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A7161 - DNA Methylation as a Driver of the Neonatal CD4+ T Cell Response to Pneumonia
Author Block: B. D. Singer1, K. Helmin1, S. Chen1, K. Anekalla1, R. Ndeh2, J. Collaco3, F. D'Alessio3, S. McGrath-Morrow4; 1Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, United States, 2Johns Hopkins University, Baltimore, MD, United States, 3Johns Hopkins Hosp, Baltimore, MD, United States, 4Pediatric Pulmonary, Johns Hopkins School of Medicine, Baltimore, MD, United States.
Rationale: Neonates with pneumonia have poorer outcomes than older children. CD4+ T cells, including T helper and regulatory T (Treg) cells, play a critical role in coordinating the immune response to pediatric pneumonia in murine models. We hypothesized that DNA methylation—a developmentally dynamic regulator of T cell identity and function—drives a unique CD4+ T cell program in neonates compared with juveniles exposed to lower respiratory tract E. coli. Methods: Male neonatal (~5 days) and juvenile (~15 days) C57BL/6NJ mice received 2.8 million colony forming units of E. coli or PBS via aspiration. Lung tissue was harvested 48 hours later for CD4+ T cell isolation by magnetic bead pre-enrichment and fluorescence-activated cell sorting. RNA and genomic DNA were purified and subjected to transcriptional profiling (RNA-sequencing) and genome-wide DNA methylation profiling (modified reduced representation bisulfite sequencing). Differential gene expression and methylation analysis were performed using the R/Bioconductor packages edgeR and DSS as well as the SeqMonk platform with downstream analysis performed in R. Results: Neonates suffered higher mortality than juveniles. We identified 3,932 differentially expressed genes across the four groups using a q-value cutoff of 0.05 in an ANOVA-like test. Unbiased techniques revealed an attenuated lung CD4+ T cell transcriptional response to pneumonia among neonates compared with juveniles. In contrast to neonates, juveniles up-regulated a robust set of immune response genes, including important pathway components of Th1, Th17, and Treg cells. DNA methylation profiling demonstrated 28,623 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. Association analysis revealed that gene promoter methylation and gene expression carried an inverse correlation. Based on these data, we performed methylation difference filtering for promoter CpGs with a methylation directionality (hyper- or hypo-methylated by 25%) opposite to the corresponding gene expression directionality (down- or up-regulated) between groups. This process revealed 643 genes whose promoter methylation status passed the filter. Of these 643 genes, we identified important Th1, Th17, and Treg cell pathway components including Tbx21, Ahr, Ikzf3, and Ikzf4. Conclusions: Compared to juveniles, neonatal mice display poorer outcomes and a limited lung CD4+ T cell transcriptional response to pneumonia. DNA methylation within the promoters of a core set of T cell response genes is statistically likely to regulate the maladaptive gene expression pattern seen in neonates. Future work with pharmacologic and epigenetic editing tools may help uncover novel therapeutic pathways for pediatric pneumonia.