CD226 knockout reduces the development of CD8+ T by impairing the TCR sensitivity of double-positive thymocytes.

The costimulation molecule CD226 is widely involved in T cell differentiation, activation and immune functional regulation in peripheral immune tissues. CD226-deficient mice have impaired immune response capacity. The function of CD226 in regulating T cell development in the thymus, a central immune organ, is not yet fully understood. We investigated the development of thymocytes using CD226 knockout mice and single-cell sequencing techniques. CD226 began to be expressed in the second half of thymocyte development, with a gradual increase from the double-positive (DP) to single-positive (SP) phase and higher levels of CD226 on CD8+ T cells than on CD4+ T cells from the SP phase to mature T cells. In the thymus of CD226KO mice, the proportion of DPT at the quiescent phase (DPT-Q) increased, of which the Gzma+ cluster that tends to be CD8+ T cells and CD5+ cluster that is undergoing positive selection decreased dramatically. Afterward, the proportion of mature CD8+ T cells reduced dramatically. Depletion of CD226 impaired TCR activation signalling and diminished AKT/ERK/NF-κB/p38 phosphorylation levels. The diminished TCR responsiveness of DPT cells impeded their positive selection process and influenced the maturation of CD8+ T cells. In mechanism, CD226 knockout enhanced DPT cell apoptosis via impairing AKT phosphorylation. These results suggest that CD226 plays a significant role in T cell thymic development via modulation of TCR signalling, affecting CD8+ T cell maturation.

CD226 deficiency exacerbated intestinal immune dysregulation in mice with dinitrochlorobenzene-induced atopic dermatitis.

Intestinal mucosal immunity plays a pivotal role in host defence. In this study, we found that cluster of differentiation 226 (CD226) gene knockout (KO) led to more severe atopic dermatitis (AD)-related skin pathologies and bowel abnormalities in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. Following DNCB administration, the expression of CD226 was elevated in intestinal mucosal tissues, including group 3 innate lymphoid cells (ILC3s) and CD4+ T cells of Peyer's patches (PPs). CD226 deficiency led to an overactive intestinal immune response in the AD-like mice, as evidenced by increased inflammation and Th1/Th2-related cytokine levels as well as increased Paneth cell numbers and antimicrobial peptide (AMP) expression, which was likely due to the higher interleukin (IL)-22 production in the lamina propria. Additionally, CD226 deficiency increased the production of IL-4 and IL-17 in mesenteric lymph nodes as well as the number of PPs and expression of immunoglobulin (Ig) A in B cells. Moreover, insufficient expression of CD226 affected the characterization of intraepithelial and lamina propria lymphocytes in the intestinal mucosa. Finally, the number of PPs was increased in CD4+ T cell-specific CD226 KO and regulatory T (Treg) cell-specific CD226 KO mice; thus, loss of CD226 in Treg cells resulted in impaired Treg cell-suppressive function. Therefore, our findings indicate that CD226 deficiency alters intestinal immune functionality in inflammatory diseases.

Competitive binding of CD226/TIGIT with poliovirus receptor regulates macrophage polarization and is involved in vascularized skin graft rejection.

End-stage organ failure often requires solid organ transplantation. Nevertheless, transplant rejection remains an unresolved issue. The induction of donor-specific tolerance is the ultimate goal in transplantation research. In this study, an allograft vascularized skin rejection model using BALB/c-C57/BL6 mice was established to evaluate the regulation of the poliovirus receptor signaling pathway using CD226 knockout or T cell immunoglobulin and ITIM domain (TIGIT)-crystallizable fragment (Fc) recombinant protein treatment. In the TIGIT-Fc-treated and CD226 knockout groups, graft survival time prolonged significantly, with a regulatory T cell proportion increase and M2-type macrophage polarization. Donor-reactive recipient T cells became hyporesponsive while responding normally after a third-party antigen challenge. In both groups, serum interleukin (IL)-1ß, IL-6, IL-12p70, IL-17A, tumor necrosis factor-α, interferon gamma, and monocyte chemoattractant protein-1 levels decreased, and the IL-10 level increased. In vitro, M2 markers, such as Arg1 and IL-10, were markedly increased by TIGIT-Fc, whereas iNOS, IL-1ß, IL-6, IL-12p70, tumor necrosis factor-α, and interferon gamma levels decreased. CD226-Fc exerted the opposite effect. TIGIT suppressed TH1 and TH17 differentiation by inhibiting macrophage SHP-1 phosphorylation and enhanced ERK1/2-MSK1 phosphorylation and nuclear translocation of CREB. In conclusion, CD226 and TIGIT competitively bind to poliovirus receptor with activating and inhibitory functions, respectively. Mechanistically, TIGIT promotes IL-10 transcription from macrophages by activating the ERK1/2-MSK1-CREB pathway and enhancing M2-type polarization. CD226/TIGIT-poliovirus receptor are crucial regulatory molecules of allograft rejection.

