Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites ß-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

Fungi and tumors: The role of fungi in tumorigenesis (Review).

Fungi inhabit different anatomic sites in the human body. Advances in omics analyses of host­microbiome interactions have tremendously improved our understanding of the effects of fungi on human health and diseases such as tumors. Due to the significant enrichment of specific fungi in patients with malignant tumors, the associations between fungi and human cancer have attracted an increasing attention in recent years. Indeed, cancer type­specific fungal profiles have been found in different tumor tissues. Importantly, fungi also influence tumorigenesis through multiple factors, such as host immunity and bioactive metabolites. Microbiome interactions, host factors and fungal genetic and epigenetic factors could be involved in fungal enrichment in tumor tissues and/or in the conversion from a commensal fungus to a pathogenic fungus. Exploration of the interactions of fungi with the bacterial microbiome and the host may enable them to be a target for cancer diagnosis and treatment. In the present review, the associations between fungi and human cancer, cancer type­specific fungal profiles and the mechanisms by which fungi cause tumorigenesis were discussed. In addition, possible factors that can lead to the enrichment of fungi in tumor tissues and/or the conversion of commensal fungi to pathogenic fungi, as well as potential therapeutic and preventive strategies for tumors based on intratumoral fungi were summarized.

Desulfovibrio vulgaris interacts with novel gut epithelial immune receptor LRRC19 and exacerbates colitis.

BACKGROUND: The overgrowth of Desulfovibrio, an inflammation promoting flagellated bacteria, has been found in ulcerative colitis (UC) patients. However, the molecular mechanism in promoting colitis remains unestablished. METHODS: The relative abundance Desulfovibrio vulgaris (D. vulgaris) in stool samples of UC patients was detected. Mice were treated with dextran sulfate sodium to induce colitis with or without administration of D. vulgaris or D. vulgaris flagellin (DVF), and the severity of colitis and the leucine-rich repeat containing 19 (LRRC19) signaling were assessed. The interaction between DVF and LRRC19 was identified by surface plasmon resonance and intestinal organoid culture. Lrrc19-/- and Tlr5-/- mice were used to investigate the indispensable role of LRRC19. Finally, the blockade of DVF-LRRC19 interaction was selected through virtual screening and the efficacy in colitis was assessed. RESULTS: D. vulgaris was enriched in fecal samples of UC patients and was correlated with the disease severity. D. vulgaris or DVF treatment significantly exacerbated colitis in germ-free mice and conventional mice. Mechanistically, DVF could interact with LRRC19 (rather than TLR5) in colitis mice and organoids, and then induce the production of pro-inflammatory cytokines. Lrrc19 knockdown blunted the severity of colitis. Furthermore, typhaneoside, a blockade of binding interfaces, blocked DVF-LRRC19 interaction and dramatically ameliorated DVF-induced colitis. CONCLUSIONS: D. vulgaris could promote colitis through DVF-LRRC19 interaction. Targeting DVF-LRRC19 interaction might be a new therapeutic strategy for UC therapy. Video Abstract.

LNCGM1082 in Gut Epithelial Cells Promotes Expulsion of Infected Epithelial Cells and Release of IL-18.

Inflammasome NLRC4 (NLR family CARD domain containing 4) can protect mucosal barriers such as intestine from invading bacterial pathogens. However, it was incompletely clear how NLRC4 was activated in intestinal epithelial cells. In this study, we demonstrated that LNCGM1082 could mediate the activation of NLRC4 via binding NLRC4 with protein kinase C (PKC)δ. LNCGM1082 knockout (KO) mice had reduced resistance against Salmonella Typhimurium infection, as well as impaired expulsion of infected gut epithelial cells and release of IL-18 upon exposure to S. Typhimurium. Similar to NLRC4 KO and PKCδ knockdown gut organoids, there also was impaired expulsion of gut epithelial cells and release of IL-18 in LNCGM1082 KO gut organoids. Furthermore, there also was reduced activation of caspase-1 and caspase-8 in these LNCGM1082 KO, NLRC4 KO, and PKCδ knockdown gut organoids upon exposure to S. Typhimurium. Our results show that LNCGM1082 in the ICEs plays a critical role in mediating activation of NLRC4 through binding NLRC4 and PKCδ and promoting expulsion of infected epithelial cells and release of IL-18 upon exposure to bacteria such as S. Typhimurium.

