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Investigation of IL-1-beta response and caspase-1 activity in preterm neonates Jen, Roger 2012

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INVESTIGATION OF IL-1-BETA RESPONSE AND CASPASE-1 ACTIVITY IN PRETERM NEONATES by Roger Jen B.Sc., The University of British Columbia, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2012  © Roger Jen, 2012  Abstract One of the body’s most vital functions lies in its ability to fight off invasions from microbes. Preterm neonates born less than 32 weeks of gestation represent the highest-risk patient group in terms of morbidity and mortality resulting from infections. However, data about the function of the immune system of preterm infants is deeply lacking. The interleukin-1β pro-inflammatory cytokine is a powerful pyrogen and inflammatory mediator implicated in several preterm diseases. IL-1β is produced as a precursor protein (pro-IL-1β) following the trigger of Toll-like receptors and secreted upon cleavage by Caspase-1 within the multiprotein NALP3 inflammasome complex. We sought to investigate early life developmental regulation mechanisms by which the production and secretion of IL-1β is limited before term of gestation. Our data show that preterm neonatal cord blood (CD14+) monocytes are substantially impaired in their ability to secrete IL-1β upon stimulation of Toll-like receptors using lipopolysaccharide. Using flow cytometry, we confirmed sufficient accumulation of the intracellular pro-IL-1 precursor protein in preterm cord blood monocytes. However, caspase-1 activity was markedly decreased in infants born before 29 weeks of gestation and was undetectable in some preterms born at the beginning of the second trimester (24+ weeks), leading to an impaired cleavage and secretion of pro-IL-1. Our data reveal a major mechanism responsible for the attenuation of inflammation before the term of gestation. This developmentally-regulated inhibition of Caspase-1 activity may be an important mechanism to prevent potentially harmful excessive inflammatory responses in early life.  ii  Preface The following study was reviewed and approved prior by the UBC Children’s and Women’s Research Ethics Board. Certificate number: H07-02681  iii  Table of Contents Abstract ......................................................................................................................................................ii Preface ...................................................................................................................................................... iii Table of Contents ...................................................................................................................................... iv List of Tables.............................................................................................................................................. vi List of Figures ........................................................................................................................................... vii List of Abbreviations ................................................................................................................................. ix Acknowledgments ...................................................................................................................................... x Chapter 1: Introduction ............................................................................................................................ 1 1.1. Introduction: why study the neonatal immune system? .............................................................. 1 1.2. The fetal, neonatal and early infancy development of the immune system ................................ 2 1.2.1. Hematopoiesis in fetal life ...................................................................................................... 2 1.2.3. The adaptive immune system in neonates ............................................................................. 5 1.2.4. The innate immune system in neonates ................................................................................. 6 1.2.5. Monocyte/Macrophages ........................................................................................................ 6 1.3. Interleukin 1 .................................................................................................................................. 7 1.3.1. Discovery of IL-1 cytokines ...................................................................................................... 7 1.3.2. IL-1β pathway in monocytes and macrophages ..................................................................... 8 1.4. The inflammasomes .................................................................................................................... 11 1.5. Methods and limitation of studying neonate immunity ............................................................. 13 1.6. Rationale and hypothesis ............................................................................................................ 14 Chapter 2: Results ................................................................................................................................... 16 2.1. Preterm neonates CMBCs produce significantly lower amount of IL-1β compared to adult PBMCs.................................................................................................................................................. 16 2.2. The lack of IL-1β secretion in preterm neonates, compared to adults, is not restored by costimulation with ATP ........................................................................................................................... 17 2.3. Monocytes are the main source of IL-1β in preterm infants ...................................................... 18 2.4. Reduced monocyte-IL-1β production is responsible for the low amounts of IL-1β production in preterm infants ................................................................................................................................... 19 2.5. Preterm CD14+ monocytes produce adequate levels of intracellular pro-IL-1β/IL-1β compared to adults ............................................................................................................................................... 19 2.6. A new fluorescent labelled inhibitor of caspases (FLICA) flow cytometry assay capable of quantifying inflammasome activity in CD14+ monocytes ................................................................... 20 2.7. Preterm neonates display reduced caspase-1 activity compared to adults ............................... 21 Chapter 3: Discussion .............................................................................................................................. 34 iv  Chapter 4: Future Directions................................................................................................................... 41 Chapter 5: Concluding Remarks .............................................................................................................. 46 Chapter 6: Materials and Methods ......................................................................................................... 47 6.1. Blood sample collection .............................................................................................................. 47 6.2. Peripheral/cord blood mononuclear cell extraction ................................................................... 47 6.3. Incubation and stimulation conditions ....................................................................................... 48 6.3.1. Culture conditions ................................................................................................................. 48 6.3.2. Ligands/Stimulants ............................................................................................................... 48 6.3.3. Harvesting of supernatant and cells ..................................................................................... 48 6.4. Staining and flow cytometry ....................................................................................................... 49 6.4.1. Preparing cells for surface staining....................................................................................... 49 6.4.2. Surface staining .................................................................................................................... 49 6.4.3. Intracellular staining ............................................................................................................. 49 6.4.4. Fluorescent labelled inhibitors of caspases (FLICA) staining ................................................ 50 6.5. ELISA ............................................................................................................................................ 51 6.6. Monocyte enrichment ................................................................................................................. 51 6.7. Western blots .............................................................................................................................. 51 6.8. IL-1β quantitative polymerase chain reaction ............................................................................ 53 6.8.1. Preparation ........................................................................................................................... 53 6.8.2. RNA extraction and isolation and conversion into cDNA...................................................... 53 6.8.3. Quantitative polymerase chain reaction .............................................................................. 54 6.9. Statistical analysis ........................................................................................................................ 55 References............................................................................................................................................... 58 Appendices .............................................................................................................................................. 70  v  List of Tables Table 3.1.1. List of surface markers used in identifying IL-1β positive population ........................... 37 Table 6.4.1. List of the concentrations of antibodies used to stain cells (surface stain). .................. 49 Table 6.4.2. List of the concentration of antibody used to stain cells (intracellular stain). ............... 50 Table 6.4.2. List of the western blot antibodies................................................................................. 52 Table 6.8.1. List of primer sequences made and credited makers. ................................................... 54 Table A.1. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for IL-1β ELISA supernatant readings for Figure 2.1.1. ......................................................... 70 Table A.2. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for IL-1β ELISA supernatant readings for Figure 2.1.2. ......................................................... 71 Table A.3. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for IL-1β ELISA supernatant readings for Figure 2.2.1. ......................................................... 72 Table A.4. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for IL-1β ELISA supernatant readings for Figure 2.2.2. ......................................................... 73 Table A.5. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for IL-1β ELISA supernatant readings for Figure 2.4.2. ......................................................... 74 Table A.6. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for monocytes percentage for Figure 2.4.1........................................................................... 75 Table A.7. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for intracellular IL-1β MFI readings for Figure 2.5.1. ............................................................ 76 Table A.8. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s posthoc test for intracellular IL-1β MFI readings for Figure 2.5.2. ............................................................ 77 Table A.9. T-values obtained and significance determined using Mann Whitney test for Figure 2.7.1. ............................................................................................................................................................. 78 Table A.10. T-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for Caspase-1 activity comparison for Figure 2.5.2. ..................................................... 78 Table A.11. Monocytic populations were enriched from PBMCs and CBMCs. Graph reveals that the average monocyte percentage are approximately the same amongst the 3 test groups.................. 79  vi  List of Figures Figure 1.2.1. A visual breakdown of the cellular components in blood of preterm infants, term infants, and adults.1.2.2. The Developing Neonatal Immune System ................................................. 4 1.2.2. The Developing Neonatal Immune System ................................................................................ 