Large Intestinal Lamina Propria Research Articles

Toll-like receptor 7 protects against intestinal inflammation and restricts the development of colonic tissue-resident memory CD8+ T cells.

The maintenance of intestinal homeostasis depends on a complex interaction between the immune system, intestinal epithelial barrier, and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover, persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA viruses, and ATG16L1, which facilitates the delivery of viral nucleic acids to the autolysosome endosome, are required for anti-viral immune responses. However, the role of the enteric virome in IBD is still poorly understood. Here, we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state, Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice, reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore, Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index, histoscore, and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. In conclusion, the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants.

A43 MECHANISMS OF UNIQUE COLONIZATION DYNAMICS AND IMMUNE RESPONSES TO MURIBACULACEAE

Abstract Background Commercially available probiotics have a limited ability to alter the gut microbiome and establish within a pre-existing microbial community. Increases in osmolality, a condition prevalent in intestinal diseases, depletes the highly abundant bacterial family Muribaculaceae (Mb) from the gut microbiome in a mouse model of short-term osmotic laxative use. Excitingly, despite microbial community re-equilibration in its absence, Mb supersedes other bacterial colonizers to its original abundance if reintroduced, suggesting unique colonization attributes and a potential privileged immune interaction. Aims We aimed to investigate whether and how Mb colonization affects intestinal innate and adaptive immune homeostasis, including the generation of Mb-specific antibodies. Methods To examine interactions between Mb and the immune system that allows for efficient colonization, we used the osmotic laxative polyethylene glycol (PEG) to clear Mb and then generated mice with complex microbiomes that either lack Mb completely (Mb naïve) or were introduced to 8 murine-derived Mb strains (Mb+). The same strains were then introduced to Mb naïve mice (primary exposure to the immune system). To model secondary exposure, Mb+ mice were treated with PEG, and Mb re-introduced 3 weeks later. Leukocytes from the small and large intestinal lamina propria and gut-draining mesenteric lymph nodes (mLNs) were profiled over 21 days to characterize colonization and recolonization dynamics. Results High-throughput 16SrRNA sequencing showed no significant difference between relative abundance of Mb isolates during colonization or recolonization, indicating that Mb is capable of establishing in mice that have never been colonized with it before, making up to 20% of the relative abundance. Broad immunophenotyping demonstrated robust CD11b-CD103+ dendritic cell and Germinal Centre B cell responses in the mLNs of mice seeing Mb for the first time, yet no subsequent plasmablast accumulation in the mLN or lamina propria. We also observed significantly increased type two innate lymphoid cells in the small intestine upon primary exposure to Mb. Recolonization elicited no detectable changes in the monitored populations. Conclusions The lack of immune reactivity upon reintroduction to Mb suggests Mb may evade immune detection upon repeat exposure and contribute to its robust colonization abilities. Determining mechanisms involved in Mb colonization and its impact on host immune responses could increase our understanding of colonization dynamics and highlights the potential of Mb as a model for microbiota reintroduction in therapeutic applications. Funding Agencies Weston Family Foundation

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

A173 ELUCIDATING THE EFFECTS OF NOD2-MEDIATED SIGNALLING ON INTESTINAL RESIDENT-MEMORY T-CELL FORMATION AND FUNCTION

Abstract Background Aberrant resident memory T-cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T-cell responses. However, the role of NOD2 in establishing memory T-cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Purpose It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific T-cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Method To address the effects of NOD2-signalling on intestinal T-cell priming in vivo, wildtype (WT) mice were adoptively transferred 50,000 naïve LCMV-specific (SMARTA) CD4+ T-cells. Mice were subsequently infected with LCMV-Armstrong in the presence or absence of the NOD2 agonist; MDP. 5-days following infection, the numbers and percentage of LCMV-specific T-cells in the mesenteric lymph nodes and spleen were examined. To examine the effects of NOD2 on intestinal TRM generation, littermate WT and NOD2 KO mice were infected with LCMV-Armstrong. Thirty-six-days following infection, the percentage and number of LCMV-specific CD4+ T-cells were profiled in the small and large intestinal lamina-propria by means of gp66-77 class-II MHC-tetramer staining. In another set of experiments, littermate WT and NOD2 KO mice were re-infected with LCMV-C13 30-days following LCMV-Armstrong immunization, and the interferon-response in the small intestine was profiled by quantitative PCR to assess the effect of NOD2-deficiency on antigen recall responses. Result(s) NOD2-stimulation by means of MDP injection increased the percentage and number of adoptively transferred SMARTA CD4+ T-cells in the mesenteric lymph nodes upon LCMV infection. Furthermore, NOD2-deficiency did not alter intestinal LCMV-specific CD4+ TRM seeding in the small and large intestinal lamina propria 36 days after infection. However, in vivo antigen recall experiments showed a decreased intestinal IFN response in NOD2 KO mice. Conclusion(s) Our findings reveal a potential role of NOD2 in the intestinal CD4+ T-cell priming and subsequent Ag-specific memory response. Disclosure of Interest None Declared

