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The role of microRNAs in the early life human innate immune response Cho, Patricia 2013

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THE ROLE OF MICRORNAS IN THE EARLY LIFE HUMAN INNATE IMMUNE RESPONSE by Patricia Cho B.Sc., The University of British Columbia, 2010  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) June 2013  © Patricia Cho, 2013  Abstract Neonates are more susceptible to infections than adults which suggests a suboptimal host immune defense. The innate immune response is the first line of defense against pathogens and must be tightly regulated. Recently, microRNAs (miRNAs) have emerged as important regulators of many biological processes. In the innate immune response, miRNAs positively and negatively regulate signalling to promote or dampen the immune response. The role of miRNAs in the development of the innate immune response, particularly in neonatal host defense, is not well understood. In this thesis work, we comprehensively profiled miRNAs in order to decipher a potential role for miRNAs in innate immune ontogeny. To this end, we contrasted miRNA expression in unstimulated versus stimulated monocytes from adult versus cord blood (CB) donors. Six immune responsive miRNAs were selected as candidates for additional studies, three of which were novel miRNAs that have not been previously investigated in innate immunity. Over-expression of these candidates in human monocytes resulted in significant changes in cytokine and chemokine production. While this verified their functional regulatory role in innate immunity, none of these cytokines/chemokines were predicted to be regulated by the selected miRNAs leaving the intermediate targets to be determined. These six candidate miRNAs were further characterized for expression kinetics. In adults, miRNA expression was rapidly induced within the first 8 hours of TLR stimulation and returned to near basal levels by 24 hours post stimulation. In CB monocytes however, miRNA expression was only observed to gradually increase, if at all, over 24 hours and expression levels never matched that observed in adult monocytes. These age-dependent differences in miRNA expression kinetics may relate to an overall  ii  inability in CB monocytes to process miRNAs into the biologically functional form. In summary, my work has shown that there are clear differences in miRNA expression between adult and CB monocytes, and this may be a result of deficiencies in the miRNA biogenesis machinery or a delay in expression in CB monocytes. Further work can now proceed to determine the underlying molecular mechanism(s) and functional implication(s) of these striking age-dependent differences in miRNA expression in innate immunity.  iii  Preface This thesis is original, unpublished work by the author, P. Cho. Studies which included the use of human primary cells were approved by the University of British Columbia Children’s and Women’s Research Ethics Board (Certificate Number H06-03339). Data acquisition by RNA-Seq in Chapter 3.1 was collected in collaboration with the laboratory of Dr. Stephen Smale at the University of California, Los Angeles.  iv  Table of Contents Abstract.............................................................................................................................. ii! Preface............................................................................................................................... iv! Table of Contents .............................................................................................................. v! List of Tables ................................................................................................................... vii! List of Figures................................................................................................................. viii! List of Abbreviations ....................................................................................................... ix! Acknowledgements .......................................................................................................... xi! 1 Introduction.................................................................................................................... 1! 2 Materials and Methods................................................................................................ 13! 2.1 Isolation of purified primary monocytes and in vitro stimulation .......................... 13! 2.2 RNA-Seq profiling and analysis ............................................................................. 13! 2.3 miScriptTM miRNA PCR array and quantitation .................................................... 14! 2.4 miRNA target prediction by miRWalk ................................................................... 15! 2.5 miRNA over-expression in THP-1 cells ................................................................. 15! 2.6 Luminex assay and analysis.................................................................................... 16! 2.7 Quantitative real-time PCR..................................................................................... 17! 2.8 Statistical analysis................................................................................................... 18! 3 Results ........................................................................................................................... 19! 3.1 Five candidate pri-miRNAs of interest were identified by RNA-Seq .................... 19! 3.1.1 Fifty-nine pri-miRNAs were detected in unstimulated adult monocytes and 57 pri-miRNAs were detected in unstimulated CB monocytes ............................ 20! 3.1.2 Eight of the upregulated pri-miRNAs and 5 of the downregulated pri-miRNAs in adult monocytes stimulated with LPS for 1 hour remained up- or downregulated after 6 hours ............................................................................. 22! 3.1.3 Seventeen of the upregulated pri-miRNAs and 10 of the downregulated primiRNAs in CB monocytes stimulated for 1 hour with LPS remained up- or downregulated after 6 hours ............................................................................. 26! 3.1.4 Pri-miR-611, -632, -922, -147b and -155 were identified by RNA-Seq as candidates of interest for further studies .......................................................... 29! 3.2 Mature miR-222 was identified as a candidate of interest by qPCR ...................... 35! 3.2.1 Two hundred sixteen mature miRNAs were detected in unstimulated adult monocytes and 294 mature miRNAs were detected in unstimulated CB monocytes......................................................................................................... 35! 3.2.2 Two mature miRNAs were upregulated and 3 mature miRNAs were downregulated by overnight R848 stimulation in both adult and CB monocytes .......................................................................................................................... 38! 3.2.3 Two miRNAs were upregulated and three miRNAs were downregulated by overnight LPS stimulation in both adult and CB monocytes ........................... 45! 3.2.4 One of the statistically significant, TLR-responsive mature miRNAs (miR222-3p) was selected as a candidate miRNA ................................................... 51! v  3.3 Candidate mature miRNA expression kinetics in CB monocytes was dissimilar to that in adult monocytes............................................................................................ 54! 3.4 Over-expression of candidate miRNAs changed cytokine and chemokine production in response to TLR stimulation ............................................................. 56! 3.4.1 Transfection of miRNA mimics into monocytic cells is not toxic .................. 58! 3.4.2 CCL2, CCL3, CCL4, IL-8 and TNF-! expression were affected by the overexpression of candidate miRNAs ..................................................................... 59! 4 Discussion ..................................................................................................................... 62! 5 Conclusion .................................................................................................................... 83! References........................................................................................................................ 85! Appendices....................................................................................................................... 93! Appendix A: Pri-miRNAs detected in unstimulated adult monocytes ......................... 93! Appendix B: Pri-miRNAs detected in unstimulated CB monocytes ............................ 94! Appendix C: Pri-miRNAs detected in adult monocytes stimulated with LPS for 1 hour ................................................................................................................................. 95! Appendix D: Pri-miRNAs detected in adult monocytes stimulated with LPS for 6 hours ................................................................................................................................. 96! Appendix E: Pri-miRNAs detected in CB monocytes stimulated with LPS for 1 hour 97! Appendix F: Pri-miRNAs detected in CB monocytes stimulated with LPS for 6 hours ................................................................................................................................. 98! Appendix G: Mature miRNAs detected in unstimulated adult monocytes................... 99! Appendix H: Mature miRNAs detected in unstimulated CB monocytes ................... 101! Appendix I: Mature miRNAs detected in R848-stimulated adult monocytes ............ 104! Appendix J: Mature miRNAs detected in R848-stimulated CB monocytes............... 107! Appendix K: Mature miRNAs detected in LPS-stimulated adult monocytes ............ 110! Appendix L: Mature miRNAs detected in LPS-stimulated CB monocytes ............... 114!  vi  List of Tables Table 1. Over- and under-expressed pri-miRNAs in unstimulated CB monocytes compared to unstimulated adult monocytes.............................................................. 21! Table 2. Induced and repressed pri-miRNAs in adult monocytes stimulated with LPS for 1 hour compared to unstimulated adult monocytes .................................................. 22! Table 3. Induced and repressed pri-miRNAs in adult monocytes stimulated with LPS for 6 hours compared to unstimulated adult monocytes................................................. 24! Table 4. Induced and repressed pri-miRNAs in CB monocytes stimulated with LPS for 1 hour compared to unstimulated CB monocytes ........................................................ 26! Table 5. Induced- and repressed pri-miRNAs in CB monocytes stimulated with LPS for 6 hours compared to unstimulated CB monocytes ...................................................... 28! Table 6. Statistically significant under-expressed pri-miRNAs in CB monocytes stimulated with LPS for 1 hour compared to adult monocytes stimulated with LPS for 1 hour................................................................................................................... 30! Table 7. Statistically significant under-expressed pri-miRNAs in CB monocytes stimulated with LPS for 6 hours compared to adult monocytes stimulated with LPS for 6 hours ................................................................................................................. 30! Table 8. Over- and under-expressed mature miRNAs in unstimulated CB monocytes compared to unstimulated adult monocytes.............................................................. 37! Table 9. Induced and repressed mature miRNAs in overnight R848-stimulated adult monocytes compared to unstimulated adult monocytes ........................................... 39! Table 10. Induced and repressed mature miRNAs in overnight R848-stimulated CB monocytes compared to unstimulated CB monocytes .............................................. 40! Table 11. Over- and under-expressed miRNAs in CB monocytes stimulated with R848 compared to adult monocytes stimulated with R848................................................ 44! Table 12. Induced and repressed mature miRNAs in overnight LPS-stimulated adult monocytes compared to unstimulated adult monocytes ........................................... 45! Table 13. Induced and repressed mature miRNAs in overnight LPS-stimulated CB monocytes compared to unstimulated CB monocytes .............................................. 47! Table 14. Over- and under-expressed mature miRNAs in overnight LPS-stimulated CB monocytes compared to overnight LPS-stimulated adult monocytes ....................... 50! Table 15. Relative pri- and mature candidate miRNA expression in adult (A) and CB monocytes in response to LPS stimulation at early (1h or 6h) and late (24h) time points......................................................................................................................... 56! Table 16. Cytokines and chemokines significantly affected by the over-expression of candidate miRNAs in response to LPS stimulation.................................................. 59!  vii  List of Figures Figure 1. The miRNA biogenesis pathway in the cell. ....................................................... 7! Figure 2. Candidate miRNA selection strategy. ............................................................... 32! Figure 3. Five candidate pri-miRNAs of interest were identified by RNA-Seq............... 34! Figure 4. miR-3683 and miR-4301 were upregulated in both adult and CB monocytes after overnight R848 stimulation. ............................................................................. 42! Figure 5. miR-7-2-3p, miR-15b-5p and miR-3908 were downregulated in both adult and CB monocytes after overnight R848 stimulation...................................................... 43! Figure 6. miR-146a-5p and miR-4301 were upregulated in both adult and CB monocytes after overnight LPS stimulation. ............................................................................... 48! Figure 7. miR-7-2-3p, miR-25b-5p and miR-3908 were downregulated in both adult and CB monocytes after overnight LPS stimulation. ...................................................... 49! Figure 8. miR-222 is upregulated by LPS and R848 stimulation in CB monocytes. ....... 53! Figure 9. Mature candidate miRNAs were rapidly induced in adult monocytes but only very gradually increased in expression in CB monocytes over 24 hours. ................ 55! Figure 10. Transfection of miRNA mimics into THP-1 cells is not cytotoxic. ................ 58! Figure 11. CCL2, CCL3, CCL4, IL-8 and TNF-! protein levels were affected by the over-expression of candidate miRNAs mimics in monocytes.................................. 61!  viii  List of Abbreviations AGO  Argonaute  AP-1  Activator protein 1  CB  Cord blood  CBMC  Cord blood mononuclear cell  CCL  Chemokine (C-C motif) ligand  CD  Cluster of differentiation  CMV  Cytomegalovirus  Ct  Cycle threshold  DGCR8  DiGeorge syndrome critical region gene 8  GFP  Green fluorescent protein  GM-CSF  Granulocyte-macrophage colony-stimulating factor  IFN  Interferon  IL  Interleukin  IKK  I"B kinase  IRAK  Interleukin-1 receptor-associated kinase  IRF  Interferon regulatory factor  LDH  Lactate dehydrogenase  LPS  Lipopolysaccharide  MAPK  Mitogen-activated protein kinase  MCP  Monocyte chemotactic protein  MDC  Macrophage-derived chemokine  MIP  Macrophage inflammatory protein  ix  miRNA  microRNA  MyD88  Myeloid differentiation primary response gene 88  NF-"B  Nuclear factor kappa B  nt  Nucleotides  PAMP  Pathogen-associated molecular pattern  PBMC  Peripheral blood mononuclear cell  pDC  Plasmacytoid dendritic cell  poly(I:C)  Polyinosinic:polycytidylic acid  PRR  Pattern recognition receptor  RPKM  Reads per kilobase per million mapped reads  SHP  Src homology 2 domain-containing protein tyrosine phosphatase  SOCS  Suppressor of cytokine signalling  STAT  Signal transducer and activator of transcription  TAB  TGF-# activated kinase 1 binding protein  TIR  Toll/interleukin-1 receptor  TNF  Tumor necrosis factor  TLR  Toll-like receptor  TRAF  TNF receptor-associated factor  TRIF  TIR-domain-containing adapter-inducing interferon-#  UTR  Untranslated region  x  Acknowledgements First and foremost, I would like to give my gratitude to my supervisor, Dr. Tobias Kollmann for all of his support and guidance during this work. Many thanks to all the members of the Kollmann Lab, especially to Dr. Edgardo Fortuno III for his mentorship and to Laura Gelinas, Kinga Smolen, and Bing Cai for their technical assistance and for being the best possible lab mates. I would like to thank the members of my Supervisory Committee Drs. David Speert, Keith Humphries, and Pascal Lavoie for helpful discussion. Finally, I would like to thank my friends and family for all of their support throughout these years.  xi  1 Introduction Neonates have a higher susceptibility to infection than adults and children,1 and worldwide, this leads to millions of deaths.2 This suggests suboptimal immune mediated defense in early life.3 Though functional, neither the innate nor adaptive immune responses perform as optimally in newborns as in adults.4-7 Several vaccines given at birth are efficacious,8 but many are not.9 In order to better protect this highly vulnerable age group, we must first decode the mechanisms that lead to age-dependent differences in immune mediated protection.  Protection from pathogens in both adults and neonates first requires the successful initiation and activation of the innate immune response, before the subsequent activation of the adaptive immune response. The innate immune system, the first line of defense against pathogens, is activated when germline-encoded pattern recognition receptors (PRRs) recognize conserved molecular motifs in microbes collectively known as pathogen-associated molecular patterns (PAMPs).10 The sensing of PAMPs by PRRs triggers a series of events which culminates in the eventual clearance of infection in healthy human adults. This requires the successful coordination of molecular events by multiple cell subsets, which must be tightly regulated to avoid excessive activation and maintain homeostasis.  TLR signalling and regulation Toll-like receptors (TLRs) are the best characterized class of PRRs. They are type I transmembrane proteins with an extracellular domain that binds PAMPs, a  1  transmembrane domain and an intracellular Toll-interleukin (IL)-1 receptor (TIR) homology domain that facilitates the binding of TIR domain-containing adapter proteins.11 Twelve TLRs have been identified, and they can be located on either the cellular surface or on endosomal surfaces. Cell surface TLRs that have been characterized are as follows: TLR2/1 and TLR2/6 heterodimers recognize diacyl lipopeptides and triacyl lipopeptides, respectively; TLR4 recognizes the lipopolysaccharide (LPS) component of gram-negative bacteria; and TLR5 recognizes flagellin, a monomer of bacterial flagella.12 TLRs that are located on endosomal membranes are: TLR3, the receptor for viral double-stranded RNA; TLR7 and TLR8, which are needed for the recognition of viral single-stranded RNA; and TLR9, which is the receptor for unmethylated CpG DNA.12 In all cases, dimerization of the receptors (homo- or hetero- depending on TLR) follows ligand binding, and the resultant conformational changes allow for the recruitment of adapter proteins such as myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapterinducing interferon (IFN) -# (TRIF).13 The binding of adapter proteins recruits additional signalling molecules which ultimately result in the activation and nuclear translocation of transcription factors such as nuclear factor "B (NF-"B) and IFN regulatory factors (IRFs) to induce inflammatory cytokine production.14 The cytokines induced are dependent on the TLRs engaged and are primarily targeted to the pathogen encountered (ex. bacterial or viral).  The two major types of agonists used as innate stimulants in this thesis work were ligands for the cell surface receptor TLR4 and for the endosomal receptors TLR7 and TLR8.  2  TLR4 is the best characterized PRR, but it is also the most complex being the only TLR which activates both the MyD88-dependent and MyD88-independent, or TRIFdependent, pathways.15 Once LPS is bound, in the MyD88-dependent pathway TLR4 signals via interleukin-1 receptor-associated kinases (IRAKs) and TNF receptorassociated factor (TRAF) 6 to activate transcription factors such as NF-"B which induce proinflammatory cytokines.16 On the other hand, the MyD88-independent pathway requires the recruitment of the adapter protein TRIF, which signals using TRAF3 and receptor-interacting serine/threonine-protein kinase (RIP) 1, to activate NF-"B and also IRF3 to induce the production of type I IFNs.16 TLR7/8, on the other hand, signals exclusively via the MyD88-dependent pathway.17 Stimulation of cells with ligands for TLR4 and TLR7/8 produce a very robust immune response in myeloid cells, and for this reason, were chosen as the main stimulants used to assay for the specific regulatory component of the innate immune response, the miRNAs (see below).  Monocytes and the immune response Monocytes are a vital immune cell subset that is of particular importance because of their role in bridging the innate and adaptive immune responses. Upon stimulation with the TLR4 ligand LPS or the TLR7/8 ligand R848, monocytes produce a robust response even in neonates (although this is not exactly the same as in adults).18 Circulating monocytes in blood can differentiate into macrophages and dendritic cells both in vivo and in vitro.19 Monocytes migrate to sites of infections where they become macrophages, which have higher expression of PRRs and scavenger receptors, are more highly phagocytic, and help in the clearing of pathogens. Classical activation of macrophages can be achieved in  3  vitro by stimulation with LPS and is characterized by increased microbicidal activity and proinflammatory cytokine production.20 The main functions performed by macrophages are phagocytosis, antigen presentation and cytokine production.21 The phagocytic abilities of macrophages allows them to engulf and digest debris as well as pathogens. Because of this, macrophages are well equipped for antigen presentation, a process which is essentially to the activation of the adaptive immune response.22 Finally, the recognition of PAMPs by PRRs on macrophages triggers the production of proinflammatory cytokines which are required for inflammation and the clearance of pathogens.23 These properties make monocytes (/macrophages) an integral component of the immune system, and therefore were examined with respect to their age-dependent differences in this thesis.  