5 Figure 2.1.1. CBMCs from preterm infants have significantly lower levels of IL-1β in supernatants compared to adult PBMCs when stimulated for 5 hours with LPS. .................................................... 22 Figure 2.1.2. Levels of supernatant IL-1β in 24-hour LPS-stimulated PBMCs and CBMCs................. 23 Figure 2.2.1. Levels of supernatant IL-1β in 5-hour LPS-stimulated and 1-hour ATP-stimulated PBMCs and CBMCs. ............................................................................................................................. 24 Figure 2.2.2. Levels of supernatant IL-1β in 24-hour LPS-stimulated and 1-hour ATP-stimulated PBMCs and CBMCs.. ............................................................................................................................ 25 Figure 2.3.1. Representative sample displaying positive staining of defining monocyte markers on IL-1β+ cells............................................................................................................................................ 26 Figure 2.4.1. Preterm infants display similar population percentages of CD14+ monocytes amongst extracted and unstimulated CBMCs compared to adult PBMCs term infant CBMCs. ........................ 27 Figure 2.4.2. IL-1β levels in supernatants of LPS and ATP stimulated on enriched (through negative selection) monocytes. ......................................................................................................................... 28 Figure 2.5.1. Intracellular IL-1β expression in CBMCs (term and preterm) or PBMCs (adult) upon LPS stimulation. Intracellular pro-IL-1β was quantified by use of MFI in CD14+ Monocytes. .................. 29 Figure 2.6.1. Activation of caspase-1 following LPS exposure as detected by FLICA analysis. .......... 30 Figure 2.6.2. Caspase-1 activity in CD14+monocytes as analysed using the Amnis flow cytometer. 31 Figure 2.7.1. Lower percentages of FLICA-positive CD14+ monocytes in preterm cord blood. ......... 32 Figure 2.7.2. Caspase-Inflammasome-1 activity in relation to age group/gestational age. .............. 33 Figure 4.1.1. Preliminary findings revealed that IL-1β is not released till approximately 3 to 5 hours after initial incubation with LPS (10 ng/mL) stimulation only. ........................................................... 43 Figure 4.1.2. IL-1β was capable of being released at earlier time-points then was indicated in Fig 3.2.1 with the addition of ATP (5mM) stimulation. ............................................................................ 44 Figure 4.1.3. Preliminary qPCR data of IL-1β copy numbers in adult blood showed that Il-1β mRNA were immediately transcribed within half an hour of LPS stimulation. ............................................. 45 Figure 6.3.1. Experimental layout for Results section 2.1.................................................................. 56 Figure 6.3.2. Experimental layout for Results section 2.2.................................................................. 56 Figure 6.3.3. Experimental layout for Results section 2.3.................................................................. 57 Figure 6.3.4. Experimental layout for Results section 2.5, 2.6, and 2.7. ............................................ 57 Figure A.1. Western blots of Adult PBMCs with 5 hour LPS stimulation and 1 hour ATP stimulation. Each lane represents 100,000 cells worth of proteins. Western blot showing higher presence of pro-IL-1β in adult PBMCs that were stimulated with LPS. Only in the presence of LPS and ATP stimulants do we detect the presence of the 17 kDa mature IL-1β in cell samples. .......................... 80 Figure A.2. Stimulation with various TLR ligands showed lower IL-1β levels in Preterm CBMCs. ..... 81  vii  Figure A.3. Analyses of PBMC and CBMC populations reveal that although there are population differences amongst adult PBMCs and term/preterm CBMCs, the IL-1β producing cells are CD14+ in all 3 representative subjects. .............................................................................................................. 82 Figure A.4. Western blot of pro- and mature IL-1β reveals that mature IL-1β is not released unless cells are stimulated with ATP (caspase-1 activator). .......................................................................... 83  viii  List of Abbreviations AP-1 – Activating Protein-1 ATP – Adenosine Triphosphate CBMC – Cord Blood Mononuclear Cell DAMP – Danger Associated Molecular Pattern ELISA – Enzyme-Linked Immunosorbent Assay FACS – Fluorescent Activated Cell Sorting FLICA – Fluorescent Labelled-Inhibitors of Caspases IL – Interleukin LPS – Lipopolysaccharide MyD88 – Myeloid-Differentiation-Primary-Response Gene 88 NALP – NACHT Leucine-Rich Domain and Pyrin-containing Protein NF-κB – Nuclear Factor Kappa B NLR – NOD-Like Receptor NOD – Nucleotide Oligomerization Domain PAMP – Pathogen-Associated Molecular Pattern PBMC – Peripheral Blood Mononuclear Cell PBS – Phosphate Buffer Saline PRR – Pathogen Recognition Receptor TLR – Toll-like Receptor TNF-α – Tumor Necrosis Factor α  ix  Acknowledgments First and foremost, my gratitude and a big thank you to Dr. Pascal Lavoie for his continuingguidance and support during these past 2 years. Without him, I would not have had the opportunity to pursue a Master’s degree on the fascinating subject of the neonatal immune system. As well, to be able to push me to finish a Master’s thesis that I am proud of in two years must not have been an easy feat. Once again, thank you! I would like to thank Ashish Sharma, my laboratory partner-in-crime, for many things. First, thank you for introducing me to Dr. Lavoie’s lab and helping me find this position. Secondly, thank you for all the times you helped me with my experiments, troubleshot, and brainstormed for ideas. And most of all, thank you for being an awesome friend during these past two years. Thank you Mihoko Ladd, the lab manager, for always keeping our workplace in perfect order and always finding time to help me with my experiments. Many of these experiments would not have been possible without your help. I would like to acknowledge the various Professors and their laboratory members that have aided me throughout my study: Drs. David Speert, Laura Sly, Stuart Turvey, Tobias Kollmann, Rusung Tan, and Hélène Côté. Lastly, I would like to thank my family and friends who have helped me through my study. You know who you are. Research was funded by the Child and Family Research Institute, Sick Kids Foundation, BC Children’s Hospital, and the Canadian Institutes of Health Research. Main Contributors: Dr. Pascal Lavoie and lab – Guidance Professor: Assisted greatly with analysis and strategizing of project direction and experimental designs. Mihoko Ladd – Lab Technician: A certain portion of the ELISAs and blood kinetics study was performed with her aid. Overall, she helped in all experiments performed. Ashish Sharma – Graduate Student: Assisted greatly with flow cytometry techniques and preterm infant recruitment as well as experimental designs and analysis. Dr. Laura Sly and lab – Committee member: Aided with the Western blot techniques. Dr. Stuart Turvey and lab – Committee member: Special mentions to Anthony Tang as the main individual who introduced me to the usage of the FLICA stain. Dr. David Speert and lab – The Professor who sparked my interest in the study of the caspase-1 /inflammasome pathway. Initial protocols for LPS and ATP stimulations were obtained from his laboratory. Dr. Hélène Côté and lab – Collaborator: Special mentions to Izabella Gadawska for teaching and assisting me with the preliminary studies with quantitative PCR. Dr. Tobias Kollmann – Committee Chair. Dr. Rusung Tan – External Committee member. Special mentions to Lisa Xu, our Flow cytometry technician x  To my parents, brother, and grandparents.  xi  Chapter 1: Introduction 1.1. Introduction: why study the neonatal immune system? Understanding the body and the functions underlying human health has been a constant effort by many researchers. For decades, a large majority of research has been performed on fullydeveloped adult populations, furthering our knowledge of the human anatomy and physiology. However, with recent evidence, the journey to understanding the human body has begun to deviate from the general populace of fully developed adults, to the polar extremes of the age spectrum: babies and elders. In particular, the study of infants and the growth and development of their systems provides insight into how our bodies work. One of the areas of mounting interest is the fetal and neonatal immune system. Neonates are undoubtedly one of the highest risk age groups for mortality and morbidity from infections. According to the World Health Organization (WHO), over 4,000,000 children less than five years of age die of infectious causes each year worldwide – 40% of these children are newborns.1 Neonates born prematurely (<37 weeks of gestation), especially extreme preterm infants born before 28 weeks of gestation, are at even higher risk. Over 30,000 neonates are born extremely premature each year in North America. Nearly one-third of these neonates develop an invasive bacterial or fungal infection (i.e. bacteremia, pneumonia or meningitis) in their first weeks of life. 2,3,4,5 This means that one extreme preterm infant suffers a serious infection each hour, leading to over 8,500 infants with serious invasive infections each year. The proportion of infants born prematurely has increased over the last few decades due to medical advances and modern socio-cultural changes.6 A recent study in the United Kingdom has revealed an alarming trend: unlike respiratory distress-related mortalities, which have shown a  1  constant decline, the mortality from infection in newborns has increased over the last 20 years. 7 Medical consequences of these complications impose a considerable pressure on health care resources, adding an estimated 10 hospitalization days and approximately $25,000 dollars per episode of infection/infant, out of the multibillion dollar annual economic impact of prematurity in modern societies.8,9 Unfortunately, neonatal infection often also causes irreversible damage to preterm infants’ organs (like lungs, brain, and intestines) making it one of the main contributors towards long-term neurodevelopmental impairment in this age group.10 Often times, the damage suffered from the disease is the result of uncontrolled inflammation, rather than infection itself. Although there have been studies that shown preterm and term infants produce a reduced inflammatory response, recent clinical studies have revealed that diseased preterm infants produce large amounts of IL-1, IL-6, and TNF-α in infected and diseased tissues.11,12 In fact, researchers are beginning to correlate inflammation with clinical outcome of diseases in preterm infants, but the research knowledge remains scarce.11,12 Overall, the medical consequences of infection in preterm newborns are broad and serious, and there is a need to strengthen our research knowledge base in this field to orient the development of novel therapies and treatments.  1.2. The fetal, neonatal and early infancy development of the immune system 1.2.1. Hematopoiesis in fetal life The full gestational period of a baby from conception to birth is approximately 40 weeks or just over 9 months. This time period is separated into three trimesters that marks the development of the embryo or fetus. The first trimester, lasting about 13-14 weeks, involves embryogenesis  2  followed by early fetal development. Into the second trimester, the fetal development stage lasts for the remainder of the pregnancy; the third trimester begins at 26-27 weeks.13 The development of the fetal immune system begins with hematopoiesis during pregnancy. Hematopoiesis, the formation of cellular components of the blood, in an embryo and fetus is grossly different from the process that occurs in an adult. The first disparity is the location of the process, which transitions throughout the pregnancy. During early embryogenesis, the first formation of blood cells originates from the yolk sac as early as 30 days into pregnancy. The yolk sac, in mammalian development, eventually disappears and hematopoietic stem cells (HSCs) originating from the aortagonad-mesonephros (AGM) region of the embryonic mesoderm begins to replace hematopoietic functions at approximately 6 weeks of gestation. From the AGM, it has been found that the HSCs will migrate to the fetal liver and the spleen, allowing the organs to take over erythropoiesis and myelopoiesis.14 It is important to note that the majority of myeloid lineage cells are formed in the bone marrow in contrast to the red blood cells. Finally, near the end of pregnancy, blood cell formation occurs in bone marrow, the final site of adult homeostatic blood formation.13,15–18 The second distinction is the composition of the blood cells that are formed during different stages. During early fetal development, the majority of the blood is comprised of erythrocytes and granulocyte/monocyte progenitor cells14. By approximately the 25th week of gestation or the beginning of the third trimester, differentiation of subsets becomes more distinct and clear. Figure 1.2.1 displays a comprehensive estimate of cell population makeup in preterm infants, term infants, and adults.19–24  3  Credits: Ashish Sharma, Roger Jen, Pascal Lavoie (unpublished 2012), 2012, by permission.25–35,36–48 Figure 1.2.1. A visual breakdown of the cellular components in blood of preterm infants, term infants, and adults.  