Inhibition of the TCF12/VSIG4 axis by palbociclib diminishes the proliferation and migration of glioma cells and decreases the M2 polarization of glioma-associated microglia.

The CDK4/CDK6 inhibitor palbociclib has shown the encouraging promise in the treatment of glioma. Here, we elucidated how palbociclib exerts suppressive functions in the M2 polarization of glioma-related microglia and the progression of glioma. Xenograft experiments were used to evaluate the function in vivo. The mRNA levels of transcription factor 12 (TCF12) and VSIG4 were detected by RT-qPCR, and their protein levels were assessed by immunoblotting. Cell migration was tested by wound-healing assay. Cell cycle distribution and M1/M2 microglia phenotype analysis were performed by flow cytometry. The levels of IFN-γ, TNF-α, IL-6，and TGF-ß were measured by ELISA. The TCF12/VSIG4 association was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. In U251 and LN229 glioma cells, TCF12 and VSIG4 were overexpressed, and palbociclib reduced their expression levels. TCF12 upregulation enhanced the proliferation and migration of glioma cells and the M2 polarization of glioma-associated microglia in vitro as well as the tumorigenicity of U251 glioma cells in vivo, which could be reversed by palbociclib. Mechanistically, TCF12 could enhance VSIG4 transcription and expression by binding to the VSIG4 promoter. TCF12 deficiency led to repression in glioma cell proliferation and migration as well as microglia M2 polarization, which could be abolished by increased VSIG4 expression. Our study reveals the novel TCF12/VSIG4 axis responsible for the efficacy of palbociclib in combating glioma, offering a rationale for the application of palbociclib in glioma treatment.

Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types.

V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8+ T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.

VSIG4 interaction with heparan sulfates inhibits VSIG4-complement binding.

V-set and immunoglobulin domain-containing 4 (VSIG4) is a complement receptor of the immunoglobulin superfamily that is specifically expressed on tissue resident macrophages, and its many reported functions and binding partners suggest a complex role in immune function. VSIG4 is reported to have a role in immune surveillance as well as in modulating diverse disease phenotypes such as infections, autoimmune conditions, and cancer. However, the mechanism(s) governing VSIG4's complex, context-dependent role in immune regulation remains elusive. Here, we identify cell surface and soluble glycosaminoglycans, specifically heparan sulfates, as novel binding partners of VSIG4. We demonstrate that genetic deletion of heparan sulfate synthesis enzymes or cleavage of cell-surface heparan sulfates reduced VSIG4 binding to the cell surface. Furthermore, binding studies demonstrate that VSIG4 interacts directly with heparan sulfates, with a preference for highly sulfated moieties and longer glycosaminoglycan chains. To assess the impact on VSIG4 biology, we show that heparan sulfates compete with known VSIG4 binding partners C3b and iC3b. Furthermore, mutagenesis studies indicate that this competition occurs through overlapping binding epitopes for heparan sulfates and complement on VSIG4. Together these data suggest a novel role for heparan sulfates in VSIG4-dependent immune modulation.

Identification of a novel immune checkpoint molecule V-set immunoglobulin domain-containing 4 that leads to impaired immunity infiltration in pancreatic ductal adenocarcinoma.

