Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice

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.

IL-35 inhibits CD8+ T cells activity by suppressing expression of costimulatory molecule CD28 and Th1 cytokine production

“…Zhang et al . reported that IL-35 inhibits Th1 cytokine secretion under normal conditions in mice ( 32 ). Moreover, Choi et al .…”

Pretreatment with interleukin 35-engineered mesenchymal stem cells protected against lipopolysaccharide-induced acute lung injury via pulmonary inflammation suppression

“…The retroviral vector pMSCV-IL-35-GFP reported in our previous study carried the murine IL-35 fragment covering Ebi3 linked with Il12a (Zhang et al 2018c). One day before transfection, HEK293T cells were plated, and the cell fusion rate was approximately 80% after 24 h. HEK293T cells were transfected using FuGENE ® HD Transfection Regent (Promega, USA) according to the manufacturer's instructions.…”

Immunomodulatory cytokine interleukin-35 and immune thrombocytopaenia

“… 56 IL-35 can also mediate the conversion of T h 2 cells to T reg cells, although this can be blocked by IFN-γ. 56 Furthermore, IL-35, via thymic stromal lymphopoetin, also inhibited DC priming of T h 2 responses. 37 …”