Oral Pathobionts Promote MS-like Symptoms in Mice

Dysbiotic oral microbiota has been associated with multiple sclerosis. However, the role and mechanism of oral microbiota in the development of multiple sclerosis are still elusive. Here, we demonstrated that ligature-induced periodontitis (LIP) aggravated experimental autoimmune encephalomyelitis (EAE) in mice, and this was likely dependent on the expansion of T helper 17 (Th17) cells. LIP increased the splenic richness of Enterobacter sp., which was able to induce the expansion of splenic Th17 cells and aggravate EAE in mice. LIP also led to enrichment of Erysipelotrichaceae sp. in the gut and increased Th17 cells in the large intestinal lamina propria of EAE mice. Fecal microbiota transplantation from EAE mice with LIP also promoted EAE symptoms. In conclusion, periodontitis exacerbates EAE, likely through ectopic colonization of oral pathobionts and expansion of Th17 cells.

Rap1 prevents colitogenic Th17 cell expansion and facilitates Treg cell differentiation and distal TCR signaling

T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+ Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+ Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+ T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+ T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.

A Soluble Fiber Diet Increases Bacteroides fragilis Group Abundance and Immunoglobulin A Production in the Gut.

Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Previous studies have shown that Bacteroidetes, the major phylum of gut microbiota together with Firmicutes, impact IgA production. However, the relative abundances of species of Bacteroidetes responsible for IgA production were not well understood. In the present study, we identified some specific Bacteroidetes species that were associated with gut IgA induction by hsp60-based profiling of species distribution among Bacteroidetes The levels of IgA and the expression of the gene encoding activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were increased in soluble high-fiber diet (sHFD)-fed mice. We found that Bacteroides acidifaciens was the most abundant Bacteroidetes species in both sHFD- and normal diet-fed mice. In addition, the gut IgA levels were associated with the relative abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciens Conversely, the ratio of B. acidifaciens to other Bacteroidetes species was reduced in insoluble high-fiber diet fed- and no-fiber diet-fed mice. To investigate whether B. acidifaciens increases IgA production, we generated B. acidifaciens monoassociated mice and found increased gut IgA production and AID expression. Collectively, soluble dietary fiber increases the ratio of gut Bacteroides fragilis group, such as B. acidifaciens, and IgA production. This might improve gut immune function, thereby protecting against bowel pathogens and reducing the incidence of inflammatory bowel diseases.IMPORTANCE Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Gut microbiota impact IgA production, but the specific species responsible for IgA production remain largely elusive. Previous studies have shown that IgA and Bacteroidetes, the major phyla of gut microbiota, were increased in soluble high-fiber diet-fed mice. We show here that the levels of IgA in the gut and the expression of activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were correlated with the abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciensB. acidifaciens monoassociated mice increased gut IgA production and AID expression. Soluble dietary fiber may improve gut immune function, thereby protecting against bowel pathogens and reducing inflammatory bowel diseases.

Toll-like receptor 7 protects against intestinal inflammation and restricts the development of tissue-resident memory CD8+ T cells

Abstract The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier, and the immune system. Alteration in of one of these components could lead to the development of inflammatory bowel diseases (IBD). Norovirus infection of mice with a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal inflammation. Moreover, persistent norovirus infection leads to intestinal virus-specific CD8+ T cells responses. However, the role of the enteric virome in IBD is still poorly understood. Toll-like receptor 7 (TLR7) recognizes single-stranded RNA viruses. Here, we investigate the role of TLR7 in intestinal homeostasis and inflammation. At steady state, Tlr7−/− mice have an approx. 10-fold increase in small and large intestinal lamina propria (LP) granzyme B+ tissue-resident memory (Trm) CD8+ T cells compared to WT mice (WT: 5.5, Tlr7−/−: 59.5 %, p < 0.005), reminiscent of persistent norovirus infection. Furthermore, Tlr7−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe inflammation (Histoscore: WT: 7.6, Tlr7−/−: 12.7, p < 0.005), increased disease activity index (WT: 5.5, Tlr7−/−: 7.4, p < 0.05), and increased secretion of IFNg (WT: 5.2, Tlr7−/−: 24.2 ng/ml, p < 0.005) and TNFα (WT: 108.6, Tlr7−/−: 191.8 pg/ml, p < 0.05). Increased colonic inflammation was associated with increased LP Trm CD8+ T cells (WT: 3.9, Tlr7−/−: 42.0 %, p < 0.005). Our data shows that TLR7-deficiency promotes the development of LP Trm CD8+ T cells and increases susceptibility to DSS colitis. In conclusion, TLR7 plays an important role in maintaining immune response to intestinal viruses and protects against development of colitis.