Interleukin-6 absence triggers intestinal microbiota dysbiosis and mucosal immunity in mice.

Interleukin-6 (IL-6) in mucosal immune cells is involved in post-injury intestinal regeneration, inflammation responses, and gastric homeostasis. However, the interaction between IL-6 and the dynamic balance of gut microbiota (GM) remains unexplored. Intestinal pathology was assessed by hematoxylin and eosin and periodic acid-Schiff staining in wild-type (WT) and IL-6 gene knockout (KO) C57BL/6J mice. GM profiles were established via high-throughput sequencing of the fecal bacterial 16S rRNA gene. Intestinal α- and ß-defensins were measured by quantitative real-time PCR; further, flow cytometry was performed to analyze isolated intraepithelial lymphocytes (IELs). Compared with the WT, IL-6 KO did not obviously change gut structures, but significantly reduced GM diversity, resulting in reduced metabolic pathways with decreased gram-positive but elevated gram-negative bacteria. More taxa alterations included differences at the phyla (e.g., increased Verrucomicrobia and decreased Firmicutes) and genera (e.g., increased Akkermansia and decreased Lactobacillus) levels. Absence of IL-6 also significantly increased intestinal expression of defensins α3 and α4 (Defa3 and Defa4) and the percentage of natural TCRγδ+ IELs, providing a molecular basis for triggering mucosal immune response. Therefore, IL-6 loss remodels GM composition and alters IEL maintenance, identifying IL-6 as a crucial cytokine for GM dysbiosis and mucosal immunity.

CD226 deficiency promotes glutaminolysis and alleviates mitochondria damage in vascular endothelial cells under hemorrhagic shock.

Hemorrhagic shock (HS) is common in clinical emergencies, leading to millions of deaths each year globally. CD226 is a costimulatory adhesion molecule expressed on both immune cells and endothelial cells (ECs) to regulate their metabolic activity and function. As endothelial dysfunction occurs after HS, the roles CD226 plays in vascular EC metabolism were investigated. CD226fl/fl Tekcre mice were adopted to achieve vascular EC-specific knockout of CD226, and subjected to HS modelling. Serum levels of crucial intermediate metabolites were evaluated through liquid chromatography-mass spectrometry analysis. Human umbilical vein ECs (HUVECs) were used to study the effects of CD226 under hypoxia in vitro. Seahorse analysis evaluated the cellular glycolysis and mitochondria bioenergetics. Results showed that CD226 deficiency in vascular ECs alleviated HS-induced intestinal damage and inflammatory response in mice. Animal studies indicated an improved energy metabolism when CD226 was knocked out in ECs after HS, as evidenced by enhanced glutamine-glutamate metabolism and decreased lactic acid levels. Glut-1 was upregulated in mouse vascular ECs after HS and HUVECs under hypoxia, combined with decreased CD226. Moreover, HUVECs with CD226 knockdown exhibited relieved mitochondrial damage and early apoptosis under hypoxia, whereas CD226 overexpression showed opposite effects. Seahorse analysis showed that downregulated CD226 significantly increased mitochondrial ATP production and glucose uptake in HUVECs under hypoxia. Additionally, Erk/PHD2 signaling-mediated HIF-1α/Glut-1 and HIF-2α/ASCT2 pathways were involved in CD226 regulation on HUVEC glutaminolysis after hypoxia. Hence, CD226 deficiency promotes bypass energy supply to vascular ECs under ischemic or hypoxic stress, to ameliorate the stress-mediated metabolic disturbance.