Immune regulatory networks The timely resolution of inflammation and TLR signalling must be regulated in order to avoid excessive activation and immune mediated damage. Uncontrolled TLR signalling for example can result in sepsis-like syndrome and contribute to the pathogenesis of diseases such as atherosclerosis, asthma, arthritis, and systemic lupus erythematosus.24 Multiple levels of regulation are present to curb hyperactivation of TLR signalling, and a better understanding of negative regulation continues to emerge. For example, soluble receptors for TLRs exist to antagonize TLR binding by ligands. Functional soluble receptors for TLR2, TLR4, and TLR9 have been identified, and in their presence, stimulation with ligands results in a decreased production of proinflammatory cytokines such as tumor necrosis factor (TNF)-!.25-27 Splice variants of different innate immune  4  genes have also been observed to act as negative regulators of TLR signalling. A variant of the myeloid differentiation factor 2 (MD2), which is required for LPS signalling via TLR4, is able to interact with LPS and TLR4 but does not induce NF-kB activity28 thereby negatively regulating TLR4 activity at the onset of signalling. Splice variants for signalling molecules such as MyD88,29 IRAK130 and translocating chain-associated membrane31 have also been found to negatively regulate innate immune signalling. Numerous proteins have negative feedback roles in the innate immune response. Phosphoinositide-dependent kinase, for example, inhibits TLR-induced NF-"B activity in macrophages.32 IkB kinase (IKK) ! has been shown to limit the inflammatory response to Gram-positive infection in mice.33 The Src homology 2 domain-containing protein tyrosine phosphatase (SHP) 2 is a well characterized phosphatase that inhibits TRIFdependent signalling to reduce IFN-# production.34 A member of the tripartite-motif proteins, TRIM30!, which is induced in an NF-"B-dependent manner by several TLR ligands, negatively regulates TLR signalling by targeting the adaptor proteins TGF-# activated kinase 1 binding protein (TAB) 1 and TAB2 for degradation.35 Numerous members of the suppressor of cytokine signalling (SOCS) family of proteins are particularly well characterized to negatively impact cytokine signalling, particularly JAKSTAT signalling in addition to TLR signalling.36 The proteins mentioned here are critical to the proper control of the immune response, but many additional layers of control remain to be determined.  Furthermore, a class of post-transcriptional regulators, the microRNAs (miRNAs), are also centrally involved in regulating the innate immune response; they form the focus of  5  this thesis research and will be discussed in detail below.  miRNA function and biogenesis miRNAs were first identified in C. elegans two decades ago and have since been observed to be conserved in eukaryotic organims.37 They are short (~22 nucleotides (nt)), single-stranded, non-coding RNAs that regulate target gene expression posttranscriptionally either by sequestering and degrading mRNA or by inhibition of translation.38 miRNAs in general can be found in most types of cells, but some are expressed in a tissue-specific manner.39 miRNAs regulate gene expression in almost all biological processes, so unsurprisingly, miRNAs also figure prominently into the regulation of the innate immune response.  The biogenesis of mature, functionally relevant miRNAs is a complex one. Most miRNAs are intergenic, although some can be found in introns, and are primarily transcribed by RNA polymerase II with a 5’ cap and 3’ poly(A) tail (Figure 1).40, 41 While still in the nucleus, these primary transcripts (pri-miRNA) are cleaved by the RNase III enzyme Drosha and its cofactor DiGeorge syndrome critical region gene 8 (DGCR8) protein into ~70 nt long pre-miRNA transcripts that contain a hairpin loop.42 Pre-miRNAs are then exported out of the nucleus into the cytoplasm by the Expotin-5 protein coupled with the GTP-bound cofactor Ran.43 Once in the cytoplasm, the RNase III enzyme Dicer cleaves the hairpin loop from the pre-miRNAs to yield the ~22 nt miRNA:miRNA* duplex.44  6  Figure 1. The miRNA biogenesis pathway in the cell. Pri-miRNA transcripts are transcribed mainly by RNA polymerase II (Pol II) and cleaved into pre-miRNA transcripts by Drosha and DGCR8. These pre-miRNA transcripts are exported out of the nucleus by Exportin-5, where they are then cleaved into short double stranded miRNA duplexes by Dicer. One strand of the miRNA duplex acts as the biologically functional mature miRNA and is loaded onto the RISC complex. Target gene regulation by miRNA occurs by transcriptional control (destabilization and degradation) or translational control (inhibition of translation). Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology, copyright 2009.  The functional miRNA guide strand has a sequence which is complementary to the target gene, while the passenger strand (or *strand) is degraded. The guide strand is then loaded onto the RNA-induced silencing complex (RISC), which contains a number of  7  proteins including members of the Argonaute (AGO) family45. AGO proteins are the catalytic components of RISC, and they facilitate the effecter functions of RISC.45, 46 Some sequence complementarity between the mature miRNA and the 3’ untranslated region (UTR) of the target gene is required, and this ultimately results in control of transcript levels (by destabilization and degradation of mRNA) or protein levels (by inhibition of translation).47  Nomenclature for miRNAs will be summarized briefly here before continuing, as confusion can easily arise. The miRNA duplex gives rise to two short RNAs, the guide strand and the passenger or * strand. They can also be denoted as the -5p or -3p strand (ex. miR-100-5p), which signifies the strand that was more proximal to the 5’ or 3’ end of the original primary transcript, respectively. miRNAs which are nearly identical in sequence except for one or two nucleotides are annotated with lowercase letters (ex. miR146a vs. miR-146b). Pre-miRNAs which originate from different places in the genome, but which give rise to the same mature miRNAs are annotated with an additional number at the end (ex. miR-123-1 and miR-123-2). And finally, the “seed” sequence of a mature miRNA refers to the 7 nt minimum sequence (nucleotides 2-8 from the 5’ end) that must be complementary to some part of the target gene UTR. The remaining nucleotides do not necessarily need to be complementary.  miRNAs in the innate immune response miRNAs play critical roles in the immune response. They have been shown to be  8  essential for the maturation and differentiation of different immune cell subsets, including B cells, T cells and myeloid cell progenitors, and in particular for the regulation of the innate immune response.48 Importantly, a number of miRNAs have been defined as TLR responsive and act in negatively regulating the innate immune response.  One of the first miRNAs which was identified to be TLR responsive was miR-146a. It was observed to be LPS responsive in the THP-1 monocytic cell line by Taganov et al. in an NF-"B-dependent manner.49 In that study, mature miR-146a was demonstrated to be rapidly induced within the first 8 hours at which point expression plateaued for up to 16 hours post stimulation. It was also upregulated by TNF-! and IL-# treatment. miR-146a has been shown to have significant roles in endotoxin tolerance and cross-tolerance in human monocytes.50, 51 In miR-146a knock-out mice, LPS challenge leads to exaggerated inflammatory responses, and bone-marrow derived macrophages from these mice produced significantly more of the proinflammatory cytokines TNF-!, IL-6 and IL1#. Targets of miR-146a include IRAK1 and TRAF6.49 These data would suggest that miR-146a is required to control the proinflammatory response by regulating some of the key players in TLR signalling.  miR-155 is another well-studied example of regulators for the innate immune response. In the same study whereby miR-146a was determined to be NF-"B-dependent, miR-155 was also identified to be induced by endotoxin in human monocytes.49 Additional studies have since shown that miR-155 is also responsive to the TLR3 ligand polyinosinic:polycytidylic acid (poly (I:C)) and IFN-#.52 Its expression pattern is not  9  unlike that of miR-146a in that rapid upregulation was also observed within the first 6 hours post LPS stimulation before plateauing for up to 24 hours.53 miR-155 was shown to significantly affect the IL-1 and IL-6 signalling pathways.53 Like miR-146a, miR-155 also plays a role in endotoxin tolerance.54 By infecting macrophages with an RNA virus, Wang et al. showed that miR-155 was inducible in a MyD88-independent pathway to promote type I IFN signalling.55 Type I IFN production in plasmacytoid dendritic cells has also been shown to be regulated by miR-155.56 In monocyte-derived dendritic cells, Lu et al. observed that miR-155 regulated cell development, apoptosis and IL-12 production.57 Validated targets of miR-155 include the signalling proteins SOCS1,55, 58 TAB253, 56 and CCAAT-enhancer-binding protein #.59  miR-146a and mir-155 may be two of the most well studied, if not the most well studied, miRNAs in the immune response, but novel miRNA regulators of the immune response continue to rapidly emerge. miR-147b is another LPS-responsive miRNA which requires both MyD88 and TRIF signalling pathways, although to date only one study (in mouse macrophages) has shown this.60 miR-125b expression is greater in macrophages than in other leukocytes, and an over-expression of miR-125b corresponds with increased costimulatory molecule expression and increased responsiveness to IFN-$.61 In another study, miR-125b expression is downregulated in murine macrophages following LPS treatment, which inversely correlates with the production of TNF-!, a predicted target of miR-125b, suggesting that this downregulation is necessary for proper TNF-! production.54 miR-21 is another NF-"B- and MyD88-induced miRNA that positively regulates IL-10 production in response to LPS by targeting the proinflammatory protein  10  programmed cell death protein 4.62 So while it is evident that miRNAs have important roles in the fine tune control of the immune response by regulating protein production and feedback loops, research remains to be done to determine how this is achieved.  miRNAs and the neonate Precious little is known about miRNA regulation of the neonatal innate immune response. This is curious considering that miRNAs have been acknowledged as having critical roles in development in general and in the regulation of the immune response in particular. The few studies that have been published on the role of miRNAs in neonatal innate immune regulation do not allow to extract the overarching and deep insight necessary to see the forest for the trees. In the first instance in which miRNAs were studied in the context of neonatal immunology, the authors were only interested in comparing miRNA expression profiles in different CB lineages (cluster of differentiation (CD) 34+ stem cells, monocytes, T cells, and granulocytes) as well as between CB, peripheral blood, and bone marrow stem cells.63 In another early study, Lederhuber et al. were specifically interested in miR-146a/b expression in cord blood (CB) monocytes as it had already been established to be LPS responsive in human monocytes.64 In that study, the authors observed miR-146b to be similarly upregulated in both adult and CB monocytes after LPS stimulation. miR-146a was also upregulated in both adult and CB monocytes, but expression was significantly higher in CB monocytes. However, no functional studies were pursued. More recently, miR-125b expression was studied in CB monocytes after LPS treatment.65 In contrast to other studies54, adult monocytes had low miR-125b expression which was upregulated after LPS stimulation while CB monocytes  11  had high basal miR-125b levels which was downregulated after stimulation. The authors suggested that this correlated with the higher TNF-! mRNA and protein levels after LPS stimulation in the CB monocytes. Finally, Takahashi et al. published a limited study on a very similar research focus as this thesis (unstimulated and stimulated adult and CB monocyte comparisons).66 Their research focused on 69 specific miRNAs rather than the entire miRNome. The observed ‘hits’ that were identified provide additional rational for our project.  In summary, miRNA regulation of the innate immune response has been well documented and is acknowledged as a critical player in the immune response to pathogens and stimulation. Yet given their importance, miRNA research on their role in the neonate has been limited leaving a significant gap in our knowledge of their role in innate immune regulation. This thesis research will attempt to provide a comprehensive view, contributing to clarity on this matter, and will also lay out concrete, experimental stepping-stones towards a better understanding of the similarities and differences in immune regulation in the neonate as well as in the adult.  12  2 Materials and Methods 2.1 Isolation of purified primary monocytes and in vitro stimulation This study was approved by the Ethics Review Board at the University of British Columbia. Written, informed consent was obtained from healthy adult volunteers or women undergoing elective Caesarean section at BC Children’s Hospital (for cord blood). Peripheral blood from healthy adult donors and CB immediately following birth was collected by venipuncture. Peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were isolated immediately after sample procurement by Ficoll-Paque density gradient centrifugation (GE Healthcare, Baie d’Urfe, QC). Primary monocytes were isolated from PBMCs/CBMCs by positive selection of CD14+ cells using magnetic-activated cell sorting (MACS; Miltenyi Biotec Inc., Auburn, CA). Purified monocytes were resuspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% human AB serum (Gemini Bio-Products, Sacramento, CA). Primary cells were stimulated with the TLR4 ligand E. coli 0111:B4 LPS (10 ng/ml; InvivoGen, San Diego, CA), or the TLR7/8 ligand R848 (5 µM; InvivoGen) for times indicated.  2.2 RNA-Seq profiling and analysis Monocytes stimulated with LPS were lysed in Buffer RLT provided with the RNeasy Mini Kit (Qiagen) and then spun through a QIAshredder column to homogenize the lysates. Lysates were stored at -80°C and shipped out for sequencing on dry ice. Poly (A) tailed RNA was selected from 120 ng of total RNA and fragmented into 200-300 bp fragments. A strand-specific cDNA library was produced from these fragments and  13  sequenced on an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA). Alignments were restricted to uniquely mapping reads. Transcript levels were quantified using the reads per kilobase of exon per million mapped reads (RPKM) method.67 As stated by Mortazavi et al., “The RPKM measure of read density reflects the molar concentration of a transcript in the starting sample by normalizing for RNA length and for the total read number in the measurement.” In other words, the RPKM value was calculated as follows:  RPKM =  total exon reads exon length (kb) " mapped reads(10 6 )  where for a!given exon the total number of sequence reads that are aligned to it is divided by the product of the length of the transcript (in kilobases) and the total number of mapped reads (in millions) in the sample. Longer transcripts, by virtue of being longer, should have more mapped reads than shorter transcripts, and the total number of mapped reads varies between samples. The RPKM method allows for the direct comparison of transcript abundance between different mRNAs and different samples since the calculated RPKM is a value normalized for both transcript length and number of total mapped reads per sample.  2.3 miScriptTM miRNA PCR array and quantitation Cells were lysed in Lysis/Binding Buffer provided with the miRVanaTM miRNA Isolation Kit (Ambion Inc., Austin, TX) and stored at -80°C. Lysates were thawed at room temperature, and total RNA was isolated as per the manufacturer’s protocol. Purified RNA was eluted in 50 µl nuclease-free water and treated with DNase I (Invitrogen). 1 µg  14  of total RNA was reverse transcribed using the miScript II RT Kit (Qiagen, Valencia, CA) in 20 µl reactions. Small RNAs were polyadenylated in parallel to reverse transcription. cDNA was diluted to a final volume of 330 µl. miRNA expression was determined by SYBR Green using the Human miRNome miScript miRNA PCR Array (v16.0, 384-well; Qiagen). Plate set-up and cycling conditions were as per the manufacturer’s protocol. Transcripts with cycle threshold (Ct) values less than 35 were considered expressed. Analysis was performed using the %%Ct method. Of the six housekeeping small RNAs provided on each plate, five were consistently expressed. The arithmetic mean of these five small RNAs (SNORD61, SNORD68, SNORD95, SNORD96A and RNU6-2) was used for normalization.  2.4 miRNA target prediction by miRWalk For selected miRNA candidates, target prediction was performed using miRWalk68, an online database of several algorithms used to predict miRNA targets. Target prediction is based on sequence complimentarity of miRNAs to target genes, with each algorithm varying slightly on match criteria (for example some allow for mismatched pairs, GU wobbles or different minimum seed lengths). Five popular prediction algorithms were used: miRWalk68, Diana-microT (v3.0)69, miRanda70, miRDB71 and TargetScan (v6.2)72.  2.5 miRNA over-expression in THP-1 cells The monocytic leukemia cell line THP-1 (ATCC, Manassas, VA) was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) at a density  15  between 2 " 10 5 and 1 " 10 6 cells/ml. 1 " 10 5 cells were plated in 200 µl complete growth medium in 48-well plates. Cells were transfected by the addition of 40 µl Opti-MEM ! ! ! (Gibco/Invitrogen) containing miRVana miRNA mimics (50 nM final concentration;  Ambion Inc.) and LipofectamineTM 2000 (1 µg/well; Invitrogen). A pmaxGFP plasmid (Amaxa, Allendale, NJ) encoding green fluorescent protein (GFP) driven by a cytomegalovirus (CMV) promoter (200 ng/well) was transfected as a control for transfection efficiency. THP-1 cells were transfected for 24 hours at 37°C with 5% CO2. Expression of GFP was determined by visual inspection. Cells were then stimulated with LPS (10 ng/ml) for 24 hours. Cell cytotoxicity was determined using a lactate dehydrogenase (LDH) assay (Roche, Mannheim, Germany) in comparison to cells treated with 2% Triton-X as a positive control (100% cytotoxic). Supernatant was collected and stored at -80°C.  2.6 Luminex assay and analysis Determination of cytokine production in cells was via the multiplexed bead-based Luminex assay (Luminex Corp., Austin, TX). Cytokines and chemokines that were expected to be produced by monocytes based on literature were chosen for the multiplexed panel (Millipore, Billerica, MA). The cytokines assayed were IFN-$, IL-1!, IL-1#, IL-1Ra, IL-6, IL-7, IL-8, IL-10, granulocyte macrophage colony-stimulating factor (GM-CSF), chemokine (C-C motif) ligand (CCL) 2 (also known as monocyte chemotactic protein (MCP) 1), CCL3 (also known as macrophage inflammatory protein (MIP) 1!), CCL4 (also known as MIP-1#), CCL7 (also known as MCP-3), CCL22 (also known as macrophage-derived chemokines (MDC)) and TNF-!. Previously collected  16  supernatant was thawed at room temperature. The beads, biotinylated detection antibody and streptavidin phycoerythrin were diluted 1:1 with the assay buffer provided with the kit. The supernatant was incubated with beads for two hours at room temperature, and the rest of the assay was performed as per the manufacturer’s protocol with the only modification being an additional wash step prior to the addition of the streptavidin phycoerythrin. Plates were read using the Luminex 200 Total System, and data acquisition and preliminary analysis was performed using MasterPlex CT and QT (MiraiBio, San Francisco, CA), respectively. Standard curves were calculated using five parameter logistics with the lowest and highest points on the curve being13.72 and 10 000 pg/ml, respectively. Cytokine concentration was calculated from the median fluorescence intensity value.  2.7 Quantitative real-time PCR Total RNA isolation from cells previously lysed and stored at -80°C followed by cDNA synthesis were done as described above under methods section 2.3. miRNA primers were purchased (Qiagen) and reconstituted in 10% Tris-EDTA buffer (pH 8.0). miRNA expression was detected by SYBR Green. miRNA expression was normalized to the expression of the small nucleolar RNA SNORD68, and data was analyzed using the %%Ct method. Samples in which two peaks were observed in the dissociation curve, indicating primer dimers, were omitted from analysis.  17  2.8 Statistical analysis Statistical analyses were performed using Microsoft Excel or GraphPad Prism (GraphPad Software Inc., La Jolla, CA). General statistical tests used were Student’s t-test (paired or un-paired depending on comparisons) with p < 0.05 considered significant. Groups compared and detailed statistical analyses are as outlined in the results section.  18  3 Results Comprehensive miRNA expression profiles in neonatal immune cells have not been previously described either at basal levels or in response to stimulation. To better establish what miRNA host genes were LPS responsive, pri-miRNA expression was first assayed at early time points (Section 3.1); this allowed us to measure gene expression changes downstream of TLR engagement. As pri-miRNAs are then sequentially cleaved in multiple steps into mature biologically functional miRNAs, a process which requires time, we also profiled mature miRNA expression at a later time point (which will be discussed in detail in Section 3.2). Together, such time-courses would provide a glimpse of the likely dynamic miRNA-dependent changes in innate immune responses.  