Interleukin-35 protein inhibits osteoclastogenesis and attenuates collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features include synovial inflammation, bone erosion, and joint structural damage. Our previous studies have shown that interleukin (IL)-35 is involved in the pathogenesis of bone loss in RA patients. In this study, we are further evaluating the efficacy of IL-35 on collagen-induced arthritis (CIA) in the mouse model. Male DBA/1J mice (n = 10) were initially immunized, 2 µg/mouse IL-35 was injected intraperitoneally every week for 3 weeks after the establishment of the CIA model. Clinical arthritis, histopathological analysis, and three-dimensional micro-computed tomography (3D micro-CT) were determined after the mice were anesthetized on the 42th day. In vitro, RANKL/M-CSF induced mouse preosteoclasts (RAW264.7 cells line) was subjected to antiarthritis mechanism study in the presence of IL-35. The results of clinical arthritis, histopathological analysis, and 3D micro-CT, the expression of RANK/RANKL/OPG axis, inflammatory cytokines, and osteoclastogenesis-related makers demonstrated decreasing severity of synovitis and bone destruction in the ankle joints after IL-35 treatment. Furthermore, IL-35 attenuated inflammatory cytokine production and the expression of osteoclastogenesis-related makers in a mouse preosteoclasts cell line RAW264.7. The osteoclastogenesis-related makers were significantly reduced in IL-35 treated RAW264.7 cells line after blockage with the JAK/STAT1 signaling pathway. These results demonstrated that IL-35 protein could inhibits osteoclastogenesis and attenuates CIA in mice. We concluded that IL-35 can exhibit anti-osteoclastogenesis effects by reducing the expression of inflammatory cytokines and osteoclastogenesis-related makers, thus alleviating bone destruction in the ankle joint and could be a potential therapeutic target for RA.

Immunomodulatory effects of iTr35 cell subpopulation and its research progress.

The spotlight in recent years has increasingly focused on inducible regulatory T cells 35 (iTr35), a novel subpopulation of regulatory T cells characterized by phenotypic stability, heightened reactivity, and potent immunosuppressive function through the production of IL-35. Despite being in the exploratory phase, research on iTr35 has garnered significant interest. In this review, we aim to consolidate our understanding of the biological characteristics and immunomodulatory mechanisms of iTr35, offering fresh perspectives that may pave the way for its potential applications in disease diagnosis and treatment.

Estimating the salivary levels of IL-35 in smokers with periodontitis: A cross sectional study.

Background: A refined network and integrated host immune response to bacteria are formed by anti-inflammatory cytokines and the cells that they are associated to IL-35 has been recognized as having strong suppressive activity in chronic inflammatory diseases, together with IL-10 and TGF-ß. It is believed that inflammatory diseases like periodontitis trigger the inducible Treg population to express IL-35, expanding regulatory responses by increasing infection. Aim: The objective is to estimate and compare the salivary IL-35 levels in Periodontally healthy subjects, smokers and non-smokers with Periodontitis in order to educate on the consequences of periodontal health among the diseased patients. Materials and Methods: Totally 42 subjects were included and they were categorized into Group 1 (n = 14) as Periodontally healthy subjects, Group 2 (n = 14) as systemically healthy non-Smokers with periodontitis and Group 3 (n = 14) as systemically healthy smokers with periodontitis. Each subject was assessed for clinical parameters such as Plaque index, Gingival index, Probing depth, clinical attachment. A polypropylene tube was used to collect unstimulated saliva and centrifuged it at 800 × g for 10 min. Supernatants were collected and stored at -80◦C. A commercially available enzyme-linked immunosorbent assay kit was used to analyse levels of human salivary IL-35. Results: The average age of the subjects in Group 1, Group 2 and Group 3 were 50.53, 52.93 and 52.07 years respectively. All three groups showed a statistically significant difference in clinical parameters including Plaque index, Gingival index, Probing depth and clinical attachment. The salivary IL-35 level was found to be elevated in non-smokers who have periodontitis compared to smokers with periodontitis and healthy individuals. Despite this, the salivary IL-35 levels were found to be statistically significant among three groups at P < 0.001. Conclusion: The salivary levels of IL-35 were found to increase in Periodontitis patients with/without smoking, along with increased clinical parameters. IL-35 is considered a influential biomarker for periodontal disease.

