Ontogeny of RORγt+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis

Background Neonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated. Results We examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity. Conclusions Our data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

The small and large intestine contain transcriptionally related mesenchymal stromal cell subsets that derive from embryonic Gli1+ mesothelial cells

Intestinal fibroblasts (FB) play essential roles in intestinal homeostasis. Here we show that the small and large intestinal lamina propria (LP) contain similar FB subsets that locate in specific anatomical niches and express distinct arrays of epithelial support genes. However, there were tissue specific differences in the transcriptional profile of intestinal FB subsets in the two sites. All adult intestinal LP mesenchymal stromal cells (MSC), including FB, smooth muscle cells (SMC) and pericytes derive from Gli1-expressing embryonic precursors which we identify as mesothelial cells. Trajectory analysis suggested that adult SMC and FB derive from distinct embryonic intermediates, and that adult FB subsets develop in a linear trajectory from CD81+ FB. Finally, we show that colonic subepithelial PDGFRαhi FB comprise several functionally and anatomically distinct populations that originate from an Fgfr2-expressing FB intermediate. Collectively our results provide novel insights into MSC diversity, location, function and ontogeny, with implications for our understanding of intestinal development, homeostasis and disease.

The small and large intestine contain transcriptionally related mesenchymal stromal cell subsets that derive from embryonic Gli1+ mesothelial cells

Intestinal fibroblasts (FB) play essential roles in intestinal homeostasis. Here we show that the small and large intestinal lamina propria (LP) contain similar FB subsets that locate in specific anatomical niches and express distinct arrays of epithelial support genes. However, there were tissue specific differences in the transcriptional profile of intestinal FB subsets in the two sites. All adult intestinal LP mesenchymal stromal cells (MSC), including FB, smooth muscle cells (SMC) and pericytes derive from Gli1-expressing embryonic precursors which we identify as mesothelial cells. Trajectory analysis suggested that adult SMC and FB derive from distinct embryonic intermediates, and that adult FB subsets develop in a linear trajectory from CD81+ FB. Finally, we show that colonic subepithelial PDGFRαhi FB comprise several functionally and anatomically distinct populations that originate from an Fgfr2-expressing FB intermediate. Collectively our results provide novel insights into MSC diversity, location, function and ontogeny, with implications for our understanding of intestinal development, homeostasis and disease.

Lactococcus lactis subsp. Cremoris C60 restores T Cell Population in Small Intestinal Lamina Propria in Aged Interleukin-18 Deficient Mice

Lactic acid bacteria (LAB), a major commensal bacterium in the small intestine, are well known beneficial bacteria which promote establishment of gut-centric immunity, such as anti-inflammation and anti-infection. In this report, we show that a LAB strain Lactococcus lactis subsp. Cremoris C60 possess an ability to activate antigen presenting cells, such as dendritic cells (DCs), and intestinal T cells which possibly support to maintain healthy intestinal immunological environment in aging process. We found that CD4+ T cells in the small intestine are dramatically decreased in aged Interleukin-18 knock out (IL-18KO) mice, associated with the impairment of IFN-γ production in the CD4+ T cells, especially in small intestinal lamina propria (LP). Surprisingly, heat killed-C60 (HK-C60) diet completely recovered the CD4+ T cells population and activity in SI-LP and over activated the population in Peyer’s patches (PPs) of IL-18KO mice. The HK-C60 diet was effective approach not only to restore the number of cells, but also to recover IFN-γ production in the CD4+ T cell population in the small intestine of IL-18-deficient mice. As a possible cause in the age-associated impairment of CD4+ T cells activity in IL-18KO mice, we found that the immunological activity was downregulated in the IL-18-deficient DCs. The cytokines production and cellular activation markers expression were downregulated in the IL-18-deficient bone marrow derived dendritic cells (BMDCs) at the basal level, however, both activities were highly upregulated in HK-C60 stimulation as compared to those of WT cells. Antigen uptake was also attenuated in the IL-18-deficient BMDCs, and it was significantly enhanced in the cells as compared to WT cells in HK-60 stimulation. An in vitro antigen presentation assay showed that IFN-γ production in the CD4+ T cells was significantly enhanced in the culture of IL-18-deficient BMDCs compared with WT cells in the presence of HK-C60. Thus, we conclude that HK-C60 diet possesses an ability to restore T cells impairment in the small intestine of IL-18-deficient environment. In addition, the positive effect is based on the immunological modification of DCs function which directory influences into the promotion of effector CD4+ T cells generation in the small intestine.

Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program

Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria (LP) and intraepithelial (IE) compartments, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we addressed the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+ T cells from mLN, LP and IE segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile. Treg fate–mapping coupled with RNA– and ATAC–sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4 lineage–defining transcription factor ThPOK results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Thus, coordinated replacement of circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting.

OP39 Treatment of ulcerative colitis With AMT-101, a novel oral interleukin-10 immunomodulatory fusion biologic that traffics across the intestinal epithelium

Background While different chronic inflammatory diseases can be correlated with distinct pro-inflammatory cytokines, interleukin-10 (IL-10) represents a central anti-inflammatory cytokine capable of modulating many pro-inflammatory signals. Previous clinical efforts to capture the benefit of IL-10 in suppressing the pro-inflammatory state in inflammatory bowel disease (IBD) patients have been limited by dose-limiting systemic side effects. Methods We have designed a chimaera of human IL-10 genetically fused to a non-toxic and poorly immunogenic fragment of the cholix exotoxin, termed AMT-101, that demonstrated rapid receptor-mediated transcytosis in vitro and in vivo and could activate phospho-STAT3 in cells within the lamina propria following luminal administration (data not shown). Mice with oxazolone-induced colitis were dosed by oral gavage with AMT-101 daily for 12 days, at which time colon tissue and serum were examined for hallmarks of inflammation. Enteric-coated capsules were used to deliver either 1 or 5 mg of AMT-101 to the distal ileum of cynomolgus monkeys; serum was collected to examine PK and PD outcomes in this non-inflamed model. Results Histological changes of colonic tissue associated with oxazolone-induced colitis was blocked by the oral gavage of AMT-101. Increases in serum levels of pro-inflammatory cytokines IL-1b, IL-6, and IL-17A were blunted by AMT-101 treatment. Remarkably, endogenous IL-10 increased in this model in an attempt to correct inflammation, but this was also decreased by the delivery of AMT-101. Cynomolgus monkeys dosed orally with AMT-101 capsules showed very low serum levels compared with those observed after IV injection of 0.5 mg/kg AMT-101. Strikingly, serum levels of IL-1 receptor antagonist (IL-1RA) as an anti-inflammatory PD marker were increased to a greater extent following oral capsule dosing compared with IV administration. Conclusion These studies provide strong pre-clinical evidence that AMT-101 can effectively reach the intestinal lamina propria to delivery biologically-active IL-10 following transcytosis across the intestinal epithelium. Importantly, the gut-selective nature of the responses observed suggests AMT-101 may alleviate the previous issues of dose-limiting side effects observed with systemic administration of IL-10 and point to the intestinal lamina propria as a critical site of IL-10’s immunomodulatory actions. AMT-101 has advanced to the clinic and is currently being evaluated in a Phase1b trial in patients with active ulcerative colitis.