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Ontogeny of cytokine respones following cytoplasmic pattern recognition receptor stimulation Bedi, Harjot Kaur 2014

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Ontogeny of Cytokine Responses following Cytoplasmic Pattern Recognition Receptor Stimulation  by  Harjot Kaur Bedi B.Sc. Hons., Queen’s University, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2014  © Harjot Kaur Bedi, 2014   i  Abstract  The innate immune system of newborns is biased towards the production of cytokines that are largely anti-inflammatory or promote development of Th2- and Th17-type of immunity, i.e., an immune response focused against pathogens in the extracellular milieu, and in particular, mucous membranes. This leads to an increased susceptibility to infections by intracellular pathogens, whose clearance depends primarily on effective cell-mediated Th1-type immunity. These insights were gleaned from the analysis of cytokines produced in neonatal and infant blood in response to Toll-like receptor (TLR) stimulation. However, TLRs are only one of the classes of a broader repertoire of molecules called pattern recognition receptors (PRRs). To our knowledge, PRRs that are specialized for the detection of intracellular pathogens – namely, cytoplasmic PRRs – have not been systematically and comprehensively analyzed in the context of the newborn immunity. Delineating the response of cytoplasmic PRRs in the newborn to pathogen-associated molecular patterns (PAMPs) typical of intracellular pathogens represents a vital first step towards identifying strategies aimed to ameliorate the newborn’s greater susceptibility to intracellular pathogens. We, therefore, stimulated adult and cord blood with PAMPs typical of cytoplasmic pathogens and followed cytokine production both globally (secreted into the culture supernatant) as well as on the cellular level (intracellular cytokine cytometry (ICC)). We were surprised to find that neonatal blood secreted significantly more IFNα, IP-10, IL-8 and MCP-1 compared to adult blood, especially following stimulation of the PRR Stimulator of Interferon genes (STING). However, we were unable to detect any response to these PAMPs using ICC; this likely stems from the requirement of positive cytokine feedback mechanism necessary for production of type 1 interferons that was inhibited in our ICC set-up. Our data, taken together with the emergence of STING as a key PRR in the detection of intracellular pathogens, e.g.    ii  Listeria and several viruses, led us to hypothesize that altered response to STING may be functionally related to the known exquisite sensitivity of newborns to infection with such pathogens. Additional studies aimed at identifying the biological role of activation of cytoplasmic pattern recognition receptors will be conducted to understand the age-related differential immune response to intracellular infections.                 iii  Preface  The idea of this research project was the brainchild of my supervisor, Dr. Tobias Kollmann. He analyzed previous research findings in the lab to suggest a project that struck my interest.   I developed the experimental plan, performed the assays and analyzed the data with guidance from Drs. Mathieu Garand and Bing Cai in our lab.  The study was conducted with approval from the UBC Research Ethics Board under the project title “Neonatal Immune Responses”; Certificate #H13-00347.              iv  Table of Contents Abstract ............................................................................................................................................ i Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii Abbreviations ................................................................................................................................ xii Acknowledgements ...................................................................................................................... xiv Dedication ..................................................................................................................................... xv 1 Introduction ............................................................................................................................. 1 1.1 Role of the innate immune system ................................................................................................ 1 1.2 Immunity of the neonate ............................................................................................................... 2 1.3 Pattern recognition receptors (PRRs): location predicts function ................................................. 3 1.3.1 RIG-I-like Receptors (RLRs) ................................................................................................ 4 1.3.2 Stimulator of Interferon Genes (STING) .............................................................................. 5 1.4 Aim and hypothesis: ..................................................................................................................... 7 2 Methods ................................................................................................................................... 8 2.1 Cytoplasmic PRR plates................................................................................................................ 8 2.2 Blood sample processing and in vitro stimulation ........................................................................ 8 2.3 Staining, acquisition and analysis ................................................................................................. 9 2.4 Luminex-based assessment of cytokines in culture supernatant ................................................. 10 2.5 Statistical analysis ....................................................................................................................... 11 3 Pilot Results ........................................................................................................................... 12 3.1 Ligand choice .............................................................................................................................. 12 3.1.1 Objective ............................................................................................................................. 12 3.1.2 Results ................................................................................................................................. 13 3.1.3 Conclusion .......................................................................................................................... 14 3.2 Dose-response ............................................................................................................................. 14 3.2.1 Objective ............................................................................................................................. 14 3.2.2 Results: ................................................................................................................................ 15 3.2.3 Conclusion .......................................................................................................................... 16    v  3.3 Time course – Luminex .............................................................................................................. 17 3.3.1 Objective ............................................................................................................................. 17 3.3.2 Results ................................................................................................................................. 17 3.3.3 Conclusion .......................................................................................................................... 18 3.4 Impact of using freeze-thawed ligands compared to fresh ligands ............................................. 18 3.4.1 Objective ............................................................................................................................. 18 3.4.2 Results ................................................................................................................................. 19 3.4.3 Conclusion .......................................................................................................................... 19 3.5 Luminex pilot .............................................................................................................................. 19 3.5.1 Objective ............................................................................................................................. 19 3.5.2 Results ................................................................................................................................. 20 3.5.3 Conclusion .......................................................................................................................... 20 3.6 Flow cytometry pilot ................................................................................................................... 21 3.6.1 Objective ............................................................................................................................. 21 3.6.2 Results ................................................................................................................................. 21 3.6.3 Conclusion .......................................................................................................................... 22 3.7 Role of Lyovec in flow cytometry .............................................................................................. 23 3.7.1 Objective ............................................................................................................................. 23 3.7.2 Results ................................................................................................................................. 23 3.7.3 Conclusion .......................................................................................................................... 23 3.8 Summary of results from pilot studies ........................................................................................ 24 4 Results of Main Study ........................................................................................................... 25 4.1 Higher IFNα produced in response to STING and MDA5 stimulation by neonatal blood compared to adult blood .......................................................................................................................... 25 4.2 Higher IP-10/CXCL10 produced by cord blood in response to STING and MDA5 ligand stimulation compared to adult blood ....................................................................................................... 26 4.3 Significantly higher MCP-1 produced by cord blood stimulated with any cytoplasmic PRRs or R848 compared to adult whole blood. .................................................................................................... 26 4.4 R848 and cytoplasmic PRR stimulated cord blood produced higher IL-8 compared to stimulated adult blood .............................................................................................................................................. 27 4.5 Single-cell analysis showed significantly higher TNFα production by adult cDCs, pDCs and monocytes compared to cord cells. ......................................................................................................... 28 4.6 Significant production of IL-12 and IL-6 by R848-stimulated cDCs and monocytes in both adult and cord blood......................................................................................................................................... 29    vi  4.7 Plasmacytoid dendritic cells (pDCs) are the source of IFNα production in R848-stimulated adult and cord cells .......................................................................................................................................... 29 5 Discussion .............................................................................................................................. 31 5.1 Discussion of results ................................................................................................................... 31 5.2 Clinical implications ................................................................................................................... 37 5.3 Future directions ......................................................................................................................... 41 6 Tables..................................................................................................................................... 44 7 Figures ................................................................................................................................... 48 8 References ............................................................................................................................. 72                vii  List of Tables Table 1. Summary of the final ligands selected for the study, their product information, target receptors and concentrations used. ............................................................................................... 44 Table 2. List of all ligands and their receptor targets tested for their efficacy in whole blood. ... 45 Table 3. List of all cytokines and chemokines tested for in the 23-plex luminex assay to detect differential responses from neonatal and adult whole blood assays. ............................................ 46 Table 4. Comprehensive summary of lasers, photomultiplier tube (PMT), long pass (LP) and band pass (BP) filters, target fluorochromes and their clones used for the flow cytometry panel for this study ................................................................................................................................. 47               viii  List of Figures Figure 1. Test of different concentrations of 3'3'-cGAMP (STING ligand) complexed with Lyovec showed a dose-dependent increase in TNF-alpha (a) and IL-1beta (b) production in two adult subjects. ................................................................................................................................ 48 Figure 2. Different concentrations of poly(I:C) were tested with and without Lyovec (+L indicates presence of Lyovec). IFN-alpha (a) and gamma (b) were produced in the presence of Lyovec but only baseline concentrations of the two cytokines were detected without Lyovec. n=1 (adult) for each cytokine tested. .................................................................................................... 