A glance on the role of IL-35 in systemic lupus erythematosus (SLE).

It is well known that systemic lupus erythematosus (SLE) is an auto-inflammatory disease that is characterized by chronic and widespread inflammation. The exact pathogenesis of SLE is still a matter of debate. However, it has been suggested that the binding of autoantibodies to autoantigens forms immune complexes (ICs), activators of the immune response, in SLE patients. Ultimately, all of these responses lead to an imbalance between anti-inflammatory and pro-inflammatory cytokines, resulting in cumulative inflammation. IL-35, the newest member of the IL-12 family, is an immunosuppressive and anti-inflammatory cytokine secreted mainly by regulatory cells. Structurally, IL-35 is a heterodimeric cytokine, composed of Epstein-Barr virus-induced gene 3 (EBI3) and p35. IL-35 appears to hold therapeutic and diagnostic potential in cancer and autoimmune diseases. In this review, we summarized the most recent associations between IL and 35 and SLE. Unfortunately, the comparative review of IL-35 in SLE indicates many differences and contradictions, which make it difficult to generalize the use of IL-35 in the treatment of SLE. With the available information, it is not possible to talk about targeting this cytokine for the lupus treatment. So, further studies would be needed to establish the clear and exact levels of this cytokine and its related receptors in people with lupus to provide IL-35 as a preferential therapeutic or diagnostic candidate in SLE management.

Interplay of IL-33 and IL-35 Modulates Th2/Th17 Responses in Cigarette Smoke Exposure HDM-Induced Asthma.

Cigarette smoke (CS) facilitates adverse effects on the airway inflammation and treatment of asthma. Here, we investigated the mechanisms by which CS exacerbates asthma. The roles of IL-33 and IL-35 in asthma development were examined by treatment with IL-33 knockout (IL-33 KO) or transfection of adenovirus encoding IL-35 (Ad-IL-35) in a murine model of cigarette smoke-exposure asthma. Furthermore, the involvement of IL-33 and IL-35 in regulating DCs and Th2/Th17 cells was examined in a coculture system of DCs with CD4+ T cells. Additionally, we observed the effect of CpG-ODNs on the balance of IL-33 and IL-35. We show that CS and house dust mite (HDM) exposure induced IL-33 and suppressed IL-35 levels in cigarette smoke-exposure asthma in vivo and in vitro. Treatment with IL-33 KO or Ad-IL-35 significantly attenuated airway hyperreactivity, goblet hyperplasia, airway remodelling, and eosinophil and neutrophil infiltration in the lung tissues from asthmatic mice. Furthermore, we demonstrated reciprocal regulation between CS and HDM-modulated IL-33 and IL-35. Mechanistically, IL-33 KO (or anti-ST2) and Ad-IL-35 attenuated Th2- and Th17-associated inflammation by downregulating TSLP-DC signalling. Finally, administration of CpG-ODNs suppressed the expression of IL-33/ST2 and elevated the levels of IL-35, which is mainly derived from CD4+Foxp+ Tregs, to alleviate Th2- and Th17-associated inflammation by inhibiting the activation of BMDCs. Taken together, the IL-33/ST2 pathway drives the DC-Th2 and Th17 responses of cigarette smoke-exposure asthma, while IL-35 has the opposite effect. CpG-ODNs represent a potential therapeutic strategy for modulating the balance of IL-33 and IL-35 to suppress allergic airway inflammation.