CD226 Deficiency Alleviates Murine Allergic Rhinitis by Suppressing Group 2 Innate Lymphoid Cell Responses.

Allergic rhinitis (AR) is an immunoglobulin E-mediated type 2 inflammation of the nasal mucosa that is mainly driven by type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s). CD226 is a costimulatory molecule associated with inflammatory response and is mainly expressed on T cells, natural killer cells, and monocytes. This study is aimed at elucidating the role of CD226 in allergic inflammatory responses in murine AR using global and CD4+ T cell-specific Cd226 knockout (KO) mice. AR nasal symptoms were assessed based on the frequency of nose rubbing and sneezing. Hematoxylin and eosin and periodic acid-Schiff staining and quantitative real-time PCR methods were used to determine eosinophils, goblet cells, and ILC2-associated mRNA levels in the nasal tissues of mice. CD226 levels on ILC2s were detected using flow cytometry, and an immunofluorescence double staining assay was employed to determine the number of ILC2s in the nasal mucosa. The results showed that global Cd226 KO mice, but not CD4+ T cell-specific Cd226 KO mice, exhibited attenuated AR nasal symptoms. Eosinophil recruitment, goblet cell proliferation, and Th2-inflammatory cytokines were significantly reduced, which resulted in the alleviation of allergic and inflammatory responses. ILC2s in the murine nasal mucosa expressed higher levels of CD226 after ovalbumin stimulation, and CD226 deficiency led to a reduction in the proportion of nasal ILC2s and ILC2-related inflammatory gene expression. Hence, the effect of CD226 on the AR mouse model may involve the regulation of ILC2 function rather than CD4+ T cells.

CD226 knockout alleviates high-fat diet induced obesity by suppressing proinflammatory macrophage phenotype.

Obesity is associated with chronic low-grade inflammation, contributing to an increasing prevalence of chronic metabolic diseases, such as insulin resistance, non-alcoholic fatty liver disease (NALFD), and steatohepatitis. Macrophages are the predominant immune cells in adipose tissues. Adipose tissue macrophages (ATMs) would switch to pro-inflammatory M1 state during obesity, causing local and systemic inflammation. However, the regulatory mechanism of ATMs has not yet been well described within this process. Using a high-fat diet (HFD)-induced mouse obesity model, we found that the costimulatory molecule CD226 was highly expressed on ATMs and knockout (KO) of CD226 alleviated obesity caused by HFD. Loss of CD226 reduced the accumulation of ATMs and hindered macrophage M1 polarization, with lower serum proinflammatory cytokine levels. Furthermore, deficiency of CD226 on ATMs decreased the phosphorylation levels of VAV1, AKT, and FOXO1 and thereby upregulated PPAR-γ. Further administration of PPAR-γ inhibitor restored M1 phenotype in CD226KO ATMs. In summary, loss of CD226 alleviates the HFD-induced obesity and systemic inflammation through inhibition of the accumulation and M1 polarization of ATMs in which PPAR-γ-dependent signaling pathway is involved, suggesting that CD226 may be identified as a potential molecular target for the clinical treatment of obesity.

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity.

This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.

[Expression of CD226 in the small intestinal group 3 innate lymphoid cells (ILC3) in mice].

Objective To observe the expression of adhesion molecule CD226 on the small intestinal group 3 innate lymphoid cells (ILC3) in mice. Methods The bioinformatics was used to analyze the expression of CD226 on murine ILCs. Small intestinal mucosal lamina propria lymphocytes (LPL) were isolated from wild-type C57BL/6J mice, and the expression of CD226 on ILC1 and ILC3 was detected by flow cytometry. A mouse model of dextran sulfate sodium (DSS)-induced colitis was constructed to observe the changes in the expression of CD226 on ILC3. Results Both ILC1 and ILC3 in the mice small intestine expressed CD226 molecules; the proportion of ILC3 was reduced, while the expression level of CD226 on ILC3 was increased in the colitis model. Conclusion CD226 is expressed on the small intestines of mice, and although the proportion of ILC3 decreases in the DSS-induced colitis, the expression of CD226 on ILC3 increases.

