Induction of kawasaki disease-like phenomena by bacillus Calmette-Guerin injection in PD-1 gene knockout mice

Abstract

[한글]

[영문]Background: Programmed cell death-1 (PD-1) is a negative co-stimulatory receptor, which binds with either of the two ligands, PD-L1 or PD-L2 (B7-DC). This inhibitory receptor plays a role in regulating self tolerance and autoimmunity.The pathogenesis of Kawasaki disease (KD) is not revealed yet but a certain defect among series of apoptotic processes after infectious stimuli has been suspected as one of the possible causes. Recently, various apoptotic defects have been explored in autoimmune vasculitis including KD. Among these apoptotic components, PD-1-PD-L pathway is emerging as a major process in the pathogenesis of autoimmune vasculitis. The role of PD-1 in the pathogenesis of KD, however, has not yet been explored.This study was focused to investigate if KD is related with the defect of PD-1-PD-L pathway. To investigate the associative role of PD-1 in the pathogenesis of KD, the KD-like features of PD-1 knockout (PD-1KO) mice after stimulation with immunizing agents, and possible single nucleotide polymorphism regions of PD-1 geneMethods: For the induction of the KD animal model, two times of intradermal injection of either bacillus Calmette-Guerin (BCG) or heat shock protein 65 (HSP65) was performed to 3-4 week (wk)-old PD-1KO mice of BALB/c and C57BL/6 background. Daily body temperature of each mouse was recorded in all experimental groups and the changes in their clinical features were monitored. After 2, 4, 8, 12, 16 and 24 wks of injection, mice were sacrificed and their organs including heart, liver, gallbladder, and kidneys, their serum, and isolated lymphocytes from spleen were examined for analysis of histological and immunological changes.For the analysis of single nucleotide polymorphisms (SNPs) of PD-1 gene (PDCD1) in patients with KD, rs41386349 and rs2227981, two most possible SNPs in Asian ethnic descent from about 30 known SNPs, were chosen and examined in 73 patients with KD and 100 healthy children.Results: After second injection of BCG, PD-1KO mice showed Kawasaki-like features such as prolonged fever for more than 5 days, erythematous edema on 4 soles, anal desquamation, and gallbladder (GB) hydrops. Inflammation in coronary arteries of at least more than one artery were found in all PD-1KO mice while no coronary lesion was found in wild type (WT) mice. Coronary arteritis seemed to develop from the time of second injection of BCG because no lesion was found after the first injection of BCG. By immunohistochemical staining, CD3, CD4, CD8, CD19 and CD31 positive cells were found in the lesions of coronary arteritis. IgG and complement C3 were positive in coronary arterial lesions by immunofluorescent staining. Coronary arterial lesions were aggravated as long as 6 months after 2nd injection of BCG by serial observations. Initially inflammatory cells were infiltrated in adventitia layer or aggregated on the surface of endothelial cells. One to two months (mo) later, intimal proliferation was observed. And 3 to 4 mo later, disruption of the internal elastic lamina and fibrotic changes were observed. Moreover, even without BCG challenges, PD-1KO mice showed coronary arterial lesions spontaneously since 4 mo of age. When the PD-1KO mice were challenged with HSP65, coronary arterial lesions similar to those seen when stimulated with BCG were observed. Likewise, inflammatory reactions in other organs including hepatic arteries, renal arteries, and biliary arteries were also involved in an animal model of KD. In the splenic cytotoxic T cells of PD-1KO mice, significantly increased interferon-γ and IL-6 secretion was observed when they were stimulated with anti-CD3 antibody. PD-L1 expression was observed in both WT and PD-1KO and no difference was found between both groups.For the analysis of human PD-1 gene polymorphisms, higher T allelic frequency of rs41386349 was found in the KD group than in the normal group (P = 0.007, odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.2-2.9). PD-1 rs2227981 polymorphism was not significant in patients with KD compared with the control group (P = 0.4, OR = 1.2, 95% CI = 0.8-1.9). Furthermore, no difference between patients with coronary artery dilatation (CAD) and those without CAD was found in terms of PD-1 polymorphism.Conclusion: As a first attempt to elucidate the associative role of PD-1 in the pathogenesis of KD, the induction of animal model of KD using BCG or HSP65 was tried in the PD-1KO mice in this study. The PD-1KO mice showed features mimicking findings seen in KD. In addition, SNP analysis of a specific site for PD-1 gene in human showed significant differences between patients with KD and normal population. These results suggest that PD-1 may play a critical role in the development of KD.