3.1 Five candidate pri-miRNAs of interest were identified by RNA-Seq To assay for pri-miRNA expression, primary monocytes were isolated from 3 adult peripheral blood and 3 cord blood donors and stimulated with LPS (10 ng/ml) for 1 or 6 hours. Following stimulation, total RNA was extracted using the Qiagen RNeasy kit. RNA purification using this kit is dependent on the selective binding of RNAs longer than 200 bases to a silica-gel membrane. As per Qiagen’s handbook, the proportion of nucleotides smaller than 200 bases recovered, including miRNAs and pre-miRNAs, is comparable to that recovered from centrifugation through a CsCl cushion where small RNAs are not expected to sediment efficiently. Additionally, RNAs with poly(A) tails were selected for sequence library synthesis. Therefore, pri-miRNA transcripts are the primary source of miRNA signal in this RNA-Seq data set. Of note, for some primary transcripts, there are different splice variants, and this difference is detectable by RNA-  19  Seq. Different variants for the same transcript will be indicated by an accompanying accession ID in the data presented.  3.1.1 Fifty-nine pri-miRNAs were detected in unstimulated adult monocytes and 57 pri-miRNAs were detected in unstimulated CB monocytes Using RNA-Seq technology, pri-miRNA transcripts with RPKM values greater than 4 were considered expressed. In unstimulated adult monocytes, a total of 59 different primiRNA transcripts were detected (Appendix A). Of these 59 different primary transcripts, 10 were detected in just 1 adult donor, 9 were detected in 2 adult donors and 40 were detected in all 3 adult donors. In unstimulated CB monocytes, 57 different primiRNA transcripts were detected (Appendix B). 14 of these primary transcripts were detected in only 1 donor, 22 were detected in 2 donors and 21 were detected in all 3 CB donors.  The pri-miRNA expression in unstimulated CB monocytes was compared to pri-miRNA expression in unstimulated adult monocytes by dividing the mean RPKM values in CB donors by the mean RPKM values in adult donors. In considering pri-miRNA transcripts that were more highly expressed in CB donors compared to in adult donors, pri-miRNAs had to be detected (mean RPKM value of at least 4) in CB donors while their expressions did not necessarily have to be detected in adult donors. When considering pri-miRNA transcripts that were lower in expression in CB donors compared to in adult donors, those transcripts had to be detected in adult donors although they did not necessarily have to be  20  detected in CB donors. Only 1.5-fold differences in expression were considered. These same cut-offs/inclusion criteria were used throughout the RNA-Seq data analyses.  Three pri-miRNAs were over-expressed and 14 pri-miRNAs were under-expressed in unstimulated CB monocytes when compared to unstimulated adult monocytes (Table 1). Table 1. Over- and under-expressed pri-miRNAs in unstimulated CB monocytes compared to unstimulated adult monocytes miRNA  Average RPKM (CB)  miR-1238 miR-4648 miR-5010 miR-147b miR-1227 miR-4751 miR-761 miR-3916 miR-497HG miR-1181 miR-142 miR-3918 miR-1282 miR-4271 miR-632 miR-3655 miR-611  4.09 5.13 5.12 5.50 5.64 3.94 4.36 6.33 7.41 1.42 9.42 1.13 38.25 1.44 0.54 0.98 0.62  Average RPKM (adult) 1.59 2.04 2.16 10.53 10.81 7.80 8.85 13.33 17.37 4.64 34.96 4.86 201.67 8.95 10.24 19.33 68.49  Fold Change (CB/adult)  p-value  2.58 2.51 2.36 -1.91 -1.92 -1.98 -2.03 -2.10 -2.34 -3.26 -3.71 -4.31 -5.27 -6.20 -19.06 -19.63 -110.19  0.440 0.475 0.317 0.268 0.187 0.286 0.146 0.020 0.026 0.085 0.071 0.015 0.007 0.008 0.024 0.035 0.018  The expression of pri-miRNAs was compared between adult and CB monocytes by using unpaired Student’s t-tests. Statistically significant differences in expression were observed for 8 pri-miRNAs that were all under-expressed in unstimulated CB monocytes in comparison to unstimulated adult monocytes.  21  The main focus of this research was on innate immune responsive miRNAs. The next 2 sections (3.1.2 and 3.1.3) will focus on LPS-responsive pri-miRNA transcripts in each age group separately. That is, pri-miRNAs that were induced/repressed by LPS in adult monocytes were compared to pri-miRNAs in unstimulated adult monocytes in Section 3.1.2, and pri-miRNAs that were induced/repressed by LPS in CB monocytes were compared to pri-miRNAs in unstimulated CB monocytes in Section 3.1.3. Following that, differences in pri-miRNA expressions between the two age groups were compared in Section 3.1.4.  3.1.2 Eight of the upregulated pri-miRNAs and 5 of the downregulated pri-miRNAs in adult monocytes stimulated with LPS for 1 hour remained up- or downregulated after 6 hours In adult monocytes stimulated with LPS for 1 hour, 66 different pri-miRNA transcripts were detected. 14 of these primary transcripts were detected in only 1 donor, 7 were detected in 2 donors and 45 were detected in all 3 donors (Appendix C). Using a 1.5-fold change in expression cut-off, 15 pri-miRNAs were induced by 1 hour of LPS stimulation (Table 2). Statistical analyses were performed using paired Student’s Table 2. Induced and repressed pri-miRNAs in adult monocytes stimulated with LPS for 1 hour compared to unstimulated adult monocytes miRNA miR-147b miR-22 miR-4657 miR-4648 miR-22HG (NR_028504)  Average RPKM (1h LPS) 449.45 84.71 53.70 10.04 61.33  Average RPKM (unstimulated) 10.53 12.97 9.32 2.04 14.86  Fold Change (1h LPS /unstim) 42.69 6.53 5.76 4.92 4.13  p-value 0.060 0.001 0.054 0.116 0.008  22  miRNA miR-22HG (NR_028505) miR-22HG (NR_028503) miR-22HG (NR_028502) miR-4751 miR-4647 miR-21 miR-3661 miR-4420 miR-3614 miR-4680 miR-761 miR-4800 miR-3605 miR-1306 miR-4312 miR-4517 miR-4298 miR-421 miR-373 miR-142 miR-1248 miR-573  Average RPKM (1h LPS) 62.24  Average RPKM (unstimulated) 15.36  Fold Change (1h LPS /unstim) 4.05  p-value  58.67  15.18  3.86  0.010  51.32  14.64  3.51  0.010  26.21 46.90 8.95 85.84 8.70 38.18 43.30 5.78 20.29 9.65 5.76 82.15 8.95 2.74 2.51 5.54 2.25 8.39 1.67  7.80 15.09 3.15 37.02 4.91 21.64 27.75 8.85 33.56 16.15 9.96 159.19 17.46 5.54 5.49 13.33 9.27 34.96 10.21  3.36 3.11 2.84 2.32 1.77 1.76 1.56 -1.53 -1.65 -1.67 -1.73 -1.94 -1.95 -2.02 -2.19 -2.40 -4.12 -4.17 -6.11  0.091 0.004 0.006 0.027 0.138 0.112 0.006 0.344 0.167 0.105 0.005 0.025 0.090 0.040 0.006 0.017 0.026 0.077 0.028  0.008  t-tests with p < 0.05 considered significant. Of these pri-miRNAs, 9 changes in expression were statistically significant. Twelve pri-miRNAs were downregulated by at least 1.5-fold. Of these transcripts, seven were statistically significant.  Following 6 hours of LPS stimulation, 72 different pri-miRNA transcripts were detected in adult monocytes. 15 of these pri-miRNAs were detected in only 1 donor, 15 were detected in 2 donors and 42 were detected in all 3 donors (Appendix D). Using a 1.5-fold  23  cut-off, 32 pri-miRNAs were induced in adult monocytes stimulated with LPS for 6 hours compared to unstimulated adult monocytes (Table 3). Using a paired Table 3. Induced and repressed pri-miRNAs in adult monocytes stimulated with LPS for 6 hours compared to unstimulated adult monocytes miRNA miR-210HG miR-147b miR-155 (NR_001458) miR-155 (NR_030784) miR-5188 miR-21 miR-3945 miR-146a miR-614 miR-4485 miR-4645 miR-4482-2 miR-1304 miR-4482-1 miR-4751 miR-3671 miR-3614 miR-1260b miR-4257 miR-612 miR-4657 miR-1181 miR-324 miR-555 miR-922 miR-4648 miR-4700 miR-4632 miR-4647 miR-3652 miR-3656 miR-22 miR-4517  Average RPKM (6h LPS) 11.48 5161.94 42.39  Average RPKM (unstimulated) 0.02 10.53 0.13  Fold Change (6h LPS /unstim) 563.78 490.33 336.88  p-value  27.25  0.74  37.02  0.004  6.21 57.62 12.74 5.12 4.34 7.00 15.95 7.19 5.13 8.10 54.42 4.27 108.26 4.99 16.71 5.71 31.97 13.17 9.39 6.59 40.94 4.50 11.98 252.42 30.75 4.95 11.47 20.37 3.50  0.28 3.15 0.83 0.40 0.35 0.70 1.71 0.79 0.63 1.10 7.80 0.64 21.64 1.01 3.40 1.28 9.32 4.64 3.38 2.80 17.46 2.04 5.49 122.27 15.09 2.87 6.80 12.97 5.54  22.48 18.32 15.41 12.67 12.51 9.96 9.32 9.13 8.10 7.36 6.98 6.63 5.00 4.94 4.92 4.47 3.43 2.84 2.78 2.36 2.35 2.20 2.18 2.06 2.04 1.72 1.69 1.57 -1.58  0.113 0.052 0.057 0.084 0.242 0.026 0.057 0.019 0.033 0.023 0.178 0.070 0.023 0.073 0.055 0.284 0.040 0.319 0.113 0.066 0.035 0.513 0.028 0.088 0.014 0.621 0.355 0.146 0.168  0.198 0.037 0.018  24  miRNA miR-5047 miR-4775 miR-3916 miR-4420 miR-4709 miR-1227 miR-7-1 miR-3064 miR-4658 miR-3940 miR-611 miR-142 miR-3655 miR-3658 miR-4761 miR-5193 miR-761 miR-1228 miR-4680 miR-1248 miR-223 miR-4800  Average RPKM (6h LPS) 96.72 8.38 7.50 2.65 344.81 5.21 4.42 186.22 3.65 6.67 28.21 13.93 6.71 1.85 3.24 6.85 2.67 1.69 6.02 2.01 2.44 0.54  Average RPKM (unstimulated) 159.19 14.67 13.33 4.91 642.02 10.81 9.27 402.91 8.63 16.07 68.49 34.96 19.33 5.37 9.52 21.78 8.85 5.62 27.75 10.21 33.56 16.15  Fold Change (6h LPS /unstim) -1.65 -1.75 -1.78 -1.85 -1.86 -2.07 -2.10 -2.16 -2.37 -2.41 -2.43 -2.51 -2.88 -2.90 -2.94 -3.18 -3.32 -3.33 -4.61 -5.08 -13.74 -29.83  p-value 0.003 0.028 0.026 0.312 0.004 0.080 0.119 0.009 0.284 0.103 0.262 0.152 0.089 0.006 0.043 0.006 0.109 0.036 0.011 0.037 0.011 0.039  Student’s t-test for analysis, the increase in expression of 12 of these pri-miRNA transcripts were statistically significant. The expression of 23 pri-miRNA transcripts were decreased in adult monocytes following 6 hours of stimulation with LPS compared to unstimulated cells. Thirteen of these 23 downregulated pri-miRNAs were statistically significant differences in expression.  The majority of the differentially regulated pri-miRNAs between adult monocytes stimulated with LPS for 1 hour or 6 hours are distinct. Eight of the upregulated primiRNAs and 5 of the downregulated pri-miRNAs by LPS stimulation for 1 hour remain up- or downregulated after 6 hours of LPS stimulation.  25  3.1.3 Seventeen of the upregulated pri-miRNAs and 10 of the downregulated primiRNAs in CB monocytes stimulated for 1 hour with LPS remained up- or downregulated after 6 hours Exactly as described above for adult monocytes, CB monocytes were also stimulated with LPS (10 ng/ml) for 1 or 6 hours. In CB monocytes stimulated with LPS for 1 hour, 59 different pri-miRNAs were detected (Appendix E). Seventeen pri-miRNAs were detected in 1 CB donor, 16 were detected in 2 donors and 26 were detected in all 3 donors. CB monocytes stimulated with LPS for 6 hours expressed detectable levels of 66 different pri-miRNA transcripts (Appendix F). Twenty-six pri-miRNAs were detected in 1 donor, 12 were detected in 2 donors and 28 were detected in all 3 CB donors.  CB monocytes stimulated with LPS for 1 hour upregulated 24 genes by at least 1.5-fold in comparison to unstimulated CB monocytes (Table 4). The differential expression of Table 4. Induced and repressed pri-miRNAs in CB monocytes stimulated with LPS for 1 hour compared to unstimulated CB monocytes miRNA miR-155 (NR_001458) miR-147b miR-3945 miR-221 miR-146a miR-155 (NR_030784) miR-24-2 miR-22 miR-22HG (NR_028504) miR-22HG (NR_028505)  Average RPKM (1h LPS) 26.75  Average RPKM (unstimulated) 0.04  Fold Change (1h LPS /unstim) 663.74  p-value  661.70 17.65 12.83 9.33 6.39  5.50 0.40 0.44 0.41 0.47  120.31 43.95 29.26 22.57 13.63  0.093 0.080 0.038 0.132 0.205  6.58 86.95 89.97  0.56 9.86 15.89  11.75 8.81 5.66  0.320 0.068 0.007  93.97  16.67  5.64  0.008  0.168  26  miRNA miR-27a miR-4751 miR-22HG (NR_028503) miR-4657 miR-22HG (NR_028502) miR-4645 miR-21 miR-4647 miR-3661 miR-3614 miR-612 miR-3620 miR-3656 miR-4648 miR-761 miR-3605 miR-1228 miR-142 miR-5047 miR-570 miR-5193 miR-223 miR-4632 miR-4761 miR-4658 miR-7-1 miR-4517 miR-4700 miR-922 miR-5010 miR-4800 miR-1248  Average RPKM (1h LPS) 5.20 22.04 81.14  Average RPKM (unstimulated) 0.93 3.94 15.45  Fold Change (1h LPS /unstim) 5.62 5.59 5.25  p-value  57.45 68.36  11.15 14.51  5.15 4.71  0.015 0.004  4.58 12.79 44.67 81.28 60.98 5.07 82.98 12.16 8.26 2.87 5.18 2.67 5.93 80.56 2.88 13.51 14.95 78.82 4.72 5.44 3.90 2.61 2.43 5.53 1.73 3.56 1.32  1.07 3.42 16.12 34.04 26.64 2.41 46.09 7.20 5.13 4.36 8.08 4.18 9.42 128.94 4.77 22.64 25.40 139.72 9.72 11.30 8.55 5.83 5.55 13.20 5.12 12.40 11.57  4.28 3.74 2.77 2.39 2.29 2.10 1.80 1.69 1.61 -1.52 -1.56 -1.57 -1.59 -1.60 -1.65 -1.68 -1.70 -1.77 -2.06 -2.08 -2.19 -2.23 -2.29 -2.39 -2.96 -3.48 -8.76  0.272 0.111 0.068 0.080 0.002 0.112 0.041 0.142 0.255 0.195 0.183 0.185 0.515 0.141 0.278 0.265 0.227 0.177 0.234 0.184 0.029 0.218 0.301 0.162 0.198 0.226 0.050  0.440 0.272 0.003  8 of these upregulated primary transcripts were statistically significant as determined by paired Student’s t-tests. 18 pri-miRNAs were downregulated by LPS stimulation in CB  27  monocytes after 1 hour, but only 1 of these differences was statistically significant (p < 0.05).  Stimulation of CB monocytes with LPS for 6 hours upregulated 23 and downregulated 18 primary miRNA transcripts (Table 5). Seven of the upregulated genes and 2 of the downregulated pri-miRNAs in comparison to unstimulated CB monocytes were statistically significant changes in expression. Table 5. Induced- and repressed pri-miRNAs in CB monocytes stimulated with LPS for 6 hours compared to unstimulated CB monocytes miRNA miR-147b miR-210HG miR-155 (NR_001458) miR-21 miR-155 (NR_030784) miR-3945 miR-4751 miR-146a miR-4645 miR-612 miR-4632 miR-4657 miR-3605 miR-4700 miR-3614 miR-22 miR-555 miR-570 miR-22HG (NR_028504) miR-22HG (NR_028505) miR-3620 miR-4648  Average RPKM (6h LPS) 5059.60 15.84 14.79  Average RPKM (unstimulated) 5.50 0.03 0.04  Fold Change (6h LPS /unstim) 919.96 490.29 367.12  p-value  55.79 6.72  3.42 0.47  16.31 14.34  0.061 0.042  5.50 52.59 4.48 8.67 12.17 455.39 35.77 21.34 13.12 59.94 21.34 7.15 8.34 25.37  0.40 3.94 0.41 1.07 2.41 139.72 11.15 8.08 5.55 26.64 9.86 3.44 4.77 15.89  13.69 13.33 10.84 8.09 5.05 3.26 3.21 2.64 2.36 2.25 2.16 2.08 1.75 1.60  0.088 0.267 0.063 0.032 0.354 0.117 0.042 0.201 0.105 0.039 0.058 0.174 0.291 0.105  26.43  16.67  1.59  0.118  71.08 7.88  46.09 5.13  1.54 1.54  0.017 0.267  0.020 0.231 0.043  28  miRNA miR-22HG (NR_028503) miR-3940 miR-3658 miR-497HG miR-7-1 miR-1228 miR-3916 miR-1227 miR-5047 miR-4658 miR-4680 miR-3064 miR-4517 miR-4761 miR-4420 miR-223 miR-5193 miR-1248 miR-4800  Average RPKM (6h LPS) 23.61  Average RPKM (unstimulated) 15.45  Fold Change (6h LPS /unstim) 1.53  p-value  10.41 3.46 4.70 5.32 2.48 3.72 3.07 65.96 4.84 9.43 198.55 2.31 3.13 1.46 4.88 2.77 1.24 0.38  15.79 5.26 7.41 8.55 4.18 6.33 5.64 128.94 11.30 22.32 499.41 5.83 9.72 5.30 25.40 22.64 11.57 12.40  -1.52 -1.52 -1.58 -1.61 -1.69 -1.70 -1.84 -1.96 -2.33 -2.37 -2.52 -2.52 -3.11 -3.62 -5.21 -8.17 -9.30 -32.77  0.350 0.099 0.093 0.076 0.271 0.157 0.046 0.128 0.216 0.054 0.054 0.171 0.252 0.137 0.128 0.235 0.043 0.186  0.127  In total, 17 pri-miRNAs that were upregulated by LPS stimulation after 1 hour in CB monocytes remain upregulated after 6 hours, and 10 pri-miRNAs that were downregulated at the 1 hour time point remain downregulated after 6 hours.  3.1.4 Pri-miR-611, -632, -922, -147b and -155 were identified by RNA-Seq as candidates of interest for further studies Comparisons of pri-miRNAs in adult monocytes stimulated with LPS versus in CB monocytes stimulated with LPS for the same duration of time similarly yielded a large number of differentially expressed pri-miRNAs. Statistical analyses between the two age groups were performed using unpaired Student’s t-tests, and a significance cut-off of 0.05  29  was used. A comparison of unstimulated monocytes between adult and CB donors was already presented (Table 1). Comparisons of stimulated adult monocytes and stimulated CB monocytes also produced long lists of differentially expressed pri-miRNAs. Therefore, only pri-miRNAs that were statistically different between the age groups being compared are listed below.  After 1 hour of LPS stimulation, differential expression was observed for 6 pri-miRNAs. For all 6 pri-miRNAs, lower expression was observed in stimulated CB monocytes than in stimulated adult monocytes (Table 6). At the 6 hour time point, all 12 pri-miRNAs identified had lower expression in CB monocytes compared to stimulated adult monocytes (Table 7). No pri-miRNAs had significantly higher expression in CB monocytes than in adult monocytes at either time point. Table 6. Statistically significant under-expressed pri-miRNAs in CB monocytes stimulated with LPS for 1 hour compared to adult monocytes stimulated with LPS for 1 hour miRNA miR-761 miR-497HG miR-4800 miR-1282 miR-3655 miR-611  Average RPKM (CB) 2.87 6.92 3.56 36.21 0.77 1.05  Average RPKM (adult) 5.78 18.56 9.65 249.82 13.47 61.00  Fold Change (CB/adult) -2.01 -2.68 -2.71 -6.90 -17.44 -58.31  p-value 0.027 0.007 0.031 0.018 0.020 0.020  Table 7. Statistically significant under-expressed pri-miRNAs in CB monocytes stimulated with LPS for 6 hours compared to adult monocytes stimulated with LPS for 6 hours miRNA miR-3614 miR-3916 miR-155 (NR_001458)  Average RPKM (CB) 59.94 3.72 14.79  Average RPKM (adult) 108.26 7.50 42.39  Fold Change (CB/adult) -1.81 -2.02 -2.87  p-value 0.046 0.016 0.013  30  miRNA miR-922 miR-497HG miR-155 (NR_030784) miR-4482-1 miR-4482-2 miR-4257 miR-4485 miR-4271 miR-3655  Average RPKM (CB) 13.26 4.70 6.72  Average RPKM (adult) 40.94 14.99 27.25  Fold Change (CB/adult) -3.09 -3.19 -4.05  p-value  1.52 1.31 2.64 1.10 1.54 0.61  8.10 7.19 16.71 7.00 11.27 6.71  -5.33 -5.47 -6.33 -6.38 -7.32 -11.01  0.009 0.007 0.028 0.009 0.010 0.033  0.029 0.005 0.001  The main research focus of this thesis was in differential miRNA expression between CB and adult monocytes. From this data set, only pri-miRNAs in which statistically significant differences were observed (either between unstimulated and stimulated cells or between CB and adult monocytes or both) were further considered (Figure 2). Furthermore, for many of the pri-miRNAs sequenced very little was known following their discovery. For some, this included a complete lack of predicted gene targets because of their recent addition to the miRNA database (www.mirbase.org; currently version 19). The MiRWalk database68, which is a collection of multiple third-party prediction algorithms, was used to predict genes that could potentially be regulated by the candidate miRNAs of interest. Each algorithm operates on the principle of Watson-Crick base pairing between the sequence of the candidate miRNA and the 3’ UTR sequences of genes, although each have slightly varied criteria for gene target prediction (ex. allowance of mismatched pairs). miRNAs for which target genes could not be predicted (and would therefore be a dead end) were omitted from further consideration.  31  Figure 2. Candidate miRNA selection strategy. LPS responsive pri-miRNAs in adult monocytes (circle A) were compared with LPS responsive pri-miRNAs in CB monocytes (circle B). Pri-miRNAs which were differentially expressed between adult and CB monocytes (circle C) were then compared to these LPS responsive pri-miRNAs. Pri-miRNAs that were selected for further consideration were LPS responsive and/or differentially expressed between adult and CB monocytes.  Based on these combined considerations, 5 candidate miRNAs were identified from the RNA-Seq data. The candidate miRNAs were selected for various reasons, that is, their patterns of expression were not the same. For example, pri-miR-611 and pri-miR-632 were detected in adult monocytes but not in CB monocytes (Figures 3A and 3B). In unstimulated cells, pri-miR-611 was 110-fold higher (p < 0.05) in adult monocytes. Pri-  32  miR-611 expression in adult monocytes after 1 hour of LPS stimulation was still significantly higher (58-fold) than in CB monocytes stimulated with 1 hour of LPS. Expression of pri-miR-611 was still higher in adult monocytes after 6 hours of stimulation, although this was no longer statistically significant. Expression of pri-miR632 was 19-fold higher in unstimulated adult monocytes than it was in unstimulated CB monocytes (p < 0.05). Following 1 and 6 hours of LPS stimulation, pri-miR-632 was still more highly expressed in adult monocytes than in CB monocytes although these were not statistically significant. Pri-miR-922 was upregulated in response to LPS stimulation after 6 hours in adult monocytes but not in CB monocytes (Figure 3C). Expression in unstimulated monocytes was fairly similar between adult and CB donors. Pri-miR-922 was upregulated by 2.3-fold in adult monocytes stimulated with LPS for 6 hours but it was not upregulated in CB monocytes stimulated with LPS for 6 hours. On the other hand, pri-miR-147b was highly upregulated in both adult and CB monocytes in response to LPS stimulation (Figure 3D). The average RPKM values for both groups following 6 hours of LPS stimulation was ~5000 while the average RPKM value for both unstimulated groups was less than 11. Both pri-miR-155 transcripts were upregulated in both adult and CB monocytes in response to LPS (Figures 3E and 3F). Unlike pri-miR147b, the upregulation of pri-miR-155 (both transcripts) was greater in LPS-stimulated adult monocytes than in LPS-stimulated CB monocytes.  33  Figure 3. Five candidate pri-miRNAs of interest were identified by RNA-Seq. Monocytes were isolated 3 from adult peripheral blood and 3 CB donors and stimulated with the TLR 4 ligand LPS (10 ng/ml) for 1 or 6 hours. Pri-miRNA expression was detected by RNA-Seq. Paired and unpaired Student’s t-tests were used for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001. A) pri-miR-611 B) primiR-632 C) pri-miR-922 D) pri-miR-147b E) pri-miR-155 (NR_001458) F) pri-miR155 (NR_ 030784)  34  3.2 Mature miR-222 was identified as a candidate of interest by qPCR Next, to establish mature miRNA expression profiles in unstimulated and stimulated cells, monocytes were isolated from 3 healthy adult peripheral blood and 3 healthy CB donors and stimulated with LPS. Since it was expected that the mature miRNAs would need time to be processed from pri-miRNAs, a later time point was chosen as opposed to an early time point for pri-miRNA profiling. Therefore, monocytes were stimulated overnight. This study was also expanded to include overnight stimulation with the TLR7/8 ligand R848, which is also known to induce robust immune responses in monocytes. The mature miRNAs expressed were profiled using the miScript miRNA qPCR array (Qiagen). This is a SYBR Green PCR assay that profiles 1066 of the most abundantly expressed human miRNAs over three 384-well plates per array.  