4  1.2.2. The developing neonatal immune system The immune system of a full-term newborn baby is different compared to the mature immune system of an adult. Although the functionality of many neonatal immune cell types may arguably appear immature compared to an adult’s, it is likely adapted to major critical transitional phases in early life. In fact, the immune systems of full-term healthy babies are well adapted to suit the barrage of microbes, both pathogenic and ones that may eventually become part of the normal flora, when they come out of the maternal womb. However, there are key differences that can cause a baby to be susceptible to infections and diseases.  1.2.3. The adaptive immune system in neonates Healthy newborns lack an educated adaptive immune system needed for the rapid clearance of several infections.49 The humoral immune system of a neonate is also immature, mainly in its response to T cell independent antigenic reponses.43–47 The majority of the humoral response in infants at birth comes from the pre-transferred maternal Immunoglobulin(Ig)-G antibodies that are shuttled from the placenta to the infants throughout the third trimester of pregnancy. Following birth, Ig-A antibodies can be obtained from the ingestion of breast milk.50–52 This evolutionary mechanism helps compensate for the late development of the Thymus-independent immune response.53 The other component of the adaptive immune system is comprised of a limited repertoire of naïve T cells, lacking antigen specific memory.14 Newborns harbour TH2 responses, skewing away from the possibly harmful but infection-fighting TH1 responses.54,55 A population of cytotoxic leukocytes (CTLs or CD8+ T-cells) are also functionally different in full term newborns.56,57  5  Differences in the adaptive immune system of full-term newborns can partially explain their decreased ability to fight against infections. However, this does not fully explain why premature infants are more susceptible to infections and show higher morbidity rates than full-term babies.  1.2.4. The innate immune system in neonates The major first-line defence that aids in the protection of newborns is the innate immune system. The innate immune system is comprised of cellular and non-cellular components that protect against infections from microorganisms.58 While the non-cellular component seeks to neutralize pathogens upon recognition, cellular immunity provides the major source of communication in our bodies, signalling for the presence of danger and triggering proper pathways to deal with the infection. During the onset of an infection, various receptor proteins located on and in immune cells are able to detect signs of infection and damage in the body. These molecular warning signals are referred to as pathogen associated molecular patterns (PAMPs) while the detection proteins on or in the immune cells are named pathogen recognition receptors (PRRs)59,60. The detection of PAMPs by PRRs causes immune cells to secrete chemical signals called cytokines, which acts as beacons for other cells to respond appropriately to the infection59,60. In contrast to a healthy term baby, these signals are often skewed and regulated differently in premature babies, suggesting possible reasons for their increased susceptibility to infections.61–63  1.2.5. Monocyte/Macrophages Monocytes and macrophages are among the most important of the cellular innate immune system.64 Their importance is evident when looking at the developmental hematopoietic differences in foetuses. During early stages of hematopoiesis, the first cells to be derived are erythrocytes and 6  monocytic progenitors.65–68 Circulating monocytes in the blood eventually give rise to mature tissue specific macrophages. Macrophages are regularly involved in normal body processes. One of these processes is the clearing of apoptotic cells and the recycling of erythrocytes. 69 However, it also assumes the role as one of the most vital inflammatory cells in our body. Monocytes and macrophages detect exogenous danger signals (PAMPs) through Toll-like receptors (TLRs), NOD-like intracellular receptors, and IL-1 receptors (IL-1R).70 These signals transverse into the cell, most through the myeloid-differentiation-factor 88 (MyD88) adaptor molecule, and culminates in the activation of various inflammatory transcription factors, such as NF-κB and AP-1.70 The transcription factors boost the production of various inflammatory cytokines (IL-1, IL-6, TNF-α). A chain reaction occurs following the secretion of the cytokines, as many effector cells become activated. It stands to reason that if issues arise from the stimulation and activation of monocytes, an immune response will not be mounted properly.  1.3. Interleukin 1 1.3.1. Discovery of IL-1 cytokines The history of IL-1 cytokines began during the 1960s. At this point in time, immunologists analysing “factors” from biological fluids discovered a substance that induced various biological activities amongst human blood cells.71 It was found that this substance causes the production of collagenase and prostaglandin E2 from synovial (joint) cells, indicating its role in inflammation and rheumatoid arthritis.71 In 1979, the term interleukin-1 was first coined for this substance and further discoveries were made, including the finding that IL-1 is mainly secreted by monocytes and macrophages.72  7  The IL-1 cytokine is a family of proteins comprised of 11 different variants, named IL-1F1 to IL1F11. Of the 11 variants, IL-1F1 (IL-1α) and IL-1F2 (IL-1β), the first two discovered members of this family, are major mediators of the innate and adaptive immune system. The major role of the two cytokines is to initiate the pro-inflammatory response in the presence of foreign pathogen-associated molecular patterns (PAMPs) from bacteria or viruses and endogenous danger-associated molecular patterns (DAMPs) arising from damaged tissue. Of the two cytokines, IL-1β is arguably the more potent of the two due to its ability to cause systemic effects, while the effects of IL-1α are more autocrine/paracrine in nature.73  1.3.2. IL-1β pathway in monocytes and macrophages Activation Imagine hypothetically that a knife wound was exacerbated by coating it with a layer of dirt. Bacteria and various pathogenic organisms have breached the skin, a physical immune barrier. Endotoxins, specifically lipopolysaccharide (LPS), from Gram-negative bacteria are shed into the tissue surroundings by the bacteria. Monocytes and macrophages are recruited from the bloodstream and nearby tissues and begin detecting and reacting to the LPS through means of the Toll-like receptor 4 (TLR4) receptor pathway74. Important receptors and proteins that are integral to the initial interaction of LPS by monocytes and macrophages include LPS-binding protein (LBP), CD14, Myeloid Differentiation Factor2 (MD-2), and TLR475–78. Although not required, LBP is a soluble protein that enhances the affinity of LPS to TLR479. Its role is thought to consist of extracting LPS from the bacterial membrane and delivering it to CD14 on the surface receptors of monocytes79. With the formation of the LPS-LBPCD14 complex, interaction with soluble secreted glycoprotein MD-2 allows further increased affinity  8  of LPS to TLR480. Despite the fact that MD-2 is a soluble protein, it is often associated extracellularly with the ectodomain of TLR4. The LPS-MD-2-TLR4 forms a dimer with another identical multimer resulting in the activation of the downstream signalling cascade 80.  Signalling The MD-2 undergoes tyrosine phosphorylation by Src-kinase Lyn81. This phosphorylation was shown to significantly increase the activation of NF-κB downstream81. Following the activation of the TLR4 dimer through MD-2 phosphorylation, a signalling cascade involving TIRAP, MyD88, IRAK1/IRAK4, and IKK eventually culminates in the activation of the NF-κB transcription factor80.  Transcription Although the NF-κB transcription factor is the most common route of IL-1 transcription, studies have shown that there are numerous transcription factors that can induce the production of IL-1β82,83,84,85,86. The IL-1β promoter regulatory regions are distributed and span across several thousand basepairs upstream of the gene73. Other promoter elements that has been discovered in the promoter region includes endotoxin-induced Activating protein-1 (AP-1) sites and cAMP response element (CRE)-NFIL-6 sites83. Although there are numerous promoters for the induction of IL-1β gene transcription, there is a dissociation between IL-1β transcription and protein translation73. Stimulants such as hypoxia, complement component C5a, adherence of monocyte and macrophages to surfaces, or the clotting of blood can induce IL-1β gene transcription but not necessarily translation84.  9  Translation Translation of the IL-1β transcript into the propeptide form is dictated mainly by the stability of the mRNA. It has been shown that even a small amount of LPS stimulation is capable of stabilizing the AU-rich 3’ untranslated region of the transcript87. AU-rich regions are sites of instability that allows cellular enzymes to bind and encourage poly-A tail removal on the transcript88. It has also been found that IL-1 can act upon itself on a feedback signalling loop that prevents the deadenylation of its transcripts89. This helps preserve the poly-A tail on the mRNA, allowing it to extend its half-life. The effect of the IL-1 on deadenylation can also be seen on the transcripts of other cytokines, such as IL-8 and gro-α90.  Post-translational modification IL-1β is first made as a 37 kDa propeptide. The pro-IL-1β does not contain a leader signal to tag it for endoplasmic reticulum/Golgi apparatus vesicle secretion91. Instead, pro-IL-1β remains within the cytoplasm of the cell. The maturation of pro-IL-1β is facilitated by caspase-1. Caspase-1 cleaves the pro-IL-1β into its secreted and bioactive 17 kDa form at the Asp116 and Ala117 site92. The mechanism and process behind the secretion of the mature IL-1β peptide is still currently unknown. A few theories involve the shuttling of the mature protein into micro-vesicles which are then exported out of the cell93. Other theories describe the process of pyroptosis, a process in which the activation of caspase-1 causes the death of the cell, resulting in IL-1β being leaked through the porous cellular membrane94.  10  1.4. The inflammasomes The IL-1β protein is strongly regulated at multiple levels. As discussed in the previous section, the IL-1β gene is regulated by numerous transcriptional elements and sites in its promoter region 95. On the other hand, various physiological phenomena such as cellular adherence and blood clotting can induce the production of IL-1β transcripts in monocytes and macrophages95. It was realized that the IL-1β protein is first translated in a 37 kDa pro-peptide form which further undergoes downstream processing to reach its final secreted bioactive 17 kDa form96. This process is mediated by the NALP3 inflammasome97. The inflammasomes are multiprotein oligomers that aid in the activation of downstream caspase 1 and 5 enzymes, ultimately promoting the maturation of IL-1β, IL-18, and IL-3397. The 4 most widely characterized inflammasomes are NALP3, NALP1, IPAF, and AIM298. Largely belonging in the NOD-like receptor (NLR) family of proteins, their role is to act as intracellular detectors of either PAMPs or DAMPs99. The IPAF multimer is directed largely towards the detection of flagellin 100. The AIM2 protein, not part of the NLR family, detects foreign double stranded DNA 101. The NALP3 and NALP1 inflammasome complex are capable of detecting a low intracellular potassium concentration102. Changes in potassium concentration can be induced by the introduction of adenosine triphosphate (ATP) to the extracellular milieu 103. The NALP3 inflammasome differs from the NALP1 inflammasome by its cytoplasmic placement while the latter was found to be localized near or within the nucleus104.  Activation of the NALP3 inflammasome. The detection of ATP is mediated by P2X-purinocepter 7 receptor (P2X7R), an ATP(ligand)gated cation channel105,106. Whether the potassium cation (K+) exits through this channel is still being  11  debated. It has been argued that the actual release of K+ is through the P2X7R activated pannexin-1 channel that forms after the activation of the P2X7R107. Whichever mode of release, the eventual result is the decreased concentration of intracellular K+, signalling for the formation of the NALP3 inflammasome.  