P160 γδ T CELLS, IL-22, THE PROBIOTIC, LACTOBACILLUS DELBRUECKII, AND THE ROLE THEY PLAY IN ATTENUATION OF ALCOHOL INDUCED EXACERBATION OF DSS-COLITIS

Abstract Ulcerative colitis is characterized by cycles of active disease flare and inactive disease remission. During UC remission, IL-22 expression can be upregulated, acting as a hallmark of entrance into a UC remission period. Recently, we found that in our mouse model of binge alcohol consumption after DSS-induced colitis, alcohol increases severity of UC flare symptoms. In this study, we assessed whether alcohol influenced IL-22 expression and thereby perpetuates UC flare. Male C57BL/6 mice received 2% DSS or water ad libitum for 5 days. On day 5, DSS was removed to mimic entrance into remission. Additionally on day 5, DSS and Sham mice were subdivided into mice gavaged with ethanol (∼3g/kg) or with water on days 5, 6, and 7. Three hours after the last gavage on day 7, mice were humanely euthanized. Large intestine lamina propria (LP) cells were isolated. The percentage of total IL-22+ LP cells was significantly decreased (p<0.05) in DSS Ethanol compared to DSS Vehicle. No differences in IL-22+ T cells, Innate Lymphoid Cells Type 3, or neutrophils were observed. Examination of I γδ T cells revealed DSS Vehicle treated mice had a significantly increased percentage of IL-22+ γδ T cells, while DSS Ethanol treated mice were unable to mount this response. Therefore, we hypothesized that by re-establishing IL-22, through either rIL-22 or a probiotic, we could alleviate the alcohol-induced exacerbation of UC. Firstly, rIL-22 administration substantially restored weight loss of DSS Ethanol treated mice back to that of DSS Vehicle (∼12.5% back to ∼6% on day 7). Increased colonic shortening (p<0.001) and increased Enterobacteriaceae copy number were also attenuated following binge alcohol and colitis with IL-22 treatment. Knockout of STAT3 in IECs resulted in loss of IL-22 protection, demonstrating STAT3 is required for protection. Secondly, we utilized Lactobacillus delbrueckii, a common probiotic known to play a role in IL-22 release. Treatment with Lacto attenuated both weight loss and colon length in DSS Ethanol mice back to levels of DSS alone (p<0.01 and p<0.001, respectively). Additionally, Lacto treatment mitigated increases in Enterobacteriaceae copy number seen in DSS Ethanol mice and trended towards an increase in IL-22 in DSS Ethanol + Lacto mice. Levels of pSTAT3 were decreased in DSS Ethanol treated mice compared to DSS Vehicle, but administration of Lacto in DSS Ethanol mice increased levels of pSTAT3 back to that of the DSS Vehicle group. Treatment with Lacto supernatant alone was not sufficient to mitigate the exacerbation of UC following ethanol. Our findings suggest that both rIL-22 and Lactobacillus delbrueckii utilize the IL-22/pSTAT3 signaling pathway to attenuate alcohol-induced increases in ulcerative colitis symptoms. (R21AA022324, R21AA025806, T32AA013527, F30AA027442, F31AA025536)

P160 γδ T CELLS, IL-22, THE PROBIOTIC, LACTOBACILLUS DELBRUECKII, AND THE ROLE THEY PLAY IN ATTENUATION OF ALCOHOL INDUCED EXACERBATION OF DSS-COLITIS

