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Background: Gut microbiota contributes to the regulation of host immune response and homeostasis. Bile acid (BA) derivatives from gut microbiota can affect the differentiation and function of the immune cells. However, it is incompletely clear for the regulation of BA metabolites in these immune cells. Here we investigated the effects the BA metabolites on the macrophages. Results: We here find that BA metabolites can regulate sensitivity of macrophages to LPS and or Gram-negative bacteria. BA derivatives could induce lncRNA57RIKexpression through sphingosine-1-phosphate receptor 2 (S1PR2) in the macrophages of mice and humans, which play a critical role in Gram-negative bacteria mediated IL-1βmaturation and pyroptosis of macrophages. This lncRNA57RIK could bind intracellular proteases caspase-4/11 with guanylate-binding protein 1 (GBP1) in the human and mice together to cause LPS mediated activation of caspase-4/11. Murine or human lncRNA57RIK knockout (KO) macrophages did not produce response(s) to LPS or Gram-negative bacteria. LncRNA57RIK KO mice had also reduced inflammatory responses to LPS or Salmonella typhimurium (S.T) infection. Conclusion: Taken together, gut microbiota derived BA metabolites mediated lncRNA57RIK is necessary for LPS induced caspase-4/11 activation.

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Figure S1. Characterization of inflammatory cytokine and tumor mediated MDSCs. Figure S2. Characteristics of lnc-C/EBPβ. Figure S3. Sequences of mouse lnc-C/EBPβ and humanlnc-C/EBPβ. Figure S4. The suppressive MDSC isolated from tumors may express high levels of both Lnc-C/EBPβ and C/EBPβ. Figure S5. The tumor associated factors mediated Lnc-C/EBP� are dependent on STAT3. Figure S6. Gene microarray of lnc-C/EBPβ shRNA/letiviruses and lnc-C/EBPβ/lentiviruses transduced cells. Figure S7. Lnc-C/EBPβ impedes differentiation of M-MDSCs. Figure S8. Lnc-C/EBPβ also impacts survival of MDSCs. Figure S9. The binding of Lnc-C/EBPβ to C/EBPβ LIP did not affect the transcriptional activity of C/EBPβ LAP-LAP* or LAP-LAP.

Supplementary Materials and methods

Background and Aims Increased E. coli in the colon are related to the occurrence and development of multiple diseases. Chemokines are shown to possess potential antimicrobial activity, including against Gram-positive and -negative bacterial pathogens. We here investigated function[s] of chemokine CXCL9 expressed in the gut epithelial cells, and mechanism[s] of CXCL9 by which to kill E. coli. Methods We generated CXCL9fl/flpvillin-creT mice [pvillin-cre positive mice] and their control CXCL9fl/flpvillin-crewmice [pvillin-cre negative mice], and then employed a dextran sulphate sodium [DSS]-mediated colitis model to determine the sensitivity of CXCL9fl/flpvillin-creT mice. We analysed the composition of the gut microbiota by using 16S ribosomal RNA [V3-V4 variable region] sequencing and shotgun metagenomic analyses. We generated E. coli ΔFtsX [FtsX-depleted E. coli] and E. coli ΔaceE [aceE-depleted E. coli] by using a bacterium red recombining system to investigate the mechanism[s] of CXCL9 by which to kill E. coli. Results CXCL9 fl/flpvillin-creTmice were more sensitive to chemically induced colitis than their control littermates, CXCL9fl/flpvillin-crewmice. After DSS treatment, there were markedly increased gut E. coli [Escherichia-Shigella] in the colonic contents of CXCL9fl/flpvillin-creT mice as compared with control CXCL9fl/flpvillin-crew mice. The increased E. coli could promote colitis through NLRC4 and caspase 1/11-mediated IL-18, which was derived from gut epithelial cells. We finally demonstrated that CXCL9 expressed in gut epithelial cells could kill the overgrown E. coli. E. coli expressed Ftsx and PDHc subunits aceE. E.coliΔaceE but not E. coliΔFtsX were resistant to CXCL9-mediated killing. Conclusions Gut epithelial cells-derived CXCL9 can kill the expanded E. coli through aceE, to remain gut homeostasis.

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.

