Innate immune cells, such as intestinal epithelial cells, dendritic cells (DCs), macrophages, granulocytes, and innate lymphoid cells provide a first line of defence to enteric pathogens. To study the role of CX3CR1+ DCs and macrophages in host defence, we infected CX3CR1-GFP animals with Citrobacter rodentium. When transgenic CX3CR1-GFP animals are infected with the natural mouse pathogen C. rodentium, CX3CR1−/− animals showed a delayed clearance of C. rodentium as compared with (age- and sex-matched) wild-type B6 animals. The delayed clearance of C. rodentium is associated with reduced interleukin (IL)-22 expression. In C. rodentium-infected CX3CR1-GFP animals, IL-22 producing lymphoid-tissue inducer cells (LTi cells) were selectively reduced in the absence of CX3CR1. The reduced IL-22 expression correlates with decreased expression of the antimicrobial peptides RegIIIβ and RegIIIγ. The depletion of CX3CR1+ cells by diphtheria toxin injection in CX3CR1-GFP × CD11c.DOG animals confirmed the role of CX3CR1+ phagocytes in establishing IL-22 production, supporting the clearance of a C. rodentium infection.
CD69 is highly expressed by lymphocytes at mucosal surfaces. We aimed to investigate the role of CD69 in mucosal immune responses. The expression of CD69 by CD4 T cells isolated from the spleen, mesenteric lymph nodes, small intestinal lamina propria, and colonic lamina propria was determined in specific pathogen-free B6 and TCR transgenic animals, as well as in germ-free B6 mice. Transfer colitis was induced by transplanting RAG−/− mice with B6 or CD69−/−CD45RBhigh CD4 T cells. CD69 expression by CD4 T cells is induced by the intestinal microflora, oral delivery of specific Ag, and type I IFN (IFN-I) signals. CD4 T cells from CD69−/− animals produce higher amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IL-21, whereas the production of TGF-β1 is decreased. CD69-deficient CD4 T cells showed reduced potential to differentiate into Foxp3+ regulatory T cells in vivo and in vitro. The transfer of CD69−/−CD45RBhigh CD4 T cells into RAG−/− hosts induced an accelerated colitis. Oral tolerance was impaired in CD69−/− and IFN-I receptor 1-deficient mice when compared with B6 and OT-II × RAG−/− animals. Polyinosinic-polycytidylic acid treatment of RAG−/− mice transplanted with B6 but not CD69−/− or IFN-I receptor 1-deficient CD45RBhigh CD4 T cells attenuated transfer colitis. CD69 deficiency led to the increased production of proinflammatory cytokines, reduced Foxp3+ regulatory T cell induction, impaired oral tolerance, and more severe colitis. Hence, the activation Ag CD69 plays an important role in regulating mucosal immune responses.
Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4+ T cells and/or CD4− cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69−/− CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69−/− CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69−/− mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69−/− CD45RBhigh CD4 T cells into RAG−/− hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.
Dendritic cells (DCs) and macrophages populate the intestinal lamina propria to initiate immune responses required for the maintenance of intestinal homeostasis. To investigate whether CX3CR1(+) phagocytes communicate with CD4 T cells during the development of transfer colitis, we established an antigen-driven colitis model induced by the adoptive transfer of DsRed OT-II cells in CX3CR1(GFP/+) × RAG(-/-) recipients challenged with Escherichia coli expressing ovalbumin (OVA) fused to a cyan fluorescent protein (CFP). After colonization of CX3CR1(GFP/+) × RAG(-/-) animals with red fluorescent E. coli pCherry-OVA, colonic CX3CR1(+) cells but not CD103(+) DCs phagocytosed E. coli pCherry-OVA. Degraded bacterial-derived antigens are transported by CD103(+) DCs to mesenteric lymph nodes (MLNs), where CD103(+) DCs prime naive T cells. In RAG(-/-) recipients reconstituted with OT II cells and gavaged with OVA-expressing E. coli, colonic CX3CR1(+) phagocytes are in close contact with CD4 T cells and presented bacterial-derived antigens to CD4 T cells to activate and expand effector T cells.
Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS–) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS– strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS– strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.
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