Ulcerative colitis is characterized by cycles of active disease flare and inactive disease remission. During UC remission, IL-22 expression can be upregulated, acting as a hallmark of entrance into a UC remission period. Recently, we found that in our mouse model of binge alcohol consumption after DSS-induced colitis, alcohol increases severity of UC flare symptoms. In this study, we assessed whether alcohol influenced IL-22 expression and thereby perpetuates UC flare. Male C57BL/6 mice received 2% DSS or water ad libitum for 5 days. On day 5, DSS was removed to mimic entrance into remission. Additionally on day 5, DSS and Sham mice were subdivided into mice gavaged with ethanol (∼3g/kg) or with water on days 5, 6, and 7. Three hours after the last gavage on day 7, mice were humanely euthanized. Large intestine lamina propria (LP) cells were isolated. The percentage of total IL-22+ LP cells was significantly decreased (p<0.05) in DSS Ethanol compared to DSS Vehicle. No differences in IL-22+ T cells, Innate Lymphoid Cells Type 3, or neutrophils were observed. Examination of I γδ T cells revealed DSS Vehicle treated mice had a significantly increased percentage of IL-22+ γδ T cells, while DSS Ethanol treated mice were unable to mount this response. Therefore, we hypothesized that by re-establishing IL-22, through either rIL-22 or a probiotic, we could alleviate the alcohol-induced exacerbation of UC. Firstly, rIL-22 administration substantially restored weight loss of DSS Ethanol treated mice back to that of DSS Vehicle (∼12.5% back to ∼6% on day 7). Increased colonic shortening (p<0.001) and increased Enterobacteriaceae copy number were also attenuated following binge alcohol and colitis with IL-22 treatment. Knockout of STAT3 in IECs resulted in loss of IL-22 protection, demonstrating STAT3 is required for protection. Secondly, we utilized Lactobacillus delbrueckii, a common probiotic known to play a role in IL-22 release. Treatment with Lacto attenuated both weight loss and colon length in DSS Ethanol mice back to levels of DSS alone (p<0.01 and p<0.001, respectively). Additionally, Lacto treatment mitigated increases in Enterobacteriaceae copy number seen in DSS Ethanol mice and trended towards an increase in IL-22 in DSS Ethanol + Lacto mice. Levels of pSTAT3 were decreased in DSS Ethanol treated mice compared to DSS Vehicle, but administration of Lacto in DSS Ethanol mice increased levels of pSTAT3 back to that of the DSS Vehicle group. Treatment with Lacto supernatant alone was not sufficient to mitigate the exacerbation of UC following ethanol. Our findings suggest that both rIL-22 and Lactobacillus delbrueckii utilize the IL-22/pSTAT3 signaling pathway to attenuate alcohol-induced increases in ulcerative colitis symptoms. (R21AA022324, R21AA025806, T32AA013527, F30AA027442, F31AA025536)

TNFRSF25 and CD25 Stimulation Expands Tregs and ILC2s in the GI Tract: Recipient Modulation Pre-HSCT

Our lab has shown that using a 2-pathway strategy of stimulating TNFRSF25 and CD25 with a TL1A-Ig fusion protein (FP) and low dose IL-2, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased. Tregs and innate lymphoid cells (ILCs) are critical to maintaining immune homeostasis and generating tolerance in the GI tract. In addition to a highly diverse population of Tregs, the GI also contains various populations of ILCs, recently shown to modulate GVHD (Bruce, 2017). ILCs express CD25 as well as TNFRSF25 and respond individually to IL-2 and TL1A administration respectively. Here we asked how our 2-pathway (FP+IL-2) approach affected Tregs and ILCs in the GI of healthy mice. First, we examined Tregs from B6-Nur77^GFP or B6-Nur77^GFPFoxP3^RFP mice to assess the Treg TcR signaling status in different tissues. Mice were administered FP, IL-2, or FP+IL-2 over 1 wk. The frequency of Treg/CD4 in the lamina propria (LP) of the large intestine (LI) reached levels >60% (1A). This elevation included FoxP3⁺RORyt⁻ and FoxP3⁺RORyt⁺ Tregs. In contrast to our previous findings in the LHC, treatment with FP alone elevated levels to the same extent as the FP+IL-2 in the LI/LP, whereas IL-2 treatment alone had only a modest effect on elevating the frequency of Tregs (1A), suggesting that the activation status of Tregs differs based on compartmental location. The frequency of GFP⁺ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LHC (<35%) (1C). An independent experiment verified these findings and found that only conjunctival Tregs had a similar activation (GFP) phenotype (1D). Interestingly, without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon (1B). Next, we evaluated FP+IL-2 treated B6-Nur77^GFP mice. Tregs - <i>but not CD8 T-cells</i> in the LHC had elevated GFP by frequency and MFI vs untreated mice (1E). Notably, we also found that FP+IL-2 treatment <i>in vivo</i> increases the relative frequency of both ILC2s and ILC3s (1F). Regarding ILC2s, FP+IL-2 increased their frequency greater than either TNFRSF25 or CD25 stimulation alone in the GI tract (1G). We then asked if our FP+IL-2 approach could be used to augment these populations as a potential strategy to protect HSCT recipients. FP+IL-2 stimulation prior to TBI in BALB/c mice significantly augmented Tregs and ILC2s in the GI tract for at least 1 wk post-irradiation (2A,2B). In total, these findings demonstrate 1) basal Treg activation status differs depending on the compartment and mucosal Tregs express highest TcR activation, 2) FP alone vs. FP+IL-2 stimulation results in equivalent expansion of GI Tregs, 3) only FP+IL-2 effectively expand ILC2s and 4) pre-TBI FP+IL-2 treatment results in elevated Treg and ILC2 levels post-irradiation. Ongoing experiments are examining GI GVHD and the role of Tregs/ILCs in TNFRSF25 +/- CD25 stimulated aHSCT recipients.