3.2.1 Two hundred sixteen mature miRNAs were detected in unstimulated adult monocytes and 294 mature miRNAs were detected in unstimulated CB monocytes Due to the large number of miRNAs assayed in this array, the most reasonable and feasible method of analysis was the %%Ct method. This method of analysis assumes that the primer efficiencies for this commercially designed assay was nearly perfect. It was also assumed that the efficiencies of primers for the mature miRNAs were very similar to that of the primers for the small RNAs which were included in the panel as normalizing genes. miRNAs with raw Ct values less than 35 were considered expressed. For the purpose of analysis, miRNAs with Ct values greater than 35 were assigned a value of 35.  35  In unstimulated adult monocytes, 216 different mature miRNAs were detected (Appendix G). Of these miRNAs, 54 were detected in all 3 donors, 59 were detected in 2 donors and 103 were detected in only 1 of the 3 donors. In unstimulated CB monocytes, 294 different mature miRNAs were detected (Appendix H). 96 miRNAs were detected in all 3 CB donors, 96 were detected in 2 donors and 102 were detected in only 1 of the 3 donors.  In all samples (stimulated and unstimulated; adult and CB), 5 of the 6 small RNAs included in the array as reference genes for normalization were consistently detected across all qPCR plates. The arithmetic mean of these five small RNAs on each plate was used for the normalization of each miRNA on the given plate. Changes in the expression of miRNAs were calculated as fold changes by dividing the mean normalized expression value of one sample group by the second sample group of interest. According to the Qiagen miScript analysis software: miRNAs with Ct values less than 25 were considered highly expressed; miRNAs with Ct values between 25 and 30 were considered expressed at a moderate level; and miRNAs with Ct values between 30-35 were considered expressed at a low level. miRNAs with Ct values greater than 35 were not considered to be expressed. A cut-off of a 1.5-fold change (up or down) was imposed. When considering miRNAs that were more highly expressed in CB monocytes than in adult monocytes, miRNAs must have had mean Ct values corresponding to reasonable expression (i.e. less than 30) for the CB samples, while Ct values could have been less than or greater than 30 for adult samples. Conversely, when considering miRNAs that had lower expression in CB samples than in adult samples, mean Ct values had to be less  36  than 30 for the adult samples. miRNAs with Ct values just over 30 (but not greater than 31) were also considered. All miRNAs with at least a 1.5-fold difference in expression between unstimulated CB and unstimulated adult monocytes are presented (Table 8). Unpaired student’s t-tests were used for comparison, and p-values less than 0.05 were considered significant. Table 8. Over- and under-expressed mature miRNAs in unstimulated CB monocytes compared to unstimulated adult monocytes miRNA miR-142-3p miR-1471 miR-19b miR-20b miR-19a miR-103a miR-29c miR-16 miR-17 miR-20a miR-27a miR-106b miR-15a miR-142-5p miR-424 miR-195 miR-18a miR-30b miR-30e miR-3607-3p miR-221 miR-93 miR-3607-5p miR-30c miR-425 miR-223 miR-26a miR-191 miR-1280 let-7a* miR-1260  Average Ct (CB) 27.00 29.14 28.71 29.48 28.23 26.58 29.74 26.28 29.21 27.54 27.95 28.25 29.99 29.34 29.66 27.94 30.68 29.57 29.49 29.49 30.85 29.65 30.79 28.80 29.89 24.74 28.07 28.15 27.33 30.44 28.55  Average Ct (adult) 33.67 35.00 34.56 34.97 33.62 31.95 34.94 31.37 34.28 32.57 32.96 33.07 34.80 34.10 34.11 32.35 34.99 33.86 33.74 33.64 34.88 33.59 34.73 32.69 33.76 27.06 30.38 30.27 28.97 29.53 30.10  Fold Change (CB/adult) 10.65 6.28 6.05 4.71 4.39 4.36 3.87 3.58 3.48 3.42 3.38 2.96 2.94 2.84 2.29 2.22 2.09 2.05 2.00 1.85 1.77 1.61 1.59 1.55 1.54 -1.91 -1.92 -2.19 -2.96 -3.06 -3.14  p-value 0.015 0.374 0.011 0.034 0.025 0.023 0.062 0.039 0.079 0.011 0.028 0.018 0.064 0.033 0.617 0.037 0.370 0.045 0.089 0.855 0.587 0.168 0.668 0.206 0.153 0.246 0.278 0.213 0.134 0.235 0.107  37  miRNA miR-720 miR-15b miR-223* miR-15b* miR-26b miR-7-2* miR-23a miR-21 miR-23a* miR-22* miR-26b* miR-489 miR-191* let-7a miR-328 miR-628-3p miR-3908 miR-3190 miR-1253 miR-3683 miR-361-5p miR-3605-5p miR-4267 miR-548m  Average Ct (CB) 28.97 28.88 24.74 28.88 29.15 29.11 28.11 25.73 28.11 24.74 29.15 31.38 28.15 30.44 28.23 32.40 30.87 32.99 33.06 25.76 26.31 35.00 35.00 30.52  Average Ct (adult) 30.36 30.31 27.06 30.31 29.95 29.74 28.58 26.18 28.58 27.06 29.95 30.90 30.27 29.53 27.04 30.93 29.15 30.93 30.59 22.94 22.60 30.07 28.79 23.55  Fold Change (CB/adult) -3.51 -3.53 -3.88 -4.93 -5.48 -5.95 -6.90 -6.96 -7.27 -7.33 -7.43 -13.25 -16.66 -17.81 -21.04 -26.53 -31.64 -38.45 -51.26 -67.88 -124.33 -293.04 -711.61 -1152.96  p-value 0.298 0.033 0.117 0.241 0.076 0.576 0.144 0.234 0.228 0.228 0.108 0.115 0.082 0.240 0.373 0.019 0.375 0.349 0.327 0.322 0.149 0.116 0.169 0.272  In total, 25 miRNAs were more highly expressed in unstimulated CB monocytes than in unstimulated adult monocytes, and 12 of these were statistically significant (p < 0.05). 30 miRNAs were under-expressed in unstimulated CB monocytes, and only 2 of these differences were statistically significant (p < 0.05).  3.2.2 Two mature miRNAs were upregulated and 3 mature miRNAs were downregulated by overnight R848 stimulation in both adult and CB monocytes We were interested in studying the differential expression of miRNAs between CB and adult monocytes in response to innate immune stimuli. As mentioned above, mature miRNA profiling was expanded to include stimulation with the TLR7/8 ligand R848 in  38  addition to stimulation with LPS overnight since both are known to induce robust cellular responses in human monocytes. The cut-offs, inclusion criteria for analysis and statistical analyses performed were as described earlier (Chapter 3.2.1) for unstimulated cells.  In adult monocytes stimulated with R848, 343 mature miRNAs were detected (Appendix I). Of these miRNAs, 72 were detected in all 3 adult donors, 103 were detected in 2 of the 3 donors and 168 were detected in only 1 of the 3 donors. miRNA expression in the R848-stimulated adult monocytes was compared to unstimulated adult monocytes. Five miRNAs were upregulated and 17 were downregulated in all donors in response to TLR7/8 stimulation (Table 9). Table 9. Induced and repressed mature miRNAs in overnight R848-stimulated adult monocytes compared to unstimulated adult monocytes miRNA miR-3683 miR-4301 miR-3154 miR-30a-5p miR-223-3p miR-15b-5p miR-1260a miR-23a-3p miR-21-5p let-7a-5p miR-489 miR-628-3p miR-548m miR-7-2-3p miR-380-5p miR-3190-3p miR-1253 miR-328 miR-3605-5p miR-3908 miR-4267  Average Ct (R848) 18.62 28.53 29.19 30.87 25.50 28.84 28.81 27.28 24.95 28.41 30.56 30.64 24.76 31.78 32.45 33.38 33.06 31.28 34.38 35.00 35.00  Average Ct (Unstimulated) 22.94 32.78 32.23 33.73 27.06 30.31 30.10 28.58 26.18 29.53 30.90 30.93 23.55 29.74 30.24 30.93 30.59 27.04 30.07 29.15 28.79  Fold Change (R848/Unstim) 3.96 3.73 1.62 1.58 -1.55 -1.66 -1.81 -1.87 -1.96 -2.11 -3.62 -3.76 -10.30 -18.19 -20.55 -24.27 -24.74 -83.99 -100.48 -292.74 -374.93  p-value 0.441 0.135 0.422 0.682 0.474 0.368 0.418 0.526 0.951 0.761 0.393 0.710 0.314 0.314 0.440 0.359 0.348 0.368 0.119 0.374 0.170  39  Although 21 total miRNAs were differentially expressed following R848 stimulation, none of these reached statistical significance.  In R848-stimulated CB monocytes, 301 different mature miRNAs were detected (Appendix J). 50 miRNAs were detected in all 3 donors, 69 were detected in 2 donors and 182 were detected in only 1 of the 3 CB donors. When R848-stimulated CB monocytes were compared to unstimulated CB monocytes, 9 miRNAs were upregulated and 20 miRNAs were downregulated (Table 10). Table 10. Induced and repressed mature miRNAs in overnight R848-stimulated CB monocytes compared to unstimulated CB monocytes miRNA miR-548m miR-361-5p miR-222-3p miR-3683 miR-221-3p miR-4301 miR-1280 miR-328 miR-106b-5p miR-7-2-3p let-7g-5p miR-15a-5p miR-425-5p miR-26b-5p miR-3607-5p miR-18a-5p miR-15b-5p miR-142-5p miR-30c-5p miR-17-5p miR-26a-5p miR-103a-3p miR-3607-3p miR-20b-5p miR-4286  Average Ct (R848) 23.49 24.32 30.50 24.20 30.95 30.74 27.92 29.00 30.22 31.13 31.47 32.02 31.93 31.20 32.50 32.81 31.01 31.49 30.96 30.98 30.31 28.86 31.37 31.92 31.61  Average Ct (Unstimulated) 30.52 26.31 31.81 25.76 30.85 30.64 27.33 28.23 28.25 29.11 29.44 29.99 29.89 29.15 30.79 30.68 28.88 29.34 28.80 29.21 28.07 26.58 29.49 29.48 29.46  Fold Change (R848/Unstim) 334.01 10.17 6.38 5.71 2.39 1.81 1.70 1.50 -1.52 -1.58 -1.58 -1.59 -1.60 -1.62 -1.69 -1.71 -1.71 -1.73 -1.73 -1.77 -1.84 -1.89 -1.91 -2.11 -2.30  p-value 0.350 0.394 0.021 0.630 0.117 0.192 0.141 0.455 0.121 0.984 0.303 0.052 0.095 0.304 0.054 0.224 0.308 0.156 0.109 0.175 0.253 0.156 0.375 0.042 0.051  40  miRNA let-7i-5p miR-1471 miR-3908  Average Ct (R848) 32.70 32.61 35.00  Average Ct (Unstimulated) 29.99 29.14 30.87  Fold Change (R848/Unstim) -2.56 -4.31 -9.08  p-value 0.171 0.374 0.374  Of all the 28 differentially expressed miRNAs, only 2 were statistically significant differences between the R848-stimulated and unstimulated CB monocytes. miR-222-3p was induced in CB monocytes following overnight R848 stimulation by 6.38-fold (p = 0.021). miR-20b-5p was the only miRNA to have a statistically significant downregulation in expression by R848 stimulation (2.11-fold; p = 0.042).  Only 2 miRNAs were upregulated in both adult and CB monocytes in response to R848 stimulation when compared to their respective unstimulated cells (Figure 4). miR-3683 and miR-4301 were both induced by overnight R848 stimulation in adult and CB monocytes. Two additional miRNAs were upregulated by R848 stimulation in adult monocytes, while 6 additional miRNAs were upregulated in CB monocytes. Although 1.5-fold changes in expression were detected (at least), significant increases were only detected in CB monocytes for miR-222-3p (p = 0.021) as mentioned above (Table 10).  41  Figure 4. miR-3683 and miR-4301 were upregulated in both adult and CB monocytes after overnight R848 stimulation. Monocytes isolated from 3 adult peripheral and 3 cord blood donors were stimulated with R848 (5 µM) overnight. miRNA expression in stimulated cells was compared to unstimulated cells in their respective age groups. Fold changes were determined by the %%Ct method. miRNAs with at least a 1.5-fold increase in expression are listed.  A number of miRNAs were also downregulated by R848 stimulation in adult monocytes. Similarly, miRNAs were also downregulated by R848 stimulation in CB monocytes (Figure 5). In adult monocytes, 17 miRNAs were downregulated by at least 1.5-fold, and 20 miRNAs were downregulated by at least 1.5-fold in CB monocytes. However, only three miRNAs responded similarly in adult and CB monocytes, namely miR-7-2-3p, miR-15b-5p and miR-3908 (Figure 5). And as mentioned above, only downregulated miR-20b-5p in response to R848 stimulation in CB monocytes was statistically significant (p = 0.042) (Table 10).  42  Figure 5. miR-7-2-3p, miR-15b-5p and miR-3908 were downregulated in both adult and CB monocytes after overnight R848 stimulation. Monocytes were isolated from adult peripheral blood and cord blood and stimulated with R848 (5 µM) overnight. miRNA expression in stimulated cells was compared to the unstimulated cells in the respective age groups. Fold change was determined by the %%Ct method. miRNAs with at least a 1.5-fold decrease in expression are listed.  miRNAs expressed in overnight R848-stimulated CB monocytes were also directly compared to miRNAs expressed in overnight R848-stimulated adult monocytes (ie. considering only what was present and not what was induced or repressed in response to stimulation) by the %%Ct method. In R848-stimulated CB monocytes, 15 mature miRNAs had at least a 1.5-fold greater expression than in R848-stimulated adult monocytes (Table 11). Twenty-five mature miRNAs in R848-stimulated CB monocytes had at least a 1.5-fold lower expression than in adult monocytes stimulated with R848 (Table 6).  43  Table 11. Over- and under-expressed miRNAs in CB monocytes stimulated with R848 compared to adult monocytes stimulated with R848 miRNA miR-19a-3p miR-142-3p miR-328 miR-19b-3p miR-20a miR-22-3p miR-27a-3p miR-548m miR-16-5p miR-29c-3p miR-195-5p miR-106b-5p miR-17-5p miR-103a-3p miR-222-3p miR-30a-5p miR-92a-3p miR-489 let-7g-5p miR-720 miR-25-3p miR-191-5p let-7i-5p let-7d-5p miR-26a-5p miR-15b-5p miR-23a-3p miR-21-5p miR-320a miR-4301 miR-26b-5p miR-628-3p miR-744-5p let-7a-5p let-7f-5p let-7b-5p miR-3173-3p miR-361-5p miR-3154 miR-3683  Average Ct (CB) 29.26 28.84 29.00 30.63 29.08 30.55 29.04 23.49 28.22 30.58 29.75 30.22 30.98 28.86 30.50 31.92 31.42 31.83 31.47 28.89 32.00 29.76 32.70 32.48 30.31 31.01 29.51 27.30 32.73 30.74 31.20 33.69 33.91 31.60 32.85 34.23 34.35 24.32 33.48 24.20  Average Ct (Adult) 32.56 31.57 31.28 32.75 30.93 32.01 30.50 24.76 29.14 31.33 30.42 30.72 31.74 29.30 30.92 30.87 30.31 30.56 30.18 27.51 30.58 28.05 30.94 30.72 28.21 28.84 27.28 24.95 30.38 28.53 28.30 30.64 30.80 28.41 29.17 30.33 30.69 19.87 29.19 18.62  Fold Change (CB/Adult) 12.20 8.22 6.00 5.39 4.43 3.40 3.40 2.98 2.33 2.07 1.96 1.75 1.72 1.68 1.66 -1.68 -1.75 -1.95 -1.97 -2.10 -2.16 -2.63 -2.74 -2.75 -3.46 -3.64 -3.79 -4.13 -4.13 -4.52 -6.04 -6.69 -7.00 -7.41 -10.34 -12.04 -12.48 -17.71 -19.09 -47.09  p-value 0.145 0.046 0.381 0.068 0.047 0.513 0.098 0.461 0.430 0.491 0.377 0.067 0.514 0.325 0.401 0.140 0.162 0.659 0.378 0.396 0.207 0.199 0.232 0.383 0.179 0.105 0.177 0.367 0.320 0.093 0.107 0.337 0.146 0.292 0.159 0.365 0.345 0.310 0.360 0.399  44  Amongst these miRNAs, the differential expression of two miRNAs (both of which were upregulated) was statistically significant. miR-142-3p expression was 8.22-fold higher in CB monocytes (p = 0.046), and miR-20a was 4.43-fold higher in CB monocytes (p = 0.047) (Table 11). Interestingly, miR-142-3p and miR-20a expression was already 10.65-fold (p = 0.015) and 3.42-fold (p = 0.011) higher in unstimulated CB monocytes than in unstimulated adult monocytes so it would appear that these differences in expression were independent of stimulation.  3.2.3 Two miRNAs were upregulated and three miRNAs were downregulated by overnight LPS stimulation in both adult and CB monocytes The same adult and CB donors assayed for mature miRNA expression after R848 stimulation were also assayed for mature miRNA expression after LPS stimulation by qPCR. Analysis was performed as described in Chapter 3.2.2 for comparisons between unstimulated and stimulated cells and for comparisons between age groups.  In adult monocytes stimulated with LPS overnight, 372 different mature miRNAs were detected (Appendix K). Two hundred six miRNAs were detected in all 3 adult donors, 98 were detected in 2 donors and 68 were detected in just 1 of the 3 donors. When compared to unstimulated cells, LPS-stimulated adult monocytes upregulated 6 mature Table 12. Induced and repressed mature miRNAs in overnight LPS-stimulated adult monocytes compared to unstimulated adult monocytes miRNA miR-4301 miR-1184 miR-221-3p miR-146a-5p  Average Ct (LPS) 27.56 30.06 30.38 30.69  Average Ct (Unstimulated) 32.78 34.84 34.88 35.00  Fold Change (LPS/Unstim) 5.04 4.54 3.74 2.73  p-value 0.144 0.376 0.057 0.192  45  miRNA miR-24-3p miR-17-5p miR-26a-5p miR-15b-5p miR-1280 miR-223-3p let-7a-5p miR-21-5p miR-1260a miR-628-3p miR-489 miR-548m miR-3683 miR-7-2-3p miR-380-5p miR-361-5p miR-1253 miR-328 miR-3190-3p miR-4267 miR-3605-5p miR-3908  Average Ct (LPS) 28.76 30.75 28.16 28.09 27.11 24.96 27.60 24.40 29.24 29.87 30.28 23.80 23.01 30.15 31.28 23.42 31.80 28.94 32.96 31.69 33.52 35.00  Average Ct (Unstimulated) 32.59 34.28 30.38 30.31 28.97 27.06 29.53 26.18 30.10 30.93 30.90 23.55 22.94 29.74 30.24 22.60 30.59 27.04 30.93 28.79 30.07 29.15  Fold Change (LPS/Unstim) 1.97 1.56 -1.55 -1.56 -1.67 -1.69 -1.90 -2.10 -3.32 -3.47 -4.69 -7.21 -7.73 -8.02 -12.45 -12.77 -14.00 -22.51 -24.70 -55.00 -80.52 -426.16  p-value 0.211 0.384 0.556 0.376 0.494 0.430 0.752 0.898 0.207 0.908 0.395 0.998 0.453 0.308 0.350 0.232 0.342 0.371 0.365 0.174 0.135 0.374  miRNAs and downregulated 20 mature miRNAs by at least 1.5-fold (Table 12). None of these were statistically significant.  CB monocytes stimulated with LPS overnight expressed 543 different mature miRNAs (Appendix L). Eighty-five miRNAs were detected in all 3 CB donors, 71 miRNAs were detected in 2 donors and 387 were detected in only 1 of the 3 donors. Comparison of LPS-stimulated CB monocytes with unstimulated CB monocytes showed that 11 miRNAs were upregulated and 14 miRNAs were downregulated by at least 1.5-fold (Table 13). The induction of miR-29a-3p and miR-222-3p by 3.35-fold (p = 0.047) and  46  Table 13. Induced and repressed mature miRNAs in overnight LPS-stimulated CB monocytes compared to unstimulated CB monocytes miRNA miR-548m miR-615-5p miR-634 miR-146a-5p miR-361-5p miR-29a-3p miR-4301 miR-222-3p miR-3651 miR-27a-3p miR-29c-3p miR-16-5p miR-106b-5p miR-7-2-3p miR-142-3p miR-19b-3p miR-142-5p miR-103a-3p miR-15b-5p miR-425-5p miR-30b-5p miR-3607-5p miR-15a-5p miR-3908 miR-1471  Average Ct (LPS) 21.53 29.16 29.33 31.01 23.95 29.16 28.86 29.88 30.37 26.56 28.49 26.29 28.30 29.61 27.18 28.91 29.55 26.88 29.19 30.35 30.08 31.75 30.73 35.00 33.43  Average Ct (Unstimulated) 30.52 34.55 32.38 34.39 26.31 31.50 30.64 31.81 31.50 27.95 29.74 26.28 28.25 29.11 27.00 28.71 29.34 26.58 28.88 29.89 29.57 30.79 29.99 30.87 29.14  Fold Change (LPS/Unstim) 454.48 37.54 7.42 6.92 3.41 3.35 2.85 2.53 1.81 1.74 1.58 -1.52 -1.56 -1.58 -1.70 -1.72 -1.74 -1.86 -1.87 -2.07 -2.15 -2.35 -2.52 -21.24 -21.73  p-value 0.393 0.372 0.390 0.114 0.528 0.047 0.081 0.020 0.317 0.182 0.091 0.164 0.368 0.519 0.076 0.419 0.205 0.150 0.391 0.032 0.064 0.116 0.042 0.374 0.374  2.53-fold (p = 0.020), respectively were statistically significant. Furthermore, only the repression of miR-425-5p and miR-15a-5p by 2.07-fold (p = 0.032) and 2.52-fold (p = 0.042), respectively were statistically significant.  Compared to the unstimulated cells in their respective age groups, miR-146a-5p and miR-4301 were upregulated by overnight LPS stimulation in both adult and CB monocytes (Figure 6). miR-4301 upregulation in response to LPS stimulation was not  47  statistically significant in either adult or CB donors. However, it was also observed to be upregulated in response to overnight R848 stimulation in both adult and CB monocytes suggesting that it was indeed responsive to TLR stimulation.  Figure 6. miR-146a-5p and miR-4301 were upregulated in both adult and CB monocytes after overnight LPS stimulation. Monocytes isolated from 3 adult peripheral and 3 cord blood donors were stimulated with LPS (10 ng/mL) overnight. miRNA expression in stimulated cells was compared to unstimulated cells in their respective age groups. Fold changes were determined by the %%Ct method. miRNAs with at least a 1.5-fold increase in expression are listed.  Three mature miRNAs were downregulated by LPS stimulation in adult and CB monocytes compared to unstimulated monocytes in their respective age groups (Figure 7). The downregulation of miR-7-2-3p, miR-15b-5p and miR-3908 was not statistically  48  Figure 7. miR-7-2-3p, miR-25b-5p and miR-3908 were downregulated in both adult and CB monocytes after overnight LPS stimulation. Monocytes isolated from 3 adult peripheral and 3 cord blood donors were stimulated with LPS (10 ng/mL) overnight. miRNA expression in stimulated cells was compared to unstimulated cells in their respective age groups. Fold changes were determined by the %%Ct method. miRNAs with at least a 1.5-fold increase in expression are listed.  significant (Tables 12 and 13). The same 3 miRNAs were also downregulated by R848 stimulation in adult and CB monocytes (Figure 5), although they were also not statistically significant (Tables 9 and 10).  The miRNAs expressed in CB monocytes were directly compared to miRNAs expressed in adult monocytes by the %%Ct method using their normalized Ct values. Twenty-eight mature miRNAs were observed to have greater expression in CB monocytes and 27 mature miRNAs were observed to have lower expression in CB monocytes than in adult monocytes (Table 14). While none of these observations were statistically significant,  49  Table 14. Over- and under-expressed mature miRNAs in overnight LPS-stimulated CB monocytes compared to overnight LPS-stimulated adult monocytes miRNA miR-634 miR-142-3p miR-615-5p miR-19a-3p miR-27a-3p miR-19b-3p miR-20a miR-29c-3p miR-106b-5p miR-103a-3p miR-3607-3p miR-29a-3p miR-18a-5p miR-548m miR-424-5p miR-4286 miR-93-5p miR-195-5p miR-20b-5p miR-16-5p miR-17-5p miR-181a-5p miR-30c-5p miR-425-5p miR-140-3p miR-3651 miR-142-5p miR-30e-5p miR-25-3p miR-191-5p miR-146a-5p miR-21-5p miR-197-3p miR-361-5p miR-489 miR-532-3p miR-378a-3p let-7d-5p miR-26b-5p miR-23a-3p  Average Ct (CB) 29.33 27.18 29.16 27.47 26.56 28.91 27.28 28.49 28.30 26.88 29.49 29.16 30.25 21.53 29.56 29.55 29.32 27.78 29.34 26.29 29.16 30.45 28.71 30.35 30.60 30.37 29.55 29.48 30.23 27.75 31.01 24.72 31.37 23.95 30.90 31.03 31.73 30.79 28.40 26.97  Average Ct (Adult) 35.00 31.56 33.24 31.65 29.76 32.08 30.34 31.49 31.15 29.68 31.84 31.79 32.87 23.80 31.93 31.56 31.53 29.94 31.45 28.30 30.75 32.34 30.57 32.18 32.42 31.84 31.33 31.12 30.12 27.62 30.69 24.40 30.94 23.42 30.28 30.31 30.98 30.00 27.52 26.09  Fold Change (CB/Adult) 29.89 10.52 9.93 9.12 4.65 4.55 4.22 4.01 3.62 3.51 3.23 3.11 3.11 2.84 2.61 2.56 2.34 2.24 2.17 2.03 1.90 1.86 1.83 1.80 1.78 1.76 1.73 1.57 -2.14 -2.17 -2.47 -2.48 -2.67 -2.85 -3.05 -3.26 -3.33 -3.41 -3.63 -3.64  p-value 0.372 0.100 0.382 0.290 0.157 0.285 0.114 0.327 0.160 0.162 0.220 0.544 0.260 0.378 0.497 0.248 0.192 0.326 0.173 0.111 0.165 0.195 0.306 0.381 0.289 0.267 0.508 0.319 0.342 0.129 0.213 0.380 0.175 0.420 0.473 0.381 0.135 0.359 0.130 0.182  50  miRNA miR-4301 miR-185-5p miR-15b-5p miR-23b-3p let-7e-5p let-7f-5p miR-490-3p miR-320a let-7a-5p miR-637 let-7b-5p miR-744-5p miR-3683 miR-628-3p miR-1184  Average Ct (CB) 28.86 31.56 29.19 32.07 32.49 30.41 33.01 32.33 29.73 31.90 32.32 32.56 25.99 32.65 34.27  Average Ct (Adult) 27.56 30.53 28.09 30.79 30.92 28.83 30.94 30.35 27.60 29.72 29.95 30.14 23.01 29.87 30.06  Fold Change (CB/Adult) -3.88 -4.06 -4.24 -4.83 -5.90 -5.96 -7.14 -7.83 -8.63 -8.96 -10.21 -10.63 -12.43 -13.66 -31.58  p-value 0.146 0.142 0.100 0.156 0.246 0.153 0.153 0.141 0.279 0.356 0.319 0.228 0.222 0.282 0.372  several of these were on trend with what was previously observed when comparing unstimulated (Table 8) and R848-stimulated (Table 11) adult and CB monocytes.  