BACKGROUND: Checkpoint-based immunotherapy has failed to elicit responses in the majority of patients with pancreatic cancer. In our study, we aimed to identify the role of a novel immune checkpoint molecule V-set Ig domain-containing 4 (VSIG4) in pancreatic ductal adenocarcinoma (PDAC). METHODS: Online datasets and tissue microarray (TMA) were utilized to analyze the expression level of VSIG4 and its correlation with clinical parameters in PDAC. CCK8, transwell assay and wound healing assay were applied to explore the function of VSIG4 in vitro. Subcutaneous, orthotopic xenograft and liver metastasis model was established to explore the function of VSIG4 in vivo. TMA analysis and chemotaxis assay were conducted to uncover the effect of VSIG4 on immune infiltration. Histone acetyltransferase (HAT) inhibitors and si-RNA were applied to investigate factors that regulate the expression of VSIG4. RESULTS: Both mRNA and protein levels of VSIG4 were higher in PDAC than normal pancreas in TCGA, GEO, HPA datasets and our TMA. VSIG4 showed positive correlations with tumor size, T classification and liver metastasis. Patients with higher VSIG4 expression were related to poorer prognosis. VSIG4 knockdown impaired the proliferation and migration ability of pancreatic cancer cells both in vitro and in vivo. Bioinformatics study showed positive correlation between VSIG4 and infiltration of neutrophil and tumor-associated macrophages (TAMs) in PDAC, and it inhibited the secretion of cytokines. According to our TMA panel, high expression of VSIG4 was correlated with fewer infiltration of CD8+ T cells. Chemotaxis assay also showed knockdown of VSIG4 increased the recruitment of total T cells and CD8+ T cells. HAT inhibitors and knockdown of STAT1 led to decreased expression of VSIG4. CONCLUSIONS: Our data indicate that VSIG4 contributes to cell proliferation, migration and resistance to immune attack, thus identified as a promising target for PDAC treatment with good prognostic value.

Therapeutic modulation of V Set and Ig domain-containing 4 (VSIG4) signaling in immune and inflammatory diseases.

Inflammation is the result of acute and chronic stresses, caused by emotional or physical trauma, or nutritional or environmental pollutants, and brings serious harm to human life and health. As an important cellular component of the innate immune barrier, the macrophage plays a key role in maintaining tissue homeostasis and promoting tissue repair by controlling infection and resolving inflammation. Several studies suggest that V Set and Ig domain-containing 4 is specifically expressed in tissue macrophages and is associated with a variety of inflammatory diseases. In this paper, we mainly summarize the recent research on V Set and Ig domain-containing 4 structures, functions, function and roles in acute and chronic inflammatory diseases, and provide a novel therapeutic avenue for the treatment of inflammatory diseases, including nervous system, urinary, respiratory and metabolic diseases.

Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH.

BACKGROUND: The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear. METHODS: Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4+ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining. RESULTS: Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163+ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4+-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4+-KCs than in those cocultured with lipotoxic KCs. CONCLUSIONS: A subgroup of Vsig4+ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.

VSIG4-expressing tumor-associated macrophages impair anti-tumor immunity.

VSIG4, a newly identified co-inhibitory molecule belonging to the B7-related family, is exclusively expressed on tissue-resident macrophages and is involved in the suppression of T cell proliferation and cytokine production. We sought to characterize the role of VSIG4 in anti-tumor immunity in the tumor microenvironment, focusing on VSIG4-expressing tumor-associated macrophages (TAMs). We found that VSIG4-expressing TAMs negatively regulated antigen-specific T cell proliferation and cytokine production through direct inhibition via cell cycle arrest, but not apoptosis, as well as through their arginase 1 activity. Furthermore, VSIG4-expressing TAMs suppress tumor-specific CD8+ T cell cytotoxicity. Therefore, our results suggest that VSIG4-expressing TAMs could be a negative cellular regulator of anti-tumor immunity in the tumor microenvironment.

VSIG4/CRIg directly regulates early CD8+ T cell activation through its counter-receptor in a narrow window.

T-cell responses are fine-tuned by positive and negative co-signal molecules expressed on immune cells and adjacent tissues. VSIG4 is a newly identified member of the B7 family of ligands, which negatively regulates innate inflammatory and CD4+ T cell-mediated responses. However, little is known about the direct effects of VSIG4, which are exerted through an unidentified counter-receptor on CD8+ T cells. We investigated the binding of the VSIG4-Ig fusion protein during CD8+ T cell activation, and the functional involvement of VSIG4 pathway, using VSIG4-Ig and VSIG4-transfectants. VSIG4-Ig binding to CD8+ T cells was temporally observed in the CD44high phenotype during initial activation. VSIG4-Ig binding was observed earlier than the induction of PD-1, LAG3, and TIM-3, which are immune checkpoint receptors for exhausted CD8+ T cells. Immobilized VSIG4-Ig inhibited anti-CD3/CD28 mAb-induced CD8+ T cell activation, as indicated by proliferation and IFN-γ production, similar to the downregulation of T-bet and Eomesodermin transcription factors. VSIG4 on FcγR+ P815 or specific antigen-presenting E.G7 cells inhibited the generation of effector CD8+ T cells, as indicated by proliferation, IFN-γ and TNF-α expression, and granule degradation, compared to parental cells. However, the window for the regulatory function of VSIG4 was narrow and dependent on the strength of TCR (and CD28)-mediated signals. Our results suggested that VSIG4 directly delivers co-inhibitory signals via an as-yet unidentified counter-receptor on activated CD8+ T cells. VSIG4-mediated CD8+ T cell tolerance might contribute to the steady-state maintenance of homeostasis.

