their passaging to the immune cells7. Tuft cells: Tuft cells are chemosensory epithelial cells in the small intestine and colon, responsible for recognizing luminal pathosymbionts and helminths, and secrete IL-25 as an inducer of ILC2 expansion18. Figure 1.1. Representative small intestinal epithelium cell types Enterocytes are the most abundant type of IECs responsible for nutrient absorption. Paneth cells are located at the bottom of intestinal crypts and produce AMPs such as RegIII-b, RegIII-g, and a-defensin. Goblet cells are secretory cells responsible for the production of AMP and mucin, forming a mucus barrier. Enteroendocrine cells are responsible for producing hormones. M cells contribute to trapping pathogens and passage them to immune cells. Reprinted with permission from Allaire et al7. 5 1.2 Development of the intestinal niche 1.2.1 Prenatal intestinal mucosal development Though all vertebrates follow a similar embryogenic gut tissue development process, the length of the gestational period and tissue maturity at birth is entirely different between species19. Pre-existing knowledge regarding the development of the intestinal immune system derives largely from mouse studies. The short duration of gestation in mice (about 20 days) leads to incomplete maturation of intestinal mucosal surfaces, requiring drastic functional and structural changes in the postnatal period. Conversely, the human gut mucosal surface is more holistically developed and differentiated at birth due to the longer gestational period. As a result, studying murine models to reveal the process of gut development may not be appropriate to fully understand the gut development process and the ontogeny of host-microbial cross-talks in humans. In both mice and humans, a safe transition from the comparatively sterile in utero environment to communication with commensal and pathogenetic microbes after birth must be established. Nevertheless, due to ethical and medical concerns, accessing human mucosal tissue samples from both healthy and diseased newborns for research studies is limited2. In the following paragraphs, I summarized the current knowledge of fetal intestinal development based on mice work, and when available, from human studies. In mice, small intestinal villi appear between gestational days 15 (E15) and E17; proliferating epithelial cells are confined in the intervillous region20. While the overall tissue structure is formed in fetal mice, constant renewal of epithelium including enterocyte migration and exfoliation at the villus tip is not fully established. Moreover, crypt-based Paneth cells are 6 absent in the murine fetal intestine compared to adult mice. These two factors make neonatal mice susceptible to enteric pathogens in the absence of constant surface renewal and secretion of antimicrobial peptides21. Intestinal crypts, including stem cells and transient amplifying cells, develop 10-12 days after birth in newborn mice5. There are about four to six intestinal stem cells per crypt in the small intestine; these stem cells can differentiate into four types of cells, including enterocytes, enteroendocrine cells, Paneth cells, and goblet cells5. In adult mice, small intestinal crypts offer a proper niche for intestinal stem cells and proliferating transient amplifying cells. There is a rapid turnover of transient amplifying cells along the crypt-villus axis and exfoliation at the villus tip2. However, in neonatal mice, the renewal of IECs is not as consistent as in adult counterparts, and the proliferation, migration, and exfoliation speeds of epithelial cells are very slow22. The intestine of newborn mice is devoid of Paneth cells and a lack of AMPs produced by Paneth cells, such as a-defensin, b-defensin, and lysozyme exists. Instead, Cathelicidin-related antimicrobial peptide (CRAMP) is expressed by neonatal IECs, which can provide protection from enteric pathogens and fill the transition during the postnatal period to the presence of mature Paneth cells23. During the weaning period, production of CRAMP is decreased as mature Paneth cells begin to produce defensin24. 7 1.2.1.1 Fetal Intestinal mucosal development in human Due to a longer gestational period, newborn humans have a mature intestinal crypt-villus architecture with consistent stem cell proliferation and differentiation to subtypes of IECs24. Although many RNA sequencing (RNA-seq) studies have focused on the diversity and state of intestinal cell types in the adult intestine, the timing and origin of intestinal development are not completely understood. Recent single cell RNA-sequencing (scRNA-seq) studies have investigated intestinal cell composition and their anatomical locations25,26. Elmentaite et al. studied the development of intestinal cell types throughout life by providing scRNA-seq on distinct tissue regions of the second trimester and adult intestine and draining mesenteric lymph nodes (MLN). A developmental switch was shown in epithelial cell populations from fetal tissues as compared to pediatric and adult ones, with an increased number of epithelial stem cells and goblet cells at the fetal stage26. Moreover, Tuft cells were found to impact immune sensing due to their expression of FCGR2A, which encodes a receptor-activated in response to IgG. IgG can be transported to the utero via the placenta and can provide a potential route to activate tuft cells26. Another scRNA-seq study characterizing human intestinal morphogenesis showed that despite similar types of IECs observed in fetal and adult tissues, the proportion of immature cells was higher in the fetal tissue compared to the adult tissue. This frequency of immature cells might might contribute to premature infant diseases such as necrotizing enterocolitis (NEC)25. 8 1.2.2 Prenatal intestinal immune system development In both murine and human neonates, secondary lymph structures, such as MLN and Peyerâs patches (PP), are formed before birth. However, the size and development of germinal centers are dependent on the postnatal microbial colonization of the gut24,27,28. Additionally, cryptopatches and isolated lymphoid follicles appear in mice after birth24. In parallel with bacterial localization, combinations of endogenous and exogenous factors mediate the localization of lymphocytes to the gut mucosa. During the first four weeks after birth, innate immune lymphocytes (ILCs), natural killer (NK) cells, and T helper (Th)2-like cells migrate from the murine fetal liver to the gut mucosa via endogenous signals29. The interaction between ILCs and stromal cells leads to the development of mucosal lymphoid tissue. ILCs are classified into three types and are regulated by two main transcription factors, retinoic acid receptor-related orphan receptor gt (RORgt) and Inhibitor of DNA binding 2 (Id2), to express chemokines such as C-X-C motif chemokine receptor 5 (CXCR5) and CC-chemokine receptor 7 (CCR7). Exposure to exogenous factors such as maternal-derived microbes affects the intestinal development of the fetus. For example, maternal microbial products can activate aryl hydrocarbon (AhR), detected in the placenta and breast milk, stimulating gut development30. 1.2.3 Intestinal mucosal maturation during the postnatal age The anatomical organization of the epithelium and localization of innate immune signaling pathways associated with toll-like receptor (TLR) and IL-1 receptor families show drastic age-dependent differences, especially in mice due to the shorter gestational period compared to 9 human22,31. In neonate mice, at days 10-12 of postnatal age, the development of crypt stem cells and transient amplifying cells lead to the initiation of epithelium renewal. However, epithelial cell migration along the crypt-villus axis is relatively slow compared to adult mice and there is no continuous epithelial turnover in neonates5. Upon development of the crypts, crypt-based Paneth cells appear and are responsible for producing a variety of AMPs, including a-defensin and cryptdin-related sequence peptides32. Production of c-type lectin regenerating islet-derived protein IIIg (RegIIIg) contributes to host-microbial homeostasis and increases remarkably during the postnatal age2. Although intestinal mucin genes are expressed before birth, goblet cells are not fully developed, expression of major mucins such as MUC2, 3, and 5 is limited after birth, and a protective mucus barrier does not form in early postnatal age. As a result, the poorly developed mucus layer affects host-microbial homeostasis, particularly in premature babies, leading to an increased risk of infection8,22. In diseases that affect premature infants such as bacterial meningitis, the lack of a well-developed mucus layer allows E.coli and group B Streptococci to translocate out of the intestinal tract. It has also been shown there are fewer intestinal goblet cells and less mucus in babies with NEC33,34. 1.2.4 Postnatal development of the intestinal immune system 1.2.4.1 Maturation of the intestinal innate immune system during postnatal age While there are significant differences in neonatal and adult innate immune recognition, the neonate immune system is described to be âdifferentially adaptedâ rather than âimmatureâ22. 10 Directly after birth, exposure to environmental microorganisms regulates the ability of epithelial cells to activate innate immune pathways35. Upon exposure of microbiota to epithelial cells, IECs express innate immune receptor molecules such as TLR, Rig I-like receptor, nucleotide-binding oligomerization domain (NOD)-like receptor, and the inflammasome family36. These innate immune receptors facilitate recognition of the conserved microbial structure and induce the production of AMPs. Studies in both neonate mice and human neonatal IECs show low expression of double-stranded RNA (dsRNA) TLR3 associated with age-dependent susceptibility to rotavirus infection24,37. Flagellin receptor TLR5 expression is induced in neonatal mice epithelium in the pre-weaning period and affects the composition of microbiota throughout life38. The sensitivity of the intestinal epithelial surface to environmental microbial ligands is suppressed by developmental and adaptive mechanisms. Postnatal epithelial cells exposed to exogenous endotoxins such as lipopolysaccharide (LPS) from Gram-negative bacteria induce upregulation of microRNA 146a (miR146a). High levels of miR146a, along with endogenous mediator S100A8/9, lead to post-transcriptional downregulation of the innate immune signaling molecule IL-1-associated kinase1(Irak1). This behaves as a negative regulatory mechanism for toll-like receptor 4 (TLR4) to induce immune tolerance which can facilitate microbial colonization during the early stages of life39â41. 11 1.2.4.1.1 Innate lymphoid cells The neonate thymus is significantly dominated by group 3 ILCs (ILC3), which are involved in protecting against invasion or colonization of pathogens and establishing intestinal commensal microorganisms42,43. ILCs are recognized as an important moderator of tissue homeostasis and protect against microorganisms via tissue remodeling after damage44. ILCs develop from common lymphoid progenitor cells (CLP) and share similar morphology with lymphocytes. ILCs are classified into three groups subject to the expression of surface markers, transcription factors, and cytokines45. Group 1 ILCs (ILC1) rely on T-bet and secrete IFNg (similar to Th1 cells) after stimulation with IL-12, IL-15, and IL-1846,47. In both humans and mice, ILC1 is mainly located in the lamina propria(LP)48. In the presence of transcription factors RAR-related Orphan Receptor alpha (RORat) and GATA binding protein 3 (GATA3) and following stimulation with IL-25 and thymic stromal lymphopoietin (TSLP), Group 2 ILC (ILC2) can secrete Th-2-like cytokines IL-5 and IL-1349. ILC2 are mainly found in fetal gut tissue and, in later stages, in the skin, lung, intestinal submucosa, and lymphoid organs, protecting against helminth infection50. ILC3 produce T helper17 (Th17) and Th22-like cytokines IL-17, IL-22, and granulocyte macrophage-colony stimulating factor (GM-CSF) after stimulation with IL-23, IL-1b, RORgt, and AHR transcription factors51. ILC3 is mainly located in the intestinal mucosal tissue, protecting the intestinal mucosa from infections and maintaining intestinal homeostasis. The protective role of ILC3, similar to that of Th17 cells, is primarily mediated through secretion of IL-17, IL-22, and GM-CSF, activating epithelial cells to produce AMPs such as RegIIIb and RegIIIg to remove pathogens4. ILC3 also regulates T cell responses to commensal bacteria through the 12 expression of MHC-II molecules, supporting the tolerance function of intestinal dendritic cells (DC) via GM-CSF secretion, and adjusting epithelial glycosylation35 (Table 1.1). 1.2.4.1.2 Dendritic cells Dendritic cells (DCs) are important in triggering and shaping the immune response in mucosal tissues. They are distributed in mucosal surfaces and reside in gut-associated lymphoid tissues (GALT) including Peyerâs patches (PPs) and solitary isolated lymphoid tissue (SILT) or LP52. Human DCs are defined with CD103+ CD11b+ markers53. DCs in the intestinal mucosa can acquire antigens from M cells and produce IL-23 upon activation. IL-23 production promotes Th17 differentiation and expression of IL-17 and IL-22 in ILC3 and Th17 cells. In the resting condition, DCs sample antigens and produce IL-10 to establish a non-inflammatory environment. However, during infection, IECs produce a large quantity of the chemokine CCL20 to recruit DCs into the epithelial layer of PPs. DCs subsequently produce co-stimulatory molecules, leading to naïve T cell differentiation into effector cells. DCs are also found in the LP, where they sample luminal antigens and present them to naïve T cells in the MLN. In a homeostatic environment, the encounter between migrating DCs and naïve T cells in the MLNs leads to the differentiation of naïve T cells to regulatory FOXP3+ Treg cells, particularly in the presence of TGFb, which are returning to LP to suppress the production of inflammatory responses to harmless antigens in food 24,52â54. 13 Table 1.1 The innate lymphoid cell family ILCs Transcription factors Cytokines Effector cytokines ILC1 T-bet IL-12, IL-15, IL-18 IFNg, TNFa ILC2 GATA3 IL-25, IL-33 IL4, IL-5, IL-9, IL-13, Arg ILC3 RORgT IL-1b, IL-23, AHR ligands IFNg, TNFa, IL-22, GMCSF 1.2.4.2 Maturation of intestinal adaptive immune system during postnatal age In adults at the steady state, an immense number of antigen-experienced T cells and plasma cells are found in the intestinal tract, while the adaptive immune system in the rest of the body is restricted to the secondary lymphoid organs and circulatory system 22. Thus, this activation of enteric proinflammatory responses in the gut epithelium involving non-pathogenic bacteria is called âphysiological inflammationâ22. Under homeostatic conditions, this occurs when an appropriate compensatory inhibitory control is not detrimental to the host. It is suggested the presence of active lymphocytes is linked to balanced interaction between the adaptive immune system and luminal antigens including microbiota, diet, and environment-derived antigens22,55. After birth, neonates begin to reach the same level of adult âphysiological inflammationâ once solid food is introduced. In mice, this transition begins at the age of 2 weeks and ends at week 6; the transition occurs in humans during the first two years of life22. 14 1.2.4.2.1 Regulatory T cells Forkhead box protein 3 (FOXP3)+ regulatory T (TReg) cells are recruited to the gut mucosa following bacterial colonization to secrete anti-inflammatory IL-10, preserving the epithelial barrier56,57. Tregs contribute to the establishment of tolerance (oral immunity) to microbiota, diet, and environment-derived antigens. The population of thymus-derived Treg cells that establish peripheral tolerance to self-antigens is found in the neonatal small intestine of mice in the 4th week of life. This Treg cell population expresses transcription factor RORgt 22,58. Previous studies in human fetuses demonstrate the high frequency of Treg cells in fetal lymph nodes, including MLNs, that continue until infancy. One study suggested migratory DCs and epithelial cells can play a role as local antigen-presenting cells (APCs). It is proposed they induce the proliferation of Foxp3+ Treg cells by presenting antigens to the T cells and, in the presence of retinoic acid and TGF-b, increase Treg pools. Treg cells are also induced by gut commensals, such as Lactobacillus, Bifidobacterium, Bacteroides, Clostridium, and Streptococcus, and bacterial metabolites, including butyric acid and propionic acid 59â61. Studies in murine pups showed a large population of B cells and T cells leave the primary lymphoid tissue at birth and reside in secondary lymphoid tissues, such as the draining MALT and GALT, but are not found at effector sites. However, the maturation of neonatal CD4+ TCRab+ cells is delayed throughout postnatal age despite the rapid appearance of microbiota and antigen exposure62. Two main factors that suppress the responsiveness of CD4+ T cells during the postnatal period at the mucosal surface are maternal secretory immunoglobulin A (sIgA) and neonatal Tregs. Torow et al. showed that in mice breast milk-derived sIgA prevents 15 immune stimulation of CD4 T cells by reducing the translocation of luminal antigens. This study also described how Treg cells located in the neonateâs PPs inhibit intestinal CD4+ T-lymphocyte maturation. Moreover, germinal center formation in PPs, due to constant exposure to microbiota and food-derived antigens, does not occur in neonates and the translocation of activated T cells to the LP and the epithelium-associated compartment starts around weaning63 (Figure 1.2). Figure 1.2. Intestinal mucosal immune system development At birth, the small intestine is colonized by a low-diversity microbiota that induces innate immune cells. T and B cells leave the thymus and bone marrow and reside in secondary lymphoid tissue (SLO), including mesenteric lymph nodes and GALT. Maternal IgA and neonatal thymus derived Treg (tTreg) cells form the initial state of the adaptive immune system. During the weaning period, ingestion of solid food leads to an increase in microbial diversity. Antimicrobial producing Paneth cells are formed, and the production of mucus is upregulated by goblet cells. Goblet cells also begin to introduce microbial antigens to dendritic cells (DCs) in the lamina propria. In SLO, DCs present antigens to naïve T cells and trigger adaptive immune activation characterized by the production of pro-inflammatory 16 cytokines such as TNFa, and IFNg. Simultaneously, RORgt+ Tregs are induced, promoting tolerance and regulating the mucosal immune system to have appropriate responsiveness to immune stimuli later in life. After the weaning period, the composition of the microbiota is stabilized and less sensitive to endogenous stimuli such as incoming pathogens or antibiotic treatment. In homeostatic conditions, luminal antigens play a role as antigen-presenting cells, while the Tregs cells population is dominated to induce IgA production by plasma cells. Reprinted with permission from Hornef et al22. 