The Location of CD4+FoxP3+ Cells with Regard to CD25 and TNFRSF25 Receptor Signals Matters: Different Requirements for GI and Peripheral Tregs

Regulatory T cells (Tregs) are critical to maintaining immune homeostasis and generating tolerance in the gastrointestinal (GI) tract. GI complications play a prominent role following allogeneic HSCT (aHSCT) and we are interested in manipulating GI Tregs to regulate GVHD. Previously our lab has shown that using a two-pathway strategy of stimulating the TNFRSF25 and CD25 receptors with a TL1A-Ig fusion protein (FP) and IL-2low dose, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased (Wolf, BBMT 2017; Copsel, BBMT 2018). As a consequence of microbes and food antigens, Treg populations in the GI have a relative highly diverse composition compared to the lymphohematopoietic Treg compartment. This includes, but is not limited to, the presence of a stable and suppressive FoxP3+RORyt+ Treg population. Additionally, the GI tract contains various populations of innate lymphoid cells (ILCs) that interact with Tregs. ILCs express CD25 as well as TNFRSF25, and respond individually to IL-2 and TL1A administration respectively (Danny, JCI 2017; Verneris, Blood 2013). Based on the extensive differences between the lymphohematopoietic and GI Treg compartments, we evaluated how our two-pathway strategy may be differentially affecting the levels and activation status of Tregs in the GI tract. To address this question, we initially utilized B6-Nur77GFP mice, where GFP expression occurs with activation of the Nur77 promoter. However, since Nur77 is only produced following TcR engagement and not inflammatory signals, the strength of TcR stimulation in all T cell populations can be monitored by GFP levels. We first examined Tregs from 2-pathway treated B6-Nur77GFP mice and subsequently generated B6-Nur77GFPFoxP3RFP mice to readily assess the TcR signaling status of Tregs in different compartments. Mice were systemically administered FP, IL-2, or FP + IL-2 over a 1 wk period. The frequency of Tregs (FoxP3+CD4+) / Tcon (CD4+FoxP3-) in the lamina propria (LP) of the large intestine (LI) reached levels &gt;60%. (1A). This elevation of Treg / Tcon frequency included FoxP3+RORyt- Tregs as well as FoxP3+RORyt+ double positive Treg populations. In contrast to our previous findings in the LHC, treatment with TL1A-Ig FP alone elevated levels to the same extent as the combination of FP + IL-2 (1A). Importantly, IL-2 treatment alone - as reported in the LHC - again had only a modest effect on elevating the frequency of Tregs in the LI/LP (1A). These observations suggest that the activation status of Tregs may differ based on the compartmental location. To asses activation status, we evaluated 2-pathway treated B6-Nur77GFP mice. Tregs in the LN/spleen had elevated frequency of GFP+ Tregs and higher GFP MFI than untreated mice. This elevated TcR stimulation was present in peripheral Tregs - but not CD8 - T cells (1B). Without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon cells (1B). The frequency of GFP+ Tregs and the GFP MFI was clearly highest (&gt;45%) in the SI and LI LP vs. LN/spleen (&lt;35%) (1C). In an independent experiment, we verified these findings and learned that only conjunctival Tregs demonstrated similarly increased frequency of GFP+ Tregs with elevated GFP MFI (1D). We also found that TL1A-Ig + IL-2 treatment in vivo increases the relative frequency of both innate ILC2 and ILC3 lymphoid populations (2A). The combination also increased the frequency of ILC2 cells greater than either reagent alone in the GI tract (2B). In total, our results objectively validate selective Treg vs. Tcon targeting via TNFRSF25. These findings also demonstrate that basal Treg activation status differs depending on the compartment. Notably, Tregs in mucosal vs. LHC tissue expressed higher TcR activation levels. Such Tregs have the potential for co-stimulation via TNFRSF25. Moreover, since IL-2 is required to maintain Tregs under both basal and activated conditions, our findings suggest a local source of IL-2 is present to maintain GI Tregs. ILC3 cells are recently reported to generate IL-2 (Zhou L, Nature 2019). We hypothesize that TNFRSF25 stimulation in ILC3 cells results in a local source of IL-2 which can account for our finding that TL1A-Ig alone vs. TL1A-Ig + IL-2 stimulation results in equivalent levels of GI Tregs. Ongoing experiments are examining how GI GVHD in aHSCT recipients are affected by TNFRSF25 +/- CD25 Treg/ILC stimulation. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Thymically-derived Foxp3+ regulatory T cells are the primary regulators of type 1 diabetes in the non-obese diabetic mouse model.

Regulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets. Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation. Deletion of CNS1 impaired in vitro induction of Foxp3 in naïve NOD CD4+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in RORγt+ Tregs and corresponding increase in Helios+ Tregs was observed in NOD CNS1-/- mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1-/- mice. Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.

Murine Fecal Microbiota Transplantation Alleviates Intestinal and Systemic Immune Responses in Campylobacter jejuni Infected Mice Harboring a Human Gut Microbiota.

Human campylobacteriosis constitutes a zoonotic food-borne disease and a progressively rising health burden of significant socioeconomic impact. We have recently shown that conventional mice are protected from Campylobacter jejuni infection, which was not the case for human microbiota associated (hma) mice indicating that the host-specific gut microbiota composition primarily determines susceptibility to or resistance against C. jejuni infection. In our present preclinical intervention study we addressed whether gut microbiota changes in stably C. jejuni infected hma mice following murine fecal microbiota transplantation (mFMT) could alleviate pathogen-induced immune responses. To accomplish this, secondary abiotic C57BL/6 mice were generated by broad-spectrum antibiotic treatment, perorally reassociated with a complex human gut microbiota and challenged with C. jejuni by gavage. Seven days later C. jejuni infected hma mice were subjected to peroral mFMT on 3 consecutive days. Within a week post-mFMT fecal pathogenic burdens had decreased by two orders of magnitude, whereas distinct changes in the gut microbiota composition with elevated numbers of lactobacilli and bifidobacteria could be assessed. In addition, mFMT resulted in less C. jejuni induced apoptotic responses in colonic epithelia, reduced numbers of macrophages and monocytes as well as of T lymphocytes in the large intestinal mucosa and lamina propria and in less distinct intestinal pro-inflammatory cytokine secretion as compared to mock challenge. Strikingly, inflammation dampening effects of mFMT were not restricted to the intestinal tract, but could also be observed systemically as indicated by elevated serum concentrations of pro-inflammatory cytokines such as TNF-α, IL-12p70, and IL-6 in C. jejuni infected hma mice of the mock, but not the mFMT cohort. In conclusion, our preclinical mFMT intervention study provides evidence that changes in the gut microbiota composition which might be achieved by pre- or probiotic formulations may effectively lower intestinal C. jejuni loads, dampen both, pathogen-induced intestinal and systemic inflammatory sequelae and may represent a useful tool to treat continuous shedding of C. jejuni by asymptomatic carriers which is critical in the context of food production, hospitalization and immunosuppression.

The Combination of Bifidobacterium breve and Three Prebiotic Oligosaccharides Modifies Gut Immune and Endocrine Functions in Neonatal Mice