VISTA Stimulation of VSIG4-Positive Macrophages Strongly Suppresses T Cell Proliferation via Excessive Nitric Oxide Production in Sepsis.

Organ damage and immune deficiency are important problems in sepsis caused by an excessive immune response. There is controversy about the cause of immune suppression. In this study, we investigated the roles of macrophages that exhibit excessive activity on T cell immunity. Peritoneal macrophages from mice with cecal ligation and puncture (CLP)-induced sepsis migrated to different organs. In particular, V-set immunoglobulin (Ig)-domain-containing 4 (VSIG4) positive macrophages appeared in the spleen 48 h after CLP induction. When cocultured with splenic T cells, VSIG4(+) cells inhibited the proliferation of activated T cells through the release of nitric oxide (NO) compared to VSIG4(-) cells. Stimulation of VSIG4(+) cells with V-domain Ig suppressor of T cell activation (VISTA) antibody increased the expression of several cytokine genes and the release of NO, but not phagocytosis, compared to those of hamster IgG-stimulated VSIG4(+) cells. When cocultured with splenic T cells, VISTA-stimulated VSIG4(+) cells induced excessive T cell suppression via more NO secretion compared to hamster IgG-stimulated VSIG4(+) cells. Taken together, the current study demonstrates that VSIG4(+) peritoneal macrophages play important roles in inducing immunosuppression and that VISTA acts as a costimulatory receptor in these cells. These data suggest that blocking the migration of VSIG4(+) cells might alleviate excessive immune activity and that blocking VISTA on VSIG4(+) macrophages might play a crucial role in the development of new therapies to prevent T cell suppression in sepsis.

Immunoregulatory protein V-set and immunoglobulin domain-containing 4 is overexpressed in patients with endometriosis.

AIM: V-set and immunoglobulin domain-containing 4 (VSIG4) is a potent negative regulator of T-cell responses and is suggested to regulate antitumor immunity. This study investigates whether VSIG4 is significantly expressed in endometriosis patients and the association between VSIG4 levels and serum cancer antigen (CA)-125 levels, VSIG4 levels and endometriosis severity. METHODS: Tumor tissues and peripheral blood samples were obtained during surgery from 42 endometriotic cyst and 21 nonendometriotic tumor patients. The levels of VSIG4 mRNA, VSIG4 protein expression in tumor tissue and serum soluble VSIG4 concentration were compared between the two groups. After dividing the cohort using the optimized cut-off values obtained by receiver operating characteristic curve analysis, we examined the association between VSIG4 levels and serum CA-125 levels, VSIG4 levels and the factors indicating endometriosis severity. RESULTS: The expressions of VSIG4 mRNA, VSIG4 protein and serum VSIG4 concentration were significantly increased in the endometriotic cyst group compared with the control group (P = 0.001, 0.002 and 0.049, respectively). The optimized VSIG4 cut-off values for endometriosis prediction were 0.71, 0.32 and 144.37 pg/mL, respectively. After cohort division using these values, high VSIG4 levels group showed significantly elevated CA-125 compared with low VSIG4 level group (P = 0.010, 0.043 and 0.039, respectively). There was no association between VSIG4 levels and the factors indicating endometriosis severity. CONCLUSION: The expression of VSIG4 in endometriosis patients is increased compared with nonendometriotic tumor patients, and higher VSIG4 levels are significantly associated with higher serum CA-125 levels. VSIG4 may be importantly involved in the immunological alteration of endometriosis.