1.2.4.2.2 Th17 cells CD4 T cells are predominant in LP that are activated by DCs and express memory markers such as CD45RO. Th17 are dominant in the intestinal mucosa, secreting important cytokines for the local defense such as IL-17 and IL-2264. Effector CD8 T cells are also found in LP at the steady state, which is responsible for the production of cytokines, and cytotoxic activity in response to pathogen invasion65. Th17 cells are primarily known for their role in the pathogenicity of inflammatory disorders. However, Th17 cells are residents within the intestinal tissue after interaction with the gut microbiota to induce a homeostatic environment. Gut microbiota plays an essential role as a potential stimulus of Th17 differentiation. In mice, segmented filamentous bacteria (SFB) can orchestrate the differentiation of Th17 cells in the presence of nuclear transcription factor RORgt to produce IL-17A, IL-17F, IL-21, and IL-22. By binding IL-17A/F to receptors on epithelial and mesenchymal cells, the two cytokines induce CXCL chemokines and cytokines, such as IL-6 and G-CSF, which promote neutrophil recruitment and differentiation, respectively66â68. Germ-free mice and newborn mice lack Th17 cells in LP, emphasizing the role of microbiota in the differentiation of these cells69,70. Thus, the appearance of Th17 cells in the LP can be 17 associated with the colonization of commensal bacteria during weaning. Gut microbiota, such as SFB, induce IL-1b secretion of intestinal macrophages that bind to T cell-expressed IL-1 receptor to stimulate Th17 cell differentiation66. SFB is well-established to be attributed to a commensal bacterium that induces Th17 cells in murine LP but the existence of an analogue bacteria in humans is still debatable. One study identified Bifidobacterium adolescent (B. adolescent) as a human symbiont bacterial species that can induce Th17 cells in a mouse gut71. In the next two sections, a discussion of two key cytokines produced by ILC3, and Th17 cells will be expanded upon. 1.2.4.3 IL-17 1.2.4.3.1 Members of the IL-17 family The IL-17 cytokine is secreted by different types of cells, including CD4+ T cells, gd T cells, ILCs, and natural killer (NK) cells72. The IL-17 family includes six members, defined as IL-17A to IL-17F. IL-17A and IL-17 F share 50% of amino acid sequence homology and are the most well-characterized subtypes of the IL-17 family. These two subtypes make homo- or heterodimers and bind to expressed IL-17 receptors to stimulate downstream signaling pathways and activate inflammatory responses73. Other members of the IL-17 family are produced by cell types that include chondrocytes (IL-17B), epithelial cells (IL17C), skeletal muscle, brain, heart, lung tissue (IL-17D), and mononuclear leukocytes (IL17E)74. IL-17C aids in promoting cytokines and AMPs production in the gastrointestinal tract, protecting against microbial infection and pathogenesis of autoimmune diseases including psoriasis and multiple sclerosis75. IL-17C mediates barrier 18 protection by promoting the expression of the tight junction molecule occludin by colonic epithelial cells76. IL-17D is also found in different organisms and is only produced by resting CD4+ T cells and CD19+ B cells. The biological function of IL-17B and IL-17D remains poorly understood77. IL-17E (also known as IL-25) promotes the production of Th2 cytokines including IL-4, IL-5, and IL-13 to recruit eosinophils and is involved in host defense against helminth and parasitic infections77,78. 1.2.4.3.2 IL-17 functional roles in the intestine In general, IL-17 has an inflammatory role; IL-17 receptors (IL-17R) are highly expressed by stromal cells and fibroblasts, suggesting a general role of IL-17 in the inflammatory response. Thus, IL-17 is detected during early chronic inflammation and autoimmunity in the serum79. IL-17 concurrently with IL-22 is responsible to protect against pathogens by maintaining the strength of the intestinal mucosal barrier and by the induction of AMPs including b-defensin and RegIIIg. Nevertheless, upon pathogenic infection and loss of barrier integrity, IL-17 is produced by ILC3 or Th17 cells, targeting epithelial cells to induce the expression of CXCL9 and CXCL10 chemoattractants, which bind to CXCR3 expressed by IFNg-producing cells. IL-17 also enhances secretion of CXCL8 (IL-8), CXCL1, CXCL2, and CXCL5 and growth factors such as G-CSF and GM-CSF to protect the host from pathogenic infection80,81. These chemokines recruit neutrophils to the site of infection for a rapid response to clear fungal or bacterial infection including Citrobacter rodentium, Salmonella Typhimurium, and Candida albicans72. 19 Commensal bacteria in mice, such as SFB, can induce Th17 cell development in the steady-state, protecting them against Citrobacter rodentium infection82. In addition to neutrophil recruitment, in the gut epithelium, IL-17 is also capable of inducing antimicrobials such as claudin1 and claudin2, forming tight junctions, and maintaining intestinal integrity70. 1.2.4.4 IL-22 Il-22 is a member of the IL-10 family; its receptor complex is composed of the IL-22RA1 subunit, shared with IL-20 and IL-24, and the shared IL-10RB (IL-22R2) subunit, used by IL-10 and IL-26 and IFNl. Although IL-10RB is expressed by a broad range of cells, IL-22RA expression is constrained to the epithelial cells in the lung, gastrointestinal tract, thymus, skin, pancreas, liver, and kidney83. IL-22 is produced by Th2, Th22, Th17, CD8+ T cells, gd T cells, NK cells, and ILC3s. IL-22 production is induced by proinflammatory cytokines secreted by myeloid cells such as IL-1b, IL-6, IL-23, and TNFa84. 1.2.4.4.1 Functional roles of IL-22 in the intestine In mouse models, IL-22R1 is expressed by intestinal epithelial lineages, induces proliferation, and expands the size of intestinal organoids85. ILC3 derived-IL-22 is an additional trigger of goblet cell hyperplasia, which leads to the production of mucin via the Signal Transducer and Activator of Transcription 3 (STAT3)-dependent pathway. A study showed that the lack of IL-22 in mice resulted in a reduced level of goblet cells after infection86. IL-22 is also involved in promoting glycosylation of mucins and induces expression of the fucosyltransferase 2 (FUT2) gene in the intestines86. 20 Upon infection of epithelial cells with pathogens such as Klebsiella pneumoniae, Citrobacter rodentium, and vancomycin-resistant Enterococcus, IL-22 induces signaling pathways linked with producing various types of antimicrobial peptides87,88. Paneth cells which are found in the small intestine are the main source of AMPs89. AMPs within the mucosa include the following: calprotectin and lipocalin, involved in limiting iron availability in the gut, S100A8, and S100A9, which heterodimerize to form calprotectin that can limit access of pathogens to zinc, and other microbial nutrients. Il-22 induces epithelial claudin2 which is associated with epithelial tight junctions leading to water efflux, diarrhea, and removing pathogens in the intestine90. Additionally, IL-22 induces the production of C-type lectin RegIIIb and RegIIIg from Paneth cells, which directly interact against Gram-positive bacteria by targeting bacterial peptidoglycan72. 1.2.5 Role of the microbiome During pregnancy, the maternal immune system maintains tolerance against semi-allogeneic fetal tissues to prevent rejection and subsequent miscarriage. Despite the poor understanding of the interactions between microbiota and the immune system, studies have shown different factors, such as pre-gestational mass index, gestational weight gain, disease including gestational diabetes and allergy, and also antibiotic consumption, can affect the microbiota of mother and fetus30. Several studies have investigated the influence of maternal diet on the newborn microbiota composition and the development of the immune system. Maternal and postnatal dietary fat levels affect the microbiome composition of offspring in both human and Macaca fuscata 21 (Japanese macaque)91. Studies in mice and humans have revealed the critical role of maternal microbiota in shaping the immune system of the offspring. Birth is a transitional stage for neonates from the fetal environment to a new environment enriched with microorganisms, nutrients, and other exogenous substrates. The first encounter with the microbiota occurs immediately after vaginal delivery when the urogenital microbial population is transmitted to the newborn. However, in Cesarian sections, the composition of microbes in the infantâs intestine resembles the environmental bacterial community which includes the maternal skin microbiota24,92. In mice, this critical time period of microbiota colonization is named âthe window of opportunityâ during the weaning period. Although the bacterial density in the neonatal intestinal tract reaches the same number as adults within a day of birth, adult-like microbial diversity and established composition are not reached until approximately 2-3 years in humans and 3-4 weeks in mice. During the early phases of colonization, there is a rapid increase in the number of microbes that can colonize the gut, due to the lack of bacterial competition and the nutrient-rich environment22,93. Two main categories of factors impact the newborn microbiota composition: 1) Exposure to specific microbes due to the geographical location, and presence of siblings or pets in the household. 2) Exposure to antibiotics or dietary factors like breast milk that can suppress or induce the growth of specific bacteria. 22 Some studies have shown that the treatment of newborn mice with antibiotics or colonization of germ-free (GF) mice after weaning leads to increased susceptibility to allergy in the gut and lung94â96. However, exposure of mice to microbiota at birth reduced their susceptibility to colitis-associated tumorigenesis in adulthood97,98. Additionally, host factors such as gestational age (GA) and genetic elements can directly or indirectly affect microbiota expansion22. 1.2.5.1 Role of microbiota during fetal life Whether there is a role in the microbiota during fetal development is unclear. Recent studies have challenged the original paradigm regarding the sterile environment of the womb. The presence of viable commensal bacteria in the meconium, umbilical cord blood of healthy human neonates99, and human amniotic fluid by C-section suggests a transfer of maternal bacteria to the fetus100. Another study on pregnant women who received Bifidobacterium lactis (B. lactis) in combination with Lactobacillus rhamnoses GG (LGG) or placebo for 14 days revealed the presence of DNA from these bacteria in the placenta samples after C-section, in addition to changes in Toll-Like Receptor (TLR) gene expression in meconium and placenta samples101. However, it is important to note that fetal environmental microbiota is extremely low and some studies have reported no distinguishable differences between bacteria from the placenta and amniotic fluid with contamination controls, suggesting that most of the bacteria may reflect contamination102,103 . In mice, the transfer can be studied using genetically labeled bacteria100. In humans, it is difficult to investigate how bacteria are transferred from the mother to the fetus due to ethical and technical challenges. In mouse models, it has been speculated that dendritic cells are able 23 to sample the bacteria and environmental microorganisms without disturbing the epithelial barrier function, and can transport them to the lymph nodes for initiating a proper immune response104. Human studies showed that the source of placental and amniotic microbes is most likely the oral microbiota rather than vaginal, skin, or nasal microbiota, including non-pathogenic bacteria such as Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla105. It has been proposed that microbes can be established through the hematogenous spread of oral microbiota, likely during early vascularization and placentation. In addition, oral bacteria can reach the bloodstream and induce the labor process105. The possible beneficial effects of microbes on birth and the mechanism involved in accessing the microbes to the gestational cavity are still debated30. Many studies emphasize the role of maternal vaginal, oral, or even placenta microbiota in premature delivery. However, it is not certain which immune processes are involved. Suggested mechanisms include the effect of pro-inflammatory molecules from microbes or direct exposure to microbial antigens that are translocated from bleeding gingiva, causing an autoimmune response leading to premature delivery30. 1.2.6 Breast milk as a modulator of postnatal intestinal maturation Breastfeeding is the main source of neonatal environmental exposure in early life, facilitating adequate microbial colonization and diverse immune system maturation. In comparison to formula feeding, breastfeeding results in a lower incidence of morbidity and mortality in infants, as well as a lower frequency of gastrointestinal infections, inflammatory, respiratory, and allergic diseases106,107. Breast milk contains a balance of essential components for neonatal 24 growth including lipids, proteins, carbohydrates and there is a wealth of antimicrobial and anti-inflammatory compounds that support the vulnerable infant after delivery and maturation of the neonatal immune system. 1.2.6.1 Breast milk Microbiota The predominant bacteria detected in human breast milk include Staphylococcus, Streptococcus, Propionibacterium, and probiotic genera such as Bifidobacterium and Lactobacillus. These bacteria are involved in facilitating infant digestion, improving intestinal barrier function through the induction of mucin, competing with pathogens to provide a protectant environment, and reducing intestinal permeability108. Bacteria in breast milk stimulate the development of effector immune responses in infants in such a way that influences the shift from an intrauterine predominant type of Th2 immune response to a balanced Th1/Th2 response109. In addition, a recent study showed that the term breastfeeding is associated with about a two-fold increase in Treg cells and a reduction of inflammatory cytokines in blood as compared to formula-fed infants107. 1.2.6.2 Human milk oligosaccharides Human milk oligosaccharides (HMOs) are one of the most crucial biologically active components of breast milk; HMOs are either fucosylated or sialylated. HMOs act as a prebiotic in the establishment of beneficial microbiota in the newborn gut and inhibit the expansion of pathogenic microbes such as B. Streptococci106. Studies also show the role of HMOs in protecting against NEC in premature babies and a negative correlation between the level of HMOs in breast milk and the incidence of this disease110,111. HMOs affect the production of 25 intestinal mucin levels which leads to the decreased attachment of bacteria to the gut epithelium and reduced risk of developing NEC111. HMOs support the development of the newborn immune system by promoting the maturation of monocyte-derived dendritic cells (moDCs), which lead to the stimulation of Treg cells, and boost immune tolerance, a preventive role in developing immune-mediated diseases in infants112. 1.2.6.3 Protein and peptides in breast milk A wide range of peptides and proteins found in breast milk have antimicrobial properties (immunoglobulins, lactoferrin, and cytokines) and growth factors (EGF))106. As the immune system of newborns is largely naïve and inexperienced, passive immunization through breast milk and transportation of specific maternal antibodies, mainly in the form of soluble IgA (sIgA), is necessary113. sIgA is essential to block the adhesion of microbes including bacteria, fungi, viruses, and parasites to epithelial cells and to avoid inflammatory reactions that may cause tissue damage114. Transfer of maternal sIgA to the infant decreases as the infantâs endogenous antibody levels increase115. The level of secreted IgA in the breast milk of mothers after preterm delivery is significantly higher than in term babies, confirming the adaptation of breast milk to induce more support of premature infants116. 1.2.6.4 Immune cells and other immunological compounds The main active live type of cells found in breast milk are leukocytes including macrophages (40-50% of total leukocytes), neutrophils (40-50% of total leukocytes), and lymphocytes (5-10% of total leukocytes)117. T cells make up nearly 80% of the lymphocyte population, most active, and motile, and provide active immunity to infants118. Recent studies revealed the ILC1 26 as the most abundant type of ILCs available in human milk106. The role of ILCs in breast milk is not yet determined but ILC1 and ILC3 are generally established to be involved in protecting against bacteria and preserving epithelial homeostasis, while ILC2s are crucial in defending against parasitic infection at mucosal surfaces119. Soluble CD14 (sCD14) is a pattern-recognition receptor that forms the LPS-recognition complex with TLR4, also found in breast milk. sCD14 is shown to be one of the most important modulators of intestinal homeostasis which plays a role in an infantâs intestinal defense120. Breast milk, especially colostrum, contains different types of cytokines such as IL-1b, IL-6, IL-8, IL-10, and TGF-b. IL-10 is associated with the inhibition of blood lymphocyte proliferation and a mutation in the receptor of IL-10 leads to early-onset colitis with hyperinflammation of the infant mouse intestine 121,122. TGF- b can reduce inflammation by inhibiting of pro-inflammatory cytokines, induction of sIgA production, and regulation of T cells activation123. 1.3 Necrotizing enterocolitis: intestinal disease of prematurity Necrotizing enterocolitis (NEC) is a serious and common gastrointestinal (GI) complication affecting about 5% of preterm neonates110. It is a major cause of neonatal mortality affecting approximately 5-10% of preterm infants34. NEC is characterized by excessive inflammation in response to commensal bacteria in the intestinal mucosa in preterm neonates, leading to ischemic bowel injury, barrier disruption, and apoptosis of intestinal epithelial cells124. This overwhelming inflammation shows that the neonatal immune system is not immature or less responsive but rather âdifferentially adaptedâ as compared to adult counterparts22. Excess 27 inflammation and hemodynamic instability during NEC can also adversely affect other organs, associated with worsened long-term neurodevelopment110. 1.3.1 NEC clinical manifestations Classic signs of NEC in infants include feeding intolerance, abdominal distention, and bloody stool at 8-10 days of age. Suspicious early signs of NEC include dilated loops of the bowel, a paucity of gas, and gas-filled loops of the bowel that are constant on repeated examinations110. Symptoms can have rapid progression within an hour; subtle signs include changes to abdominal discoloration, intestinal perforation, and peritonitis which results in systemic hypotension, requiring surgical supports110. 1.3.2 Affected age group Although NEC is mainly acknowledged as a preterm infant disease, symptoms can occur in late preterm and full-term infants. In more mature neonates, the disease usually appears in the first week after birth and is associated with additional problems not evident in the classic version of the disease, including intestinal anomalies (e.g., aganglionosis or atresias), congenital heart disease, and perinatal stress that can affect enteric blood flow125. 1.3.3 Contributing factors in developing NEC Epidemiologic observations suggest NEC is a multifactorial disease with a combination of genetic predisposition, premature birth, intestinal immaturity, abnormal microbial colonization of bacteria, formula feeds, antibiotics, and impaired mucosal protective mechanisms. Factors will be further detailed in the following sections. 28 1.3.3.1 Prematurity The time between birth to the beginning of NEC is inversely relative to the length of gestation. A study on 84 infants showed a non-linear relationship between gestational age and the onset of NEC. NEC appears around 29-32 weeks postmenstrual age126. In general, the prematurity of infants causes immature motility, digestion, absorption, and immune defense that lead to an increased risk of intestinal injury110. 1.3.3.2 Intestinal dysbiosis Intestinal dysbiosis has an important role in NEC development. However, the identification of specific causative microbes or metabolites is not yet determined. As NEC does not occur until 8-10 days postpartum, it is assumed that inappropriate initial colonization with pathogens in the gut leads to the development of NEC127. Studies have shown that germ-free animals do not develop NEC, and human infants with NEC also have concomitant bacteremia and endotoxemia128. Although the specific species of pathogens which cause NEC remains unknown, studies confirmed the disease is associated with both unusual species of bacteria and reduced diversity of microbiota110. 