Microbial DNA enrichment promotes liver steatosis and fibrosis in the course of non-alcoholic steatohepatitis.

AIM: Low-grade inflammation is the hallmark of non-alcoholic fatty liver diseases (NAFLD) and non-alcoholic steatohepatitis (NASH). The leakage of microbiota-derived products can contribute to liver inflammation during NAFLD/NASH development. Here, we assessed the roles of gut microbial DNA-containing extracellular vesicles (mEVs) in regulating liver cellular abnormalities in the course of NAFLD/NASH. METHODS: We performed studies with Vsig4-/- , C3-/- , cGAS-/- , and their wild-type littermate mice. Vsig4+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut mEVs were adoptively transferred into Vsig4-/- , C3-/- , cGAS-/- , or littermate WT mice, and hepatocyte inflammation and HSC fibrogenic activation were measured in these mice. RESULTS: Non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis development was concomitant with a diminished liver Vsig4+ macrophage population and a marked bacterial DNA enrichment in both hepatocytes and HSCs. In the absence of Vsig4+ macrophages, gut mEVs translocation led to microbial DNA accumulation in hepatocytes and HSCs, resulting elevated hepatocyte inflammation and HSC fibrogenic activation. In contrast, in lean WT mice, Vsig4+ macrophages remove gut mEVs from bloodstream through a C3-dependent opsonization mechanism and prevent the infiltration of gut mEVs into hepatic cells. Additionally, Vsig4-/- mice more quickly developed significant liver steatosis and fibrosis than WT mice after Western diet feeding. In vitro treatment with NASH mEVs triggered hepatocyte inflammation and HSC fibrogenic activation. Microbial DNAs are key cargo for the effects of gut mEVs by activating cGAS/STING. CONCLUSION: Accumulation of microbial DNAs fuels the development of NAFLD/NASH-associated liver abnormalities.

Accumulation of microbial DNAs promotes to islet inflammation and ß cell abnormalities in obesity in mice.

Various microbial products leaked from gut lumen exacerbate tissue inflammation and metabolic disorders in obesity. Vsig4+ macrophages are key players preventing infiltration of bacteria and their products into host tissues. However, roles of islet Vsig4+ macrophages in the communication between microbiota and ß cells in pathogenesis of obesity-associated islet abnormalities are unknown. Here, we find that bacterial DNAs are enriched in ß cells of individuals with obesity. Intestinal microbial DNA-containing extracellular vesicles (mEVs) readily pass through obese gut barrier and deliver microbial DNAs into ß cells, resulting in elevated inflammation and impaired insulin secretion by triggering cGAS/STING activation. Vsig4+ macrophages prevent mEV infiltration into ß cells through a C3-dependent opsonization, whereas loss of Vsig4 leads to microbial DNA enrichment in ß cells after mEV treatment. Removal of microbial DNAs blunts mEV effects. Loss of Vsig4+ macrophages leads to microbial DNA accumulation in ß cells and subsequently obesity-associated islet abnormalities.

Hepatocyte-derived exosomes from early onset obese mice promote insulin sensitivity through miR-3075.

In chronic obesity, hepatocytes become insulin resistant and exert important effects on systemic metabolism. Here we show that in early onset obesity (4 weeks high-fat diet), hepatocytes secrete exosomes that enhance insulin sensitivity both in vitro and in vivo. These beneficial effects were due to exosomal microRNA miR-3075, which is enriched in these hepatocyte exosomes. FA2H is a direct target of miR-3075 and small interfering RNA depletion of FA2H in adipocytes, myocytes and primary hepatocytes leads to increased insulin sensitivity. In chronic obesity (16-18 weeks of a high-fat diet), hepatocyte exosomes promote a state of insulin resistance. These chronic obese hepatocyte exosomes do not directly cause impaired insulin signalling in vitro but do promote proinflammatory activation of macrophages. Taken together, these studies show that in early onset obesity, hepatocytes produce exosomes that express high levels of the insulin-sensitizing miR-3075. In chronic obesity, this compensatory effect is lost and hepatocyte-derived exosomes from chronic obese mice promote insulin resistance.