Serum soluble VSIG4 as a surrogate marker for the diagnosis of lymphoma-associated hemophagocytic lymphohistiocytosis.

Lymphoma-associated haemophagocytic lymphohistiocytosis (L-HLH) is characterized by excessively activated macrophages and cytotoxic T lymphocytes, but few reliable markers for activated macrophages are available clinically. This study, designed to discover novel biomarkers for the diagnosis of lymphoma patients with L-HLH, was initiated between 2016 and 2018. Fifty-seven adult lymphoma patients were enrolled - 39 without HLH and 18 with HLH. The differential serum protein expression profile was first screened between lymphoma patients with and without L-HLH by a quantitative mass spectrometric approach. Soluble V-set and immunoglobulin domain-containing 4 (sVSIG4), specifically expressed by macrophages, was significantly upregulated in the L-HLH group. Subsequently, sVSIG4 concentration was confirmed by enzyme-linked immunosorbent assay to be significantly increased in lymphoma patients with L-HLH. When it was exploited for the diagnosis of lymphoma patients with L-HLH, the area under a receiver operating characteristic curve was 0·98 with an optimal cut-off point of 2195 pg/ml and the corresponding sensitivity and specificity were 94·44% and 94·87% respectively. In addition, the one-year overall survival was significantly worse in patients with a sVSIG4 concentration above 2195 pg/ml compared with those below 2195 pg/ml (5·3% vs. 72·2%, P < 0·0001). sVSIG4 may be a surrogate marker of activated macrophages for the diagnosis of lymphoma patients with L-HLH.

Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response.

Ischemic stroke is a leading cause of death among human in the world, and a critical cause for long-term disability. Accumulating studies have indicated that inflammatory response regulated by microglia contributes a lot to neuronal death, but the molecular mechanism still remains unclear. V-set and immunoglobulin domain-containing 4 (Vsig4), a complement receptor of the immunoglobulin superfamily (CRIg) that specifically expresses in resting tissue-resident macrophages, plays a critical role in regulating various inflammatory diseases via multiple signaling pathways. However, the effects of Vsig4 on ischemic stroke have not been investigated. In this study, we identified that Vsig4 expression was decreased after cerebral ischemic injury induced by middle cerebral artery occlusion (MCAO). Immunofluorescence staining showed that Vsig4 was co-localized with Iba1 in microglial cells from the infarct region of MCAO-operated mice. After over-expressing Vsig4 in mice, MCAO-induced infarction area and neurological deficits score were markedly attenuated. In addition, neurological dysfunction due to MCAO surgery was improved by Vsig4 over-expression. Microglial M1 polarization was detected in mice with MCAO surgery, which was markedly inhibited by Vsig4 over-expression, as evidenced by the markedly reduced expression of CD16, CD11b, inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6); however, the expression of M2-like phenotype hallmarks such as arginase 1 (Arg1), CD206, IL-10 and Ym-1 was significantly up-regulated. Mechanistically, the anti-inflammatory role of Vsig4 was mainly through the blockage of toll-like receptor 4/nuclear factor kappa B (TLR4/NF-κB) signaling via the in vivo and in vitro experiments. Also, we found that microglial TLR4 expression in the cerebral infarct area of MCAO mice was highly suppressed by Vsig4 over-expression. In vitro, the neuron-glial mixed culture by fluorescent staining showed that oxygen glucose deprivation (OGD) treatment led to significant cell death, while being attenuated by Vsig4 over-expression in primary microglial cells. Finally, we showed that Vsig4 could interact with TLR4 and repress its expression, subsequently alleviating ischemic stroke. Collectively, our findings demonstrated that microglial Vsig4 protected against post-stroke neuro-inflammation mainly through interacting with TLR4.

Expression of Vsig4 attenuates macrophage-mediated hepatic inflammation and fibrosis in high fat diet (HFD)-induced mice.