Tanshinone IIA (Tan IIA) possesses potent anti-atherogenic function, however, the underlying pharmacological mechanism remains incompletely understood. Previous studies suggest that oxidized LDL (oxLDL)-induced NLRP3 (NOD-like receptor (NLR) family, pyrin domain-containing protein 3) inflammasome activation in macrophages plays a vital role in atherogenesis. Whether the anti-atherogenic effect of Tan IIA relies on the inhibition of the NLRP3 inflammasome has not been investigated before. In this study, we found that Tan IIA treatment of high-fat diet fed ApoE-/- mice significantly attenuated NLRP3 inflammasome activation in vivo. Consistently, Tan IIA also potently inhibited oxLDL-induced NLRP3 inflammasome activation in mouse macrophages. Mechanically, Tan IIA inhibited NF-κB activation to downregulate pro-interleukin (IL) -1β and NLRP3 expression, and decreased oxLDL-induced expression of lectin-like oxidized LDL receptor-1 (LOX-1) and cluster of differentiation 36 (CD36), thereby attenuating oxLDL cellular uptake and subsequent induction of mitochondrial and lysosomal damage - events that promote the NLRP3 inflammasome assembly. Through regulating both the inflammasome 'priming' and 'activation' steps, Tan IIA potently inhibited oxLDL-induced NLRP3 inflammasome activation, thereby ameliorating atherogenesis.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils and monocytes with potent immunosuppressive activity that regulate immune responses in the tumor microenvironment. We identified a novel long noncoding RNA (lncRNA), named as lnc57Rik, in the MDSCs that controls their immunosuppressive functions. Lnc57Rik was induced in in vitro and in vivo inflammatory settings and upregulated the genes related to MDSC-mediated immunosuppression, including Arg-1, NOS2, NOX2, and COX2. Furthermore, Lnc57Rik can not only bind with the C/EBPβ isoform liver-enriched activator protein to activate C/EBPβ but also with the methyltransferase WD repeat-containing protein 5 that enables the enrichment of histone H3 trimethylated lysine 4 marks on the promoter regions of Arg-1, NOS2, NOX2, and COX2, eventually resulting in their transcriptional activation. Furthermore, the conserved human lnc57Rik has a similar function as murine lnc57Rik. Taken together, upregulation of lnc57Rik in the tumor microenvironment promotes the immunosuppressive function of MDSCs.

Summary Inflammatory macrophages play a critical role in gut and extra-gut inflammatory disorders, which may be promoted through the dysbiosis of gut microbiota. However, it is poorly understood how gut microbiota affect inflammatory macrophages. Here, we found that increased Escherichia coli (E. coli) in inflamed colon may induce inflammatory macrophages in gut and extra-gut tissues. These E. coli are different from other commensal and pathogenic E. coli in genomic components and also in ability to induce inflammatory responses. Dominant E. coli from colitic tissues induce gut inflammatory macrophages through a regulating network consisted of IL-18, IFN-γ, IL-12, and IL-22 in gut tissues. These E. coli also directly activate macrophages. Cytosolic inflammasome components PCKδ, NLRC4, caspase8, and caspase1/11 are involved in E. coli-mediated activation in both gut epithelial cells and macrophages. These disclose a novel mechanism for how dysbiosis of gut microbiota in colitis cause inflammatory macrophages related to multiple diseases., Graphical Abstract, Highlights • Increased commensal E. coli in colitis induce inflammatory macrophages • Colitic E. coli are different from other commensal and pathogenic E. coli • Gut inflammatory macrophages by E. coli need IL-18, IFN-γ, IL-12, and IL-22 • PCKδ, NLRC4, caspase8, and caspase1/11 are required for E. coli-mediated activation, Biological Sciences; Pathophysiology; Immunology; Microbiology

