Interleukin-12 family cytokines have emerged as critical regulators of immunity with some members (IL-12, IL-23) associated with disease pathogenesis while others (IL-27sue injury by promoting the expansion of regulatory B and T-cell subsets [2,3].Discovery of IL-23 in 2000 [4] led to the reevaluation of IL-12 and IL-23 in autoimmune diseases. For example, therapeutic targeting of IL-12p40 decreases pathology in many mouse models of autoimmune diseases [5], while disease is exacerbated in IL-12p35-deficient mice [6,7]. Thus, IL-23 rather than IL-12 was * These authors contributed equally to this work as first authors.* * These authors contributed equally to this work as senior authors. Eur. J. Immunol. 2016. 46: 1343-1350 found to be the critical cytokine for autoimmune inflammation including experimental immune-mediated disease [6][7][8][9][10]. Currently, at least ten therapeutic agents targeting IL-23 are being tested in the clinic for more than 17 human immune-mediated diseases [11]. Both IL-27 and IL-35 have immune-suppressive activities and are also cytokines with strikingly diverse influences on the immune response so that viable therapeutic targets may also be exploited for treatment of human inflammatory diseases [12,13]. Thus, understanding immunobiology of IL-12 family cytokines would undoubtedly provide valuable knowledge that can be exploited therapeutically. The IL-12 family cytokines are α/β heterodimers consisting of one α subunit (IL-23p19, IL-27p28, IL-12p35) and one β chain (IL-12p40, Ebi3) [14,15]. Although there are currently four known members in the family, the predictable range of combinations is six and it is conceivable that additional pairings such as IL-23p19/Ebi3 are possible [12,[14][15][16][17]. In this study, we sought to discover additional IL-12 members that might exist in nature. By combining different alpha and beta IL-12 subunit proteins in vitro we detected a novel stable p19/Ebi3 heterodimeric complex by immunoprecipitation. We have characterized the p19/Ebi3 cytokine (IL-39) and demonstrated that it possesses biological activities in vitro and in vivo. Results IL-23p19 (p19) and Ebi3 form a composite factor (IL-39)To examine whether p19 can form a stable complex with Ebi3, we mixed equal amounts of the two proteins and immunoprecipitation (IP)/Western blot analyses revealed formation of a stable human p19/Ebi3 complex (Fig. 1A). We could not detect the p19/Ebi3 following IP with isotype IgG or anti-c-Jun antibody, providing suggestive evidence for potential bona fide p19/Ebi3 cytokine. To confirm our finding in another animal species, we genetically engineered and expressed mouse p19 and Ebi3 subunits in CHO cells (Fig. 1B). IP of supernatants derived from transfectants with anti-p19 mAb and followed by Western blot analysis using anti-Ebi3 mAb confirmed coexpression p19 and Ebi3 and formation of a stable p19/Ebi3 heterodimer (Fig. 1C). We further confirmed this observation by reciprocal IP with anti-Ebi3 mAb and Western blotting with anti-p19 mAb and the p19/Ebi3 comple...

Sepsis is an excessive inflammatory condition with a high mortality rate and limited prediction and therapeutic options. In this study, for the first time, to our knowledge, we found that downregulation and/or blockade of T cell Ig and mucin domain protein 3 (Tim-3), a negative immune regulator, correlated with severity of sepsis, suggesting that Tim-3 plays important roles in maintaining the homeostasis of sepsis in both humans and a mouse model. Blockade and/or downregulation of Tim-3 led to increased macrophage activation, which contributed to the systemic inflammatory response in sepsis, whereas Tim-3 overexpression in macrophages significantly suppressed TLR-mediated proinflammatory cytokine production, indicating that Tim-3 is a negative regulator of TLR-mediated immune responses. Cross-talk between the Tim-3 and TLR4 pathways makes TLR4 an important contributor to Tim-3–mediated negative regulation of the innate immune response. Tim-3 signaling inhibited LPS–TLR4–mediated NF-κB activation by increasing PI3K–AKT phosphorylation and A20 activity. This negative regulatory role of Tim-3 reflects a new adaptive compensatory and protective mechanism in sepsis victims, a finding of potential importance for modulating innate responses in these patients.

High mobility group protein B1 (HMGB1) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of HMGB1 in plasma and sputum of patients with asthma and COPD across disease severity needs to be defined. The objective of the study was to examine the induced sputum and plasma concentrations of HMGB1 in COPD and asthmatic patients to determine differences in HMGB1 levels between these diseases and their relationship with airway obstruction and inflammatory patterns. A total of 147 participants were enrolled in this study. The participants included 34 control subjects, 61 patients with persistent asthma (according to the Global Initiative for Asthma [GINA] guidelines) and 47 patients with stable COPD (stratified by Global Initiative for Chronic Obstructive Lung Disease [GOLD] status). Spirometry was performed before sputum induction. HMGB1 levels in induced sputum and plasma were determined by enzyme-linked immunosorbent assay. Sputum and plasma concentrations of HMGB1 in patients with asthma and COPD were significantly higher than concentrations in control subjects and were significantly negatively correlated with forced expiratory volume in 1 s (FEV 1 ), FEV 1 (% predicted) in all 147 participants. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthma patients and healthy controls (P < 0.001). This difference was present even after adjusting for sex, age, smoking status, daily dose of inhaled corticosteroids and disease severity. There were no significant differences in HMGB1 levels between patients with eosinophilic and noneosinophilic asthma. HMGB1 levels in asthmatic and COPD patients were positively correlated with neutrophil counts and percentage of neutrophils. In multivariate analysis, the two diseases (asthma and COPD) and disease severity were independent predictors of sputum HMGB1, but not smoking, age or use of inhaled corticosteroids. In conclusion, these data support a potential role for HMGB1 as a biomarker and diagnostic tool for the differential diagnosis of asthma and COPD. The importance of this observation on asthma and COPD mechanisms and outcomes should be further investigated in large prospective studies.