Gut microbiota: A double-edged sword in immune checkpoint blockade immunotherapy against tumors.

Tumor cells can evade immune surveillance by expressing immune checkpoint molecule ligands, resulting in effective immune cell inactivation. Immune checkpoint blockades (ICBs) have dramatically improved survival of patients with multiple types of cancers. However, responses to ICB immunotherapy are heterogeneous with lower patient response rates. The advances have established that the gut microbiota can be as a promising target to overcome resistance to ICB immunotherapy. Furthermore, some bacterial species have shown to promote improved responses to ICBs. However, gut microbiota is critical in maintaining gut and systemic immune homeostasis. It not only promotes differentiation and function of immunosuppressive immune cells but also inhibits inflammatory cells via gut microbiota derived products such as short chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites, which play an important role in tumor immunity. Since the gut microbiota can either inhibit or enhance immune against tumor, it should be a double-edged sword in ICBs against tumor. In this review, we discuss the effects of gut microbiota on immune cells and also tumor cells, especially enhances of gut microbiota on ICB immunotherapy. These discussions can hopefully promote the development of ICB immunotherapy.

Gut fungal mycobiome: A significant factor of tumor occurrence and development.

A variety of bacteria, viruses, fungi, protists, archaea and protozoa coexists within the mammalian gastrointestinal (GI) tract such as that fungi are detectable in all intestinal and colon segments in almost all healthy adults. Although fungi can cause infectious diseases, they are also related to gut and systemic homeostasis. Importantly, through transformation of different forms such as from yeast to hyphae, interaction among gut microbiota such as fungal and bacterial interaction, host factors such as immune and host derived factors, and fungus genetic and epigenetic factors, fungi can be transformed from commensal into pathogenic lifestyles. Recent studies have shown that fungi play a significant role in the occurrence and development of tumors such as colorectal cancer. Indeed, evidences have shown that multiple species of different fungi exist in different tumors. Studies have also demonstrated that fungi are related to the occurrence and development of tumors, and also survival of patients. Here we summarize recent advances in the transformation of fungi from commensal into pathogenic lifestyles, and the effects of gut pathogenic fungi on the occurrence and development of tumors such as colorectal and pancreatic cancers.

Gut microbiota derived bile acid metabolites maintain the homeostasis of gut and systemic immunity.

Bile acids (BAs) as cholesterol-derived molecules play an essential role in some physiological processes such as nutrient absorption, glucose homeostasis and regulation of energy expenditure. They are synthesized in the liver as primary BAs such as cholic acid (CA), chenodeoxycholic acid (CDCA) and conjugated forms. A variety of secondary BAs such as deoxycholic acid (DCA) and lithocholic acid (LCA) and their derivatives is synthesized in the intestine through the involvement of various microorganisms. In addition to essential physiological functions, BAs and their metabolites are also involved in the differentiation and functions of innate and adaptive immune cells such as macrophages (Macs), dendritic cells (DCs), myeloid derived suppressive cells (MDSCs), regulatory T cells (Treg), Breg cells, T helper (Th)17 cells, CD4 Th1 and Th2 cells, CD8 cells, B cells and NKT cells. Dysregulation of the BAs and their metabolites also affects development of some diseases such as inflammatory bowel diseases. We here summarize recent advances in how BAs and their metabolites maintain gut and systemic homeostasis, including the metabolism of the BAs and their derivatives, the role of BAs and their metabolites in the differentiation and function of immune cells, and the effects of BAs and their metabolites on immune-associated disorders.