Effect of T-regulatory cells and interleukin-35, interleukin-10, and transforming growth factor-beta on diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma that affects B lymphocytes. It can develop in the lymph nodes and can be localized or generalized. Despite DLBCL being considered potentially curable, little research has been conducted on the relationship between the body's immune response and DLBCL. AIM: To study the expression and significance of T-regulatory cells (Tregs) interleukin (IL)-35, IL-10, and transforming growth factor-beta (TGF-ß) in DLBCL. METHODS: Data from 82 patients with DLBCL who were initially admitted to The First Affiliated Hospital of Ningbo University (Zhejiang Province, China) between January 2017 and June 2022 and treated with standard first-line regimens were reviewed. Three patients were lost to follow-up; thus, 79 patients were included in the statistical analysis and then divided into three groups according to the evaluation of clinical efficacy: Incipient (new-onset and treatment-naïve), effectively treated, and relapsed-refractory. Thirty healthy individuals were included in the control group. The expression of peripheral blood T lymphocytes and their associated factors IL-35, IL-10, and TGF-ß in the four groups were observed. RESULTS: In contrast to the successfully treated and normal control groups, both the incipient and relapse-refractory groups exhibited greater proportions of CD4-positive (+) Tregs (P < 0.05), whereas the proportion of CD8+ Tregs did not differ substantially between the groups. Serum levels of IL-35 and IL-10 in the incipient and relapsed-refractory groups were higher than those in the effectively treated and normal control groups (P < 0.05). There was no statistically significant distinction in the expression level of TGF-ß between the groups (P > 0.05). The correlation between IL-35 and IL-10 concentrations was significantly positive, with a correlation coefficient of 0.531 (P < 0.05). The correlation between IL-35 and TGF-ß concentration was significantly positive, with a correlation coefficient of 0.375 (P < 0.05). The correlation between IL-10 and TGF-ß concentration was significantly positive, with a correlation coefficient of 0.185 (P < 0.05). The expression concentrations of IL-35, IL-10 and TGF-ß were apparently and positively correlated (P < 0.05). CONCLUSION: Tregs IL-35, and IL-10 may be closely associated with the occurrence and development of DLBCL and the detection of related indices may be helpful in the analysis of disease prognosis.

Differential expression of interleukin-35 receptor distinguishes different subsets of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in a mouse endometriosis model.

The immune system contributes to the pathophysiology of endometriosis. The role of ThGM cells, which produce granulocyte macrophage-colony-stimulating factor (GM-CSF), in the pathogenesis of endometriosis remains unknown. To analyze the features of ThGM cells in endometriosis, a mouse endometriosis model was established. ThGM cells in the spleen, peritoneal fluid (PF), and endometriotic lesions (EL) were measured by flow cytometry, based on the expression of surface markers and intracellular proteins. Live ThGM cells were sorted according to chemokine receptor expression profiles and their effects on other CD4+ T cell subsets were determined by co-culture assays. An adoptive transfer assay was performed to characterize the effect of ThGM cells on endometriosis. We found that ThGM cells were present in endometriotic PF and EL. Live EL ThGM cells were enriched in CD4+CXCR3-CCR8-CCR4+CCR10+ T cells. EL ThGM cells differentially express interleukin-35 receptor (IL-35R), consisting of an IL-35R+ subset and an IL-35R- subset. The IL-35R+ subset expressed less GM-CSF, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α) and proliferated slower than the IL-35R- subset. Meanwhile, the IL-35R+ subset was weaker than the IL-35R- subset in promoting the functions of Th1 and Th17 cells. ThGM cell transfer did not influence EL development but significantly alleviated pro-inflammatory cytokines in PF and ELs. Interleukin-35 (IL-35), the ligand of IL-35R, suppressed ThGM cell function and proliferation in an IL-35R-dependent manner. In summary, ThGM cells in the PF and ELs might exacerbate endometriotic inflammation. IL-35 might suppress the function of ThGM cells via IL-35R.

Interleukin-35 and Thymoquinone nanoparticle-based intervention for liver protection against paracetamol-induced liver injury in rats.