Adipose Tissue Macrophages Modulate Obesity-Associated ß Cell Adaptations through Secreted miRNA-Containing Extracellular Vesicles.

Obesity induces an adaptive expansion of ß cell mass and insulin secretion abnormality. Expansion of adipose tissue macrophages (ATMs) is a hallmark of obesity. Here, we assessed a novel role of ATMs in mediating obesity-induced ß cell adaptation through the release of miRNA-containing extracellular vesicles (EVs). In both in vivo and in vitro experiments, we show that ATM EVs derived from obese mice notably suppress insulin secretion and enhance ß cell proliferation. We also observed similar phenotypes from human islets after obese ATM EV treatment. Importantly, depletion of miRNAs blunts the effects of obese ATM EVs, as evidenced by minimal effects of obese DicerKO ATM EVs on ß cell responses. miR-155 is a highly enriched miRNA within obese ATM EVs and miR-155 overexpressed in ß cells impairs insulin secretion and enhances ß cell proliferation. In contrast, knockout of miR-155 attenuates the regulation of obese ATM EVs on ß cell responses. We further demonstrate that the miR-155-Mafb axis plays a critical role in controlling ß cell responses. These studies show a novel mechanism by which ATM-derived EVs act as endocrine vehicles delivering miRNAs and subsequently mediating obesity-associated ß cell adaptation and dysfunction.

MiR-690, an exosomal-derived miRNA from M2-polarized macrophages, improves insulin sensitivity in obese mice.

Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both in vivo and in vitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.

CRIg+ Macrophages Prevent Gut Microbial DNA-Containing Extracellular Vesicle-Induced Tissue Inflammation and Insulin Resistance.

BACKGROUND & AIMS: Liver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown. METHODS: We performed studies with CRIg-/-, C3-/-, cGAS-/-, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA-containing extracellular vesicles (mEVs) were adoptively transferred into CRIg-/-, C3-/-, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated. RESULTS: Gut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA-mediated inflammatory responses. CONCLUSIONS: Deficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

IL-37 suppresses the sustained hepatic IFN-γ/TNF-α production and T cell-dependent liver injury.

T cell-dependent liver injury is an important reason for the massive hepatic damage and cirrhosis. So far it is unclear whether the development of the disease could be efficiently suppressed by anti-inflammatory cytokine that modulates innate immune cells. Here we report that anti-inflammatory cytokine IL-37 could efficiently suppress the sustained hepatic expression of IFN-γ and TNF-α, two critical cytokines for inducing hepatocyte apoptosis and liver fibrosis in T cell-dependent liver injury. IL-37 could directly suppress IFN-γ/TLR4 ligand-induced M1 activation of macrophages, thus reducing the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-12. Moreover, IL-37 attenuated Th1 response in vivo and increased the expression of Th2 cytokines IL-4 and IL-13, which in turn promoted M2 activation of macrophages in the liver. The increase of M2 activation not only further reduced TNF-α, IL-1ß and IL-12 expression, but also increased IL-10 and IL-1Ra expression in macrophages, thus more efficiently suppressing the hepatic IFN-γ expression. By suppressing IFN-γ/TNF-α expression, IL-37 suppressed the up-regulation and activation of MLKL that drives hepatocellular necrosis in T cell-dependent liver damage. Accordingly, IL-37 efficiently reduced liver injury and hepatic inflammation after the repeated ConA challenge and the induction of autoimmune hepatitis, and also suppressed hepatic fibrosis resulting from the sustained liver damage. This study showed that the direct and indirect effect of IL-37 on macrophages could reduce the hepatic TNF-α expression, and also modulate IL-1ß/IL-12 and IL-10/IL-1Ra expression to suppress the hepatic IFN-γ expression, thus suppressing the development of T cell-dependent liver injury such as autoimmune hepatitis.

BML-111 suppresses TGF-ß1-induced lung fibroblast activation in vitro and decreases experimental pulmonary fibrosis in vivo.

