Changes and significance of histone acetylation associated with forkhead box P3 gene during the acute phase of Kawasaki disease

Abstract

Objective To investigate the histone acetylation status of forkhead box P3(Foxp3) gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Forty-two children with KD and 32 age-matched healthy children consented to participate in this study as a control group.Co-immunoprecipitation and real-time PCR were performed to determine acetylation levels of histone H4 associated with Foxp3 gene and binding abilities of Kruppel like factor 10(KLF10) and p300/CBP-associated factor (PCAF) with Foxp3 gene in CD4+ T cells.The proportion of CD4+ CD25high Foxp3+ cells (Treg) and protein levels of Foxp3, KLF10, PCAF, phosphated SMAD family member 2/3(pSmad2/3) and itchy E3 ubiquitin protein ligase(Itch) were analyzed by flow cytometry.Quantitative real-time PCR was used to evaluate the levels of Foxp3, transforming growth factor-β1 receptor Ⅱ (TGF-βRⅡ), transforming growth factor-β1 receptor Ⅰ(TGF-βRⅠ), tyrosine kinase 2(Tyk2), SH2 domain-containing protein-tyrosine phosphatase-2(SHP2) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) mRNA in Treg.Plasma concentrations of TGF-β and interleukin-6(IL-6) were measured by enzyme-linked immunosorbent assay. Results (1)Compared with the control group, the proportion of Treg, expression levels of Foxp3 and TGF-β, and histone acetylation levels of Foxp3 promoter decreased remarkably during acute KD, and the differences were all statistically significant(all P<0.05), and restored after IVIG therapy(all P<0.05). Meanwhile, the 5 indexes aforementioned in KD patients with coronary artery lesions (KD-CAL+ ) were lower than those without coronary artery lesions(KD-CAL-), and the differences were all statistically significant (all P<0.05). (2)Compared with the control group, protein levels of KLF10 and PCAF in Treg cells, and their binding abilities with Foxp3 gene were significantly down-regulated during acute KD, and the differences were all statistically significant(all P<0.05), and restored to some extent after IVIG treatment(all P<0.05). Positive correlations between protein levels of KLF10 and PCAF, and mRNA level of Foxp3 were detected in acute KD patients (r=0.47, 0.59, all P<0.05). Furthermore, protein levels of KLF10 and PCAF and their binding abilities with Foxp3 gene in KD-CAL+ group were lower than those in KD-CAL- group, and the differences were all statistically significant(all P<0.05). (3) Compared with the control group, the plasma concentration of TGF-β and expressions of TGF-βRⅡ/Ⅰ, pSmad2/3, Itch, CTLA4 and SHP2 in Treg were down-regulated during the acute phase of KD, and the differences were all statistically significant(all P<0.05), as well as plasma concentration of IL-6 and expression its downstream molecule Tyk2 increased remarkably in patients with acute KD(all P<0.05). All the indexes mentioned before restored significantly after IVIG treatment (all P<0.05). Simultaneously, the 7 former indexes in KD-CAL+ group were found to be lower than those in KD-CAL- group, while 2 indexes of the latter in KD-CAL+ group were higher than those in KD-CAL- group, and the differences were all statistically significant(all P<0.05). Conclusion Histone hypoacetylation of Foxp3 promoter might be one of the important factors contributing to insufficiency and dysfunction of regulatory T cells during acute Kawasaki disease. Key words: Kawasaki disease; Foxp3; Histone; Acetylation; Immune