3.2.4 One of the statistically significant, TLR-responsive mature miRNAs (miR-2223p) was selected as a candidate miRNA Altogether, the lists of differentially expressed miRNAs was extensive when considering the different conditions (unstimulated, R848-stimulated and LPS-stimulated) and age groups (adult and CB). Therefore, this list was first restricted to a set of statistically significant and TLR-responsive mature miRNAs in either adult or CB monocytes (Tables 9-10, 12-13). These mature miRNAs were: miR-20b-5p (downregulated by R848 in CB monocytes only) miR-29a-3p (upregulated by LPS in CB monocytes only) miR-222-3p (upregulated by LPS and R848 in CB monocytes only) miR-15a-5p (downregulated by LPS in CB monocytes only)  51  miR-425-5p (downregulated by LPS in CB monocytes only)  Of these mature miRNAs, miR-222-3p (or simply miR-222 from this point forward) was the only miRNA that was both induced by TLR stimulation and for which a statistically significant difference in expression was observed between adult and CB monocytes. Coincidentally, none of the TLR-responsive mature miRNAs in adult monocytes were statistically significantly changed in expression.  In adult monocytes, mature miR-222 was not induced by overnight LPS or R848 stimulation. miR-222 expression in CB monocytes was first compared relative to miR222 expression levels in unstimulated adult monocytes (Figure 8A). miR-222 expression in unstimulated CB monocytes was lower than in unstimulated adult monocytes. Upon overnight LPS stimulation, expression of miR-222 in CB monocytes was significantly induced and comparable to expression in unstimulated adult monocytes. When CB monocytes were stimulated with R848 overnight, miR-222 expression was significantly higher than in unstimulated CB monocytes and exceeded the expression observed in adult monocytes. miR-222 expression in TLR-stimulated CB monocytes was next compared relative to unstimulated CB monocytes (Figure 8B). The change in miR-222 expression was greater than 2.5-fold in LPS-stimulated cells and almost 6.4-fold in R848-stimulated cells. Both of these fold-changes in expression were statistically different than the change in expression in adult monocytes (in which miR-222 was not induced).  52  Figure 8. miR-222 is upregulated by LPS and R848 stimulation in CB monocytes. Adult and CB monocytes were stimulated with LPS (10 ng/mL) or R848 (5 µM) overnight. Differences in miRNA expression between groups was compared by the %%Ct method and presented as fold change. Paired Student’s t-tests were performed when comparing stimulated cells to unstimulated cell in the respective age groups, and unpaired Student’s t-test were used when comparing the same stimulation conditions between the two age groups. * p < 0.05, ** p < 0.01, *** p < 0.001 A) Expression of miR-222 in all sample groups was compared to miR-222 expression in unstimulated adult monocytes B) Expression of miR-222 in each age group was compared to their respective unstimulated controls  53  3.3 Candidate mature miRNA expression kinetics in CB monocytes was dissimilar to that in adult monocytes Of all the candidate miRNAs chosen (pri-miRNAs and mature miRNAs), only miR-155 expression, and to a much smaller extent miR-147b, had been previously described in the context of innate immunity. Furthermore, half of the candidate miRNAs (miR-611, miR632 and miR-922) had not been characterized in any setting, let alone as age-dependent differences. Since so little was known about most of the candidate miRNAs, their expressions were further characterized in adult and CB monocytes. Monocytes from 4 adult and 3 CB donors were isolated and stimulated with LPS (10 ng/ml) for 0, 1, 2, 6, 8 and 24 hours. Total RNA was purified, and mature candidate miRNA expression was assayed by SYBR Green qPCR assay. Samples in which primer dimers were observed (as identified from melting curve analyses) were omitted. Data was normalized to the small RNA SNORD68 and analyzed using the %%Ct method.  For all six mature candidate miRNAs assayed, rapid upregulation and a peak in expression was observed in adult monocytes at early time points following LPS stimulation. However, in CB monocytes, upregulation of all mature candidate miRNAs was much more gradual (if at all) over the 24 hour time period (Figures 9A-F). The increase in expression also never reached the levels observed in stimulated adult monocytes. This expression pattern was generally true for all adult and CB donors assayed. Pri-miRNA and mature miRNA expression for all six candidate miRNAs are summarized in Table 15 below. Interestingly, pri-miR-147 was similarly upregulated by both adult and CB monocytes in response to LPS treatment (RNA-Seq data, Chapter  54  Figure 9. Mature candidate miRNAs were rapidly induced in adult monocytes but only very gradually increased in expression in CB monocytes over 24 hours. Monocytes were isolated from 4 adult peripheral and 3 CB donors and stimulated with LPS (10 ng/ml) for 0, 1, 2, 6, 8 and 24 hours. Mature miRNA expression was assayed by SYBR Green qPCR. Data was normalized to SNORD68 expression and analyzed using the %%Ct method. Data is presented as relative expression (fold change) in comparison to the unstimulated controls for each donor. Solid lines and symbols represent adult donors. Dashed lines and open symbols represent CB donors. A) miR-222 B) miR-611 C) miR-632 D) miR-922 E) miR-147b F) miR-155  55  3.1.4; Figure 3D), while the mature miR-147b assayed here showed a disparity in expression (only upregulated in adult monocytes). The difference in pri-miRNA and mature miRNA expression seemed to indicate that for miR-147b there was a difference in miRNA biogenesis between CB and adult monocytes. Table 15. Relative pri- and mature candidate miRNA expression in adult (A) and CB monocytes in response to LPS stimulation at early (1h or 6h) and late (24h) time points Candidate miRNA miR-611 miR-632 miR-147b miR-922 miR-155 miR-222  primature primature primature primature primature primature  Unstimulated A ! CB ACB A ! CB ACB ACB ACB ACB ACB ACB ACB -  Stimulated (early) A ! CB A ! CB A ! CB A ! CB A ! CB ! A ! CB A ! CB A ! CB A !! CB! A ! CB -  A-  A ! CB -  CB -  Stimulated (late) A-  CB -  A-  CB -  A-  CB -  A-  CB -  A-  CB -  A-  CB !  Overall, this difference in mature miRNA expression kinetics was true for all candidate miRNAs assayed suggesting that there may be a global difference in miRNA processing between adult and CB monocytes.  3.4 Over-expression of candidate miRNAs changed cytokine and chemokine production in response to TLR stimulation The candidate miRNAs were shown to be induced by TLR stimulation, but it remained unknown whether they had any relevant biological functions in immune cells (except for miR-155 and miR-147b which were already known to have roles in immunology).  56  miRNAs were known to regulate one or more target genes either at the mRNA level (by destabilization and degradation of transcripts) or at the protein level (by inhibition of translation or elongation); but either way, a change in the protein level was expected. Without knowing the direct target gene(s) for each candidate miRNA, deciphering targets/pathways to pursue would be difficult. To narrow down the list of targets to pursue, an innate immune phenotype needed to be established. In the literature, it has been established that many signalling mediators are targets of miRNAs, and this would have obviously affected the downstream phenotype observed. Cytokine/chemokine production was the furthest downstream, quantifiable innate immune phenotype in monocytes that was affectable by any changes in upstream mediators. In this way, any observed changes in the phenotype as a result of changes in candidate miRNA expression would have more likely highlighted pathways and targets worth pursuing. A panel of innate cytokines and chemokines were selected to represent the expected innate immune phenotype: IFN-$, IL-1!, IL-1Ra, IL-6, IL-7, IL-8, IL-10, GM-CSF, CCL2 (MCP-1), CCL3 (MIP-1!), CCL4 (MIP-1#), CCL7 (MCP-3), CCL22 (MDC) and TNF-!. According to literature, these selected cytokines and chemokines were expected to be produced in response to TLR stimulation in human monocytes. If by changing the expression of candidate miRNAs, a change in protein levels was observed (for one or more cytokines/chemokines) then this would indicate that genes were affected, although the target genes could be signalling mediators or the cytokines/chemokines themselves.  57  3.4.1 Transfection of miRNA mimics into monocytic cells is not toxic In order to determine if candidate miRNAs affected protein expression, miRNA mimics were transfected into THP-1 cells, a human monocytic cell line. These monocytes were then subsequently stimulated with LPS (10 ng/mL) for 24 hours. Transfection efficiency, as determined by the expression of a transfected reporter plasmid encoding GFP driven by a CMV promoter, was determined to be ~50%. Cytotoxicity as a result of lipofectamine transfection was determined by assaying for the activity of LDH, an enzyme released upon cell death. Cells treated with 2% Triton X-100 were 100% toxic and untreated cells were used for background subtraction. Cytotoxicity as a result of transfection was low (~10%) (Figure 9). 100 90 80 70 60 50 40 30 20 10 0 Untreated 2% TX  LPS  -  -  -  +  Negative miR-155 Control mimic  +  +  Figure 10. Transfection of miRNA mimics into THP-1 cells is not cytotoxic. THP-1 cells were transfected with miRNA mimics using lipofectamine for 24 hours and then stimulated with LPS (10 for 24 hours. mimic Cell treated with 2% Triton XUntreated 2% Triton-X - ng/ml) Negative miR-155 Ctrl 100 (TX) were 100% cytotoxic and untreated, unstimulated cells were used for background subtraction. miR-155 mimic is presented here as a representative of a targeting miRNA. A negative control (non-targeting miRNA) was also transfected.  58  3.4.2 CCL2, CCL3, CCL4, IL-8 and TNF-! expression were affected by the overexpression of candidate miRNAs THP-1 cells were transfected with mimics for the candidate miRNAs (50 nM) for 24 hours and then stimulated with LPS (10 ng/ml) for an additional 24 hours. Supernatant was collected and measured for cytokine and chemokine production using the multiplexed bead-based Luminex assay. Protein expression and secretion by cells transfected with candidate miRNA mimics was compared to cells transfected with nontargeting mimics as a negative control.  The protein secretion of 5 of the selected cyokines and chemokines was affected by the over-expression of candidate miRNAs. IFN-$, IL-1!, IL-6, IL-7 and GM-CSF were not detected at all under any condition. The chemokines CCL7 and CCL22 were not significantly affected by the over-expression of any of the candidate miRNAs. Cytokines and chemokines that were significantly up- or downregulated compared to the negative control using Student’s t-tests (p < 0.05) are listed in Table 16 and detailed in Figure 10. Table 16. Cytokines and chemokines significantly affected by the over-expression of candidate miRNAs in response to LPS stimulation Candidate miRNA overexpressed miR-222 miR-611 miR-632 miR-922 miR-147b miR-155  Cytokine/Chemokine significantly affected Upregulated Downregulated CCL2, CCL3, TNF-! CCL4 None CCL3, CCL4 None CCL3, CCL4, TNF-! IL-8, CCL2, TNF-! CCL3 None CCL3, CCL4 IL-8, CCL2, CCL3, TNF-! None  Depending on the miRNA being over-expressed, cytokines/chemokines were over- or under-expressed in comparison to cells transfected with non-targeting negative controls.  59  CCL2 expression was significantly increased by the over-expression of miR-222, miR922 and miR-155 mimics (Figure 10A) in response to LPS stimulation in THP-1 cells. CCL3 expression in response to LPS stimulation was significantly increased by the overexpression of miR-222 and mir-155 mimics, but was significantly decreased by the overexpression of miR-611, miR-632, miR-922 and miR-147b mimics (Figure 10B). CCL4 was downregulated by the over-expression of miR-222, miR-611 and miR-147b mimics (Figure 10C). Over-expression of miR-922 and miR-155 mimics resulted in a significant increase in IL-8 protein levels in response to LPS stimulation in THP-1 cells (Figure 10D). And finally, TNF-! level was upregulated by the over-expression of miR-222, miR-922 and miR-155 mimics, but downregulated by the over-expression of miR-632 mimic in response to 24 hours of LPS stimulation (Figure 10E). Overall, this data indicated that these candidate miRNAs regulated the production of certain cytokines and chemokines. However, none of the cytokines and chemokines affected were predicted to be direct targets of any of the candidate miRNAs by miRWalk. This suggested an indirect mechanism impacting production and/or secrection of the respective cytokines/chemokines.  60  A  B  CCL2 2500  **  300  1500  **  **  100 Media  (-)'ve Control  -  0  miR-222 miR-922 miR-155  +  C  +  +  +  LPS  Media (-)'ve miR- miR- miR- miR- miR- miRControl 222 155 611 632 922 147b  -  +  +  +  D  CCL4 600  *  **  *  400 300  **  **  +  +  +  +  IL-8 400  500  ** *  300 200  200  100  100  LPS  ***  200  500  0  *  **  **  1000  LPS  *  *  400  2000  0  CCL3 500  Media  (-)'ve Control  miR-222  -  +  +  E  miR-611 miR-147b  +  +  0  LPS  Media  (-)'ve Control  miR-922  miR-155  -  +  +  +  TNF-! 175  **  150 125 100  *  75  ** *  50 25 0  LPS  Media  -  (-)'ve miRControl 222  +  +  miR922  miR155  miR632  +  +  +  Figure 11. CCL2, CCL3, CCL4, IL-8 and TNF-! protein levels were affected by the over-expression of candidate miRNAs mimics in monocytes. THP-1 cells were transfected with candidate miRNA mimics (50 nM) using lipofectamine for 24 hours followed by LPS stimulation (10 ng/ml) for 24 hours. Supernatant was collected and assayed for cytokines and chemokines by Luminex. Protein levels in cells transfected with candidate miRNA mimics was compared to cells transfected with a non-targeting negative ((-)’ve) control by Student’s t-test. Data is presented as mean and standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 A) CCL2 B) CCL3 C) CCL4 D) IL-8 E) TNF-!  61  4 Discussion Regulation of immune response genes is vital to homeostasis of the immune system. Dysregulated immune responses could result in serious risk for infection or immune mediated damage and autoimmune diseases.73 Regulation of the immune response by miRNAs has recently been identified as a prominent mechanism involved in posttranscriptional regulation of immune gene expression.74 miRNA regulation of the innate immune response in particular has been shown to be of central importance, with miRNAs having both positive as well as negative regulatory roles in the innate immune response.38 Indeed, miRNAs regulate, and are themselves regulated by, the same inflammatory transcription factors, signalling molecules, kinases and cytokines that regulate the innate immune system in general.49, 52, 59, 75-77 This suggests complex regulatory networks functioning in innate immunity, of which miRNAs are an important component.  In this thesis work, miRNA expression was assayed in primary monocytes isolated from adult peripheral blood and cord blood. Experiments were performed on these cells in vitro, thereby making this an artificial system. Additionally, purified cells were used which cannot reflect the interactions between different cell types in vivo. However, blood in the umbilical cord (and vessels in the placenta) is composed of elements of whole blood and circulates in the fetus. Cord blood therefore provides the most accessible source of human neonatal immune cells which is easily collectible and ethically justifiable. In vitro experiments do provide an indication of the functional capabilities of the cells in adults and in neonates, and therefore can be used as an extrapolation of in vivo function.  62  Research into the roles of miRNAs in the post-transcriptional regulation of immune responses in neonatal cells has been limited in scope. Most studies in neonatal cells have centered around miR-146a and miR-155, two of the most studied miRNAs in the context of immunology. For instance, miR-146a and miR-155 expression were higher in CB plasmacytoid dendritic cells (pDCs) than in adult pDCs.78 Despite the authors’ speculation that their increased expression in CB pDCs resulted in impaired TLR9 signalling, and ultimately decreased IFN-! production, no direct correlations between elevated miRNA levels and impaired function were investigated. In another study, elevated levels of miR-125b was suggested to regulate TNF-! levels in neonatal monocytes stimulated with LPS for 2 or 6 hours.65 In the same study, miR-155 was also more highly expressed in adult monocytes than in neonatal monocytes. In one other study, miR-155 and miR-18 were the only two miRNAs upregulated in response to LPS stimulation in CB CD14+ monocytes.66 And finally, in the most broad study to date, only 69 miRNAs were profiled in unstimulated and stimulated adult and CB monocytes. This is surprising given that more than 1000 miRNAs have already been discovered.66 This random approach to investigation of particular miRNAs has so far negated a comprehensive analysis of age-dependent differences in miRNA regulation of innate immunity.  Here, miRNA expression in unstimulated versus TLR-stimulated adult and CB monocytes was investigated in an unbiased, comprehensive approach. First, primary transcripts (pri-miRNAs) were assayed following stimulation to gain insight into the full pattern of TLR-inducible miRNAs. Next, the processed, mature miRNAs were profiled  63  following the same stimulation to provide a measure of biologically functional miRNAs present during the innate immune response. Furthermore, the expression kinetics of select candidate miRNAs were characterized to assess miRNA processing kinetics in detail. Finally, it was shown that these selected TLR-induced candidate miRNAs do in fact play a role in regulating the immune response.  I will here provide a more detailed discussion of the 6 miRNAs that displayed the most striking age-dependent difference, namely miR-155, miR-147b, miR-222, miR-611, miR632 and miR-9.  miR-155 in the innate immune response The primary miR-155 transcript was induced by LPS treatment in both adult and CB monocytes after 6 hours; however, upregulation was significantly higher in adult monocytes than in CB monocytes. This was true for both the shorter primary transcript (accession ID NR_030784) which maps to the 65 bp hairpin loop portion of the miR-155 transcript, and the longer primary transcript (accession ID NR_001458) which maps to the remainder of the 1500 bp host gene.  miR-155 was originally identified to be LPS responsive in THP-1 cells49 and soon after was identified to be responsive to other TLR ligands in murine macrophages.52 miR-155 is known to have substantial roles in the immune response, and differences in miR-155 expression between adult and CB monocytes could have considerable effects. It is inducible by TLR ligands via MyD88- and TRIF-dependent pathways, as well as by  64  cytokines including TNF-!, IFN-# and IFN-$.52-54 Despite being one of the best characterized immune responsive miRNAs, how miR-155 itself is regulated as well as what is regulated by miR-155 remains unsolved. Contradicting evidence exists which argues that miR-155 is not NF-"B regulated79, although most recent publications show a positive correlation between NF-"B and miR-155 expression.80, 81 Alignment of human and mouse miR-155 host gene promoters has shown that there is a 75 nucleotide homology region which is centered around a TATA box and sandwiched by activator protein 1 (AP-1) and E-twenty six 1 binding sites.82 A putative binding site for NF-"B was found in each promoter, but they were not positionally conserved between humans and mouse. In response to B-cell receptor engagement, only the AP-1 binding site was required for promoter activity while mutation of the human NF-"B binding site did not alter promoter activity. Taken together, this would suggest that the direct comparison of regulation between human and mouse miR-155 is not entirely straightforward. Morever, Kluiver et al. suggested that there were multiple levels of miR-155 regulation: one at the transcriptional level by protein kinase C and NF-"B, and one at the level of maturation from primary transcript into mature miR-155 via a yet unknown mechanism.83 In this thesis research, significant differences in primary transcript levels already existed 6 hours post TLR stimulation so it is likely that there are differences in transcriptional control between adult and CB monocytes. There additionally appeared to be a disparity in miR155 at the level of maturation for adult and CB monocytes. These results further supports that there may be multiple levels of miR-155 regulation.  Regarding downstream targets, it was shown that THP-1 cells transfected with miR-155  65  mimics and stimulated with LPS produce significantly more IL-8, CCL2, CCL3 and TNF-!. Increased TNF-! production in miR-155 over-expressing cells was consistent with literature,54 while IL-8 production has been previously observed to be decreased by miR-155 over-expression though in a different cell type.80 Needless to say, none of the upregulated cytokines/chemokines are direct targets of miR-155 since the direct interaction of miRNAs with their gene targets would be expected to result in a decrease in protein expression.  