EBI3 Research Articles

GPR183-Oxysterol Axis in Spinal Cord Contributes to Neuropathic Pain.

Neuropathic pain is a debilitating public health concern for which novel non-narcotic therapeutic targets are desperately needed. Using unbiased transcriptomic screening of the dorsal horn spinal cord after nerve injury we have identified that Gpr183 (Epstein-Barr virus-induced gene 2) is upregulated after chronic constriction injury (CCI) in rats. GPR183 is a chemotactic receptor known for its role in the maturation of B cells, and the endogenous ligand is the oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC). The role of GPR183 in the central nervous system is not well characterized, and its role in pain is unknown. The profile of commercially available probes for GPR183 limits their use as pharmacological tools to dissect the roles of this receptor in pathophysiological settings. Using in silico modeling, we have screened a library of 5 million compounds to identify several novel small-molecule antagonists of GPR183 with nanomolar potency. These compounds are able to antagonize 7α,25-OHC-induced calcium mobilization in vitro with IC50 values below 50 nM. In vivo intrathecal injections of these antagonists during peak pain after CCI surgery reversed allodynia in male and female mice. Acute intrathecal injection of the GPR183 ligand 7α,25-OHC in naïve mice induced dose-dependent allodynia. Importantly, this effect was blocked using our novel GPR183 antagonists, suggesting spinal GPR183 activation as pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify this receptor as a potential target for therapeutic intervention. SIGNIFICANCE STATEMENT: We have identified several novel GPR183 antagonists with nanomolar potency. Using these antagonists, we have demonstrated that GPR183 signaling in the spinal cord is pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify it as a potential target for therapeutic intervention.

Abstract 4550: Increased IL-27 is associated with poor prognosis in renal cell carcinoma and supports use of SRF388, a first-in-class IL-27p28 blocking antibody, to counteract IL-27-mediated immunosuppression in this setting

Abstract IL-27 is a heterodimeric member of the IL-12/IL-23 cytokine family that consists of two subunits, IL-27p28 and Epstein-Barr virus-induced gene 3 (EBI3), and binds to a heterodimeric receptor composed of the IL-27 receptor subunit alpha (IL-27RA) and glycoprotein 130 (gp130). IL-27 signals through the JAK-STAT pathway to limit the duration and intensity of T cell-mediated immunity by altering immunoregulatory receptor expression and proinflammatory cytokine secretion during infection and cancer. Analysis of data from The Cancer Genome Atlas (TCGA) revealed that IL-27p28, EBI3, and IL-27RA transcript levels are often elevated in tumors from patients with renal cell carcinoma (RCC) and that high levels of these genes are associated with poor clinical outcome. Using an IL-27 gene signature derived from activated CD4+T cells, several genes downstream of IL-27 signaling were found to be coordinately regulated in a subset of RCC patients and associated with poor prognosis. Moreover, plasma levels of the EBI3 subunit of IL-27 were found to be elevated in a subset of RCC patients and inversely correlated with both disease-free and overall survival. The ability of IL-27 to dampen immune responses and the association of elevated levels of this cytokine with poor clinical outcome in RCC suggest that IL-27 blockade may represent a promising strategy to treat cancer in patients with high circulating EBI3. SRF388 is a first-in-class IL-27p28 antibody that blocks the interaction of IL-27 with IL-27RA and inhibits IL-27 signaling in primary human immune cells. This mechanism results in diminished inhibitory receptor expression and increased cytokine production. Exogenous IL-27 inhibited the activity of PD-1 blockade in vitro by counteracting the increased cytokine production observed after treatment of activated human PBMCs from healthy donors and RCC patients. Furthermore, treatment with SRF388 stimulated increased cytokine production in activated PBMCs from patients with RCC in the absence of exogenous IL-27; this proinflammatory response was further enhanced when combined with PD-1 blockade. Finally, SRF388 demonstrated single-agent anti-tumor activity in a murine orthotopic model of RCC. These data indicate that blockade of IL-27 can potentiate anti-tumor responses by counteracting IL-27-mediated immune escape and may represent a promising strategy for treating cancer patients. Citation Format: Matthew Rausch, Jing Hua, Devapregasan Moodley, Kerry F. White, Katherine H. Walsh, Christine E. Miller, Gege Tan, Benjamin H. Lee, Isabelle Cousineau, Jean-Baptiste Lattouf, John Stagg, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill. Increased IL-27 is associated with poor prognosis in renal cell carcinoma and supports use of SRF388, a first-in-class IL-27p28 blocking antibody, to counteract IL-27-mediated immunosuppression in this setting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4550.