12  1.5. Methods and limitation of studying neonate immunity Not surprisingly, the depth and breadth of literature on premature neonatal immunity is very limited. This is largely due to ethical limitations related to the invasiveness of studies involved in vulnerable neonate populations. Unlike adult samples, neonatal cells are derived mainly from umbilical cord blood. The umbilical cord is formed during the fifth week of gestation, replacing the yolk sac that originally provided the nutrients for the embryo. It is comprised of two arteries (deoxygenated blood away from the fetal heart) and a vein (oxygenated blood to the fetal heart) connected to the placenta. Since the blood of the fetus does not mix with the mother’s, cord blood is thought to mimic the peripheral circulating blood of the new-born infant. Cord blood cells are not identical to the cells in peripheral blood, however. The hormones and temperature introduced through child-birth can act upon the blood. Examples include raised levels of serotonin and thromboxane levels as well as increased sensitivity to oxytocin in the tissues of the umbilical cord.109 Studies have shown that phenotypes of monocytes can be altered simply through the mode of delivery 72. It was found that labour and vaginal birth can induce the expression of TLR2/4 on monocytes, while babies delivered via Caesarean-sections showed lower levels of the receptors.110 There are other methods for analysing different cellular and tissue parameters in infants such as amniotic fluid sampling and tracheobronchial aspiarate fluid collection. Amniotic fluids are used as pre-natal measurement samples. Since the fluid is produced essentially by the baby, it can be used to determine the general health of the child. However, fetal cells present in the amniotic fluid are sparse in comparison to the cells that can be extracted from cord blood. Tracheobronchial aspirate fluid (TBAF) is a common biological fluid collected for the measurement of cytokines and metabolites from premature infants. Fetal lungs of premature infants 13  are vulnerable to inflammatory damage and infection due to the use of mechanical ventilators and oxygen-supplemented air as emergent rescue techniques111. The fluid can be used to analyse levels of lung PMNs and incubation of the fluid with ex-vivo immune cells can mimic the response of fetal lung. However, once again, the amount of cells that can be attained for stimulation and experiments is limited. Of the various techniques accessible for use, we felt that analysing mononuclear cells from cord blood would provide the best in vitro model for our study.  1.6. Rationale and hypothesis Overview: Preterm neonates are prone to infections from bacteria, viruses, and fungi. The high infection rate is thought to be due to lower immune-associated cytokine production such as IL-1, IL-6, TNF-α, and IFN-α, amongst others. Although the production of these pro-inflammatory cytokines have been proven to be decreased in preterm neonates in numerous in vitro experiments, clinical results have shown that tissue damage in some diseases are caused through an overt inflammatory response. Therefore, we wish to investigate the two contradicting claims and provide a reason as to why this might be the case. One of the cytokines that was found to be associated with infections, increased tissue damage and preterm labour is IL-1β. Our aims were to investigate if preterm neonates are able to produce IL-1β, and if so, to examine mechanisms that could potentially explain why clinical studies have shown that IL-1β is present in preterm infants with diseases. Hypothesis: Preterm neonatal monocytes produce lower amounts of IL-1β compared to adults or term infants and regulatory mechanisms prevent the production or secretion of IL-1β in preterm infants. 14  The hypothesis will be addressed through the following aims: Aim #1: To test if preterm cord blood mononuclear cells are capable of producing IL-1β when stimulated with lipopolysaccharide and ATP. Aim #2: To determine if regulatory mechanisms (specifically the NALP3 inflammasome) play a role in attenuation of the secretion of IL-1β in preterm infants.  15  Chapter 2: Results 2.1. Preterm neonates CMBCs produce significantly lower amount of IL-1β compared to adult PBMCs Preterm neonates, as discussed in Chapter 1, have an increased susceptibility to infections in parts due to an immature immune system response. As previously seen in our lab, preterm neonates have diminished inflammatory cytokine responses that potentially explain their vulnerability to infections from microorganisms112. Hence, to further the investigation, one potential inflammatory cytokine that was of interest was IL-1β. IL-1β was chosen due to its importance as a systemic cytokine and its unique caspase-1 dependent secretion mechanism113,95,114. Cord-blood mononuclear cells (CBMCs) from preterm neonates less than 32 weeks of gestation and full term neonates (>37 weeks), and peripheral blood mononuclear cells (PBMCs) from adults’ blood were collected and stimulated with lipopolysaccharide (LPS). The range of effective LPS stimulation found in literature ranged from 50pg/mL to 1μg/mL115,116. We chose a mid-range concentration of 10ng/mL and 100ng/mL of LPS as a starting point to experiment on the PBMCs/CBMCs. For the first experiment, 500,000 PBMCs/CBMCs were stimulated for 5 hours and 24 hours with either 10ng/mL or 100ng/mL of LPS or remained unstimulated. Supernatants were collected at the end of the incubation and analyzed using enzyme-linked immunosorbent assays (ELISAs). As seen in Figure 2.1.1, a disparate rift was already seen in the presence of IL-1β in the supernatant of adult PBMCs versus term and preterm CBMCs. Kruskal-Wallis analysis and post-hoc Dunn’s test revealed that the amount of IL-1β produced/secreted by preterm and term CBMCs was significantly lower compared to adult PBMCs. This strongly suggested that preterm infants do have  16  significantly lower abilities to produce or secrete IL-1β. Conversely, although the levels of IL-1β in terms were higher, we were unable to detect a statistical difference likely due to our low sample size (P>0.05). Statistical analyses are detailed in Table A.1. Supernatants analyzed at 24 hours (Figure 2.1.2) revealed that preterm infants still have the lowest concentrations of IL-1β when stimulated with LPS. Although the same trend was observed at 24 hours, the mean level of IL-1β in preterm CBMCS was not significantly different to adult PBMCs (Table A.2) and this may be due to a small sample size and greater signal variability. In previous analysis, we showed that IL-1β levels were consistently lower in preterm CBMCs stimulated with various TLR ligands (Figure A.2). This suggested that the regulation of IL-1β is potentially controlled on a post-translational level. To this end, we decided to investigate the role of caspase-1 pathways using ATP in the secretion of IL-1β.  2.2. The lack of IL-1β secretion in preterm neonates, compared to adults, is not restored by costimulation with ATP The release of IL-1β is controlled by two mechanisms: the rate at which IL-1β is produced and the rate at which it is cleaved and released from the cells96,117,92. The cleaving of IL-1β into its active form is mediated by caspase-1, which is activated by the NALP3 inflammasome91,118. By using adenosine-5'-triphosphate (ATP), we can induce the activation of the NALP3 inflammasome complex in the PBMCs and CBMCs and can therefore expect higher levels of IL-1β secretion when detected in the culture supernatant99,119 (Figure A.1 displays production of pro-IL-1β with LPS and the cleaving of the protein when costimulated with ATP). Given that preterm infants and term infants produced lower amounts of IL-1β in the presence of LPS stimulation, we wondered whether preterm infants  17  may produce sufficient amount of the pro-IL-1β protein to restore high IL-1β levels following addition of ATP. PBMCs and CBMCs were plated and stimulated with 100 or 10 ng/mL of LPS for 5 and 24 hours. Costimulation with 5mM of ATP was added 1 hour prior to the end of stimulation. Addition of ATP caused an increase in IL-1β when cells were stimulated with LPS (Figures 2.2.1 and 2.2.2). Although increased levels of IL-1β were detected in LPS-stimulated preterm CMBCs, these levels were much lower than term neonates and adults at 5 hours of stimulation (P<0.05).  2.3. Monocytes are the main source of IL-1β in preterm infants In the previous sections, our results show that preterm CBMCs stimulated with LPS and ATP for 5 hours showed significantly lower amounts of IL-1β compared to adults. For this reason, we speculated various possibilities as to why we do not see the adult levels of IL-1β production/secretion in preterm CBMCs. Monocytes are the main producers of IL-1β in adult blood.120 Whether this held true when applied to preterm infants was questionable since cell type population composition may differ in early-life. In order to clarify the specific cell population that was responsible for the production of IL1β, we decided to use flow cytometry to analyse specific surface markers associated with monocytes. A selection of myeloid cell markers (CD14, CD33, CD11c, HLA-DR, and CD16) was used to stain and identify PBMCs/CBMCs that were IL-1β positive (Figure 2.3.1). It was determined that a majority of the IL-1β positive population was comprised of CD14+/CD33+/HLA-DR+ cells even in preterm CBMCs. Differences in expression of CD11c and CD16 were not significant. As well, it was shown that no other populations besides CD14+ cells were producing IL-1β (Figure A.3).  18  Therefore, the main producer of IL-1β in preterm CBMCs was verified as CD14+/CD33+/HLADR+ monocytes. CD14+ gating was used in later experiments with CD33 and HLA-DR as safeguard markers to check for correct monocyte population gating in flow cytometry analyses.  2.4. Reduced monocyte-IL-1β production is responsible for the low amounts of IL-1β production in preterm infants Preterm infants have lower counts of monocytes in circulating blood compared to term infants.121 In order to account for this reduction in circulating monocytes, we compared the size of the CD14+/CD33+ cell population between the different age groups. In Figure 2.4.1, results indicated that terms have the largest average percentage of CD14+ cells. Unexpectedly, preterms were found to have a high monocyte population, compared to adults. Next, we performed monocyte enrichment techniques on the samples and subjected them to the same LPS and ATP treatments as before. Once again, preterm samples enriched to 70% monocytes population composition (Table A.11) produced/secreted significantly lower amounts of IL-1β than monocyte-enriched adult and term PBMCs/CBMCs (Figure 2.4.2). Altogether, these data show that the lack of IL-1β found in supernatant of preterm CBMCs is not due to alternate production sources or lack of secreting cells (i.e. monocytes).  2.5. Preterm CD14+ monocytes produce adequate levels of intracellular pro-IL-1β/IL-1β compared to adults So far, we showed that preterm infants’ CBMCs produce/secrete less IL-1β when compared to adults. We also confirmed that the “defect” is intrinsic to monocytes. Our next series of experiments aimed to compare the production of intracellular pro-IL-1β in monocytes of the different age groups. 19  Since IL-1β is not secreted in large amounts unless it is cleaved into its mature form (Figure A.4)122, it is possible to use intracellular stains on LPS-, but not ATP-, stimulated cells to see relative amounts of proIL-1β production in the different subjects. Using two sets of flow cytometry experiments internally calibrated to provide comparable measures of protein using the Mean Fluorescence Intensity, we found that LPS-stimulated preterm CBMCs contain comparable intracellular pro-IL-1β compared to term neonates or adults (Figures 2.5.1). This result is very important as it suggests that the functional difference resulting in the lack of secreted IL-1β in preterm monocytes lies downstream to the production of pro-IL-1β.  2.6. A new fluorescent labelled inhibitor of caspases (FLICA) flow cytometry assay capable of quantifying inflammasome activity in CD14+ monocytes Since it was observed that pro-IL-1β was produced inside CD14+ monocytic cells of premature infants, it was hypothesized that the lack of mature IL-1β was due to a lack of active caspase-1. Immature pro-caspase-1 is cleaved into its active form by the ATP-activated NALP3 inflammasome complex. A new FLICA assay was developed to quantify and examine levels of caspase-1 inside the monocytes. Figure 2.6.1 shows the differences of caspase-1 staining with FLICA in different conditions. It is interesting that the detection of caspase-1 activity followed a bimodal distribution at all age groups, suggesting heterogeneity in the ability of a portion of CD14+ monocytes to activate caspase-1 and secrete IL-1β upon exposure to ATP and LPS. The Amnis ImagestreamX flow cytometer allows not only analysis of cells through quantitative parameters, but also permitted us to examine qualitative fluorescent microscopic pictures. We viewed the monocytes in the samples that were stained positively for the FLICA assay dye. 20  Interestingly, what was found in FLICA positive monocytes were singular bright dots. The bright singularities likely represent a functioning NALP3 inflammasome. Figure 2.6.2 shows examples of preterms infants displaying weak caspase-1 activity (25 and 26 weeker preterms) whereas strong caspase-1 activity was detected in more mature infants as well as adults.  