49 Figure 3. Poly(I:C), when complexed with Lyovec, induced production of IFN-alpha and IP-10 in whole blood assay compared to poly(I:C) without Lyovec, which showed baseline level of cytokine produced for each cytokine. n=7 (cord blood) for both cytokines. ................................ 50 Figure 4. Production of IFN-alpha (a), IL-8 (b) and MCP-1 (c) were quantified in a time-course study of adult and cord whole blood stimulated with various cytoplasmic pattern recognition receptor ligands. Supernatants were collected at 6, 12, 18 and 24hrs. (numbered in each graph). A time-dependent increase in cytokine production was seen in all three cytokines detected. n=3 for each group (adult and cord) for this pilot study. ..................................................................... 53 Figure 5. Similar levels of TNF-alpha (a) and IL-1beta (b) were produced by whole blood stimulated with freshly prepared and freeze-thawed ligands. The results ensured comparable efficacy of fresh and freeze-thawed ligands, hence, allowing more stringent quality control in terms of ligands used for each donor in the study. n=1 (adult) for each cytokine. ....................... 54 Figure 6. Neonatal whole blood produced higher IFN-alpha levels when stimulated with MDA5 and STING ligands compared to adult whole blood. The data shown represents mean values with standard error bars for n=5 for each group (adult and cord). ........................................................ 55 Figure 7. Neonatal whole blood produced higher IP-10 levels when stimulated with MDA5 and STING ligands compared to adult whole blood. The data shown represents mean values with standard error bars for n=5 for each group (adult and cord). ........................................................ 56 Figure 8. Neonatal whole blood stimulated with cytoplasmic pattern recognition receptors produced higher IL-8 compared to adult whole blood.  High background IL-8 production was also observed for R848 stimulated adult and cord blood. The data represents mean values with standard error bars of n=5 for each group (adult and cord). ......................................................... 57 Figure 9. Neonatal whole blood stimulated with cytoplasmic pattern recognition receptors produced higher MCP-1 compared to adult whole blood. The background levels of MCP-1 for Lyovec-only stimulated adult and cord blood were also high. The data represents mean values with standard error bars of n=5 for each group (adult and cord). ................................................. 58    ix  Figure 10. Gating strategy for detection of innate immune cells using flow cytometric assay. Exdoublet peak is done to ensure analysis of only single cells. The cells are then gated on size to exclude dead cells and debris. CD66 marker is used to exclude granulocytes (CD66+). CD66- cells are then expanded and gated using HLA-DR and CD14. HLA-DR negative cells are characterized as Antigen Presenting cells – (APC-) and further gated on to get NK, NK T and γδ cells.  HLA-DR positive cells are characterized as APC+ and further gated for B cells and plasmacytoid and conventional monocytes. CD14 cells are gated as monocytes/macrophages in the assay. This gating strategy was used for all cells analyzed for intracellular cytokines in the study. ............................................................................................................................................. 59 Figure 11. Relative frequency of cells (percent positive) expressing cytokines ((a) represents IFN-alpha and (b) represents TNF-alpha) in adult cells using intracellular staining. The percent positive cells were determined based on the gating for unstimulated cells that were stimulated with RPMI-alone. We saw IFN-alpha and TNF-alpha production for cells stimulated with R848 (TLR7/8 ligand) but not for cells stimulated with cPRR ligand 3’3’-cGAMP (STING activator)........................................................................................................................................................ 60 Figure 12. Little or no IFN-alpha detected in both neonatal and adult plasmacytoid dendritic cells (pDCs) stimulated with cytoplasmic pattern recognition receptor ligands. Both stimulated adult and neonatal pDCs showed similar levels of IFN-alpha produced. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848 and LPS, and Lyovec for all the cytoplasmic pattern recognition receptors). n=5 for both groups and the data represents mean values with standard error bars............................................................................................ 61 Figure 13. Similar amounts of IL-6 (a) and TNF-alpha (b) were produced in adult and cord monocytes and conventional dendritic cells (cDCs), respectively, stimulated with R848 and LPS. Adult and cord monocytes and cDCs showeed no cytokine production when stimulated with cytoplasmic pattern recognition receptor ligands. The data depicted shows valued subtracted from the medium (RPMI for R848 and LPS, and Lyovec for all cytoplasmic pattern recognition receptors). n=5 was used for each group and the data shows mean values with standard error bars. ............................................................................................................................................... 62 Figure 14. Similar levels of IL-6 were produced in adult plasmacytoid dendritic cells (pDCs) stimulated with both LPS and Lyovec or LPS alone. The x-axis represents the time when Brefeldin A (BFA) was added to the whole blood assay for a total 6hr incubation. The data shows the percentage of IL-6 produced when BFA was added to the assay at a later time. n=1 (adult). ........................................................................................................................................... 63 Figure 15. Significantly higher IFNalpha was produced by cord blood stimulated with MDA5 and STING ligands compared to adult blood. No significant difference was observed in neonatal and adult blood stimulated with R848 or RIG-I ligand. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848, and Lyovec for cytoplasmic pattern    x  recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ........................................................................ 64 Figure 16. Significantly higher IP-10 levels were produced by neonatal cells stimulated with MDA5 and STING ligands compared to adult cells. No difference in IP-10 production for adult and neonatal whole blood stimulated with RIG-I ligand and R848. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ........................... 65 Figure 17. Significantly higher MCP-1 was produced by neonatal whole blood stimulated with R848 and cytoplasmic pattern recognition receptors (except 3'3'-cGAMP). The data shown in (a) depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). Unsubtracted values are shown in (b) to show the high background MCP-1 production by whole blood stimulated with Lyovec alone. The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). .................................................................................................................. 66 Figure 18. Neonatal whole blood produced higher IL-8 when stimulated with MDA5, RIG-I and STING ligands compared to adult whole blood. The data shown in (a) depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor (cPRR) ligands). Unsubtracted values are shown in (b) to show the high background IL-8 production by whole blood stimulated with Lyovec alone. Lyovec-induced IL-8 production was higher compared to cPRR-stimulated adult whole blood. The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ... 67 Figure 19. Adult conventional dendritic cells (cDCs) produced significantly higher percentage of TNF-alpha compared to neonatal cDCs stimulated with R848. Both adult and neonatal cDCs stimulated with cytoplasmic pattern recognition receptors showed little or no TNFalpha production. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ....................................................................................................................................... 68 Figure 20. Similar percentages of IL-6 produced in adult and neonatal (a) conventional dendritic cells (cDCs) and (b) monocytes both stimulated with R848. In contrast, no IL-6 was produced by adult or neonatal cDCs or monocytes stimulated with cytoplasmic pattern recognition receptors.    xi  The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ... 69 Figure 21. Adult monocytes stimulated with R848 produced significantly higher IL-12 compared to neonatal monocytes. Little or no IL-12 was produced by both adult and cord monocytes stimulated with cytoplasmic pattern recognition receptors. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). ........................................................ 70 Figure 22. Adult plasmacytoid dendritic cells (pDCs) produced significantly higher IFN-alpha when stimulated with R848 compared to cord pDCs. Little or no IFN-alpha produced by both adult and cord pDCs stimulated with cytoplasmic pattern recognition receptors. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001). .......................................... 71             xii  Abbreviations 2'3'-cGAMP cyclic [G(2',5')pA(3'5')p] 3'3'-cGAMP cyclic [G(3',5')pA(3'5')p] 5'ppp-dsRNA 5'-triphosphate double stranded RNA Ag Antigen Alum Aluminum hydroxide and potassium salts APC Allophycocyanin APC Antigen Presenting Cell ATP Adenosine triphosphate BFA Brefeldin A Cardif CARD adaptor inducing IFNβ cDCs Conventional Dendritic cells CDNs cyclic di-nucleotides cGAS cGAMP synthase DNA Deoxyribonucleic acid ECMV encephalomyelocarditis virus EDTA Ethylenediamietetraacetic acid ER Endoplasmic reticulum FACS Fluorescence-activated cell sorting HCV Hepatitis C Virus ICS Intracellular staining IFN Interferon IFNα/IFN-alpha Interferon-alpha IFNβ/IFN-beta Interferon-beta IFNγ/IFN-gamma Interferon-gamma IKK-i IkB kinase IL-8 Interleukin-8 IP-10 Interferon-gamma induced protein 10 IPS-1 IFNβ promoter stimulator 1 IRF3 IFN regulatory factor 3 ISG IFN-stimulating genes JAK Janus Kinase JEV Japanese encephalitis virus LGP2 Laboratory of genetics and physiology 2 Lm Listeria monocytogenes MAVS Mitochondrial antiviral signalling protein MC Mononuclear cells MCP-1 Monocyte chemotactic protein 1 MDA5 Melanoma differentiation-associated protein 5 MHC Major histocompatibility complex    xiii  MyD88 Myeloid differentiation primary response gene 88 NDV Newcastle disease virus NK cells Natural Killer cells NK T cells Natural Killer T cells NOD Nucleotide oligomerization domain PAMP Pathogen-associated molecular pattern PBS Phosphate Buffer solution pDCs Plasmacytoid Dendritic cells PE  Phycoerythin PerCP Peridinin chlorophyll protein Poly(dA:dT) poly(deoxyadenylic-deoxythymidylic) acid Poly(I:C) Polyinosinic-polycytidylic acid PRR Pattern recognition receptor RIG-I Retinoic acid-inducible gene 1 RLR RIG-I-like receptor RNA Ribonucleic acid RSV Respiratory syncytial virus SeV Sendai Virus STAT Signal transducer and activator of transcription STING Stimulator of Interferon genes TBK1 TANK-binding kinase 1 Th1 T helper cell type 1 Th17 T helper cell type 17 Th2 T helper cell type 2 TLR Toll-like Receptor TNFα/TNF-alpha Tumor necrosis factor alpha Trif TIR-domain-containing adaptor-inducing IFNβ Unstim Unstimulated VISA Virus-induced signalling adaptor VSV Vesicular stomatitis virus WB Whole Blood WT Wild-type       xiv  Acknowledgements First and foremost, I would like to acknowledge my supervisor, Dr. Tobias Kollmann, for his endless kindness, support and encouragement, and the members of my supervisory committee, Dr. Laura Sly and Dr. Soren Gantt. This would not have been possible without your valuable insight.  I would like to thank all the members of the Kollmann lab for providing me with guidance, support, positive criticism and feedback, and moreover, a positive space to learn and grow as a researcher. I would especially like to thank Drs. Mathieu Garand and Bing Cai for being patient with me while I made mistakes and learnt from them throughout the time in the lab. I am beyond grateful to Dr. Tobias Kollmann for taking me into his lab and for being an amazing mentor; for providing me with the advice and insight to successfully complete my project; and for the endless support and encouragement to help me take a step closer to fulfilling my dreams.  Special thanks to my family and friends for the love, care and moral support through my academic ventures; and to Mr. Sanjeet Sahota for all his help, with Microsoft Excel and formatting of my thesis, and the continuous words for encouragement to help me achieve my goals. I am forever grateful. Thank you.        xv  Dedication    To my parents and grandparents     1  1 Introduction 1.1 Role of the innate immune system Humans are constantly surrounded by microorganisms, many of which are capable of causing disease. Yet most of us manage to avoid the otherwise fatal effects often accompanying infection with such pathogens. A major detriment to the ability of pathogens to infect and cause disease is the presence of a functional immune system – specifically, the innate arm of the immune system. The innate immune system serves to recognize pathogens within minutes or hours and effectively prevents them from successfully establishing infection (1,2). The other branch of immune system, adaptive immunity, has a more specific response to pathogens, but responds relatively delayed (1,2).  The innate immune system represents the first wall of defense against pathogens that manage to breach anatomical barriers. It consists of preformed factors, enzymes, cells, and receptors that broadly recognize pathogens that enter the human body (1-3). Preformed factors kill pathogens or weaken their effects, and include antimicrobial enzymes as well as the complement system present in blood and extracellular fluid. The cells and receptors of the innate immune system recognize foreign material and activate various intracellular pathways aimed to eliminate the infections. Among these receptors is a group commonly referred to as pattern recognition receptors (PRRs) because of their role in the recognition of conserved molecular patterns of microbes (1,3). PRRs are present on and within cells of the innate immune system, such as neutrophils, macrophages, dendritic cells and monocytes. The recognition of specific ligands by PRRs activates a series of downstream signalling molecules, which in turn lead to the production of effector molecules (e.g. cytokines) that orchestrate the elimination of pathogens. The success of the immune response – both the    2  innate and any ensuing adaptive response – requires the combination of cytokines produced to be finely coordinated.  