Abstract 1050: Serum interleukin-27 serves as an early biomarker predicting development of hepatocellular carcinoma (HCC) in two large prospective cohorts

Abstract Background: Interleukin-27 (IL-27), which consists of p28 and Epstein-Barr virus induced gene 3 (EBI3) subunits, is a heterodimeric member of the IL-12/IL-23 cytokine family. Overexpression of IL-27 increases T cell expression of inhibitory receptors including PD-1, LAG3, TIGIT, and TIM3 that could limit intensity and duration of T cell responses to infection and cancer. IL-27 is overexpressed in HCC. The purpose of the study was to examine the role of IL-27 in the development of HCC in humans. Methods: Two parallel studies were conducted. The discovery study included 100 HCC cases and 100 individually matched controls from the Singapore Chinese Health Study, which enrolled 32,000 participants with blood samples free of cancer at entry into the study and subsequently followed up for >15 years. The validation study also included 100 HCC cases and 100 matched controls from the Shanghai Cohort Study, a prospective cohort of 18,000 participants who were free of cancer at entry and similarly followed up for >20 years. Human IL-27 protein was quantified in sera that were collected, on average, 4.5 year prior to the diagnosis of HCC. Conditional logistic regression method was used to estimate odds ratio (OR) and 95% confidence interval (CI) of HCC for higher tertiles of IL-27 with adjustment for hepatitis B surface antigen (HBsAg), alcohol intake, body mass index and cigarette smoking. The Cancer Genome Atlas (TCGA) data were analyzed for the IL-27 gene expression in liver and other cancers. Results: Serum IL-27 levels were significantly higher in persons who subsequently developed HCC than controls who remained free of HCC in both studies. In the discovery study, persons in the 2nd (1.39-1.95 ng/mL) and 3rd tertile of IL-27 (>1.95 ng/ml) had 9.3 (95% CI = 1.07-80.36) and 51.7 (95% CI = 5.1-527.8) times the risk of developing HCC, respectively, compared with the lowest IL-27 (<1.39 ng/ml). These novel results were confirmed in the validation study; the corresponding ORs (95% CIs) for HCC were 4.9 (1.2-19.6) and 16.3 (3.5-77.4) (both Ptrend < 0.001). When IL-27 and HBsAg were jointly analyzed, the OR (95% CI) of HCC was 24.73 (3.23-189.09) for lowest IL-27 & positive HBsAg, 45.52 (8.71-237.91) for highest IL-27 & negative HBsAg, and 265.19 (40.69-1728.45) for highest IL-27 & positive HBsAg, compared with lowest IL-27 & negative HBsAg. The TCGA analysis revealed that the IL-27 mRNA levels in primary liver cancer was at least 20 times higher than that of any other cancer. Conclusion: Higher serum levels of IL-27 are associated with significantly higher risk of HCC development. Implications: IL-27 can be further developed as a biomarker for identification of individuals at high risk for HCC and for diagnosis of HCC at an early stage, enabling more frequent surveillance. Anti-IL-27 agents may be developed for primary prevention of HCC. The blockade of IL-27 alone or in combination with other anti-immunosuppressive agents such as anti-PD1, anti-PD-L1, IL-2 and anti-LAG3 may represent novel immunotherapy strategies for HCC. Citation Format: Jian-Min Yuan, Wang Yue, Renwei Wang, Jennifer Admas-Haduch, Woon-Puay Koh, Yu-Tang Gao, Michael T. Lotze. Serum interleukin-27 serves as an early biomarker predicting development of hepatocellular carcinoma (HCC) in two large prospective cohorts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1050.

