B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10 −/−, Tlr2 −/−, and Myd88 −/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra −/− T cells. B. breve treatment of Tlr2 −/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4+ T cells from wild-type mice, but not Il10 −/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo.

The gastrointestinal tract harbors numerous commensal bacteria, referred to as the microbiota, that benefit host health by digesting dietary components and eliminating pathogens. The intestinal microbiota maintains epithelial barrier integrity and shapes the mucosal immune system, balancing host defense and oral tolerance with microbial metabolites, components, and attachment to host cells. To avoid aberrant immune responses, epithelial cells segregate the intestinal microbiota from immune cells by constructing chemical and physical barriers, leading to the establishment of host-commensal mutualism. Furthermore, intestinal immune cells participate in the maintenance of a healthy microbiota community and reinforce epithelial barrier functions. Perturbations of the microbiota composition are commonly observed in patients with autoimmune diseases and chronic inflammatory disorders. An understanding of the intimate interactions between the intestinal microbiota, epithelial cells, and immune cells that are crucial for the maintenance of intestinal homeostasis might promote advances in diagnostic and therapeutic approaches for various diseases.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.