Paracetamol (PAR) is a commonly used antipyretic and analgesic agent, but its excessive usage can induce liver damage and major health consequences. Interleukin-35 (IL-35) is utilized to treat immunological disorders, intestinal illness, arthritis, allergic disease, hepatitis, and cancer. Thymoquinone (THYO) is also effective against a wide range of disorders. Consequently, this study sought out to explore the ameliorative effects of IL-35 and THYO against PAR-induced hepatotoxicity in rats. Sixty male rats were separated into six groups (10 rats/group): I control (0.5 mL NaCl, 0.9%/rat via oral gavage); II (IL-35), and III (TYHO) received intraperitoneal (i.p) injection of IL-35 (200 ng/kg) or THYO (0.5 mg/kg), respectively. Group IV (PAR) received 600 mg/kg of PAR orally; V (PAR + IL-35) and VI (PAR + TYHO); rats received 600 mg/kg of PAR orally and i.p injection of IL-35 (200 ng/kg) or THYO (0.5 mg/kg), respectively. Administration of IL-35 or THYO markedly mitigated the increasing in the levels of liver parameters triggered by PAR and noticeable enhancement of antioxidant and immunological markers were observed. Additionally, IL-35 or THYO decreased TNF-α, NF-κB, IL-10, IL-6 and IFN-γ in contrast to the PAR control group. Moreover, levels of Capase-3, and cytochrome C were significantly reduced by THYO or IL35, while, levels of Bcl-2 were markedly increased. Furthermore, significant downregulation of IL1-ß, TNF-α, TGF-ß, and Caspas-3 genes, as well as significant upregulation of Bcl-2 and IL-10 expression were detected. In conclusion, IL-35 and THYO insulated liver from PAR toxicity by mitigating oxidative stress, tissue damage, inflammation, and apoptosis.

Impaired Regulation by IL-35 in Systemic Sclerosis.

This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 ± 5.6 vs. 143 ± 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF-ß (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 ± 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 ± 9.2 vs. 83.4 ± 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 ± 1.5 vs. 4.8 ± 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 ± 2.3 vs. 10.2 ± 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 ± 30.5 pg/mL vs. 37.4 ± 6.4 pg/mL, p < 0.001) and TGF-ß (4194 ± 777 pg/mL vs. 2413 ± 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF-ß (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 ± 8.0 vs. 832.3 ± 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target.

IL-35 inhibits adipogenesis via PPARγ-Wnt/ß-catenin signaling pathway by targeting Axin2.

Interleukin (IL)-35, a member of the IL-12 family, functions as an immunosuppressive cytokine that plays a crucial role in the regulation of immune-related disorders and inflammatory diseases. Adipose tissue, which is now recognized as an immune organ, is regulated by immunocytes through various signaling pathways, including the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) pathway and the Wnt/ß-actin pathway. However, there is limited research regarding the effects of IL-35 on adipogenesis. Our current findings indicated that IL-35 impedes the proliferation and promotes the cytotoxicity of 3T3-L1 preadipocytes. Furthermore, IL-35 inhibited the adipogenic differentiation, as well as suppressed triglyceride and lipid accumulation. Additionally, the expression of PPARγ and C/EBPα, two key regulators of adipogenesis, were both down-regulated with IL-35 treatment. In order to explicate the mechanisms underlying the effects of IL-35, we conducted an investigation into the expression of Axin2, an intracellular inhibitor of Wnt/ß-catenin signaling, in 3T3-L1 preadipocyte cells. Gene silencing of Axin2 through small interfering RNAs (siRNAs) enhanced PPARγ and C/EBPα expression while decreasing nuclear ß-catenin levels in the presence of IL-35. Furthermore, in IL-35-treated cells, Axin2 knockdown boosted adipogenic differentiation (as measured by increased Oil Red O staining). These findings imply that IL-35 regulates Axin2 expression and thereby plays an important role in adipocyte development.

Interleukin 35: protective role and mechanism in type 1 diabetes.

Interleukin 35 (IL-35), a cytokine secreted by regulatory T (Treg) cells from the differentiation of conventional CD4+ T cells, is a member of the IL-12 family. The IL-12 family of cytokines exhibits an anti-inflammatory property. IL-35 has recently been shown to influence the immune modulation in various diseases, including inflammatory bowel disease, Graves' disease, rheumatoid arthritis, colitis, psoriasis, and type 1 diabetes (T1D). T1D is an immune-related disease caused by destruction of pancreatic ß cells, characterized by an absolute lack of insulin. Recently, studies have suggested that protective effects of IL-35 work by improving blood glucose levels and preventing an attack of inflammatory factors on the islets. The protective mechanism may be closely related to the anti-inflammatory properties of IL-35, which include regulating macrophage phenotype, suppressing T cell proliferation, decreasing the differentiation of Th17 cells, increasing the Treg cell population, and inducing IL-35-producing regulatory T cells (iTr35). Here, we review the protective effects and mechanisms of action of IL-35 in T1D.

Interleukin-35 expression promotes hepatocellular carcinogenesis by inducing γδ T-cell exhaustion.