LNCGM1082-mediated NLRC4 activation drives resistance to bacterial infection.

The activation of NLRC4 is a major host response against intracellular bacteria infection. However, NLRC4 activation after a host senses diverse stimuli is difficult to understand. Here, we found that the lncRNA LNCGM1082 plays a critical role in the activation of NLRC4. LNCGM1082 in macrophages affects the maturation of interleukin (IL)-1ß and pyroptotic cell death only after exposure to an NLRC4 ligand. Similar to NLRC4-/- mice, LNCGM1082-/- mice were highly sensitive to Salmonella Typhimurium (S. T) infection. LNCGM1082 deficiency in mouse or human macrophages inhibited IL-1ß maturation and pyroptosis. Mechanistically, LNCGM1082 induced the binding of PKCδ with NLRC4 in both mice and humans. In contrast, NLRC4 did not bind PKCδ in LNCGM1082-/- macrophages. The activity of the lncRNA LNCGM1082 induced by S. T may be mediated through TLR5 in the macrophages of both mice and humans. In summary, our data indicate that TLR5-mediated LNCGM1082 activity can promote the binding of PKCδ with NLRC4 to activate NLRC4 and induce resistance to bacterial infection.

Gut-Microbiota-Derived Metabolites Maintain Gut and Systemic Immune Homeostasis.

The gut microbiota, including bacteria, archaea, fungi, viruses and phages, inhabits the gastrointestinal tract. This commensal microbiota can contribute to the regulation of host immune response and homeostasis. Alterations of the gut microbiota have been found in many immune-related diseases. The metabolites generated by specific microorganisms in the gut microbiota, such as short-chain fatty acids (SCFAs), tryptophan (Trp) and bile acid (BA) metabolites, not only affect genetic and epigenetic regulation but also impact metabolism in the immune cells, including immunosuppressive and inflammatory cells. The immunosuppressive cells (such as tolerogenic macrophages (tMacs), tolerogenic dendritic cells (tDCs), myeloid-derived suppressive cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Breg) and innate lymphocytes (ILCs)) and inflammatory cells (such as inflammatory Macs (iMacs), DCs, CD4 T helper (Th)1, CD4Th2, Th17, natural killer (NK) T cells, NK cells and neutrophils) can express different receptors for SCFAs, Trp and BA metabolites from different microorganisms. Activation of these receptors not only promotes the differentiation and function of immunosuppressive cells but also inhibits inflammatory cells, causing the reprogramming of the local and systemic immune system to maintain the homeostasis of the individuals. We here will summarize the recent advances in understanding the metabolism of SCFAs, Trp and BA in the gut microbiota and the effects of SCFAs, Trp and BA metabolites on gut and systemic immune homeostasis, especially on the differentiation and functions of the immune cells.

Klebsiella pneumoniae Induces Inflammatory Bowel Disease Through Caspase-11-Mediated IL18 in the Gut Epithelial Cells.

BACKGROUND & AIMS: Klebsiella pneumoniae (KLP), a Gram-negative bacterium belonging to the family of Enterobacteriaceae, is a common cause of antimicrobial-resistant opportunistic infections in hospitalized patients. KLP can colonize in the human gastrointestinal tract, especially in patients with inflammatory bowel diseases. However, effects of KLP on the onset and development of inflammatory bowel disease remain unclear. METHODS: We analyzed the relationship between Mayo indexes of ulcerative colitis and KLP using quantitative reverse-transcription polymerase chain reaction and endoscopy. Using caspase-1/11-/-, NLRP3-/-, NLRC4-/-, interleukin (IL)18-/-, and IL22-/- mice, we showed that KLP could induce colitis through caspase-11-mediated release of mature IL18. Through in vitro gut organoid culture, we determined the mechanism for KLP to induce colitis. RESULTS: We first found that there was a positive relationship between the Mayo indexes of ulcerative colitis and KLP. Then, we isolated a strain of KLP, named Klebsiella pneumoniae J (KLPJ), from the colon tissues of patients with colitis. This strain of bacteria could induce the production of mature IL18 in colon epithelial cells and gut organoids, and also induce colitis and promote dextran sodium sulfate-mediated colitis. Using caspase-1/11-/-, NLRP3-/-, NLRC4-/-, IL18-/-, and IL22-/- mice, we showed that KLPJ-mediated colitis occurred through activation of caspase-11, and was dependent on IL18 and partly on IL22. Our data also showed that lipopolysaccharide from KLPJ could bind with caspase-11 to induce mature IL18 in mouse and human colon organoids. CONCLUSIONS: KLPJ from the colon tissues of patients with ulcerative colitis can colonize the colon, activate caspase-11 inflammasomes, and contribute to intestinal inflammation.