1.3.3.3 Antibiotic therapy Antibiotic consumption is a known cause of changing the microbial ecology of a preterm infantâs GI tract. Antibiotic treatment can lead to reduced microbial diversity and change in microbial species, leading to colonization resistance reduction due to the absence of rich protective intestinal microflora129. 29 1.3.4 The molecular mechanism involved in NEC development Several studies utilized mouse models to show the pathophysiology of NEC on IECs. Colonizing bacteria generate metabolites and products, such as LPS, that interact with TLRs in the IECs. This leads to the activation of several signaling pathways including the NF-kB pathway. Many other inflammatory cytokines and chemokines are also activated, triggering a violent inflammatory storm34. Other studies in rat, mouse models, and a resected intestine from infants with NEC confirmed a higher number of TLR4 surface receptors in these infants compared to the full-term infants or animals without NEC130. Egan et al. showed an association between the exaggerated expression of TLR4 in the intestinal epithelium of premature newborns, the induction of lymphocyte chemoattractant CCL25, and the increased level of pSTAT3, followed by the over-expression of TLR4124. Investigations on the role of immune cells in the pathology of NEC reveal an imbalance of Treg and Th17 cells which occurs due to increased activity of pSTAT3 followed by induction of a Th17 inflammatory phenotype and decreased activity of FOXP3+ Treg cells124. A study on the human fetal intestine revealed infiltration of TNF-a producing CD4+T cells and overexpression of TNF- a induced gene in the inflamed LP in intestine131. 1.3.5 Current treatment strategies Medical interventions involve abdominal decompression, bowel rest, and a broad range of antibiotics. Surgical intervention may be required for NEC-affected infants with intestinal perforation, worsening clinical or biochemical states such as shock or decreasing platelet count, and neutrophil count. Surgical procedures can include drain placement, exploratory laparotomy with resection of diseased bowel, and enterostomy with the creation of a stoma110. 30 Since none of these interventions are completely safe for preterm neonates, there is an urgent need to better understand the pathogenesis of this major neonatal disease. 1.4 Thesis rationale and objectives The overall objective of this thesis is to identify the molecular mechanism underlying why neonatal Th17 cells are skewed towards low IL-17 production and to characterize the physiological effect of IL-22 on IECs. I hypothesized that neonatal Th17 cells ability to produce a significant level of IL-22 is beneficial to IECs proliferation and differentiation and also to establish a gut homeostatic environment upon microbe colonization. The overall working hypothesis will be evaluated in through the following objectives: â¢ Objective 1 (Chapter 2): To determine the molecular mechanism underlying the lack of IL-17 production by neonatal Th17 cells compared to their adult counterparts. â¢ Objective 2 (Chapter 3): To establish an ex vivo human infant intestinal organoid model to study the developmental changes of IECs during the early stages of life. â¢ Objective 3 (Chapter 4): To determine the potential effects of neonatal Th17 cell-derived cytokines on infant intestinal organoids. 31 Chapter 2: Neonatal T-helper 17 responses are skewed towards an immunoregulatory interleukin-22 phenotype 2.1 Introduction Candida invasion at the skin and mucosal surfaces lead to mucocutaneous candidiasis (MC) in neonates, manifesting as oral thrush and diaper rashes132. However, why neonates are particularly susceptible to fungal infections is not well understood on a molecular level133. Th17 cells are pivotal in fighting against Candida, evidenced by increased susceptibility to MC in individuals with loss-of-function mutations in IL-17, and related genes (e.g. IL-17 receptor)132. In full-term neonates, Candida species trigger robust in vitro innate immune responses134. Although there is a study showed that neonatal T cells are also able to produce strong IL-17 responses in lymph nodes and the gut135, others showed that peripheral blood naïve T cells produce low Th17 responses136,137, and IL-17-producing cells in the blood circulation are limited to a subset of CD45RO+CCR7-CD25low CCR6+ effector memory (Tem) cells138. Thus, it is of interest to investigate the mechanisms involved in developmental changes in CD4 Th17 responses in humans. Also, how these differences affect the ability of neonates to respond to pathogens such as Candida species. Studies on cord blood as a representative of the neonatal immune system showed a high level of Treg and Th2 cells following immune stimulation whereas, Th1 production is limited in the early stage of life139. Earlier studies have established that fetal CD4 T cells are transcriptionally distinct from their adult counterparts140. However, to our knowledge, there have been few studies directly comparing gene expression profiles in term neonatal and adult naïve T cells, at the genome-wide level. 32 Although IL-6 and Transforming Growth Factor beta (TGF-β) are sufficient for Th17 cell differentiation in mice141, human Th17 cells have different cytokine requirements for differentiation, where IL-1β and IL-23 are sufficient for the Th17 differentiation142. In light of these findings, Acosta-Rodriguez et al. proposed that TGF-β is not essential during human Th17 cell differentiation143. Zhang et al. also showed that TGF-β is not required for Th17 differentiation using adult blood but interestingly, it is essential when using cord blood144. This raises the possibility that previous observations reflect age-dependent TGF-β requirements during the Th17 differentiation of human naïve CD4 T cells. In CD4 T cells, phosphorylation of STAT3 is a result of IL-6 and IL-23 binding to their receptors. Then pSTAT3 induces activation of RORC, the gene encoding the master Th17 transcriptional regulator RAR-related Orphan Receptor gamma (RORgt). Basic Leucine Zipper ATF-Like Transcription Factor (BATF) and Interferon Regulatory Factor 4 (IRF4) cooperate to increase the accessibility of chromatin to STAT3 and RORgt141,145. RORC is down-regulated in naïve neonatal CD4 T cells compared to their adult counterparts136. Though these data were obtained in absence of stimulation, such differences may suggest that the mechanisms regulating Th17 cell responses during the neonatal period may lie upstream of RORgt. During Th17 cell differentiation, STAT3 is associated with TGF-β signaling, resulting from the activation of two serine/threonine kinase transmembrane receptors: type I and type II TGF-β receptors146. Signaling is mediated through the Small Mothers Against Decapentaplegic (SMAD) proteins. Notably, SMAD2 and SMAD3 cooperate to regulate not only the expression of the RORC gene but also the expression of the IL17A gene through SMAD4. 33 Here, we aim to understand the transcriptional and molecular events regulating the lack of Th17 cell response in human neonatal CD4 T cells. To this end, we compared neonatal and adult naïve CD4 T cells in an unbiased genome-wide gene expression analysis. This analysis uncovered major developmental changes in gene networks between T cells from these two age groups, beyond what can be expected from cells of the same lineage. These changes encompassed basic cellular, but also T cell-specific domains such as Th differentiation, IL-7-dependent homeostatic proliferation, and TGF-β signaling. These observations were functionally supported, through experiments revealing that the requirement for TGF-β during the differentiation of human Th17 cells is age-dependent. Experiments also implicate developmental changes in the activity of STAT3 and SMADs, biasing neonatal T cells towards an immunoregulatory Th22, rather than a Th17 cell phenotype. Our findings, in primary human newborn T cells, support developmentally regulated transcriptional networks programmed to limit systemic Th17 cell responses during the newborn period, at the same time increasing newbornsâ susceptibility to MC. 2.2 Material and Methods 2.2.1 Blood sample collection Cord blood samples were collected in sodium heparin anti-coagulated Vacutainers (Becton Dickinson, Canada) from healthy full term (>38-41 weeks of gestation) neonates born by cesarian section without labor at the Childrenâs & Womenâs Health Centre of British Columbia (C&W), after parental informed consent. In some cases, samples collected by Canadian Blood Service (CBS) were used with parentsâ consent. Our studies were approved by the Childrenâs & Womenâs Research Ethics Board (#H14-03199). 34 Peripheral blood samples from healthy adult donors (20-40 years old) were also collected and the samples were processed within 4 hours of collection (#H07-02681). 2.2.2 Naïve T cell purification Mononuclear cells were extracted from peripheral or cord blood based on a previously published protocol 147. Whole blood was diluted with PBS buffer and gently added to the Ficoll-paque density gradient solution and centrifuged for 30-40 minutes at 2300 rpm without brake. As a result, the non-nucleated red cells and granulocytes were separated from the mononuclear cell fraction. Naïve CD4+ T cells were purified by either Fluorescent Activated Cell sorting (FACS) or using a negative selection kit (EasySepTM Naïve CD4+ T cell Isolation Kit; StemCell Technologies, Vancouver, Canada). Briefly, all the mononuclear cells except naïve CD4 T cells are recognized by tetrameric antibody complexes and dextran-coated magnetic particles. Then the labeled cells are separated using the EasySepTM magnet and naïve CD4 T cells remained intact. The purity of isolated naïve CD4+ T cells was checked regularly using flow cytometry gating for CD3+ CD4+ CD45RO- CCR7+ CD25- (Figure 2.1). For the FACS approach, naïve CD4 T cells are gated for CD3+ CD4+ CD45RO- CCR7+ CD25- using the following antibodies: anti-CD3 FITC (clone UCHT1; eBioscience, Canada) or PE (clone HIT3a; BD Biosciences, Canada), anti-CD4 BV605 (clone OKT4; BioLegend, CA USA), anti-CCR7 Alexa Fluor 647 (clone 3D12; BD Biosciences, Canada), anti-CD25 PE-Cy7 (clone M-A251; BD Biosciences, Canada), anti-CD45RO Alexa Fluor 700 (clone UCHL1; BioLegend, CA USA) with or without anti-CD235 FITC (clone 10F7MN; eBioscience, Canada) on a BD FACS Aria (Figure 2.2). 35 Figure 2.1. Representative flow cytometry analysis of naïve CD4 T cells Magnetic bead-purified naïve adult and neonatal CD4 T cells used to confirm â¥99.5% purity after staining for CD3, CD4, and CD45RA. 2.2.3 RNA extraction and gene expression profiling Frozen FACS-isolated T cell pellets were used to extract total RNA using RNeasy Mini Kit (Qiagen). Next, the RNA samples were purified using RNA Clean & Concentrator kit (Zymo Research Corporation, USA). RNA samples were quantified using NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Samples integrity was evaluated via Agilent RNA 6000 Nano kit and Agilent 2100 Bioanalyzer (both Agilent, USA) before loading on bead chip arrays. Only samples with RNA Integrity Numbers (RIN) greater than 9.3 were included. RNA was hybridized to the Illumina HumanHT-12-v4-BeadChip array. Data was transferred to GenomeStudio (Illumina); 36 Figure 2.2. Representative of gating strategy and flow cytometry analysis of naïve adult and neonatal CD4 T cells Isolation of naïve CD4 T cells via fluorescence-activated cell sorting (FACS) method used in Illumina genome-wide gene expression experiments. In the lower graphs, red overlays are Fluorescence minus one (FMO) control. Lumi and Limma packages from R were used for further processing and normalizing the data. Adjustment for multiple testing was done using the Benjamini & Hochberg method and a false discovery rate (FDR) of < 5% was considered significant. The FACS isolation and data analysis strategies for gene expression experiments using naïve CD4 T cells data are depicted in Figure 2.2 as an example of the gating strategy that was used in FACS. 37 2.2.4 Real-time qPCR To validate the microarray data, STAT3 gene was used for confirmation by qPCR from separate neonatal and adult subjects. First, total RNA samples were converted to cDNA using Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific, USA). The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 62°C for 60 s in a ViiA 7 real-time PCR system (Life Technologies, Canada). The primer used for qPCR confirmation is listed in (Table 2.1) with primer efficiencies >92%. Real-time PCR was performed using Power SYBR Green (Thermo Fisher Scientific, USA). Average Ct values were calculated using the 2-ÎÎCt method. 2.2.5 Stimulation of naïve T cells Isolated naïve CD4+T cells were stimulated with a 1:1 ratio of anti-CD3/CD28 beads (Thermo Fisher Scientific, DE, USA). RPMI1640 medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin. For differentiation of naïve CD4+ T cells to Th17 cells, the following cytokine polarizing conditions were used: IL-6 (10 ngâmlâ1), IL-23 (10 ngâmlâ1), IL-1β (10ângâmlâ1) Â± TGF-β (3ângâmlâ1) (all purchased from Peprotech, NJ, USA). The following cytokine polarizing conditions were used for other types of T cell stimulation: Th0 (no exogenous cytokines), and Th1: IL-12 (10 ngâmlâ1) (all purchased from Peprotech, NJ, USA). IL-10 inhibition was achieved in presence of an anti-IL-10 (5âµgâmlâ1) and anti-IL-10 receptor, alpha subunit (5âµgâmlâ1) blocking antibodies (both from R&D systems, Ontario, Canada). STAT3 inhibition was achieved using Stattic (10 µM, Cayman Chemical, MI, USA). Naïve CD4+ T cells were seeded at the concentration of 5x105 cells per well in U-bottom 96 well plates. The medium was changed on day 3 and replaced with fresh medium including 38 previously mentioned cytokines. T cells were cultured for 6-7 days. For T cell stimulation in the presence of antigen-presenting cells, neonatal or adult T cells were stimulated with a 1:1 ratio of allogeneic CD3-depleted neonatal or adult carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific, cat# C34554)-labeled antigen-presenting cells in the presence of anti-CD3 (OKT3; 0.5 µg ml-1) with or without an anti-CD28 antibody (1âµgâmlâ1; Invitrogen, cat# 11131D) for 6 days. 2.2.6 STAT3 phosphorylation For STAT3 phosphorylation, mononuclear cells were starved in XH-vivo media without serum at a concentration of 1-2 x106 cells/ml overnight at 37°C. The next day, starved mononuclear cells were stimulated with recombinant IL-6 at a final concentration of 100 ng/ml for 15 minutes at 37°C. Stimulated mononuclear cells were then fixed using pre-warmed CytofixTM buffer (BD Biosciences, Canada), permeabilized using Perm/wash buffer (BD Biosciences, Canada), and stained using the following fluorescence-conjugated antibodies: anti-CD3-PE (clone UCHT1; BioLegend, USA), anti-CD4-FITC (clone PRA-T4; BioLegend, USA), anti-CD45RA-PE-Cy7 (clone HI100; BioLegend, USA), and pSTAT3-Alexa Fluor 647 (clone 13A3-1, BioLegend, USA). Samples were then analyzed by flow cytometry on a 4-laser LSR II or Fortessa flow cytometers (BD Biosciences, USA). Data were analyzed using FlowJo v10 (Ashland, USA) with gating on CD3+CD4+CD45RA+ based on fluorescent minus one (FMO) staining controls. 2.2.7 STAT3 gene cloning After extracting total RNA from adult peripheral blood mononuclear cells, the human STAT3 gene was amplified via RT-PCR (Table 2.1). The gene was then cloned into a pIRES2-eGFP 39 plasmid (http://www.dmlim.net/vectors/pIRES2-EGFP/pIRES2-EGFP-map.pdf). Integrity of the plasmid insert with flanking regions was confirmed by Sanger sequencing (Figure 2.3). Table 2.1 List of primers used for STAT3 gene analysis Gene/Primer name Direction Sequence STAT3 (cloning) forward 5â²-TGCTAGCAGGATGGCCCAATGGAATCAG-3â² reverse 5â²-TGAGCTCTCACATGGGGGAGGTAGCGC-3â² STAT3 (qPCR) forward 5â²-ATGCCACTTTGGTGTTTCAT-3â² reverse 5â²- AATCTCCATTGGCTTCTCAA-3â² GAPDH (normalization primers) forward 5â²- AAGGTGAAGGTCGGAGTCAA-3â² reverse 5â²- AATGAAGGGGTCATTGATGG-3â² Figure 2.3. Genetic map of cloning human STAT3 gene in pIRES2EGFP vector Gene map is created using Benchling software 2.2.8 STAT3 Transfection The STAT3-GFP or GFP only (âempty vectorâ, EV) plasmids were transfected at a concentration of 5ug/ml into neonatal naïve CD4+ T cells using a Lonza Nucleofector 2b 40 device (Basel, Switzerland). An optimized electroporation program was used (Pulse 2100v, pulse width 015ms, pulse number 02). Prior to transfection, the purity of naïve T cells (>99.5% CD45RA+ cells) was verified by flow cytometry staining for the CD3, CD4, and CD45RA surface markers. After overnight cell culture at 37°C for recovering transfected cells, dead cells were removed with a Dead Cell Removal kit (Miltenyi Biotech, # 130-090-101). Then, the efficiency of transfection was assessed by green fluorescence protein expression measured by flow cytometry comparing STAT3-transfected and empty-vector-transfected cells as a negative control. Naïve CD4+ transfected cells were stimulated with a 1:1 ratio of anti-CD3/CD28 beads and polarized in the presence of Th17 cytokines for 3 days. Production of IL-17 was measured using a high sensitivity IL-17 ELISA kit (Thermo Fisher Scientific, DE, USA). IL-17 cytokine expression data were normalized to the percentage of GFP-expressing cells in each sample. 2.2.9 SMAD2/3 phosphorylation For SMAD2/3 phosphorylation, magnetic isolated naïve CD4 T cells were starved in RPMI media without serum overnight at a density of 2 million cells per ml. The next day, cells were stimulated with recombinant TGF-b (R&D systems; cat# 240-B-002/CF) at a final concentration of 20 ng/ml for one hour at 37°c and then fixed using pre-warmed CytofixTM buffer (BD Biosciences, Canada), permeabilized using Perm III buffer (BD Biosciences, Canada), and stained using the following fluorescence-conjugated antibodies: anti-CD4-FITC (clone PRA-T4; BioLegend, USA), anti-CD45RA-PE-Cy7 (clone HI100; BioLegend, USA), and anti-Smad2 (pS465/pS467)/Smad3 (pS423/pS425)-PE (clone 072-670; BD Biosciences, 41 Canada). Samples were then analyzed by flow cytometry, gating on CD4+CD45RA+ based on fluorescent minus one staining controls. 2.2.10 Western Blot Magnetic bead-purified neonatal and adult naïve CD4 T cells were lysed in RIPA buffer supplemented with phosphatase/protease inhibitors (Santa Cruz Biotechnology, #sc-24948). Measurement of protein concentration was performed using Pierce 660 nm protein assay (Thermo Fisher Scientific, #22662). Lysates were boiled in 4X Laemmeli buffer (Bio-Rad) supplemented with 2-mercaptoethanol, ran on a 4-20% Tris-Glycine mini protein gradient gel (Thermo Fisher Scientific, #XP04202BOX), and transferred to a PVDF membrane (Bio-Rad, 1620177). The membrane was incubated with β-Actin (8H10D10) mouse mAb (Cell Signaling Technology) and Smad2/3 (D7G7) XP® rabbit mAb (Cell Signaling Technology) as primary antibodies and IRDye® 680RD Goat anti-Mouse IgG (LI-COR) and IRDye® 800CW Donkey anti-Rabbit IgG (LI-COR) as secondary antibodies. The blot was imaged using a LI-COR Odyssey 9120 machine and the image was processed using Image Studio Lite ver. 5.2 software. 2.2.11 Data analysis Cytokine levels were compared using 2-tailed Mann-Whitney U tests (for independent groups), Wilcoxon tests (for comparison of non-parametric data between stimulating conditions within same age groups) or paired t-tests (for comparison of parametric data between stimulating conditions within same age groups), or in some instances, 95% confidence intervals (for non-inferential, exploratory group comparisons where data was deemed to be normally distributed). Effects of age, stimulating cytokine conditions, and T cell or antigen- 42 presenting cell age were assessed using 2-way ANOVAs as specified. Inferential group comparisons were adjusted for multiple testing using the Benjamini-Hochberg method and a 5% false-discovery rate, whenever indicated and as specified. A p value of <0.05 was considered significant. All statistical analyses were conducted using GraphPad Prism 8 or 9 (San Diego, CA). 