1.2 Immunity of the neonate Human newborns undergo a drastic transition from the relatively sterile environment in utero to the microbe-rich world outside (4). The human neonatal immune system is known to be biased against the Th1-type cytokine response, and instead favours the Th2/Th17-type cytokine responses (4-7). This may be to avoid an overactive immune response at the maternal-placental semiallogeneic interface (4).  The diminished capacity of neonates to produce pro-inflammatory or Th1-polarized cytokine responses leads to a higher probability of acquiring infections, especially from intracellular microbes, such as Listeria monocytogenes (Lm) (4,8,9). Further compounding their greater susceptibility to infection is their lower responsiveness to many vaccinations.  This is due in part to the newborn’s adaptive immune cells being relatively naïve, i.e., requiring stronger/longer stimulation in order to become activated and develop immune memory (6,8,10). Besides the adaptive immune system, similar limitations (e.g., less responsiveness) were also previously thought to manifest in the newborn’s innate immunity to invading microbes (5). However, several studies have now shown that neonatal (cord) blood or cord blood mononuclear cells (CBMCs) produce higher levels of particular cytokines during TLR stimulation (11-13). For example, our group and other studies have previously identified higher levels of IL-23, IL-6 and IL-10 production in CBMCs after stimulation with several Toll-like receptor (TLR) ligands (14-16). While the molecular reason for this has not fully been delineated, it appears unlikely to be due to differential    3  expression of PRRs, and more likely the result of specific regulatory elements in the signalling cascades downstream of TLRs.  1.3 Pattern recognition receptors (PRRs): location predicts function Of the known PRR classes, Toll-like receptors (TLRs) have been best characterized. TLRs are present on the cell membrane or in endosomes, (i.e., all facing an extracellular milieu), where they recognize PAMPs present outside the cell or those taken up from the extracellular milieu and phagocytosed (5,6,11-13). Eleven distinct TLRs have been identified in humans, with each playing a crucial role – individually and via cross-talk – in orchestrating the response to invading microbes (5,11,13,17).  Several other classes of PRRs are designed to detect PAMPs present in the cell cytoplasm. These include nucleotide oligomerization domain (NOD)-like receptors, retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), and stimulator of interferon genes (STING). These cytoplasmic PRRs (cPRRs) respond specifically to PAMPs produced by intracellular or, more specifically, cytoplasmic pathogens (18,19). The list of PAMPs that specifically activate the above-mentioned PRRs are outlined in table 2.  Given the varied location of PRRs, it is interesting to note that each class of PRRs can also activate differing signalling cascades to drive differential production of effector molecules and cytokines. For example, cPRRs are known to be particularly powerful at inducing type 1 interferons, a class of cytokines commonly considered to have ‘anti-viral’ properties for their ability to release an avalanche of effector mechanisms targeting viruses and other cytoplasmic microbes.     4  1.3.1 RIG-I-like Receptors (RLRs) Given their host-cell adapted life cycle, the majority of PAMPs released by viruses relate to their replication, i.e., nucleic acids. The class of cPRRs that has been most studied are the RIG-I-like receptors (RLRs). The RLR family consists of three members: retinoic acid-inducible gene-I (RIG-I), melanoma differentiation factor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) (18,20-24). All three RLRs share a DExD/H (Asp-Glu-x-Asp/Histidine)-box helicase domain and two N-terminal caspase-recruitment domains (CARDs) (21-24). RIG-I is stimulated by 5’triphosphate double stranded RNA (5’ppp-dsRNA) (25-28). The 5’triphosphate (5’ppp)-end is present in the cytoplasm over the course of replication of viral dsRNA in the cytoplasm and has been shown to be essential for activation of RIG-I (22). Kato et al. (2006) showed that RNA with 5’ppp-end has a 126-fold higher affinity for RIG-I compared to RNA with a hydroxyl end (as would be seen in host cell-derived mRNA) (22,29). MDA5, on the other hand, is activated by long dsRNA, or its synthetic dsRNA analogue, polyinosine-polycytidylic acid (poly(I:C)) (29). Studies have shown that poly(I:C) plays a role in activating RIG-I as well as TLR3 (21). When activated, RIG-I recruits a CARD-containing adaptor protein, IPS-1 (also known as MAVS, VISA or Cardif) (21,23). IPS-1 leads to activation of kinases TBK1 and IKK-i, which triggers the phosphorylation of interferon-regulatory factors (IRF) 3 and 7. Phosphorylated IRF 3 and 7 translocate to the nucleus and lead to the transcription of type-1 IFNs (22,30).  However, knock-out studies for RIG-I, MDA5, and Trif (a downstream protein in TLR3-mediated pathway) have shown that MDA5 is essential for poly(I:C) mediated Interferon-α (IFNα) production (22,29). As such, RIG-I and MDA5 are believed to play a crucial role in the recognition of different RNA viruses present or transcribed in the cytoplasm and mediate an anti-viral state via the production of type 1 IFNs.    5  1.3.2 Stimulator of Interferon Genes (STING) STING is an endoplasmic-reticulum (ER)-associated protein with five transmembrane motifs (31), and is essential in the production of type 1 IFN after recognition of double-stranded (ds) DNA, or viral DNA. However, STING also recognizes cyclic di-nucleotides (CDNs) that have been found to be produced by cytoplasmic microbes such as Lm and secreted into the cytoplasm apparently as part of their communicative system (31-33). Recognition of CDNs in the cytoplasm leads to activation of STING, which then associates with TANK-binding kinase 1 (TBK1) and relocates to perinuclear vesicles containing exocyst components Sec 3 and Sec 5 (31,33-35). STING then triggers the activation of both nuclear factor-kappa B (NK-kB) and IRF3 transcription pathways, giving rise to the production of pro-inflammatory cytokines, especially type 1 IFNs (31,33,36).  STING appears to play a key role in the initiation of an effective antiviral response, evident from how mouse embryonic fibroblasts (MEFs) from STING knock-out (STING-/-) mice were incapacitated in their control of Vesicular Stomatitis Virus (VSV) compared to wild-type mice (31). Moreover, the addition of STING to STING-/- mice was able to resume their control of VSV infection (31). These studies demonstrate that STING mediates necessary anti-viral, protective responses in the human body. Ishikawa and Barber (2008) have also shown that mice lacking STING produce less IFNβ when infected with Sendai virus (SeV), a negative sense, single-stranded RNA virus that was previously believed to activate only RIG-I (31). The attenuation in IFNβ production was not observed when fibroblasts were activated with poly(I:C) (31). This indicates that STING plays an important role downstream of RIG-I, but not MDA5, in an infected cell. Therefore, STING is centrally involved in the recognition of both viral DNA and RNA present in the    6  cytoplasm and in the activation of downstream effector mechanisms that mediate production of anti-viral, type I IFNs.  In addition to mediating anti-viral responses following the recognition of pathogenic DNA or RNA in the cytoplasm, STING has also been shown to mediate type 1 IFN production upon recognition of bacterial CDNs in the cytoplasm. Cyclic di-AMP and cyclic di-GMP are bacterial CDNs that function as second messengers for bacterial cells to regulate motility, expression of genes etc. (37). In particular, cytoplasmic bacteria, such as Lm, are known to release CDNs into the cytoplasm during their cytoplasmic life cycle. These CDNs then lead to the production of type 1 IFNs mediated through the STING-TBK1-IRF3 pathway (38-41). It remains unclear how CDNs are detected and lead to activation of STING, i.e., whether they bind directly or have partners that mediate such binding.  A prominent role of STING in the response against Lm infection has been indicated by the finding that STING knock-out mice suffer higher bacterial burden after Lm infection compared to wild-type mice (39). However, contrary to studies that have shown protection by the STING-mediated innate response via production of type 1 IFNs following detection of Lm-released CDNs in the cytoplasm, an inhibitory effect on the adaptive immune system has also been observed. Archer et al. demonstrated that STING-mediated type 1 IFN production inhibits cell-mediated immunity (CMI) (42). CMI is necessary for the development of immune memory, which serves to enhance the immune response and consequent protection during re-infections (43,44). They showed that a secondary lethal infection after a lower immunization dose of attenuated Lm led to better clearance of listeria in STING-deficient mice compared to wild-type (WT) mice (42), suggesting either that STING-deficient mice develop more effective adaptive immunity and protection compared to WT mice, or that    7  STING-mediated type 1 IFN production following Lm infection inhibits adaptive immune memory function.  Flow cytometry analysis showed that IFNα, TNFα and IL-2 producing cytokines are produced at significantly higher levels in CD8+ and CD4+ T cells compared to wild-type mice (42). This demonstrates that in the absence of STING, CD4+ and CD8+ T cells expand, produce pro-inflammatory cytokines and clear infection more readily than in the presence of STING. Further investigations demonstrated that STING-independent production of type 1 IFNs also correlated with increased Lm bacterial load. Taken together, the available data regarding cPRR signalling and type 1 IFN production in the context of Lm infection suggest that STING-mediated production of type 1 IFN increases early innate resistance to Lm infection, but at the same time also inhibits the formation of optimal and long-term protective cell-mediated immunity (42). 1.4 Aim and hypothesis: Given the well-documented higher risk of the human newborn to suffer severely following infection by viruses or cytoplasmic bacteria, we hypothesize that cPRRs function suboptimally in the neonate as compared to the adult. However, given the conflicting role that IFNs appear to play in the host’s innate vs. adaptive defense against intracellular pathogens, it is unclear whether cPRR signalling leading to type 1 IFN production in the newborn would be significantly higher or lower as compared to the adult. The aim of my study was, therefore, to compare the quantitative relationship of cPRR signalling focused on inducing type 1 IFN in newborns and adults.      8  2 Methods 2.1 Cytoplasmic PRR plates The ligands and their concentrations used, outlined in Table 1, were diluted according to manufacturer’s instructions to the concentration mentioned in the table. Further dilutions of each reagent were made using RPMI medium (Invitrogen). Ten times concentrated sample for each cPRR – (Poly(I:C), 5’ppp-dsRNA, 3’3’-cGAMP, 2’3’-cGAMP, Poly(dA:dT)) – was made using Lyovec as the transfection reagent and stored at room temperature for 15mins before proceeding further. The ligands were prepared at 10 times the final desired concentration in sterile conditions under a hood with laminar airflow. Prepared concentrated ligands were stored in two deep-96-well (VWR) source plates – with or without Brefeldin A (BFA; Sigma-Aldrich). Source plates were sealed using sterile aluminum plate sealers (USA Scientific) and preparation of plates proceeded immediately. Evolution P3 Precision Pipetting Platform (PerkinElmer) was used to dispense 20μL of the reagents from each well in the source plate into each well of the recipient 96-well round bottom polystyrene plates (Corning) under a laminar airflow hood under sterile conditions. The recipient plates were then sealed with aluminum plate sealers and stored at -80°C until use. 2.2 Blood sample processing and in vitro stimulation The study was approved by the Ethics Review Board at University of British Columbia. Blood was drawn from healthy adult individuals via sterile venipuncture into sodium-heparin vacutainers (Becton Dickinson). Cord blood was collected from healthy, full-term elective caesarean sections without labour into sodium-heparin vacutainers. Adult and cord blood was stored at room temperature and was processed within 4hrs of blood draw. Whole blood (WB) was diluted 1:1 with sterile, pre-warmed (37°C) RPMI. 180μL of WB mixed 1:1 with RPMI    9  was added to premade (thawed in 37°C in 5% CO2 incubator for 10mins followed by a spin down) 96-well plates. For each donor, WB mixed 1:1 with RPMI was added to two plates – first plate with BFA and second plate without BFA. For the ICS assay, plates with BFA were incubated for 6hrs in 37°C in 5% CO2 incubator. After the completion of 6hrs, EDTA was added to each well to achieve a final concentration of 2mM EDTA. The cells were resuspended seven times to detach any adherent cells and re-incubated for 10mins. The cells were then resuspended and added to 1400μL of 1X BD FACS Lysing Solution in 2mL microtubes (Corning #430909; preferred) or deep 96-well plates and stored at -80°C until staining. For the luminex assay, plates without BFA were incubated for 24hrs in 37°C in 5% CO2 incubator. At 24hrs, these plates were spun down. 100μL of supernatant was collected from each well, making sure no cells were present in the collected sample. The samples were collected in round-bottom 96-well plates and stored at -80°C. 2.3 Staining, acquisition and analysis Antibodies, and their clones and sources, for staining are shown in table 4. Dilutions were performed before the assay was run to optimize the level of each antibody. Frozen samples were completely thawed in a 37°C water bath. The samples were spun, aspirated and pellets were resuspended in 150μL of PBSAN (PBS containing 0.5% bovine serum albumin and 0.1% sodium azide).The cells were washed twice followed by addition of 150μL FACS Permeabilizing solution (BD). The cells were then incubated at room temperature for 10mins. Following permeabilization, the cells were washed twice again before the addition of antibodies to stain the cells. In addition to cell-staining antibodies, compensation beads (Comp Beads, Becton Dickinson) were also prepared – 4μL of positive and negative each of anti-mouse Ig Comp Beads and 91μL of PBSAN were pipetted into the plate in addition to    10  1μL of each antibody. The comp beads were added to the plate and they were treated as cells from there on. The plates were stored in the dark for 30-60mins after the addition of antibodies to the cells. After the 30mins incubation, 100μL of PBS was added to the wells in use. The sample was then spun, aspirated and washed another time with PBS. Strepavidin, diluted 1:1000 in PBS, was then used to stain all wells in use. The cells were incubated in the dark again for 15mins. 100μL of PBSAN was added to the wells in use to stop the staining and the sample was spun down again. The samples were washed twice using PBSAN. The cells were re-suspended in 200μL of PBSAN immediately followed by flow acquisition. For WB, we aimed to collect 300,000 events. The samples were collected uncompensated from BD LSRII 4 Laser – blue, red, UV and violet. Compensation was done later using FlowJo (TreeStart, ON) and all samples were compensated.  