Costimulatory Molecule CD226 Regulates Atopic Dermatitis in a Mouse Model.

This study investigated the role of CD226 in a 2,4-dinitrochlorobenzene (DNCB)-induced mouse model of atopic dermatitis. The results showed that the lack of CD226 (global and CD4+ T-cell specific) significantly increased ear thickness, reddening, swelling, and scaling of the skin as well as inflammatory cell and mast cell infiltration. RT-qPCR results demonstrated that the mRNA expressions of atopic dermatitis-related inflammatory cytokines and chemokines were markedly increased in the draining lymph nodes and lesioned ear skin tissues of global and CD4+ T-cell-specific CD226-deficient mice compared with that in control mice. In vitro assessment revealed that CD226 directly modulates TGFß-mediated regulatory T (Treg) cell differentiation and proliferation. Notably, Treg cell-specific deletion of CD226 (Cd226fl/flFoxp3cre mice) resulted in more severe dermatitis and epidermal thickening than those observed in littermate mice upon DNCB treatment. Subsequent analysis showed that the infiltration of Treg cells in ear lesions and the number of Tregs in the spleen were significantly reduced in Cd226fl/flFoxp3cre mice after DNCB treatment. In addition, the lack of CD226 induced apoptosis of Treg cells through the activation of caspase 3. Therefore, these results suggest that CD226 has potential efficacy in atopic dermatitis, correlating with Treg cell inhibition.

[Knockout of CD226 alleviates depression-like behavior induced by chronic restraint stress in mice by modulating the ratio of immune cells in spleen and intestine].

Objective To investigate the immunoregulatory effects of CD226 on the chronic restraint stress (CRS)-induced depression-like behavior and its underlying mechanism in mice. Methods Male C57/BL6J mice and CD226 gene knockout (KO) mice with the same strain (4-6 week old) were adopted to establish CRS models. The stress-induced depression scores of mice were evaluated through behavioral testing such as forced swimming test and sucrose preference test. Flow cytometry was used to analyze the differences of the intraepithelial lymphocytes in the spleens, peyer's patches, and intestines between the two groups. Results Compared with WT CRS group, mice in CD226KO CRS group showed significantly decreased immobility time in forced swimming test and increased sucrose preference rate. The ratio of CD4+ T cells to CD8+ T cells in spleen was significantly reduced, combined with the remarkably elevated proportion of TCRαß and TCRαßCD8αß cells in the small intestinal IELs of CD226 KO mice with CRS. Conclusion Knockout of CD226 alleviates CRS-induced depression-like behavior in mice, alters the proportion of immune cells in murine spleen and intestine, and improves the overall immune status of mice under stress.

Ablation of CD226 on CD4+ T cells modulates asthma progress associated with altered IL-10 response and gut microbiota.

To investigate the role of the costimulatory molecule CD226 in asthma pathogenesis, we produced a CD4+ T-cell-specific CD226 knockout mice model (Cd226ΔCD4) and induced airway allergic inflammation by administering ovalbumin (OVA). Our results revealed alleviated lung inflammation, decreased levels of OVA-specific IgE, and increased levels of IL-10 in the serum of Cd226ΔCD4 mice (P < 0.05). Moreover, IL-10 levels in CD4+ T cells were significantly elevated in the mediastinal lymph node, spleen, and Peyer's patches in the Cd226ΔCD4 mice compared with those in controls (P < 0.05 to P < 0.01). Notably, there was a significantly higher IL-10 mRNA levels in the large intestine of the mice (P < 0.05). The protective effect of CD226 deficiency is also associated with the accumulation of gut TCRγδ+ intraepithelial lymphocytes and reversion of the gut microbiome dysbiosis. The Bacteroidetes-to-Firmicutes ratio and the abundance of Akkermansia increased in the absence of CD226 after OVA treatment. Our data reveal the synchronous changes in the lung and intestine in OVA-treated CD226-knockout mice, supporting the gut-lung axis concept and providing evidence for novel therapeutic approaches for asthma.

CD226 maintains regulatory T cell phenotype stability and metabolism by the mTOR/Myc pathway under inflammatory conditions.