Novel tumor-associated macrophage populations and subpopulations by single cell RNA sequencing.

Tumor-associated macrophages (TAMs) are present in almost all solid tumor tissues. 16They play critical roles in immune regulation, tumor angiogenesis, tumor stem cell activation, tumor invasion and metastasis, and resistance to therapy. However, it is unclear how TAMs perform these functions. With the application of single-cell RNA sequencing (scRNA-seq), it has become possible to identify TAM subpopulations associated with distinct functions. In this review, we discuss four novel TAM subpopulations in distinct solid tumors based on core gene signatures by scRNA-seq, including FCN1 +, SPP1 +, C1Q + and CCL18 + TAMs. Functional enrichment and gene expression in scRNA-seq data from different solid tumor tissues found that FCN1 + TAMs may induce inflammation; SPP1 + TAMs are potentially involved in metastasis, angiogenesis, and cancer cell stem cell activation, whereas C1Q + TAMs participate in immune regulation and suppression; And CCL18 + cells are terminal immunosuppressive macrophages that not only have a stronger immunosuppressive function but also enhance tumor metastasis. SPP1 + and C1Q + TAM subpopulations can be further divided into distinct populations with different functions. Meanwhile, we will also present emerging evidence highlighting the separating macrophage subpopulations associated with distinct functions. However, there exist the potential disconnects between cell types and subpopulations identified by scRNA-seq and their actual function.

FABP4 in Paneth cells regulates antimicrobial protein expression to reprogram gut microbiota.

Antimicrobial proteins possess a broad spectrum of bactericidal activity and play an important role in shaping the composition of gut microbiota, which is related to multiple diseases such as metabolic syndrome. However, it is incompletely known for the regulation of defensin expression in the gut Paneth cells. Here, we found that FABP4 in the Paneth cells of gut epithelial cells and organoids can downregulate the expression of defensins. FABP4fl/flpvillinCreT mice were highly resistance to Salmonella Typhimurium (S.T) infection and had increased bactericidal ability to pathogens. The FABP4-mediated downregulation of defensins is through degrading PPARγ after K48 ubiquitination. We also demonstrate that high-fat diet (HFD)-mediated downregulation of defensins is through inducing a robust FABP4 in Paneth cells. Firmicutes/Bacteroidetes (F/B) ratio in FABP4fl/flpvillinCreT mice is lower than control mice, which is opposite to that in mice fed HFD, indicating that FABP4 in the Paneth cells could reprogram gut microbiota. Interestingly, FABP4-mediated downregulation of defensins in Paneth cells not only happens in mice but also in human. A better understanding of the regulation of defensins, especially HFD-mediated downregulation of defensin in Paneth cells will provide insights into factor(s) underlying modern diseases.Abbreviations: FABP4: Fatty acid binding protein 4; S. T: Salmonella Typhimurium; HFD: High-fat diet; Defa: α-defensin; 930 HD5: Human α-defensin 5; HD6: Human α-defensin 6; F/B: Firmicutes/Bacteroidetes; SFB: Segmental filamentous bacteria; AMPs: Antimicrobial peptides; PPARγ: Peroxisome proliferator-activated receptor γ; P-PPAR: Phosphorylated PPAR; Dhx15: DEAD-box helicase 15; 935 EGF: Epidermal growth factor; ENR: Noggin and R-spondin 1; CFU: Colony forming unit; Lyz1: Lysozyme 1; Saa1: Serum amyoid A 1; Pla2g2a: Phospholipase A2, group IIA; MMP-7: Matrix metalloproteinase; AU-PAGE: Acid-urea polyacrylamide gel electrophoresis; PA: Palmitic 940 acid; GPR40: G-protein-coupled receptor; GF: Germ-free; EGF: Epidermal growth factor; LP: Lamina propria; KO: Knock out; WT: Wild-type.

