Sophie Kirschner

• Environmental exposures during susceptible early-life developmental periods can have the ability to model and shape individuals immunological responses in adulthood. This has been partly demonstrated in animal models, highlighting the long-term consequences of early-life exposure to bacterial infection in adulthood but the mechanisms driving and maintaining the immune early life programming are not yet fully understood. In this thesis, we investigated the epigenetic mechanisms, mainly DNA methylation, controlling the establishment/maintenance of early-life programming by bacterial and viral exposure in mice. Initially, it was important to carefully consider the sequencing method which would deliver a genome-wide DNA methylation profile of high quality to be able to identify LPS-programming specific methylation signature. For this purpose, we investigated the cutting pattern of restriction enzymes used for reduced representation CpG sequencing in order to control the number of dynamically regulated CpG sites interrogated. CpGs in CGI and shelf/shore could be enriched by the enzymes MspI, HhaI and BstUI, particularly in gene bodies for all genomic regions, promoters (TSS1500, TSS200), intra- (1st exon, gene body, 3’UTR, 5’UTR) and inter-genic regions. The enzyme HpyCH4IV mainly enriched CpG elements in the open sea for all genomic elements. This study allowed us to make an informed decision about the enzymes best fitted to identify DNA methylation patterns associated with LPS and H1N1-programming. To understand the mechanisms controlling the establishment/maintenance of early-life programming in mice, we characterised LPS-programmed lymphocytes in vivo and in vitro. A single exposure to LPS at post-natal day 14 elicited stable, sex specific, long-term hypo responsiveness of both the in-vivo and ex-vivo immune response to a homotypic LPS re-exposure in adulthood. In addition, both the HPA and HPG axes were concurrently programmed with blunted corticosterone after an acute stress and reduced circulating testosterone levels. Genome wide DNA methylation analysis identified a similar number of hyper- and hypo-methylated loci in LPS-programmed mice, spread across the genome specifically in intragenic regions. The programming associated phenotype was trans-generationally inherited in an oscillatory manner to the two subsequent generations. This suggests compensation efforts from the offspring phenotype to match and best adapt to their living environment. Several sperm miRNAs were found differentially expressed in LPS-programmed males, suggesting a probable route for the transmission of programming to next generations. Next, we adopted a similar approach to investigate the consequences of neonatal exposure to influenza virus. Viral respiratory tract infections are highly prevalent during early-life and have a long-lasting, profound, impact on both neurodevelopment and the subsequent risk for developing allergy and asthma. Little is known about the long-term effects on the innate immune system. BALB/c mice exposed to Influenza A (H1N1, A/Puerto Rico/8/1934) at PND14 had increased serum IL-6, MIP-β and RANTES (p<0.05) when re-exposed to H1N1 in adulthood, confirming long-term immune programing. Similar effects were observed after re-stimulating H1N1 programed mice with polyI:C and homotypic polyI:C programming and re-stimulation, suggesting preferential programming of the innate immune system. Reduced-representation epigenome sequencing identified a network of methylation changes common to both early life polyI:C and H1N1 programming, leaving a mechanistic ‘trace’ that remained visible throughout life. Taken together our data suggest that neonatal exposure to infections induces an early activation of the innate immune system which coincides with cellular developmental peaks, hence epigenetically and transcriptionally programming cellular functions until adulthood. Early-life infection with either LPS or H1N1 was shown to lead to profound DNA methylation and transcriptional re-programming of immune cells. The pathway and network analysis of differentially methylated genes revealed that the immune system was at the centre of a number of transcriptionally modified clusters, in accordance with the diverse consequences of early-life LPS programming on the immune, neurological and hormonal systems.