2.7. Preterm neonates display reduced caspase-1 activity compared to adults We used the FLICA assay to analyse the functionality of the NALP3 inflammasome on a per-cell basis. Strikingly, we found that the proportion of CD14+ monocytes displaying positive levels of caspase-1 activity was much lower in preterm infants of lower gestational age at birth. Figure 2.7.1 shows examples of flow microscopy data showing caspase-1 activity across different age groups. When larger numbers of subjects were analyzed, we found that preterm neonates (N=16) displayed significantly less caspase-1 activity in CD14+ monocytes compared to adults (N=18) (Figure 2.7.1). Finally, there was a significant correlation between lower amount of active caspase-1 and gestational age of the preterm infants at birth (Figure 2.7.2).  21  IL-1b (pg/mL)  3000  Adult Term Infant Preterm Infant  *** *  *** *  2000  1000  0 Control  LPS10  LPS100  Conditions Figure 2.1.1. CBMCs from preterm infants have significantly lower levels of IL-1β in supernatants compared to adult PBMCs when stimulated for 5 hours with LPS. IL-1β is markedly reduced in the supernatants of 5 hour LPS stimulated preterm CBMCs compared to adult PBMCs. Significant differences were found between Adult and Preterm (***) for both LPS treatment conditions. Significant differences were also found between Adult and Term Infants (*). N=12 for all Adult, Term, and Preterm. LPS10=LPS 10ng/mL. LPS100=LPS 100ng/mL. See Table A.1 for T-values. Error bars represent standard error mean. Non-detectable levels are represented with N.D.  22  5000  Adult Term Infant Preterm Infant  IL-1b (pg/mL)  4000  3000  2000  1000  0  N.D.  N.D.  Control  N.D.  LPS10  LPS100  LPS Concentration (ng/mL) Figure 2.1.2. Levels of supernatant IL-1β in 24-hour LPS-stimulated PBMCs and CBMCs. IL-1β levels are reduced in the supernatant of 24 hour LPS stimulated preterm CBMCs compared to adult PBMCs. However, differences were found to not be significant using Kruskal-Wallis test. N=6 for all Adult, Term, and Preterm. LPS10=LPS 10ng/mL. LPS100=LPS 100ng/mL. See Table A.2 for T-values. Error bars represent standard error mean. Non-detectable levels are represented with N.D.  23  * 20000  ** Adult Term Infant Preterm Infant  IL-1b (pg/mL)  15000  10000  5000  0  *** *  *** *  LPS10  LPS100  N.D. N.D.  Control  ATP  ATPLPS10  ATPLPS100  Conditions  Figure 2.2.1. Levels of supernatant IL-1β in 5-hour LPS-stimulated and 1-hour ATP-stimulated PBMCs and CBMCs. The addition of ATP promotes the release of IL-1β from PBMCs and CBMCs in all 3 subject groups. Once again, the trend of preterm infants having lower IL-1β levels compared to adults and terms is still present. IL-1β levels from LPS10 and LPS100 stimulated preterm CBMCs were significantly lower than adult PBMCs (***). Preterm supernatant IL-1β levels are also significantly lower than the adult IL-1β supernatant levels with ATP and LPS stimulation as well (ATPLPS10**/ATPLPS100*). N=12 for all Adult, Term, and Preterm. LPS10=LPS 10ng/mL. LPS100=LPS 100ng/mL. ATP=5mM of ATP, added at the 4th hour. See Table A.3 for T-values. Error bars represent standard error mean. Non-detectable levels are represented with N.D.  24  Adult Term Infant Preterm Infant  IL-1b (pg/mL)  15000  10000  5000  0  N.D. N.D. N.D.  Control  N.D.  LPS100  LPS10  ATP  ATPLPS100 ATPLPS10  Conditions Figure 2.2.2. Levels of supernatant IL-1β in 24-hour LPS-stimulated and 1-hour ATP-stimulated PBMCs and CBMCs.. The trend of preterm infants having lower IL-1β levels compared to adults and terms is still present. However, no significant differences were noted for all 3 sample groups. N=6 for all Adult, Term, and Preterm. LPS10=LPS 10ng/mL. LPS100=LPS 100ng/mL. ATP=5mM of ATP, added at the 23rd hour. See Table A.4 for T-values. Error bars represent standard error mean. Non-detectable levels are represented with N.D.  25  Negative IL-1β cell gate  Positive IL-1β cell gate  FITC  APC  PerCP-Cy5.5  eFluor450  PE  PE-Cy7  Figure 2.3.1. Representative sample displaying positive staining of defining monocyte markers on IL-1β+ cells. IL-1β+ cells were stained strongly for CD14, CD33, and HLA-DR while minimal increases were found for CD16 and CD11c markers. Markers for population containing IL-1β cytokine are consistent amongst Adult, Term, and Preterm neonates. 26  8  Percentage (%)  6  4  2  0 Adult  Term  Preterm  Figure 2.4.1. Preterm infants display similar population percentages of CD14+ monocytes amongst extracted and unstimulated CBMCs compared to adult PBMCs term infant CBMCs. Differences are not statistically significant. N=11 for adults and term, N=12 for preterms. See Table A.6 for statistics. Error bars represent standard error mean.  27  50000  Adult Term Preterm  IL-1b (pg/mL)  40000  30000  20000  10000  0  N.D. N.D.  Control  LPS10  ATP  ATPLPS10  Conditions Figure 2.4.2. IL-1β levels in supernatants of LPS and ATP stimulated on enriched (through negative selection) monocytes. PBMCs and CBMCs subjected to monocyte enrichment were stimulated with 10ng/mL of LPS and/or 5mM ATP 5mM for 5 hours and 1 hour respectively. Differences in the capacity to produce and secrete IL-1β into the supernatant are apparent when cells are stimulated with both ATP and LPS. Significance is found between adult and preterm IL-1β levels when treated in the ATPLPS10 condition. N=8 for adults and terms, N=3 for preterms. See Table A.5 for T-values. See Table A.11 for average percentage of monocytes per group. Error bars represent standard error mean. Non-detectable levels are represented with N.D.  28  Mean Fluorescence Index (IL-1B FITC)  1500  Adults Term Preterm  1000  500  Group 2 LPS100  Group 2 LPS10  Group 2 Control  Group 1 LPS100  Group 1 LPS10  Group 1 Control  0  Figure 2.5.1. Intracellular IL-1β expression in CBMCs (term and preterm) or PBMCs (adult) upon LPS stimulation. Intracellular pro-IL-1β was quantified by use of MFI in CD14+ Monocytes. Graph represents two separate sets of experiment. Cells were stimulated with varying doses of LPS for 5 hours. The amount of intracellular IL-1β was detected using intracellular staining. Overall, no significant differences in intracellular IL-1β were found. N=6 for adults, N=5 for terms, and n=8 for preterms for Group 1. N=6 for adults, N=6 for terms, and N=4 for preterms for Group 2. See Table A.7 for statistics. Error bars represent standard error mean .  29  Unstimulated  LPS 10ng/mL  LPS 10ng/mL with ATP 5mM  Inflammasome Positive Sample  Inflammasome Negative Sample  Figure 2.6.1. Activation of caspase-1 following LPS exposure as detected by FLICA analysis. An adult sample representative of an inflammasome-positive subject and a preterm sample representative of an inflammasome-negative subject were selected for this figure.  30  Figure 2.6.2. Caspase-1 activity in CD14+monocytes as analysed using the Amnis flow cytometer. Analysis of caspase-1 activity reveals a bright stained dot (majority of cells display one, but a small percentage display up to 3-4 dots) in each cell. Preterm infant subjects that were FLICA negative lack the presence of the bright dot when cells were analysed. Samples were incubated with 10 ng/mL LPS for 5 hours and stimulated with 5 mM ATP for 1 hour before harvest.  31  Percentage Monocytes FLICA+ (%)  80  60  40  20  0 Adult  Term  Preterm  Age Groups  Figure 2.7.1. Lower percentages of FLICA-positive CD14+ monocytes in preterm cord blood. Preterm infants have lower percentages of monocytes displaying inflammasome activity compared to adults or term infants. Differences between adults and preterm groups were statistically significant (p<0.05). N=18 for adults, N=3 for terms,and N=16 for preterms. See Table A.9 for statistics. Error bars represent standard error mean.  32  80  60  40  24-27 wks  27-29 wks  29-33 wks  0  Term  20  Adult  Caspase-1 activity (% of CD14+cells)  *p<0.05  *  100  Figure 2.7.2. Caspase-Inflammasome-1 activity in relation to age group/gestational age. Caspase-1 activity was calculated by determining the percentage of CD14+ cells that were stained positively for FLICA stain. Significant differences were found between adult levels of caspase-1 and extreme preterms born between 24-29 weeks of gestation. See Table A.10 for statistical analysis.  33  Chapter 3: Discussion In this study, we sought to investigate the innate immune system in preterm neonates. Particularly, our focus was on studying the IL-1β cytokine in preterm infants. Preterm neonates have an immature immune system compared to their adult counterpart. These differences in the innate immune system, arguably the main defence available during early life, are the reasons behind the susceptibility of neonates to life-threatening infections. Our data indicate that IL-1β secretion is impaired in preterm infants born less than 32 weeks of gestational age. The analysis of preterm CBMCs using a combination of ELISAs and flow cytometry has revealed important differences in the post-translational regulation of the IL-1β cytokine early in life. Namely, we show that extreme preterm neonates are capable of producing the IL-1β precursor intracellularly. However, cleavage and secretion of the latter is markedly impaired due to a lack of caspase-1 activity.  Within the IL-1 cytokine family, IL-1α, IL-1β, IL-1 receptor antagonist, and more recently, IL-18 are the best characterized cytokines123. IL-1β is an important systemic cytokine that has pleotropic roles in the human body’s immune system. Unlike other growth factors and cytokines, IL-1β affects nearly every cell type through the IL-1 receptor (IL-1RI). Therefore, one can see how such a cytokine is important to investigate in premature infants. We have shown with our first experiments that preterm infants secreted significantly less IL-1β than term infants and adults when stimulated with varying concentrations of LPS (Figure 2.1.1 and 2.1.2). High concentrations of IL-1β have been found to be deleterious to the growth of embryos and infants124. Similar to preterms, term CBMCs displayed lower supernatant IL-1β levels in our first stimulation experiments. In spite of the lower production of IL-1β in term infants, the group secreted higher average levels of IL-1β than preterm infants. As an important systemic cytokine that can induce various bodily changes, such as fever, angiogenesis, 34  neutrophilia and more, the human body has numerous mechanisms that control the levels of extracellular IL-1 to prevent signalling leading to potentially harmful inflammatory responses123,125. Examples of these mechanisms include the release of the IL-1SRII inhibitor, which binds to IL-1β with 50 times higher affinity than to IL-1α, and IL-1Ra, a competitive inhibitor of IL-1126–132. Given the critical role of IL-1β in the regulation of inflammatory responses, we hypothesize that this lack of IL-1β at the earliest viable times in life represents an evolutionary mechanism that protects the fetus from untoward effects of inflammation.  Although it has been suggested that prolonged exposure to high levels of IL-1β encourages cellular recycling of the cytokine, we believe this is not the case in our experiments since the recycling is mediated by IL-1RII-polymorphonuclear cell (neutrophils) interactions133; these cell types were selected against during the Ficoll-Paque centrifugation step. Hence, the most important mechanism arguably preventing the release of IL-1β intracellularly is the NALP3 inflammasome and caspase-1 machinery, which is in charge of cleaving pro-IL-1β into its mature bioactive form134. The regulation of caspase-1 in fetal/neonatal development has not been investigated previously, to our knowledge. However, it is known that caspase-1 activity in the intra-amniotic fluid is reduced with increasing gestational age and that high caspase-1 activities correlate with intrauterine infections in preterm infants as well as labour in healthy term infants135. In other words, the process of inflammation seems to be closely linked with parturition and term labour.136,137 Unlike term infants, however, caspase-1 levels are more closely correlated with infections in preterm infants135. Altogether, these data suggest that a tight regulation of fetal caspase-1 activity may be required in order to avoid premature labour.  35  The amniotic fluids in preterm infants with chorioamnionitis have high levels of IL-1β and caspase-1 levels. Surprisingly, preterm infants of mothers without intra-uterine infections were found to have the lowest amniotic concentrations of caspase-1, even when compared to the amniotic fluid from mothers of healthy term infants135. We originally hypothesized that supernatant levels of IL-1β in LPS and ATP-stimulated premature CBMCs would be comparable to baseline levels of IL-1β seen in our first experiments. In other words, the presence of ATP would not induce an increase in IL-1β supernatant levels even when preterm CBMCs are costimulated with LPS. In Figures 2.2.1 and 2.2.2, we discovered that, on the contrary, preterm CBMCs were capable of producing significantly higher amounts of IL-1β with only 1 hour of ATP stimulation. Although the number of fold increase was not as dramatic as the increase seen when adult PBMCs were stimulated with ATP, this raised critical questions. Were there cellular factors that affected the secretion of IL-1β from preterm cells? If not, does that mean the preterm CBMCs were capable of producing pro-IL-1β but lacked the machinery to process the cytokine to its mature form?  