2.4 Luminex-based assessment of cytokines in culture supernatant Supernatant were thawed at room temperature and spun down to settle any cell. Samples were then diluted 1:1 in RPMI 1640. A custom, eBiosciences ProcartaPlex custom 23-plex immunoassay kit (#EPX230-14055-810) was used for the analysis. Standards were prepared by combining all the standards for the plate and making a serial 4-fold dilution of the top standard. Beads were diluted 1:3 in assay diluent and incubated on the plate secured on top of a magnetic plate for 2mins to allow the beads to settle. Beads were then washed twice and samples and standards were put on the plate. The plate, sealed with a plate sealer, was allowed to stay on a shaker for 90mins at room temperature and then incubated overnight at 4°C. The following day, detection antibody and streptavidin were added to the plate with half hour incubations and washes after each step on the shaker. After another wash, reading buffer    11  was added and the plates were read using BioPlex 200 system (Bio-Rad). The analysis was performed on Multiplex software and using Excel.  2.5 Statistical analysis Graphs were prepared using Excel (Microsoft) and Prism (GraphPad). Statistical analysis of cytokine production between the two groups – adults and neonates – for both global cytokine response and cell-specific cytokine production was done using the non-parametric Mann-Whitney U test (for two groups). A p value of <0.05 was considered statistically significant.  Statistical analysis was not performed on the results obtained from the pilot studies because the same size was small for most of them. For the results obtained from global cytokine analysis, the values of cytokines detected in the medium used for ligand preparation (negative controls for the ligand) were subtracted from the total cytokines detected to obtain the subtracted values depicted in the figures. This was essential, especially for luminex assays, because several different plates were run to analyze all the samples. The standards on each plate had different sensitivities, and thus different upper and lower detection limits. Subtraction of the baseline detection level was used to standardize and hence, compare the cytokine levels throughout all samples in the study. Similar to luminex assay, baseline levels of cells producing cytokines in unstimulated and Lyovec-only samples were subtracted from the values obtained from stimulated cells. This allowed for a comparison that was feasible across all donors in the study.       12  3 Pilot Results Prior to commencing the work to test our hypothesis, I first set out to determine the optimal experimental set-up. To this end, I investigated several ligands and their dose and length of incubation for analysis of global cytokine response (using Luminex) and cell-type specific response (using flow cytometry). In addition, pilot studies were also conducted to determine effective cytokine read-outs and specific conditions for flow cytometry. Although the sample sizes were small for most pilots, they served their purpose in guiding our procession towards next step of the study.  3.1 Ligand choice 3.1.1 Objective Several different ligands for cPRRs are available on the market manufactured by various companies. The main objective of this pilot was to explore the effectiveness and conditions of these ligands in a whole blood (WB) assay. Some ligands, such as poly(I:C) and curdlan, are either toxic or ineffective in inducing a cytokine response in WB. Therefore, the ligands of interest, which lead to activation of cPRRs were first screened in adult WB to narrow the range of ligands feasible to be used for the main study. Besides testing the efficacy of these reagents to induce a measurable cytokine response in WB, the second criteria for inclusion of ligands in the final study was to observe the existence of a difference in the cytokine response between adult and cord blood. This was necessary to allow us to address the main objective of the study, which was to explore differences in cPRR signalling between adults and neonates.    13  3.1.2 Results I performed extensive research to screen various ligands from different manufacturers and compare their efficacy in WB. The list of all ligands tested is included in table 2. One of the main concerns for this pilot was to determine the right experimental conditions of use for each ligand. Since the purpose of most ligands is to activate cPRRs, it was essential for them to enter the cytoplasm. Hence, we also tested Lyovec as a transfection reagent. One of the kits tested was the nucleotide oligomerization domain (NOD)-ligand kit. The ten ligands present in the kit specifically activated NOD1, NOD2 or both receptors. However in our hands, they all failed to elicit a response across a wide range of different concentrations tested in WB. These ligands have been shown to activate specific purified cell types or cell-lines. The response of such cells may have been masked in our WB assay because of other cell types or factors present in the plasma.  Another class of ligands discarded from the final choice were the inflammasome inducers where ATP and Alum were two potential ligands of high interest. Alum was excluded because although it showed a response, the response between adult and cord blood were not strikingly different and hence, less potential to be a pathway of concern for the objective of the study. And although ATP showed a differential response for some cytokines for adult and cord WB, its downstream pathway has been well-characterized in the past. Therefore, we excluded it to focus our study on ligands that have not been explored extensively in the past in our previous work (45).  After extensive research and testing, we chose to include 5 cPRRs ligands in the main study: 5’ppp-dsRNA (RIG-I ligand), Poly(I:C) (MDA5 ligand); 3’3’-cGAMP (canonical    14  STING ligand), 2’3’-cGAMP (non-canonical STING ligand) and Poly(dA:dT) (STING + RIG-I ligand). The ligands mimic nucleic acids that would be released by viruses and cytoplasmic bacteria into the cell. The ligands were complexed with Lyovec, a transfection reagent, which allowed the ligands to enter the cell’s cytosolic compartment and activate PRRs present there. Besides the cytoplasmic PRRs mentioned above, two wells were added as negative controls – unstimulated sample (RPMI medium only) and Lyovec only, to monitor the effects of the transfection reagent. R848 was also added to the final ligand list as a positive control since it has been extensively studied previously in our and other labs and its response for most cytokines has been characterized previously. The final list of all reagents included in the study is outlined in table 1. 3.1.3 Conclusion The ligands selected for this study – 5’ppp-dsRNA, Poly(I:C), 3’3’-cGAMP, 2’3’-cGAMP and Poly(dA:dT) – were focused on RLRs and STING because of their role in detection of viral RNA and DNA present in the cytoplasm, as well as CDNs released into the cytoplasm by intracellular bacteria such as Lm.  3.2 Dose-response  3.2.1 Objective Most of the ligands included in the analysis for this study had not been previously tested in a WB assay. Therefore, we tested a wide range of concentrations of each ligand to determine the best possible concentration in our in vitro model. In addition to testing different concentrations, we also tested poly(I:C) with and without Lyovec. Poly(I:C) is known to activate through two main pathways – TLR3 and MDA5. This pilot was conducted    15  to determine the best concentrations for each ligand and determine which pathways were being activated based on the given doses. 3.2.2 Results: a) CDN: Three different concentrations – 100μg/mL, 10μg/mL and 0.1μg/mL – were tested for 3’3’-cGAMP and 2’3’-cGAMP, both with Lyovec (Fig.1 a and b). Almost no TNFα or IL-1β was produced for the WB assay stimulated with 0.1μg/mL of the CDN. The other two concentrations showed a dose-dependent increase in the production of TNFα and IL-1β, with no or very minute production of the two cytokines for the unstimulated or Lyovec-only stimulated samples.  b) 5’ppp-dsRNA: 5’ppp-dsRNA is a specific ligand for RIG-I. It was complexed with Lyovec at a concentration suggested by the manufacturer (data not shown).  c) Poly(dA:dT): Poly(dA:dT) was tested with and without Lyovec for various concentrations - 10μg/mL, 1μg/mL and 100ng/mL (data not shown). There were almost no cytokines produced for the samples stimulated with only poly(dA:dT) without Lyovec. However, we observed a dose-dependent response when Lyovec was added to different concentrations of poly(dA:dT). d) Poly(I:C): Poly(I:C) has the ability to enter the endosomes and activate the TLR3 pathway, or activate MDA5 when it is present in the cytoplasm. Since the main objective of this study was to observe the response in adult and neonatal WB to activation of cytosolic PRRs, MDA5-mediated activation of cells was of interest. Therefore, we tested two different concentrations of poly(I:C) - 10μg/mL and 1μg/mL – with and without Lyovec to observe the dose-dependent and Lyovec-mediated difference in IFNα and IFNγ production (Fig. 2a and b, respectively). This was tested in only one subject to observe if there was any difference    16  with the presence of Lyovec. Similar to poly(dA:dT), we observed almost no cytokines produced in WB stimulated without Lyovec; this is consistent with our previous data where WB responses without Lyovec were only observed at concentrations of or above 50μg/mL (11). Dose-dependent difference in cytokine production was, however, seen in WB stimulated with poly(I:C) at the lower concentration when complexed with Lyovec. This suggests a likely preferential induction of MDA5 in this set up. To confirm the effect of Lyovec, we then performed this pilot in 6 cord samples using 1μg/mL of poly(I:C) with and without Lyovec. Production of IFNα and IP-10 confirmed our previous results (Fig. 3a and b, respectively).  3.2.3 Conclusion The final concentrations of all ligands decided from a panel of tests are outlined in table 1. All cytosolic PRRs required the use of Lyovec to ensure that the ligands had entered the cell and were activating cytosolic PRRs. Although we saw a dose-dependent increase in cytokine production after stimulation with different concentrations of ligand, the lowest concentration with a consistent and robust cytokine response reactivity was chosen for each ligand; this was to ensure that the ligands used were not toxic to the cells and to conserve the amount of reagent used (as they are expensive).  In addition to determining adequate stimulating doses of the ligands, we also determined the role of Lyovec for poly(I:C). In WB stimulated with a very small concentration of poly(I:C) alone, there was almost no or baseline secretion of IFNα and IFNγ  detectable in the supernatant. In contrast, when poly(I:C) was allowed to form a complex with Lyovec before stimulating WB, we saw significantly higher production of the two cytokines with no major difference between the two concentrations. This Lyovec-dependent increase in    17  cytokine production indicates that at the given low concentrations of poly(I:C) complexed with Lyovec, MDA5 was more likely activated. Previous studies, both in our lab and in the published literature, had shown that very high concentrations of poly(I:C) alone are required to see any cytokine production in WB. However, when added at a high concentration, poly(I:C) is more likely to activate the TLR3 pathway and  is very toxic to the cells.  Therefore, we used Lyovec as a transfection reagent for all cytoplasmic PRRs and determined the adequate concentrations of each ligand to induce cytokine production in WB.  3.3 Time course – Luminex 3.3.1 Objective Luminex assay measures the cytokines released by the cells in the supernatant (plasma) of the WB culture set-up. The kinetics of cytokines released from the cells is different depending on both cell types and ligands. Therefore, we performed a time-course study to determine the best time to collect supernatant from the WB assay.  3.3.2 Results The pilot study was set up to obtain supernatants from incubated samples every 6hrs for 24hrs. Therefore, supernatants were collected at 6, 12, 18 and 24hrs and analyzed using 3 basic Luminex kits for IFNα, IL-8 and MCP-1 (Fig 4a, b and c, respectively). We found that, for all three cytokines, adult and cord WB stimulated with all ligands produced more cytokines at 24hrs.  Besides the levels of cytokines for stimulated cells, we also observed a cytokine-dependent increase in background levels produced by WB stimulated with only Lyovec. High amounts of IL-8 and MCP-1 were present in Lyovec only stimulated samples at 24hrs.    18  Also, the increase in Lyovec-associated background was time-dependent. Amounts of IL-8 and MCP-1 gradually increased from 6 to 24hrs.   3.3.3 Conclusion The trend in data obtained from IL-8, MCP-1 and IFNα all favoured collection of supernatants from samples at 24hrs. The levels of cytokines produced were the highest and there was a clear difference in levels of cytokines produced between adult and neonatal WB at 24hrs. Therefore, supernatants were collected at 24hrs for all subjects in the study.  