Epstein-Barr virus-induced gene 3 (EBI3) single nucleotide polymorphisms and their association with central obesity and risk factors for cardiovascular disease: The GEA study

Obesity, a chronic low-grade inflammation metabolic abnormality, is related to high proinflammatory cytokines concentrations. Epstein-Barr virus-induced gene 3 (EBI3) encodes for the EBI3 beta subunit that constitutes interleukin (IL) 27 and 35. Our objective was to assess the association of three EBI3 single nucleotide polymorphisms (SNPs) with the presence of central obesity in a group of Mexican subjects. The rs428253, rs4740, and rs4905 EBI3 SNPs were genotyped in 1323 individuals (1092 central obese and 231 non-central obese). We also analyzed IL-6, IL-27, and IL-35 concentrations. Under different models, the rs4740 (OR = 0.384, Precessive = 0.010; OR = 0.404, Pcodominant 2 = 0.019) and rs4905 (OR = 0.380, Precessive = 0.009; OR = 0.404, Pcodominant 2 = 0.018) were related with a low risk of central obesity. In central obese subjects, the SNPs were related to lower risk of hypoalphalipoproteinemia (rs4740) and with high IL-6 concentrations (rs428253, rs4740, and rs4905), whereas in non-central obese individuals, the rs428253 was related with low risk of increased visceral abdominal fat and hypertriglyceridemia. Interleukin-6, IL-27 and IL-35 concentrations were similar in both groups and no relation was noticed with the studied genotypes. Our results suggest an association of EBI3 SNPs with a low risk of central obesity and with a few risk factors for cardiovascular disease in individuals with and without central obesity.

Interleukin-35 pretreatment attenuates lipopolysaccharide-induced heart injury by inhibition of inflammation, apoptosis and fibrotic reactions

Previous studies have demonstrated that targeting inflammation is a promising strategy for treating lipopolysaccharide (LPS)-induced sepsis and related heart injury. Interleukin-35 (IL-35), which consists of two subunits, Epstein–Barr virus-induced gene 3 (EBI3) and p35, is an immunosuppressive cytokine of the IL-12 family and exhibits strong anti-inflammatory activity. However, the role of IL-35 in LPS-induced heart injury reains obscure. In this study, we explored the role of IL-35 in heart injury induced by LPS and its potential mechanisms. Mice were treated with a plasmid encoding IL-35 (pIL-35) and then injected intraperitoneally (ip) with LPS (10 mg/kg). Cardiac function was assessed by echocardiography 12 h later. LPS apparently decreased the expression of EBI3 and p35 and caused cardiac dysfunction and pathological changes, which were significantly improved by pIL-35 pretreatment. Moreover, pIL-35 pretreatment significantly decreased the levels of cardiac proinflammatory cytokines including TNF-α, IL-6, and IL-1β, and the NLRP3 inflammasome. Furthermore, decreased number of apoptotic myocardial cells, increased BCL-2 levels and decreased BAX levels inhibited apoptosis, and LPS-induced upregulation of the expression of cardiac pro-fibrotic genes (MMP2 and MMP9) and fibrotic factor (Collagen type I) was inhibited. Further investigation indicated that pIL-35 pretreatment might suppressed the activation of the cardiac NF-κBp65 and TGF-β1/Smad2/3 signaling pathways in LPS-treated mice. Similar cardioprotective effects of IL-35 pretreatment were observed in mouse myocardial fibroblasts challenged with LPS in vitro. In summary, IL-35 pretreatment can attenuate cardiac inflammation, apoptosis, and fibrotic reactions induced by LPS, implicating IL-35 as a promising therapeutic target in sepsis-related cardiac injury.

Interleukin-35 promotes the differentiation of regulatory T cells and suppresses Th2 response in IgG4-related type 1 autoimmune pancreatitis.