The innate immune response contributes to hepatic steatosis and nonalcoholic fatty liver disease (NAFLD). However, the pathogenic mechanism of NAFLD is still poorly understood. The costimulatory molecule V-set and immunoglobulin domain-containing protein-4 (Vsig4), which is exclusively expressed on macrophages, shows significant role in regulating macrophage-mediated inflammation. Here, we attempted to explore if Vsig4 expression was involved in high fat diet (HFD)-induced NAFLD. The results indicated that Vsig4 expression was markedly down-regulated in fatty livers of NAFLD patients and obese mice. Vsig4 knockout accelerated HFD-induced metabolic dysfunction. In addition, the loss of Vsig4 significantly promoted insulin resistance and lipid deposition in liver samples of HFD-challenged mice. Furthermore, HFD-induced inflammation was apparently accelerated in Vsig4 knockout mice by further activating nuclear factor-κB (NF-κB) signaling pathway. Also, Vsig4 deficient mice exhibited greater collagen accumulation in hepatic samples in HFD-challenged mice compared to the WT mice, which was through promoting transforming growth factor-ß1 (TGFß1) signaling. Importantly, we found that lipopolysaccharide (LPS)- or TGFß1-stimulated inflammation and fibrosis in primary hepatocytes and hepatic stellate cells, respectively, were markedly exacerbated by co-culture with condition medium from bone marrow-derived macrophages (BMDMs) with Vsig4 deficiency. Finally, transplantation of bone marrow cells from control mice to Vsig4-knockout mice restored the severity of steatosis, inflammation and fibrosis after HFD feeding. Therefore, loss of Vsig4 accelerated the severity of lipid deposition, fibrosis and the inflammatory response. Vsig4 could be a therapeutic target for NAFLD treatment.

NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis.

Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Epstein-Barr virus-encoded latent membrane protein 1 induces epithelial to mesenchymal transition by inducing V-set Ig domain containing 4 (VSIG4) expression via NF-kB in renal tubular epithelial HK-2 cells.

The epithelial to mesenchymal transition (EMT), a hallmark of chronic kidney disease, is a key event in the conversion from tubular epithelial cells to myofibroblasts in renal fibrosis. Epstein-Barr virus (EBV) is a γ-herpes oncovirus associated with chronic kidney disease. However, the relationship between EBV and the EMT process in renal tubular epithelial cells is not well understood. Among EBV-latent genes, EBV-encoded latent membrane protein 1 (LMP1) induces EMT by regulating a variety of molecules in EBV-induced oncogenic transformation. In this study, we investigated EBV-encoded LMP1 and EMT process markers in human proximal tubule epithelial cell line HK-2. LMP1 overexpression induces cell morphological changes via the epithelial to mesenchymal process in HK-2 cells, and these changes accelerate cell proliferation, cell motility, and invasion. Furthermore, VSIG4 upregulation by EBV-LMP1 induced LMP1-mediated EMT, cell motility, and invasion. VSIG4 upregulation by LMP1 was regulated at the transcriptional level via the NF-kB signaling axis. These results suggest that EBV-encoded LMP1 regulates EMT through the NF-kB-VSIG4 axis in HK-2 cells, and VSIG4 is a potential target in EBV-induced chronic kidney diseases.

Complement receptor of the immunoglobulin superfamily reduces murine lupus nephritis and cutaneous disease.

Complement activation takes place in autoimmune diseases and accounts for tissue inflammation. Previously, complement inhibition has been considered for the treatment of SLE. Complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the alternative pathway of complement and a soluble form reverses established inflammation and bone destruction in experimental autoimmune arthritis. We asked whether specific inhibition of the alternative pathway could inhibit autoimmunity and/or organ damage in lupus-prone mice. Accordingly, we treated lupus-prone MRL/lpr mice with a soluble form of CRIg (CRIg-Fc) and we found that it significantly diminished skin lesions, proteinuria and pyuria, and kidney pathology. Interestingly, serum levels of anti-DNA antibodies were not affected despite the fact that serum complement 3 (C3) levels increased significantly. Immunofluorescent staining of kidney tissues revealed a reduction in staining intensity for C3, IgG, and the macrophage marker Mac-2. Thus our data show that inhibition of the alternative pathway of complement controls skin and kidney inflammation even in the absence of an effect on the production of autoantibodies. We propose that CRIg should be considered for clinical trials in patients with systemic lupus erythematosus.