Tumor Environment Promotes Lnc57Rik-Mediated Suppressive Function of Myeloid-Derived Suppressor Cells.

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/EBPß isoform liver-enriched activator protein to activate C/EBPß 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.

Gut Microbiota-Derived Tryptophan Metabolites Maintain Gut and Systemic Homeostasis.

Tryptophan is an essential amino acid from dietary proteins. It can be metabolized into different metabolites in both the gut microbiota and tissue cells. Tryptophan metabolites such as indole-3-lactate (ILA), indole-3-acrylate (IAC), indole-3-propionate (IPA), indole-3-aldehyde (IAID), indoleacetic acid (IAA), indole-3-acetaldehyde and Kyn can be produced by intestinal microorganisms through direct Trp transformation and also, partly, the kynurenine (Kyn) pathway. These metabolites play a critical role in maintaining the homeostasis of the gut and systematic immunity and also potentially affect the occurrence and development of diseases such as inflammatory bowel diseases, tumors, obesity and metabolic syndrome, diseases in the nervous system, infectious diseases, vascular inflammation and cardiovascular diseases and hepatic fibrosis. They can not only promote the differentiation and function of anti-inflammatory macrophages, Treg cells, CD4+CD8αα+ regulatory cells, IL-10+ and/or IL-35+B regulatory cells but also IL-22-producing innate lymphoid cells 3 (ILC3), which are involved in maintaining the gut mucosal homeostasis. These findings have important consequences in the immunotherapy against tumor and other immune-associated diseases. We will summarize here the recent advances in understanding the generation and regulation of tryptophan metabolites in the gut microbiota, the role of gut microbiota-derived tryptophan metabolites in different immune cells, the occurrence and development of diseases and immunotherapy against immune-associated diseases.

A Special Network Comprised of Macrophages, Epithelial Cells, and Gut Microbiota for Gut Homeostasis.

A number of gut epithelial cells derived immunological factors such as cytokines and chemokines, which are stimulated by the gut microbiota, can regulate host immune responses to maintain a well-balance between gut microbes and host immune system. Multiple specialized immune cell populations, such as macrophages, dendritic cells (DCs), innate lymphoid cells, and T regulatory (Treg) cells, can communicate with intestinal epithelial cells (IEC) and/or the gut microbiota bi-directionally. The gut microbiota contributes to the differentiation and function of resident macrophages. Situated at the interface between the gut commensals and macrophages, the gut epithelium is crucial for gut homeostasis in microbial recognition, signaling transformation, and immune interactions, apart from being a physical barrier. Thus, three distinct but interactive components-macrophages, microbiota, and IEC-can form a network for the delicate and dynamic regulation of intestinal homeostasis. In this review, we will discuss the crucial features of gut microbiota, macrophages, and IEC. We will also summarize recent advances in understanding the cooperative and dynamic interactions among the gut microbiota, gut macrophages, and IEC, which constitute a special network for gut homeostasis.

Gut microbiota-derived metabolite 3-idoleacetic acid together with LPS induces IL-35+ B cell generation.

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. Video Abstract.

Gut Epithelial-derived CXCL9 Maintains Gut Homeostasis Through Preventing Overgrown E. coli.

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.

Identification and characterization of B220+/B220- subpopulations in murine Gr1+CD11b+ cells during tumorigenesis.