IgG4-related disease (IgG4-RD) is a systemic inflammatory disease, which includes type 1 autoimmune pancreatitis (AIP). Interleukin-35 (IL-35) exhibits immunosuppressive effects in several autoimmune diseases. However, the expression of IL-35 had not been reported so far in type 1 AIP. We evaluated the association between IL-35 and several cytokines, which mediate the function of Tregs in type 1 AIP. Plasma was collected from patients with type 1 AIP, alcoholic chronic pancreatitis (ACP), and healthy controls (HC) and assayed for cytokine expression. Total mRNA separated from peripheral blood was isolated from naïve Tregs (nTregs) and effector Tregs (eTregs). EBI3 and IL-12p35 gene expressions were tested in these cells by quantitative PCR. In addition, expression of IL-35 subunits in the pancreatic tissues of patients with type 1 AIP and ACP was analyzed by immunohistochemistry. IL-35 was significantly elevated in type 1 AIP (n = 32) plasma compared with ACP (n = 16) and HC (n = 22), but IL-27 was not. We also detected many cells expressing both EBI3 and IL-12p35 in type 1 AIP tissues. Moreover, in peripheral blood lymphocyte, the percentage of nTregs and eTregs of CD4+ T cells in patients with type 1 AIP (n = 14) compared with HC (n = 15) was significantly decreased and increased, respectively. There were no significant differences of gene expression in patients with type 1 AIP and HC. This study identified elevated expression of plasma IL-35 and tissue IL-35 subunits in patients with type 1 AIP. This might lead to inflammation suppression via activated eTregs. IL-35 might be associated with this anti-inflammatory role, especially against the Th2 response through several cytokines and the differentiation of Tregs in type 1 AIP.

7α,25-Dihydroxycholesterol Suppresses Hepatocellular Steatosis through GPR183/EBI2 in Mouse and Human Hepatocytes.

Nonalcoholic fatty liver disease is a chronic inflammatory liver disease. It is associated with obesity and type 2 diabetes. Oxycholesterols are metabolites of cholesterol, and several of them can act on the G protein-coupled receptor, G protein-coupled receptor 183 (GPR183)/Epstein-Barr virus-induced gene 2. We found expression of GPR183 in human hepatoma cell lines and in vivo induction of GPR183 expression in mouse livers after high-fat diet feeding. Therefore, the role of oxycholesterols and GPR183 in hepatocytes was studied using a model of hepatic steatosis induced by liver X receptor (LXR) activation. LXR activation by T0901317 resulted in fat accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by 7α,25-dihydroxycholesterol, the most potent agonist of GPR183. The protective effects of 7α,25-dihydroxycholesterol were suppressed by a specific GPR183 antagonist, NIBR189 [(2E)-3-(4-Bromophenyl)-1-[4-4-methoxybenzoyl)-1-piperazinyl]-2-propene-1-one]. T0901317 treatment induced expression of the major transcription factor for lipogenesis, sterol regulatory element-binding protein 1c (SREBP-1c). 7α,25-Dihydroxycholesterol inhibited the induction of SREBP-1c proteins in a GPR183-dependent manner. Using inhibitors specific for intracellular signaling molecules, 7α,25-dihydroxycholesterol-induced suppression of hepatocellular steatosis was shown to be mediated through Gi/o proteins, p38 mitogen-activated protein kinases, phosphoinositide 3-kinase, and AMP-activated protein kinase. In addition, the inhibitory effect of 7α,25-dihydroxycholesterol was validated in HepG2 cells and primary mouse hepatocytes. Therefore, the present report suggests that 7α,25-dihydroxycholesterol-GPR183 signaling may suppress hepatocellular steatosis in the liver. SIGNIFICANCE STATEMENT: Oxycholesterols, which are metabolites of cholesterol, act on the G protein-coupled receptor, G protein-coupled receptor 183 (GPR183)/Epstein-Barr virus-induced gene 2, which is expressed in human hepatoma cell lines, and its expression is induced in vivo in mouse livers after high-fat diet feeding. Activation of GPR183 inhibits fat accumulation in primary mouse hepatocytes and HepG2 cells through Gi/o proteins, p38 mitogen-activated protein kinases, phosphoinositide 3-kinase, and AMP-activated protein kinase.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

Interleukin 39: a new member of interleukin 12 family.

Interleukin (IL)-12 family member is a heterodimer glycoprotein, composed of two covalently linked subunits, α and β chains. The α subunit consists of IL-23p19, IL-27p28, and IL-12p35, and the β subunit includes IL-12p40 and Epstein-Barr virus-induced gene (Ebi3). IL-39 is a new heterodimeric IL-12 family member composed of IL-23p19 and Ebi3 subunits. IL-39 is secreted by lipopolysaccharide-stimulated B cells. Other immune cells, such as dendritic cells and macrophages, express IL-39 mRNA. In lupus-like mice, GL7+B cells and CD138+plasma cells are highly activated and widely expressed, promoting high expression of IL-39. IL-39 mediates inflammatory responses through binding to a heterodimer of IL-23R/gp130 receptor and activation of signal transducer and activator of transcription (STAT)1/STAT3 signal molecules. The serum levels of IL-39 were significantly increased in patients with acute coronary syndrome compared with patients with normal coronary arteries. This review discusses the biological characteristics, receptor, and signal pathway as well as biological activity of IL-39 and its potential role in inflammation and other diseases.