Interleukin (IL)-35, a new member of the IL-12 family, exerts immunosuppressive effects in the hepatic microenvironment. Innate immune cells, such as γδ T cells, have vital roles in hepatic diseases, including acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). In the current study, we focused on the effects and mechanisms of IL-35 on the local immune status of γδ T cells, especially in liver tumors. Based on CCK8 assay and immunofluorescence results, we showed that exogenous IL-35 stimulation of γδ T cells attenuated proliferative ability and killing functions against Hepa1-6 or H22 cells. Flow cytometry results showed that exogenous IL-35 stimulated the expression of programmed cell death 1 (PDCD1) and lymphocyte activation gene 3 (LAG3) in γδ T cells. The exogenous IL-35 stimulated group also showed impairment of cytotoxic cytokine secretion. In addition, stat5a revealed a significant increase after IL-35 stimulation of γδ T cells screened via transcription factor based on PCR array analysis. Furthermore, bioinformatics analysis revealed that stat5a-related tumor-specific genes were mainly involved in immune regulatory pathways. Correlation analysis indicated that stat5a expression was significantly and positively correlated with tumor immune cell infiltration, and pdcd1 and lag3 expression. Finally, bioinformatics analysis using the TCGA and GSE36376 HCC datasets corroborated the significant positive correlation between IL-35 and stat5a. Taken together, overexpressed IL-35 promoted exhaustion and impaired the anti-tumor function of γδ T cells in HCC. Targeting IL-35 might be a promising means to enhance the antitumor therapy of γδ T cells, which would significantly improve prognosis.

Regulatory B cells induced by interleukin-35 inhibit inflammation and alveolar bone resorption in ligature-induced periodontitis.

BACKGROUND: Regulatory B cells (Bregs) have been reported to suppress immune responses and alveolar bone loss in murine periodontitis models. These cells could be induced by interleukin (IL)-35 which is increased upon periodontal inflammation. Thus, this study aimed to explore the role of Bregs induced by IL-35 in periodontitis. METHODS: Experimental periodontitis was induced in mice by ligature. Two weeks after ligation, the test group was systemically treated with IL-35 for 1 week. Four weeks after ligation, all mice were euthanized, and alveolar bone loss was evaluated by microcomputed tomography. Cytokines associated with periodontitis were analyzed using reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Bregs in spleens, cervical lymph nodes, and periodontal tissues were detected by flow cytometry and immunofluorescence staining. RESULTS: In the mouse model of periodontitis, IL-35 induced the expansion of CD1dhi CD5+ B10 cells with increased interleukin-10 (IL-10) and IL-35 production. IL-35 administration also attenuated alveolar bone loss and reduced the levels of proinflammatory cytokines in situ. CONCLUSIONS: Following ligature-induced periodontitis in mice, IL-35 inhibited periodontal inflammation and alveolar bone resorption at least partially through the induction of B10 cells and IL-35+ Bregs.

Interleukin-35 Enhances Regulatory T Cell Function by Potentially Suppressing Their Transdifferentiation into a T Helper 17-Like Phenotype in Kawasaki Disease.

Interleukin-35 (IL-35) modulates immune cell activity in inflammation and autoimmune disorders. However, its specific effects on regulatory T cells (Tregs) in Kawasaki disease remain ambiguous. We enrolled 37 patients with Kawasaki disease and 20 healthy controls in this study. The percentages of CD4+CD25+CD127dim/- Tregs and CD4+IL-17A+ T helper 17 (Th17) cells were determined via flow cytometry. Tregs were enriched and stimulated by recombinant IL-35. Immunosuppressive activity of Tregs was via co-culture with autologous CD4+CD25- T cells. Purified Tregs were cultured for Th17 polarization, and the influence of IL-35 on Tregs transdifferentiation into a Th17-like phenotype was determined. The percentage of Tregs was elevated in patients with Kawasaki disease and positively correlated with C-reactive protein levels. There was no significant difference in the percentage of Th17 cells between the two groups. IL-35 stimulation increased the percentage of Tregs in both groups, but decreased the percentage of Tregs Th17 cells in affected patients. IL-35 enhanced the immunosuppressive activity of Tregs in both groups, resulting in decreased cellular proliferation and increased IL-35 subunit mRNA relative levels in co-culture system. IL-35 did not affect the immune checkpoint molecule expression in Tregs, but inhibited the transdifferentiation of Tregs into a Th17-like phenotype in affected patients, indicating by the down-regulations of C-C motif chemokine receptor-4/6 expression, retinoid-related orphan nuclear receptor γt mRNA levels, and IL-17 secretion. IL-35 contributes to the immunosuppressive function of Tregs by inhibiting the cellular proliferation and transdifferentiation of Tregs into a Th17-like phenotype, which may be a protective mechanism against Kawasaki disease.