The role of SPI1/VSIG4/THBS1 on glioblastoma progression through modulation of the PI3K/AKT pathway.

INTRODUCTION: Glioblastoma multiforme (GBM) poses a significant challenge in terms of treatment due to its high malignancy, necessitating the identification of additional molecular targets. VSIG4, an oncogenic gene participates in tumor growth and migration in various cancer types. Nevertheless, the precise process through which VSIG4 facilitates the malignant progression of glioma remains to be elucidated. OBJECTIVES: This research aims to explore the function and molecular mechanism involving VSIG4 in the malignant progression of glioma. METHODS: The amount of VSIG4 was measured using qPCR, western blotting, and immunohistochemistry. Lentivirus infections were applied for upregulating or downregulating molecules within glioma cells. The incorporation of 5-ethynyl-20-deoxyuridine, Transwell, cell counting kit-8, and clone formation experiments, were applied to assess the biological functions of molecules on glioma cells. Dual luciferase reporter gene, RNA immunoprecipitation, and chromatin immunoprecipitation assays were used to explore the functional relationship among relevant molecules. RESULTS: The upregulation of VSIG4 was observed in GBM tissues, indicating an adverse prognosis. Silencing VSIG4 in glioma cells resulted in a decrease in cell viability, invasion, proliferation, and tumorigenesis, an increase in cell apoptosis, and a stagnation in the cell cycle progression at the G0/G1 phase. Mechanistically, SPI1-mediated upregulation of VSIG4 expression led to binding between VSIG4 and THBS1 protein, ultimately facilitating the malignant progression of glioma cells through the activation of the PI3K/AKT pathway. The inhibited proliferative and invasive capabilities of glioma cells were reversed by overexpressing THBS1 following the knockdown of VSIG4. CONCLUSION: Our findings provide evidence for the role of VSIG4 as an oncogene and reveal the previously unidentified contribution of the SPI1/VSIG4/THBS1 axis in the malignant progression of glioma. This signaling cascade enhances tumor growth and invasion by modulating the PI3K/AKT pathway. VSIG4 as a potential biomarker may be a viable strategy in the development of tailored molecular therapies for GBM.

VSIG4 ameliorates intestinal inflammation through inhibiting macrophages NLRP3 inflammasome and pyroptosis.

Inflammatory bowel disease (IBD) is one of the most common diseases in the digestive system related to aberrant inflammation. V-set and immunoglobulin domain-containing 4 (VSIG4), a type I transmembrane receptor exclusively expressed in a subset of tissue-resident macrophages, has been reported to exert anti-inflammatory activity in immune-related diseases, which has been not explored in IBD yet. This study aims to explore the role and the potential mechanism of VSIG4 in IBD. Clinical samples were obtained from IBD patients and were examined by immunohistochemical staining. THP-1 cells were differentiated into macrophages, and then stimulated with IL-4 plus IL-13 or LPS to induce pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. Cell transfection was conducted to overexpress VSIG4. Western blot and immunofluorescence assays were performed to assess NLRP3 inflammasome- and pyroptosis-related proteins. Cytokines were measured using ELISA. A cell co-culture model of Caco-2 cells and VSIG4-mediated macrophages were established. Cell viability and apoptosis was examined by CCK-8 and flow cytometry assays, respectively. VSIG4 was downregulated in IBD and was negatively correlated with NLRP3 inflammasome. M1 macrophages exhibited higher levels of NLRP3 inflammasome, pyroptosis and inflammatory response than M2 macrophages, while VSIG4 overexpression efficiently reversed these changes in M1 macrophages. In addition, VSIG4 overexpression partly abolished M1 macrophages-induced cell viability loss, inflammatory response, apoptosis and pyroptosis in Caco-2 cells. Collectively, VSIG4 might alleviate intestinal inflammation through regulating M1/M2 macrophages, providing novel insights for the treatment of human IBD.