IL-35, a double-edged sword in cancer.

Interleukin 35 (IL-35), a cytokine mainly produced by regulatory T cells (Treg cells), is composed of an Epstein-Barr virus-induced gene 3 β-chain and an IL-12 p35 α-chain. IL-35 causes tumorigenicity in cancer, protects cancer cells against apoptosis, and facilitates cancer progression. However, a few reports have referred to its contradictory roles in cancer prevention. Therefore, the exact purpose of this cytokine in cancer development has become a fundamental question that needs to be answered. In this review, we explain the structure of IL-35 and its receptors and their different signaling pathways. Finally, the function of IL-35 in some cancers and the possible application of this cytokine in approaches for cancer therapy have been discussed.

Cloning and Expression of EBI3 and p28 Subunits of Human Interleukin 27 in E. coli

Background: Interleukin-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by Antigen Presenting Cells( APCs ) .The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 (EBI3) and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. Methods: The IL-27 subunit genes were constructed into plasmid vectors, they sub-cloned into pETDeut-1an expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was done by IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. Results: The 28kDa and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique.

Immunoregulatory Functions of the IL-12 Family of Cytokines in Antiviral Systems.

Members of the interleukin 12 (IL-12) family have been known to be inflammatory factors since their discovery. The IL-12 family consists of IL-12, IL-23, IL-27, IL-35, and a new member, IL-39, which has recently been identified and has not yet been studied extensively. Current literature has described the mechanisms of immunity of these cytokines and potential uses for therapy and medical cures. IL-12 was found first and is effective in combatting a wide range of naturally occurring viral infections through the upregulation of various cytokines to clear the infected cells. IL-23 has an essential function in immune networks, can induce IL-17 production, and can antagonize inhibition from IL-12 in the presence of T helper (Th) 17 cells, resulting in type II IFN (IFN-γ) regulation. IL-27 has a competitive relationship to IL-35 because they both include the same subunit, the Epstein–Barr virus-induced gene3 (EBi3). This review provides a simple introduction to the IL-12 family and focuses on their functions relevant to their actions to counteract viral infections.

EBI3 impacts Leishmania braziliensis-induced inflammation in localized cutaneous leishmaniasis

Abstract Localized Cutaneous Leishmaniasis (LCL) can ultimately progresses to chronic ulcerated lesion with strong local inflammatory reaction. We hypothesized that regulatory cytokines, such as Interleukin-27 (IL-27) and interleukin-35 (IL-35) can play a role in susceptibility to Leishmania braziliensis infection. IL-27 and IL-35 are members of the interleukin-12 (IL-12) family structurally related by sharing Epstein–Barr virus-induced gene 3 (EBI3), which forms a heterodimer either with the p28 subunit to build IL-27 or with IL-12p35 to form IL-35. We used RT-PCR and Immunohistochemistry analysis to detect high expression of IL-27 and IL-35 subunits in the patients biopsies compared to healthy controls, suggesting that these cytokines can be involved in immunopathology induced by the infection. Moreover, L. braziliensis infection also induced IL-27/35 mRNA expression both in vitro and in vivo. We here analysed the function of EBI3 during infection with the intracellular parasite Leishmania braziliensis. We showed that EBI3 defficient mice displayed bigger lesion and an elevated inflammatory immune response in the ear compared to Wild-type mice with similar parasites levels in the ear and lymph nodes. The lack of EBI3 led to an increased expression of T helper type 1(Th1-), Th2- and Th17-derived cytokines in the infected ears. So far, our results demonstrate that EBI3 acts as a regulator of inflammatory immune responses in experimental cutaneous leishmaniasis, although, it is not required for control of L. braziliensis infection. Financial Support: CAPES, FAPESP, CRID.