Pancreatic cancer: Emerging field of regulatory B-cell-targeted immunotherapies.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is characterized by a high mortality rate and poor prognosis. Current treatments for PDAC, are ineffective due to a prominent immunosuppressive PDAC tumor microenvironment (TME). Although B lymphocytes are highly infiltrated into PDAC, the importance of B lymphocytes in tumorigenesis is largely neglected. B cells play a dual role in the PDAC tumor microenvironment, acting as either anti-tumorigenic or pro-tumorigenic depending on where they are localized. Tumor-infiltrating B cells, which reside in ectopic lymph nodes, namely tertiary lymphoid structures (TLS), produce anti-tumor antibodies and present tumor antigens to T cells to contribute to cancer immunosurveillance. Alternatively, regulatory B cells (Bregs), dispersed inside the TME, contribute to the dampening of anti-tumor immune responses by secreting anti-inflammatory cytokines (IL-10 and IL-35), which promote tumor growth and metastasis. Determining the role of Bregs in the PDAC microenvironment is thus becoming increasingly attractive for developing novel immunotherapeutic approaches. In this minireview, we shed light on the emerging role of B cells in PDAC development and progression, with an emphasis on regulatory B cells (Bregs). Furthermore, we discussed the potential link of Bregs to immunotherapies in PDAC. These current findings will help us in understanding the full potential of B cells in immunotherapy.

Correlation analysis of systemic immune inflammatory index, Serum IL-35 and HMGB-1 with the severity and prognosis of sepsis.

Objective: To investigate the correlation between systemic immune inflammatory (SII) index, serum interleukin-35 (IL-35) and high mobility Group-Box one (HMGB-1) with the severity and prognosis of sepsis. Methods: A retrospective analysis was performed on the clinical data of 209 patients with sepsis admitted to Ganzhou City People's Hospital from October 2019 to October 2021. One hundred eighteen patients in Group-A had common sepsis, and 91 patients in Group-B had septic shock, which were subdivided into the survival Group-And the mortality Group-According to the 28d prognosis. The levels of SII, IL-35 and HMGB-1 in different groups were compared, and their correlation with the severity and prognosis of sepsis was analyzed. Result: The levels of SII, IL-35 and HMGB-1 in Group-A were significantly lower than those in Group-B (p<0.05). The levels of SII, IL-35 and HMGB-1 in the survival group were significantly lower than those in the death group (p<0.05). Spearman correlation analysis showed that the levels of SII, IL-35, and HMGB-1 were significantly positively correlated with the severity of sepsis (p<0.05), and significantly positively correlated with the prognosis of patients with sepsis (p<0.05). Conclusion: SII, IL-35 and HMGB-1 are remarkably correlated with the severity and prognosis of patients with sepsis. With the increasing in SII, IL-35 and HMGB-1 levels, patients will suffer from severe disease progression and poor prognosis, which require more clinical attention.

JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

BACKGROUND: Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. METHODS: In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. RESULTS: HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. CONCLUSIONS: A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.

Inducible nitric oxide synthase-expressing myeloid-derived suppressor cells regulated by interleukin 35 contribute to the pathogenesis of psoriasis.