2.3 Results 2.3.1 Neonatal T cells are inherently biased towards high IL-22 cytokine production To better understand age-specific T cell differentiation biases, we isolated naïve CD4 T cells (CD3+CD4+CD45RO-CCR7+CD25-) from cord blood (neonatal) and healthy adult peripheral blood using FACS (Figure 2.2). Naïve T cells were stimulated using anti-CD3/CD28 beads in the presence of either Th17-polarizing (IL-1β, IL-6, and IL-23), Th1-polarizing (IL-12), or no cytokines (Th0) for 6 days. Luminex data analysis showed that neonatal T cells in âno cytokineâ condition (Th0) produced lower levels of Th1 (interferon-g, IFN-g) and Th17 (IL-17 and IL-21) cytokines compared to adult counterparts. For T cells stimulated in Th1 and Th17 conditions, we observed a high level of IL-10 IL-13, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), and also IL-22 in the neonatal cells ( Figure 2.4A). These results confirmed the previously proven Th2 bias of neonatal T cells and showed an inherent bias towards the production of IL-22 rather than IL-17. Flow cytometry experiments by intracellular cytokine staining showed that IL-22-producing cells were a distinct subset from IL-10 producing cells, and largely not IL-17-producing cells. 43 Previous studies showed that neonatal APCs such as dendritic cells produce an abundant level of IL-1β, IL-6, and IL-2148. To determine whether Th17 cell differentiation in neonates is dependent on a signal from neonatal APCs, we stimulated neonatal and adult T cells (using CD3) in the presence of allogenic adult or neonatal CD3-depleted mononuclear cells without any exogenous cytokines. Of note, this crossover experimental design also allowed the mitigation of effects due to expected age-related differences in the proportion of subsets of bona fide antigen-presenting cells among blood mononuclear cells149,150. Regardless of whether neonatal or adult APCs were used, there was a significantly lower level of IL-17 and IFN-g produced by neonatal T cells than by adult T cells. Neonatal T cells particularly produced a greater level of IL-22 in the presence of neonatal antigen-presenting cells Figure 2.4B-D). Neonatal APCs generally express a lower level of the CD28 ligands B7.1/2 which might lead to poor CD28 co-stimulation139 and co-stimulation through ICOS induced IL-22 production. Thus, to resolve the role of low APC-mediated CD28 co-stimulation, we repeated the experiments in the presence and absence of an anti-CD28 antibody. We noticed that neonatal IL-17 production was decreased in the presence of anti-CD28 antibody while it promoted IFN-g production regardless of the APCs cell used ( Figure 2.4E). 44 Figure 2.4. Neonatal T cells are inherently biased against Th17 responses independent of antigen-presenting cell signals. (A) Radar plots of cytokines produced by adult or neonatal naïve CD4 T cells 6 days post-stimulation (anti-CD3/CD28 beads) in Th0- (no exogenous cytokines), Th1- (IL-12) or Th17- (IL-1β, IL-6 and IL-23) polarizing conditions. Data are presented on a log-10 scale (pg mlâ1) averaged from two separate sets of experiments that include a total of 6-12 subjects per cytokine polarizing condition per age group; P values for each cytokine were calculated by two-tailed Mann-Whitney U test using non-logarithmic values and correction was applied for multiple comparisons using the Benjamini-Hochberg method; *p<0.05; **p<0.01; ***p<0.001; (B) Intracellular IL-17 and IFN-g detection (by flow cytometry) in adult (red) or neonatal (blue) naïve CD4 T cells stimulated using soluble anti-CD3 antibody (OKT3) for 6 days in presence of carboxyfluorescein succinimidyl ester (CFSE)-labeled allogeneic A B C D E 45 neonatal (N) or adult (A) antigen-presenting cells (APCa = adult APCs, APCn = neonatal APCs, Ta = adult T cells, Tn = neonatal T cells). Data are aggregated from 11 independent experiments each using 2 freshly collected (same day) neonatal and adult samples, with p values for the effect of T cell or APC age on cytokine response, using a 2-way ANOVA; (C) Corresponding cytokine measurement in supernatants (ELISA); means with 95% CIs (D) Representative flow cytometry gating for intracellular IL-17 and IFN-g expression in naïve adult or neonatal CD4 T cells stimulated with anti-CD3 (OKT3; 0.5 µg ml-1) in the presence of allogeneic neonatal (N) or adult (A) CD3-depleted mononuclear cells (used as APCs). (E) Effect of CD28 co-stimulation on Th1/Th17 responses in adult or neonatal naïve CD4 T cells. Intracellular IL-17 and IFN-g detection (by flow cytometry) in adult or neonatal naïve CD4 T cells stimulated using soluble anti-CD3 (OKT3) for 6 days in presence of CFSE-labeled allogeneic neonatal (N) or adult (A) CD3-depleted mononuclear cells (used as APCs) with or without exogenous anti-CD28 antibody co-stimulation (1 µg mlâ1). P values represent effect from T cell and CD28 co-stimulation on cytokine responses, using a 2-way ANOVA. 2.3.2 Unbiased transcriptome analyses reveal altered TGF-β signaling in neonatal T cells We performed a genome-wide transcriptomic analysis of ex vivo FACS isolated naïve CD4 T cells (CD3+CD4+CD45RO-CCR7+CD25-) from 12 full term neonatal and 12 healthy adult subjects to examine the developmental transcriptional basis underlying the lack of Th17 differentiation of neonatal T cells. The gene expression profile demonstrated a significant separation between the two age groups of adults and neonates (Figure 2.5A). Figure 2.5B shows a large number of differentially expressed genes (n=5,976; FDR <5%) which are corresponding to ~46% of the detectably expressed unique annotated transcripts. One third of (n=2,282) the differentially expressed genes had >1.2-fold expression change between neonatal and adult T cells. Gene Ontology analysis mapped differentially expressed genes to broad cellular domains regulating cell cycle, post-transcriptional events, and purine nucleoside monophosphate metabolism. 46 The gene expression profile of neonatal T cells showed upregulation of genes involved in the regulation of Th2 differentiation including increased baseline expression of GATA3 , SGK1 (a positive regulator of Th2 cells)151, and SATB1 (a negative Th17 regulator)152. On the other hand, there was a decreased expression of Th1-regulating genes such as IL12RB1, IFNGR2 and STAT4 (Figure 2.5C-E). There was also a reduced expression level of the genes responsible to drive the balance of Treg towards Th17 responses including BATF and RORC 152,153. Gene Set Enrichment Analysis (GSEA) identified a Th22 gene signature within STAT3 targets in neonatal T cell, supporting a biased neonatal response (Figure 2.5F). GSEA analysis also revealed enrichment in TGF-β signaling genes including the TGF-β receptors TGFBR3, TGFBR2, Furin, and also SMAD2, SMAD4 and SMAD7. Expression of the SMAD4 gene was decreased, while expression of the TGF-β-signaling inhibitor SMAD7146 was increased, in neonatal T cells. We also showed that the expression of phosphorylated (active form) of both SMAD2 and SMAD3 were increased in neonatal T cells (Figure 2.6A-C) even though we did not observe an increased expression of SMAD2 and SMAD3 at the gene level. Additionally, GSEA showed enrichment in the expression of target genes for both SMAD2 and SMAD3 (Figure 2.6D and E). Overall, data supported developmental changes in several key regulators of T cell differentiation and TGF-β signaling. 47 Figure 2.5. Genome-wide gene expression comparison identifies a unique developmental Th22 signature at baseline in naïve neonatal CD4 T cells. (A) Principal Component Analysis using above-intensity gene expression probes between unstimulated adult (red) and neonatal (blue) naïve CD4 T cells (B) Volcano plot of fold-change and unadjusted p values of genes expressed differentially in neonatal and adult naïve CD4 T cells. TGF-β- and Th17-related genes with higher expression in adults are shown in red whereas genes with higher expression in neonates are shown in blue; Expression heatmap with hierarchical clustering of genes from GSEA between neonatal and adult naïve CD4 T cells (C) IL-7-dependent homeostatic T cell expansion (D) TGF-b signaling (F) Heatmap of genes overlapping between STAT3 target genes from the Chromatin Immunoprecipitation Enrichment Analysis (ChEA) database, and upregulated / down-regulated genes in Th22 cells compared to Th1, Th2 and Th17 cells based on154. Red and blue colors in the heatmap represent Z-scores for normalized gene expression levels. Refer to table S2 in the published paper for complete data on significant levels of gene expression155. -100 -50 0 50 100-150-100-50050100PC1 (22.3%)PC2 (13.9%)-4 -2 0 2 40102030log2 [FC neonate vs adult]-log 10[P value]AUTS2IGF2BP3CDCA7NR3C2BCL11ATSHZ2NREPGZMACACNA1INR3C2YBX3SMC4MT1FTGFBR3GBP5SERPINB6KLRB1KLF9BCL11AIL7RSOX4CTLA4UCP2STAT4p300TSPO YY1BATFc-MafRoraSGK1IL27RASGK1RoraSTAT3CTCFSMAD4 GATA3SMAD7STAT3STAT2MAD7RARAFURINSMAD7TGFBR2SMAD2SMAD7TGFBR3RoraRora4RO ARORARorcTGFB3AdultNeonateIL-7-dependent homeostatic T cell expansionCTGF-β signalingNeonate AdultA B C D E F Adult NeonateSTAT3 targets up in Th22 cellsSTAT3 targets down in Th22 [binary]â3â2â10123AS286AS287AS294AS296AS299AS300AS319AS298AS314AS316AS317AS318TS174TS184TS185TS186TS187TS188TS165TS166TS169TS171TS172TS173AS286AS287AS294AS296AS299AS300AS319AS298AS314AS316AS317AS318TS174TS184TS185TS186TS187TS188TS165TS166TS169TS171TS172TS173 â3â2â10123AS286AS287AS294AS296AS299AS300AS319AS298AS314AS316AS317AS318TS174TS184TS185TS186TS187TS188TS165TS166TS169TS171TS172TS173AS286AS287AS294AS296AS299AS300AS319AS298AS314AS316AS317AS318TS174TS184TS185TS186TS187TS188TS165TS166TS169TS171TS172TS173ANKRD28BCL7ABRSK1CACNB3CMIPDLGAP4GALNT6HEATR3HIPK2HPGDLMNAMYO1GPAG1PFKLPPFIB 1TOB1ABLIM1ACTN1AFF3APBB1IPAPPL2ATICE2F3EPB41FYNGIMAP4GSPT2GYPCICOSIRF4ITGA4LYRM4PACS1PASKPDE9APITPNC1PLAC8PRKAR1BPRKCBRHOHRIN3SATB1SNRNP35TCF7ZNF518BZNF69â3â2â10123AS286AS287AS294AS296AS299AS300AS319AS298AS314AS316AS317AS318TS174TS184TS185TS186TS187TS188TS165TS166TS169TS171TS172TS173 48 2.3.3 The requirement for TGF-β during the differentiation of human Th17 cells is age-dependent The changes that we observed at the transcriptomic level in TGF-β signaling between adult and neonatal T cells persuaded us to investigate whether there is an age-dependent requirement for TGF-β signaling during human Th17 cell differentiation. Thus, we stimulated magnetic bead-purified adult and neonatal naïve CD4 T cells with anti-CD3/CD28 beads in the presence of a combination of Th17-required cytokines IL-1b, IL-6, IL-23, and TGF-β cytokines for 6 days and measured the level of produced IL-17 and IL-22 (Figure 2.7A and B). Three main results were obtained from this experiment: first, regardless of the cytokine polarizing condition, neonatal Th17 cells produced lower IL-17 but significantly higher IL-22 compared to their adult counterparts. This bias was supported by reduced RORgt expression in neonatal T cells (Figure 2.7C, D). Second, the requirement of cytokines differed to some extent between adult and neonatal T cells during Th17 cell differentiation: IL-1b and IL-23 seemed sufficient in adult T cells to produce IL-17, however, IL-6 and TGF-b were required for neonatal T cells to achieve the maximum level of IL-17 production. We also observed that exogenous TGF-b increased IL-17 production in neonates but had no effect on IL-17 levels in the adult counterparts. Third, adult T cells produced more IL-22 in the presence of IL-1b and IL-23, but IL-6 was essential for the maximal level of IL-22 production by neonatal T cells. Finally, TGF-b suppressed IL-22 production in both age groups (Figure 2.7A and B). All these data clearly demonstrated the different effects of TGF-b on the production of IL-17 and IL-22 in these two age groups. 49 Figure 2.6. Expression and activity of SMAD2 and SMAD3. (A) Western blot detection of native SMAD2 and SMAD3 in magnetic bead-purified neonatal and adult naïve CD4 T cells (3 independent samples per age group), and corresponding signal intensity for SMAD2 quantification relative to β-actin (lower panel). (B) SMAD2 and SMAD3 phosphorylation at serine residues 465 & 467, and serine residues 423 & 425 sites, respectively, by flow cytometry, in isolated naïve CD4 T cells (gated on CD4+CD45RA+ cells) after one-hour incubation with TGF-β. (C) Representative flow cytometry data of (B), with histograms for cells incubated with TGF-β in red and histograms in absence of TGF-β in blue. (D&E) Leading edge genes of the gene set enrichment analysis of transcription factors from the ChEA (Chromatin Immunoprecipitation Enrichment Analysis) database are presented. Red and blue colors show gene expression on a Z-score scale. A B C D E 50 Figure 2.7. Neonatal Th17 cells differ from adult T cells in their cytokine responses and are skewed toward high IL-22 production IL-17 (A) and IL-22 (B) were measured in adult (red) and neonatal (blue) magnetic bead-isolated naïve CD4 T cells stimulated using anti-CD3/CD28 beads for 6 days in the presence of the indicated cytokine combinations. Data are combined from 8 separate experiments using cells from different subjects per age group, with cytokines measured in a single batch/assay; undetectable cytokines are shown as 0.01 pg ml-1; IL-17 (A) and IL-22 (B) production were significantly different between neonatal and adult T cells, as assessed using a 2-way ANOVA (age effects: p=0.023 for IL-17 and p=0.002 for IL-22); where statistical significance for the differences in cytokine stimulation conditions within age groups were adjusted for multiple comparisons using a 5% false-discovery rate; *p<0.05; **p<0.01; ***p<0.001; (C) Representative gating (on live cells) of RORgt expression in IL-17-producing cells from neonatal and adult naïve CD4 T cells differentiated for 6 days in the presence of IL-1β, IL-6, IL-23 and TGF-β, followed by a 5-hour PMA/Ionomycin stimulation (to bring up IL-17 expression), and (D) cumulative data from 6 independent neonatal and adult samples. 2.3.4 Reduced STAT3 limits Th17 cell differentiation of neonatal T cells Reduced STAT3 gene expression in neonatal naïve CD4+ T cells was confirmed by qPCR analysis in another set of neonatal and adult samples (Figure 2.8A). Although the expression of IL-6 receptor in neonatal naïve T cells was similar to their adult counterparts (Figure 2.8B), STAT3 phosphorylation assay confirmed the reduced activity of pSTAT3 at tyrosine residue 705 (Y705) after stimulation of naïve T cells with IL-6 (Figure 2.8 C,D). Th0IL-6IL-1b, IL-23IL-1b, IL-6, IL-23IL-1b, IL-6, IL-23, TGF-bTh0IL-6IL-1b, IL-23IL-1b, IL-6, IL-23IL-1b, IL-6, IL-23, TGF-b0.010.11101001000IL-17A (pg ml-1)******************Th0IL-6IL-1b, IL-23IL-1b, IL-6, IL-23IL-1b, IL-6, IL-23, TGF-bTh0IL-6IL-1b, IL-23IL-1b, IL-6, IL-23IL-1b, IL-6, IL-23, TGF-b0.010.1110100IL-22 (ng ml-1)***************AdultNeonate010RORγt (% cells)0.0381B C A D RORgt-AlexaFluor488IL-17-BV650Neonate Adult 51 However, as the production of IL-22 by human T cells is also largely STAT3-dependent83, additional experiments confirmed that neonatal T cells show some functionally relevant residual STAT3 activity, as indicated by a decrease in IL-22 production in the presence of a STAT3 inhibitor (Figure 2.9A). To determine if STAT3 activity is reduced in neonates and if this could limit the production of IL-17 and as a result Th17 differentiation capacity, we cloned the human STAT3 gene into pIRES2-eGFP plasmid (Figure 2.3). The integrity of the plasmid insert with flanking regions was confirmed by Sanger sequencing. Figure 2.8. STAT3 expression is down-regulated in neonatal Th17 cells. (A) qPCR confirmation of STAT3 gene expression in 8 neonatal and 8 adults naïve CD4 T cell samples (normalized on GAPDH; p value by unpaired t test). (B) IL-6 receptor is expressed on naïve neonatal CD4 T cells. Expression of IL-6 receptor, or CD126 was determined by flow cytometry on blood mononuclear cells (gated on CD3+CD45RA+CCR7+CD25- cells). (C) STAT3 phosphorylation (intracellular flow cytometry) measured in neonatal and adult naïve CD4 T cells (gated on CD3+CD4+CD45RA+ cells) after 15 minutes stimulation with IL-6 (100 ngâmlâ1). Data are combined from 6 donors per age group in 2 separate experiments (p value by unpaired t test). (D) Representative gating strategy for STAT3 phosphorylation (by flow cytometry) in neonatal and adult naïve CD4 T cells (gated on CD3+CD4+CD45RA+ cells) after 15 minutes of stimulation with IL-6 (100 ngâmlâ1). Colors represent fluorescence-minus one control (grey), unstimulated (blue) and stimulated (red) samples. B C A D 52 Next, using nucleofector 2b and 4D devices (Lonza), we optimized the insertion of STAT3- pIRES2-eGFP plasmid transfection into the nucleus of naïve CD4 T cells. Testing several transfection settings and concentrations of STAT3 pIRES2-eGFP plasmid, the highest efficiency of nucleofection was achieved by using 10 µg of STAT3 pIRES2-eGFP and program U014 on a Lonza 2bTM transfection machine. Figure 2.9B and C show the transfection efficiency of the STAT3 gene into the nucleus of naïve CD4+ T cells using %GFP-expressing cells and Figure 2.9D shows the viability percentage of transfected naïve T cells. To determine the effect of the exogenous STAT3 on the differentiation of naïve CD4+ T cells into Th17 cells, we stimulated naïve CD4+ T cells with anti-CD3/CD28 in the presence of IL-1β, IL-6, and IL-23 for 3 days. Transient overexpression of STAT3 increased neonatal IL-17 production which supports an important role for altered STAT3 signaling in limiting neonatal Th17 cell differentiation (Figure 2.9E). Notably, STAT3 overexpression also increased IL-22 production, which at the same time remained suppressible by TGF-β (Figure 2.9F). IL-10 cytokine, which is abundantly expressed by neonatal T cells, can also phosphorylate STAT3 through the IL-10 receptor but inhibits IL-17 production. To exclude the inhibitory effect of IL-10 on IL-17 production in neonatal T cells, we blocked the IL-10 cytokine as well as its high-affinity receptor IL-10R1 which did not detectably increase IL-17 production in neonatal CD4 (Figure 2.10). Thus, STAT3 is recognized as an important developmental modulator of Th17 cell differentiation in human neonatal T cells. 53 Figure 2.9. Reduced STAT3 expression limits neonatal Th17 cell differentiation. (A) IL-22 production in neonatal T cells after 3 days of stimulation (anti-CD3/CD28 beads) in the presence of IL-1β, IL-6, and IL-23, with or without STAT3 inhibition (Stattic, 10µM). (B) Representative cell viability (7-AAD) and transfection efficiency (GFP-positive cells) after transfection of neonatal naïve CD4 T cells with empty vector (EV) or STAT3-expressing vector; aggregated data for (C) cell viability and (D) GFP-expression from 9 separate transfection experiments using separate cord blood donors. (E) IL-17 and (F) IL-22 production (normalized to %GFP+ live cells) in non-transfected, control (empty) GFP vector- and STAT3/GFP vector-transfected neonatal naïve CD4 T cells stimulated for 3 days (anti-CD3/CD28 beads) with IL-1β, IL-6, and IL-23 Â± TGF-β; data are from 7 independent experiments, with lines connecting cells from the same blood donations; p-value by paired t-test or Wilcoxon test. IL-1b, IL-6, IL-23IL-1b, IL-6, IL-23, TGF-b0200040006000IL-22 (pg ml-1)p=0.022p=0.031UntransfectedEmpty vectorSTAT3 vectorp=0.023IL-1b, IL-6, IL-23, TGF-b0200400600800IL-22 (pg ml-1)p=0.023C D A B E F 54 Figure 2.10. Effect of IL-10 blocking on IL-17 production in neonatal and adult naïve CD4 T cells T cells were stimulated with anti-CD3/CD28-coated beads for 6 days without exogenous cytokines (Th0) or in the presence IL-1β, IL-6, IL-23 and TGF-β Â± anti-IL-10 and anti-IL-10 receptor antibodies. P values were calculated by paired Wilcoxon test (within age groups) or Mann Whitney (between age groups). Data are from one experiment with 8 subjects per group. *p<0.05; **p<0.01. 