Su1885 Colitis Vaccine: Flagellin-Elicited Immunity Keeps Motile Bacteria In-Check and Protects Against Intestinal Inflammation

Su1884 Influence of the Microbiome on Epigenetic Mechanisms in Inflammatory Bowel Disease (IBD) Tiago Medina, Alexander J. Murison, Sebastian Scheer, Michelle I. Smith, Mark S. Silverberg, Colby Zaph, Mathieu Lupien, Cheryl Arrowsmith, Daniel DeCarvalho Inflammatory Bowel Disease is thought to occur as a result of a complex interplay between genetics and environmental factors such as the intestinal microbiota, which leads to a dysregulated immune response. Here, we describe a model that allowed us to evaluate the importance of early exposure of the offspring to their mother`s microbiota and determined how microbiota may alter the epigenetic program of infiltrating CD4 T cells. In this model, the microbiota of Foxp3-EGFP reporter breading mice was depleted with an antibiotic cocktail from the time of their weaning through the weaning on their offspring. The use of antibiotics prevented the transfer of a conventional maternal microbiota to the offspring who are then separated from the mother at weaning and given normal drinking water. At 8 weeks of age the offspring were sacrificed, their splenic naïve CD4 T cells were isolated via FACS and transferred into RAG1-/- mice, which lack T and B cells. A control group of breading mice were not given antibiotics and the splenic naïve CD4 T cells of their offspring were also transfer into RAG1-/- mice. RAG1-/- mice that received a T cell adoptive transfer from the offspring of antibiotic treated parents experienced intense gut inflammation as measured by histology and weight loss, while those that received the control group cells did not. This suggests that the inflammation was due to a microbiome difference and not the transfer itself. As further evidence, cohousing the offspring of antibiotic-treated and nontreated parents prior to adoptive transfer restored the microbiota of the treated offspring, as determined by qPCR of dominant taxa, and prevented their RAG1-/- recipient mice from developing intestinal inflammation. CD4 T cells from the offspring of antibiotic treated mothers strongly proliferated after in vitro stimulation compared to the control group. With the transfer of CD4 T cells into RAG1-/- mice, we saw a drastic change in the methylome between treated naïve CD4 T cells prior to transfer and those recovered from the RAG1-/mice after transfer, a change not observed in the control group naïve CD4 T cells. Preventing exposure of the offspring to their mother's microbiota by maternal antibiotic treatment was also able to modulate epigenetic changes in the CD4 T cells of the offspring, promoting the hypermethylation of anti-inflammatory genes, such as IL-10RB, ICOSL, IL-4R, HLA-G SOCS1, GRP83 (Treg inductor) and NKIRAS2 (NF- kB inhibitor) and the hypomethylation of pro-inflammatory genes, such as IFNG, IFNAR2, IL-17RB, IL-15RA, IL1RL1, GZMB, CD27, CD70, ALCAM (adhesion molecule), CCR4 and CCR5. In conclusion, the exposure of the offspring to their mother's microbiota during the parturition induces an epigenetic program capable of repressing pro-inflammatory genes and induces tolerogenic genes in CD4 T cells, preventing the inflammatory outcome.

Fig. 1. Circadian variation in microbiome in mice under control (CTRL, C), light SF (Light, L) and dark SF (Dark, D). Total reads for all microbiota (A). Select genera across conditions (B).

Su1885 Fig. 2. Ten days of light SF markedly suppresses mesenteric lymph node (MLN) cytokine gene expression. MLNs were pooled (4-5 mice/group), RNA isolated and cytokine profiles determined by qPCR array. Data are mean fold change relative to control (non-SF) mice for CCR5 and its ligands (A), Cxcr5/Cxcl13 (B) and multifunctional cytokine Aimp1 (C).