Our next series of experiments were performed to check if cellular interactions played a role in IL-1β production and if monocytes were the main cells producing IL-1β in CBMCs. Monocyte, macrophages, and dendritic cells are the major producers of IL-1β in the blood138,139. There have been reports that platelets constitute a small population that produces IL-1β. However, this likely does not confound our data since IL-1 produced from platelets (removed by the Ficoll-Paque procedure) is generally bound to the cell surface and not secreted into the supernatant140,141. Using a negative selection kit for monocytes, we enriched for the population in our samples to see if the response to LPS and ATP were similar to what we saw in our previous experiments. Figure 2.3.1 and Figure 2.4.2 reveals that monocytes were the main producers of IL-1β in the adult PBMCs and term 36  CBMCs. Interestingly, the concentration of IL-1β detected in the supernatant of preterm monocytes stimulated with LPS and ATP was much reduced (to insignicant levels) compared to term neonates or adults. To ensure that monocytes were the main source of IL-1β-producing cells in preterm cord blood as well, we used flow cytometry. In literature, population description of myeloid progenitor and monocytes in preterm infants is sparse. The markers that were chosen are markers associated with myeloid and monocytic cells (Table 3.1.1).  Surface Marker  Description of biological relevance  CD11c  Integrin protein, most associated with dendritic cells but also on monocytes, macrophages, and neutrophils.142  HLA-DR  MHC class II cell surface receptor protein, expressed highly in macrophages, B-cells, and dendritic cells.143  CD16  Fc receptor, expressed on monocytes, macrophages, natural killer (NK) cells, and neutrophils.144  CD33  Transmembrane lectin receptor expressed on cells of myeloid lineage.144  CD14  A pattern recognition receptor that works in conjunction with TLR4, expressed on macrophages, monocytes, and dendritic cells.145  Table 3.1.1. List of surface markers used in identifying IL-1β positive population  When we compared the surface markers of IL-1β positive cells of preterm and term CBMCs and adult PBMCs, we found little difference in the surface marker expression amongst the groups. This is highly suggestive that the phenotypically similar subset of cells producing IL-1β in preterms, terms, and adults were monocytes. Given that the population that produced IL-1β were the same amongst the three samples, we wanted to determine if there were any differences in the size of the monocyte population. Our results (Figure 2.4.1) show that both preterm and term cord blood have comparable levels of CD14+ monocytes to adult’s blood. In term infants, monocyte populations are larger in  37  proportion compared to adults, as in our studies146,147. These data reassured us that the differences in IL-1β production between preterm, term neonates and adults are not due to changes in the absolute number of monocytes in mononuclear cells. As to why preterm monocyte enriched populations were incapable of increasing IL-1β (Figure 3.3.1), we theorized two possibilities: either preterm monocytes cannot produce IL-1β alone in the absence of other cell types or the intracellular caspase-1 mechanisms were not functioning.  IL-1β is first formed as an inactive pro-peptide in the cell and requires caspase-1 to convert pro-IL-1β to its bioactive and secreted form. In our first experiments (Figure 2.1.1 and 2.1.2), we noticed that preterm infants do have the capacity to produce pro-IL-1β. In Figure 2.5.1 and 2.5.2 we confirmed that preterm infants were capable of translating significant amounts of pro-IL-1β intracellularly. It may seem counterintuitive but preterm infants are capable of producing inflammatory cytokines when the right stimulation is provided. Previous studies in our lab have shown that responses to certain ligands elicit stronger cytokine responses. For example, preterm CBMCs generated a reduced IL-6 response when stimulated with Pam3CSK (TLR1/2), R-FSL (TLR2/6), and LPS (TLR4). In spite of this, when the same CBMCs were stimulated with 3M-013 (TLR7) and 3M003 (TLR7/8), they produced high levels of IL-6 cytokine, comparable to term infants, in response to the ligands. This suggests that contrary to the popular dogma, preterm infants have a highly regulated and controlled innate immune system. This illustration becomes apparent when we analyse the FLICA expression in preterm monocytes. As seen in our last experiments, there was a decreased amount of monocytes possessing the capabilities to form the NALP3 inflammasome. Therefore, we concluded that the lack of IL-1β in preterms is to a large extent due to lower levels of NALP3 inflammasome complexes within monocytes. 38  Our findings may help explain why preterm infants are so vulnerable to neonatal infection and sepsis. It is widely believed that preterm infants have a reduced capacity to respond to bacterial products such as LPS. Preterm infants develop infections because the body is unable to respond appropriately to the invasions of microbes. However, despite studies showing decreased signalling (Toll-like receptor/NF-κB) and cytokine response in vitro, preterm infants may still be able to produce IL-1β in certain clinical situations. For example, studies in preterm infants reported higher inflammatory cytokine production in peripheral tissues. In necrotizing enterocolitis (NEC), a largely preterm-associated disorder where the lack of blood flow reduces mucous production in the gut, researchers have found extremely high levels of IL-1, IL-6, and TNF-α cytokine production in diseased tissues and blood sera148,149. With such high levels of pro-inflammatory cytokines detected in clinical preterm subjects, some studies have suggested that complications arise not necessarily from the colonization of microbes or the infection of pathogens, but rather from the preterm infant’s inability to resolve the inflammatory response. In fact, researchers have correlated low levels of counterinflammatory cytokines (IL-8, IL-1Ra, and IL-10) with adverse clinical outcomes in infants with NEC150. Other preterm disorders that can be linked with hyperinflammation include encephalitis, meningitis, pneumonia, and blood infections151–153. These diseases confer an enormous burden on the preterm infant through damage caused, not necessarily by invading microorganisms, but by the inflammation that is induced.  In literature, the neonatal system, at least in full-term infants, is often described to be preferentially skewed towards the TH2 response.154,155 This is misleading since the description is more appropriately applied to the immune system of the mother, rather than the infant. Since the notion of the TH2 response implies the assistance of cytokine productions from CD4+ T cells, it is not particularly accurate to apply such a category onto neonates, considering the naivety of their T-cell 39  repertoire. Rather, it is more precise to state that due to a lack of a T H1 response, neonates are preferentially characterized to have a TH2 response. The IL-1 cytokine is considered to be part of the TH1 response. The TH1 response is regarded as a cell-mediated immunity. The main roles behind this response is the activation of various cells (CD8+ cytotoxic T-cells, NK cells, macrophages) that can facilitate the destruction of intracellular pathogens. As such, T H1 responses are often regarded as extremely damaging when compared to the humoral-associated TH2 response. Therefore, for the benefit of the infants, a heavy TH2 maternal response is beneficial for the growth and development of infants before and after birth.154–158 Pro-IL-1β was found to accumulate in the monocytes of premature infants in our study, which is also seen in adult and term monocytes. However, the preterm infants did not have high levels of the NALP3 inflammasome to allow the activation of pro-IL1β. One might think that having an inability to secrete cytokines would be a disadvantageous for the preterm infants. However, on the contrary, we believe that this is a mechanism that serves to protect infants from disease.  40  Chapter 4: Future Directions In future studies, we plan to complete the analyses presented in this thesis on larger sample size, wherever indicated, and investigate the mechanisms underlying the IL-1β/caspase-1/NALP3 inflammasome transcription and translation. Two alternate possibilities may explain why extreme preterm infants lack caspase-1 activity and those are that either the preterm monocytes do not express caspase-1 or other essential components of the NALP3 inflammasome , or caspase-1 fails to activate following assembly of the Inflammasome complex after ATP stimulation. Again, different reasons may explain why caspase-1 fails to activate, including the lack of essential Inflammasome components or the presence of inhibitors (e.g. IL-1R antagonists, proteinase inhibitor 9, caspase-12, pyrin, etc.). So far, our experiments do not allow us to detect the caspase-1 protein pre-activation. However, we have used caspase-1 antibodies in Western blot analyses, in preliminary studies, although the signal obtained from the amount of caspase-1 protein present in preterm neonates’ primary cells is too weak to be interpreted (Roger J et al., unpublished work). Also, we have designed PCR primer pairs to detect expression of IL-1β, caspase-1, and NLRP3 (NALP3 gene) mRNA transcripts. Using the same experimental conditions, we will quantify the expression of these components, as well as other Inflammasome proteins at the mRNA levels using real-time PCR. Preliminary studies are already underway to troubleshoot the different protocols needed to process the samples. Figure 4.1.1 through 4.1.3 display the preliminary data that has been obtained in a kinetics study looking at IL-1. Alongside this study, our project was originally meant as a translational research topic and plan to investigate the effects of the perinatal environment on cord blood responses (e.g. chorioamnionitis, exposure to immunosuppressive drugs such as antenatal corticosteroids), including IL-1 production and caspase-1 activity, how long these innate immune “deficits” persist during  41  neonatal life and how they more directly affect the risk of infection. Finally since my project took a different approach from its original purpose, LPS was the main and only stimulant for my study. It would be interesting to develop an infectious disease model to investigate the more specific role of IL1 in bacterial infections affecting preterm infants. As well, it would be interesting to examine the correlation between other clinical outcomes (e.g. bronchopulmonary dysplasia, necrotizing enterocolitis) and the preterm infants’ ability to produce these major inflammatory cytokines.  42  1000  Adult Term Preterm  IL-1b (pg/mL)  800  600  400  200  0 0 m in  15 m in  30 m in  1 hr  2 hrs  3 hrs  5 hrs  Time  Figure 4.1.1. Preliminary findings revealed that IL-1β is not released till approximately 3 to 5 hours after initial incubation with LPS (10 ng/mL) stimulation only. IL-1β ELISA readings on LPS stimulated whole blood diluted 1:1 with phosphate buffer saline (PBS). N=5 for adults, N=6 for term infants, and N=1 for preterm infants. Error bars represent 1 standard deviation.  43  50000  Adult Term Preterm  IL-1b (pg/mL)  40000  30000  20000  10000  0 0 m in  2 hrs  3 hrs  5 hrs  Time  Figure 4.1.2. IL-1β was capable of being released at earlier time-points then was indicated in Fig 3.2.1 with the addition of ATP (5mM) stimulation. Stimulation was performed in whole blood diluted 1:1 with phosphate buffer saline (PBS). LPS (10 ng/mL) was added at time 0. ATP was added one hour before supernatant was taken (e.g. the 2 hour sample was stimulated with LPS at time 0 and ATP was added at 1 hour). N=5 for adults, N=6 for term infants, and N=1 for preterm infants. Error bars represent 1 standard deviation.  44  Copy number Ratio (Il-1-beta/beta-actin)  8000  Unstimulated LPS 10 ng/mL *  6000 *  4000 *  2000 *  0 0 m in  15 m in 30 m in 1 hr  2 hrs  3 hrs  5 hrs  Time Figure 4.1.3. Preliminary qPCR data of IL-1β copy numbers in adult blood showed that Il-1β mRNA were immediately transcribed within half an hour of LPS stimulation. Data was obtained from one adult. Bars labelled with (*) are calculated using copy number values that were interpolated from within the standard curve of the experiment.  45  Chapter 5: Concluding Remarks In our study, we have demonstrated that preterm infants are capable of producing IL-1β cytokine in its propeptide form. However, we observe a gestational age-dependent reduction in infants’ ability to secrete IL-1β due to reduced caspase-1/NALP3 inflammasome activities. In our opinion, the ‘dysfunction’ of the NALP3 inflammasome pathway serves as a safety gate to prevent a dramatic uncontrolled IL-1β cytokine storm in preterm neonates. For future studies, we hypothesize that although the inflammasome plays an important role in IL-1β secretion, transcription rates might possibly contribute to controlling IL-1β production as well. Future experiments proposed will examine NALP3 inflammasome pathway and the kinetics of IL-1β production through quantitative polymerase chain reaction experiments. The experiments will give us more insight into the specific traits of IL-1β control mechanisms in preterm infants as well as help us better understand neonatal infections and diseases.  