Besides highest cytokine production from stimulated cells, cells incubated with only the transfection reagent, Lyovec, also showed increased IL-8 and MCP-1 production at 24hrs. This was striking because Lyovec is marketed as an innate transfection reagent, hence, should not activate the cells in any way. The exact mechanism of Lyovec’s activity should be explored to better understand its role in production of specific cytokines when added alone to the cells. At the present time, this issue has not been resolved. 3.4 Impact of using freeze-thawed ligands compared to fresh ligands 3.4.1 Objective We obtain blood from adult and cord donors on different days over the course of several months, yet want to ensure consistency of the reagents used to maintain the highest level of quality control. The source company recommended addition of freshly diluted ligands to the WB assay. However, this would have introduced another variable in our study. Human populations display a large variability in their response and it would have been very difficult to deduce whether differences in cytokine levels were due to human variability or variability in ligand preparation, i.e. physiological or artefact. Using the same reagents prepared once,    19  frozen and thawed just prior to use would possibly reduce this risk. Therefore, we performed a pilot study to test for the effects of using fresh vs. frozen ligand preparations for the same donor. 3.4.2 Results The results of the pilot study demonstrated that there was no significant difference in the levels of TNFα and IL-1β produced with fresh and freeze-thawed ligands for all of the stimulations tested.  3.4.3 Conclusion Given the sentinel nature of the innate immune system, it is highly sensitive to technical artefacts. Therefore, it is necessary to control all variables to ensure that the difference in cytokines produced is due to biologically meaningful variables, not technical variables. We compared freshly prepared and freeze-thawed ligands and found that there was no significant difference between TNFα and IL-1β produced in response to the two preparations. Therefore, to maintain quality control for all the subjects in the study, ligands were prepared in one large batch and frozen in plates at -80°C until use.  3.5 Luminex pilot 3.5.1 Objective The main objective of this study was to determine which cytokines are produced in significantly different amounts by adult and cord WB when stimulated with various cytoplasmic PRRs. We used a 23-plex luminex kit to determine which cytokines differ in response to the stimulation. This allowed us to narrow down our focus on the selected few cytokines that showed any difference in production between stimulated adult and cord blood cells.     20  3.5.2 Results Supernatants were collected at 24hrs for a small sample size (n=5 for each group) and analyzed using the broader (and more expensive) 23-plex luminex assay. The luminex assay consisted of pro- and anti-inflammatory cytokines, all of which are outlined in table 3. Observing an overall response of most commonly studied cytokines was essential to determine the role of cytoplasmic PRR stimulation in both adult and cord blood. Luminex analysis showed very little or no response for many cytokines present in the kit. Examples of cytokines that showed very little response are IFNβ, IL-1α, IL-1β, IL-12p70, IL-6, MIP-1β, GM-CSF, ENA78, M-CSF and MIG (data not shown). There were other cytokines that were produced in detectable amounts by both adult and cord blood, however, there was no clear difference in cytokine production in the two groups, for example, IL-10, IFNγ, TNFα, IL-12p40, IL-18, IL-23, MCP-3 and GRO-alpha (data not shown). There were very few cytokines that showed both detectable levels of cytokine production and an obvious difference in cytokine levels in both groups. These cytokines included IFNα, IP-10, MCP-1, IL-8 (Figures 6-9) and IL-1RA (data not shown). Statistics were not performed on the results because these experiments were only used to get a direction for further experiments. 3.5.3 Conclusion The main purpose of this pilot study was to determine the best luminex read-outs to detect a difference between adult and cord blood stimulated with cytoplasmic PRRs. From the small sample size of n=5 (per group), we observed a clear difference in cytokine levels for IFNα, IP-10, MCP-1 and IL-8. We used the difference in mean and standard deviation values in each group for these cytokines and performed power calculations to determine the    21  final sample size needed to show significant results. This pilot study, therefore, served a crucial role in determining the specific cytokines and sample size for the whole study. 3.6 Flow cytometry pilot 3.6.1 Objective The main objective of this pilot study was to test the 13 antibodies with markers for cell subsets (anchor) and intracellular cytokine staining. The antibodies used for this study (outlined in table 3) had been previously outlined and shown to be the most effective to detect innate immune cells and intracellular cytokines produced by cells in adult and cord WB stimulated with various TLR ligands (11). Hence, this pilot was performed to ensure the effectiveness of the antibodies for WB stimulated by cPRR ligands. In addition to efficient functional response, another aim was to observe differences in cytokine production from individual cell types present in stimulated adult and cord WB in response to cPRR stimulation.  3.6.2 Results The first observation of this pilot study was that all 13 antibodies, for cell surface markers and intracellular cytokines, worked effectively in the assay. The gating strategy used for all samples is outlined in figure 10. The relative frequency (percent positive) of cells expressing a particular cytokine was expressed as a subtracted percentage from the unstimulated (RPMI-only) or Lyovec levels detected. The gating strategy for detecting percent positive cells is shown in figure 11 (Fig 11a demonstrates IFNα detection, whereas fig 11b demonstrates TNFα detection for unstim, R848 and 3’3’-cGAMP (cPRR ligand). The cytokine levels detected by R848 (targeting TLR7/8) stimulated adult and cord WB cells were comparable to    22  those seen and published previously in our lab. For example, we see almost equal percentage of IFNα cells in adult and neonatal pDCs (Fig. 12). Therefore, we confirmed that there were no major differences in the staining or acquisition protocol for the assay in my hands. The other major and striking observation from the pilot was that there were almost no cytokines detected from adult and cord blood cells stimulated with any of the cPRR ligands. This is demonstrated in figures 13 a and b, which show production of IL-6 by monocytes and TNFα by conventional dendritic cells (cDCs).  This was surprising because the same samples of WB, from the same donors, were shown to produce large amounts of several different cytokines when stimulated for 24hrs (as seen in fig. 13) (but without the Golgi blocker BFA that is used in ICC). The lack of cytokine production was consistent for all cell types observed. 3.6.3 Conclusion The first conclusion of this pilot was that the antibodies used for cell surface and intracellular cytokines were effective in my hands. Secondly, instead of observing a difference in cytokine production parallel to our observations from the pilot study for luminex assay, we observed little or no cytokine production from adult and cord cells stimulated with cytoplasmic PRR ligands. Direct comparison of luminex and flow cytometry assays is not feasible because flow cytometry is a 6hr incubation of cells and ligands with BFA. For the luminex assay, on the other hand, supernatants are collected from cells stimulated for 24hrs and without BFA. Given these two key differences between luminex and ICC, we ran two additional pilots to explore the effects of time and BFA on ICC. In addition, another possibility for the lack of cytokines detected in cells in flow cytometry in response to cPRRs but not TLRs could have been because of Lyovec.  Lyovec was added to the assays to    23  allow ligands to enter the cell, but it could have had an effect on the cell membrane that may have allowed the cytokines to be released (leaked out) from the cells. 3.7 Role of Lyovec in flow cytometry 3.7.1 Objective The pilot study performed to test for intracellular cytokine production from stimulated adult and cord cells demonstrated lack of cytokine production for all cell types stimulated with cPRR ligands but not for R848 stimulated cells. One of the main differences between R848 and cytoplasmic PRRs is the presence of Lyovec as a transfection reagent for cytoplasmic PRRs. Therefore, we wanted to test whether the lack of intracellular cytokines detected due to the presence of Lyovec during the incubation.  3.7.2 Results To test for the effect of Lyovec on adult and cord blood cells, a direct comparison was done by stimulating the same sample of WB with lipopolysaccharide (LPS; TLR4 target), with or without Lyovec (Fig. 14). In addition, cytokine production from LPS stimulated cells, with and without Lyovec, were tested under different conditions – BFA added at 0hr, 1hr, 2hrs and 3hrs for a total of 6hr assay. This allowed us to determine whether BFA impacted Lyovec’s effect on the cells. From all the conditions, we observed no difference in cytokine production in several cells when comparing LPS stimulated WB with or without Lyovec. Similar production of IL-6 in cDCs regardless of the presence of Lyovec has been demonstrated in figure 14.  3.7.3 Conclusion We did not observe differences in levels of cytokines produced in LPS-stimulated cells regardless of the presence (or absence) of Lyovec. This demonstrates that the lack of    24  cytokines produced by all cell types stimulated with cytosolic PRR ligands in the flow cytometry pilot study was not because of a cell membrane-destroying effect of Lyovec. 3.8 Summary of results from pilot studies After extensive research and testing, I was able to choose the cPRR ligands and determine their concentration and incubation conditions for each ligand, and all of this while maintaining stringent quality control for the study. Luminex pilot enabled us to select IFNα, IP-10, MCP-1 and IL-8 for our final read-outs. In addition to testing the efficacy of flow cytometry assay in my hands, I also found that inhibition of release of cytokines from the cell, possibly because of BFA and not Lyovec, may play a role in the lack of cytokines detected in cPRR stimulated blood using ICC analysis.             25  4 Results of Main Study 4.1 Higher IFNα produced in response to STING and MDA5 stimulation by neonatal blood compared to adult blood Contrary to the dampened IFNα response seen in cord blood and mononuclear cells in response to TLR ligands (11), we observed a striking and significantly higher production of IFNα by cord blood in response to stimulation with several STING and MDA5 ligands (Fig. 15). The canonical STING activator, 3’3’-cGAMP, which is a cyclic dinucleotide produced by bacteria, induced the highest IFNα response in cord blood, a median of about 750pg/mL, compared to adult blood, which induced only about 10pg/mL of IFNα. 2’3’-cGAMP, the noncanonical STING activator produced by cyclic-GAMP synthase (cGAS), and poly(dA:dT), a synthetic analog of B-DNA, also activate STING. They both also produced significantly more IFNα in cord blood (about 150pg/mL) than adult blood (about 10pg/mL). Poly(I:C) complexed with Lyovec, known to activate MDA5 (previously shown in Figs. 2 and 3), also showed higher production of IFNα in cord blood compared to adult blood. Cord blood stimulated with poly(I:C) complexed with Lyovec produced about 215pg/mL of IFNα compared to only about 7pg/mL in adult WB. 5’ppp -dsRNA, RIG-I activator, showed slightly less IFNα production by cord blood. R848 (TLR7/8 ligand) stimulated adult and cord blood showed no significant difference (145pg/mL vs. 148pg/mL, respectively) in IFNα production. Overall, all cPRR ligands, except 5’ppp-dsRNA, induced greater production of IFNα from neonatal WB compared to adult WB.     26  4.2 Higher IP-10/CXCL10 produced by cord blood in response to STING and MDA5 ligand stimulation compared to adult blood Adult and cord WB stimulated with R848 (TLR7/8 ligand) and 5’ppp-dsRNA (RIG-I ligand) produced comparable amounts of IP-10 in adult and neonatal WB (Fig. 16). R848-stimulated WB induced about 150pg/mL, and 5’ppp-dsRNA-stimulated WB led to the production of about 80pg/mL of IP-10. However, cord blood stimulated with 3’3’-cGAMP and 2’3’-cGAMP, poly(dA:dT) and poly(I:C) produced significantly higher IP-10 compared to stimulated adult blood. 3’3’-cGAMP-stimulated neonatal and adult whole blood showed the greatest difference in IP-10 production – about 250pg/mL of IP-10 was detected in stimulated neonatal WB compared to 105pg/mL in adult WB. 2’3’-cGAMP-stimulated whole blood showed a similar difference – 238pg/mL of IP-10 in stimulated neonatal WB compared to 100pg/mL in adult WB. Neonatal WB also produced higher IP-10 compared to adult WB stimulated with poly(I:C) (about 217pg/mL vs. 115pg/mL) as well as with poly(dA:dT) (about 228pg/mL vs. 96pg/mL). Hence, neonatal WB produced more IP-10 compared to adult WB stimulated when stimulated with cPRRs.   4.3 Significantly higher MCP-1 produced by cord blood stimulated with any cytoplasmic PRRs or R848 compared to adult whole blood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that recruits monocytes, memory T cells and dendritic cells to the sites of inflammation or infection in the body. Therefore, it is an important factor to recruit immune cells to the site of infection. Cord blood stimulated with any of the cPRRs or R848 (TLR7/8) showed significantly higher MCP-1 production compared to adult blood (Fig. 17a). Fig. 17a demonstrates the mean values calculated by subtracting the MCP-1 produced in the medium used for ligand preparation –    27  RPMI for R848-stimulated blood and Lyovec for cPRR-stimulated blood – from the total MCP-1 produced in WB. Subtracted mean values showed the greatest difference in MCP-1 production in the two groups stimulated with RLRs. Both MDA5 and RIG-I ligands induced about 200-300pg/mL of MCP-1 in neonatal WB compared to about 45pg/mL in MDA5-stimulated and about 80pg/mL in RIG-I-stimulated adult WB. Poly(dA:dT) stimulated neonatal WB produced about 340pg/mL of MCP-1 compared to only 66pg/mL in adult WB. The canonical and non-canonical STING ligands, 3’3’-cGAMP and 2’3’-cGAMP, showed similar levels of MCP-1 present in supernatant for both adult and neonatal WB– about 0pg/mL. Therefore, stimulated cPRRs, such as MDA5, RIG-I and STING, induced higher MCP-1 production in neonatal whole blood. In addition to higher MCP-1 produced by stimulated cells, we also observed higher MCP-1 production in both adult and cord blood incubated with the transfection reagent, Lyovec (shown in Fig. 17b with the unsubtracted data). The background MCP-1 production by Lyovec only-stimulated adult WB was higher than the MCP-1 levels detected by 3’3’-cGAMP and 2’3’-cGAMP.   