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

Introduction

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). Unlike the pro-inflammatory properties of other IL-12 family members, IL-35 plays potent immunosuppressive roles partly by means of Tregs expansion, which is essential for the maintenance of immune tolerance (Collison et al., 2010). 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). Our team also proved the therapeutic application of IL-35 in dextran sulfate sodium (DSS)-induced colitis (Zhang et al., 2018). Thus, the anti-inflammatory ability makes IL-35 a promising intervention agent in inflammation, infection and other immune-related disorders.

T cells especially CD4⁺ T helper (Th) cells and CD4⁺ CD25⁺ Tregs are critical for immune-regulation in cellular immunity and immunity homeostasis (Xiao et al., 2012; McQuillan, Lynch & Mills, 2010). Cytokine profiles released by these cells also take part in and accelerate their action (Choi et al., 2015; Egwuagu et al., 2015). Th1 cells together with secreted IL-2 and interferon-γ (IFN-γ) have been well-known to mediate inflammation and related diseases, which got significantly inhibited by IL-35 in animal disease models (Ma et al., 2014; Guo et al., 2017). Th2 and Tregs, in favor of anti-inflammation partly depending on the production of IL-10 and IL-35, respectively, received effective increase post IL-35 administration (Guo et al., 2017; Zhao et al., 2017; Bettini & Vignali, 2009). Another CD8⁺ T cells, whose function relies on cytotoxin release including Granzyme B (Gzmb) and perforin 1 (Prf1) upon antigen stimulation, also received effective suppression following IL-35 use (Wong & Pamer, 2003; Milstein et al., 2011). However, most of these findings about the immune-regulation capacity of IL-35 were discovered based on disease modeling animals, the in vivo effects on normal animals have not been well described. In the present study, a plasmid carrying recombinant mouse IL-35 sequence was intravenously injected into normal mice and the short-term effect of general overexpression of IL-35 heterodimer on immunological status, particularly the differentiation of T-cell subsets, was evaluated. The results could facilitate illuminating the underlying therapeutic mechanisms of IL-35.

Materials and Methods

Results

The constructed plasmid gained successful gene expression of IL-35

As shown in Fig. 1A, a recombinant plasmid carrying IL-35 gene composted of Ebi3 and IL12a fragments was constructed. The expression of IL-35 was further confirmed by HEK293T transient transfection assay. SDS-PAGE and western blot for IL-12 p35 were conducted and the results both showed strong bands at about 52 KDa in DNA transfected cell lysates (Fig. 1B), indicating the successful expression of IL-35.

IL-35 inhibited CD4⁺ T cells and promoted CD4⁺ CD25⁺ Tregs generation in vitro

It has been reported that IL-35 could act on phenotype differentiation of CD4⁺ T cells (Guan et al., 2017; Huang et al., 2017). In the present study, splenic T cells were isolated and directly treated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h in the presence of anti-CD3/CD28 ex vivo. The cells were then collected to assess the proportion of T-cell subsets by FACS. The results showed that CD4⁺ T cell population substantially raised upon activation, which got effectively reduced post rIL-35 supplement (Fig. 2A). Also, the CD4⁺CD25⁺ Tregs proportion decreased when exposed to anti-CD3/CD28, but increased after recombinant IL-35 treatment (Fig. 2B). In addition, the rIL-35 affect showed some dose-dependence, indicating that IL-35 could inhibit CD4⁺ T cells proliferation and generate CD4⁺CD25⁺ Tregs.

IL-35 facilitated Th2 and Tregs function in vitro

Cytokines produced by Th and Treg cells also take part in and play important roles in immune-regulation (Ma et al., 2014). The total RNA of cultured T cells mentioned above were extracted and the cytokine expression involved Th1 (Il2), Th2 (Il10) and Tregs (Foxp3) were examined by real time PCR. Data indicated that rIL-35 administration significantly reduced Il2 level and sharply boosted Il10 and Foxp3 expression in a dose-dependent manner (Fig. 3), which suggested that IL-35 treatment facilitated Th2 and Tregs function.

IL-35 overexpression inhibited CD4⁺ and CD8⁺ T cells but enhanced CD4⁺CD25⁺ Tregs in vivo

To further investigate the effect of IL-35 on the T cells differentiation in vivo, wild type C57BL/6J mice received an intravenous injection of the plasmid pMSCV-IL-35-GFP. After 72 h, peripheral blood was sampled to test the expression of Ebi3 and Il12a fragments by real time PCR. Spleen tissues were used to estimate the proportion of T-cell subsets by FACS. The results demonstrated both significant increases in Ebi3 and Il12a levels in mice received pMSCV-IL-35-GFP injection, suggesting the expression of exogenous gene sequence and upregulation of IL-35 (Fig. 4). The same as Fig. 2 described, FACS analysis showed obviously lowered percentage of total CD4⁺ and CD8⁺ T cells (Figs. 5A–5D) but considerably amplified CD4⁺CD25⁺ Tregs (Figs. 5E, 5F) compared to PBS group. Consistent with previous reports, Treg cells proliferated in response to IL-35 upregulation (Castellani et al., 2010).

