Transcriptome-wide profiling of the neonatal monocyte response to E. coli and S. epidermidis Emma de Jong, B.Sc. (Hons) This thesis is presented for the degree of Doctor of Philosophy School of Veterinary & Life Sciences 2015 Declaration I declare that this thesis is my own account of my research, and contains as its main content work that has not previously been submitted for a degree at any other tertiary education institution. .................................... (Emma de Jong) i ii Abstract Preterm infants are extremely vulnerable to life-threatening invasive infections (particularly with Staphylococcus epidermidis and Escherichia coli) however our understanding of their innate immune defences is limited. Furthermore, prenatal exposure to histologic chorioamnionitis (HCA) complicates 40–70% of preterm births and is known to modulate the risk for sepsis, yet the impact of HCA on the development of innate immunity is largely unknown. We hypothesised that inadequate monocyte activation by neonatal pathogens results in impaired innate immune responses thereby increasing preterm infants’ susceptibility to invasive infection, and that prenatal exposure to HCA overrides these impairments. Cell sorting and bacterial stimulation methodologies were developed and optimised specifically for working with human infant cord blood samples. RNA-sequencing was performed on purified cord blood monocytes from very preterm (≤31 weeks gestational age (GA)) and term infants (37–40 weeks GA) following challenge with live S. epidermidis or E. coli to identify gene/pathway differences specific to the preterm infant. Protein levels of inflammatory cytokines and chemokines were measured in paired monocyte culture supernatants. Preterm infants displayed a quantitative monocyte deficiency compared to term infants, manifesting as reduced frequencies of classical monocytes with significantly reduced CD14 expression. However monocytes from preterm infants did not exhibit an intrinsically deficient transcriptional or protein response to stimulation with either pathogen. Prenatal exposure to HCA resulted in the transcriptional reprograming of a subset of genes towards a hyporesponsive phenotype in response to S. epidermidis, but not E. coli. The major transcriptional changes induced by either pathogen were highly conserved across infant groups and between stimuli, highlighting a conserved neonatal monocyte response to infection that was largely mediated by pattern recognition receptor/NF-B signalling. In addition, we observed an interferon/anti-viral immune signature that was specific to monocyte stimulation with E. coli. This is the first transcriptome-wide analysis of the neonatal monocyte response to E. coli and S. epidermidis. This data provides novel insights into the functionality of preterm and term infant monocytes and confirms that exposure to HCA may impact on the development on neonatal immunity. iii iv Table of contents Declaration………………………..……………………………………………………...i Abstract…………………………………………………………………………………iii Acknowledgements……………………………………………………………………..ix Abbreviations…………………………………………………………………………...xi List of Figures……………………………………………………………………….…xiv List of Tables……………………...……………………………………………….….xvii CHAPTER 1 LITERATURE REVIEW ............................................................................................... 1 1.1 AN INTRODUCTION TO PRETERM BIRTH .......................................................................................... 1 1.1.1 Epidemiology ......................................................................................................................... 1 1.1.2 The significance of preterm birth ........................................................................................... 2 1.1.3 Risk factors for preterm birth................................................................................................. 3 1.2 INFECTIONS AND PRETERM BIRTH ................................................................................................... 3 1.2.1 Intrauterine infection and inflammation ................................................................................ 4 1.2.2 Early-onset neonatal sepsis ................................................................................................... 6 1.2.3 Late-onset neonatal sepsis ..................................................................................................... 6 1.3 PATHOGENESIS OF NEONATAL SEPSIS ............................................................................................. 8 1.3.1 Regulation of inflammation in neonatal sepsis ...................................................................... 9 1.3.2 Pathogen-specific responses to sepsis pathogens ................................................................ 11 1.4 DEVELOPMENT OF NEONATAL INNATE IMMUNE DEFENCES ARE CRITICAL FOR PROTECTION AGAINST BACTERIAL PATHOGENS ........................................................................................................... 11 1.4.1 Maturation of innate immunity in neonates ......................................................................... 13 1.4.2 Monocytes and immune-regulation ...................................................................................... 17 1.5 HOW DOES EXPOSURE TO HISTOLOGIC CHORIOAMNIONITIS IMPACT NEONATAL MONOCYTE FUNCTION? ............................................................................................................................................. 27 1.6 TRANSCRIPTIONAL PROFILING OF IMMUNE RESPONSES IN SEPSIS .................................................. 28 1.6.1 DNA microarrays and RNA sequencing .............................................................................. 28 1.6.2 Relevant transcriptomics studies in the field of sepsis and innate immunity ....................... 30 1.7 SUMMARY .................................................................................................................................... 32 1.8 AIMS AND HYPOTHESES OF THIS THESIS........................................................................................ 33 CHAPTER 2 MATERIALS AND METHODS ................................................................................. 35 2.1 STUDY PARTICIPANTS ................................................................................................................... 35 2.2 BLOOD SAMPLE COLLECTION AND PROCESSING ............................................................................ 35 2.3 MICROBIOLOGY ............................................................................................................................ 36 2.3.1 Strain selection .................................................................................................................... 36 2.3.2 Preparation of bacterial broths ........................................................................................... 36 2.3.3 Growth and storage of live mid-log bacterial stocks ........................................................... 36 v 2.3.4 Determining bacterial viability of frozen stocks ................................................................... 37 2.4 CELL CULTURE METHODOLOGY .................................................................................................... 37 2.4.1 Thawing of cryopreserved mononuclear cells ...................................................................... 37 2.4.2 CBMC surface receptor staining .......................................................................................... 38 2.4.3 Purification of monocytes by cell sorting ............................................................................. 39 2.4.4 Monocyte stimulation with live E. coli or S. epidermidis ..................................................... 41 2.4.5 Quantitative detection of cytokines/chemokines in culture supernatants ............................. 41 2.5 CELL DEATH ASSAYS ..................................................................................................................... 43 2.5.1 LDH assay ............................................................................................................................ 43 2.5.2 Apoptosis assessment by flow cytometry .............................................................................. 43 2.6 MOLECULAR STUDIES ................................................................................................................... 44 2.6.1 Purification of total RNA from cultured monocytes ............................................................. 44 2.6.2 cDNA synthesis ..................................................................................................................... 44 2.6.3 Primer design ....................................................................................................................... 44 2.6.4 Conventional PCR for primer optimisation .......................................................................... 45 2.6.5 Sequencing of PCR products ................................................................................................ 45 2.6.6 Real-time polymerase chain reaction ................................................................................... 47 2.7 NEXT-GENERATION SEQUENCING OF MRNA ................................................................................. 48 2.7.1 Experimental design ............................................................................................................. 48 2.7.2 RNA sequencing by AGRF ...................................................................................................