Long noncoding RNA (lncRNA) plays a critical role in many biological processes, such as cell differentiation and development. However, few studies about lncRNAs regulating the differentiation and development of myeloid-derived suppressor cells (MDSCs) exist. In this study, we identified a lncRNA pseudogene, Olfr29-ps1, which was expressed in MDSCs and upregulated by the proinflammatory cytokine IL6. The Olfr29-ps1 in vertebrates is conserved, and the similarity between the Olfr29-ps1 and human OR1F2P sequence is 43%. This lncRNA promoted the immunosuppressive function and differentiation of monocytic (Mo-)MDSCs in vitro and in vivo. It directly sponged miR-214-3p to downregulate miR-214-3p, which may target MyD88 to modulate the differentiation and development of MDSCs. The functions of Olfr29-ps1 were dependent on IL6-mediated N6-methyladenosine (m6A) modification, which not only enhanced Olfr29-ps1, but also promoted the interaction of Olfr29-ps1 with miR-214-3p. Thus, our results demonstrated that the pseudogene Olfr29-ps1 may regulate the differentiation and function of MDSCs through a m6A-modified Olfr29-ps1/miR-214-3p/MyD88 regulatory network, revealing a mechanism for the regulation of myeloid cells and also providing potential targets for antitumor immunotherapy.

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.

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.

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 complement-mediated 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., Qi et al. show that gut complement factor D (CFD) plays an important role in eliminating Escherichia coli, using gut specific CFD null mice. They find that E. coli isolated from inflamed guts are sensitive to both CFD and Reg4-mediated attack complexes. This study provides insights into how the level of E. Coli is kept low in the gut to prevent its inflammation.

Myeloid-derived suppressor cells (MDSC) are regulators of immune responses in cancer. The differentiation and function of these MDSCs may be regulated through multiple factors, such as microRNAs. However, the effect of long noncoding RNAs (lncRNA) on the differentiation and function of MDSCs is poorly understood. We identified a long noncoding RNA (lncRNA) named lnc-C/EBPβ in MDSCs, which may control suppressive functions of MDSCs. Lnc-C/EBPβ could be induced in in vitro and in vivo tumor and inflammatory environments. It regulated a set of target transcripts, such as Arg-1, NOS2, NOX2, and COX2, to control immune-suppressive function and differentiation of MDSCs. This lncRNA was also able to bind to the C/EBPβ isoform LIP to inhibit the activation of C/EBPβ. We also found that the conserved homologue lnc-C/EBPβ has a similar function to murine lnc-C/EBPβ. These findings suggest a negative feedback role for lnc-C/EBPβ in controlling the immunosuppressive functions of MDSC in the tumor environment. Cancer Immunol Res; 6(11); 1352–63. ©2018 AACR.

Myeloid-derived suppressor cells (MDSCs) are major regulators of immune responses in cancer. Both C/EBP homologous protein (CHOP) and C/EBPβ play a critical role in regulating immunosuppressive function of MDSCs. In this study, we identified a novel long noncoding RNA termed as lnc-chop in MDSCs, which may interact with CHOP and the C/EBPβ isoform liver-enriched inhibitory protein. The binding of lnc-chop with both CHOP and the C/EBPβ isoform liver-enriched inhibitory protein promoted the activation of C/EBPβ and upregulated the expression of arginase-1, NO synthase 2, NADPH oxidase 2, and cyclooxygenase-2, which are related to the immunosuppressive function of MDSCs in inflammatory and tumor environments. Additionally, lnc-chop also promoted the enrichment of H3K4me3 on the promoter region of arginase-1, NO synthase 2, NADPH oxidase 2, and cyclooxygenase-2. These findings suggest an important role of lnc-chop in controlling immunosuppressive function of MDSCs in the tumor environment.

Gut mucosal layers are crucial in maintaining the gut barrier function. Gut microbiota regulate homeostasis of gut mucosal layer via gut immune cells such as RORγt (+) IL-22(+) ILC3 cells, which can influence the proliferation of mucosal cells and the production of mucin. However, it is unclear how gut microbiota execute this regulation. Here we show that lactobacilli promote gut mucosal formation by producing L-Ornithine from arginine. L-Ornithine increases the level of aryl hydrocarbon receptor ligand L-kynurenine produced from tryptophan metabolism in gut epithelial cells, which in turn increases RORγt (+)IL-22(+) ILC3 cells. Human REG3A transgenic mice show an increased proportion of L-Ornithine producing lactobacilli in the gut contents, suggesting that gut epithelial REG3A favors the expansion of L-Ornithine producing lactobacilli. Our study implicates the importance of a crosstalk between arginine metabolism in Lactobacilli and tryptophan metabolism in gut epithelial cells in maintaining gut barrier., Qi, Li, Yun, Zhang et al.show that lactobacilli promote the generation of gut mucosa by facilitating the conversion of arginine to L-Ornithine. This study highlights the importance of a crosstalk between arginine metabolism in Lactobacilli and tryptophan metabolism in gut epithelial cells in maintaining a healthy gut barrier.

