The expansion of Enterobacteriaceae, such as E. coli is a main characteristic of gut inflammation and is related to multiple human diseases. However, how to control these E. coli overgrowth is not well understood. Here, we demonstrate that gut complement factor D (CFD) plays an important role in eliminating E. coli. Increased E. coli, which could stimulate inflammatory macrophages to induce colitis, were found in the gut of CFD deficient mice. We also showed that gut Reg4, which is expressed in gut epithelial cells, stimulated complementmediated attack complexes to eliminate E. coli. Reg4 deficient mice also had increased E. coli. The dominant E. coli were isolated from colitis tissues of mice and found to be sensitive to both CFD-and Reg4-mediated attack complexes. Thus, gut Reg4-and CFD-mediated membrane attack complexes may maintain gut homeostasis by killing inflammatory E. coli.

Background IL-35–producing Bregs and Treg cells critically regulate chronic illnesses worldwide via mechanisms related to disrupting the gut microbiota composition. However, whether the gut microbiota regulates these IL-35+ cells remains elusive. We herein investigated the regulatory effects of the gut microbiota on IL-35+ cells by using genetically modified mouse models of obesity. Results We first found that gut Reg4 promoted resistance to high-fat diet-induced obesity. Using 16S rRNA sequencing combined with LC-MS (liquid chromatography–mass spectrometry)/MS, we demonstrated that gut Reg4 associated with bacteria such as Lactobacillus promoted the generation of IL-35+ B cells through 3-idoleacetic acid (IAA) in the presence of LPS. HuREG4IECtg mice fed a high-fat diet exhibited marked IL-35+ cell accumulation in not only their adipose tissues but also their colons, whereas decreased IL-35+ cell accumulation was observed in the adipose and colon tissues of Reg4 knockout (KO) mice. We also found that Reg4 mediated HFD-induced obesity resistance via IL-35. Lower levels of IAA were also detected in the peripheral blood of individuals with obesity compared with nonobese subjects. Mechanistically, IAA together with LPS mediated IL-35+ B cells through PXR and TLR4. KO of PXR or TLR4 impaired the generation of IL-35+ B cells. Conclusion Together, IAA and LPS induce the generation of IL-35+ B cells through PXR and TLR4.

Macrophages are mainly divided into two populations, which play a different role in physiological and pathological conditions. The differentiation of these cells may be regulated by transcription factors. However, it is unclear how to modulate these transcription factors to affect differentiation of these cells. Here, we found that lncLy6C, a novel ultraconserved lncRNA, promotes differentiation of Ly6Chigh inflammatory monocytes into Ly6Clow/neg resident macrophages. We demonstrate that gut microbiota metabolites butyrate upregulates the expression of lncLy6C. LncLy6C deficient mice had markedly increased Ly6Chigh pro-inflammatory monocytes and reduced Ly6Cneg resident macrophages. LncLy6C not only bound with transcription factor C/EBPβ but also bound with multiple lysine methyltransferases of H3K4me3 to specifically promote the enrichment of C/EBPβ and H3K4me3 marks on the promoter region of Nr4A1, which can promote Ly6Chigh into Ly6Cneg macrophages. As a result, lncLy6C causes the upregulation of Nr4A1 to promote Ly6Chigh inflammatory monocytes to differentiate into Ly6Cint/neg resident macrophages.

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The activation of NLRC4 is a major host response against the infection by intracellular bacteria. However, there still remain challenges in understanding the activation upon sensing of diverse stimuli. We here found that lncRNA LNCGM1082 plays a critical role in the activation of NLRC4. LNCGM1082 in macrophages could affect maturation of interleukin (IL)-1β and pyroptotic cell death only after exposed to NLRC4 ligand. Similar to NLRC4-/mice, LNCGM1082-/mice were high sensitive to Salmonella typhimurium infection. LNCGM1082 de ciency in mouse or human macrophages had reduced IL-1β maturation and pyroptosis. Mechanistically, LNCGM1082 could induce the combination of PKCδ with NLRC4 in both mice and human. There was absence of binding of NLRC4 with PKCδ in LNCGM1082-/macrophages. This lncRNA could be induced by Salmonella typhimurium through TLR5 in the macrophages of both mice and human. Thus, our data indicate that LNCGM1082 induced by TLR5 can mediate the binding of PKCδ with NLRC4 to cause the activation of NLRC4.

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The activation of NLRC4 is a major host response against the infection by intracellular bacteria. However, there still remain challenges in understanding the activation upon sensing of diverse stimuli. We here found that lncRNA LNCGM1082 plays a critical role in the activation of NLRC4. LNCGM1082 in macrophages could affect maturation of interleukin (IL)-1β and pyroptotic cell death only after exposed to NLRC4 ligand. Similar to NLRC4-/- mice, LNCGM1082-/- mice were high sensitive to Salmonella typhimurium infection. LNCGM1082 deficiency in mouse or human macrophages had reduced IL-1β maturation and pyroptosis. Mechanistically, LNCGM1082 could induce the combination of PKCδ with NLRC4 in both mice and human. There was absence of binding of NLRC4 with PKCδ in LNCGM1082-/- macrophages. This lncRNA could be induced by Salmonella typhimurium through TLR5 in the macrophages of both mice and human. Thus, our data indicate that LNCGM1082 induced by TLR5 can mediate the binding of PKCδ with NLRC4 to cause the activation of NLRC4.

Background IL-35 producing B and Treg cells are critical regulators in worldwide chronic illnesses, which are related to disruptions of gut microbiota composition. Whether gut microbiota regulate these IL-35+ cells maintains elusive. Here we investigated the regulation of gut microbiota in the IL-35+ cells through genetically modified mouse models against obesity. Results Here, we first found that gut Reg4 can promote resistance to high-fat diet induced obesity. Using 16S rRNA gene-based microbiota analysis combined with LC-MS (Liquid chromatography-mass spectrometry)/MS, we demonstrate that gut Reg4 associated microbiota such as lactobacillus can promote generation of the IL-35+ B cells through producing 3-idoleacetic acid (IAA) in the presence of LPS. HuREG4IECtg mice had accumulation of IL-35+ cells not only in adipose but also in colon after feeding high-fat diet; whereas decreased IL-35+ cells in adipose and colon tissues could be detected in Reg4 knockout (KO) mice. Lower levels of IAA were also detected in the peripheral blood of individuals with obesity. Mechanistically, IAA with LPS mediated IL35+ B cells are through PXR and TLR4. PXR KO or TLR4 KO impaired generation of IL-35+B cells. Conclusion IAA with LPS can induce generation of the IL-35+B cells through PXR and TLR4.