Interleukin-35 Promotes Macrophage Survival and Improves Wound Healing After Myocardial Infarction in Mice.

Targeting inflammation has been shown to provide clinical benefit in the field of cardiovascular diseases. Although manipulating regulatory T-cell function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. IL (interleukin)-35, an immunosuppressive cytokine mainly produced by regulatory T cells, is a novel member of the IL-12 family and is composed of an EBI3 (Epstein-Barr virus-induced gene 3) subunit and a p35 subunit. However, the role of IL-35 in infarct healing remains elusive. This study aimed to determine whether IL-35 signaling is involved in healing and cardiac remodeling after myocardial infarction (MI) and, if so, to elucidate the underlying molecular mechanisms. IL-35 subunits (EBI3 and p35), which are mainly expressed in regulatory T cells, were upregulated in mice after MI. After IL-35 inhibition, mice showed impaired infarct healing and aggravated cardiac remodeling, as demonstrated by a significant increase in mortality because of cardiac rupture, decreased wall thickness, and worse cardiac function compared with wild-type MI mice. IL-35 inhibition also led to decreased expression of α-SMA (α-smooth muscle actin) and collagen I/III in the hearts of mice after MI. Pharmacological inhibition of IL-35 suppressed the accumulation of Ly6Clow and major histocompatibility complex IIlow/C-C motif chemokine receptor type 2- (MHC IIlow CCR2-) macrophages in infarcted hearts. IL-35 activated transcription of CX3CR1 (C-X3-C motif chemokine receptor 1) and TGF (transforming growth factor) β1 in macrophages by inducing GP130 signaling, via IL12Rβ2 and phosphorylation of STAT1 (signal transducer and activator of transcription family) and STAT4 and subsequently promoted Ly6Clow macrophage survival and extracellular matrix deposition. Moreover, compared with control MI mice, IL-35-treated MI mice showed increased expression of α-SMA and collagen within scars, correlating with decreased left ventricular rupture rates. IL-35 reduces cardiac rupture, improves wound healing, and attenuates cardiac remodeling after MI by promoting reparative CX3CR1+Ly6Clow macrophage survival.

Interleukin-12p35 Deficiency Reverses the Th1/Th2 Imbalance, Aggravates the Th17/Treg Imbalance, and Ameliorates Atherosclerosis in ApoE-/- Mice.

Interleukin- (IL-) 35, a novel functional cytokine of regulatory T cells (Treg) comprised of the IL-12p35 subunit and the other subunit Epstein-Barr virus-induced gene 3 (EBI3), regulates the activity of CD4+ T cells and macrophages, thereby playing a critical role in inflammatory and autoimmune diseases. Previous studies demonstrated that both recombinant mice and human IL-35 attenuated atherosclerosis in ApoE-/- mice. Additionally, EBI3 deficiency enhanced the activation of macrophages and increased atherosclerotic lesions in LDLR-/- mice. This study generated double-deficient mice for ApoE and IL-12p35 (ApoE-/- IL-12p35-/- mice) and investigated the effect of IL-12p35 deficiency on atherosclerosis. IL-12p35 deficiency alleviated Th1/Th2 imbalance, aggravated Th17/Treg imbalance, and attenuated atherosclerotic plaque formation in ApoE-/- mice. Additionally, exogenous rIL-35 treatment reversed the imbalance of Th17/Treg and attenuated atherosclerosis in ApoE-/- mice. These findings suggest that IL-12p35 deficiency ameliorates atherosclerosis in ApoE-/- mice, partially, via attenuating the Th1/Th2 imbalance, although IL-12p35 deficiency aggravates the Th17/Treg imbalance.

Interleukin-35 is associated with the tumorigenesis and progression of prostate cancer.