Regulatory T (Treg) cells exhibit immunosuppressive phenotypes and particular metabolic patterns with certain degrees of plasticity. Previous studies of the effects of the co-stimulatory molecule CD226 on Treg cells are controversial. Here, we show that CD226 primarily maintains the Treg cell stability and metabolism phenotype under inflammatory conditions. Conditional deletion of CD226 within Foxp3+ cells exacerbates symptoms in murine graft versus host disease models. Treg cell-specific deletion of CD226 increases the Treg cell percentage in immune organs but weakens their immunosuppressive function with a T helper 1-like phenotype conversion under inflammation. CD226-deficient Treg cells exhibit reduced oxidative phosphorylation and increased glycolysis rates, which are regulated by the adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/myelocytomatosis oncogene (Myc) pathway, and inhibition of Myc signaling restores the impaired functions of CD226-deficient Treg cells in an inflammatory disease model of colitis. This study reveals an Myc-mediated CD226 regulation of Treg cell phenotypic stability and metabolism, providing potential therapeutic strategies for targeted interventions of Treg cell-specific CD226 in inflammatory diseases.

Identification of CaMK4 as a sex-difference-related gene in knee osteoarthritis.

Background: Osteoarthritis (OA) is a common degenerative joint disease with a higher prevalence in females than in males. Sex may be a key factor affecting the progression of OA. This study aimed to investigate critical sex-difference-related genes in patients with OA and confirm their potential roles in OA regulation. Methods: OA datasets GSE12021, GSE55457, and GSE36700 were downloaded from the Gene Expression Omnibus database to screen OA-causing genes that are differentially expressed in the two sexes. Cytoscape was used to construct a protein-protein interaction network and determine hub genes. Synovial tissues of patients (male and female) with OA and female non-OA healthy controls were obtained to confirm the expression of hub genes and screen the key genes among them. Destabilization of the medial meniscus (DMM)-induced OA mice model was established to verify the screened key genes. Hematoxylin and eosin (HE) staining and Safranin O-fast green dye staining were employed to observe synovial inflammation and pathological cartilage status. Results: The abovementioned three datasets were intersected to obtain 99 overlapping differentially expressed genes, of which 77 were upregulated and 22 were downregulated in female patients with OA. The hub genes screened were EGF, AQP4, CDC42, NTRK3, ERBB2, STAT1, and CaMK4. Among them, Ca2+/calmodulin-dependent protein kinase-4 (CaMK4) was identified as a key sex-related gene for OA. It was significantly higher in female OA patients than in the cases of male patients. Moreover, CaMK4 was significantly increased in female patients with OA compared with the female non-OA group. These results suggest that CaMK4 plays an important role in the progression of OA. OA mouse models demonstrated that CaMK4 expression in the mice knee joint synovial tissue elevated after DMM, with aggravated synovial inflammation and significant cartilage damage. Cartilage damage improved after intraperitoneal administration of the CaMK4 inhibitor KN-93. Conclusions: CaMK4 is a key sex-related gene influencing the progression and pathogenesis of OA and may be considered as a new target for OA treatment.

Loss of CD226 protects apolipoprotein E-deficient mice from diet-induced atherosclerosis.

CD226 is a costimulatory molecule that regulates immune cell functions in T cells, natural killer cells, and macrophages. Because macrophage-derived foam cell formation is a crucial factor contributing to the development of atherosclerosis, we aimed to evaluate the potential roles of CD226 in the pathogenesis of atherosclerosis. The effects of CD226 on atherosclerosis were investigated in CD226 and apolipoprotein E double-knockout (CD226-/- ApoE-/-) mice fed with a high-cholesterol atherogenic diet. CD226 expression in macrophages was evaluated using flow cytometry. Histopathological analysis was performed to evaluate the atherosclerotic lesions. Inflammatory cell infiltration was detected using immunofluorescence staining. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PEMs) were isolated from the mice and used to explore the mechanism in vitro. The in vivo results indicated that CD226 knockdown protected against atherosclerosis in ApoE-/- mice, evidenced by reduced plaque accumulation in the brachiocephalic artery, aortic roots, and main aortic tree. CD226 gene-deficient macrophages showed reduced foam cell formation under ox-low density lipoprotein stimulation compared with wild-type (WT) cells. CD226 deficiency also decreased the expression of CD36 and scavenger receptor (SR)-A (responsible for lipoprotein uptake) but increased the expression of ATP-binding cassette transporter A1 and G1 (two transporters for cholesterol efflux). Therefore, loss of CD226 hinders foam cell formation and atherosclerosis progression, suggesting that CD226 is a promising new therapeutic target for atherosclerosis.