2.4 Discussion In this study, we performed a side-by-side comparison of Th17 cell differentiation and cytokine requirements between neonatal and adult CD4 T cells. Our results showed that neonatal CD4 T cells exhibited non-stereotypic Th17 responses, evidenced by producing a high level of the immunoregulatory cytokines IL-22 and IL-10. Our unbiased transcriptomic and functional analysis revealed developmental changes in STAT3 and TGF-b signaling networks in neonatal CD4 T cells. The cytokine profile analysis showed the age-dependent requirements of TGF-b, with a dependency mostly revealed in cord blood which was not found in adult peripheral blood naïve CD4 T cells. This observation can be used to re-interpret 55 our previous knowledge about the STAT3/TGF-b functional and gene network data, emphasizing the significance of donor age in human Th17 differentiation studies145,156. Different studies showed that an excessive level of IL-17 production can be linked to necrotizing enterocolitis, a serious gastrointestinal disease in the newborn period124. Also, systematic blockade of IL-17 reduced mortality of neonatal mice from polymicrobial sepsis, suggesting a detrimental effect of IL-17 during neonatal sepsis157. In order to maintain local homeostasis during the early stage of life, naïve CD4 T cells may preferentially acquire an immunoregulatory Th22 phenotype rather than a stereotypic Th17 phenotype157. IL-17 and IL-22 are the two main cytokines produced by Th17 cells. Both of these cytokines affect epithelial mucosal cells in the skin, liver, gut, and lung84. IL-22 is a member of the IL-10 family of known anti-inflammatory cytokines. In contrast to IL-17, IL-22 has a more protective and proliferative effect on gut epithelial cells, inducing regeneration, proliferation, enhancing mucin production, and the post-translational fucosylation of sugar moieties on mucin proteins, which promotes beneficial gut microbial colonization84,158. Also, IL-22 induces antibody-mediated immunity and pathogenic bacteria via iron scavenger hemopexin159,160. As a result, we postulated that the preference of neonatal naïve CD4 T cells towards the production of a high level of IL-22 is advantageous to limit the systemic consequences of mucosal inflammation during de novo gut colonization of commensal microbes. 56 Data reveal age-related differences in STAT3 activity and in the requirements for the TGF-β cytokine during Th17 cell differentiation in humans. Gene expression data closely implicate differential activity in SMADs. Mechanistic studies are required to elucidate how these changes operate to alter TGF-β signaling in neonatal T cells. However, we can speculate on this matter here: on the one hand, SMAD2 phosphorylation enhances RORC gene expression161. On the other hand, SMAD4 can indirectly suppress RORC expression by interacting with SKI, a transcriptional repressor that controls histone acetylation/de-acetylation at the RORC locus. TGF-β signaling causes the degradation of SKI, which then becomes unavailable to help SMAD4 suppress the RORC locus146,162. Therefore, our data support a model whereby increased SMAD2 activity in the context of reduced SMAD4 renders naïve neonatal CD4 T cells more dependent on TGF-β signaling during the Th17 cell differentiation, despite lower STAT3 activity. In addition, mechanisms common to both neonatal and adult T cells, such as the transcription factor c-Maf that suppresses IL-22 production by binding directly to its proximal gene promoter region163, may explain how TGF-β suppresses IL-22 production in both of these cell types. Although there is limited STAT3 activity in neonatal T cells, STAT3 expression levels appear to be sufficient to produce a robust IL-22 response despite the lack of IL-17 response. IL-21 also provides positive feedback to amplify the precursor frequency of Th17 cells along with TGF-β164,165. Together with expression of the transcriptional regulator of Th22 cells, aryl hydrocarbon receptor (AhR), IL-21 can also activate STAT3 to induce IL-22, but not IL-17, production166. However, the comparable IL-21 expression between neonatal and adult T cells in Th1 or Th17-polarizing conditions argues against a developmental effect through this 57 cytokine. Intracellular flow cytometry cytokine staining experiments showed that the majority of cells between day 5 and day 21 produce either IL-17 or IL-22 rather than both cytokines. However, these preliminary experiments were suboptimal due to the differences in the timing of intracellular detection for these two cytokines, and further studies are needed to determine whether a small subset of cells can express both cytokines, under specific T cell receptor/cytokine stimulatory conditions. Studies are also needed to understand how human T cell gene networks are regulated developmentally. Considering that the action of transcription-factor networks is context-dependent during the lineage differentiation of CD4 T cells167, it is plausible that broad developmental-specific epigenetic changes underlie the changes in gene expression observed in this study. The implication of epigenetic changes regulating the network of active transcription factors, as well as the amount of TGF-β signaling that forms the basis of the continuum of âclassicalâ to âalternativeâ Th17 cells, reinforce the notion that lineage-regulating transcription factors influence the final effector phenotype of Th17 cells, potentially through developmentally altered changes in chromatin accessibility168. 58 Chapter 3: Establishment of human infant intestinal organoid model 3.1 Introduction As detailed in Chapter 1, the neonatal intestinal mucosa differs functionally from the adult intestinal mucosa, in adaptation to early life conditions. For example, the neonatal epithelium contains fewer differentiated sub-types of IECs, lower renewal capacity, fewer microvascular folds, and a lack of diversity of the intestinal microbiome169â171. This may result in functionally immature mucosal barriers and vastly different immune interactions compared to adults. Both animal and human studies have revealed important size, structure, and functional differences between the neonatal and adult gut and intestinal epithelial layers172. However, biological differences between species warrant human studies. Because of the major ethical and practical difficulties in studying the human pediatric gut, there is a need to establish in vitro human gut models. Organoids are three-dimensional (3D) models recapitulating the physiological structure and function of the original tissue initially derived from; in this study, the intestinal epithelium10. In general, organoids can be generated from two cellular sources including (1) pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), and (2) organ-specific adult stem cells (ASCs). Organoid cultures are composed of either epithelial cells or both epithelial and mesenchymal cells173. Over the past decade, intestinal epithelial organoids, also known as enteroids, have been utilized as a suitable ex vivo model to investigate the role of IECs in the intestinal mucosal system and their interactions with the microbiome or immune cells174. The enteroid system contains intestinal stem cells that can differentiate into different subtypes of IECs through activation of related signaling 59 pathways175. However, until recently, this had never been successfully attempted in human infants, with the intention of studying the developing intestine and intestinal epithelium. During my Ph.D., I developed one of the first successful human infant intestinal organoid ex vivo models. Intestinal biopsies were collected from infants less than 6 months of age. To construct mini-guts, small intestinal crypts or single intestinal stem cells (ISCs), expressing Lgr5 are embedded in a 3D matrix called Matrigel, which closely resembles the extracellular matrix (ECM) in an in vitro environment176. Matrigel acts as a basement membrane matrix containing ECM molecules including laminin, collagen IV, and enactin176. Organoid culture media are supplemented by defined stem cell niche factors such as epithelial growth factor (EGF), Noggin (inhibitor of BMP signaling), and R-spondin-1 (an enhancer of Lgr5 in the Wnt signaling pathway), which mediate the proliferation of IECs and formation of intestinal crypts177. Four main signaling pathways that function cooperatively for the proliferation and maintenance of ISCs are described below. These signaling pathways are also essential for the growth, proliferation, and maintenance of enteroid models. i) the Wnt/b-catenin signaling pathway, a key regulator of transient amplifying and intestinal stem cell maintenance178. The R-spondin induces the activation of Wnt by suppressing Rnf42/Znrf3 receptors that then degrade Wnt receptor179. ii) the Bone Morphogenic protein (BMP) signaling is involved in the differentiation of IEC subtypes and suppression of ISCs through the expression of BMP downstream 60 molecules such as SMAD1, SMAD5, and SMAD8. Noggin acts as an antagonist of BMP signaling and thus, is required for long-term culturing of intestinal organoids10. iii) EGF signaling that mediates intestinal crypt proliferation180. iv) the Notch signaling pathway involved in the maintenance of ISCs and Transit Amplifying cells. Inhibition of Notch signaling induces differentiation of secretory-lineage cells in the intestine179. Organoid culture media is supplemented by stem cell niche factors such as EGF, Noggin (inhibitor of BMP signaling), and R-spondin-1 (enhancer of Lgr5 in Wnt signaling pathway) that mediates proliferation of IECs and formation of intestinal crypts177. In Chapter 3, I herein describe the protocol that I have established in my Ph.D., to isolate, grow, and optimize human infant enteroids. This organoid model was used to study the developmental changes of intestinal epithelial cells during the early stages of life. To the best of my knowledge, this is the first study using human infant intestinal organoids to research the development of IECs; previous studies focused mostly on pup mice181 or human fetal-derived organoid models182. 3.2 Material and Methods 3.2.1 Ethics statement All samples and biopsies in this study were collected in accordance with guidelines established by the University of British Columbiaâs/Childrenâs & Womenâs Health Centre of British Columbia Research Ethics Board. Parental written informed consent was obtained from the 61 parent or legal guardian of all babies, before the procedure and specimen biopsy (#H19-02989). 3.2.2 Inclusion criteria Babies younger than 6 months of age who required an intestinal procedure as part of standard clinical care for the following reasons were included: 1) Babies that required rectal biopsy for suspected Hirschsprungâs disease (received outpatient treatment in the surgery or GI clinic, or cared for in the Neonatal Intensive Care Unit (NICU)) 2) Babies born with an anorectal malformation requiring stoma creation 3) Babies with intestinal obstruction requiring surgery for operative repair 4) Babies that required diagnostic colonoscopy with biopsies 3.2.3 Exclusion criteria Babies with severe bleeding disorders were excluded. 3.2.4 Patient recruitment Eligible babies were identified by the neonatologist, surgeon, or gastroenterologist involved in the patientâs care. Most of the infants were hospitalized and screened for study eligibility by a surgeon/gastroenterologist after consent had been obtained for the clinical procedure. Only after this did the overseeing clinician briefly introduce the study and inquire if families would be interested in learning about the study from a member of the research team. After a research team member discussed the study and consent, the babyâs parents or legal guardians were 62 provided time (usually 24-72h) to consider the study, ask questions, and sign the consent form electronically if they agreed to participate in the study. 3.2.5 Sample collection Intestinal biopsy samples were obtained at the time of the intestinal procedure that was already planned for each baby. Biopsy samples were obtained by the neonatal surgeon performing the clinical procedure. No research team staff were involved in obtaining the biopsies. For patients undergoing surgeries for abdominal exploration with bowel resection, two biopsies were obtained at that time, or patients undergoing diagnostic colonoscopy had two additional biopsies taken for the study (approximately 2 X 2 mm of tissue total). All procedures posed a minimal additional risk to the patients as they were already undergoing an intestinal resection. The de-identified specimens were then transferred to the research team at BC Childrenâs Hospital Research Institute. 3.2.6 Clinical Data Collection Collected baseline information included sex, age (weeks), intestinal location for biopsy, and reason for colonoscopy. The age of the patient was used to qualitatively assess major differences across the relatively wide age range of the infant group. Information regarding the location of biopsy may provide insights to optimize organoid procedures including stem cell isolation from intestinal crypts, proliferation, and differentiation into mature intestinal epithelial cells (IECs). Information was provided by the surgeon at the time of delivery to the research lab. The site of the biopsy was important for documentation as it could affect the quality of the biopsy or the results from the organoids (Table 3.1). 63 3.2.7 Isolation and growth of infant intestinal organoids Based on previously established methods, tissueâderived epitheliumâonly organoids were generated183. Biopsy samples were washed and resuspended in a cold chelating solution (CCS) with 0.5 M EDTA and 100 µL of Gentamicin. Biopsies were incubated at 4°C on a shaker for 45 minutes (600 strokes/minute), finely chopped, and resuspended in MEM+++ (advanced DMED/F12 supplemented with 1% 1X Penicillin/Streptomycin, 1% 1X Glutamax and 1% 1M HEPES). After washing, the supernatant was removed and cold 1:1 Matrigel (Corning) was added (40 µL/well). Matrigel domes of 30 µL were placed in the center of wells in a pre-warmed 24-well culture plate. Plates were incubated at 37°C for 15-30 min to ensure solidification of Matrigel. 500 µL of organoid growth medium was added to each well and incubated at 37°C, 5% CO2. Organoid growth medium containing base media (advanced DMEM/F12, supplemented with 1% Penicillin/Streptomycin, 1% Glutamax, and 1M HEPES) and 50 % WRN (Wnt3a, R-spondin-3, and Noggin) supplemented with N2 (Invitrogen), B27 (Invitrogen), N-acetyl-cysteine (Sigma-Aldrich), nicotinamide (Sigma-Aldrich), mEGF (Invitrogen), A83-01 (Tocris), SB 202,190 (Sigma-Aldrich), Y-27632 (AbMole) was added (400 µL/well) and changed every two days. Organoids were passaged every 7 to 10 days, depending on growth patterns until an appropriate size for further experiments184. 3.2.8 Optimization of infant intestinal organoids To optimize the growth of infant enteroids, two different splitting methods were used: trypsinization and mechanical splitting. TryplE was used to digest the tight junction of the cells 64 and dissociate them into single cells. Mechanical or physical methods were used to split organoids into smaller clusters of IECs. 3.2.8.1 Enzymatical splitting Organoid media was removed after 6-7 days of growth. Organoids were disrupted, resuspended in TrypLE express (Gibco), and incubated at 37°C with 5% CO2 for 2x5 minutes. Organoids were then gently pipetted using a p1000 tip to dissociate them into the single cell suspension. 3.2.8.2 Mechanical splitting Organoid media was removed once organoids were determined to be large enough to passage. Organoids suspended in MEM+++ were mechanically dissociated into smaller fragments of IECs using 2-3 sets of vigorous up and down pipetting, 50x per set using 200 µl pipet cut tips. 3.2.9 Growth media and passaging After splitting the organoids with either of the above methods, the resuspended solution was centrifuged for 5 min at 1600 RPM at 4°C, and the supernatant was discarded. A mixture of dissociated, diluted organoids and Matrigel suspension at a ratio of 1:2 was applied to the center of the well in pre-warmed 24-well culture plates. To passage, Matrigel domes were disrupted with cold PBS including 5 mM EDTA and incubated at 4°C for 30 min. Organoids were then washed twice with cold media and broken with vigorous pipetting (50x, 3 rounds) with vortexing. Disrupted organoids were centrifuged, the supernatant removed, then resuspended in the proper volume of Matrigel. Organoids 65 utilized in experiments were developed for 3-5 passages. Bright-field (BF) images of organoids were obtained with an Olympus microscope. The budding number and organoid area for each organoid were measured using Fiji ImageJ software. 5-10 organoids and 30 organoids per condition were randomly selected for the measurements of buds and surface area, respectively. 3.2.10 Harvesting and fixation of human enteroids for immunohistochemistry Organoids were harvested using previously described methods185. In brief, Matrigel domes were disrupted using cold 5 mM EDTA in PBS, spun, and washed in PBS. Harvested enteroids were fixed with formalin, resuspended in melted 3% agarose with PBS, solidified, and placed in 70% ethanol until further histology processing. 3.2.11 Immunofluorescence and staining of enteroid cultures As previously described11, immunofluorescence staining was conducted with the following primary antibodies/lectins: mucin 2, Ki67, and epithelial cadherin. Secondary antibodies included Alexa Fluor 488 or 568. ProLong Gold antifade reagent containing 40,6-diamidino-2-phenylindole (DAPI, Invitrogen) was used to mount slides. A Zeiss Axiomager Z1 (Carl Zeiss, Oberkochen, Germany) at 20X magnification and an AxioCam HRm camera with AxioVision software were used to view and image slides. 3.3 Results 3.3.1 Collecting infant intestinal biopsies and establishing a biobank Fourteen biopsy samples were collected and used to develop the organoid model, over the two-year study period. Samples originated from different locations of small and large intestines 66 including the ileum, rectum, sigmoid, and duodenum. The range of age for the babies used in this study was between 1-19 weeks old. However, only biopsies derived from the ileum region were chosen for study experiments, as NEC develops in this region of the small intestine124. 3.3.2 Human infant intestinal organoid cultures Six out of the fourteen samples were qualified for study, passing the isolation and growth processes successfully. Ileum-derived enteroids were focused upon due to NEC development occurring exclusively in infantsâ ileum region of the small intestine. The remaining samples had inconsistent or slow growth patterns, including a sample from a baby with a chromosomal abnormality, making those unsuitable downstream experiments (Table 3.1). Table 3.1 Data set of human infant enteroids ID Sex Gestational age at birth (weeks) Postnatal Age (weeks) Location of biopsy Organoids growth N001 F 32 9 Ileum good N002 M 33 12 Ileum good N003 F 38 2 Rectum good N004 F - 13 Sigmoid Inconsistent or weak N005 F 38 10 Ileum good N006 F 33 6 Ileum Inconsistent or weak N007 M 40 8 Duodenum Slow N008 F 40 1 Sigmoid Inconsistent or weak N009 F 26 11 Ileum Inconsistent or weak N010 F 40 9 Ileum Slow N011 M 33 19 Ileum good N012 F 37 7.5 Ileum good N013 M 36 12 Ileum Not recovered N014 M 29 10 Ileum Slow recovering 67 3.3.3 Optimization of human infant intestinal organoid cultures Brightfield images displayed clear differences between infant enteroids developed using the two different splitting methods described in method section 3.2.8 (Figure 3.1). Enteroids split using the mechanical method were of significantly larger size compared to the enteroids that were trypsinized (Figure 3.1 and Figure 3.2A). Also, trypsinized enteroids had a more cystic phenotype and were less differentiated compared to mechanically split enteroids, which showed more budding and crypt-like structures (Figure 3.1 and Figure 3.2B). H&E images also confirmed that mechanically split enteroids had an increased thickness of IECs compared to trypsinized counterparts (Figure 3.3A and C). Additionally, immunostained images for Ki67, a marker for IEC proliferation, were more distinctive in the enteroids treated with mechanical splitting (Figure 3.3B and D). After comparing the phenotypes of enteroids generated with the different splitting methods, it was decided that all following experiments would use the mechanical splitting method. 