Colitis Vaccine: Flagellin-Elicited Immunity Keeps Motile Bacteria In-Check and Protects Against Intestinal Inflammation Benoit Chassaing, Hao Tran, Andrew T. Gewirtz BACKGROUND: Inflammatory Bowel Disease (IBD) is driven by a breakdown in the normally mutually beneficial host-microbiota relationship. Manipulating the host portion of this relationship, particularly antagonizing immune-promoting cytokines, has improved IBD management. We recently reported that disturbance of the intestinal microbiota using three different approaches (infectious agent, genetic predisposition and environmental factor) leads to a microbiota with increased pro-inflammatory potential and development of intestinal inflammation. Approaches to impact the microbiota to ameliorate IBD are not well developed despite the appreciation that microbiota composition has a great impact on disease outcome. GOAL: We hypothesize that manipulating the microbiota so as to make it inherently less pro-inflammatory (i.e. reduce levels of innate immune activators) may ultimately provide a novel approach to prevent and/or treat chronic intestinal inflammation. METHODS: Mice were treated with either PBS or purified flagellin (10µg) weekly by intraperitoneal injection. Feces were collected weekly and used for downstream analysis: fecal flagellin and LPS via cell-based reporter assay, inflammatory marker lipocalin-2 via ELISA, fecal and serum antiflagellin IgA and IgG via ELISA, and microbiota composition via 16S RNA Illumina Miseq sequencing. 12 weeks following the initial treatment, mice were either euthanized and intestinal samples processed to measure the intestinal inflammatory state (histopathology via hematoxylin & eosin staining, myeloperoxidase enzymatic activity, bacterial localization via confocal microscopy), or subjected to anti-IL-10 receptor treatment before tissue collection. RESULTS: Weekly treatment of mice with flagellin led to a strong induction of serum anti-flagellin IgG and fecal anti-flagellin IgA. Moreover, such immunization against bacterial flagellin was associated with strong modification of fecal microbiota composition that associated with a decrease in pro-inflammatory potential, as revealed by a decrease in the level of fecal bioactive flagellin. Such flagellin immunization and microbiota composition alteration protected mice against intestinal inflammation induced by anti-IL-10 receptor antibody treatment, as revealed by decreased spleen weight as well as decrease colon weight/length ratio in immunized animals compared to PBS-treated group. CONCLUSIONS: Flagellin inoculation alters microbiota composition and decreases level of fecal flagellin, and associated with protection against intestinal inflammation development. The mucosal immune system holds the power to reshape the microbiota and protect against inflammatory bowel disease.

Su1883 Loss of Tumor Necrosis Factor Receptor 1 Induces Severe Early-Onset Colitis in IL10 Knockout Mice in a Microbiome-Dependent Manner Sharon S. Tam, Philip E. Dubé, Rabea Alhosh, Shivesh Punit, Nandini Girish, Kay Washington, D. Brent Polk Tumor necrosis factor (TNF) is a therapeutic target in inflammatory bowel disease (IBD) and TNF receptor 1 (TNFR1) polymorphisms have been described in IBD patients. In fact, synergistic effects between TNFR1 and interleukin 10 (IL10) variants in early-onset IBD have been described. Although TNF and TNFR1 have been implicated in IBD pathogenesis, TNF also provides protective physiological roles in host defense and inflammatory resolution, suggesting that TNFR1 may regulate inflammatory responses to the commensal microbiome in IBD. Il10-/- mice provide a spontaneous model of colitis driven by the lack of immune tolerance to normal gut microbiota. We hypothesized that TNFR1 protects against colitis in Il10-/- mice by restraining dysbiosis and inflammation. Il10-/-Tnfr1+/- mice were crossed to generate Il10-/-Tnfr1-/- mice and control Il10-/-Tnfr1+/+ littermates. Co-housed mice underwent colonoscopy and barrier function assay (FD4 absorption) prior to euthanasia at 8 wk of age. A separate group of 8 wk-old Il10-/-Tnfr1-/- mice were treated with either antibiotics (neomycin and metronidazole) or water for 2 wk. Mice underwent colonoscopy before and after antibiotics, and endoscopic appearance was scored. Colon tissues were analyzed for histologic scoring, and mucosal immune cell populations were characterized by flow cytometry. Il10-/-Tnfr1-/- mice developed severe colitis in a highly reproducible manner by 8 wk of age, evidenced by both endoscopic and histologic scoring (p<0.01). Il10-/-Tnfr1-/- mice presented with extreme mucosal thickening and granularity by colonoscopy, and florid inflammation, crypt abscesses, marked enterocyte loss, and epithelial hyperplasia by histology, whereas Il10-/- littermates appeared healthy. FD4 absorption was increased in Il10-/Tnfr1-/- mice (p<0.0001). There were increases in colonic macrophages (F4/80+) and dendritic cells (MHCII+CD11c+) in mice lacking TNFR1. Furthermore, survival was reduced in Il10-/Tnfr1-/- mice vs. Il10-/- littermates (p<0.001), with 50% mortality by 12 wk. Antibiotics significantly improved colitis in Il10-/-Tnfr1-/- mice, inducing remission with reduced endoscopic (p<0.01) and histologic (p<0.01) scores. Antibiotics decreased mononuclear cells in

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the lamina propria, crypt abscesses, and hyperplasia. Il10-/-Tnfr1-/- mice develop early-onset severe colitis and antibiotic depletion rapidly induces remission. TNFR1 may inhibit colitis by restraining dysbiosis and mucosal immune responses, potentially macrophages and/or dendritic cells. Studies will determine how TNFR1 regulates disparate cell types in colitis and how these impact host-microbial interactions and which microbial factors determine disease severity in this model. Understanding how TNFR1 contributes to protection from colitis will lead to nuanced therapeutic avenues for IBD, particularly for early-onset IBD.