Although all murine MDSCs are defined as Gr1+CD11b+, their true immunophenotype remains elusive. In this study, we found murine Gr1+CD11b+ cells can be divided into two subsets: Gr1+CD11b+B220- and Gr1+CD11b+B220+, especially in the spleen tissues. Unlike the dominant B220- subset, the B220+ subpopulation was not induced by tumor in vivo. Moreover, Gr1+CD11b+B220+ cells from tumor-bearing mice spleens were unable to induce arginase 1 and inducible nitric oxide synthase expression, inhibit T cell proliferation, or promote tumor growth in primary tumor site. Nevertheless, these cells suppressed tumor metastasis in vivo and reduced cancer cell motility in vitro, while Gr1+CD11b+B220- cells from tumor-bearing mice spleens promoted tumor metastasis and enhanced cancer cell motility. Furthermore, both the polymorphonuclear (PMN-MDSCs) and monocytic MDSCs (Mo-MDSCs) could be further divided into B220- and B220+ subsets; interestingly, tumor only induced the expansion of B220- PMN-MDSCs and B220- Mo-MDSCs, but not the B220+ counterparts. Compared with B220- PMN-MDSCs and B220- Mo-MDSCs, the Ly6G+Ly6C-CD11b+B220+ and Ly6G-Ly6C+CD11b+B220+ cells from tumor-bearing mice spleens exhibited a more mature phenotype without immunosuppressive activity. Additionally, IL-6 deficiency attenuated the tumor-induced accumulation of MDSCs, B220- MDSCs and B220- PMN-MDSCs but increased the percentages of Gr1+CD11b+B220+, Ly6G+Ly6C-CD11b+B220+, and Ly6G-Ly6C+CD11b+B220+ cells, indicating the opposing roles of the IL-6 signaling pathway in the expansion of B220- MDSCs and their B220+ counterparts. Taken together, our findings indicate that the B220+ subset is a distinct subset of Gr1+CD11b+ cells functionally different from the B220- subpopulation during tumorigenesis and induction of MDSCs to B220+ cells may be helpful for cancer therapy.

LRRC19 Promotes Permeability of the Gut Epithelial Barrier Through Degrading PKC-ζ and PKCι/λ to Reduce Expression of ZO1, ZO3, and Occludin.

BACKGROUND: A dysfunctional gut epithelial barrier allows the augmented permeation of endotoxins, luminal antigens, and bacteria into the bloodstream, causing disease. The maintenance of gut epithelial barrier integrity may be regulated by multiple factors. Herein we analyze the role of leucine-rich repeat-containing protein 19 (LRRC19) in regulating the permeability of the gut epithelial barrier. METHODS: We utilized Lrrc19 knockout (KO) mice and clinical samples through transmission electron, intestinal permeability assay, Western blot, and immunofluorescence staining to characterize the role of LRRC19 in the permeability of the gut epithelial barrier. RESULTS: We found that LRRC19, which is expressed in gut epithelial cells, impairs gut barrier function. Transmission electron micrographs revealed a tighter junction and narrower gaps in the colon epithelium cells in LRRC19 KO mice. There were lower levels of serum lipopolysaccharide and 4 kDa-fluorescein isothiocyanate-dextran after gavage in LRRC19 KO mice than in wild-type mice. We found that LRRC19 could reduce the expression of zonula occludens (ZO)-1, ZO-3, and occludin in the colonic epithelial cells. The decreased expression of ZO-1, ZO-3, and occludin was dependent on degrading protein kinase C (PKC) ζ and PKCι/λ through K48 ubiquitination by LRRC19. The expression of LRRC19 was also negatively correlated with ZO-1, ZO-3, occludin, PKCζ, and PKCι/λ in human colorectal cancers. CONCLUSIONS: The protein LRRC19 can promote the permeability of the gut epithelial barrier through degrading PKC ζ and PKCι/λ to reduce the expression of ZO-1, ZO-3, and occludin.