Although psoriasis is classified as a T cell-mediated inflammatory disease, the contribution of myeloid cells to the pathogenesis of psoriasis is not fully understood. In the present study, we demonstrated that the expression of the anti-inflammatory cytokine interleukin-35 (IL-35) was significantly increased in patients with psoriasis with a marked increase in the number of myeloid-derived suppressor cells (MDSCs). Similar results were obtained in an imiquimod-induced psoriasis mouse model. IL-35 reduced the total number of MDSCs and their subtypes in the spleens and psoriatic skin lesions, ameliorating psoriasis. IL-35 also reduced the expression of inducible nitric oxide synthase in MDSCs, although it had no significant effect on interleukin-10 expression. Adoptive transfer of MDSCs from imiquimod-challenged mice aggravated the disease and weakened the effect of IL-35 in the recipient mice. In addition, mice transferred with MDSCs isolated from inducible nitric oxide synthase knockout mice had milder disease than those with wild-type MDSCs. Furthermore, wild-type MDSCs reversed the effects of IL-35, while MDSCs isolated from inducible nitric oxide synthase knockout mice did not affect IL-35 treatment. In summary, IL-35 may play a critical role in the regulation of iNOS-expressing MDSCs in the pathogenesis of psoriasis, highlighting IL-35 as a novel therapeutic strategy for patients with chronic psoriasis or other cutaneous inflammatory diseases.

Interleukin-35 dampens T helper 22 phenotype shift in CD4+CD25+CD127dim/- regulatory T cells in primary biliary cholangitis.

The phenotype shift in regulatory T cells (Tregs) contributes to immunopathogenesis of autoimmune diseases. The current study was aimed to investigate the regulatory function of interleukin-35 (IL-35) to T helper 22 (Th22) cell phenotype shift in Tregs in primary biliary cholangitis (PBC). Fifty-five PBC patients and twenty-four controls were enrolled. CD4+CD25+CD127dim/- Tregs and Th22 cells were investigated by flow cytometry. Forkhead box P3 (FoxP3) and aryl hydrocarbon receptor (AhR) mRNA levels were assessed by real-time polymerase chain reaction. Plasma IL-10 and IL-22 levels were measured by ELISA. Purified Tregs were stimulated with exogenous IL-35, and were co-cultured with autologous CD4+CD25- T cells. Cellular proliferation and cytokine production was measured. Purified Tregs were also cultured into Th22 condition in the presence or absence of exogenous IL-35, and Th22 phenotype were assessed. PBC patients had lower levels of Treg percentage, FoxP3 mRNA, and plasma IL-10, while had higher levels of Th22 proportion, AhR mRNA, and plasma IL-22. Tregs from PBC patients showed reduced immunosuppressive activity, which presented as increased cellular proliferation, interferon-γ production and decreased IL-35/IL-10 secretion in co-culture system. Tregs shifted into Th22 phenotype in PBC patients with elevated CCR4, CCR6, and CCR10 expression as well as increased IL-22 production. IL-35 not only enhanced inhibitory function of Tregs but also suppressed phenotype shift of Tregs into Th22 phenotype in PBC patients. This process was accompanied by elevation of IL-10 and transforming growth factor-ß1 secretion by Tregs from PBC patients. The present data suggested that reduced IL-35 might be insufficient to maintain Tregs function and phenotype shift from Tregs into Th22 phenotype in PBC patients.

Suppressive mechanisms of regulatory B cells in mice and humans.

B cells include immune-suppressive fractions, called regulatory B cells (Bregs), which regulate inflammation primarily through an interleukin 10 (IL-10)-mediated inhibitory mechanism. Several B-cell fractions have been reported as IL-10-producing Bregs in murine disease models and human inflammatory responses including autoimmune diseases, infectious diseases, cancer and organ-transplant rejection. Although the suppressive functions of Bregs have been explored through the hallmark molecule IL-10, inhibitory cytokines and membrane-binding molecules other than IL-10 have also been demonstrated to contribute to Breg activities. Transcription factors and surface antigens that are characteristically expressed in Bregs are also being elucidated. Nevertheless, defining Bregs is still challenging because their active periods and differentiation stages vary among disease models. The identity of the diverse Breg fractions is also under debate. In the first place, since regulatory functions of Bregs are mostly evaluated by ex vivo stimulation, the actual in vivo phenotypes and functions may not be reflected by the ex vivo observations. In this article, we provide a historical overview of studies that established the characteristics of Bregs and review the various suppressive mechanisms that have been reported to be used by Bregs in murine and human disease conditions. We are only part-way through but the common phenotypes and functions of Bregs are still emerging.