Several types of oligosaccharides are used in infant formula to improve the gut microbiota of formula-fed infants. We previously reported that a combination of 3 oligosaccharides (lactulose, raffinose, and galacto-oligosaccharides; LRG) and Bifidobacterium breve effectively increased B. breve numbers, acetate, and the expression of several immune- and gut hormone-related mRNAs in neonatal mice gut. We investigated whether changes in neonatal gut microbiota alter gut immune and endocrine development. We first compared postnatal day (PD) 14 with PD21 in C57BL/6J male mouse pups to identify the physiologic immune and endocrine changes during development. In a separate study, we administered phosphate-buffered saline (control group; CON), B. breve M-16V (M-16V), or M-16V+LRG to male mouse pups from PD6 to PD13, and analyzed the gut microbiota and immune and endocrine parameters on PD14 to evaluate whether M-16V+LRG accelerates gut immune and endocrine development. The proportion of regulatory T (Treg) cells in the CD4+ cells of large intestinal lamina propria lymphocytes (LPLs) was significantly increased (63% higher) at PD21 compared with PD14. The serum glucagon-like peptide (GLP)-1 tended to be lower (P=0.0515) and that of GLP-2 was significantly lower (58% lower) at PD21 than at PD14. M-16V+LRG significantly increased the Treg proportion in large intestinal LPL CD4+ cells (20% and 29% higher compared with CON and M-16V, respectively) at PD14. M-16V+LRG also caused significant changes in expression of large intestinal mRNAs that are consistent with developmental progression, and increased serum concentrations of GLP-1 (207% and 311% higher compared with CON and M-16V, respectively) and GLP-2 (57% and 97% higher compared with CON and M-16V, respectively) at PD14. Neonatal administration of M-16V+LRG alters the gut microbiota and enhances gut immune and endocrine development in suckling mice.

Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function.

Dendritic cells (DCs), primary antigen-presenting cells, are now well known as an immunoregulator of many aspects of immune responses including inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis. We have reported that PIR-A/Bhigh cDCs (conventional DCs) appeared in dextran sodium sulfate (DSS)-induced colitis and serve as a negative immunoregulator in an animal model of IBD. The immunoregulatory role of PIR-A/B+ cDCs was confirmed in both an in vitro culture system and an in vivo transfer experiment. Here, we have investigated the differentiation process of PIR-A/B+ cDCs in an in vitro inflammatory environment and examined their functions. cDCs were isolated from the large intestinal lamina propria from C57BL/6 mice and cultured in an inflammatory environment (IL-1, IL-6, TNFα, and LPS). The appearance of PIR-A/B+ cDCs was determined after 24h, and the in vitro-induced PIR-A/B+ cDCs were functionally and genetically examined. PIR-A/B+ cDCs were detected after a 24-h culture only in the inflammatory environment, and the cells acted as a negative immunoregulator when examined in an allogenic mixed leukocyte reaction (MLR). The message level of IL-27 was highly upregulated in PIR-A/B+ cDCs, while that of high mobility group box 1 protein (HMGB1) was downregulated in these cells. This was well in accordance with the fact that PIR-A/B+ cDCs showed a suppressive function against activated T cells. We found that PIR-A/B+ cDCs produced IL-27, as verified by an ELISA assay, and that the inhibitory effect by PIR-A/B+ cDCs was, at least partially, due to IL-27. Furthermore, CD85d+ cells, a human counterpart of mouse PIR-A/B+ cDCs, were found in the lamina propria of the colon of the patients with ulcerative colitis, but not in the similar part of the non-inflammatory area of colon specimens from patients with colon cancer. PIR-A/B+ cDCs induced in an in vitro inflammatory environment model showed a suppressive function against activated T cells by producing an inhibitory cytokine.

The Probiotic Compound VSL#3 Modulates Mucosal, Peripheral, and Systemic Immunity Following Murine Broad-Spectrum Antibiotic Treatment.

There is compelling evidence linking the commensal intestinal microbiota with host health and, in turn, antibiotic induced perturbations of microbiota composition with distinct pathologies. Despite the attractiveness of probiotic therapy as a tool to beneficially alter the intestinal microbiota, its immunological effects are still incompletely understood. The aim of the present study was to assess the efficacy of the probiotic formulation VSL#3 consisting of eight distinct bacterial species (including Streptococcus thermophilus, Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, and L. delbrueckii subsp. Bulgaricus) in reversing immunological effects of microbiota depletion as compared to reassociation with a complex murine microbiota. To address this, conventional mice were subjected to broad-spectrum antibiotic therapy for 8 weeks and perorally reassociated with either VSL#3 bacteria or a complex murine microbiota. VSL#3 recolonization resulted in restored CD4+ and CD8+ cell numbers in the small and large intestinal lamina propria as well as in B220+ cell numbers in the former, whereas probiotic intervention was not sufficient to reverse the antibiotic induced changes of respective cell populations in the spleen. However, VSL#3 application was as efficient as complex microbiota reassociation to attenuate the frequencies of regulatory T cells, activated dendritic cells and memory/effector T cells in the small intestine, colon, mesenteric lymph nodes, and spleen. Whereas broad-spectrum antibiotic treatment resulted in decreased production of cytokines such as IFN-γ, IL-17, IL-22, and IL-10 by CD4+ cells in respective immunological compartments, VSL#3 recolonization was sufficient to completely recover the expression of the anti-inflammatory cytokine IL-10 without affecting pro-inflammatory mediators. In summary, the probiotic compound VSL#3 has an extensive impact on mucosal, peripheral, and systemic innate as well as adaptive immunity, exerting beneficial anti-inflammatory effects in intestinal as well as systemic compartments. Hence, VSL#3 might be considered a therapeutic immunomodulatory tool following antibiotic therapy.