46  Chapter 6: Materials and Methods 6.1. Blood sample collection Cord blood samples were obtained with parental consent from BC’s Children Hospital in Vancouver, Canada. Cord blood samples were collected directly from the umbilical cords of the placentas no more than 30 minutes after being disconnected from the neonates. All term (>37-weeks of gestation) samples were from Caesarean-delivered infants. Premature cord blood samples (<32 weeks of gestation) were obtained through the neonatal program from BC’s Children Hospital. Preterm infants were either delivered through natural vaginal or Caesarean birth. Ethical consent was obtained from and after the surgical recovery of the mother. Adult blood samples were obtained from random healthy donors at the Child and Family Research Institute and BC’s Children Hospital. All blood samples were collected in sodium heparin tubes. Ethical consents were obtained from all participants in the study.  6.2. Peripheral/cord blood mononuclear cell extraction Peripheral/cord blood mononuclear cells (PBMCs/CBMCs) were extracted by using Ficoll-Paque (Catalogue # 17-1440-02) density centrifugation technique. Blood from adults were first diluted 1:1 with phosphate buffer saline (PBS). Blood from preterm and term samples were diluted 2:1 with PBS. Diluted blood was layered carefully upon 20 mL of Ficoll-Paque reagent. The samples were spun down at 2200 rpm for 20 minutes in a centrifuge with slow acceleration and no brakes at 23°C. Following separation, PBMCs/CBMCs were carefully obtained and separated from the Ficoll-Paque by suction. The cells were washed and spun down twice with copious amounts of phosphate buffer saline solution (PBS). Cells were then resuspended in cRPMI, filtered through 70μm microfilters to  47  eliminate cell clumps, and counted using crystal violet stain and a haemocytometer. Cells were diluted to a working concentration of 2.5 million cells/mL. In the end, 200μL of cells were used for the experiments in 96 well plates, allowing for 500,000 cells per condition.  6.3. Incubation and stimulation conditions 6.3.1. Culture conditions PBMCs were incubated in GIBCO® RPMI Media 1640 supplemented with 10% human Ab serum and 1 unit/mL of Pen-Strep. This concoction will be referred to as cRPMI from henceforth. Incubations were at 37°C with 5% CO2. 6.3.2. Ligands/Stimulants InvivoGen Ultra Pure E. coli 0111:B4 lipopolysaccharide (Catalogue # tlrl-pelps) was used for the stimulation of TLR4 at varying concentrations. ATP was added one hour before the processing of culture samples to activate the NALP3 inflammasome. One gram of adenosine-5’-triphosphate hydrate (ATP) (Catalogue #150266) from MP Biomedical was reconstituted in 0.1M NaOH solution and titrated with 10M NaOH to a pH of 7.5, aliquoted and stored in -20 degrees Celsius. The ATP was then diluted to 250mM working concentration and final concentration in cell culture medium was 5mM. For experimental layout of specific experiments in the Results, please refer to Figures 4.3.1-4.3.4. 6.3.3. Harvesting of supernatant and cells At the end of an incubation, plates were spun down at 1400 rpm for 5 minutes to pellet cells. 100 uL of supernatant was collected from each condition and stored at -80°C for ELISA studies. Remaining supernatant was aspirated and cells were fixed in 100uL of 1x BD FACS Lysing Solution (Catalogue #349202) and stored in -80°C. 48  6.4. Staining and flow cytometry 6.4.1. Preparing cells for surface staining If prepared fresh from culture, then cells were washed twice with PBS and ready for staining. If cells were saved in BD FACS Lysing Solution, then samples were taken out of -80°C and placed into 37°C incubator for 10 minutes. Immediately after thawing, 150 uL of PBS was gently added to each sample for a first wash. After centrifugation at 1400 rpm for 10 minutes, a second PBS wash was performed. Cells were ready for intracellular and/or surface staining afterwards. 6.4.2. Surface staining Samples were stained in a cold PBS antibody cocktail at a maximum concentration of 20 million cells/mL for 45 minutes. Cells were washed twice at the end of the incubation with 4x staining volume of PBS (e.g. 50 uL stain would need 200 uL of PBS). Cells were re-suspended in 300 uL of PBS if flow cytometry was performed immediately after staining. If flow cytometry was performed at a later time, then cells were resuspended in 2% paraformaldehyde-PBS solution and stored at 4°C. Antibody – conjugate Fluorochrome  Concentration used  Company  Antibody type  Clone  CD14 – PeCy7  1/100  eBioscience  Mouse IgG1,k  61D3  CD33 – PE  1/200  eBioscience  IgG1,k  WM-53  HLA-DR – PerCpCy5.5  1/100  BD Parmingen Custom  IgG2b,k  TU36  CD16 – eFluor450  1/100  eBioscience  IgG1  eBioCB16  CD11c – APC  1/100  eBioscience  IgG1,k  3.9  Table 6.4.1. List of the concentrations of antibodies used to stain cells (surface stain). 6.4.3. Intracellular staining Cells were surface stained first, washed in PBS once and fixed in 2% paraformaldehyde(PFA)-PBS solution. PFA served as a fixing agent to prevent the loss of staining antibodies through the 49  subsequent steps. Cells were permeabilized using BDTM Perm Buffer II, diluted to 2% (originally 10% in original stock) with water, for 10 minutes. After, cells were washed twice using PBS and resuspended in PBS with intracellular antibodies for 40 minutes. At the end of the incubation, cells were washed in PBS, spun down, and resuspended in 300 uL of PBS.  Antibody – conjugate Fluorochrome IL-1β  Concentration used 1/50  Company  Antibody type  Clone  eBioscience  Mouse IgG1  CRM56  Table 6.4.2. List of the concentration of antibody used to stain cells (intracellular stain).  6.4.4. Fluorescent labelled inhibitors of caspases (FLICA) staining The FLICA stain (Abd Serotec) is a unique stain in that it does not utilize antibody tagging to detect active caspase-1. The FLICA stain is a peptide sequence (Z-YVAD) linked to a fluoromethyl ketone (FMK) followed by a conjugation to a fluorochrome (fluorescein isothiocyanate). Z-YVAD (GlutamineTyrosine-Valine-Alanine-Aspartic Acid) is the peptide sequence recognized by the caspase-1 active site. Particularly, the alanine-aspartic acid residue link, the sequence also found on the IL-1β cleavage site, is the target that is cleaved by active caspase-1 in normal circumstances. The fluoromethyl ketone (FMK) acts as an inhibitor by covalently binding and inactivating the enzyme. The FLICA stain was provided in a powder form. When resuspended in 50 uL of DMSO, the initial concentration of FLICA was at 600x stock concentration. We diluted the stain (1:20) to a working concentration of 30x. Ideally, 6.67 uL of FLICA stain would be added to 200 uL of our cell suspension. However, we found that 9 uL of FLICA stain gave a stronger and clearer stain. The FLICA stain was added 1 hour prior to the end of the incubation period. 50  6.5. ELISA ELISAs were performed using and following the protocol suggested by eBioscience’s Ready-Set-Go!® ELISA kit. Samples were prepared at varying dilutions to fit within readouts of the standard dilutions.  6.6. Monocyte enrichment The EasySep® Human Monocyte Enrichment Kit without CD16 Depletion (catalogue#19058) from Stemcell was used to help enrich for monocytes from PBMCs and CBMCs. The process was performed as suggested by the kit (Manual EasySep™ Protocol Using The Purple EasySep™ MAGNET (CATALOG #18000)). The antibody selection cocktail consisted of antibodies against CD2, CD3, CD19, CD20, CD56, CD66b, CD123, glycophorin A and dextran-coated magnetic particles. A few modified adjustments were used to accommodate the usage of the kit with CBMCs. As listed in the protocol, a maximum concentration of 5x107 cells/mL is allowed before the enrichment. Higher concentrations would result in lower purity of enriched cells. In CBMCs, the amount of contaminating nucleated red blood cells (RBCs) is extremely high due to their low density and hence their inability to traverse the Ficoll-Paque separation gradient. In order to compensate for this contamination, red blood cells were counted in the initial cell count to assure that the concentration of CBMCs+RBCs do not surpass 5x10 7 cells/mL. The rest of the protocol was followed as normal afterwards. After enrichment, cells were stained and analysed through the BD LSRII flow cytometer for CD14+ purity. A live population of 70% CD14+ cells was considered a successful enrichment.  6.7. Western blots After stimulation, cells were washed once with PBS to exclude proteins from media. Cells were lysed and proteins deactivated and denatured using 10% w/v SDS PAGE sample buffer with β51  mercaptoethanol. Samples were heated at 95°C for 2 minutes before stored at -80°C. When running samples on gel, 100,000 cells (20uL) per condition were ran on 12.5% SDS PAGE gels for 90 minutes at 120V and wet transferred onto polyvinylidene fluoride membranes (PVDF) for 90 minutes at 90V. PVDF membranes were washed in water after transfer for 1 minute and allowed to air dry until PVDF membranes became opaque. Membranes were placed in 5% Bovine Serum Albumin (BSA) Tris Buffer Saline – Tween (TBS-T) blocking buffer overnight at 4°C. Following blocking, primary antibody stains were used at 1:1000 concentrations for 3 hours. PVDF membranes were washed 3 times for 10 minutes with TBS-T followed by secondary HRP-conjugated antibody incubation for 1 hour (1:10 000). The membranes were subjected to a final 3 times 10 minute TBS-T wash before prepared for enhanced chemiluminescence. Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen catalogue #WP20005) was used on the membranes. Kodak® films were exposed to membranes for 1, 5, 20 and 60 minute intervals.  Antibody – conjugate Fluorochrome  Concentration used  Company  Antibody type  Primary IL-1β Cleaved (Asp116) #2021  1/1,000  Cell Signalling  Rabbit  Primary IL-1β #2022  1/1,000  Cell Signalling  Rabbit  Secondary anti-Rabbit Goat IgG (H+L) Human IgG adsorbed HRP conjugated  1/10,000  BioRad  Goat  Table 6.4.2. List of the western blot antibodies.  52  6.8. IL-1β quantitative polymerase chain reaction 6.8.1. Preparation Blood samples were diluted 1:1 with cRPMI before plating (200uL/well). After stimulation and incubation, blood was frozen immediately at -80°C in eppendorf tubes. 6.8.2. RNA extraction and isolation and conversion into cDNA Blood samples were taken out of freezer and immediately defrosted using 1mL of room temperature PBS. Since red blood cells were lost through the freeze-thaw process, the samples were spun down at 10,000 rpm for 1 minute to collect PBMCs and CBMCs. Supernatant was aspirated and cell pellets were lysed open using 500uL of TRIzol® Reagent. 200 uL of chloroform was added to the TRIzol-lysed cell solution and allowed to rest on ice for 15 minutes. Sample was spun down at 10,000 rpm for 10 minutes at 4°C. After centrifugation, the top aqueous layer where the RNA resided was carefully taken without disturbing the interphase and bottom organic phase. 400 uL of isopropanol was added to the separated aqueous layer and left on ice for 10 minutes to precipitate out the RNA from solution. Glycogen (Invitrogen) was added to help with the visualization of the RNA pellet. Sample was then centrifugated at 10,000 rpm for 15 minutes, at 4°C the isopropanol aspirated, and the cell pellet underwent a 75% ethanol wash (1mL). After a final centrifugation at 10,000 rpm for 15 minutes, the ethanol was aspirated and cell pellet was allowed to air dry for 30 minutes. After the pellet was allowed to dry, 20 uL of RNAse free water was added to resuspend the RNA pellet. The RNA sample was taken through a DNAse treatment step using DNAse I (Invitrogen) kit. The RNA was subjected to the High Capacity cDNA Reverse Transcription Kit (Invitrogen) to convert the RNA sample into cDNA.  53  6.8.3. Quantitative polymerase chain reaction The system of qPCR that we utilized was the SYBR-Green I (Light Cycler 480 SYBR Green I Master Mix) detection approach. Since SYBR-Green binds to double stranded DNA without discrimination, it was important that our PCR primers were of perfect match and high fidelity to our target mRNA transcript sequence (i.e. intron spanning and absence of homologies to other genes or transcripts of interest). Our primers were tested out and confirmed to be specific and suitable. Thermal program was set as follow: 95°C for 10 minutes followed by 95°C for 15 seconds and 60°C for 1 minute for 40 cycles followed by a heat dissociation step to check products. Copy numbers are calculated from standard curve based on max peak second derivative calculations.  Transcript name IL-1β  Primer Sequences (F) 5’ CCT AAA CAG ATG AAG TGC TCC 3’  Created by Roger Jen – P. Lavoie’s Lab  (R) 5’ GGT GCT CAG GTC ATT CTC CTG G 3’ Β-Actin  (F) 5’ TCC TAT GTG GGC GAC GAG G 3’ (R) 5’ GGT GTT GAA GGT CTC AAA CAT G 3’  NLRP3  (F) 5’ ATG CAA GAA TAT GCC TGT TCC TGT G (R) 5’ AGA AGG ATC TCT TCA CTT CCT GCC 3’  Caspase-1  (F) 5’ GAG ATG AGC CGA AGT GGG GTT C 3’ (R) 5’ GAC GGG CAT TCC TGT CTT CAA TGC 3’  Izabella Gadawska – H. Côté’s Lab Anaïs Nedelec and Roger Jen– P. Lavoie’s Lab Anaïs Nedelec and Roger Jen– P. Lavoie’s Lab  Table 6.8.1. List of primer sequences made and credited makers.  