Abstract
Pancreatic islet transplantation is a promising treatment for type 1 diabetes (T1D). Interleukin-35 (IL-35) is a recently discovered cytokine that exhibits potent immunosuppressive functions. However, the role of IL-35 in islet transplant rejection remains to be elucidated. In this study, we isolated islet cells of BALB/c mouse and purified CD4+ T cell subsets of a C57BL/6 mouse. The model for islet transplantation was established in vitro by co-culture of the islet cells and CD4+ T cells. IL-35 (20 ng/ml) was administered every other day. Following co-culture, the islet function and Treg/Th17 ratio were analyzed on days 1, 3, and 5. Furthermore, the Th17/Treg ratio was modulated (1:0–2), and the function of islet cells as well as proliferation of Th17 cells were analyzed. T cell sorting was performed using the magnetic bead sorting method; Treg and Th17 count using flow cytometry; cell proliferation detection using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method, and islet function test using the sugar stimulation test. Results showed that Th17 counts increased in the co-culture system. However, after administration of IL-35, the number of Treg cells increased significantly compared to that in the control group (50.7% of total CD4+ T cells on day 5 in IL-35 group vs. 9.5% in control group) whereas the proliferation rate of Th17 cells was significantly inhibited (0.3% in IL-35 group vs. 7.2% in control group on day 5). Reducing the Th17/Treg ratio significantly improved the function of transplanted islets. Treg inhibited Th17 proliferation and IL-35 enhanced this inhibitory effect. IL-35 mitigates the function of murine transplanted islet cells via regulation of the Treg/Th17 ratio. This might serve as a potential therapeutic strategy for in-vivo islet transplant rejection and T1D.
Highlights
The absolute counts of T helper 17 (Th17) showed only a slight increase, their ratio in CD4+ T cells was significantly decreased in the IL-35 group compared to that in the control group (0.3% vs. 7.2% on day 5, P
A remarkable difference in the ratio of Th17/regulatory CD4 +CD25+Foxp3+ T cell (Treg) in CD4+ T cells was observed under these conditions, on day 5 after co-culture (1.4% in control group vs. 0.1% in IL-35 group, P
On day 5, with an increase of Treg ratio, the proliferation of the carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled Th17 cells was suppressed markedly; besides, stronger suppression was observed when IL-35 was added, compared to that in the control group (Fig 6)
Summary
Tregs play an anti-inflammatory role mainly by releasing inhibitory cytokines such as TGF-β and IL-10 or contact-dependent suppression on other immune cells, including CD8+, CD4+ T cells and B cells [12]. Th17 cells, mainly expressed by factors such as retinoic acid receptorrelated orphan receptor γt (RORγt), have been reported to play a potent pro-inflammatory role by producing the signature cytokine IL-17A [25,26,27,28,29]. Recent studies found that the balance between Tregs and Th17 plays an important role in the above diseases, by regulation of the immunologic homeostasis through the secretion of antior pro-inflammatory cytokines, depending on the activation of Forkhead box P3 (FoxP3) and signal transducer and activator of transcription 5 (STAT5) or RORγt and STAT3, respectively [30, 31, 33, 35, 36]
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