Upregulation of IL-35 increased Th2 cytokine production in vivo

In this study, we also measured the mRNA levels of cytokines and genes related to Th1, Th2, CD8⁺ and Tregs using real time PCR. The results showed that Th1 cytokines IL-2 and IFN-γ (Figs. 6A, 6B) as well as Gzmb and Prf1 in CD8⁺ T cells (Figs. 6D, 6E) obviously increased in mice received pMSCV plasmid. While, the generous expression of IL-35 effectively inhibited the aforementioned effector molecules. Besides, the splenic levels of Il10 (Fig. 6C) and Foxp3 (Fig. 6F) of pMSCV-IL-35 group both got remarkably elevated. Therefore, IL-35 could effectively suppress Th1 and CD8⁺ T cell function, but strengthen Th2 and Tregs.

Discussion

In the present investigation, a plasmid loading IL-35 linear gene containing Ebi3 joint with Il12a was intravenously injected into normal mice to determine the effect of IL-35 heterodimer overexpression on T cell immunity and T cell differentiation under normal condition. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibited CD4⁺ and CD8⁺ T cells proliferation and Th1 cytokine secretion. On the contrary, elevated level of IL-35 significantly induced CD4⁺ CD25⁺ Tregs and Th2 enhancement. The in vitro study provided similar results, suggesting the immunosuppressive action of IL-35 on T-cell subsets evolution. All of the data indicated the Th1 and CD8⁺ T cells suppression but Th2 and Tregs bias in the presence of IL-35.

As we all know, T cells, roughly divided into CD4⁺ Th cells, CD8⁺ cytotoxic T cells (CTLs) and Tregs according to the functional distinction, play a central role in cell-mediated immunity (Milstein et al., 2011; Overgaard et al., 2015). Cytokine profiles secreted by these cells further augment their immunoregulatory capacity and act on other subsets (Biron & Tarrio, 2015). In brief, IL-2 and IFN- γ produced by Th1 cells can enhance the cytotoxic effect of CD8⁺ T cells, which can be well inhibited by IL-10 released from Th2 cells and/or IL-35 synthesized by Tregs (Guo et al., 2017; Biron & Tarrio, 2015). The ability of Th1 and Th2 cytokines has been clearly stated, and recent studies focused on the newly discovered immunosuppressive factor IL-35.

IL-35, a member of the IL-12 family, is a heterodimeric cytokine composed of the Ebi3 and IL-12A assembled in innate Tregs (Collison et al., 2007; Niedbala et al., 2007). The anti-inflammatory capacity of IL-35 has been investigated in several inflammatory disorder models, such as infection diseases, organ transplantation rejection and autoimmunity diseases (Guan et al., 2017; Sun et al., 2015; Liu et al., 2015; Li et al., 2012). Evidence from these animal models showed that IL-35 administration suppressed inflammation related T cell differentiation. However, the existing results are mainly obtained from disease models, it still remains to be determined whether IL-35 could affect the T subsets evolution without antigen stimulation. The present study primarily aimed at expounding the impact of IL-35 on T cell differentiation and function in normal healthy mice.

Some works proposed that the Tregs and Th2 augment partly at least contributed to the therapeutic potential of IL-35 (Zhao et al., 2017). To confirm this finding, activated T cells were treated with recombinant IL-35 factor for 24 h ex vivo. The data indicated that IL-35 remarkably suppressed CD4⁺ T cell proliferation (Fig. 2A) and IL-2 release (Fig. 3A). Instead, CD4⁺CD25⁺ Tregs (Fig. 2B) and IL-10 secretion (Fig. 3B) by Th2 cells got effective increase. Foxp3 also showed a distinct growth under treatment with 50ng/mL IL-35 factor (Fig. 3C). The in vivo effect of IL-35 on T-cell subsets development was then investigated after a plasmid carrying IL-35 fragment (Fig. 1) administration in normal mice. The much higher expression of Ebi3 and IL-12p35 in mice received pMSCV-IL-35 plasmid could be observed three days later (Fig. 4). Similarly, both splenic CD4⁺ and CD8⁺ T cells performed significantly restrained ( Figs. 5A–5D), but CD4⁺CD25⁺ Tregs got well amplification (Figs. 5E, 5F). Related cytokine and effector molecule analysis further confirmed lower IL-2 and IFN-γ level (Figs. 6A, 6B) together with largely reduced Gzmb and Prf1 production (Figs. 6D, 6E) upon IL-35 expression. In contrast, both IL-10 (Fig.  6C) and Foxp3 (Fig. 6F) increased significantly. Thus, IL-35 overexpression indeed affect T cell differentiation and induced an obvious Th2 and Tregs bias.

The suppressive capacity of IL-35 has been tested in many disease models, still the influence of general IL-35 administration on normal body needs to be clarified. Data listed here based on normal healthy mice could partly help to understand how IL-35 work and the potential therapeutic mechanisms in disease cases. And it is important to note that the present study was conducted in normal animals and the outcome might have some difference from those in pathologic condition. The cure mechanisms of IL-35 treatment in various disease models still need further researches.

Conclusions

The present study explored the effect of recombinant heterodimer IL-35 on T-cell subset developing in normal mice for the first time. The results indicated that IL-35 upregulation could effectively induce Th2 and Tregs bias and inhibit Th1 and CD8⁺ T cell function in healthy animals. Meanwhile, further studies concerning long-term effect and related mechanisms are still required to elucidate in IL-35 application.

Supplemental Information