4.4  R848 and cytoplasmic PRR stimulated cord blood produced higher IL-8 compared to stimulated adult blood IL-8 is a marker of different inflammatory processes and is produced by monocytes, macrophages and other immune cells. We observed higher IL-8 production by cord blood stimulated with R848, RIG-I, MDA5 and STING ligands (Fig. 18a). Fig. 18a depicts mean values that were calculated by subtracting the background IL-8 produced in the medium used for ligand preparation alone from the total IL-8 produced in the whole blood assay. Similar to MCP-1, we observed higher background IL-8 production from both adult and cord blood    28  incubated with Lyovec only (Fig. 18b shows unsubtracted values). The most striking observation was that the amount of IL-8 produced in Lyovec only-stimulated adult WB was higher than that produced by any of the cPRR-stimulated adult WB. When subtracted, this led to getting nil values for adult WB, which affected the statistical difference seen in unsubtracted, raw values obtained from the assay. The unsubtracted values showed significant difference in IL-8 production in neonatal and adult WB when stimulated with R848 and all cPRRs. Regardless of the stimulation, stimulated adult WB produced about 50-150pg/mL, whereas stimulated cord blood led to production of 100-650pg/mL of IL-8. Therefore, neonatal WB, overall, leads to production of more IL-8 compared to adult WB when stimulated with R848 and cPRR ligands.  4.5 Single-cell analysis showed significantly higher TNFα production by adult cDCs, pDCs and monocytes compared to cord cells. As previously outlined (12), polychromatic, single-cell, high-throughput flow-cytometry based approach was used to determine the percentage of cytokines produced by innate immune cells in adult and neonatal WB. Our analysis of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) and monocytes showed significantly higher production of TNFα in response to TLR7/8 ligand R848 by adult cells compared to cord cells (Fig. 19 shows TNFα produced by cDCs; data not shown for other cell types). About 47% of adult cDCs produced TNFα compared to only 30% cord cDCs. This was in stark contrast to the complete lack of TNFα produced by cDCs, pDCs and monocytes for both adult and cord cells stimulated with all cPRR ligands. While we only studied the relative frequency of the cells (percent positive) that produced cytokines above the baseline, unstim-level, another    29  method to analyze cytokine production is to measure the mean fluorescence intensity (MFI). In addition, Shooshtari et al. have developed a quantitative approach by incorporating relative frequency and MFI to produce a generalized integrated MFI (iMFI) (46). We, however, did not determine the iMFI scores because we failed to see substantial percent positive cells in both adult and cord cells stimulated with cPRR ligands.  4.6 Significant production of IL-12 and IL-6 by R848-stimulated cDCs and monocytes in both adult and cord blood Adult and cord cDCs and monocytes stimulated with R848 (TLR7/8 ligand) produced detectable amounts of IL-12 and IL-6 by the cells. There was no significant difference in IL-6 production in adult and cord cDCs and monocytes (Fig 20 a and b). About 30% of both adult and cord cDCs, and about 20% of monocytes produced IL-6. Also, no significant difference was detected in IL-12 production in adult and cord cDCs (data not shown). However, a significantly lower percentage of monocytes present in cord blood compared to adult monocytes (about 16% vs. about 45%, respectively) produced IL-12 (Fig. 21). Despite the detection of IL-6 and IL-12 in R848-stimulated cDCs and monocytes, there was very little to no IL-6 or IL-12 detected in cytoplasmic PRR-stimulated adult and neonatal cDCs and monocytes (Figs. 20-21).   4.7 Plasmacytoid dendritic cells (pDCs) are the source of IFNα production in R848-stimulated adult and cord cells B cells, γδ cells, NK cells, NK T cells, cDCs, monocytes and pDCs were analyzed, and pDCs were the only cell type which showed significant levels of IFNα production in R848-   30  stimulated adult and cord blood (Fig. 22). Neonatal pDCs produced significantly less IFNα than adult pDCs when stimulated with R848 (about 18% in neonatal pDCs compared to about 41% in adult pDCs). However, cPRR-stimulated adult and cord pDCs produced very little to no IFNα.                       31  5 Discussion 5.1 Discussion of results The aim for this study was to investigate the role of cPRRs in induction of the immune response, measured in terms of global cytokine production (in cell supernatant) and intracellular cytokine production. In summary, we found that cPRR ligands led to significantly higher production of type 1 IFNs and pro-inflammatory cytokines in neonates compared to adult whole blood. This striking difference was, however, only seen in cell culture supernatants when analyzed using luminex. Intracellular cytokine detection performed using flow cytometric analysis showed very little to no cytokine production for both adult and neonatal cells stimulated with cPRR ligands, while TLR ligand stimulated cells of the same samples led to the expected cytokine production. Additional experiments are needed to determine the reasons for the disassociation in results from luminex and flow cytometric assays. Also, the striking difference in neonatal WB and adult WB in response to cytoplasmic PRRs, especially STING, could potentially lead a way for use of its ligand as a vaccine delivery platform for neonatal vaccines and/or help explain the increased risk of newborns to suffer and die from Lm infection. The exact cell types in WB that are responsible for this difference could not be further deduced.  Analysis of cytokine production in cell supernatants showed slightly lower production of IFNα in cord WB stimulated with 5’ppp-dsRNA (RIG-I ligand) compared to adult WB. This trend was consistent with previously published findings of IFNα detection in neonatal and adult MCs (47). Specifically, Marr et al. demonstrated that pDCs are responsible for the production of IFNα in adult and cord mononuclear cells (MCs) (47). They also contrasted this by demonstrating comparable amounts of monocyte-mediated IL-6 production in    32  neonates and adults (47). Therefore, RIG-I-dependent IFNα production is mediated by pDCs, and is reduced in neonates. In my hands with the samples used, IFNα production in R848-stimulated adult and cord blood showed very little difference between the two groups. This is not consistent with previous published results from our lab; however, Corbett et al. used 3M-003 as a TLR7/8 ligand activator, which has a different molecular structure as compared to the TLR7/8 PAMP I used in this study (11). They showed higher IFNα production in adult MCs post-stimulation with 3M-003 (11) compared to neonatal and infant MCs. Hence, the inconsistency in IFNα production could be because of the use of different ligands. We observed significantly higher IFNα production in neonates compared to adults following STING and MDA5 stimulation. The degree of difference between neonatal and adult IFNα production in response to cPRRs was surprising. It signifies that neonates either have over-active STING and MDA5 receptors, or differential regulation of the downstream signalling of STING and MDA5 in neonates and adults. This has potentially significant implications both mechanistically as well as therapeutically (see discussion of these points below).   IP-10 production following cPRR stimulation and detection through global cytokine analysis also demonstrated the same trends as IFNα. No difference was seen in IP-10 levels detected for R848 and 5’ppp-dsRNA stimulated adult and neonatal blood. However, we observed a significantly higher amount of IP-10 in neonatal WB stimulated with MDA5 and STING ligands. IP-10 is a chemokine secreted by cells in response to type 1 and II IFN stimulation, and it acts as a chemoattractant for activated T cells (48). Therefore, similar IFNα and IP-10 production results likely represent a consistency in presence of more type I and type II IFNs in stimulated neonatal cells, and hence a better antiviral immune response mediated through STING and MDA5.    33  Similar to IFNα and IP-10, neonatal WB showed greater production of both IL-8 and MCP-1 in response to STING and MDA5 and also RIG-I stimulation. IL-8 is a neutrophil chemotactic in activated cells (49). IFNγ has been shown to stimulate the production of IL-8 (49). MCP-1 is a chemoattractant that regulates the migration of monocytes and macrophages to the site of infection (50). Migration of monocytes is essential for routine surveillance, and infiltration to the site of infection signifies a potent immune response to pathogens. Therefore, higher production of IL-8 and MCP-1 in neonatal WB signifies better mobilization of the immune cells in response to STING, MDA5 and RIG-I stimulation compared to adult WB. Although RIG-I and MDA5 belong to the same family of receptors and both are activated by dsRNA in the cytoplasm, they showed differential cytokine production in response to stimulation with their respective ligands (22,29). The ligands for RIG-I and MDA5, 5’ppp-dsRNA and poly(I:C), are both synthetic analogs of dsRNA, with small variations in their size and molecular patterns. The variation in their shape and blunt end vs. 5’ppp-overhang are essential to determine which RIG-I-like receptor they activate (18,22,28,29). Therefore, despite the large similarity, poly(I:C) and 5’ppp-dsRNA specifically activate MDA5 and RIG-I, respectively. This specificity for specific nucleic acids has been confirmed in studies with mice deficient for MDA5 or RIG-I. RIG-I deficient mice are highly susceptible to infections from several RNA viruses, such as Newcastle disease virus (NDV), Sendai Virus (SeV), vesicular stomatitis virus (VSV), Japanese encephalitis virus (JEV) and Hepatitis C virus (HCV) (22,51,52). Mice lacking MDA5, however, were shown to have abrogated gene induction response for IFNβ, IP-10 and IL-6 in response to various picornaviruses, such as encephalomyelocarditis virus (ECMV), mengo virus, and Theiler’s virus (22,53).Therefore,    34  although RIG-I and MDA5 helicases belong to the same family of RLRs and detect dsRNA in the cytoplasm, their activation profile differs slightly based on the specific virus or viral analogs present. Besides the difference in RIG-I and MDA5 helicase activity, we also observed that cytokine production mediated by MDA5 ligand, poly(I:C), was consistent with STING ligands for all four cytokines for our final read-out. This was, however, not true for RIG-I ligands. There was no significant difference between IFNα and IP-10 production in neonatal and adult WB after stimulation with RIG-I ligands; but a significant increase was observed in neonatal WB for STING and MDA5 ligands. Ishikawa and Barber showed that STING-deficient (STING-/-) mouse embryonic fibroblasts (MEF) infected with VSV, which is known to activate RIG-I, have more progeny virus compared to WT mice (33). A rescue response was demonstrated, in terms of number of progeny VSV present, by the addition of STING to STING-/- MEFs. They contrasted this by demonstrating that STING did not play a significant role in IFNβ induction in cells incubated with poly(I:C), which activates MDA5 (33). Therefore, although STING has been shown to play a role downstream of RIG-I but not MDA5, our results showed the opposite trend. This could be because of the suboptimal activation or function of pDCs, which have been shown to be the predominant IFNα producers in neonates (47).  In summary, we detected a strikingly higher production of IFNα, IP-10, MCP-1 and IL-8 in neonatal WB compared to adult WB in response to STING and MDA5 ligands. MCP-1 and IL-8 are also produced in significantly higher amounts in response to RIG-I stimulation in neonates. STING, RIG-I and MDA5 detect viral and bacterial DNA and RNA directly released into the cytosolic space (21,27). Unlike TLR ligands, which are present in    35  extracellular (including endosomal) spaces, cPRR ligands detect a breach of a barrier to the intracellular environment that may pose a particularly serious threat to the newborn. Hence, the increased production of type 1 IFNs and pro-inflammatory cytokines in response to cPRR, but not TLR, ligands could be biologically important. This would further strengthen our theory that the neonatal immune system is not immature, but rather distinct from the adult immune system. These differences likely are driven by age-dependent differences in functional demands and have been selected for over the millennia of our evolution.  A concern in the global cytokine response we measured was the high background cytokine-specific response for adult and cord blood stimulated with Lyovec only. High amounts of IL-8 and MCP-1, but not IFNα or IP-10, were found from the same supernatant collected from each donor. Lyovec is a cationic lipid-based transfection reagent that is complexed with cytoplasmic PRRs to allow them to enter the cell membrane. Although the exact mechanism of Lyovec’s function is not clear, it is marketed as a reagent that couples with a neutral lipid, which helps the cell membranes after they destabilize to allow ligands into the cell. This may explain the high background production of IL-8 and MCP-1. Higher expression of MCP-1 has been observed previously in response to cationic liposomes (54).  