To investigate the effects of p300/CBP on histone acetylation of Foxp3 gene and its roles in the immunological pathogenesis of Kawasaki disease (KD).

Forty-six children with KD and twenty-eight age-matched health children were consented to participate in this study. Co-immunoprecipitation and real-time PCR were performed to detect Foxp3-associated acetylation levels of histone H4 and binding abilities of p300, CBP, pSmad3 (phosphorylated mothers against decapentaplegic homolog 3) and NF-AT (nuclear factor of activated T cells) with Foxp3 gene in CD4⁺ T cells. The percentages of CD4⁺ CD25^high Foxp3⁺ cells (Treg) and the expression of Foxp3, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), p300, CBP, TGF-βRⅡ (transforming growth factor β receptor Ⅱ) and pLAT1 at protein level were analyzed by flow cytometry. Quantitative real-time PCR was used to measure the expression of Foxp3, IL-10, TGF-β, TGF-βRⅠ, Egr-1 (early growth response protein 1), RARα (retinoic acid receptor α) and PLCγ1 (phospholipase C-γ1) in Treg cells at mRNA level. Plasma concentrations of TGF-β and retinol acid (RA) were measured by enzyme-linked immunosorbent assay.

(1) The percentages of Treg cells, levels of Foxp3 and molecules associated with suppressive function of Treg cells (TGF-β, IL-10 and CTLA4), acetylation levels of histone H4 associated with promoter, conserved non-coding DNA sequence 1 (CNS1) and CNS2 of Foxp3 gene decreased remarkably during acute KD (P<0.05), but were restored after IVIG therapy (P<0.05). Meanwhile, all of the aforementioned items in KD patients with coronary artery lesions (KD-CAL⁺ ) were lower than those without coronary artery lesions (KD-CAL^-) (P<0.05). No significant differences in histone H4 acetylation associated with CNS3 were found among different groups (P>0.05). (2) The levels of p300 and CBP in Treg cells and their binding abilities with Foxp3 gene were down-regulated significantly during acute KD (P<0.05), but were restored to some extent after IVIG treatment (P<0.05). The Foxp3-associated histone acetylation was positively correlated with the expression of p300 and CBP at mRNA level during acute KD (r=0.65, 0.42, P<0.05). Furthermore, the expression of p300 and CBP and their binding abilities with Foxp3 gene in KD-CAL⁺ group were lower than those in KD-CAL^- group (P<0.05). (3) Compared with healthy subjects, plasma concentrations of TGF-β and RA and the expression of TGF-βRⅠ/Ⅱ/Egr-1, RARα and pLAT1/PLCγ1 were down-regulated during acute KD (P<0.05); the binding abilities of pSmad3 and NFAT with Foxp3 gene were reduced remarkably in patients with acute KD (P<0.05). All the items mentioned above were restored after IVIG treatment (P<0.05). Moreover, the ten items aforementioned in KD-CAL⁺ group were lower than those in KD-CAL^- group (P<0.05). (4) Higher acetylation levels of histone H4 associated with promoter, CNS1 and CNS2, and enhanced binding abilities of p300 and CBP with Foxp3 gene were found in CD4⁺ T cells isolated from patients with acute KD after co-stimulation with TGF-β, RA and anti-CD3/CD28 antibodies as compared with those in CD4⁺ T cells without stimulation (P<0.05). However, no statistical difference in the acetylation level of histone H4 associated with CNS3 was found between the two groups (P>0.05).

Hypoacetylation of histone H4 associated with Foxp3 gene caused by insufficient expression of p300/CBP and their impaired binding abilities might be involved with immune dysfunction in KD. IVIG therapy regulates the expression of p300/CBP and their binding abilities with Foxp3 gene through up-regulating TGF-β signal.

Kawasaki disease; Foxp3; Histone acetylation; Regulatory T cell; Immunity