68 Figure 3.1. Organoid culture of epithelial cells derived from infantsâ intestines Organoids developed with the Matrigel dome as a 3D structure. (A) Representative images of TrplE-dissociated enteroids grown for 6 days (top row). (B) Representative images of mechanically dissociated enteroids grown for 6 days after splitting (bottom row). Scale bars, 500 µm. Figure 3.2. Surface area and the number of buds of infant enteroids (A) Diameter length of enteroids cultured with control media for 4 days. Each point indicates one enteroid (n=30) for one individual. (B) Quantification of the average number of buds per organoid in enteroids treated with two different methods of splitting. Statistical significance was calculated using Mann-Whitney T-test. *P< 0.05, **P<0.01, ***P<0.001. A B A 0200004000060000â±â±â±â±â±â±â±â±0.00.51.01.52.0â±â±â±â±01234â±010203040â±â±Mechanical splitting TrypsinizationAverage number of buds/organoidB 69 Figure 3.3. Representative immunohistochemistry staining of infant enteroids (A) H&E images of infant enteroids treated with two different dissociation methods, enzymatic (TrplE) (left) and mechanical (right) panel. Scale bars, 100 µm. (B) Immunohistochemistry of Ki67 (red) expressing-cells in enteroids cultured in control media using trypsinization (left) and mechanical splitting (right). Arrows show the thickness of organoid IECs. Scale bars, 100 µm. (C) Thickness measurement of IECs in split infant enteroids (D) Quantification of Ki67-positive cells per organoid for 2 independent subjects. Statistical significance was calculated using Mann-Whitney T-test. *P< 0.05, **P<0.01, ***P<0.001. B A C D 0200004000060000â±â±â±â±â±â±â±â±0.00.51.01.52.0â±â±â±â±01234â±010203040â±â±Ki67+ cells/organoid N001 N002Mechanical splitting Trypsinization0.00.51.01.52.0IECs thickness/organoid (um)â±â±â±â±N001 N002 70 3.4 Discussion In this chapter, I established a human infant intestinal organoid model (or mini-gut) as an ex vivo technique to investigate postnatal intestinal epithelium function during development. Upon birth, infants are exposed to the exterior environment, necessitating rapid protective immune responses against invading pathogens while remaining tolerant against colonizing commensal bacteria2,30. Thus, multiple external and internal factors contribute to intestinal development, in a complex intestinal environment, during the early stages of life. We posit that the use of a human infant organoid model would help understand events that lead to the hyperinflammatory response, disruption of the mucosal barrier, and development of neonatal diseases such as NEC34. In the past, different human cell lines have been used to model interactions of IECs with immune cells or microbes. However, these cell lines have inherent limitations that can be addressed using organoid models. Importantly, cell lines (2D cultures) do not necessarily reflect the complex microenvironment cells encounter in a tissue186,187. For example, the Caco-2 cell line mimics the small intestine with a villus-like enterocyte differentiation process. However, Caco-2 cell line cannot differentiate into goblet cells188â190. Moreover, the proliferation rates of cells in 3D cell culture are more realistic and can be high or low depending on the technique and the types of cells being studied as compared to 2D cell culture191. Cell lines are a powerful homogenous study tool while 3D organoid culture may better capture the heterogenicity of the samples and experimental outcomes depending on study groups192. 71 Studies using mouse and human fetal-derived intestinal organoids have demonstrated that intestinal epithelium lacks mature digestion function and host-defense mechanisms193â195. The human fetal intestinal organoid model recapitulated the gene expression pattern of the original fetal tissue and maintain the fetal gene regulation and development over the time182,196. Hence, infant tissue-derived intestinal organoid models may be a more appropriate method to study the immaturity of IECs during the early stages of post-natal life and the mechanisms involved in the pathogenesis of intestinal-related diseases175. After establishing the human infant enteroid system, the growth of infant enteroids was optimized by using two different splitting methods. Using an enzymatic method for passaging the enteroids, IECs were dissociated into a single cell while the mechanical pipetting method disrupted the cluster of IECs into a smaller group of cells. However, using BF and IHC imaging, the phenotype of enteroids generated with these two methods was observed to be significantly different, with more differentiation in mechanically-split enteroids, with a higher number of buds (Figure 3.2B) and expression of Ki67+cells (Figure 3.3D) compared to trypsinized enteroids. This could be attributed to the relatively larger size of cell clusters that are provided by mechanical splitting; embedding them into Matrigel allows for attachment and faster growth compared to single cells from the trypsinization method, which may require more time to grow. In addition, IECs thickness was significantly higher in mechanically-split enteroids, supported by H&E images which revealed a more packed arrangement of IECs (Figure 3.3A). This phenotype could be more similar to the polarized structure of IECs in the physiological environment197. 72 Several reasons might explain the slow growth of organoids in some subjects. First, biopsies received might not have cut deep enough into the intestinal epithelium to reach deep enough into the crypts to retrieve Lgr5+ cell crypts. Thus, the lack of Lgr5+ cells would prevent enteroid growth after isolation and seeding of cells. Live and functional Lgr5+ IECs were not isolated or sorted from the biopsy tissues used for embedding in Matrigel. Therefore, there is a possibility that for some of the biopsies, there was a lack of sufficient numbers of Lgr5+ containing crypts due to the inflammation of the tissues. Second, contamination of organoids with gut bacteria is another factor that can inhibit enteroid growth. As the gut contains a diverse range of bacteria, so might tissue biopsies, possibly inducing contamination and bacterial overgrowth. Third, the organoids derived from infants with genetic abnormalities showed a vastly different phenotype from the normal patient tissue-derived enteroids. These enteroids were of smaller size and contained a lower number of buds. Although it is difficult to conclude from such a small number of cases, it remains plausible that these samples could have produced vastly different results in the end. Using ISCs from individuals with different genetic backgrounds and growing them in the context of 3D culture allows for focus on IEC characteristics, function highlights differences, and recapitulates individual genetic defects. It is important to note there may be limitations to the technique I used to establish organoid models. Matrigel, a primary component of the organoid system, has a heterogenous composition of ECM proteins that can vary depending on the manufacturer and production lot. Matrigel may also lack the required signals for the structure and function of organoids198. Furthermore, there exists limited accessibility to organoids embedded into the Matrigel; organoids must be removed from Matrigel prior to fixation, immunostaining, and extraction 73 of DNA and RNA175. Though methods have been employed to limit complications, these limitations can affect the efficiency of the organoid model. 74 Chapter 4: Cross-talk between Th17 cells and intestinal epithelium promotes gut health in infants through the actions of IL-22 4.1 Introduction Prior to birth, digestion is unnecessary for the human fetus as any required nutrients are obtained from the motherâs blood via the placenta and umbilical cord1,199. Accordingly, the gastrointestinal (GI) tract of a newborn is developmentally immature at birth, but upon the infantâs first intake of breast milk or formula, the GI tract begins a prolonged course of maturation and development that can take up to 3 years to complete106. Notably, this intestinal development occurs in the context of antigenic stimulation from a rapid increase in gut microbial content, as the GI tract transitions from the in-utero environment to one containing an array of dietary and microbial antigens30. This creates the need for the neonatal adaptive immune system to discriminate between commensal bacteria and pathogens2,30. It also reflects a need for strong physical barriers to protect the infant from threats arising within the intestine22,200,201. Notably, intestinal epithelial cells (IECs), and the overlying mucus layer, provide the primary mucosal barriers in the gut, separating luminal microbes and food antigens from the underlying mucosal immune system. This segregation not only helps promote beneficial microbe-host interactions within the gut7, with many host-protective commensal microbes obtaining key nutrients from the mucus layer202, but it also limits the risk of antigens leaking out and triggering exaggerated inflammation as seen in diseases such as necrotizing enterocolitis (NEC), which is a surgical conditions encountered in prematurely born neonates110. 75 Aside from mucin-secreting goblet cells202, other specialized secretory IEC such as Paneth cells release antimicrobial peptides (AMPs) that provide further protection against luminal pathogens203. Due to their location between luminal microbes and underlying immune cells, IECs are frequently the subject of injury associated with exaggerated immune responses against gut microbes204. To deal with this, small intestinal IECs steadily proliferate, completely renewing the epithelial barrier every three to six days by migrating from the base of crypts toward the villus tip where they are sloughed205. This replacement can be accelerated in response to overt tissue damage as well as cytokine signals released by T cells. Th17 cells appear to play a central role in the pathology of NEC in premature neonates206. These cells are enriched in the intestinal LP, even under steady-state conditions207,208. IL-17 is a key cytokine produced by these cells and is known to promote inflammatory responses at mucosal surfaces72, including the recruitment of neutrophils and other inflammatory cell types209,210. Th17 cells interact with IECs, protecting the gut against bacterial and fungal pathogens while maintaining immune tolerance to commensal microbes211. On the other hand, high levels of IL-17 can also drive excessive inflammatory responses, such as those seen in individuals suffering from inflammatory bowel disease (IBD) or NEC124,212â214. In contrast, while naïve CD4 T cells isolated from the cord blood of infants have a propensity to develop into T regulatory (Treg) and T helper 2 (Th2) cells following TCR activation, their development into Th17 cells is functionally distinct, characterized by limited production of IL-17136,155, as described in Chapter 2. As such, the cross-talk between Th17 cells and the gut epithelium is likely altered in infants. This suggests that the infantâs gut relies on T cell- 76 produced IL-22 rather than IL-17 to strengthen and protect the integrity of the intestinal mucosa. In this study, we hypothesize that neonatal Th17 cells are developmentally adapted to the infant's gut and that IL-22, particularly, induces a protective response on IECs. To test this hypothesis, we developed a novel infant intestinal organoid model (also known as enteroids) to compare the baseline functions of IECs from infants (less than 6 months of age) and older children (6-14 years of age). We show that neonatal Th17 cells induce the proliferation of infant-derived enteroids, as well as mucus and AMP production, in an IL-22-dependent manner. Further genome-wide transcriptomic analyses of infant enteroids suggest that IECs pathways facilitate cross-talk with developmentally adapted Th17 to promote a healthy gut. 4.2 Material and Methods 4.2.1 Ethics statement This research was approved by the University of British Columbiaâs/Childrenâs & Womenâs Health Centre of British Columbia Research Ethics Board (#H19-02989 & #H14-03199). Parental written informed consent was obtained for the use of all biopsy specimens. 4.2.2 Naïve CD4 T cell isolation and differentiation Cord blood was collected from term deliveries by Cesarean section (>38-41 weeks of gestation) in sodium heparin anti-coagulated vacutainers (Becton Dickinson, Canada), and processed within 2-6 hours of collection. Cord blood mononuclear cells (CBMCs) were isolated from de-identified cord blood samples via density centrifugation with LymphoPrep 77 (STEMCELL Technologies), cryopreserved in PBS with 44 mg/mL human serum albumin, 2 mM EDTA, and 10% DMSO (1.0 x 106 cells/mL), and stored at -80°C. Naïve CD4+ T cells were isolated from thawed CBMCs via immunomagnetic negative selection (StemCell Kit), manually counted, and seeded in a 96-well round-bottom plate (0.5 x 106 cells per well). As previously described155,215,216, activation of CD4 T cells was achieved using anti-CD3 and anti-CD28 soluble antibody complexes (25 µL/mL; ImmunoCultâ¢ Human CD3/CD28 T Cell Activator) in complete RPMI (RPMI with 10 % human AB serum, 1 % sodium pyruvate, 1 % 1X Penicillin/Streptomycin) in the presence of human recombinant IL-6 (20 ng/mL), IL-23 (10 ng/mL), IL-1β (10 ng/mL) (Peprotech) and neutralizing IL-4 and IFN-γ antibodies (RATIO; BD Pharmingen) at 37°C (5% CO2) for seven days, with fresh media and cytokines added after 3 days. Supernatants were collected at the end of the culture and frozen at -80°C in batches. IL-22 concentration in culture supernatants was determined by ELISA (Biolegend IL-22 Kit). 4.2.3 Collection of infant and pediatric biopsies To characterize the intestinal epithelial landscape in early life, we generated human ileum-derived organoids (enteroids) from biopsies taken from infants <6 months of age at the BC Childrenâs Hospital. Ileum-derived organoids from pediatric cases (6-14 years old) served as controls. 4.2.4 Generation and maintenance of human infant and pediatric-derived enteroids Based on previously established methods, tissueâderived epitheliumâonly organoids were generated. Biopsy samples were washed and resuspended in a cold chelating solution (CCS) 78 with 0.5 M EDTA and 100 µL of Gentamicin. Biopsies were incubated at 4°C on a shaker for 45 min. (600 strokes/minute), finely chopped, and resuspended in MEM+++ (advanced DMED/F12 supplemented with 1% 1X Penicillin/Streptomycin, 1% 1X Glutamax, and 1% 1M HEPES). After washing, the supernatant was removed and cold 1:1 Matrigel (Corning) was added (40 µL/well). Matrigel domes of 30 µL were placed on the center of wells in a pre-warmed 24-well culture plate. Plates were incubated at 37°C for 15-30 min to ensure solidification of Matrigel. 500 µL of organoid growth medium was added to each well and incubated at 37°C (5% CO2). Organoid growth medium (advanced DMEM/F12 used as base media, supplemented with 1% 1X Penicillin/Streptomycin, 1% 1X Glutamax, and 1% 1M HEPES) and 50% WRN supplemented with N2 (Invitrogen), B27 (Invitrogen), N-ace-tylcystine (Sigma-Aldrich), nicotinamide (Sigma-Aldrich), mEGF (Invitrogen), A83-01 (Tocris), SB 202,190 (Sigma-Aldrich), Y-27632 (AbMole) was added (400 µL/well) and changed every two days. Organoids were passaged every 7 to 10 days until determined appropriate for further experimentation184. To passage, Matrigel domes were disrupted with cold PBS including 5 mM EDTA, and incubated at 4°C for 30 min. Organoids were then washed twice with cold media and broken with vigorous pipetting (50x, 3 rounds) and vortexing. Disrupted organoids were centrifuged, the supernatant removed, then resuspended in the proper volume of Matrigel. Organoids utilized in experiments were developed for 3-5 passages. 79 4.2.5 Stimulation of human enteroids Enteroids were passaged as described above. Following culture for 4 days, organoid media was replaced with fresh 70% organoid growth medium supplemented either with neonatal Th17 culture supernatant or cRPMI as a control condition. For conditions requiring recombinant cytokines, IL-22 was added to the growth organoids medium with 30 % of cRPMI. Recombinant Il-22 (R&D Systems) was used at 100 ng/ml. Neutralizing Il-22 (R&D Systems) was also used at 5 µg/ml. Th17 cell culture supernatants were refreshed in organoid media every 3 days for 6 days of co-culture. On Day 7, organoid cultures were collected, pooled, and harvested for further analysis. 4.2.6 Harvesting and fixation of human enteroids for immunohistochemistry Organoids were harvested as previously described185. In brief, Matrigel domes were disrupted using cold 5 mM EDTA in PBS, spun, and washed in PBS. Harvested enteroids were fixed with formalin, resuspended in melted 3% agarose with PBS, solidified, and placed in 70% ethanol until further histology processing. 4.2.7 Immunofluorescence and staining of enteroid cultures As previously described11, immunofluorescence staining was conducted with the following primary antibodies/lectins: mucin 2, Ki67, and epithelial cadherin. Secondary antibodies included Alexa Fluor 488 or 568. ProLong Gold antifade reagent containing 40,6-diamidino-2-phenylindole (DAPI, Invitrogen) was used to mount slides. A Zeiss Axiomager Z1 (Carl 80 Zeiss, Oberkochen, Germany) at 20X magnification and an AxioCam HRm camera with AxioVision software were used to view and image slides. 4.2.8 RNA Extraction and qPCR analysis of enteroid cultures Following harvest, enteroids from three to four wells were pooled, resuspended in TRIzol (Invitrogen), and lysed at room temperature for 15 minutes. RNA purification was performed with the Direct-zol RNA Microprep Kit (Zymo Research) using the manufacturerâs protocol. A NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific) was used for the quantification of isolated RNA. 4.2.9 RNA Seq analysis The Agilent 2100 Bioanalyzer was utilized for achieving sample quality control. After that, the NEBnext Ultra ii Stranded mRNA standard protocol (New England Biolabs) was followed to prepare qualifying samples. Sequencing was performed on the Illumina NextSeq2000 using Paired-End 59bp Ã 59bp reads. Illumina's bcl2fastq2 was employed to De-multiplex the sequencing data. Subsequently, the De-multiplexed read sequences were aligned to the reference sequence of Homo sapiens/Mus Musculus (PAR-masked)/hg19 or mm10) using the STAR aligner217. We estimated the assembly and differential expression by using Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) through bioinformatics apps available on Illumina Sequence Hub. Downstream analysis was conducted in R 4.1.1 with R studio. Samples were batch-corrected using ComBat_Seq. For differential gene expression analysis, a paired control-treatment 81 model was fitted using the standard edgeR workflow. Gene set enrichment analysis (GSEA) was performed on a ranked gene list with -log10(p-value)*sign(log fold change) as the input metric. Gene sets were obtained from curated databases (KEGG, Hallmark, GO) and 1000 permutations were performed to calculate enrichment. Sample level enrichment analysis (SLEA) was performed as described previously218 using marker genes previously obtained by single-cell RNA-seq analysis of the human intestine219. Multiple testing was corrected for using the Benjamini-Hochberg method and a false discovery rate of 5% was considered significant. 4.2.10 Statistical analysis Data are representative of at least 3 biological replicates in independent experiments. the results are expressed as the mean values± standard errors (SEM). Mann-Whitney U-test, t-test, and two-way ANOVA were assessed using GraphPad Prism software, version 9.4. A p value of 0.05 or less was considered significant, with asterisks denoting significance in the figures. 