VSIG4 inhibits RANKL-induced osteoclastogenesis by enhancing Nrf2-dependent antioxidant response against reactive oxygen species production.

Osteoporosis is a prevalent systemic skeletal disorder, particularly affecting postmenopausal women, primarily due to excessive production and activation of osteoclasts. However, the current anti-osteoporotic drugs utilized in clinical practice may lead to certain side effects. Therefore, it is necessary to further unravel the potential mechanisms regulating the osteoclast differentiation and to identify novel targets for osteoporosis treatment. This study revealed the most significant decline in VSIG4 expression among the VSIG family members. VSIG4 overexpression significantly inhibited RANKL-induced osteoclastogenesis and bone resorption function. Mechanistically, both western blot and immunofluorescence assay results demonstrated that VSIG4 overexpression attenuated the expression of osteoclast marker genes and dampened the activation of MAPK and NF-κB signaling pathways. Furthermore, VSIG4 overexpression could inhibit the generation of reactive oxygen species (ROS) and stimulate the expression of Nrf2 along with its downstream antioxidant enzymes via interaction with Keap1. Notably, a potent Nrf2 inhibitor, ML385, could reverse the inhibitory effect of VSIG4 on osteoclast differentiation. In line with these findings, VSIG4 overexpression also mitigated bone loss induced by OVX and attenuated the activation of osteoclasts in vivo. In conclusion, our results suggest that VSIG4 holds promise as a novel target for addressing postmenopausal osteoporosis. This is achieved by suppressing osteoclast formation via enhancing Nrf2-dependent antioxidant response against reactive oxygen species production.

VSIG4 induces the immunosuppressive microenvironment by promoting the infiltration of M2 macrophage and Tregs in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and has a poor prognosis. Despite the impressive advancements in treating ccRCC using immune checkpoint (IC) blockade, such as PD-1/PD-L1 inhibitors, a considerable number of ccRCC patients experience adaptive resistance. Therefore, exploring new targetable ICs will provide additional treatment options for ccRCC patients. We comprehensively analyzed multi-omics data and performed functional experiments, such as pathologic review, bulk transcriptome data, single-cell sequencing data, Western blotting, immunohistochemistry and in vitro/in vivo experiments, to explore novel immunotherapeutic targets in ccRCC. It was found that immune-related genes VSIG4, SAA1, CD7, FOXP3, IL21, TNFSF13B, BATF, CD72, MZB1, LTB, CCL25 and KLRK1 were significantly upregulated in ccRCC (Student's t test and p-value < 0.05; 36 normal and 267 ccRCC tissues in raining cohort; 36 normal and 266 ccRCC tissues in validation cohort) and correlated with the poor prognosis of ccRCC patients (Wald test and p-value < 0.05 in univariate cox analysis; log-rank test and p-value < 0.05 in Kaplan-Meier method; 267 patients in training cohort and 266 in validation cohort). In particular, we found the novel IC target VSIG4 was specifically expressed in inhibitory immune cells M2-biased tumor-associated macrophages (TAMs), conventional dendritic cell 2 (cDC2) cells, and cycling myeloid cells in ccRCC microenvironment. Moreover, VSIG4 showed a closely relation with resistance of Ipilimumab/Nivolumab immunotherapy in ccRCC. Furthermore, VSIG4 promoted the infiltration of M2 macrophages, Tregs, and cDC2 in ccRCC tissues. VSIG4+ TAMs and VSIG4+ cDC2s may be a kind of immune cell subtypes related to immunosuppression. VSIG4 may play similar roles with other IC ligands, as it is highly expressed on the surface of antigen-presenting cells and ccRCC cells to inhibit T cells activity and facilitate immune escape. Targeting IC gene VSIG4 may provide a novel immunotherapeutic strategy to ccRCC patients with resistance to existing targeted therapy options.