A number of predicted miR-155 targets have roles in the immune response and have been pursued by others. IKK& is a related homologue to IKK complex subunits and can activate the NF-"B and IRF3 pathways.84 It has been shown to be a direct target of miR15554, 80 and thus may restrict the magnitude of the NF-"B- and IRF3-mediated responses. Indeed, in GES-1 cells over-expressing miR-155 mimics, H. pylori infections lead to a significant reduction in NF-"B activity as well as a significantly less production of the proinflamatory cytokines IL-8 and GRO-! on the mRNA and protein levels.80 In another study, TAB2 expression was repressed on the protein level by miR-155, which corresponded with miR-155 kinetics, even as TAB2 mRNA levels continued to increase suggesting translational control.53 Because TAB2 complexes with TRAF6 to facilitate immune activation following the engagement of TLRs or IL-1 receptors, miR-155 interaction with TAB2 could attenuate the immune response. On the other hand, miR155 also targets genes which are known inhibitors of the immune response. Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a target of miR155.77, 85 SHIP1 converts the signalling molecule PIP3 back to PIP2; therefore, its  66  degradation could boost PIP3 levels which activates NF-"B and MAPK and thereby promote an inflammatory response. miRWalk target analysis performed for this thesis work revealed that several SOCS genes are predicted miR-155 targets (data not shown). SOCS1 in particular has been a validated target of miR-155.55, 57 In dendritic cells, Lu et al. observed that IL-12p70 was regulated by miR-155 expression by targeting SOCS1.57 Wang et al. showed that SOCS1 was a target for miR-155 in macrophages and additionally showed that miR-155 attenuated viral reproduction by promoting type I IFN signalling.55 From the RNA-Seq data, no difference in mRNA expression was detected for IKK&, TAB2, SHIP1 or any of the SOCS after 1 or 6 hours of LPS stimulation between adult and CB monocytes. This does not preclude a difference in mRNA level at later time points. It is also entirely possible that regulation occurs on the protein level (as has been shown for TAB2 mentioned above) and therefore differences are simply not detectable on the mRNA level. In any case, it is evident that miR-155 is needed for the fine control of both the positive and negative regulation of the immune response. Perhaps the inability of CB monocytes to induce miR-155 expression to the same levels as in adult monocytes may impair their ability to adjust the immune response accordingly.  miR-147b in the innate immune response Pri-miR-147b transcript was upregulated in both adult and CB monocytes after 6 hours of LPS stimulation. This was the first time that miR-147b has been shown to be upregulated by TLR stimulation in human primary cells. In the only previous study describing the role of miR-147b in the immune response, Liu et al. observed the increased expression of  67  human and murine miR-147b homologues following LPS treatment in the human monocytic cell line THP-1, in the murine macrophage cell line RAW264.7, and in primary murine peritoneal and alveolar macrophages.60 The authors found LPS-induced miR-147b expression to be dependent on both MyD88 and TRIF and that LPS activation required NF-"B binding sites and an IFN-$-activated site element close to the transcriptional start site. This study further showed that treatment of macrophages with TNF-!, the TLR2/1 ligand PAM3CSK4 and the TLR3 ligand poly(I:C) also increased miR-147b expression. By over-expressing miR-147b in peritoneal macrophages, both TNF-! and IL-6 production were decreased in response to LPS and poly(I:C). IL-6 production was also decreased in miR-147b over-expressing macrophages in response to PAM3CSK4. However in this thesis work, TNF-! production was not affected by overexpressing miR-147b in THP-1 cells, and IL-6 production was not detected. Analysis performed for this thesis work did not predict that either IL-6 or TNF-! were expected to be targets of miR-147b. Instead, THP-1 cells transfected with miR-147b mimics produced significantly less CCL3 and CCL4 in response to LPS stimulation. Neither CCL3 nor CCL4 are expected to be targets of miR-147b since none of the 5 third-party algorithms being used via the miRWalk database have detected sufficient sequence complementarity.  The direct gene targets that interact with miR-147b, and eventually leading to a decrease in CCL3 or CCL4 chemokine secretion, remain to be elucidated. Since miR-147b is induced by multiple TLR ligands in human and murine primary and immortal cells, pursuit of the target(s) of miR-147b represents an important task given the proposed  68  prominent role for miR-147b in the regulation of the immune response. However, unlike other prominent TLR-induced miRNAs, such as miR-146a86, 87 and miR-155,88, 89 no knock-out mouse currently exists for miR-147b so it remains unknown how critical miR147b is to the control of the immune response.  miR-222 in the innate immune response Overnight TLR stimulation of CB and adult monocytes resulted in the statistically significant induction or repression of a small number of mature miRNAs. miR-222 was the only miRNAs for which statistically significant age-dependent differences in expression was observed and was therefore the focus of additional experiments. In adult monocytes, miR-222 was not induced by overnight TLR4 stimulation with LPS or TLR7/8 stimulation with R848. CB monocytes stimulated with LPS or R848 overnight upregulated miR-222 by 2.53-fold (p = 0.020) and 6.38-fold (p=0.021), respectively. In the expression kinetics data, miR-222 expression for all 4 adult donors assayed had already returned to basal levels by 24 hours post LPS stimulation (no overnight stimulation time point was assayed). For only 2 out of 3 CB donors, miR-222 expression had just begun to increase to ~3-fold 24 hours post-stimulation. A delay in kinetics as we have postulated for CB monocytes would explain why greater miR-222 expression was detected at the overnight time point for CB monocytes than for adult monocytes since miR-222 peaked much earlier for adult monocytes.  Most research on miR-222 has been on its role in various cancer models.90-92 Its role in immunology has never been studied. My data is the first to identify miR-222 to be  69  responsive to TLR stimulation. Identification of direct miR-222 target genes has been limited and has never been investigated in the immune system. Two regulatory regions ~12 kbp upstream of miR-222 have binding sites for NF-"B, and miR-222 transcription was shown to be induced by NF-"B binding to these regions in oncogenesis.93 This indicates that differential TLR signalling upstream of NF-"B activation could potentially exist between adults and CB. Downstream targets of miR-222 which have a role in the immune response and/or result in a functional difference between adult and CB monocytes remain to be identified.  In response to LPS stimulation, miR-222-overexpressing THP-1 cells produced significantly more CCL2, CCL3 and TNF-! while producing less CCL4. Since it is expected that direct interactions between miRNAs with their gene targets result in decreased protein expression, the 3’ UTR of the one downregulated chemokine (CCL4) was analyzed for potential miR-222 binding sites using miRWalk. No binding sites for miR-222 were predicted for CCL4. This implies that the observed decrease in production was not as a result of direct interaction with miR-222. Instead, the true gene targets of miR-222 most likely lie upstream of these affected cytokines and chemokines. A number of miR-222 gene targets predicted by miRWalk have roles in immunity including IRF2, SOCS3, SOCS4, STAT2 and STAT5B amongst others. Other predicted gene targets include signalling proteins such as the Syk kinase, TRAF2, MAPK1 and MAPK10. Of these predicted gene targets, only STAT2 was differentially expressed between adult and CB monocytes (~2.4-fold higher (p = 0.017) in adult monocytes following 6 hours of LPS stimulation) in the RNA-Seq data set. This is the opposite of what we may expect  70  since miR-222 expression was higher in adult monocytes at early time points so it would be anticipated that STAT2 expression should be lower. Again, this does not indicate that miR-222 does not regulate these targets. This simply indicates that at an early time point, on the mRNA level, target gene expression was not regulated by miR-222. It is quite possible that target gene regulation occurs at the protein level or at later time points. In any case, the direct targets of miR-222 warrant further investigation since it is likely to be a TLR-induced, NF-"B-regulated miRNA with many potential targets in the immune response.  miR-146a in the innate immune response miR-146a is an additional miRNA that is worth mentioning here. It was not selected as a candidate miRNA for further characterization; however, it is one of the most well studied miRNAs in the context of immunology. miR-146a was upregulated by both adult and CB monocytes following overnight TLR4 stimulation with LPS. In adult monocytes, this was a 2.73-fold increase, and in CB monocytes an even greater increase was observed (6.92-fold). In neither cell type was statistical significance reached, hence its exclusion from further studies. However, the induction of miR-146a in both adults and CB monocytes by LPS is most likely a valid observation. More specifically, miR-146a is one of the first miRNAs that was known to respond to LPS stimulation.49 Its induction is NF"B-dependent, and it serves as a negative regulator of innate immune signalling by regulating genes such as TRAF6, IRAK1 and IRAK2.49, 51, 76 Because so much is known about the regulation of miR-146a in immunology, it is very typically the first miRNA studied in any new study. Not surprisingly then, miR-146a was the first mature miRNA  71  examined in CB monocytes versus in adult monocytes. Lederhuber et al. observed an ~3-fold increase in miR-146a expression in adult monocytes stimulated with LPS for 24 hours and an ~6-fold increase in CB monocytes stimulated with LPS for 24 hours.64 These were very similar increases in miR-146a expression as observed in this thesis work. It has already been shown that miR-146a induction is MyD88- and NF-"Bdependent,49, 62 yet it was not shown to be induced by the TLR7/8 ligand R848 in this study. Even for this well-studied miRNA, further research is required to fully understand how it is regulated. It would also be interesting to see if similar expression kinetics are observed for miR-146a.  Novel candidate miRNAs affect the immune response The other candidate pri-miRNAs of interest, pri-miR-611, pri-miR-632 and pri-miR-922, have been scarcely studied, if at all, following their discovery. For the first time in this thesis work, they have been described to have age-dependent differences in expression. Pri-miR-611 and pri-miR-632 expressions were significantly higher in unstimulated adult monocytes than in unstimulated CB monocytes. After 1 and 6 hours of LPS stimulation, both pri-miRNAs were still higher in adult monocytes, although only pri-miR-611 expression at the 1 hour time point remained statistically significant. THP-1 cells that over-expressed either mature forms, ie. miR-611 or miR-632 mimics, produced significantly less CCL3 and CCL4 in response to overnight LPS stimulation. TNF-! production was also significantly reduced in cells over-expressing miR-632 mimics in response to LPS stimulation. Of these, only CCL4 was predicted to be a direct target of miR-632 by 4 out of 5 algorithms selected on miRWalk. Thus it is possible that this  72  chemokine is directly regulated by miR-632.  Pri-miR-922 was observed to be LPS responsive but only in adult monocytes. Comparable pri-miR-922 levels were detected in unstimulated monocytes and in monocytes stimulated with LPS for 1 hour in adult and CB donors. After 6 hours of LPS stimulation, adult monocytes produced significantly more pri-miR-922 transcripts while the level of pri-miR-922 expression in CB monocytes was the same as in untreated cells. By over-expressing miR-922 mimics, THP-1 cells produced significantly more IL-8, CCL2 and TNF-! while also producing significantly less CCL3. CCL3 does not have any sites which are complementary to the mature miR-922 sequence. No previous work exists on the roles of miR-611, -632 or -922 in the immune response. Extensive research needs to be undertaken to determine how these miRNAs are regulated and how they regulate TLR responses.  Mature miRNA expression kinetics differed between adult and CB monocytes The most striking finding that resulted from this thesis research was the observed difference in mature miRNA expression kinetics between adult and CB monocytes. Overall, this was true for all candidate miRNAs assayed in all adult and CB donors assayed (Figure 8). As an example, miR-147b induction will be discussed here. Primary miR-147b transcript induction was remarkably similar between adult and CB monocytes after 1 and 6 hours of LPS stimulation (this was not necessarily the case for the other candidate miRNAs). Mature miR-147b expression in all 4 adult donors peaked within 8 hours post-stimulation, although some donor to donor variability was observed for peak  73  expression magnitude and time. By 24 hours, mature miR-147b expression returned to near basal levels for adult donors. In contrast, mature miR-147b expression in CB monocytes only increased nominally for 2 out of 3 CB donors and never reached the magnitude of induction observed in the adult donors. This expression pattern was the same for all candidate miRNAs assayed: mature miRNA expression peaked within 8 hours of stimulation for adult donors, and then declined, while expression levels were either not or only marginally induced even by 24 hours of stimulation for CB donors.  Liu et al. had observed that pri-miR147b induction was much greater than mature miR147b induction in murine macrophages, prompting them to speculate that not all of the primary transcript is processed into mature transcripts. The same observation was made in this thesis work as the magnitude of induction was far greater for primary transcripts than for mature transcripts. This supports the proposal of miRNA-specific regulation of miRNA biogenesis. However, my work adds a specific angle to this, namely that global, age-dependent differences in miRNA biogenesis and maturation in CB monocytes may exist. The miR-147b data strongly supports this since a similar induction of the primary transcript gene was observed, but induction of mature transcripts was very different. The only instance where greater mature miRNA induction was observed in CB monocytes instead of in adult monocytes was either after overnight stimulation (ex. miR-222 qPCR array; Figure 7) or 24 hours stimulation (ex. miR-222 time course; Figure8A). However, this does not contradict and in fact further supports my hypothesis of a global agedependent difference as adult mature miR-222 had already peaked and returned to basal levels by these late time points, whereas mature miR-222 expression only started to  74  gradually increase (if at all) in CB. Future investigations will dissect the molecular mechanisms underlying the global differences in the miRNA biogenesis machinery and how they are regulated in an age-dependent manner. Additionally, it would be extremely interesting to investigate how this disparity in mature miRNA expression, either because of an inability to process primary miRNA transcripts into mature miRNAs or because of a delay in kinetics, correlates with age-dependent differences in innate immune responses in CB versus in adult peripheral blood.  Limitations of study Most of the data for this thesis work was acquired using two different arrays that measured two different forms of miRNAs. These were whole transcriptome profiling by RNA-Seq, which measured pri-miRNA transcripts, and the miScript miRNA qPCR array, which measured mature miRNA expression. The technologies behind the two arrays were completely different. The first data set was acquired by whole transcriptome sequencing, while the second was a variation of the SYBR Green-based qPCR. Each assay would therefore have their own technical biases and differences in detection and sensitivity. Moreover, the different stimulation time points (1 and 6 hour for RNA-Seq versus overnight for qPCR) and the different types of transcripts assayed (primary versus mature) make the expression profiles acquired from each data set not directly comparable. And they should not be, having observed age-dependent differences in mature miRNA expression over time. The adult and CB donors assayed for each set of experiments were also different; therefore, the pri-miRNA and mature miRNA data sets did not arise from the same individuals. To better understand the dynamics between  75  primary and mature miRNA expression, future experiments should include their paired study from the same donors on the same platform. That is, the expression profiles acquired using each type of assay is not expected to overlap and should not be combined.  Another limitation to this study is the large biological variability commonly observed in studies which require human donors. As mentioned above, using different donors to assay primary and mature miRNA transcript expressions likely would have affected the number of significant findings. There are likely more miRNAs that are TLR responsive and/or differentially expressed between adult and CB cells but which were not pursued because statistical significance was not reached. For instance, miR-146a is a well established LPS responsive miRNA in monocytes and macrophages (as described above). Of particular interest was that miR-146a has been shown to be LPS-induced in CB monocytes in response to overnight LPS stimulation.64 This miRNA was expected to be upregulated by LPS stimulation in adult and CB monocytes. While this trend was observed, statistical significance was not reached. By increasing the number of subjects, this study would have benefitted by likely having additional miRNAs meet the significance threshold. Since the candidate miRNAs pursued in this study met the significance cut-off (p < 0.05) despite small sample numbers, then it would seem that these are indeed real. Finally, no consideration was given to confounding factors as a result of biological variability. Most notably, sex and race could be important factors to consider but given the limited sample size, matching was not possible. By substantially increasing sample size, these factors could be accounted for. Reproducibility of results is  76  important so expanding the study by recruiting additional donors would further confirm the observations made in this thesis work.  Future directions Because the roles of miRNAs in the neonatal innate immune response had been scarcely covered before, this thesis work was necessarily broad to start in order to establish miRNA expression profiles in unstimulated and stimulated adult and CB cells as comprehensively as possible. Once this was determined, a few candidate miRNAs that were of potential interest were further pursued in an attempt to better understand their kinetics and what, if any, functional roles they may have in the innate immune response. Still, this research merely scratched the surface of understanding, and there are many worthwhile avenues to pursue before a better understanding of miRNA regulation of the immune response is achieved, especially as pertaining to neonatal immunology.  To provide further support for the observations that were made in this thesis work, additional studies will have to be performed. To start, the perfect complement sequences for the entire 22 nt of the mature candidate miRNAs could be cloned downstream of a luciferase reporter plasmid, thereby acting as the 3’ UTR of the luciferase gene for miRNA interaction. This plasmid could then be transfected into primary adult and CB monocytes. After TLR stimulation, luciferase activity could be assayed. If TLR stimulation induces/reduces candidate miRNA expression as expected, then a corresponding decrease/increase in luciferase activity would be observed when compared to unstimulated monocytes. This could also be done over a time course to observe  77  differences in luciferase activity between adult and CB monocytes over time. If there is an inability to process miRNAs, or if there is a delay in kinetics, this would be reflected as decreased or delayed luciferase activity in CB monocytes when compared to adult monocytes.  To further support that the candidate miRNAs do in fact have biological roles, inhibitors of the candidate miRNAs could be transfected into THP-1 cells which are then LPS stimulated (just as performed for the over-expression studies already performed). Their inhibition would be expected to have the opposite effect on cytokine and chemokine expression as the over-expression of their miRNA mimics had. It would also be interesting to titrate the concentration of mimics/inhibitors so see how the presence of varying amount of miRNAs affect the immune response or if simply the presence of some minimum amount is sufficient. For many of these miRNAs, this was the first time that they have been described as being responsive to innate immune stimulation. A closer analysis of their promoter regions would perhaps highlight what factors control their transcription. More concretely, the promoter regions of the miRNAs identified by this thesis work as TLR-responsive could be cloned upstream of a luciferase reporter. Deletion mutants could be made of one or multiple consensus sequences in the promoter. These reporters would then be transfected into monocytes/macrophages and luciferase activity could be assessed following TLR stimulation to determine if the predicted transcription factors bind to and regulate the select miRNA genes as predicted. It would be especially interesting to see if any of the candidate miRNAs are regulated by NF-"B like some of the most well studied miRNAs are, and further still if a network of miRNAs  78  are similarly regulated by the same set of transcription factors.  