4.3 Results 4.3.1 Neonatal Th17 cell-derived cytokines support the growth of infant enteroids As neonatal-derived Th17 cells produce copious amounts of IL-22155 (Figure 4.1A), we sought to determine the impact of neonatal Th17 supernatant, particularly IL-22, on infant and pediatric-derived enteroids (Figure 4.1A). To this end, we isolated naïve CD4 T cells from adult and cord blood (Figure 2.1) and then stimulated them in the presence of Th17-required cytokines (see methods). Infant and pediatric 82 enteroids were allowed to establish in vitro for 3 days and then co-cultured with either neonatal Th17 supernatants or control media (cRPMI) for 4 more days. As shown in Figure 4.1B, C, and D, infant-derived enteroids grown in the presence of control media were significantly larger and more crypt-like than the smaller and more spherical pediatric-derived enteroids. Although both infant- and pediatric-derived enteroids treated with Th17 cell supernatants showed an upward growth trend in comparison to the control media-treated enteroids, the responses to the supernatant were stronger in the infant-derived enteroids (Figure 4.1B, C, and E). Brightfield images of these enteroids treated with infant Th17 cell supernatant showed a more crypt-like phenotype with budding structures as compared to the cystic phenotype of the pediatric enteroids. H&E images also confirmed a denser and thicker arrangement of IECs in the stimulated, infant-derived enteroids, as compared to pediatric enteroids (Figure 4.1C). Furthermore, brightfield images taken from infant enteroids co-cultured with rhIL-22 demonstrated similar changes to those exposed to infant-derived Th17 cell supernatants (as above), with the enteroids being larger and showing more crypt-like budding than untreated enteroids. This could be seen through H& E images of the enteroids (Figure 4.1F and G). Notably, this phenotype was reversed by treating the Th17 cell supernatants with a neutralizing-IL-22 antibody (a-IL22) and the size of infant enteroids is significantly smaller in the presence of an IL-22 inhibitor (Figure 4.1H). 83 84 Figure 4.1. Neonatal Th17 cell-derived cytokines support infant organoids growth (A) IL-22 was measured in adult (blue) and neonatal (pink) magnetic bead-purified naïve CD4 T cells activated by anti-CD3/CD38 soluble antibody complex for 7 days in the presence of recombinant IL-6 (20 ng/mL), IL-23 (10ng/mL), IL-1β (10 ng/mL), and neutralizing IL-4 and IFN-γ antibodies. (B) Representative brightfield images of human pediatric and infant enteroids grown in control media (first and third column) and in neonatal Th17 supernatant (second and fourth) for 4 days. Scale bars, 500 µm. (C) H&E images of pediatric and infant enteroids cultured with control media and neonatal Th17 supernatant, respectively for 4 days. Scale bars, 100 µm. (D) Growth curve of infant (dark grey) and pediatric enteroids (light grey) after culturing with control media for 4 days. Data are means Â± standard deviations (SDs) from n= 30 enteroids; biological N=3 human individuals. Statistical significance was calculated using two-way ANOVA.**P<0.01. (E) Growth curve of infant and pediatric enteroids after culturing with neonatal Th17 supernatant for 4 days compared to the control conditions. Data are means Â± standard deviations (SDs) from n= 30 enteroids; biological N=3 human individuals. Statistical significance was calculated using two-way ANOVA. *P< 0.05. (F) Brightfield images of infant enteroids cultured with neonatal Th17 cell sup (left), rhIL-22 (100 ng/ml) (middle), and rhIL-22 with neutralizing IL-22 (right). Scale bars, 500 µm. (G) H&E images of infant enteroids cultured in the same conditions as (F). Scale bar, 100 µm. (H) Quantification of diameter length of enteroids cultured with neonatal Th17 supernatant and rhIL-22 (100ng/ml) in the absence and presence of neutralizing IL-22 for 4 days. Technical replicates n=30; N= 3 human individuals. Statistical significance was calculated using two-way ANOVA. ***P<0.001. 4.3.2 IL-22 is the key cytokine underlying the effects of the human infant Th17 cell supernatant To explore the effect of the infant Th17 cell supernatant on IEC proliferation and differentiation, enteroids were stained for the Ki67, a transient amplifying marker. Increased proliferative capacity of infant enteroids compared to pediatric enteroids was confirmed by higher expression of Ki67 (Figure 4.2A and B). The proliferation of infant-derived enteroids (as measured by Ki67 staining) was significantly decreased when neutralizing IL-22 antibody was added to the neonatal Th17 supernatant (Figure 4.2B). To assess the differentiation of IECs to goblet cells, both infant and pediatric enteroids were immunostained for MUC2, a specific marker for goblet cells. Increased differentiation of F 85 goblet cells and mucin production was confirmed by a higher number of MUC2+ organoids and MUC2 intensity in the cultures of infant-derived enteroids treated with Th17 supernatant as compared to their pediatric counterparts (Figure 4.2C). Neutralizing IL-22 also dramatically decreased MUC2 expression as compared to Th17 sup-treated enteroids (Figure 4.2D). Although the expression of lysozyme mRNA was not significantly increased in the presence of IL-22 in our RNA-seq data, immunostaining of infant enteroids that were exposed to both IL-22 and Th17 supernatant showed increased expression of lysozyme compared to the control enteroids. Lysozyme expression demonstrated the differentiation of infant IECs to Paneth cells in the presence of IL-22 (Figure 4.2E). Overall, the obtained data revealed that the stimulated supernatants from infant-derived Th17 cells increased both the proliferation and differentiation of enteroids, with the infant-derived enteroids showing a greater impact than the pediatric-derived enteroids. These data strongly suggest that high levels of IL22 in the infant-derived Th17 cell supernatants support the growth and differentiation of infantsâ IECs toward goblet cells and Paneth cells. 86 Figure 4.2. IL-22 is the key cytokine underlying the effects of the human infant Th17 cell supernatant (A) Representative immunohistochemistry of Ki67 (red) expressing cells in enteroids cultured in control media (left), neonatal Th17 cell sup (middle), and Th17 cell sup +aIL-22 (right). Infant and pediatric enteroids were immunostained for Ki67, E-cadherin, and nuclei (DAPI). Scale bars, 100 µm. (B) Quantification of Ki67-positive cells per organoid, technical replicates n=5; N= 4 human individuals. (C) Representative immunohistochemistry of MUC2 expressing LYZ DAPIControl Media Neonate Th17 Cell Sup rh IL-22D E C Infant EnteroidsPediatric Enteroids Control MediaTh17 SupαIL-22+Th17 sup 020406080100â±â±â±â± â±â±â±â±â±â±â±â±â±â±â±â±-1.0-0.50.00.51.01.5â±â±â±â± â±â±â±â±Ki67 Positive cell/OrganoidControlTh17 supaIL-22+ Th17 supControl MediaTh17 SupαIL-22+Th17 sup 0204080100â±â±â±â± â±â±â±â±â±â±â±â±â±â±â±â±-1.0-0.50.00.51.01.5â±â±â±â± â±â±â±â±ControlTh17 supaIL-22+ Th17 supMUC2 intensity(relative to DAPI) B A 87 cells in enteroids cultured in control media (left), neonatal Th17 cell sup (middle), and Th17 cell sup +aIL-22 (right). Infant and pediatric enteroids were immunostained for MUC2, E-cadherin, and nuclei (DAPI). Scale bar, 100 µm. (D) Measurement of MUC2 intensity per organoid technical replicates n=5; N= 4 human individuals. MUC2 intensity was normalized to the control group. (E) Representative immunohistochemistry of lysozyme (green) expressing Paneth cells in infant enteroids cultured in control media (left), neonatal Th17 cell supernatant (middle), and rhIL-22 (right). Infant enteroids were immunostained for Lyz (green), and DAPI (blue). Scale bar, 100 µm. 4.3.3 IL-22 upregulates anti-microbial pathways in both infant and pediatric enteroids To understand the effects of IL-22 on human infant and pediatric-derived enteroids, we performed RNA-sequencing on ileum-derived enteroids from four infants and three pediatric subjects after four days of co-culturing with IL-22 and compared them to media-treated control enteroids. Transcriptional analysis showed the same trend in infant and pediatric enteroids treated with IL-22. 1532 genes were significantly impacted by IL-22 treatment (FDR cutoff), with similar patterns observed in pediatric and infant enteroids (Figure 4.3A). IL-22 triggers the regulation of members of the Reg gene family, with significant upregulation of REG3A, REG3G, and REG1B (Figure 4.3B). REG3A and REG3G are known IL-22 target genes associated with antimicrobial peptides in the small intestine220. Expression of Reg3a/g at the protein level is confirmed by immunostaining of infant enteroids (Figure 4.3C). The list of genes that are differentially expressed in IL-22-treated enteroids is listed (Table 4.1). SPDEF, a transcription factor involved in the differentiation of goblet cells, is significantly (Q value) more expressed in both infant and pediatric enteroids that are exposed A 88 to IL-22 compared to those not exposed to IL-22. additionally, MUC4 and REG4 genes are significantly upregulated in pediatric enteroids treated with IL-22. Figure 4.3. IL-22 induces differential transcriptomic changes in infant and pediatric enteroids (A) Heatmap of genes differentially expressed in infant and pediatric -enteroids treated with IL-22. (B) Volcano plot of fold change and adjusted p values of differentially expressed genes in the presence of IL-22 and control counterparts. (C) Representative immunohistochemistry of Reg3a/g expressing enteroids cultured in control media (left) and rhIL-22 (right). Scale bar, 100 µm. A B C B -10 -5 0 5 10 15 20051015logFC- log10(adj. p val) REG1BREG1AREG3AMUC4Reg3a/g DAPI Ecad*Control media rh IL-22 89 Table 4.1 List of genes differentially expressed in IEC subtypes in infant enteroids 4.3.4 Il-22 induces the JAK-STAT-signaling pathway and attenuates stemness Gene set enrichment analysis (GSEA) revealed significant downregulation of stemness-associated pathways such as Wnt/β-catenin- and Notch-signaling, accompanied by metabolic Gene name logFC FDR Gene name logFC FDRDifferentiation ImmunityPaneth cells CASP1 4,8436 0,1138LYZ 0,2169 0,6705 CASP4 1,5085 0,0236 **REG3A 7,1541 0,0041 ** CASP5 5,6041 0,2031REG3G 7,6326 0,0041 ** CASP8 0,5455 0,3722REG1B 18,6850 0,0090 ** CASP10 1,6247 0,0109 **REG1A 13,7494 0,0658 CCL2 -0,7274 0,6436CCL20 2,3767 0,1117Stem cells CCL25 -2,8185 0,0192 **OLFM4 2,7624 0,0245 **LGR5 -0,9564 0,0304 ** CXCL1 1,2419 0,0419 **CHGA 0,1949 0,6069 CXCL2 1,9003 0,0153 **ASCL2 -0,2779 0,6922 CXCL3 1,3784 0,0231 **CXCL5 3,0773 0,0265 **Goblet cells CXCL8 0,4679 0,7332SPDEF 1,0629 0,0264 ** CXCL11 -2,4506 0,0746MUC6 -1,9237 0,0983 CXCL14 6,1113 0,0324 **MUC2 0,7798 0,7698SPINK4 -0,3269 0,6780 IL7 2,2003 0,0109 **TFF3 0,2173 0,7994 IL18BP -0,6330 0,3796CLCA1 4,5161 0,2095 IL18 0,9733 0,1590ZG16B 0,1304 0,8601 IL10RB 0,1264 0,7122ZG16 -3,2916 0,3983 IL22RA1 -0,3524 0,3705REG4 -4,3947 0,2203 SIGIRR 0,2842 0,6814IRF1 1,9288 0,0159 **Enterocytes IRF7 1,3362 0,0131 **ALPI 0,7582 0,6618 IRF9 1,4133 0,0278 **SLC26A3 -1,4635 0,6436 STAT3 0,5749 0,0780TMEM37 0,5283 0,1463 STAT1 0,3996 0,2563FABP2 0,8732 0,4415 STAT4 0,4323 0,6500TLR2 0,0461 0,9234TLR3 1,2199 0,3888TLR4 -1,1074 0,4437TLR5 -0,2896 0,5427TNFSF10 1,0439 0,1117TNFSF13 0,5688 0,1106TNFRSF1A 0,5153 0,1358TNFAIP8 0,7381 0,1433 90 remodeling favoring catabolic pathways such as glycolysis, at the expense of quiescence-associated pathways such as fatty acid metabolism (Figure 4.4A&B). The expression of LGR5, ISCs biomarker, is downregulated significantly in both infant and pediatric enteroids after exposure to IL-22, contrary to the upregulated expression of OLFM4 (Figure 4.4B). Additionally, IL-22 led to significant induction of immune-related pathways, including known IL-22 downstream targets such as complement and JAK-STAT-signaling (Figure 4.4C). Indeed, transcription factor target analysis using the ChEA database221 showed an overrepresentation of STAT4, STAT3 and STAT1 targets (Figure 4.4D). Additionally, IRF1 and IRF8 targets were enriched among the IL-22-induced genes, further reflected by significant upregulation of interferon α and γ response pathways with IL-22 treatment (Figure 4.4D). Lastly, as IL-22 has been implicated in IEC proliferation and differentiation, we assessed whether IL-22 treatment altered the cell type composition in the enteroids. We used previously published markers derived from single-cell RNA-seq analysis of the human intestine219 to calculate sample level enrichment analysis (SLEA) scores for all samples. Strikingly, four days of culture in the presence of IL-22 led to a significant increase in SLEA scores for Paneth, goblet, progenitor, and stem cells (Figure 4.4F). These results suggest that IL-22 induces changes in the overall cellular makeup of the enteroids. By significantly inducing progenitor cells, IL-22 might favor the terminal differentiation of differentiated secretory cell types such as goblet cells and Paneth cells. 91 -2 -1 0 1 2 3 4IL 2 S T A T 5 S IG N A L IN GC O M P L E M E N TT N F A S IG N A L IN G V IA N F K BIL 6 J A K S T A T 3 S IG N A L IN GIN T E R F E R O N G A M M A R E S P O N S EIN T E R F E R O N A L P H A R E S P O N S EF A T T Y A C ID M E T A B O L IS MB IL E A C ID M E T A B O L IS MG L Y C O L Y S ISM T O R C 1 S IG N A L IN GM Y C T A R G E T S V 2M Y C T A R G E T S V 1E 2 F T A R G E T SN O T C H S IG N A L IN GW N T B E T A C A T E N IN S IG N A L IN GA P O P T O S ISImmunityMetabolismProliferationNormalized Enrichment Score (NES)WNT b Catenin NOTCH Signaling Immune signaling051015EnterocyteSLEA score- + - +Infant Pediatric4567Goblet- + - +Infant Pediatric45678Paneth- + - +Infant Pediatric3456Enteriendocrine- + - +Infant Pediatric19202122232425Progenitor- + - +Infant Pediatric2224262830TA- + - +Infant Pediatric181920212223Stem Cell- + - +Infant PediatricIL-22EnteroendocrineIL-22 ns * *** ns * ns *Age ns ns ns * ns ns nsA B E C Paneth cell IL-6 JAK STAT Signaling D F 92 Figure 4.4. Il-22 attenuates stemness-associated pathways and induces the JAK-STAT-signaling pathway (A) GSEA of differentially expressed genes in infant and pediatric enteroids following treatment with IL-22. (B) Heatmap representation of the leading genes involved in WNT b Catenin and NOTCH signaling (C) IL-6 JACK-STAT signaling (D) Immune signaling pathways and manually selected genes involved in (E) Paneth cell in infant and pediatric enteroids treated with control media and IL-22. The color scale represents Z scores. (F) SLEA scores of IECs sub-types in infant and pediatric enteroids before and after exposure to rhIL-22. 4.4 Discussion This study examined the physiological effect of neonatal Th17 cell-derived cytokines, particularly IL-22, on the phenotype, proliferation, and differentiation of intestinal epithelial cells in infants. Our findings indicate that infant enteroids treated with neonatal Th17 supernatant promoted proliferation, increased mucus production and the differentiation of IEC subtypes, including goblet and Paneth cells, in an IL-22-dependent manner. Genome-wide transcriptomic studies further identify potential differences in JAK-STAT pathways, intestinal stemness as well as in the composition of IECs. IL-22 is characterized by the dual function of inciting and inhibiting inflammation222. Both animal and human models have confirmed the constructive role of IL-22 in epithelial proliferation and regeneration223, expansion of transient amplifying (TA) cells85,224,225, production of the mucus membrane, and stimulation of AMP production225. Neonatal Th17 cells abundantly produce IL-10 and IL-22155. Th17 cell-derived IL-22 increased infant enteroid size (Figure 4.1E). Expression of Ki67, a TA marker, is dramatically declined in both infant and pediatric enteroids after treating enteroids with IL-22 inhibitor, confirming the role of IL-22 on the proliferation of IECs in the human enteroids (Figure 4.2A and B). Given the beneficial role of IL-22 in the proliferation and regeneration of IECs, our data support a role 93 for IL-22 in enhancing the integrity and development of the intestinal mucosal system in the early stages of life. This may explain the predominant role of Th17 cells in NEC as evidenced by animal experimental models and human tissue studies124. Current studies have linked the lack of IL-22 in premature mice to the development of NEC226. Moreover, previous studies confirm that IL-17 expression is associated with intestinal mucosal injury in NEC124. Thus, it can be deduced that the inability of neonatal Th17 cells to produce IL-17 and their tendency to produce IL-22 is necessary for the proliferation and differentiation of infant IECs in the early stages of life. In light of the data presented herein, we wondered whether an exaggerated or developmentally precocious strong Th17 cell bias towards the production of IL-17 creates pathological conditions that may lead to NEC in developmentally maladapted infants. Further experiments are needed to confirm my theory here. Transcriptomic data of enteroids treated with IL-22 showed upregulation of IEC sub-types, including enterocyte progenitors, goblet cells, and Paneth cells, when compared to untreated enteroids (Figure 4.4F). Infant enteroids treated with IL-22 demonstrated downregulation of LGR5 mRNA expression in RNA-seq analysis, consistent with previous studies in mouse models227,228. Conversely, when comparing human infant IL-22-treated enteroids with pediatric control enteroids, OLFM4 was significantly upregulated; the opposite trend was found in murine enteroids, highlighting potential differences between species and the importance of conducting studies using human samples 85,224. RNA-seq transcriptional analysis also revealed that IL-22 triggers the expression of genes involved with antimicrobial activity, including REG1A, REG1B, and REG3A in infant enteroids. This was also confirmed at the 94 protein level (Figure 4.3C) and is in line with a previous study on human and mouse enteroids229. IL-22 not only triggers Paneth cell differentiation but also incites the production of AMPs in intestinal stem cells, which are located at the bottom of the crypts229. Furthermore, overexpression of Reg1α and Reg1β, which are directly induced by IL-22, are involved in Crohnâs disease (CD) and ulcerative colitis (UC), confirming the dual function of IL-22 as both a trigger and inhibitor of inflammation33,34. Both Paneth cells and enterocyte subtypes are augmented in pediatrics and infant enteroids treated with IL-22. Previous studies demonstrate that IL-22 induces lysozyme production in mice and human ileum enteroids85,229. Despite increased upregulation of lysozyme expression in the human infant RNA-seq data following treatment of enteroids with IL-22, there was no significant trend in lysozyme expression. However, we confirmed the expression of lysozyme in infants treated with IL-22 and neonatal Th17 supernatant through immunohistochemistry staining of lysozyme (Figure 4.2E). This can be attributed to the early expression of lysozyme in enteroids IECs that we could not detect the lysozyme mRNA expression in RNA-seq data. IL-22 induced the expression of goblet cell genes, namely MUC2, TFF3, and SPDEF, which are essential for the differentiation of goblet cells231. Despite RNA-seq data stating the MUC2 gene was not differentially expressed in IL-22 treated enteroids, we found MUC2 expression at the protein level, evidenced by significant MUC2 intensity in Th17-treated enteroids compared to untreated enteroids and downregulated after enteroid treatment with neutralizing IL-22 in both infant and pediatric groups (Figure 4.2D). Since RNA sequencing was performed following 4 days of enteroids treatment, it is assumed that MUC2 expression decreased 95 gradually during the co-culturing period, and any detected MUC2 protein resulted from the translation of MUC2 transcripts during the early stages of co-culturing. A limitation of this study is that I only examined Th17 cells. However, other cells are known to produce IL-22. Indeed, ILC3, an analog of Th17 cells, is one of the first innate components of defense to protect the fetus and its maturation, which is closely regulated by microbial colonization at birth232. The research described how the dysregulation of ILC3 subtypes in both mice models and necrotic human tissue leads to a reduction of IL-22206. As ILC3s are the predominant source of IL-22 during an innate response, it is justifiable that the presence of ILC3 and the consequent production of IL-22 can be advantageous and have a constructive impact on the infant IECs. However, due to limitations regarding extraction and culture of ILC3s from neonatal peripheral blood, in vitro differentiated neonatal Th17 cells were chosen for this study and used to test the effects of resultant supernatant on an infant enteroid model. Thus, considering that innate immune systems mature at earlier fetal stages than adaptive responses, it is speculated that ILC3s are the preceding source of protective IL-22 in LP of infants. IL-10 is another main cytokine that is produced by neonatal Th17 cells. We did not investigate the physiological role of IL-10 in infant enteroids in this study. However, He et al. recently showed that IL-10 does not impact on the Paneth cell percentage in human enteroids229. Overall, this study showed the impact of Th17-derived cytokines, particularly IL-22 on the enhancement of proliferation of infant enteroids and triggering the differentiation to different IECs including goblet and Paneth cells. 