Knockout of IL-6 mitigates cold water-immersion restraint stress-induced intestinal epithelial injury and apoptosis.

Background: Interleukin-6 (IL-6) is essential for maintaining intestinal epithelial homeostasis. Although cold water-immersion restraint (CWIR) stress is commonly used to induce in vivo gastric injury, it also affects intestinal epithelial permeability. Although IL-6 is increased in response to acute physiological and psychological stress, its exact effects on the pathophysiology of the intestinal epithelium in response to acute CWIR stress remain unknown. Methods: We used IL-6 knockout (KO) mice with acute CWIR modeling to investigate the effect of IL-6 deficiency on intestinal epithelial morphology and pathological damage using histological staining assays under the acute stress. We detected jejunal epithelial apoptosis using TUNEL and standard molecular experiments. Results: CWIR caused intestinal epithelial damage, which was alleviated by the absence of IL-6, as evidenced by morphological changes and goblet cell and intestinal permeability alteration. IL-6 KO also reduced CWIR-mediated inflammatory levels and improved stress defense. Meanwhile, IL-6 deficiency decreased the intestinal epithelial apoptosis induced by CWIR administration. This IL-6 KO-led effect depended more on mitochondrial AIF signaling rather than the traditional caspase pathway. Conclusion: As a result, we concluded that acute CWIR-induced severe intestinal damage and jejunal epithelium apoptosis could be alleviated by IL-6 deficiency, implying a protective effect of IL-6 deficiency on the intestines under acute stress. The findings shed new light on treating CWIR-induced intestinal disorders by inhibiting IL-6 signaling.

Acute Cold Water-Immersion Restraint Stress Induces Intestinal Injury and Reduces the Diversity of Gut Microbiota in Mice.

Growing evidence has demonstrated that stress triggers gastrointestinal (GI) disorders. This study aimed to investigate how the acute cold water-immersion restraint (CWIR) stress affects intestinal injury and gut microbiota (GM) distribution. Male C57BL/6 mice were used to establish a CWIR animal model. Hematoxylin-eosin and periodic acid-Schiff staining were performed to assess intestinal histopathological changes. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and immunofluorescence staining were used to evaluate the expression of inflammatory cytokines and immune cell infiltration in the intestinal tissues. The gut permeability and intestinal occludin protein expression were determined through fluorescein isothiocyanate-dextran detection and western blot, respectively. GM profiles were analyzed via high-throughput sequencing of the fecal bacterial 16S rRNA genes. Results showed that CWIR induced more severe intestinal mucosal injury compared to the control, leading to a significant increase in tumor necrosis factor-α expression, but no infiltration of neutrophil and T cells. CWIR also resulted in GI disruption and increased the permeability of the intestinal mucosa. GM profiles showed that CWIR reduced GM diversity of mice compared with the control group. Specifically, aerobic and gram-negative bacteria significantly increased after CWIR, which was associated with the severity of gut injury under stress. Therefore, acute CWIR leads to severe intestinal damage with inflammation and disrupts the GM homeostasis, contributing to decreased GM diversity. Our findings provide the theoretical basis for the further treatment of intestinal disorders induced by CWIR.

[Preparation and identification of mouse-derived monoclonal antibodies to human ST2 molecule].