Pulmonary fibrosis is an aggressive end­stage disease. Transforming growth factor­ß1 (TGF­ß1) mediates lung fibroblast activation and is essential for the progress of pulmonary fibrosis. BML­111, a lipoxinA4 (LXA4) receptor (ALX) agonist, has been reported to possess anti­ï¬brotic properties. The present study aimed to elucidate whether BML­111 inhibits TGF­ß1­induced mouse embryo lung fibroblast (NIH3T3 cell line) activation in vitro and bleomycin (BLM)­induced pulmonary fibrosis in vivo. In vitro experiments demonstrated that BML­111 treatment inhibits TGF­ß1­induced NIH3T3 cell viability and the expression of smooth muscle α actin (α­SMA), fibronectin and total collagen. Furthermore, this suppressive effect was associated with mothers against decapentaplegic homolog (Smad)2/3, extracellular signal­regulated kinase (ERK) and Akt phosphorylation interference. In vivo experiments revealed that BML­111 treatment markedly improved survival rate and ameliorated the destruction of lung tissue structure. It also reduced interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α) and TGF­ß1 expression in the BLM intratracheal mouse model. In addition, the expression ofα­SMA and extracellular matrix (ECM) deposition (total collagen, hydroxyproline and fibronectin) were also suppressed following BML­111 treatment. However, BOC­2, an antagonist of ALX, partially weakened the effects of BML­111. In conclusion, these results indicated that BML­111 inhibits TGF­ß1­induced fibroblasts activation and alleviates BLM­induced pulmonary fibrosis. Therefore, BML­111 may be used as a potential therapeutic agent for pulmonary fibrosis treatment.

Galectin-7 promotes proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting The TGFß/Smad3 pathway.

Galectin-7 (Gal-7) has been associated with cell proliferation and apoptosis. It is known that Gal-7 antagonises TGFß-mediated effects in hepatocytes by interacting with Smad3. Previously, we have demonstrated that Gal-7 is related to CD4+ T cells responses; nevertheless, its effect and functional mechanism on CD4+ T cells responses remain unclear. The murine CD4+ T cells were respectively cultured with Gal-7, anti-CD3/CD28 mAbs, or with anti-CD3/CD28 mAbs & Gal-7. The effects of Gal-7 on proliferation and the phenotypic changes in CD4+ T cells were assessed by flow cytometry. The cytokines from CD4+ T cells were analysed by quantitative real-time PCR. Subcellular localisation and expression of Smad3 were determined by immunofluorescence staining and Western blot, respectively. Gal-7 enhanced the proliferation of activated CD4+ T cells in a dose- and ß-galactoside-dependent manner. Additionally, Gal-7 treatment did not change the ratio of Th2 cells in activated CD4+ T cells, while it increased the ratio of Th1 cells. Gal-7 also induced activated CD4+ T cells to produce a higher level of IFN-γ and TNF-α and a lower level of IL-10. Moreover, Gal-7 treatment significantly accelerated nuclear export of Smad3 in activated CD4+ T cells. These results revealed a novel role of Gal-7 in promoting proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting the TGFß/Smad3 pathway.

TLR2/4 ligand-amplified liver inflammation promotes initiation of autoimmune hepatitis due to sustained IL-6/IL-12/IL-4/IL-25 expression.

Autoimmune hepatitis (AIH), a serious autoimmune liver disease, can be a lifelong illness, leading to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). So far the mechanisms for disease initiation are largely unknown. Here we report that the amplified non-AIH liver inflammation could promote the initiation of AIH due to the sustained increase of IL-6, IL-12, IL-4, and IL-25 in the liver. The liver injury resulting from virus (adenovirus) or chemicals (CCl4) could induce an amplified (stronger/long-lasting) hepatic inflammation by releasing the ligands for TLR2/TLR4. The amplified inflammation resulted in the increase of multiple cytokines and chemokines in the liver. Among them, the sustained increase of IL-6/IL-12 resulted in the activation of STAT3 and STAT4 in hepatic CD4+CD25+ Treg cells, thus suppressing Foxp3 gene expression to reduce the suppressive function of Treg cells in the liver, but not those in the spleen. The increase of IL-12 and the impairment of Treg function promoted Th1 response in presence of self-mimicking antigen (human CYP2D6). Intriguingly, the amplified inflammation resulted in the increase of IL-4 and IL-25 in the liver. The moderate increase of IL-4 was sufficient for cooperating with IL-25 to initiate Th2 response, but inefficient in suppressing Th1 response, favoring the initiation of autoimmune response. Consequently, either adenovirus/CYP2D6 or CCl4/CYP2D6 could induce the autoimmune response and AIH in the mice, leading to hepatic fibrosis. The findings in this study suggest that the amplified non-AIH inflammation in the liver could be a driving force for the initiation of autoimmune response and AIH.

