The roles of activated hypoxia-inducible factor 1α signaling in Th17/regulatory T cells imbalance in Kawasaki disease

Abstract

Objective To investigate the roles and significance of hypoxia-inducible factor 1α(HIF-1α)signaling in Th17/Treg imbalance in Kawasaki disease(KD). Methods Thirty-six children with KD and 32 age-matched healthy children consented to participate in the study.The proportions of Th17, Treg and expression levels of forkhead box protein P3(Foxp3), IL-10, IL-17A, HIF-1α and phosphated signal transducer and activator of transcription 3(pSTAT3)in CD4+ T cells, were evaluated by flow cytometry.Quantitative real-time PCR was used to assess the transcription levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), receptor interacting protein-1(RIP1), IL-17A, RAR-related orphan receptor γt(RORγt)and p300 in CD4+ T cells.Immunoprecipitation combined with quantitative PCR and Western blot was performed to determine histone acetylation level of IL-17A gene and poly-ubiquitination status of Foxp3 protein in CD4+ T cells.Plasma concentrations of IL-1β, IL-6, IL-21, IL-23 and tumor necrosis factor α(TNF-α) were measured by enzyme-linked immunosorbent assay. Results (1)The proportion of Th17 and expression of IL-17A in patients with acute KD increased remarkably while the proportion of Treg and expressions of Foxp3 and IL-10 were found to be lower than those of the healthy controls(t=7.62, 9.60, 5.72, 6.82, 5.83; all P<0.05), which resulted in a higher ratio of Th17/Treg during acute KD(t=12.21, P<0.05), and all the data mentioned above restored significantly after intravenous immunoglobulin(IVIG) treatment(t=6.28, 8.30, 4.75, 5.02, 3.95, 9.39; all P<0.05). (2) Acetylation of the histone H3 associated with IL-17A gene and poly-ubiquitination of Foxp3 protein in CD4+ T cells increased significantly, and more higher expressions of HIF-1α, RORγt and p300 were detected during acute KD[H3: (7.32±2.53)% vs(2.60±1.08)%; Foxp3: (42.94±15.91)% vs(11.01±3.60)%; t=10.20, 11.71, 6.02, 6.92, 6.01; all P<0.05], and the 5 items decreased remarkably after IVIG therapy[H3: (4.63±2.07)% vs(7.32±2.07)%; Foxp3: (26.37±11.58)% vs(42.94±15.91)%; t=4.94, 5.05, 3.40, 5.31, 4.29; all P<0.05]. Meanwhile, positive correlations between expre-ssion of HIF-1α with histone acetylation of IL-17A gene and poly-ubiquitination of Foxp3 protein were observed in patients with acute KD(r=0.65, 0.52; all P<0.05). (3) Compared with the healthy controls, plasma concentrations of inflammatory cytokines(IL-1β, IL-6, IL-21, IL-23 and TNF-α)and expressions of TLR4 were elevated while expression levels of its downstream molecules(MyD88, pSTAT3 and RIP1)in CD4+ T cells were up-regulated during acute KD(t=11.00, 15.18, 7.21, 11.09, 9.09, 3.84, 10.07, 5.41, 8.13; all P<0.05), and restored remarkably after therapy(t=7.81, 10.59, 4.70, 6.73, 6.49, 3.03, 6.50, 3.63, 5.22; all P<0.05). Conclusion Excess activation of HIF-1α signaling may be one of the important factors contributing to the imbalance of Th17/Treg in KD. Key words: Kawasaki disease; Hypoxia-inducible factor 1α; T helper cell 17; Regulatory T cells; Imbalance