Mastering gut permeability: New roles for old friends.

Mast cells are innate immune cells that respond rapidly to infection in barrier tissues such as the skin and intestinal mucosa. Expulsion of parasitic worms in the gut involves a robust type 2 host response, and an acute mastocytosis is often generated at the site of infection. However, the role of mast cells in resistance to worm infections appears to be parasite specific. Mast cells are also involved in tissue repair, but the long-term contribution of mast cell activation after worm expulsion has not been definitively studied. In this issue of European Journal of Immunology, Sorobetea etal. [Eur. J. Immunol. 2017. 47: 257-268] demonstrate that activated mast cells persist in the large intestinal lamina propria and intraepithelial compartment long after worm expulsion, resulting in continued local and systemic presence of the mast cell protease mast cell protease 1 (MCPt-1) and enhanced intestinal permeability. In this commentary, we discuss these findings in the wider context of mast cell function in health and disease.

Abstract A080: Balance of commensal bacteria specific Th17 and RORγt+ Treg cells in intestinal homeostasis and inflammation

Abstract A number of Helicobacter species, including Helicobacter hepaticus, have been isolated as the etiological agents of ulcerative inflammatory bowel disease and cancer in their respective hosts. H. hepaticus induces severe Th17-Th1 mediated colitis in a number of immuno-compromised mouse strains, but does not usually cause disease in most wild-type mouse strains. However, how the inflammatory response is carefully contained to be harmless under homeostatic conditions and how perturbations lead to pathogenicity remain elusive questions. To understand this, we developed new research tools, including TCR transgenic mice, as well as MHCII tetramers to interrogate the regulation of H. hepaticus-specific CD4+ T cell function. Interestingly, in contrast to Segmented Filamentous Bacteria (SFB), which induces Th17 cells in the small intestinal lamina propria (SILP) of both IL10+/− and IL-10−/− mice, H. hepaticus mainly induces RORgt+ Treg cells in the large intestinal lamina propria (LILP) of IL-10-sufficient mice, but induces a Th17-Th1 response in the absence of IL-10. We propose that the balance between self/commensal antigen-induced RORgt+ Treg and Th17 cells may be a general strategy to control autoimmune inflammation. Note: This abstract was not presented at the conference. Citation Format: Mo Xu, Yi Yang, Maria Pokrovskii, Carolina Galan, Dan R. Littman. Balance of commensal bacteria specific Th17 and RORγt+ Treg cells in intestinal homeostasis and inflammation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A080.

Disturbance in the Mucosa-Associated Commensal Bacteria Is Associated with the Exacerbation of Chronic Colitis by Repeated Psychological Stress; Is That the New Target of Probiotics?

Psychological stress can exacerbate inflammatory bowel disease. However, the mechanisms underlying how psychological stress affects gut inflammation remain unclear. Here, we focused on the relationship between changes in the microbial community of mucosa-associated commensal bacteria (MACB) and mucosal immune responses induced by chronic psychological stress in a murine model of ulcerative colitis. Furthermore, we examined the effect of probiotic treatment on exacerbated colitis and MACB composition changes induced by chronic psychological stress. Repeated water avoidance stress (rWAS) in B6-Tcra-/- mice severely exacerbated colitis, which was evaluated by both colorectal tissue weight and histological score of colitis. rWAS treatment increased mRNA expression of UCN2 and IFN-γ in large intestinal lamina propria mononuclear cells (LI-LPMC). Interestingly, exacerbated colitis was associated with changes in the microbial community of MACB, specifically loss of bacterial species diversity and an increase in the component ratio of Clostridium, revealed by 16S rRNA gene amplicon analysis. Finally, the oral administration of a probiotic Lactobacillus strain was protective against the exacerbation of colitis and was associated with a change in the bacterial community of MACB in rWAS-exposed Tcra-/- mice. Taken together, these results suggested that loss of species diversity in MACB might play a key role in exacerbated colitis induced by chronic psychological stress. In addition, probiotic treatment may be used as a tool to preserve the diversity of bacterial species in MACB and alleviate gut inflammation induced by psychological stress.