[Differences in peripheral blood gene expression profiles between liver and kidney transplantation recipients].

OBJECTIVE: To investigate the differences in the gene expression profiles of the peripheral blood immune cells between liver and kidney transplantation recipients. METHODS: A dataset containing the gene expression profiles of 27 liver transplantation recipients and 25 kidney transplantation recipients (from GSE22229 and GSE28842, respectively) was downloaded from the GEO database. By combining gene set enrichment analysis (GSEA) and biological network analysis of the differentially expressed genes using Cytoscape software, we analyzed the core genes closely related to liver or kidney transplantations. RESULTS: GSEA identified 20 highly overlapping genes for liver transplantation and another 20 for kidney transplantation using leading edge analysis. Fourteen hub nodes (gene) for liver transplantation and 13 for kidney transplantation were identified by cytoscape software using interaction network analysis. Five core genes related to liver transplantation and 5 to kidney transplantation were obtained by integrating GSEA and biological network analysis. CONCLUSION: Controlling the transcription and translation of the genes of the peripheral blood immune cells is the main immune regulation mechanism in liver transplantation recipients, but in the recipients of kidney transplantation, the protein interaction network plays a more prominent role. Energy metabolism and functional regulation of the immune cells are closely related. The core genes in peripheral blood immune cells related to liver or kidney transplantation may play key roles in regulating immune functions.

Pregnancy estrogen drives the changes of T-lymphocyte subsets and cytokines and prolongs the survival of H-Y skin graft in murine model.

BACKGROUND: Estrogen as well as CD4(+)Foxp3(+) regulatory T cells were shown to have a protective role not only in maintaining maternal-fetal tolerance but also against autoimmune diseases. We aimed to investigate whether the pregnancy levels of estrogen are enough to induce transplant tolerance as to maintain fetal-maternal tolerance. METHODS: We established H-Y skin graft transplantation in C57BL/6 ovariectomized mice that reconstituted with estrogen. Subsequently, consecutive daily estrogen injection was administrated. Tregs and the cytokines in the peripheral blood were detected by flow cytometry and ELISA pre- and post-transplant. RESULTS: The results indicated that pregnancy levels of estrogen could promote Tregs in secondary lymphoid organs and peripheral blood (P < 0.05) but not thymus (P > 0.05). The estrogen-treated recipients accepted H-Y skin grafts for more than 35 days (median survival time (MST): (44.0 ± 1.2) days) compared with estrogen-untreated mice (MST: (23.0 ± 1.6) days) (P < 0.05). It was also observed that estrogen up-regulated the expression of Foxp3, but did not affect CD3(+)CD8(+) effector T-cells in non-transplant mice. While in the presence of H-Y antigens, the expression of Foxp3 was more significant and CD3(+)CD8(+) effector T cells were decreased significantly (P < 0.05). Meanwhile, the up-regulated IL-10 and IL-4, and down-regulated IFN-γ could be observed (P < 0.05). CONCLUSIONS: Pregnancy levels of estrogen may promote the conversion of peripheral Tregs in secondary lymphoid organs, but show no effect on the natural Tregs production, differentiation and maturity in central lymphoid organs. Furthermore, pregnancy levels of estrogen could significantly prolong the survivals of H-Y skin grafts by the expansion of Tregs, suppression of CD3(+)CD8(+) effector T-cells and immune shift towards Th2 cytokines.

Characterization of acute renal allograft rejection by human serum proteomic analysis.

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.

Characterization of Acute Renal Allograft Rejection by Human Serum Proteomic Analysis / 华中科技大学学报（医学）（英德文版）

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatog-raphy (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejec-tion (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differ-entially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were ex-cised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor,apolipoprotein A-Ⅳ precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor,etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into un-derstanding the mechanisms and potential treatment strategy of acute rejection.