Objective To prepare mouse-derived monoclonal antibody against human suppression of tumorigenicity 2 (ST2) molecule and make initial identification. Methods BALB/c mice were immunized with the recombinant human ST2 molecule, and the conventional B-cell hybridoma technology was used to prepare the anti-ST2 monoclonal antibodies (mAbs). Their application in western blotting, immunohistochemistry, and flow cytometry were evaluated. The sandwich ELISA detecting soluble ST2 was established to test the serum levels of ST2 in patients with heat stroke. And the ST2 luciferase reporter gene detection system was established to detect their neutralization activity. Results Thirty-eight hybridoma cell lines secreting mouse anti-human ST2 mAb were obtained and named from XA325.1 to XA325.38. Preliminary screening and identification of them showed that they can be used to identify the purified recombinant ST2 proteins and cellular expressed ST2 using western blotting and immunohistochemistry. Two of them can be used for flow cytometry to identify the exogenously transfected ST2 molecule on the cell surface. Using XA325.16 mAb coating, combined with XA325.5-labeled biotin, an ELISA kit detecting soluble ST2 in serum was established. It was found that the serum levels of ST2 in patients with heat stroke increased significantly. Moreover, XA325.5 was found with neutralizing activity which can block the biological effect of IL-33. Conclusion A set of mouse anti-human ST2 mAbs was prepared, which can be used in a variety of immunological detection techniques. Besides, XA325.5 neutralizing antibody has a potential value in clinical application.

Splenectomy improves liver fibrosis via tumor necrosis factor superfamily 14 (LIGHT) through the JNK/TGF-ß1 signaling pathway.

Splenectomy has been reported to improve liver fibrosis in patients with cirrhosis and hypersplenism. However, the mechanisms remain unclear. Tumor necrosis factor superfamily 14 (TNFSF14; also known as LIGHT) is highly expressed in the context of fibrosis and promotes disease progression in patients with fibrotic diseases such as pulmonary and skin fibrosis. Here, we determined whether splenectomy controls the production of LIGHT to improve liver fibrosis. Splenectomy reduced serum LIGHT levels in cirrhotic patients with hypersplenism and a ConA-induced liver fibrosis mouse model. Blocking LIGHT resulted in the downregulation of TGF-ß1 in RAW264.7 cells. LIGHT treatment of RAW264.7 and JS1 cells in coculture regulated transforming growth factor-ß1 (TGF-ß1) expression through the activation of JNK signaling. Small interfering RNA-mediated silencing of lymphotoxin ß receptor (LTßR) in macrophages resulted in pronounced decreases in the levels of fibrosis and αSMA in JS1 cells. These results indicated that LIGHT bound to LTßR and drove liver fibrosis in vitro. Blocking TGF-ß1 abolished the effect of LIGHT in vitro. Furthermore, the administration of recombinant murine LIGHT protein-induced liver fibrosis with splenectomy, while blocking LIGHT without splenectomy improved liver fibrosis in vivo, revealing that the decrease in fibrosis following splenectomy was directly related to reduced levels of LIGHT. Thus, high levels of LIGHT derived from the spleen and hepatic macrophages activate JNK signaling and lead to increased TGF-ß1 production in hepatic macrophages. Splenectomy attenuates liver fibrosis by decreasing the expression of LIGHT.

Silencing of HuR Inhibits Osteosarcoma Cell Epithelial-Mesenchymal Transition via AGO2 in Association With Long Non-Coding RNA XIST.

BACKGROUND: Osteosarcoma (OS) is a highly malignant and aggressive bone tumor. This study was performed to explore the mechanisms of HuR (human antigen R) in the progression of OS. METHODS: HuR expression levels in OS tissues and cells were detected by immunohistochemistry and western blotting. HuR siRNA was transfected into SJSA-1 OS cells to downregulate HuR expression, and then cell proliferation, migration, and epithelial-mesenchymal transition (EMT) were evaluated. RNA immunoprecipitation was performed to determine the association of the long non-coding RNA (lncRNA) XIST and argonaute RISC catalytic component (AGO) 2 with HuR. Fluorescence in situ hybridization analysis was performed to detect the expression of lncRNA XIST. Western blotting and immunofluorescence assays were performed to observe AGO2 expression after HuR or/and lncRNA XIST knockdown. RESULTS: Knockdown of HuR repressed OS cell migration and EMT. AGO2 was identified as a target of HuR and silencing of HuR decreased AGO2 expression. The lncRNA XIST was associated with HuR-mediated AGO2 suppression. Moreover, knockdown of AGO2 significantly inhibited cell proliferation, migration, and EMT in OS. CONCLUSION: Our findings indicate that HuR knockdown suppresses OS cell EMT by regulating lncRNA XIST/AGO2 signaling.