Interleukin (IL)-35 is a novel member of the IL-12 cytokine family, which exhibits a unique immune regulatory function. Previously, it was demonstrated that IL-35 expression is significantly increased in a wide range of tumor tissues, promoting angiogenesis and inhibiting the anti-tumor immune response. In the present study, the IL-35 protein expression levels were measured in the plasma and tumor tissue of patients with prostate cancer (PCA), and its role was determined in the occurrence and progression of PCA. Plasma IL-35 expression levels were measured using ELISA, and the associations of plasma IL-35 and clinicopathological parameters were analyzed. Receiver operating characteristic curves were plotted to analyze the role of IL-35 as a clinical biomarker for the diagnosis of PCA. Survival curve analysis demonstrated a significant decrease in the survival time in months in patients with PCA and increased expression levels of IL-35 compared with the participants with relatively lower IL-35 protein expression levels. IL-35 expression was detected using immunohistochemical staining of a human PCA tissue microarray. Plasma IL-35 expression levels in the patients with PCA were significantly increased compared with the patients with benign prostatic hyperplasia and the healthy controls. An increase in the plasma concentration of IL-35 was associated with progression of PCA stage and an increase in the Gleason score. Significant differences in AUCs for IL-35 and prostate-specific antigen were observed with regard to the presence of lymph node and distant metastases in patients with PCA. The expression levels of Epstein-barr virus-induced gene 3 (EBI3) and IL-12A (p35; the subunits which together form IL-35) were significantly increased in the tumor tissue compared with the adjacent normal tissue. An association was identified between the Gleason score and the expression of EBI3 and p35. Therefore, increased expression of IL-35 in the plasma and tumor tissues may contribute to the progression and metastasis of PCA, and IL-35 may serve as a novel prognostic biomarker or therapeutic target for the treatment of PCA.

The Roles of Cholesterol and Its Metabolites in Normal and Malignant Hematopoiesis.

Hematopoiesis is sustained throughout life by hematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation into hematopoietic progenitor cells (HPCs). There is accumulating evidence that cholesterol homeostasis is an important factor in the regulation of hematopoiesis. Increased cholesterol levels are known to promote proliferation and mobilization of HSCs, while hypercholesterolemia is associated with expansion of myeloid cells in the peripheral blood and links hematopoiesis with cardiovascular disease. Cholesterol is a precursor to steroid hormones, oxysterols, and bile acids. Among steroid hormones, 17β-estradiol (E2) induces HSC division and E2-estrogen receptor α (ERα) signaling causes sexual dimorphism of HSC division rate. Oxysterols are oxygenated derivatives of cholesterol and key substrates for bile acid synthesis and are considered to be bioactive lipids, and recent studies have begun to reveal their important roles in the hematopoietic and immune systems. 27-Hydroxycholesterol (27HC) acts as an endogenous selective estrogen receptor modulator and induces ERα-dependent HSC mobilization and extramedullary hematopoiesis. 7α,25-dihydroxycholesterol (7α,25HC) acts as a ligand for Epstein-Barr virus-induced gene 2 (EBI2) and directs migration of B cells in the spleen during the adaptive immune response. Bile acids serve as chemical chaperones and alleviate endoplasmic reticulum stress in HSCs. Cholesterol metabolism is dysregulated in hematologic malignancies, and statins, which inhibit de novo cholesterol synthesis, have cytotoxic effects in malignant hematopoietic cells. In this review, recent advances in our understanding of the roles of cholesterol and its metabolites as signaling molecules in the regulation of hematopoiesis and hematologic malignancies are summarized.

The impact on T-regulatory cell related immune responses in rural women exposed to polycyclic aromatic hydrocarbons (PAHs) in household air pollution in Gansu, China: A pilot investigation