Long noncoding RNA (lncRNA) plays a critical role in many biological processes, such as cell differentiation and development. However, few studies about lncRNAs regulating the differentiation and development of myeloid-derived suppressor cells (MDSCs) exist. In this study, we identified a lncRNA pseudogene

To reveal the innate immunity of mast cells against recombinant VP1-VP4 protein of foot-and-mouth disease virus (FMDV), mouse peritoneal mast cells (PMCs) were pulsed with recombinant VP1-VP4 protein. The supernatants harvested from PMCs cultures were applied to the high throughput ELISA array. Our results show that the expression levels of CCL19, L-selectin, CCL17, and TNF alpha released from PMCs pulsed with recombinant VP1-VP4 were significantly down-regulated compared with PMCs alone (P0.001). Surprisingly, in comparison with PMCs alone, the expression levels of CCL19, IL-15, IL-9, G-CSF, and Galectin-1 in PMCs with the mannose receptor (MR) inhibitor were significantly up-regulated (Plt;0.01), and the expression level of IL-10 was also remarkably up-regulated (Plt;0.05). Importantly, the protein expression levels in PMCs treated with MR inhibitor were higher than PMCs pulsed with VP1-VP4, including IL-10, IL-17, CCL20, IL-15, IL-9, L-selectin, CCL17, TNF alpha, and CCL19 (Plt;0.01) as well as CCL21, and G-CSF (Plt;0.05). Differential expression analysis in bioinformatics shows that both L-selectin and CCL17 were recognized as differentially expressed protein molecules (Log2(ratio)≤-1) when compared with PMCs alone. Furthermore, the up-regulation of the expression levels of CCL20, CCL19, L-selectin, and IL-15 in PMCs treated with MR inhibitor was defined as differential expression (Log2(ratio)≥1). These data indicate that PMCs are capable of secreting CCL19, L-selectin, CCL17, and TNF alpha spontaneously and the recombinant VP1-VP4 has an inhibitive potential to PMCs during their performance of innate immune response. Given the protein expression levels from PMCs pre-treated with MR inhibitor were significantly increased, it can be deduced that immunosuppression of FMDV is presumably initiated by the VP1 recognition of MR on mast cells.为揭示肥大细胞抗口蹄疫病毒VP1-VP4 蛋白的天然免疫作用，以重组口蹄疫病毒VP1-VP4 蛋白刺激小鼠腹腔肥大细胞 (Peritoneal mast cells, PMCs)，用高通量ELISA 芯片检测PMCs 的蛋白质表达谱。结果显示，VP1-VP4 蛋白刺激的PMCs (VP1-VP4 组) 表达CCL19、L-selectin、CCL17 和TNF-α 的水平极显著低于对照组 (PMCs) (Plt;0.001)，而VP1-VP4 蛋白刺激经甘露糖受体 (Mannose receptor, MR) 抑制剂预处理的PMCs (MR组) 表达CCL19、IL-15、IL-9、G-CSF 和Galectin-1 的水平则极显著高于对照组 (Plt;0.01)，IL-10 表达水平也有显著升高 (Plt;0.05)。MR 组与VP1-VP4 组相比，PMCs 表达IL-10、IL-17、CCL20、IL-15、IL-9、L-selectin、CCL17、TNF-α 和CCL19 的水平极显著升高 (Plt;0.01)，CCL21 和G-CSF 的表达也显著高于VP1-VP4 组(Plt;0.05)。生物信息学差异表达分析结果显示，与对照组相比，VP1-VP4 组PMCs 表达的L-selectin 和CCL17为下调性差异表达蛋白 (Log2(ratio)≤–1)。MR 组与VP1-VP4 相比，PMCs 表达的CCL20、CCL19、L-selectin和IL-15 为上调性差异表达蛋白 (Log2(ratio)≥1)。这表明，PMCs 可自发分泌CCL19、L-selectin、CCL17 和TNF-α，而VP1-VP4 则对PMCs 的天然免疫功能具有抑制作用。由于阻断MR 后PMCs 的蛋白质表达水平显著升高，所以VP1-VP4 对小鼠PMCs 的免疫抑制作用可能是由MR 介导的。.