Abstract
Purpose: :
IL-12 family cytokines have emerged as an important family of proteins in host immunity. IL-27 (IL27p28/EBI3) promotes the differentiation of Th1 cells while antagonizing Th17 development. On the other hand, IL-6 is a major inducer of Th17 differentiation. Both IL-27 and IL-6 exert their effects through binding and activating gp130. In this study, we have genetically engineered IL-30 (IL-27p28) and investigated whether this IL-27 subunit protein is a dominant negative regulator of gp130 signaling. Because both Th17 and Th1 cells are implicated in the development of experimental autoimmune uveitis (EAU), we also examined whether IL-30 can ameliorate EAU.
Methods: :
Full-length mouse IL-30 cDNA fragment was cloned into insect expression vector pMIB. The IL-30 protein was expressed in insect cells, purified by combination of Ni-NTA chromatography and differential centrifugation on centricon filtration units and characterized by polyacrylamide gel electrophoresis, immunoprecipitation and western blot. Biological effects of IL-30 were analyzed by RT-PCR, Thymidine incorporation, Annexin-V staining and intracellular cytokine staining assays. The effect of IL-30 on the development and progression of EAU was monitored by Fundoscopy, Flow cytometry and histopathology.
Results: :
IL-30 inhibited the proliferation of CD4+T cells and transcription of genes coding for pro-inflammatory genes (TNF-α, IFN-γ and IL-17a) and pro-apoptotic gene, ICE. IL-30 also suppressed the development and progression of EAU, in part, by inhibiting the differentiation and expansion of Th1 and Th17 cells and promoting IL-10 production.
Conclusions: :
IL-30 suppressed the development and progression of EAU, suggesting that it can be used to treat uveitis and other autoimmune diseases.
Keywords: autoimmune disease • cytokines/chemokines • protein purification and characterization