The most exciting finding from this thesis work was that primary transcript expression did not necessarily correspond to mature miRNA expression in CB monocytes. Considering the mature miRNA expression profiles of all six identified candidates, there appeared to be a more global deficiency affecting the miRNA maturation process in CB monocytes. This indicated that CB monocytes either have an inability to process primary transcripts into mature transcripts, or can only do so with delayed kinetics. To determine if this is indeed true, different primary transcripts could be transfected into primary adult and CB monocytes. For instance, pri-miR-155 was observed to be more highly induced in adult monocytes than in CB monocytes. By transfecting pri-miRNA into the adult monocytes, it can be expected that they will be processed into mature miRNAs fairly quickly since this was already observed in this thesis work. This can be assayed over a time course by qPCR and would serve as a control. Conversely, mature miR-146a expression was higher in CB monocytes after overnight stimulation. Pri-miR-146a could be transfected into primary adult and CB monocytes and then assayed for mature miRNA expression over time. We had speculated that in adult monocytes, expression would have already peaked and returned to basal levels by the time mature miRNA expression starts to increase. If this is true, mature miRNA expression would accumulate quickly in adult monocytes transfected with pri-miR-146a while expression would only very gradually increase at later time points in CB monocytes. Finally, the primary transcript for a miRNA that was not observed to be expressed in monocytes could be transfected into both adult and CB cells. In this way, it would be known whether this difference in  79  maturation is sequence (miRNA) –specific. It would also be worthwhile to assay primary and mature miRNA transcripts from the same donors/samples over a time course. PremiRNA transcript levels should be measure concurrently (all by qPCR). In this way, where differences in processing arise could be further narrowed down (i.e. between primary transcript induction and cleavage into the hairpin precursor or between the hairpin precursor and cleavage into the mature transcript). If there is in fact a difference in processing, then further mechanistic studies should be performed to figure out how these differences arise. Since regulation could occur at many steps along the biogenesis pathway, there may be additional levels of regulation to consider depending on the miRNA being studied. mRNA expression of most of the major proteins involved in biogenesis pathways is similar between adult and CB monocytes (data not shown). Evidently, more studies will need to follow.  The research focus of our lab is centered around understanding differences between neonates and adults; therefore, other future studies will examine the age-dependent differences observed in this thesis work. For example, differences in primary transcript induction were observed between adult and CB monocytes after TLR stimulation. This indicates that there are differences between TLR engagement and leading up to miRNA host gene transcription. This difference could be pursed, and by doing so would expand on the existing knowledge of how adult and CB cells differentially regulate gene expression in response to stimulation and activation of the innate immune response. Then, it was shown that there were differences in the biologically relevant mature miRNAs expressed between adult and CB monocytes in response to TLR stimulation.  80  The next step would be to deduce how these differences affect the innate immune response. The over-expression of the candidate miRNAs has already shown that they do indeed affect cytokine and chemokine production. As has been mentioned throughout the text, deciphering what target genes are regulated by these candidate miRNAs would be immensely helpful in determining what the affect of differential expression has on the neonatal innate immune response. Target gene prediction by using miRWalk is easy since the basis for most prediction algorithms is sequence complementarity. Validating these targets is however an arduous task. Targets which were predicted by at least 4 out of the 5 selected algorithms on miRWalk will be pursued first as a particular gene that was recognized by a high number of algorithms as being a target likely indicates a true target as opposed to genes which were predicted by only one or two algorithms. Ideally, an overlay of proteome data in LPS-stimulated adult and CB monocytes could be compared to the list of potential target genes. This can be determined by using 2D gels to first document differentially expressed proteins between adult and neonatal cells which can then be identified by mass spectrometry. This presents technical challenges since proteomic assays are typically not very “clean”. It would be much easier to begin by comparing mRNA gene expression profiles by microarray, even though this limits target genes to those that are regulated on the mRNA level. Once target genes have been selected, validation of these targets can be performed by using luciferase assays again. This time, the 3’ UTRs of the target genes would be cloned downstream of a luciferase reporter and co-transfected with the candidate miRNA of interest into a cell line. If the candidate miRNA does in fact regulate the target gene’s 3’ UTR as predicted, then luciferase activity would be reduced.  81  These experiments would provide much more clarity in understanding where agedependent differences in miRNA regulation arise and how they arise.  82  5 Conclusion The roles of miRNAs in all areas of research, including in immunology, has garnered vast amounts of much deserved attention as key regulators of gene expression. In innate immunology, much of the early pioneering work that has been performed has been in mouse models or in murine and human cell-lines. This thesis work continued to build upon, and expand, our knowledge of innate immune regulation by miRNAs. This is especially true for neonatal immune cells for which little previous work has been attempted.  Since age-dependent differences in miRNA expression has not been the focus of much previous research, this thesis work was necessarily broad in order to first establish resting and stimulated expression profiles in both adult and CB cells. An advantage to this study was that both primary as well as mature miRNA profiles were assayed. Whereas it would have been easily justifiable to only profile mature miRNA expression (since these are the biologically functional portions of the primary transcripts), an unexpected benefit has been the observation that primary transcript expression does not necessarily correspond to mature miRNA expression levels. And the time course studies have shown that there may be potential kinetic differences between adult and CB cells. It would be extremely interesting to pursue where differences in mature miRNA biogenesis arise in CB cells. Then, over-expression studies demonstrated that the candidate miRNAs affected cytokine and chemokine production in response to TLR stimulation. With this data set, it is evident that miRNAs do in fact have roles in the innate immune response and agedependent differences in mature miRNA expression exist. An enhanced understanding of  83  why these differences exist and what targets are affected is needed for a more comprehensive perception of immune regulation in adults and in neonates.  The potential applications for this research are substantial. In order to develop more effective vaccines for the newborn, we must first better understand what aspects of the neonatal immune response make them more vulnerable to infections than older children and adults. By investigating how miRNAs affect the immune response, we will have a better understanding of the regulation of immunology which in turn will allow us to design better vaccines. For instance, miRNAs which are suboptimally expressed in neonates, and which have a role in eliciting the appropriate response to stimuli, may be added as adjuvants to vaccines. Alternatively, genes which have differential protein expression between adults and neonates as a result of differential miRNA expression may be targeted for intervention directly. Further still, a recent study has shown that the overexpression of miR-155 in DCs increases their abilities to activate NK cells57 which suggests that miR-155 can be used to improve on DC vaccines by enhancing antigen presentation. The results obtained from this thesis work expanded on the existing knowledge of miRNAs in the innate immune response as well as on regulatory differences between adults and neonates. Further research into miRNAs is warranted and may provide novel targets to exploit in order to improve on existing vaccines.  84  References 1.  Philbin VJ, Levy O. 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Nucleic Acids Res 2011; 39(9): 3892-902.  92  Appendices Appendix A: Pri-miRNAs detected in unstimulated adult monocytesa miR-1236 (2) miR-124-2 (1) miR-1257 (1) miR-1264 (1) miR-127 (2) miR-1270-2 (2) miR-128-2 (2) miR-1281 (2) miR-1288 (2) miR-1289-2 (2) miR-129-1 (1) miR-129-2 (1) miR-1296 (2) miR-1298 (2) miR-1304 (1) miR-1305 (1) miR-1306 (3) miR-1307 (3) miR-130a (3) miR-130b (3) miR-132 (3) miR-1322 (3) miR-1323 (3) miR-1324 (3) miR-133a1 (3) miR-133a2 (3) miR-133b (3) miR-134 (3) miR-1343 (3) miR-135a1 (3) miR-135a2 (3) miR-135b (3) miR-136 (3) miR-137 (3) miR-137HG (3) miR-138-1 (3) miR-138-2 (3) miR-139 (3)  miR-140 (3) miR-141 (3) miR-142 (3) miR-143 (3) miR-144 (3) miR-145 (3) miR-1468 (3) miR-1469 (3) miR-146a (3) miR-146b (3) miR-1470 (3) miR-1471 (3) miR-147a (3) miR-147b (3) miR-148a (3) miR-148b (3) miR-149 (3) miR-150 (3) miR-17HG (1) miR-181d (1) miR-185 (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  93  Appendix B: Pri-miRNAs detected in unstimulated CB monocytesa miR-103a2 (1) miR-1182 (1) miR-1227 (2) miR-1228 (1) miR-1238 (1) miR-1248 (3) miR-1282 (2) miR-1291 (1) miR-142 (2) miR-147b (3) miR-21 (1) miR-22 (2) miR-223 (3) miR-22HG (NR_028502; 3) miR-22HG (NR_028504; 3) miR-22HG (NR_028503; 3) miR-22HG (NR_028505; 3) miR-3064 (3) miR-3605 (2) miR-3614 (3) miR-3620 (3) miR-3656 (2) miR-3658 (3) miR-3661 (2) miR-3916 (2) miR-3940 (2) miR-4257 (1) miR-4420 (2) miR-4517 (2) miR-4632 (3) miR-4639 (1) miR-4647 (3) miR-4648 (1) miR-4657 (2) miR-4658 (2) miR-4680 (3) miR-4700 (2) miR-4709 (3) miR-4721 (1) miR-4742 (3) miR-4745 (1)  miR-4751 (2) miR-4761 (2) miR-4775 (3) miR-4800 (2) miR-497HG (3) miR-5010 (2) miR-5047 (3) miR-5193 (2) miR-5586 (1) miR-570 (2) miR-590 (2) miR-671 (1) miR-7-1 (3) miR-761 (2) miR-922 (3) miR-939 (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  94  Appendix C: Pri-miRNAs detected in adult monocytes stimulated with LPS for 1 houra miR-103-2as (1) miR-1181 (2) miR-1227 (3) miR-1228 (2) miR-1238 (1) miR-1281 (1) miR-1282 (3) miR-142 (3) miR-146a (3) miR-147b (3) miR-155 (NR_001458; 3) miR-155 (NR_030784; 1) miR-1909 (1) miR-21 (3) miR-22 (3) miR-221 (3) miR-223 (3) miR-22HG (NR_028502; 3) miR-22HG (NR_028503; 3) miR-22HG (NR_028504; 3) miR-22HG (NR_028505; 3) miR-23a (3) miR-24-2 (3) miR-27a (1) miR-29c (1) miR-3064 (3) miR-324 (1) miR-3605 (2) miR-3614 (3) miR-3620 (3) miR-3652 (1) miR-3655 (3) miR-3656 (3) miR-3658 (1) miR-3661 (3) miR-3916 (3) miR-3918 (2) miR-3940 (3) miR-3945 (3) miR-4257 (1)  miR-4271 (2) miR-4420 (3) miR-4632 (3) miR-4647 (3) miR-4648 (3) miR-4657 (3) miR-4658 (3) miR-4680 (3) miR-4709 (3) miR-4742 (3) miR-4745 (2) miR-4751 (3) miR-4761 (3) miR-4775 (3) miR-4800 (3) miR-497HG (3) miR-5047 (3) miR-5193 (3) miR-570 (1) miR-590 (1) miR-611 (3) miR-632 (3) miR-671 (1) miR-761 (3) miR-922 (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  95  Appendix D: Pri-miRNAs detected in adult monocytes stimulated with LPS for 6 hoursa miR-1181 (2) miR-1227 (2) miR-1231 (1) miR-1238 (1) miR-1260b (2) miR-1282 (3) miR-1304 (2) miR-142 (3) miR-146a (2) miR-147b (3) miR-155 (NR_030784; 3) miR-155 (NR_001458; 3) miR-21 (3) miR-210HG (3) miR-22 (3) miR-22HG (NR_028502; 3) miR-22HG (NR_028503; 3) miR-22HG (NR_028504; 3) miR-22HG (NR_028505; 3) miR-29b1 (1) miR-29c (1) miR-3064 (3) miR-324 (3) miR-3605 (3) miR-3614 (3) miR-3620 (3) miR-3652 (2) miR-3655 (3) miR-3656 (3) miR-3661 (3) miR-3671 (2) miR-3916 (3) miR-3918 (2) miR-3940 (2) miR-3945 (3) miR-4253 (1) miR-4257 (3) miR-4260 (1) miR-4271 (3) miR-4482-1 (3)  miR-4485 (3) miR-4489 (1) miR-4517 (1) miR-4632 (3) miR-4645 (3) miR-4647 (3) miR-4648 (1) miR-4657 (3) miR-4658 (1) miR-4680 (3) miR-4700 (3) miR-4709 (3) miR-4726 (1) miR-4738 (1) miR-4742 (3) miR-4745 (2) miR-4751 (3) miR-4761 (1) miR-4775 (3) miR-497HG (3) miR-5047 (3) miR-5188 (2) miR-5193 (3) miR-555 (2) miR-570 (2) miR-611 (2) miR-612 (2) miR-614 (1) miR-632 (3) miR-7-1 (1) miR-922 (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  96  Appendix E: Pri-miRNAs detected in CB monocytes stimulated with LPS for 1 houra miR-1226 (1) miR-1227 (2) miR-1228 (1) miR-1238 (1) miR-1282 (3) miR-142 (2) miR-146a (3) miR-147b (3) miR-155 (NR_001458; 3) miR-155 (NR_030784; 2) miR-21 (3) miR-22 (3) miR-221 (3) miR-223 (3) miR-22HG (NR_028502; 3) miR-22HG (NR_028503; 3) miR-22HG (NR_028504; 3) miR-22HG (NR_028505; 3) miR-23a (1) miR-24-2 (1) miR-27a (1) miR-3064 (3) miR-324 (1) miR-3605 (2) miR-3614 (3) miR-3620 (3) miR-3656 (2) miR-3658 (1) miR-3661 (3) miR-3682 (1) miR-3916 (2) miR-3940 (2) miR-3945 (3) miR-4257 (1) miR-4260 (1) miR-4420 (1) miR-4632 (3) miR-4645 (2) miR-4647 (3) miR-4648 (2)  miR-4658 (2) miR-4680 (3) miR-4709 (3) miR-4742 (3) miR-4745 (1) miR-4751 (2) miR-4761 (1) miR-4775 (3) miR-4800 (2) miR-497HG (3) miR-5047 (3) miR-5193 (2) miR-5586 (2) miR-612 (2) miR-671 (1) miR-7-1 (1) miR-922 (2) miR-939 (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  97  Appendix F: Pri-miRNAs detected in CB monocytes stimulated with LPS for 6 hoursa miR-1181 (1) miR-1182 (1) miR-1227 (2) miR-1228 (1) miR-1238 (1) miR-1260b (1) miR-1282 (2) miR-1304 (1) miR-142 (2) miR-146a (2) miR-147b (3) miR-155 (NR_030784; 3) miR-155 (NR_001458; 3) miR-21 (3) miR-210HG (2) miR-22 (3) miR-223 (1) miR-22HG (NR_028502; 3) miR-22HG (NR_028503; 3) miR-22HG (NR_028504; 3) miR-22HG (NR_028505; 3) miR-24-2 (1) miR-29C (1) miR-3064 (3) miR-324 (1) miR-3605 (3) miR-3614 (3) miR-3620 (3) miR-3652 (1) miR-3656 (2) miR-3658 (1) miR-3661 (3) miR-3671 (1) miR-3916 (1) miR-3940 (3) miR-3945 (2) miR-4257 (1) miR-4260 (1) miR-4632 (3) miR-4645 (3)  miR-4648 (1) miR-4657 (3) miR-4658 (2) miR-4680 (3) miR-4700 (3) miR-4709 (3) miR-4726 (1) miR-4742 (3) miR-4745 (1) miR-4751 (3) miR-4761 (1) miR-4775 (3) miR-497HG (1) miR-5010 (1) miR-5047 (3) miR-5188 (1) miR-5193 (1) miR-555 (3) miR-570 (2) miR-612 (2) miR-614 (1) miR-671 (1) miR-7-1 (2) miR-761 (2) miR-922 (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  98  Appendix G: Mature miRNAs detected in unstimulated adult monocytesa let-7a (3) let-7b (2) let-7c (1) let-7d (2) let-7d* (2) let-7e (2) let-7f (3) let-7f-1* (1) let-7g (3) let-7i (3) miR-103a (3) miR-106b (2) miR-106b* (2) miR-1178 (1) miR-1182 (2) miR-1184 (1) miR-1207-5p (1) miR-1233 (1) miR-1236 (1) miR-1238 (2) miR-1247 (2) miR-1249 (1) miR-1253 (2) miR-1258 (1) miR-125b (1) miR-126* (3) miR-1260 (3) miR-1269 (1) miR-127-3p (1) miR-1272 (1) miR-1280 (3) miR-1281 (2) miR-1285 (1) miR-1301 (1) miR-1307 (3) miR-130b (1) miR-130b* (2)  miR-1322 (1) miR-140-3p (1) miR-140-5p (1) miR-142-3p (1) miR-142-5p (2) miR-146b-3p (1) miR-150 (2) miR-151-5p (2) miR-155 (1) miR-15a (1) miR-15a* (1) miR-15b (3) miR-16 (3) miR-16-2* (1) miR-17/miR-106a (2) miR-181a (2) miR-181b (3) miR-181c (1) miR-181d (1) miR-185 (3) miR-186 (2) miR-187* (1) miR-188-3p (1) miR-18a (1) miR-18b (1) miR-1908 (1) miR-1909* (1) miR-191 (3) miR-191* (2) miR-1914 (1) miR-192* (1) miR-193a-5p (1) miR-195 (3) miR-196b* (2) miR-197 (1) miR-19a (2) miR-19b (2)  miR-200b (1) miR-200c (1) miR-202 (1) miR-20a (2) miR-20b (1) miR-21 (3) miR-2110 (1) miR-218-1* (1) miR-219-1-3p (1) miR-22 (3) miR-221 (1) miR-222 (3) miR-223 (3) miR-223* (1) miR-224* (1) miR-2278 (1) miR-23a (3) miR-23b (3) miR-24 (3) miR-25 (3) miR-25* (1) miR-26a (3) miR-26a-1* (2) miR-26b (3) miR-26b* (1) miR-27a (3) miR-297 (1) miR-29a (1) miR-29c (1) miR-29c* (1) miR-301a (1) miR-30a (2) miR-30a* (1) miR-30b (2) miR-30c (3) miR-30d (1) miR-30e (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  99  Appendix G: Mature miRNAs detected in unstimulated adult monocytesa (continued) miR-30e* (3) miR-3131 (1) miR-3135 (2) miR-3141 (1) miR-3147 (1) miR-3154 (2) miR-3159 (3) miR-3173-3p (3) miR-3174 (1) miR-3179 (1) miR-3190 (3) miR-320a (3) miR-320b (2) miR-320d (1) miR-323b-5p (2) miR-324-3p (1) miR-326 (1) miR-328 (3) miR-338-3p (1) miR-339-5p (1) miR-340* (1) miR-342-3p (1) miR-342-5p (1) miR-3605-5p (2) miR-3607-3p (3) miR-3607-5p (1) miR-361-5p (2) miR-362-3p (1) miR-362-5p (1) miR-3647-3p (1) miR-365 (1) miR-3651 (1) miR-3652 (1) miR-3683 (2) miR-3691-5p (2)  miR-378 (1) miR-378b (1) miR-380* (3) miR-382 (3) miR-3908 (1) miR-3909 (3) miR-3943 (1) miR-3945 (1) miR-411* (1) miR-421 (2) miR-422a (2) miR-423-3p (2) miR-423-5p (2) miR-424 (2) miR-425 (2) miR-4258 (1) miR-4267 (2) miR-4274 (1) miR-4286 (2) miR-4291 (1) miR-4301 (3) miR-4310 (1) miR-433 (3) miR-449b* (2) miR-454 (2) miR-484 (2) miR-486-5p (1) miR-489 (3) miR-490-3p (3) miR-491-5p (1) miR-505 (1) miR-509-3p (1) miR-532-3p (3) miR-548b-5p (2) miR-548c-5p (1)  miR-573 (1) miR-574-3p (1) miR-591 (1) miR-593* (2) miR-608 (1) miR-615-5p (3) miR-628-3p (3) miR-628-5p (2) miR-629 (2) miR-634 (1) miR-637 (3) miR-652 (1) miR-656 (3) miR-657 (1) miR-661 (2) miR-664 (2) miR-675* (2) miR-676* (2) miR-7 (3) miR-7-2* (3) miR-720 (3) miR-744 (3) miR-766 (3) miR-877* (1) miR-885-3p (1) miR-888 (2) miR-92a (3) miR-93 (2) miR-98 (2) miR-99b (2) miR-99b* (2)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  100  Appendix H: Mature miRNAs detected in unstimulated CB monocytesa let-7a (3) let-7a* (2) let-7b (2) let-7c (1) let-7d (3) let-7d* (2) let-7e (2) let-7f (3) let-7f-1* (1) let-7g (3) let-7i (3) let-7i* (1) miR-100 (1) miR-100* (1) miR-101 (2) miR-101* (1) miR-103a (3) miR-106b (3) miR-107 (2) miR-1179 (1) miR-1207-5p (2) miR-1233 (1) miR-1247 (1) miR-1248 (2) miR-1249 (2) miR-1250 (1) miR-1253 (3) miR-1258 (1) miR-125a-5p (2) miR-125b (1) miR-126 (1) miR-126* (3) miR-1260 (3) miR-128 (3) miR-1280 (3) miR-1281 (1) miR-1306 (1) miR-1307 (3) miR-130a (2) miR-130b (2) miR-130b* (2)  miR-137 (1) miR-138-1* (1) miR-140-3p (3) miR-140-5p (3) miR-142-3p (3) miR-142-5p (3) miR-143 (1) miR-145 (1) miR-146a (1) miR-146b-5p (2) miR-1471 (1) miR-148a (3) miR-148b (2) miR-149 (1) miR-150 (2) miR-151-3p (2) miR-151-5p (2) miR-152 (1) miR-1537 (1) miR-1539 (1) miR-154 (1) miR-154* (1) miR-155 (1) miR-15a (3) miR-15b (3) miR-15b* (2) miR-16 (3) miR-16-2* (3) miR-17/miR-106a (3) miR-181a (3) miR-181a-2* (1) miR-181b (3) miR-181c (3) miR-1825 (1) miR-185 (2) miR-186 (3) miR-188-3p (1) miR-188-5p (3) miR-18a (3) miR-18a* (1) miR-18b (1)  miR-190 (1) miR-191 (3) miR-191* (1) miR-194 (1) miR-195 (3) miR-196b* (1) miR-197 (2) miR-199a-3p/miR-199b-3p (3)  miR-199a-5p (1) miR-199b-5p (3) miR-19a (3) miR-19b (3) miR-208a (1) miR-20a (3) miR-20a* (2) miR-20b (3) miR-21 (3) miR-21* (1) miR-210 (1) miR-22 (3) miR-22* (1) miR-221 (3) miR-221* (2) miR-222 (3) miR-223 (3) miR-223* (2) miR-2355-3p (2) miR-23a (3) miR-23a* (1) miR-23b (2) miR-24 (3) miR-24-2* (2) miR-25 (3) miR-26a (3) miR-26a-1* (1) miR-26b (3) miR-26b* (2) miR-27a (3) miR-27a* (1) miR-27b (3) miR-28-3p (2)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  101  Appendix H: Mature miRNAs detected in unstimulated CB monocytesa (continued) miR-28-5p (3) miR-299-5p (1) miR-29a (2) miR-29b (2) miR-29b-1* (1) miR-29c (3) miR-29c* (3) miR-301a (2) miR-301b (1) miR-30a (3) miR-30a* (2) miR-30b (3) miR-30c (3) miR-30d (3) miR-30d* (1) miR-30e (3) miR-30e* (3) miR-3129-5p (1) miR-3131 (1) miR-3141 (3) miR-3154 (2) miR-3155/miR-3155b (1) miR-3159 (3) miR-3164 (1) miR-3173-3p (2) miR-3190 (3) miR-32 (2) miR-32* (1) miR-3201 (3) miR-320a (2) miR-320b (3) miR-320d (1) miR-323b-3p (1) miR-324-3p (2) miR-324-5p (2) miR-326 (3) miR-328 (3) miR-331-3p (2) miR-335 (1) miR-337-3p (1)  miR-338-3p (3) miR-339-3p (1) miR-339-5p (3) miR-33a (2) miR-340 (2) miR-340* (2) miR-342-3p (2) miR-345 (2) miR-34a (1) miR-34b (2) miR-3605-3p (1) miR-3607-3p (3) miR-3607-5p (3) miR-361-3p (1) miR-361-5p (2) miR-362-3p (2) miR-362-5p (2) miR-3622b-3p (1) miR-363 (2) miR-3647-3p (3) miR-3647-5p (2) miR-365 (2) miR-3650 (1) miR-3651 (2) miR-3653 (3) miR-3654 (1) miR-3676 (2) miR-3683 (2) miR-370 (1) miR-373* (3) miR-374a (3) miR-374a* (2) miR-374b/miR-374c (3) miR-376c (2) miR-378 (3) miR-378* (3) miR-378b (2) miR-380* (3) miR-382 (2) miR-3907 (1)  miR-3908 (1) miR-3909 (2) miR-3916 (1) miR-411* (1) miR-421 (2) miR-423-3p (3) miR-423-5p (2) miR-424 (3) miR-424* (2) miR-425 (3) miR-425* (2) miR-4263 (1) miR-4276 (1) miR-4286 (3) miR-4289 (2) miR-4291 (2) miR-4301 (3) miR-4304 (1) miR-4306 (1) miR-4326 (1) miR-450a (2) miR-451 (2) miR-454 (2) miR-454* (1) miR-484 (3) miR-486-5p (1) miR-487b (1) miR-489 (3) miR-490-3p (3) miR-491-5p (1) miR-495 (1) miR-496 (2) miR-500a (2) miR-500a* (2) miR-501-5p (1) miR-502-3p (2) miR-502-5p (2) miR-505 (3) miR-532-3p (3) miR-532-5p (2)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  102  Appendix H: Mature miRNAs detected in unstimulated CB monocytesa (continued) miR-542-3p (1) miR-548b-5p (1) miR-548d-5p (2) miR-548i (1) miR-548k (1) miR-548l (1) miR-548m (2) miR-551a (1) miR-573 (2) miR-574-3p (2) miR-576-3p (1) miR-577 (1) miR-579 (1) miR-582-5p (1) miR-589* (1) miR-590-3p (1) miR-590-5p (2) miR-598 (1) miR-608 (2) miR-615-5p (2) miR-624* (1) miR-625* (1) miR-628-3p (3) miR-632 (1) miR-634 (1) miR-637 (3) miR-652 (2) miR-656 (3) miR-660 (2) miR-661 (3) miR-664 (2) miR-664* (2) miR-675* (1) miR-676* (2) miR-7 (2) miR-7-1* (2) miR-7-2* (3) miR-720 (3) miR-744 (2) miR-758 (1)  miR-766 (2) miR-769-5p (1) miR-770-5p (2) miR-888 (2) miR-92a (3) miR-93 (3) miR-93* (2) miR-941 (3) miR-98 (2) miR-99a (2) miR-99b (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  103  Appendix I: Mature miRNAs detected in R848-stimulated adult monocytesa let-7a (3) let-7b (2) let-7b* (1) let-7c (2) let-7d (2) let-7d* (3) let-7e (2) let-7f (3) let-7g (2) let-7i (2) miR-100 (1) miR-100* (2) miR-101 (1) miR-103a (3) miR-105* (1) miR-106b (3) miR-107 (2) miR-10b* (1) miR-1183 (1) miR-1193 (1) miR-1207-5p (2) miR-1224-5p (1) miR-1231 (1) miR-1233 (1) miR-1238 (1) miR-1245 (1) miR-1247 (3) miR-1248 (2) miR-1249 (2) miR-1253 (2) miR-1255b (1) miR-125a-5p (2) miR-126* (3) miR-1260 (3) miR-1260b (1) miR-127-3p (2) miR-128 (2) miR-1280 (3) miR-1281 (3) miR-1285 (1) miR-129-3p (1)  miR-1290 (1) miR-1293 (1) miR-1296 (1) miR-1301 (1) miR-1306 (1) miR-1307 (3) miR-130a (1) miR-130a* (1) miR-130b (1) miR-130b* (3) miR-132 (2) miR-138-1* (1) miR-138-2* (1) miR-139-5p (1) miR-140-3p (2) miR-140-5p (1) miR-142-3p (2) miR-142-5p (3) miR-143 (1) miR-145 (2) miR-146a (2) miR-146b-5p (2) miR-1471 (1) miR-147b (1) miR-148a (1) miR-148b (2) miR-150 (2) miR-151-3p (1) miR-151-5p (2) miR-152 (2) miR-1537 (1) miR-1539 (1) miR-155 (3) miR-155* (1) miR-15a (3) miR-15a* (1) miR-15b (3) miR-15b* (2) miR-16 (3) miR-16-2* (3) miR-17* (1)  miR-17/miR-106a (3) miR-181a (1) miR-181b (3) miR-181c (2) miR-181c* (1) miR-181d (1) miR-185 (2) miR-186 (2) miR-188-3p (2) miR-188-5p (2) miR-18a (1) miR-18b (1) miR-1909* (1) miR-190b (1) miR-191 (3) miR-191* (2) miR-1910 (1) miR-193a-3p (1) miR-193a-5p (1) miR-193b (1) miR-195 (2) miR-195* (1) miR-196a (1) miR-196b (1) miR-197 (2) miR-199a-3p/miR-199b-3p (1)  miR-199b-5p (1) miR-19a (1) miR-19b (1) miR-200c (1) miR-200c* (1) miR-202 (1) miR-20a (2) miR-20a* (1) miR-20b (3) miR-21 (3) miR-21* (1) miR-210 (2) miR-2114* (1) miR-214* (1) miR-219-2-3p (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  104  Appendix I: Mature miRNAs detected in R848-stimulated adult monocytesa (continued) miR-22 (2) miR-22* (2) miR-221 (2) miR-221* (1) miR-222 (2) miR-223 (3) miR-223* (3) miR-2278 (1) miR-23a (3) miR-23a* (1) miR-23b (2) miR-23c (1) miR-24 (3) miR-25 (3) miR-26a (3) miR-26b (3) miR-27a (2) miR-27a* (1) miR-27b (2) miR-28-3p (2) miR-28-5p (2) miR-29a (3) miR-29a* (1) miR-29b (2) miR-29b-1* (1) miR-29c (3) miR-29c* (2) miR-301a (1) miR-301b (1) miR-30a (3) miR-30a* (3) miR-30b (3) miR-30b* (1) miR-30c (3) miR-30d (3) miR-30e (3) miR-30e* (3) miR-3118 (1) miR-3131 (1) miR-3132 (1)  miR-3141 (2) miR-3147 (1) miR-3150a-3p (1) miR-3154 (3) miR-3155/miR-3155b (1) miR-3156-5p (1) miR-3159 (3) miR-3164 (1) miR-3173-3p (2) miR-3180-3p (2) miR-3190 (2) miR-3200-5p (1) miR-3201 (2) miR-320a (3) miR-320b (3) miR-320d (2) miR-320e (1) miR-323b-3p (1) miR-324-3p (1) miR-324-5p (1) miR-326 (2) miR-328 (3) miR-331-3p (2) miR-338-3p (1) miR-339-3p (1) miR-339-5p (1) miR-33a (1) miR-340 (2) miR-340* (3) miR-342-3p (3) miR-342-5p (1) miR-345 (2) miR-346 (1) miR-34a (1) miR-3605-5p (1) miR-3607-3p (3) miR-3607-5p (3) miR-361-5p (3) miR-362-3p (2) miR-362-5p (2)  miR-3622a-5p (1) miR-3622b-3p (2) miR-363 (1) miR-3647-3p (2) miR-365 (2) miR-3651 (3) miR-3653 (2) miR-3654 (3) miR-3655 (1) miR-3667-5p (1) miR-3679-5p (1) miR-3683 (3) miR-3687 (2) miR-3690 (1) miR-3691-5p (2) miR-370 (1) miR-3714 (1) miR-373* (2) miR-374a (1) miR-374a* (1) miR-374b/miR-374c (3) miR-375 (1) miR-378 (2) miR-378* (1) miR-378b (2) miR-380* (2) miR-382 (2) miR-3907 (2) miR-3909 (3) miR-3914 (1) miR-3916 (1) miR-3918 (1) miR-3925-5p (1) miR-3943 (2) miR-421 (1) miR-422a (2) miR-423-3p (2) miR-423-5p (3) miR-424 (2) miR-425 (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  105  Appendix I: Mature miRNAs detected in R848-stimulated adult monocytesa (continued) miR-425* (2) miR-4251 (1) miR-4257 (1) miR-4260 (1) miR-4261 (1) miR-4262 (2) miR-4263 (1) miR-4273 (1) miR-4274 (2) miR-4282 (1) miR-4286 (2) miR-4289 (2) miR-4291 (2) miR-4294 (1) miR-4296 (1) miR-4299 (1) miR-4301 (3) miR-4303 (1) miR-4304 (1) miR-4306 (1) miR-431 (1) miR-4312 (1) miR-432 (1) miR-4323 (1) miR-433 (2) miR-449b* (1) miR-454 (2) miR-484 (2) miR-486-5p (1) miR-487b (1) miR-489 (3) miR-490-3p (3) miR-491-5p (1) miR-495 (1) miR-496 (1) miR-500a* (2) miR-501-3p (2) miR-502-3p (1) miR-505 (2) miR-505* (1)  miR-509-5p (1) miR-532-3p (3) miR-532-5p (2) miR-542-5p (1) miR-548b-5p (1) miR-548m (3) miR-551a (1) miR-556-5p (1) miR-564 (2) miR-573 (1) miR-574-3p (2) miR-575 (2) miR-590-3p (1) miR-590-5p (1) miR-593* (1) miR-596 (1) miR-598 (1) miR-608 (1) miR-610 (1) miR-615-5p (3) miR-627 (1) miR-628-3p (3) miR-628-5p (2) miR-629 (1) miR-631 (1) miR-637 (3) miR-652 (2) miR-656 (2) miR-657 (1) miR-660 (1) miR-661 (2) miR-664 (3) miR-664* (2) miR-676* (3) miR-7 (2) miR-7-1* (1) miR-7-2* (3) miR-720 (3) miR-744 (3) miR-765 (2)  miR-766 (3) miR-770-5p (1) miR-877 (2) miR-877* (1) miR-885-3p (1) miR-887 (1) miR-891a (1) miR-9 (2) miR-9* (2) miR-921 (1) miR-924 (1) miR-92a (3) miR-92b* (1) miR-93 (2) miR-93* (2) miR-937 (1) miR-941 (1) miR-942 (1) miR-98 (3) miR-99b (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  106  Appendix J: Mature miRNAs detected in R848-stimulated CB monocytesa let-7a (2) let-7a* (2) let-7b (1) let-7d (2) let-7d* (1) let-7e (1) let-7f (2) let-7g (2) let-7i (1) let-7i* (2) miR-100 (1) miR-100* (2) miR-101 (3) miR-103a (3) miR-106b (3) miR-106b* (1) miR-107 (1) miR-1207-5p (1) miR-1233 (1) miR-1236 (1) miR-1237 (1) miR-1238 (1) miR-1247 (1) miR-1248 (1) miR-1249 (1) miR-1253 (3) miR-1258 (1) miR-125a-5p (1) miR-125b (1) miR-126 (1) miR-126* (1) miR-1260 (3) miR-1260b (1) miR-128 (1) miR-1280 (3) miR-1281 (2) miR-1290 (1) miR-1301 (1) miR-1307 (2) miR-130a (1) miR-130b (1)  miR-130b* (1) miR-132 (1) miR-1322 (1) miR-140-3p (2) miR-140-5p (1) miR-142-3p (3) miR-142-5p (2) miR-143 (2) miR-145 (2) miR-146a (2) miR-146b-3p (1) miR-146b-5p (2) miR-147 (1) miR-1471 (1) miR-147b (1) miR-148a (2) miR-148b (1) miR-150 (1) miR-151-3p (1) miR-151-5p (2) miR-152 (1) miR-1537 (2) miR-155 (1) miR-15a (2) miR-15a* (1) miR-15b (2) miR-15b* (1) miR-16 (3) miR-16-2* (1) miR-17* (1) miR-17/miR-106a (3) miR-181a (1) miR-181b (1) miR-181c (2) miR-182* (1) miR-185 (2) miR-186 (2) miR-188-3p (1) miR-188-5p (2) miR-18a (2) miR-18b (1)  miR-190b (1) miR-191 (3) miR-191* (1) miR-1911* (1) miR-192 (1) miR-194 (1) miR-195 (3) miR-197 (1) miR-1976 (1) miR-199a-3p/miR-199b-3p (1)  miR-199b-5p (1) miR-19a (3) miR-19b (3) miR-200b (1) miR-200c* (1) miR-205* (1) miR-20a (3) miR-20a* (1) miR-20b (3) miR-21 (3) miR-21* (2) miR-210 (1) miR-2114* (1) miR-22 (3) miR-22* (2) miR-221 (3) miR-221* (1) miR-222 (3) miR-223 (3) miR-223* (1) miR-2355-3p (1) miR-23a (3) miR-23b (1) miR-24 (3) miR-24-2* (2) miR-25 (2) miR-26a (3) miR-26a-1* (1) miR-26b (2) miR-26b* (1) miR-27a (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  107  Appendix J: Mature miRNAs detected in R848-stimulated CB monocytesa (continued) miR-27b (1) miR-28-3p (2) miR-28-5p (2) miR-29a (3) miR-29a* (1) miR-29b (1) miR-29c (3) miR-29c* (3) miR-301a (1) miR-301b (1) miR-3065-3p (1) miR-3074-3p (1) miR-30a (2) miR-30a* (2) miR-30b (3) miR-30c (3) miR-30d (3) miR-30e (2) miR-30e* (1) miR-3118 (1) miR-3130-5p (1) miR-3131 (1) miR-3141 (1) miR-3147 (1) miR-3154 (3) miR-3155/miR-3155b (1) miR-3159 (3) miR-3173-3p (2) miR-3177-3p (1) miR-3190 (1) miR-3200-5p (1) miR-3201 (1) miR-320a (2) miR-320b (2) miR-323-5p (1) miR-324-3p (1) miR-324-5p (1) miR-326 (1) miR-328 (3) miR-330-3p (1)  miR-331-3p (1) miR-335* (1) miR-337-3p (1) miR-337-5p (1) miR-338-3p (1) miR-339-3p (1) miR-339-5p (1) miR-340 (2) miR-340* (1) miR-342-3p (2) miR-345 (1) miR-34a (1) miR-34c-3p (1) miR-3605-5p (1) miR-3607-3p (2) miR-3607-5p (2) miR-361-3p (1) miR-361-5p (2) miR-3613-3p (1) miR-362-3p (1) miR-362-5p (1) miR-3622a-5p (1) miR-363 (1) miR-3647-3p (3) miR-3647-5p (1) miR-365 (1) miR-3651 (3) miR-3653 (2) miR-3654 (1) miR-3676 (1) miR-3683 (2) miR-370 (1) miR-373* (2) miR-374a (2) miR-374a* (2) miR-374b/miR-374c (3) miR-376c (1) miR-378 (1) miR-378* (1) miR-378b (1)  miR-380* (2) miR-382 (2) miR-3907 (2) miR-3909 (1) miR-3916 (1) miR-3926 (1) miR-3943 (1) miR-421 (2) miR-422a (1) miR-423-3p (3) miR-423-5p (1) miR-424 (2) miR-425 (3) miR-425* (1) miR-4286 (3) miR-4289 (1) miR-4290 (1) miR-4291 (2) miR-4299 (1) miR-4301 (3) miR-4323 (1) miR-433 (1) miR-450a (1) miR-451 (1) miR-454 (2) miR-484 (2) miR-486-5p (1) miR-487b (1) miR-489 (3) miR-490-3p (3) miR-495 (2) miR-496 (1) miR-500a (2) miR-500a* (1) miR-501-3p (1) miR-501-5p (1) miR-502-3p (1) miR-502-5p (1) miR-505 (1) miR-532-3p (3)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  108  Appendix J: Mature miRNAs detected in R848-stimulated CB monocytesa (continued) miR-532-5p (1) miR-542-3p (1) miR-542-5p (1) miR-548b-5p (1) miR-548c-5p (1) miR-548d-5p (1) miR-548e (1) miR-548m (2) miR-548q (1) miR-550a (1) miR-574-3p (1) miR-576-5p (1) miR-582-3p (1) miR-582-5p (1) miR-589 (1) miR-590-3p (1) miR-590-5p (1) miR-591 (1) miR-596 (1) miR-615-5p (3) miR-616* (1) miR-617 (2) miR-625* (1) miR-628-3p (2) miR-628-5p (1) miR-634 (1) miR-637 (3) miR-652 (3) miR-656 (2) miR-660 (1) miR-661 (3) miR-664 (2) miR-664* (1) miR-675 (1) miR-675* (1) miR-676* (2) miR-7 (1) miR-7-1* (1) miR-7-2* (3) miR-720 (3)  miR-744 (1) miR-766 (2) miR-874 (1) miR-877* (1) miR-885-5p (1) miR-888 (2) miR-9 (1) miR-92a (3) miR-92b (1) miR-92b* (1) miR-93 (2) miR-93* (1) miR-941 (2) miR-942 (1) miR-98 (1) miR-99a (1) miR-99b (2) miR-99b* (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  109  Appendix K: Mature miRNAs detected in LPS-stimulated adult monocytesa let-7a (3) let-7a* (1) let-7b (3) let-7c (2) let-7d (3) let-7d* (1) let-7e (3) let-7f (3) let-7g (3) let-7i (3) let-7i* (1) miR-100 (1) miR-100* (1) miR-101 (1) miR-103a (2) miR-103a-2* (1) miR-106b (2) miR-107 (1) miR-1184 (1) miR-1203 (1) miR-1207-5p (2) miR-1224-3p (1) miR-1224-5p (1) miR-1228* (1) miR-1233 (2) miR-1238 (2) miR-124* (1) miR-1245 (1) miR-1247 (2) miR-1248 (1) miR-1249 (2) miR-1250 (1) miR-1253 (3) miR-125a-3p (1) miR-125a-5p (2) miR-126 (1) miR-126* (3) miR-1260 (3) miR-1260b (1) miR-127-3p (2) miR-1271 (1)  miR-1277 (1) miR-128 (1) miR-1280 (3) miR-1281 (2) miR-1285 (1) miR-1290 (2) miR-1302 (1) miR-1305 (1) miR-1307 (2) miR-130a (1) miR-130b (3) miR-130b* (3) miR-132 (2) miR-132* (1) miR-1322 (1) miR-135b* (1) miR-136* (1) miR-138-1* (1) miR-139-3p (1) miR-139-5p (1) miR-140-3p (2) miR-140-5p (1) miR-141 (1) miR-142-3p (1) miR-142-5p (2) miR-143 (1) miR-145 (1) miR-146a (2) miR-146b-3p (1) miR-146b-5p (2) miR-147 (1) miR-1471 (1) miR-147b (1) miR-148a (1) miR-148b (1) miR-150 (2) miR-151-3p (1) miR-151-5p (2) miR-152 (2) miR-1537 (1) miR-1539 (1)  miR-155 (2) miR-155* (2) miR-15a (2) miR-15a* (1) miR-15b (3) miR-15b* (3) miR-16 (3) miR-16-1* (1) miR-16-2* (1) miR-17* (2) miR-17/miR-106a (3) miR-181a (2) miR-181a-2* (1) miR-181b (3) miR-181c (1) miR-181c* (1) miR-181d (3) miR-1825 (1) miR-185 (3) miR-186 (1) miR-188-3p (1) miR-188-5p (1) miR-18a (1) miR-18a* (1) miR-18b (1) miR-18b* (1) miR-190b (1) miR-191 (3) miR-191* (1) miR-192 (1) miR-193a-5p (3) miR-193b (1) miR-194 (2) miR-195 (2) miR-196b (1) miR-196b* (1) miR-197 (3) miR-199a-3p/miR-199b-3p (2)  miR-199b-5p (1) miR-19a (2) miR-19b (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  110  Appendix K: Mature miRNAs detected in LPS-stimulated adult monocytesa (continued) miR-200b (1) miR-200c (2) miR-20a (2) miR-20a* (1) miR-20b (2) miR-21 (3) miR-21* (1) miR-210 (1) miR-2113 (1) miR-212 (1) miR-218-1* (1) miR-218-2* (1) miR-22 (3) miR-22* (1) miR-221 (3) miR-221* (1) miR-222 (2) miR-223 (3) miR-223* (2) miR-224* (1) miR-23a (3) miR-23b (3) miR-24 (3) miR-24-2* (1) miR-25 (3) miR-26a (3) miR-26a-2* (1) miR-26b (3) miR-26b* (1) miR-27a (2) miR-27a* (1) miR-27b (1) miR-28-3p (2) miR-28-5p (2) miR-298 (1) miR-29a (2) miR-29a* (1) miR-29b (1) miR-29b-1* (2) miR-29b-2* (1)  miR-29c (2) miR-29c* (2) miR-301a (1) miR-301b (1) miR-302d (1) miR-30a (2) miR-30a* (2) miR-30b (2) miR-30c (3) miR-30d (2) miR-30e (2) miR-30e* (2) miR-3117-3p (1) miR-3130-5p (1) miR-3131 (1) miR-3135 (1) miR-3141 (2) miR-3150a-3p (1) miR-3154 (3) miR-3159 (3) miR-3161 (1) miR-3173-3p (3) miR-3176 (1) miR-3190 (2) miR-3191 (1) miR-3193 (1) miR-32 (1) miR-3200-5p (1) miR-3201 (2) miR-320a (3) miR-320b (2) miR-320d (2) miR-320e (1) miR-323-5p (1) miR-323b-5p (1) miR-324-3p (2) miR-324-5p (1) miR-326 (1) miR-328 (3) miR-330-3p (1)  miR-331-3p (1) miR-331-5p (1) miR-335 (1) miR-338-3p (1) miR-339-3p (2) miR-339-5p (1) miR-33a (1) miR-33a* (1) miR-33b (1) miR-340 (1) miR-340* (2) miR-342-3p (3) miR-342-5p (1) miR-345 (1) miR-346 (1) miR-34a (2) miR-34a* (1) miR-34b* (1) miR-3605-5p (1) miR-3607-3p (2) miR-3607-5p (3) miR-361-3p (1) miR-361-5p (3) miR-362-3p (1) miR-362-5p (1) miR-363 (1) miR-3647-3p (2) miR-3647-5p (1) miR-365 (2) miR-3651 (2) miR-3653 (2) miR-3654 (1) miR-3661 (1) miR-3676 (1) miR-3683 (2) miR-3691-5p (1) miR-370 (1) miR-373* (2) miR-374a (2) miR-374a* (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  111  Appendix K: Mature miRNAs detected in LPS-stimulated adult monocytesa (continued) miR-374b* (1) miR-374b/miR-374c (3) miR-375 (1) miR-376b (1) miR-376c (1) miR-378 (3) miR-378* (1) miR-378b (2) miR-380* (3) miR-382 (2) miR-3907 (2) miR-3909 (3) miR-3923 (1) miR-411 (1) miR-421 (1) miR-422a (2) miR-423-3p (2) miR-423-5p (2) miR-424 (2) miR-425 (3) miR-425* (1) miR-4258 (1) miR-4267 (3) miR-4286 (2) miR-4289 (1) miR-4291 (2) miR-4296 (1) miR-4301 (3) miR-4302 (1) miR-4304 (2) miR-431 (1) miR-431* (1) miR-4318 (1) miR-433 (3) miR-449a (1) miR-449b (1) miR-449b* (1) miR-450a (1) miR-454 (2) miR-455-3p (1)  miR-455-5p (1) miR-483-5p (1) miR-484 (3) miR-486-3p (1) miR-486-5p (2) miR-487b (2) miR-489 (3) miR-490-3p (3) miR-491-5p (1) miR-493 (1) miR-495 (1) miR-500a (1) miR-500a* (2) miR-501-3p (2) miR-501-5p (2) miR-502-3p (1) miR-502-5p (1) miR-505 (2) miR-505* (2) miR-506 (1) miR-532-3p (3) miR-532-5p (1) miR-545* (1) miR-548b-5p (1) miR-548d-5p (1) miR-548m (2) miR-555 (1) miR-556-5p (1) miR-564 (1) miR-570 (1) miR-573 (3) miR-574-3p (2) miR-576-5p (2) miR-579 (1) miR-582-5p (1) miR-590-3p (1) miR-590-5p (1) miR-596 (1) miR-598 (1) miR-608 (1)  miR-610 (1) miR-615-5p (2) miR-616 (1) miR-616* (1) miR-623 (1) miR-624* (1) miR-625* (1) miR-627 (1) miR-628-3p (3) miR-628-5p (1) miR-629 (2) miR-631 (1) miR-637 (3) miR-640 (1) miR-650 (1) miR-651 (1) miR-652 (2) miR-656 (3) miR-659 (1) miR-660 (2) miR-661 (3) miR-664 (3) miR-664* (2) miR-675* (2) miR-676* (2) miR-7 (3) miR-7-1* (1) miR-7-2* (3) miR-708 (1) miR-720 (3) miR-744 (3) miR-765 (1) miR-766 (3) miR-770-5p (2) miR-877 (2) miR-877* (1) miR-9 (1) miR-9* (1) miR-92a (3) miR-92b (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  112  Appendix K: Mature miRNAs detected in LPS-stimulated adult monocytesa (continued) miR-92b* (2) miR-93 (2) miR-93* (1) miR-937 (1) miR-941 (1) miR-942 (3) miR-98 (3) miR-99a (1) miR-99b (2)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  113  Appendix L: Mature miRNAs detected in LPS-stimulated CB monocytesa let-7a (3) let-7a* (1) let-7b (3) let-7b* (1) let-7c (1) let-7d (3) let-7d* (1) let-7e (1) let-7f (3) let-7f-1* (1) let-7f-2* (1) let-7g (3) let-7g* (1) let-7i (3) let-7i* (2) miR-100 (1) miR-100* (1) miR-101 (3) miR-103a (3) miR-103a-2* (1) miR-105* (1) miR-106b (3) miR-106b* (1) miR-107 (2) miR-1180 (1) miR-1182 (1) miR-1183 (1) miR-1184 (1) miR-1197 (1) miR-1203 (1) miR-1207-5p (1) miR-1224-5p (1) miR-1226 (1) miR-1227 (1) miR-1228* (1) miR-1233 (1) miR-1236 (1) miR-1237 (1) miR-1238 (1) miR-124 (1) miR-1247 (1)  miR-1248 (2) miR-1249 (1) miR-1253 (3) miR-1256 (1) miR-125a-3p (1) miR-125a-5p (1) miR-125b (1) miR-125b-2* (1) miR-126 (1) miR-126* (1) miR-1260 (3) miR-1260b (1) miR-1262 (1) miR-1263 (1) miR-127-3p (1) miR-1271 (1) miR-1273d (1) miR-1278 (1) miR-128 (1) miR-1280 (3) miR-1281 (2) miR-1285 (2) miR-1286 (1) miR-1289 (1) miR-129* (1) miR-1290 (1) miR-1291 (2) miR-1294 (1) miR-1296 (1) miR-1301 (1) miR-1303 (1) miR-1305 (1) miR-1307 (2) miR-130a (1) miR-130b (2) miR-130b* (1) miR-132 (1) miR-132* (1) miR-1322 (1) miR-136 (1) miR-136* (2)  miR-138-1* (1) miR-138-2* (1) miR-139-3p (1) miR-139-5p (1) miR-140-3p (3) miR-140-5p (2) miR-142-3p (3) miR-142-5p (3) miR-143 (1) miR-145 (1) miR-145* (1) miR-1468 (1) miR-146a (2) miR-146b-5p (1) miR-147 (1) miR-1471 (2) miR-147b (1) miR-148a (1) miR-148b (2) miR-150 (1) miR-151-3p (1) miR-151-5p (1) miR-152 (1) miR-1537 (1) miR-1538 (1) miR-1539 (1) miR-154* (1) miR-155 (1) miR-155* (1) miR-15a (3) miR-15a* (1) miR-15b (3) miR-15b* (1) miR-16 (3) miR-16-1* (1) miR-16-2* (1) miR-17* (1) miR-17/miR-106a (3) miR-181a (3) miR-181a-2* (1) miR-181a* (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  114  Appendix L: Mature miRNA detected in LPS-stimulated CB monocytesa (continued) miR-181b (2) miR-181c (2) miR-181d (1) miR-1825 (1) miR-183* (1) miR-185 (2) miR-186 (2) miR-187 (1) miR-187* (1) miR-188-3p (1) miR-188-5p (1) miR-18a (3) miR-18a* (1) miR-18b (1) miR-18b* (1) miR-190 (1) miR-1908 (1) miR-1909* (1) miR-190b (1) miR-191 (3) miR-191* (1) miR-1910 (1) miR-1912 (1) miR-1914* (1) miR-192 (1) miR-193a-3p (1) miR-193a-5p (1) miR-193b (1) miR-193b* (1) miR-194 (1) miR-194* (1) miR-195 (3) miR-195* (1) miR-196b (1) miR-196b* (1) miR-197 (3) miR-1976 (1)  miR-19b (3) miR-200a* (1) miR-200b (1) miR-200b* (1) miR-200c (1) miR-200c* (1) miR-20a (3) miR-20a* (1) miR-20b (3) miR-20b* (1) miR-21 (3) miR-21* (2) miR-210 (2) miR-2110 (1) miR-2113 (1) miR-2114* (1) miR-2116 (1) miR-2117 (1) miR-212 (1) miR-218-1* (1) miR-219-2-3p (1) miR-22 (3) miR-22* (1) miR-221 (3) miR-221* (1) miR-222 (3) miR-223 (3) miR-223* (1) miR-224 (1) miR-2278 (1) miR-2355-3p (1) miR-23a (3) miR-23a* (1) miR-23b (2) miR-24 (3) miR-24-2* (2) miR-25 (3) miR-199a-3p/miR-199b-3p (2) miR-26a (3) miR-199b-5p (2) miR-26a-2* (1) miR-19a (3) miR-26b (3)  miR-26b* (1) miR-27a (3) miR-27a* (1) miR-27b (2) miR-28-3p (1) miR-28-5p (3) miR-297 (1) miR-298 (1) miR-299-5p (1) miR-29a (3) miR-29a* (1) miR-29b (3) miR-29b-1* (1) miR-29b-2* (1) miR-29c (3) miR-29c* (3) miR-300 (1) miR-301a (1) miR-301b (1) miR-302d (1) miR-3065-3p (1) miR-3065-5p (1) miR-30a (3) miR-30a* (1) miR-30b (3) miR-30c (3) miR-30d (3) miR-30d* (1) miR-30e (3) miR-30e* (2) miR-31 (1) miR-3120-3p (1) miR-3130-5p (1) miR-3131 (2) miR-3135 (2) miR-3137 (1) miR-3141 (1) miR-3146 (1) miR-3147 (1) miR-3150a-3p (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  115  Appendix L: Mature miRNAs detected in LPS-stimulated CB monocytesa (continued) miR-3150b-3p (1) miR-3154 (2) miR-3156-5p (1) miR-3157-5p (1) miR-3158-3p (1) miR-3159 (2) miR-3160-3p (1) miR-3161 (1) miR-3164 (1) miR-3173-3p (3) miR-3176 (1) miR-3177-3p (1) miR-3179 (1) miR-3180-3p (1) miR-3185 (1) miR-3186-3p (1) miR-3190 (3) miR-3191 (1) miR-3199 (1) miR-32 (1) miR-32* (1) miR-3200-5p (2) miR-3201 (3) miR-320a (1) miR-320b (1) miR-320d (1) miR-320e (1) miR-323-5p (1) miR-323b-3p (1) miR-323b-5p (1) miR-324-3p (2) miR-324-5p (3) miR-326 (1) miR-328 (3) miR-329 (1) miR-330-3p (1) miR-330-5p (1) miR-331-3p (2) miR-331-5p (1) miR-335 (1)  miR-335* (1) miR-337-3p (1) miR-338-3p (3) miR-339-3p (2) miR-339-5p (2) miR-33a (1) miR-33a* (1) miR-340 (3) miR-340* (1) miR-342-3p (2) miR-342-5p (1) miR-345 (1) miR-346 (1) miR-34a (2) miR-34c-3p (1) miR-3605-5p (2) miR-3607-3p (3) miR-3607-5p (2) miR-361-3p (1) miR-361-5p (2) miR-3611 (1) miR-3613-3p (1) miR-362-3p (1) miR-362-5p (1) miR-3622a-5p (2) miR-3622b-3p (2) miR-363 (1) miR-3647-3p (2) miR-3647-5p (1) miR-365 (3) miR-3651 (3) miR-3652 (2) miR-3653 (3) miR-3654 (1) miR-3655 (1) miR-3661 (1) miR-3666 (1) miR-3667-5p (1) miR-3673 (1) miR-3676 (1)  miR-3682-3p (2) miR-3683 (3) miR-3685 (1) miR-3687 (1) miR-3689a-5p/miR-3689b/miR-3689e (1)  miR-369-3p (1) miR-3691-5p (1) miR-3692 (1) miR-370 (1) miR-3714 (1) miR-373* (3) miR-374/ miR-374c (2) miR-374a (2) miR-374a* (1) miR-374b* (1) miR-376a* (1) miR-376c (1) miR-378 (2) miR-378* (1) miR-378b (2) miR-378c (1) miR-379 (1) miR-380* (3) miR-381 (1) miR-382 (3) miR-3907 (1) miR-3909 (1) miR-3914 (2) miR-3915 (1) miR-3916 (1) miR-3918 (1) miR-3921 (1) miR-3923 (1) miR-3926 (1) miR-3941 (1) miR-3943 (1) miR-409-3p (1) miR-409-5p (1) miR-410 (1) miR-411 (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  116  Appendix L: Mature miRNAs detected in LPS-stimulated CB monocytesa (continued) miR-412 (1) miR-421 (2) miR-422a (1) miR-423-3p (2) miR-423-5p (2) miR-424 (3) miR-424* (1) miR-425 (3) miR-425* (1) miR-4252 (1) miR-4257 (1) miR-4258 (1) miR-4261 (1) miR-4263 (1) miR-4264 (1) miR-4267 (1) miR-4274 (1) miR-4279 (1) miR-4282 (1) miR-4286 (3) miR-4289 (2) miR-429 (1) miR-4291 (1) miR-4296 (1) miR-4298 (1) miR-4299 (1) miR-4301 (3) miR-4302 (1) miR-4304 (1) miR-431 (1) miR-431* (1) miR-4317 (1) miR-432 (1) miR-432* (1) miR-4323 (1) miR-4325 (1) miR-433 (2) miR-448 (1) miR-449a (1) miR-449b* (2)  miR-450a (1) miR-451 (1) miR-454 (1) miR-454* (1) miR-455-3p (1) miR-455-5p (1) miR-483-3p (1) miR-483-5p (1) miR-484 (3) miR-485-3p (2) miR-486-5p (1) miR-487b (1) miR-489 (3) miR-490-3p (3) miR-491-5p (1) miR-493 (1) miR-493* (1) miR-495 (1) miR-496 (1) miR-499-5p (1) miR-500a (1) miR-500a* (1) miR-501-3p (1) miR-501-5p (1) miR-502-3p (1) miR-502-5p (1) miR-505 (1) miR-505* (1) miR-506 (1) miR-508-5p (1) miR-509-3p (1) miR-515-5p (1) miR-519d (1) miR-520d-3p (1) miR-532-3p (3) miR-532-5p (2) miR-542-3p (1) miR-542-5p (1) miR-545 (1) miR-545* (1)  miR-548aa (1) miR-548b-3p (1) miR-548b-5p (3) miR-548c-5p (1) miR-548d-3p (1) miR-548d-5p (1) miR-548e (1) miR-548i (1) miR-548j (1) miR-548k (2) miR-548l (1) miR-548m (3) miR-548q (1) miR-548s (1) miR-548v (1) miR-549 (1) miR-551a (1) miR-551b* (1) miR-564 (1) miR-566 (1) miR-569 (1) miR-573 (1) miR-574-3p (2) miR-576-3p (1) miR-576-5p (1) miR-579 (1) miR-582-5p (1) miR-590-3p (1) miR-590-5p (2) miR-591 (1) miR-596 (1) miR-598 (2) miR-599 (1) miR-608 (1) miR-610 (1) miR-613 (1) miR-615-5p (2) miR-616* (1) miR-617 (1) miR-624* (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  117  Appendix L: Mature miRNAs detected in LPS-stimulated CB monocytesa (continued) miR-625* (1) miR-627 (1) miR-628-3p (3) miR-628-5p (1) miR-629 (1) miR-629* (1) miR-631 (1) miR-634 (2) miR-637 (3) miR-642a (1) miR-646 (1) miR-650 (1) miR-651 (1) miR-652 (2) miR-655 (1) miR-656 (1) miR-659 (1) miR-660 (3) miR-661 (2) miR-662 (1) miR-664 (3) miR-664* (2) miR-668 (1) miR-670 (1) miR-675 (1) miR-675* (1) miR-676* (3) miR-7 (1) miR-7-1* (1) miR-7-2* (3) miR-720 (3) miR-744 (1) miR-744* (1) miR-765 (2) miR-766 (1) miR-769-5p (1) miR-770-5p (1) miR-874 (1) miR-877 (1) miR-877* (1)  miR-885-5p (1) miR-887 (2) miR-888 (3) miR-891a (1) miR-9 (1) miR-9* (1) miR-92a (3) miR-92a-1* (1) miR-92b (1) miR-92b* (1) miR-93 (3) miR-93* (2) miR-941 (2) miR-942 (1) miR-943 (1) miR-98 (2) miR-99a (1) miR-99a* (1) miR-99b (3) miR-99b* (1)  a  Number in parentheses indicate the number of donors in which the miRNA was detected  118  

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