IL-35 and Rapamycin Reduce Acute Graft-Versus-Host Disease Associated with Decreased Platelet Aggregation in a Mouse Model

Introduction: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) caused by the activation of donor T lymphocytes by host antigen-presenting cells and the immune-mediated inflammatory response. Epithelial cells of the skin and mucous membranes, biliary ducts, and intestinal tract crypts are the primary tissue systems damaged during the pathobiological course of GVHD. IL-35, a member of the IL-12 family of cytokines, comprising an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). It is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development (Niedbala W, et al. European journal of immunology 2007). Rapamycin (Sirolimus; RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, has been used for the prophylaxis and treatment of several immune reactions including GVHD and solid organ rejection (Ho-Jin Shin, et al. Blood 2011). We hypothesized that IL-35 has a protective effect in aGVHD, and that its function may be increased by RAPA.

Methods: We used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2)F1 (BDF1, H-2b×d) mice as recipients to create an aGVHD model (Kuroiwa T, et al. The Journal of clinical investigation 2001). Mice were divided into five groups, including a BMT control group, aGVHD control group, aGVHD treated with IL-35 group, aGVHD treated with RAPA group and aGVHD treated with IL-35 and RAPA group. Morbidity and mortality related to aGVHD were observed, and 2 weeks after BMT, tissues from the intestine and liver were stained with hematoxylin and eosin and examined by light microscopy. To detect apoptosis in intestinal sections, a modified terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method was applied. CD4+CD25+Foxp3+ regulatory T cells were measured by flow cytometry. Quantitative RT-PCR was used to measure the production of IFN-γ, TNF-α and IL-17A in the spleen and intestine of each group of mice. We also measured platelet aggregation using a turbidimetric aggregation-monitoring device. Finally, western blotting was conducted to test the signaling pathways of IL-35.

Results: Mice receivingIL-35 exhibited a higher survival rate compared with GVHD mice as well as those mice receiving RAPA. When the two drugs were given together, the survival rate was much higher than that in the other groups. The aGVHD control group had the highest morbidity rate of aGVHD, and IL-35 plus RAPA could prevent the occurrence of aGVHD. Additionally, this treatment inhibited apoptosis of intestinal epithelial cells as well as donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by aGVHD. The importance of the inflammatory cytokine cascade in the pathogenesis of both clinical and experimental GVHD is now well accepted. We found that IL-35 and RAPA also markedly suppressed IFN-γ, TNF-α and IL-17A expression in the intestine and liver. Because studies by other have showed that Tregs have the ability to inhibit aGVHD, we measured Tregs in serum and found that IL-35 and RAPA treatment expanded serum Tregs. We further explored the relationship between IL-35 and platelet aggregation. Platelet aggregation was high in aGVHD mice, and the ratio of platelet aggregation was inhibited by IL-35 and RAPA. Finally, we found that the phosphorylation of STAT1 and STAT4 were inhibited in GVHD mice, and thatSTAT1 and STAT4 were phosphorylated when mice were treated with IL-35.

Conclusions: IL-35 may be useful for controlling aGVHD after allo-HSCT. IL-35 suppresses inflammatory cytokines and expands anti-inflammatory cells in aGVHD. IL-35 also prevents platelet aggregation in aGVHD mice, which could be helpful in treating thrombotic complications after HSCT. These results are readily translatable to the clinic in future clinical trials. IL-35 and RAPA may have potential clinical use for the prevention or treatment of aGVHD and thrombotic complications after HSCT.