54  6.9. Statistical analysis Statistical significances were tested by using Kruskal-Wallis nonparametric statistical analysis with post-hoc Dunn’s test to locate groups that displayed significance. Mann Whitney test was used to analyse the proportions of positive monocytes displaying FLICA positive staining amongst adult, term, and preterm groups. Statistical analysis calculated using GraphPad Prism 5 software.  55  Figure 6.3.1. Experimental layout for Results section 2.1  Figure 6.3.2. Experimental layout for Results section 2.2  56  Figure 6.3.3. Experimental layout for Results section 2.3.  Figure 6.3.4. Experimental layout for Results section 2.5, 2.6, and 2.7.  57  References 1.  PrabhuDas, M. et al. Challenges in infant immunity: implications for responses to infection and vaccines. Nature immunology 12, 189-94 (2011).  2.  Stoll, B.J. & Hansen, N. Infections in VLBW infants: studies from the NICHD neonatal research network. Seminars in Perinatology 27, 293-301 (2003).  3.  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Gaussian Approximation P value summary *** Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 18.60 Dunn's Multiple Comparison Test Adults vs Term Adults vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 12.21 Yes * 18.17 Yes *** 5.958 No ns  LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.0002 Exact or approximate P value? Gaussian Approximation P value summary *** Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 17.11 Dunn's Multiple Comparison Test Adults vs Term Adults vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 10.75 Yes * 17.63 Yes *** 6.875 No ns  Table A.1. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for IL-1β ELISA supernatant readings for Figure 2.1.1.  ----------------------------------------------------------------------------------------------------------------------------------------  LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.3567 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 2.062 Dunn's Multiple Comparison Test Adult vs Term  Difference in rank sum Significant? P < 0.05? Summary 1.440 No ns 70  Adult vs Preterm Term vs Preterm  4.440 3.000  No No  ns ns  LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.5569 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 1.171 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 0.7857 No ns 3.286 No ns 2.500 No ns  Table A.2. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for IL-1β ELISA supernatant readings for Figure 2.1.2.  ----------------------------------------------------------------------------------------------------------------------------------------  LPS 10 ng/mL Condition Kruskal-Wallis test P value < 0.0001 Exact or approximate P value? Gaussian Approximation P value summary *** Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 18.60 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 12.21 Yes * 18.17 Yes *** 5.958 No ns  LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.0002 Exact or approximate P value? Gaussian Approximation P value summary *** Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 17.11 Dunn's Multiple Comparison Test Adult vs Term  Difference in rank sum Significant? P < 0.05? Summary 10.75 Yes * 71  Adult vs Preterm Term vs Preterm  17.63 6.875  Yes No  *** ns  LPS 10 ng/mL and ATP (5mM) Condition Kruskal-Wallis test P value 0.0119 Exact or approximate P value? Gaussian Approximation P value summary * Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 8.855 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 5.042 No ns 12.71 Yes ** 7.667 No ns  LPS 100 ng/mL and ATP (5mM) Condition Kruskal-Wallis test P value 0.0164 Exact or approximate P value? Gaussian Approximation P value summary * Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 8.227 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 5.917 No ns 12.33 Yes * 6.417 No ns  Table A.3. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for IL-1β ELISA supernatant readings for Figure 2.2.1.  ----------------------------------------------------------------------------------------------------------------------------------------  LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.1580 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 3.691 Dunn's Multiple Comparison Test Adult vs Term  Difference in rank sum Significant? P < 0.05? Summary 3.083 No ns 72  Adult vs Preterm Term vs Preterm  5.917 2.833  No No  ns ns  LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.3722 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 1.977 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 2.167 No ns 4.333 No ns 2.167 No ns  LPS 100 ng/mL and ATP (5mM) Condition Kruskal-Wallis test P value 0.2963 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 2.433 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 1.333 No ns 4.667 No ns 3.333 No ns  LPS 10 ng/mL and ATP (5mM) Condition Kruskal-Wallis test P value 0.1345 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 4.012 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 3.333 No ns 6.167 No ns 2.833 No ns  Table A.4. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for IL-1β ELISA supernatant readings for Figure 2.2.2.  73  ----------------------------------------------------------------------------------------------------------------------------------------  LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.7463 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 0.5853 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 1.875 No ns -0.2500 No ns -2.125 No ns  LPS 10 ng/mL and ATP (5mM) Condition Kruskal-Wallis test P value 0.0034 Exact or approximate P value? Gaussian Approximation P value summary ** Do the medians vary signif. (P < 0.05) Yes Number of groups 3 Kruskal-Wallis statistic 11.38 Dunn's Multiple Comparison Test ATPLPS10 vs Term ATPLPS10 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 5.750 No ns 12.38 Yes ** 6.625 No ns  Table A.5. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for IL-1β ELISA supernatant readings for Figure 2.4.2.  ----------------------------------------------------------------------------------------------------------------------------------------  Monocytes Percentage Kruskal-Wallis test P value Exact or approximate P value? P value summary Do the medians vary signif. (P < 0.05) Number of groups Kruskal-Wallis statistic  0.8236 Gaussian Approximation ns No 3 0.3882 74  Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? -1.000 No -2.561 No -1.561 No  Summary ns ns ns  Table A.6. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for monocytes percentage for Figure 2.4.1.  ----------------------------------------------------------------------------------------------------------------------------------------  Group 1 LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.2876 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 2.492 Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary -4.400 No ns 0.3750 No ns 4.775 No ns  Group 1 LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.1706 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 3.537 Dunn's Multiple Comparison Test Group 1 LPS100 vs Term Group 1 LPS100 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary -6.000 No ns -1.000 No ns 5.000 No ns  Group 2 LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.4315 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 75  Kruskal-Wallis statistic Dunn's Multiple Comparison Test Group 2 LPS10 vs Term Group 2 LPS10 vs Preterm Term vs Preterm  1.681 Difference in rank sum Significant? P < 0.05? Summary 3.033 No ns 3.083 No ns 0.0500 No ns  Group 2 LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.7947 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 0.4596 Dunn's Multiple Comparison Test Group 2 LPS100 vs Term Group 2 LPS100 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary -1.833 No ns -0.5833 No ns 1.250 No ns  Table A.7. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for intracellular IL-1β MFI readings for Figure 2.5.1.  ----------------------------------------------------------------------------------------------------------------------------------------  Group 1 LPS10 ng/mL Condition Kruskal-Wallis test P value 0.2373 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 2.877 Dunn's Multiple Comparison Test Group 1 LPS 10 vs Term Group 1 LPS 10 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary -5.433 No ns -0.9583 No ns 4.475 No ns  Group 1 LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.0934 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 76  Kruskal-Wallis statistic Dunn's Multiple Comparison Test Group 1 LPS 100 vs Term Group 1 LPS 100 vs Preterm Term vs Preterm  4.741 Difference in rank sum Significant? P < 0.05? Summary -6.283 No ns 0.1667 No ns 6.450 No ns  Group 2 LPS 10 ng/mL Condition Kruskal-Wallis test P value 0.3361 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 2.181 Dunn's Multiple Comparison Test Group 2 LPS 10 vs Term Group 2 LPS 10 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 3.583 No ns 3.333 No ns -0.2500 No ns  Group 2 LPS 100 ng/mL Condition Kruskal-Wallis test P value 0.6246 Exact or approximate P value? Gaussian Approximation P value summary ns Do the medians vary signif. (P < 0.05) No Number of groups 3 Kruskal-Wallis statistic 0.9412 Dunn's Multiple Comparison Test Group 2 LPS 100 vs Term Group 2 LPS 100 vs Preterm Term vs Preterm  Difference in rank sum Significant? P < 0.05? Summary 0.0000 No ns 2.667 No ns 2.667 No ns  Table A.8. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for intracellular IL-1β MFI readings for Figure 2.5.2.  ----------------------------------------------------------------------------------------------------------------------------------------------------  FLICA positive monocytes Adult and Preterm Comparison Column A Adult vs vs Column C Preterm Mann Whitney test P value  0.0285 77  Exact or approximate P value? Gaussian Approximation P value summary * Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A,C 379 , 216 Mann-Whitney U 80.00 Table A.9. U-values obtained and significance determined using Mann Whitney test for Figure 2.7.1.  ----------------------------------------------------------------------------------------------------------------------------------------------------  Kruskal-Wallis test P value 0.0015 Exact or approximate P value? Gaussian Approximation P value summary ** Do the medians vary signif. (P < 0.05) Yes Number of groups 5 Kruskal-Wallis statistic 17.54 Dunn's Multiple Comparison Test Adult vs Term Adult vs 29-33 wks Adult vs 27-29 wks Adult vs 24-27 wks Term vs 29-33 wks Term vs 27-29 wks Term vs 24-27 wks 29-33 wks vs 27-29 wks 29-33 wks vs 24-27 wks 27-29 wks vs 24-27 wks  Difference in rank sum Significant? P < 0.05? Summary 5.357 No ns -0.1818 No ns 19.69 Yes * 21.82 Yes * -5.538 No ns 14.34 No ns 16.46 No ns 19.88 Yes * 22.00 Yes * 2.125 No ns  Table A.10. H-values obtained and significance determined using Krukal-Wallis test with Dunn’s post-hoc test for Caspase-1 activity comparison for Figure 2.5.2.  78  Percentage Monocytes (%)  100  80  60  40  20  0 Adult  Kruskal-Wallis test P value Exact or approximate P value? P value summary Do the medians vary signif. (P < 0.05) Number of groups Kruskal-Wallis statistic Dunn's Multiple Comparison Test Adult vs Term Adult vs Preterm Term vs Preterm  Term  Preterm  0.0726 Gaussian Approximation ns No 3 5.246 Difference in rank sum Significant? P < 0.05? Summary 6.375 No ns 2.000 No ns -4.375 No ns  Table A.11. Monocytic populations were enriched from PBMCs and CBMCs. Graph reveals that the average monocyte percentage are approximately the same amongst the 3 test groups.  79  Figure A.1. Western blots of Adult PBMCs with 5 hour LPS stimulation and 1 hour ATP stimulation. Each lane represents 100,000 cells worth of proteins. Western blot showing higher presence of proIL-1β in adult PBMCs that were stimulated with LPS. Only in the presence of LPS and ATP stimulants do we detect the presence of the 17 kDa mature IL-1β in cell samples.  80  3000  5000  IL-1beta (pg/mL)  IL-1beta (pg/mL)  Term Preterm 2000  1000  0 0.1  1  10  100  4000  3000  2000  1000  0 0.001 0.01  1000  LPS (ng/mL)  15000  IL-1beta (pg/mL)  IL-1beta (pg/mL)  Term Preterm  1000  0.1  1  0.1  1  10  100  PAM (ug/ml)  2000  0 0.01  Term Preterm  10  R-FSL (ug/ml)  100  Term Preterm  10000  5000  0 0.01  0.1  1  10  100  3M-003 (uM)  Figure A.2. Stimulation with various TLR ligands showed lower IL-1β levels in Preterm CBMCs.  81  SSC vs FSC  IL-1β Histogram  SSC vs CD14  Adult 2  3  1 – Black 2 – Grey 3 – White  3  1 – Black 2 – Grey 3 – White  3  1 – Black 2 – Grey 3 – White  1  Term 2  1  Preterm 2  1  Figure A.3. Analyses of PBMC and CBMC populations reveal that although there are population differences amongst adult PBMCs and term/preterm CBMCs, the IL-1β producing cells are CD14+ in all 3 representative subjects. CD14- populations, including a suspected myeloid progenitor population (population #2), do not produce IL-1β when stimulated with LPS. Mean fluorescence intensity reading of IL-1β FITC of population #1 is comparable to fluorescence minus one (FMO) control. 82  Figure A.4. Western blot of pro- and mature IL-1β reveals that mature IL-1β is not released unless cells are stimulated with ATP (caspase-1 activator).  83  

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