However, the lack of IFNα or IP-10 response to cell membrane destabilization process would then be surprising, especially because Lonez et al. also demonstrated IP-10 induction by some cationic liposomes (54). In our assay, most cPRR-stimulated adult blood and some stimulated cord blood samples showed lower IL-8 or MCP-1 production compared to the background levels produced by Lyovec alone. This made it extremely difficult to explain this phenomenon. Therefore, we need to conduct further research into exploring the mechanism of Lyovec’s activity and its role in inducing certain cytokines in whole blood assays.    36  Although it is difficult to deduce the cytokine-specific activity for cell cultures with Lyovec only, we were able to explore and interpret that Lyovec is not responsible for the lack of cytokine response observed for intracellular staining in cPRR-stimulated adult and cord blood. B cells, γδ cells, NK cells and NK T cells showed no significant production of any of the cytokines investigated. cDCs, pDCs and monocytes demonstrated significant production of some cytokines but only for cells stimulated with R848. Adult and neonatal cells stimulated with cPRRs showed little to no intracellular cytokine production. This is completely contradictory to the high levels of type I IFNs and pro-inflammatory cytokines detected in global cytokine assay using luminex. One of the possible reasons for the lack of cytokine detection was proposed to be the presence of Lyovec, which may allow the cytokines to escape the cell despite the addition of BFA. However, we showed that adult cells stimulated with LPS showed similar levels of intracellular cytokines in assay regardless of the presence of Lyovec. Addition of Lyovec, therefore, was not responsible for the lack of cytokine production in cPRR-stimulated cells.  Besides Lyovec, other differences in the luminex and flow assay were the total incubation time and addition of BFA. BFA is a fungal metabolite and it interferes with the Golgi and vesicular trafficking (55,56). Therefore, it does not allow the cytokines produced in the cell to be released into the supernatant. Although this is necessary for the successful detection of cytokines produced in the cell by flow cytometry, it inhibits intracellular interaction through cytokines released into the extracellular environment. This is primarily of concern for the production of IFNα. Activated RIG-I, MDA5 and STING all lead to downstream signalling that phosphorylates and induces nuclear translocation of IRF3 (21,33,34,41). IRF3 associates with various transcription factors in the nucleus and leads to synthesis of IFNβ. IFNβ is then    37  released from the cell and induces an antiviral state in the cell environment through autocrine and paracrine signalling (57). It binds to the IFNα/β receptor and activates downstream JAK/STAT pathway in self and neighbouring cells that may still be uninfected by the virus (57). Activation of JAK/STAT pathways leads to phosphorylation and nuclear translocation of STAT dimers to the nucleus where it induces the transcription of IFNα, along with various other IFN-stimulating genes (ISGs) (57). IFNα produced is then released by the cell and it acts back on the IFNα/β receptor in a positive feedback mechanism to induce production of more IFNα (57). Therefore, the production of IFNα is highly dependent on feedback mechanism from the cell, which is inhibited by the addition of BFA from the start of the assay. Lack of intracellular interaction and feedback mechanism may also have a negative effect on the production of other cytokines, and hence, they are not detected in the cells either. A pilot was performed to observe the effect of BFA and feedback mechanisms for flow cytometry. Although we observed production of some cytokines when BFA was added later to the assay, repetition of the experiment with a greater sample size would allow us to be confident about the results obtained. 5.2 Clinical implications In addition to its role in the recognition of DNA in the cytoplasm, STING has also been shown to impact the production of type 1 IFNs downstream of RIG-I and MDA5, which recognize cytoplasmic dsRNA (22,27,33). Activation in neonatal whole blood by STING ligands, CDNs or analog of B-DNA, consistently produced higher IFNα, IP-10, IL-8 and MCP-1 in supernatant compared to adult blood. The consistent difference in production of type 1 IFNs, pro-inflammatory cytokines in neonatal and adult WB assays could be attributable to differential regulation of the downstream pathways. Some possible areas of    38  differential regulation of cytokine production in adults and neonates could be: recruitment of adaptor proteins downstream of STING, phosphorylation and nuclear translocation of IRF3, presence (or absence) of certain transcription factors, or rates of transcription and translation. Further exploration of the differential STING pathway regulation in adult and neonates would shed light onto how various disease models, that activate STING, would differ in generating an immune response in adults and neonates. One such prevalent disease model is Listeria monocytogenes (Lm).   Lm is a facultative anaerobic, gram-positive microbe. It infects humans through food-product contamination. However, it has shown to be pathogenic mostly in newborns and elderly (5). Lm infection of antigen presenting cells (APCs) has been shown to activate three distinct bacterial recognition pathways: 1) a TLR/MyD88-mediated production of pro- and anti-inflammatory cytokines; 2) STING/IRF3-mediated production of type 1 IFNs; and 3) AIM-2/Caspase-1-dependent inflammasome-mediated induction of IL-1β and IL-18 (58). Our main focus is the activation of STING/IRF3 by the CDNs released into the cytoplasm by Lm. However, this mechanism was largely unknown in the past.  Way et al. demonstrated that MyD88-deficient mice were capable of inducing IFNγ-producing CD8+ and CD4+ T cells in response to intracellular Lm infection (59). Although the CD4+ T cell response was slightly higher in wild-type mice, there was no difference in CD8+ T cell response regardless of the presence of MyD88. In addition, they also showed that despite the absence of MyD88, the immune response generated to ActA-deficient Lm vaccine were protective and long-lasting (59). These findings were later confirmed to be effective in neonatal mice. Kollmann et al. demonstrated that neonatal mice immunized with a single ActA-deficient Lm vaccine showed protective immune response to later lethal Lm    39  challenge (8). They also showed effective production of CD8+ and Th1 memory response. This was different from previously published data that showed that a booster vaccination was needed in adult mice to maintain antigen-specific memory (8,60). Therefore, neonatal mice were not only better capable of inducing an innate immune response, but also more efficient at inducing an antigen-specific immune and memory response. Although the TLR/MyD88 pathway was previously known to have a dominant response in case of an infection, the absence of MyD88 pathway did not have a significant effect on clearance of infection or induction of memory response. Therefore, another pathway, possibly the STING-mediated pathway, could have been at function.  Ishikawa and Barber demonstrated that Lm infection of STING-deficient murine embryonic fibroblasts did not induce the production of IFNβ (61). Hence, STING is essential to induce a type 1 IFN production after intracellular infection with Lm.  The activation of STING by attenuated Lm vaccine suggests that Lm may be particularly effective as a vehicle for other neonatal vaccines. The Th2/Th-17- and anti-inflammatory-biased immune system of neonates puts them at a higher risk for acquisition of intracytoplasmic pathogens (4-6). However it has been shown that given the right stimulus, neonates are able to induce an adult-like immune response (58,60,62,63). Since STING activation leads to production of type 1 IFNs, which are known to induce activation of CD8+ and CD4+ T cell-mediated immunity, using attenuated Lm as an adjuvant for neonatal vaccines may be effective in generating an antigen-specific memory response.  Lm-based vaccines have been shown to be effective in delivering several anti-tumor antigens to the site of tumor or cancerous tissue. The immune suppression of the tumor microenvironment (TME) makes it extremely difficult to deliver or see the efficacy of anti-   40  tumor drugs. However, Chandra et al. have seen drastic reduction and even elimination of metastatic breast cancer by delivering a tumor-associated antigen using an attenuated Lm-based vaccine (64). They found equal efficacy for c-di-GMP, STING activator, compared to Lm-based tumor-associated antigen vaccine and c-di-GMP together. This was attributable to a STING-dependent activation of CD8+ T cells, hence making attenuated Lm-based vaccine a preferred option for cancer immunotherapy (64). In addition to cancer immunotherapy, Lm-based vaccines have also shown to effectively induce protective responses against infectious diseases, such as lymphocytic choriomeningitis virus (LCMV), Leishmania major and Francisella tularensis (65-67).Therefore, the higher production of IFNα in neonatal blood compared to adult blood resulting from STING activation suggests that neonatal vaccines using Lm as a vehicle may be particularly effective.  Although STING-mediated production of type 1 IFN production has been shown to be protective against future challenges of Lm, another recent study found contradictory results. Archer et al. reported that mice deficient in STING or its downstream effector IRF3 are better protected from lethal Lm challenges (42). They showed that immunized STING-deficient mice had lower bacterial burdens compared to immunized control mice. They also performed studies comparing the bacterial burdens in MyD88/ STING-deficient mice to MyD88-deficient mice and showed that although MyD88-deficient mice had slightly fewer bacteria in their liver, MyD88/STING-deficient mice had higher Lm-specific CD8+ T cells (42). Therefore, regardless of the presence of MyD88, STING-deficient mice produced a more effective cell-mediated immune response (42). This STING mediated suppression of cell-mediated immunity was found to be mediated by the presence of type 1 IFNs (42). Although the mechanism is unclear, very high doses of IFNβ have been shown to be    41  immunosuppressive and are used clinically for the treatment of relapsing-remitting multiple sclerosis (RRMS) (68). We speculate that the extreme susceptibility of neonates to severe Lm infection might result from a similar immunosuppressive effect of high levels of IFNα detected in stimulated neonatal WB (8,69). Significantly high type 1 IFN levels detected in global cytokine assay in neonatal whole blood may in fact lead to a suppression of cell-mediated immunity, i.e., while Lm-driven STING activation via very high IFNα production leads to an increase in innate immune mediated protection in the newborn, it leads via the same mechanism to a suppression of adaptive immunity and, with that, potential failure to fully clear Lm. This would predict that innate immunity in neonatal blood to Lm infection should be more efficient at initially reducing Lm burden, but with the failure to fully eradicate the microbe, lead to increased morbidity in the long term. While this may appear counterintuitive given the increased clinical susceptibility of Lm of the newborn, we have in fact recently observed an enhanced innate clearance of Lm in vitro in neonatal vs. adult cord blood (Dr. Ashley Sherrid; unpublished).  Furthermore, our findings suggest that instead of using Lm-delivery based vaccines, it may be more beneficial to add adjuvants to more precisely regulate the level of STING activation and subsequent production of type 1 IFNs in neonates. Regulating the levels of type 1 IFNs could help activate rather than inhibit the cell-mediated immunity and hence, lead to a more optimal immune memory response to the immunogens present in the neonatal vaccines.  5.3 Future directions The results obtained from the global cytokine analysis of neonatal and adult WB consistently showed higher production of type 1 IFNs and several pro-inflammatory    42  cytokines in neonatal WB compared to adult WB. However, the contrast of those results with the lack of cytokine production from intracellular staining assay need to be further explored. In addition to delayed addition of BFA to the assay, there are several other possible experiments that will need to be done to verify the theory of the necessity of feedback-loop mechanism for cytokine production in cytoplasmic PRR-stimulated cells. First, we could add recombinant IFNβ to the stimulated whole blood assay and then test for the presence of IFNα using intracellular cytokine staining and Luminex; this would test for age-dependent differences in sensitivity to type 1 IFNs. Intracellular cytokine staining may still not be completely effective since a positive feedback loop is essential for the enrichment of IFNα levels. However, addition of recombinant IFNβ to the assay with delayed addition of BFA to the assay may be beneficial in determining the levels of IFNα produced in the cells. Second, direct identification of IFNβ protein in adult and cord cells could be another option. However, IFNβ protein has not been detectable in the assays previously used in our lab (ELISA, Luminex). Therefore, detection of differential IFNβ mRNA expression in stimulated adult and neonatal cells using real-time polymerase chain reaction (RT-PCR) might provide a more feasible approach to identify its role in IFNα production. Third, we will definitely have to explore signalling pathways downstream of STING-activated adult vs. cord blood, including possibly differential nuclear translocation of IRF3 or IRF7. Together, these experiments will shed light on the mechanism behind the significantly higher IFNα production observed in stimulated neonatal blood compared to adult blood. We also plan to test the biological relevance of our findings in murine models of listeriosis. For this, we should alter host as well as pathogen, e.g., infect neonatal STING-deficient (vs. WT) mice with Lm  over- or under-producing CDNs, and measure the    43  production of type 1IFNs over time in parallel to the bacterial burden and adaptive immune response. Lastly, we should expand our in vitro modeling of Lm infection in human blood (cord vs. adult), focusing on the early innate responses (<24hrs.) as well as the in vitro establishment of adaptive immune responses (days). Here too, we should modulate host (e.g., via siRNA) as well as microbe (Lm hypo- or hyper-producing CDN). And as in the murine in vivo system, the in vitro-based human assays should also be coupled with measurement of type 1 IFN as well as bacterial burden. Together, these complementary in vivo and in vitro cellular and molecular approaches should allow us to establish functional cause-effect mechanisms and with that, pave the way for translation of our findings into clinical (therapeutic or prophylactic) benefit for the newborn.              