96 In summary, using infant and pediatric enteroids, we showed the effect of IL-22 on upregulation of genes special to Paneth cells and goblet cells. The population of specialized IECs such as Paneth cells and goblet cells change in infant and pediatrics enteroids treated with IL-22. Also, IL-22 as a main neonatal Th17 cytokine induces the production of AMPs such as lysozyme and Reg3a/g from Paneth cells and mucin from goblet cells in infant enteroids. 97 Chapter 5: Conclusion 5.1 Summary of findings The adaptive immune system in infancy is developmentally adapted to the early stage of life compared to adulthood233. Specifically, Th17 cells are less capable of producing inflammatory IL-17 in individuals younger than 3 months of age until the age of 12 months234. However, our results revealed that neonatal CD4 T cells produce high amounts of IL-22, not IL-17, in Th17-polarizing conditions. As an anti-inflammatory cytokine, IL-22 targets IECs and generates a protective effect on IECs development. This raised the question of what the developmental effect of IL-22 during infancy consists of. In light of these observations, the overarching goal of my thesis was to characterize the molecular mechanisms and effect of neonatal Th17 cells, with a specific emphasis on the cross-talk between Th17 cells and infant IECs, including the physiological effect of IL-22 on proliferation and differentiation of IECs. In Chapter 2, I performed a side-by-side comparison of the gene expression profiles of term neonatal and adult naïve CD4 T cells. This provided mechanistic insights into the limited capacity of neonatal naïve CD4 T cells to differentiate toward IL-17-producing Th17 cells. Th17 cells play a crucial role in fighting against Candida invasion at the skin and mucosal surfaces, mediated by the production of IL-17. Loss-of-function genetic mutations in IL-17 and receptor gene pathways132 can lead to severe Mucocutaneous Candidiasis (MC). Neonates are susceptible to MC and often manifest via diaper rashes and oral thrush. Thus, my data shows that neonatal T cells produce little IL-17 upon differentiation, making it plausible that 98 a deficit in T cell-produced IL-17 contributes to the underlying cause of young infant susceptibility to MC133. Naïve CD4 T cells (CD3+CD4+CD45RO-CCR7+CD25-) from the cord and peripheral blood were isolated and stimulated with anti-CD3/CD28 beads for 6 days, in the presence of either Th17-polarizing (IL-1b, IL-6, and IL-23), Th1-polarizing (IL-12), or no cytokine (Th0) conditions. Data showed that neonatal CD4 T cells are not only biased toward Th2, already demonstrated in previous studies, but also towards the production of IL-22 and IL-10 in comparison to adult T cells. To investigate the effect of neonatal APCs on the Th17 differentiation capacity of neonatal T cells, allogenic adult CD3-depleted mononuclear cells were used instead of neonatal APCs. However, neonatal T cells still produced substantially less IL-17 and IFN-g than adult T cells in the same condition ( Figure 2.4B). Overall, this experimental data revealed a qualitatively different differentiation profile for neonatal naïve CD4 T cells. There is controversial data regarding the potential of neonatal CD4 T cells to produce IL-17. While one study showed strong IL-17 responses in the lymph nodes and gut137, other studies revealed that peripheral T cells can produce a subtle IL-17 response, but only in memory T cells138. Some of these discrepancies may be due to trace contamination of cell cultures with differentiated (CD45RO-expressing) effector T cells. However, the methods used in this study, which carefully remove such cells by FACS prior to use, enable cultures to reflect phenotypes closer to that of naïve CD4 T cells. Since circulating CD4 T cells (i.e. from the cord or 99 peripheral adult blood) are the focus of our studies, it remains possible that other immune cells represent an important source of IL-17 in human infants in vivo. Next, genome-wide gene expression analysis recognized the Th22 gene signature within STAT3 downstream targets in neonatal T cells, supporting Th22-biased neonatal T cells responses. Additionally, this approach showed major developmental changes in key regulators of TGF-b signaling pathway, including TGFBR2, TGFBR3, furin, as well as SMAD4 and SMAD7 in neonatal naïve CD4 T cells (Figure 2.5D). Furthermore, downregulation of Th17 key mediators such as STAT3, RORgt, and BATF was also observed, which are known to drive the ratio of Th17 and Treg cells towards pathogenic Th17 responses in neonatal naïve CD4 T cells153 (Figure 2.5). Further functional studies suggested that the changes in TGF-b signaling between adult and neonatal T cells are age-dependent. While IL-1β and IL-23 alone were adequate for IL-17 production in adult T cells, the addition of IL-6 and TGF-b was required for the highest production of IL-17 in neonatal T cells. Neonatal T cells required IL-6 in addition to IL-1b and IL-23 for maximal IL-22 production, while adult T cells produced more IL-22 in the presence of only IL-1b and IL-23. Thus, these comparative experiments revealed age-related differences in the Th17 cytokine requirements of human T cells that had not been fully observed in previous studies136 (Figure 2.7). Most importantly, transient overexpression of STAT3 was found to have significantly induced the production of IL-17. Exogenous STAT3 also increased IL-22 production, while IL-22 100 remained suppressible in the presence of TGF-b (Figure 2.8). Based on my data, it remains plausible that relatively low STAT3 activity in neonatal naïve CD4 T cells can limit Th17 differentiation capacity since STAT3 inhibitors were found to reduce the activity of both IL-17 and IL-22 in neonatal T cells. Be that as it may, these results require further validation. Intriguingly, both levels of STAT3 phosphorylation and IL-22 production in neonatal T cells were quite variable between individuals, suggesting other contributing factors. This data leads us to speculate and question the purpose and function of naïve CD4 T cell preference to adopt an immunoregulatory Th22 phenotype rather than a stereotypic Th17 phenotype. This Th22 bias can help maintain local homeostasis235 since IL-17 is known as an inflammatory cytokine and has been linked to necrotizing enterocolitis, a serious gastrointestinal disease in newborns. Conversely, IL-22, another main cytokine produced by Th17 cells, is an anti-inflammatory cytokine that targets epithelial mucosal cells in the skin, liver, gut, and lung84. In support of this theory, it was exciting to discover in Chapter 4, that IL-22 appears to have protective roles in the intestine through enhancement of mucin production, IEC regeneration, Paneth cell differentiation, and post-translational phosphorylation of sugar moieties on mucin proteins, which promotes beneficial gut microbial colonization84,158. Considering the results of Chapter 4, we deduced that the preferential bias of circulating neonatal T cells towards IL-22 production is required to restrict the systemic consequences of mucosal inflammation during de novo gut colonization with commensal microbes. 101 The study is limited in the fact that Th17 differentiation differences were only assessed in two age groups (neonate and adult) and were therefore unable to characterize the developmental continuum in between these two life stages. Other studies show the reduced capacity of neonatal Th17 cells to produce IL-17 lasts for at least the first three months of life in infants234. Thus, we expect that IL-17 limitation lasts for the first year of life. Secondly, our data showed an IL-17 deficit in conventional CD4 T cells, whereas other types of immune cells can produce IL-17 in peripheral organs (e.g. the gut) âthis includes gd T cells, ILC3s124,236, and other T cells residing in peripheral tissues237. Lastly, other studies showed a strong Th17 response from peripheral blood neonatal T cells when exposed to Candida extracts154, highlighting the importance of studying neonatal T cell responses in the context of MC to understand their susceptibility to this microorganism. In Chapter 3, I established an intestinal organoid model as an ex vivo approach to investigate the developmental changes of intestinal epithelial cells throughout postnatal life. Previous studies showed that the structure, size, and function of neonatal IECs are quite different from their adult counterparts, primarily due to less diversity in the gut microbiota and differing immune responses in their gut 169â171. This is of great concern because immaturity of the intestinal epithelium and dysbiosis of colonizing microorganisms can lead to the development of lethal NEC, often in premature babies226. The novel intestinal organoid model established during this study contributed greatly to my thesis and is expected to significantly enhance the study and understanding of the role of IECs and underlying molecular mechanisms in early life intestinal diseases, such as NEC. 102 In this chapter, intestinal biopsies from infants (less than 6 months of age) were collected to develop an ex vivo human intestinal organoid model (enteroid) that was used to assess the development of IECs. Intestinal biopsy samples were obtained at the time of intestinal surgical procedures for each study individual at BC Childrenâs Hospital. After establishing a proper protocol to isolate the crypt from infant intestinal biopsies, tissueâderived epitheliumâonly organoids were generated183. Two different splitting methods were utilized, including enzymatical splitting (trypsinization) and mechanical splitting, to optimize the size and growth of infant enteroids. More budding, crypt-like structures were observed in the enteroids treated by mechanical splitting (Figure 3.2B). Additionally, immunostaining images confirmed an increased thickness of IECs generated by mechanical splitting in comparison to trypsinization (Figure 3.3A and C). The presence of Ki67+ cells in immunostaining images confirmed the differentiation status of IECs in enteroids treated by mechanical splitting (Figure 3.3D). Finally, Chapter 4 explored the development of IECs in infant-derived enteroids and investigated the physiological effects of IL-22, the major cytokine produced by neonatal Th17 cells, on the proliferation and differentiation of infant IECs. Newborns have an immature GI tract at birth, undergoing a long-term maturation process after exposure to external factors such as breast milk or formula, expanding food and nutrient varieties, and microbial antigens30. The developmental transition of the GI tract from a sterile environment in utero can take more than 6 months106. Immaturity of the adaptive immune system in newborns implies that these infants struggle to fully discriminate between commensal bacteria and invasive pathogens; this potentially results in excessive inflammation from otherwise non-pathogenic microorganisms, which in turn can lead to NEC110. 103 Infant enteroids co-cultured with neonatal Th17-derived supernatant expanded significantly than without. In comparison, although pediatric-derived enteroids demonstrated an increasing growth trend when treated with Th17 supernatant, they were not significantly expanded compared to the control enteroids (Figure 4.1C and E). Differentiation of neonatal Th17 cells led to the production of a copious amount of IL-22 compared to adult counterparts, which further demonstrated the effect of neonatal Th17 cytokines on infant enteroids was primarily IL-22-dependent. Brightfield images also showed that the phenotype of infant enteroids exposed to Th17 supernatant was crypt-like with budding structures, closely resembling those treated with IL-22 (Figure 4.1B). This starkly contrasted with the typical cystic shape of the pediatric enteroids. Ki67 expression, a transient amplifying marker, was also found to significantly increase in Th17 supernatant-treated infant enteroids compared to control counterparts (Figure 4.2A and B). Interestingly, treating enteroids of both age groups with neutralizing IL-22 suppressed enteroid growth, confirming the role of IL-22 in the proliferation and expansion of IECs, regardless of age (Figure 4.1H). Next, infant and pediatric enteroids treated with Th17 supernatant were immunostained for MUC2 and lysozyme, which are specific markers for goblet and Paneth cells, respectively. Immunostaining analysis showed the presence of lysozyme-producing Paneth cells in infant enteroids treated with IL-22 and Th17 supernatant (Figure 4.2E). Furthermore, MUC2 intensity increased significantly in infant and pediatric enteroids exposed to Th17 supernatant, 104 compared to control conditions. Notably, the intensity of MUC2 decreased considerably in the presence of neutralizing IL-22 antibody for both age groups (Figure 4.2C and D). With the confirmation of the significance of IL-22 and its role, we performed RNA-sequencing analysis of infant and pediatric enteroids treated with recombinant IL-22 and compared them to control enteroids (Figure 4.3A). These experiments identified important differentially-expressed pathways and genes that are emendable to future functional experiments, additional genes including REG3A, REG3G, and REG1B, and markers for goblet cells, supported by immunostaining. GSEA analysis showed downregulation of the ISC marker LGR5, genes involved in Wnt/β-catenin- and Notch-signaling, but upregulation of OLFM4 (Figure 4.4B). SLEA analysis revealed that IL-22 induced changes in the cell populations of IEC subtypes, including Paneth, goblet, progenitor, and stem cells, suggesting the impact of IL-22 on the differentiation of secretory cell types (Figure 4.4F). Our data support the presence of developmental cross-talk in infants between their gut epithelium and Th17 cells. Considering the high capacity of neonatal Th17 cells to produce IL-22, we postulate that the presence of IL-22 in the gut is favorable for IEC development in the early stage of life. Since previous studies revealed the detrimental role of IL-17 in NEC development, we postulated that the limited capacity of neonatal Th17 cells to produce IL-17 is protective for the neonatal mucosal system (Figure 5.1). To conclude, with the thesis data and results, the following model summarizes and outlines the postulates that the secretion of mucins and anti-microbial peptides are induced when 105 exposing infant IECs to neonatal Th17 cell supernatant containing IL-22 to ensure gut health in the context of a maturing intestine and immune system. Figure 5.1 Schematic model explaining the cross-talk between Th17 cells and IEC in the human infant intestine IL-22 produced by Th17 cells induces differentiation of Paneth cells and goblet cell, producing RORa/g and mucin, respectively. Contrarily, IL-17 induces inflammatory responses leading to barrier defunction and development of NEC. 5.2 Future directions In this thesis, I investigated the gene expression profile of neonatal naïve CD4 T cells to understand the developmental changes of naïve CD4 T cells toward differentiation to Th17 cells. It is plausible to hypothesize that the developmental differences of naïve CD4 T cells between neonates and adults are not restricted to the mRNA level, but also due to the epigenomic differences. Currently, Dr. Hirstâs team (Michael Smith Laboratories, UBC) is performing simultaneous transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation sequencing (ChIP-seq), and whole genome bisulfite sequencing) analysis of primary naïve CD4 T cells from both neonates and adults to unravel the unknown Reg3a/gLysozymeHomeostatic conditionIL-22AdultIL-17 106 mechanisms underlying the neonatal T cells bias toward IL-22 production, and their limited ability to produce IL-17 compared to adult T cells. Although IL-22 can be produced by a range of innate cells (e.g. ILC3s, Th17, and Th22), Th22 cells are characterized by producing exclusively IL-22 and is involved in various inflammatory skin diseases such as psoriasis238,239. In light of my findings, it is interesting to consider the presence and frequency of Th22 in neonates and the in vitro differentiation of naïve CD4 T cells from both neonates and adults toward Th22 cells. Transcriptomic and epigenomic analysis of Th22-differentiated cells might explain the molecular differences between Th17 and Th22 cell populations in neonates. A study showed that the presence of the Th22 subset in the peripheral blood of young children (1-6 years old) is characterized by the production of IL-22 from CD4+CD45RO+ memory Tcells240, which is remarkably lower as compared to adults. However, the existence of Th22 in the peripheral blood of neonates remains elusive. Considering the ubiquitous expression of TGF-b by Treg cells and the high portion of Treg in the cord blood, it is tempting to ask: how can IL-22 be produced in the presence of Treg when research has shown the inhibitory effect of TGF-b on the production of IL-22 in Th17 cells? As I mentioned in Chapter 4, NEC affects mostly preterm babies, and the molecular mechanism behind the pathogenesis of this disease is unclear due to the difficulty of studying and modeling the human neonatal intestine. Although several mice and human experimental models were designed and used for studying NEC124,131,181,182,226, it is still challenging to translate these findings to clinical care. Thus, more translational research is needed to bridge the knowledge gap in our understanding of how the structure and function of the human gut 107 change early in life, and more specifically, how microbiota colonization at birth influences these changes. Using sc-RNAseq technology, it is possible to study the intestinal cell types and their distribution, and ultimately garner a better understanding of their specific functions in premature-derived enteroids with NEC. As mentioned previously, NEC is characterized by excessive inflammation at the gut mucosa, possibly triggered by exposure to gut microbes124. Hence, sc-RNAseq from premature babies with NEC might show different transcriptomics (immune responses, microbial sensing receptor, antimicrobial peptides) correlating to its maturation stage, which in turn depends on the gestational age of the infant at birth. In light of our findings in Chapter 4, we noticed that infant enteroids showed a larger proportion of goblet cells and rapid regeneration/proliferation in the presence of IL-22. However, considering that high levels of inflammatory cytokines such as IL-17 and TNF-a have been associated with the inflamed intestines of preterm infants with NEC 124,131, it would be interesting to study the physiological effect of IL-17 on infant-derived enteroids by using different concentrations of recombinant human IL-17. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Lavoie, Pascal M.
Understanding T cell and gut interactions in early post-natal life
Text