Previous studies found associations between impairments of immune functions and exposure to polycyclic aromatic hydrocarbons (PAHs) in ambient air pollution in the U. S. and China. However, the results remain inconclusive due to the limitations of these studies. In this study, we aimed to examine the direction and magnitude of immune changes related to PAH exposure from household air pollution among rural women living in Gansu, China. Healthy village women (n = 34) were recruited and enrolled in the study. Questionnaires were administered. Blood and urine samples were collected and analyzed during non-heating (September 2017, “summer”) and heating (January 2018, “winter”) seasons. Urinary 1-hydroxypyrene (1-OHP) was quantified as the biomarker of PAH exposure. To evaluate Treg cell related immune functions, we examined immunoglobulin E (IgE), percent of T-regulatory (Treg) cells, and gene expression of following: forkhead box transcription factor 3 (Foxp3), transforming growth factor-β (TGF-β), interleukin 10 (IL-10), and interleukin 35 (IL-35), composed of interleukin-12 alpha (IL-12α) and Epstein-Barr-virus-induced gene 3 (EBi3). Urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) was measured to evaluate oxidative DNA damage. The results showed that the concentration of 1-OHP increased from 0.90 to 17.4 μmol mol-Cr −1 from summer to winter (p < 0.001). Meanwhile, average percent of Treg cells decreased from 5.01% to 1.15% (p < 0.001); IgE and mRNA expressions of Foxp3, TGF-β, IL-10, IL-12α and EBi3 all significantly decreased (p < 0.001); Urinary 8-OHdG increased from 12.7 to 30.3 ng mg-Cr −1 (p < 0.001). The changes in 8-OHdG, Foxp3 and TGF-β were significantly associated with the increase of 1-OHP. The results suggested that we observed a substantial increase of PAH exposure in winter, which was significantly associated with the repression on Treg cell function and oxidative DNA damage. Exposure to PAHs in household air pollution possibly induced immune impairments among rural women in northwest China.

Enzymatic interconversion of the oxysterols 7β,25-dihydroxycholesterol and 7-keto,25-hydroxycholesterol by 11β-hydroxysteroid dehydrogenase type 1 and 2

Oxysterols are cholesterol metabolites derived through either autoxidation or enzymatic processes. They consist of a large family of bioactive lipids that have been associated with the progression of multiple pathologies. In order to unravel (patho-)physiological mechanisms involving oxysterols, it is crucial to elucidate the underlying formation and degradation of oxysterols. A role of 11β-hydroxysteroid dehydrogenases (11β-HSDs) in oxysterol metabolism by catalyzing the interconversion of 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC) has already been reported. The present study addresses a function of 11β-HSD1 in the enzymatic generation of 7β,25-dihydroxycholesterol (7β25OHC) from 7-keto,25-hydroxycholesterol (7k25OHC) and tested whether 11β-HSD2 is able to catalyze the reverse reaction. For the first time, using recombinant enzymes, the formation of 7k25OHC from 7kC by cholesterol 25-hydroxylase (CH25H) and further stereospecific oxoreduction to 7β25OHC by human and mouse 11β-HSD1 could be demonstrated. Additionally, experiments using human 11β-HSD2 showed the oxidation of 7β25OHC to 7k25OHC. Molecular modeling provided an explanation for the stereospecific interconversion of 7β25OHC and 7k25OHC. Production of the Epstein-Barr virus-induced gene 2 (EBI2) ligand 7β25OHC from 7k25OHC in challenged tissue by 11β-HSD1 may be important in inflammation. In conclusion, these results demonstrate a novel glucocorticoid-independent pre-receptor regulation mediated by 11β-HSDs.

Interleukin-35 in immune-related diseases: protection or destruction.

Interleukin-35 (IL-35) is a recently identified heterodimeric cytokine in the IL-12 family. It consists of an IL-12 subunit α chain (P35) and IL-27 subunit Epstein-Barr virus-induced gene 3 (EBI3) β chain. Unlike the other IL-12 family members, it signals through four unconventional receptors: IL-12Rβ2-IL-27Rα, IL-12Rβ2-IL-12Rβ2, IL-12Rβ2-GP130, and GP130-GP130. Interleukin-35 signaling is mainly carried out through the signal transducer and activator of transcription family of proteins. It is secreted not only by regulatory T (Treg) cells, but also by CD8+ Treg cells, activated dendritic cells and regulatory B cells. It exhibits immunosuppressive functions distinct from those of other members of the IL-12 family; these are mediated primarily by the inhibition of T helper type 17 cell differentiation and promotion of Treg cell proliferation. Interleukin-35 plays a critical role in several immune-associated diseases, such as autoimmune diseases and viral and bacterial infections, as well as in tumors. In this review, we summarize the structure and function of IL-35, describe its role in immune-related disorders, and discuss the mechanisms by which it regulates the development and progression of diseases, including inflammatory bowel disease, collagen-induced arthritis, allergic airway disease, hepatitis, and tumors. The recent research on IL-35, combined with improved techniques of studying receptors and signal transduction pathways, allows for consideration of IL-35 as a novel immunotherapy target.