44  6 Tables  Table 1. Summary of the final ligands selected for the study, their product information, target receptors and concentrations used. Ligand Target Vendor Cat. # Stock conc Final conc Prep notes Unstim      RPMI only Lyovec  Invivogen Lyec-12   Lyovec only R848 TLR7/8 Invivogen Tlrl-r848-5 10mM 10μM  Poly(I:C) MDA-5 when with Lyovec Invivogen Tlrl-picw-250 20mg/mL 1 ug/mL + Lyovec  5’ppp-dsRNA RIG-I Invivogen Tlrl-3prna-100 0.1mg/mL 1μg/mL + Lyovec 3’3’-cGAMP STING Invivogen Tlrl-cga 1mg/mL 10μg/mL + Lyovec 2’3’-cGAMP STING Invivogen Tlrl-cga23-s 1mg/mL 10μg/mL + Lyovec Poly(dA:dT) RIG-I &CDS; AIM2 inflammasome inducer Invivogen Tlrl-patn-1 1 mg/mL 10 μg/mL + Lyovec              45  Table 2. List of all ligands and their receptor targets tested for their efficacy in whole blood. Ligands Target Ligands Target 5’ppp-dsRNA +L RIG-I C12-iE-DAP NOD1 3’3’-cGAMP +L STING iE-DAP NOD1 2’3’-cGAMP +L STING MDP NOD2 Poly(dA:dT) +/- L STING + RIG-I L18-MDP NOD2 Poly(I:C) +/- L TLR3/ MDA5 M-TriDAP NOD1/2 Nigericin NLRP3 inflammasome inducer M-TriLYS NOD2 Zymosan TLR2/ Dectin-1 Murabutide NOD2 ATP NLRP3 PGN-Ecndi NOD1/2 LPS+ATP NLRP3 PGN-Sandi NOD1/2 LPS+Alum NLRP3 Tri-DAP NOD1            46  Table 3. List of all cytokines tested for in the 23-plex luminex assay to detect differential responses from neonatal and adult whole blood assays. IL-1RA MIP-1β IL-23 IL-10 GM-CSF IL-8 IL-6 IFN-α IP-10 ENA78 IFN-β MCP-1 GRO-alpha IFN-γ MCP-3 IL-12p40 IL-1α M-CSF IL-12p70 IL-1β TNF-α MIG IL-18             47  Table 4. Comprehensive summary of lasers, photomultiplier tube (PMT), long pass (LP) and band pass (BP) filters, target fluorochromes and their clones used for the flow cytometry panel for this study LASER PMT LP BP TARGET-fluorochrome Clone  Violet(405) A 750 780/60 CD66 Biotin (Biolegend, custom) BV786 strepavidin (BD Cat # 563858  ASL-32  B 685 710/50    C 630 670/30  CD16 BV650 (Biolegend Cat# 302041)  3G8  D 595 610/20 HLA-DR  eflour 605 (eBio # 93-9956)  LN3  E 570 585/42     F 545 560/40      G 505 525/50 CD14 V500  (BD Cat # 561392) M5E2  H - 450/50 IL-12p40 eF450  (eB Cat # 48-7129-42) C8.6 Blue (488) A 685 695/40 IL-6 PerCPeF710 (eB custom no Cat #yet) MQ213A5  B 505 525/50 gd –TCR FITC (ebio Cat# 11-9959)  B1.1  C - 488/10   Green (532) A 750 780/60 CD123 PE-Cy7 (eB Cat # 25-1239) 6H6  B 685 710/50    C 630 670/30     D 600 610/20 CD3 PE-CF594 (BD Cat # 562310) UCHT1  E - 582/15 INFα PE  (BD Cat # 560097 7N4-1 Red (640) A 750 780/60 INFγ APC-eF780 (eB Cat # 47-7319-42) 4S.B3  B 685 720/40 TNFα Ax700 (eB Cat # 56-7349-42) Mab11  C - 670/30 CD11c APC (BD Cat # 340544) S-HCL-3     48  7 Figures Subject 1Subject 20100020003000UnstimLyovec100 ug/mL10 ug/mL0.1 ug/mLTNF-alpha (pg/mL) Subject 1Subject 202004006008001000UnstimLyovec100 ug/mL10 ug/mL0.1 ug/mLIL-1beta (pg/mL) Figure 1. Test of different concentrations of 3'3'-cGAMP (STING ligand) complexed with Lyovec showed a dose-dependent increase in TNF-alpha (a) and IL-1beta (b) production in two adult subjects.    49    Unstim/Lyovec10ug/mL+L1ug/mL+L10ug/mL1ug/mL 0100200300Unstim/Lyovec10ug/mL+L1ug/mL+L10ug/mL1ug/mLIFN-alpha (pg/mL) Unstim/Lyovec10ug/mL+L1ug/mL+L10ug/mL1ug/mL 0200400600Unstim/Lyovec10ug/mL+L1ug/mL+L10ug/mL1ug/mLIFN-gamma (pg/mL) Figure 2. Different concentrations of poly(I:C) were tested with and without Lyovec (+L indicates presence of Lyovec). IFN-alpha (a) and gamma (b) were produced in the presence of Lyovec but only baseline concentrations of the two cytokines were detected without Lyovec. n=1 (adult) for each cytokine tested.     50  UnstimLyovecPoly(I:C) + LPoly(I:C)   0100200300400UnstimLyovecPoly(I:C) + LPoly(I:C)IFN-alpha (pg/mL) UnstimLyovecPoly(I:C) + LPoly(I:C)05010015200250UnstimLyovecPoly(I:C) + LPoly(I:C)IP-10 (pg/mL) Figure 3. Poly(I:C), when complexed with Lyovec, induced production of IFN-alpha and IP-10 in whole blood assay compared to poly(I:C) without Lyovec, which showed baseline level of cytokine produced for each cytokine. n=7 (cord blood) for both cytokines.    51   020040060080010001200Unstim 6Unstim 12Unstim 18Unstim 24Lyovec 6Lyovec 12Lyovec 18Lyovec 24R848 6R848 12R848 18R848 24Poly(I:C) 6Poly(I:C) 12Poly(I:C) 18Poly(I:C) 245'ppp-dsRNA 65'ppp-dsRNA 125'ppp-dsRNA 185'ppp-dsRNA 243'3'-cGAMP 63'3'-cGAMP 123'3'-cGAMP 183'3'-cGAMP 242'3'-cGAMP 62'3'-cGAMP 122'3'-cGAMP 182'3'-cGAMP 24Poly(dA:dT) 6Poly(dA:dT) 12Poly(dA:dT) 18Poly(dA:dT) 24IFN-alpha (pg/mL) AdultCord   52   05001000150020002500Unstim 6Unstim 12Unstim 18Unstim 24Lyovec 6Lyovec 12Lyovec 18Lyovec 24R848 6R848 12R848 18R848 24Poly(I:C) 6Poly(I:C) 12Poly(I:C) 18Poly(I:C) 245'ppp-dsRNA 65'ppp-dsRNA 125'ppp-dsRNA 185'ppp-dsRNA 243'3'-cGAMP 63'3'-cGAMP 123'3'-cGAMP 183'3'-cGAMP 242'3'-cGAMP 62'3'-cGAMP 122'3'-cGAMP 182'3'-cGAMP 24Poly(dA:dT) 6Poly(dA:dT) 12Poly(dA:dT) 18Poly(dA:dT) 24IL-8 (pg/mL) AdultCord   53   Figure 4. Production of IFN-alpha (a), IL-8 (b) and MCP-1 (c) were quantified in a time-course study of adult and cord whole blood stimulated with various cytoplasmic pattern recognition receptor ligands. Supernatants were collected at 6, 12, 18 and 24hrs. (numbered in each graph). A time-dependent increase in cytokine production was seen in all three cytokines detected. n=3 for each group (adult and cord) for this pilot study. 050100150200250300350400450500Unstim 6Unstim 12Unstim 18Unstim 24Lyovec 6Lyovec 12Lyovec 18Lyovec 24R848 6R848 12R848 18R848 24Poly(I:C) 6Poly(I:C) 12Poly(I:C) 18Poly(I:C) 245'ppp-dsRNA 65'ppp-dsRNA 125'ppp-dsRNA 185'ppp-dsRNA 243'3'-cGAMP 63'3'-cGAMP 123'3'-cGAMP 183'3'-cGAMP 242'3'-cGAMP 62'3'-cGAMP 122'3'-cGAMP 182'3'-cGAMP 24Poly(dA:dT) 6Poly(dA:dT) 12Poly(dA:dT) 18Poly(dA:dT) 24MCP-1 (pg/mL) AdultCord   54    Figure 5. Similar levels of TNF-alpha (a) and IL-1beta (b) were produced by whole blood stimulated with freshly prepared and freeze-thawed ligands. The results ensured comparable efficacy of fresh and freeze-thawed ligands, hence, allowing more stringent quality control in terms of ligands used for each donor in the study. n=1 (adult) for each cytokine. 010002000300040005000600070008000TNF-alpha (pg/mL) FrozenFresh05001000150020002500IL-1beta (pg/mL) FrozenFresh   55  UnstimLyovecR848Poly(I:C)5'ppp-dsRNA3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)0500100015002000AdultCordIFN-alpha (pg/mL) Figure 6. Neonatal whole blood produced higher IFN-alpha levels when stimulated with MDA5 and STING ligands compared to adult whole blood. The data shown represents mean values with standard error bars for n=5 for each group (adult and cord).         56  UnstimLyovecR848Poly(I:C)5'ppp-dsRNA3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)0100200300400500AdultCordIP-10 (pg/mL) Figure 7. Neonatal whole blood produced higher IP-10 levels when stimulated with MDA5 and STING ligands compared to adult whole blood. The data shown represents mean values with standard error bars for n=5 for each group (adult and cord).          57  UnstimLyovecR848Poly(I:C)5'ppp-dsRNA3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)0100020003000AdultCordIL-8 (pg/mL) Figure 8. Neonatal whole blood stimulated with cytoplasmic pattern recognition receptors produced higher IL-8 compared to adult whole blood.  High background IL-8 production was also observed for R848 stimulated adult and cord blood. The data represents mean values with standard error bars of n=5 for each group (adult and cord).           58  UnstimLyovecR848Poly(I:C)5'ppp-dsRNA3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)0500100015002000AdultCordMCP-1 (pg/mL) Figure 9. Neonatal whole blood stimulated with cytoplasmic pattern recognition receptors produced higher MCP-1 compared to adult whole blood. The background levels of MCP-1 for Lyovec-only stimulated adult and cord blood were also high. The data represents mean values with standard error bars of n=5 for each group (adult and cord).          59   Figure 10. Gating strategy for detection of innate immune cells using flow cytometric assay. Exdoublet peak is done to ensure analysis of only single cells. The cells are then gated on size to exclude dead cells and debris. CD66 marker is used to exclude granulocytes (CD66+). CD66- cells are then expanded and gated using HLA-DR and CD14. HLA-DR negative cells are characterized as Antigen Presenting cells – (APC-) and further gated on to get NK, NK T and γδ cells.  HLA-DR positive cells are characterized as APC+ and further gated for B cells and plasmacytoid and conventional monocytes. CD14 cells are gated as monocytes/macrophages in the assay. This gating strategy was used for all cells analyzed for intracellular cytokines in the study.    60   Figure 11. Relative frequency of cells (percent positive) expressing cytokines ((a) represents IFN-alpha and (b) represents TNF-alpha) in adult cells using intracellular staining. The percent positive cells were determined based on the gating for unstimulated cells that were stimulated with RPMI-alone. We saw IFN-alpha and TNF-alpha production for cells stimulated with R848 (TLR7/8 ligand) but not for cells stimulated with cPRR ligand 3’3’-cGAMP (STING activator).     61  R848LPS5'ppp-dsRNAPoly(I:C)3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)-200204060AdultCord% positive IFN-alpha Figure 12. Little or no IFN-alpha detected in both neonatal and adult plasmacytoid dendritic cells (pDCs) stimulated with cytoplasmic pattern recognition receptor ligands. Both stimulated adult and neonatal pDCs showed similar levels of IFN-alpha produced. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848 and LPS, and Lyovec for all the cytoplasmic pattern recognition receptors). n=5 for both groups and the data represents mean values with standard error bars.          62  R848LPS5'ppp-dsRNAPoly(I:C)3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)-20020406080100AdultCord% positive IL-6 R848LPS5'ppp-dsRNAPoly(I:C)3'3'-cGAMP2'3'-cGAMPPoly(dA:dT)020406080AdultCord% positive TNF-alpha Figure 13. Similar amounts of IL-6 (a) and TNF-alpha (b) were produced in adult and cord monocytes and conventional dendritic cells (cDCs), respectively, stimulated with R848 and LPS. Adult and cord monocytes and cDCs showeed no cytokine production when stimulated with cytoplasmic pattern recognition receptor ligands. The data depicted shows valued subtracted from the medium (RPMI for R848 and LPS, and Lyovec for all cytoplasmic pattern recognition receptors). n=5 was used for each group and the data shows mean values with standard error bars.     63  0 1 2 30204060LPSLPS + LyovecBFA added  (Hrs)% positive IL-6 Figure 14. Similar levels of IL-6 were produced in adult plasmacytoid dendritic cells (pDCs) stimulated with both LPS and Lyovec or LPS alone. The x-axis represents the time when Brefeldin A (BFA) was added to the whole blood assay for a total 6hr incubation. The data shows the percentage of IL-6 produced when BFA was added to the assay at a later time. n=1 (adult).         64   Figure 15. Significantly higher IFNalpha was produced by cord blood stimulated with MDA5 and STING ligands compared to adult blood. No significant difference was observed in neonatal and adult blood stimulated with R848 or RIG-I ligand. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848, and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).         65   Figure 16. Significantly higher IP-10 levels were produced by neonatal cells stimulated with MDA5 and STING ligands compared to adult cells. No difference in IP-10 production for adult and neonatal whole blood stimulated with RIG-I ligand and R848. The data shown depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).       66    Figure 17. Significantly higher MCP-1 was produced by neonatal whole blood stimulated with R848 and cytoplasmic pattern recognition receptors (except 3'3'-cGAMP). The data shown in (a) depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). Unsubtracted values are shown in (b) to show the high background MCP-1 production by whole blood stimulated with Lyovec alone. The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).    67    Figure 18. Neonatal whole blood produced higher IL-8 when stimulated with MDA5, RIG-I and STING ligands compared to adult whole blood. The data shown in (a) depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor (cPRR) ligands). Unsubtracted values are shown in (b) to show the high background IL-8 production by whole blood stimulated with Lyovec alone. Lyovec-induced IL-8 production was higher compared to cPRR-stimulated adult whole blood. The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).    68   Figure 19. Adult conventional dendritic cells (cDCs) produced significantly higher percentage of TNF-alpha compared to neonatal cDCs stimulated with R848. Both adult and neonatal cDCs stimulated with cytoplasmic pattern recognition receptors showed little or no TNFalpha production. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).         69   R848Poly(I:C)+L5'ppp-dsRNA+L3'3'-cGAMP+L2'3'-cGAMP+LPoly(dA:dT)+L01020304050AdultCord% positive IL-6 Figure 20. Similar percentages of IL-6 produced in adult and neonatal (a) conventional dendritic cells (cDCs) and (b) monocytes both stimulated with R848. In contrast, no IL-6 was produced by adult or neonatal cDCs or monocytes stimulated with cytoplasmic pattern recognition receptors. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).    70   Figure 21. Adult monocytes stimulated with R848 produced significantly higher IL-12 compared to neonatal monocytes. Little or no IL-12 was produced by both adult and cord monocytes stimulated with cytoplasmic pattern recognition receptors. The data shown in depicts subtracted values from the medium used for the ligands (RPMI for R848 and Lyovec for cytoplasmic pattern recognition receptor ligands). The dashes among each scatter bar plot represent median values for n=17 for each group. Mann-Whitney U tests were performed and asterisks indicate statistical difference (*, p<0.05; **, p<0.01; ***, p<0.001).         71   Figure 22. Adult plasmacytoid dendritic cells (pDCs) produced significantly higher IFN-alpha when stimulated with R848 compared to cord pDCs. 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