Abstract
ObjectiveThe benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice.MethodsA plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR.ResultsWestern blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results.ConclusionThe results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.
Highlights
Interleukin 35 (IL-35), a heterodimer composed of Epstein-Barr-virus-induced gene 3 (Ebi3) and interleukin-12 alpha (IL-12A), secreted by natural regulatory T cells (Tregs), is a novel cytokine of the IL-12 family (Collison et al, 2007; Sawant, Hamilton & Vignali, 2015)
The results showed that Th1 cytokines IL-2 and IFN-γ (Figs. 6A, 6B) as well as Granzyme B (Gzmb) and perforin 1 (Prf1) in CD8+ T cells (Figs. 6D, 6E) obviously increased in mice received pMSCV plasmid
The present study explored the effect of recombinant heterodimer IL-35 on T-cell subset developing in normal mice for the first time
Summary
Interleukin 35 (IL-35), a heterodimer composed of Epstein-Barr-virus-induced gene 3 (Ebi3) and interleukin-12 alpha (IL-12A), secreted by natural regulatory T cells (Tregs), is a novel cytokine of the IL-12 family (Collison et al, 2007; Sawant, Hamilton & Vignali, 2015). How to cite this article Zhang et al (2018), Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice. Recent studies have demonstrated the efficacy of IL-35 in inflammatory bowel disease (Wang et al, 2018), autoimmune encephalomyelitis (Choi et al, 2017; Guan et al, 2017), collagen-induced arthritis (Li et al, 2016) and acute graft-versus-host disease (Zhang et al, 2015). The anti-inflammatory ability makes IL-35 a promising intervention agent in inflammation, infection and other immune-related disorders
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