Identifying genetic hypomethylation and upregulation of Toll-like receptors in Kawasaki disease

Abstract

Kawasaki disease (KD) is an acute febrile systemic vasculitis that occurs in children and is characterized by elevated levels of proinflammatory cytokines. Toll-like receptors (TLRs) serve as the sensor arm of the innate immune system and induce proinflammatory cytokine expressions.We recruited a total of 18 paired KD patients, before intravenous immunoglobulin (IVIG) and at least 3 weeks after IVIG treatment, 18 healthy controls, and 18 febrile controls. For TLR genes and their cytosine-phosphate-guanine (CpG) markers, we used Affymetrix GeneChip® Human Transcriptome Array 2.0 and Illumina HumanMethylation450 BeadChip to evaluate gene expression levels and methylation patterns, respectively.KD patients demonstrated a significantly differential expression of TLR mRNA levels compared to both the healthy and febrile controls, with only TLR 3 and 7 not differing between the KD patients and the controls. After patients underwent IVIG treatment, the TLR mRNA levels, except for TLR3, decreased significantly in KD patients. In contrast, the methylation status of the CpG sites of TLR1, 2, 4, 6, 8, and 9 demonstrated an opposite tendency between the two stages of both the KD samples and the controls.TLRs, particularly TLR1, 2, 4, 6, 8, and 9, may stimulate the immunopathogenesis of KD. These results are among the first to use TLRs to prove that a bacterial inflammatory response may trigger KD.

Keywords: IVIG; Toll-like receptor; kawasaki disease; methylation.

Figure 1. Comparison of toll-like receptors’ (TLRs) mRNA expressions by geneChip^® human transcriptome array 2.0 between acute-stage Kawasaki disease patients and control subjects

Asterisks denote significance (p < 0.05). Data are expressed as mean ± standard error for the three replications.

Figure 2. Comparison of toll-like receptors’ (TLRs) mRNA expressions by GeneChip^® human transcriptome array 2.0 between acute-stage Kawasaki disease patients and KD patients after receiving IVIG treatment

Asterisks denote significance (p < 0.05). Data are expressed as mean ± standard error for the three replications.

Figure 3. Comparison of toll-like receptors’ (TLRs) mRNA expressions by GeneChip^® human transcriptome array 2.0 between Kawasaki disease patients after IVIG treatment and control subjects

Asterisks denote significance (p < 0.05). Data are expressed as mean ± standard error for the three replications.

Figure 4. Comparison of methylation patterns of CpG markers on toll-like receptors (TLRs)

A CpG marker was defined to be on a gene when located within the −5000 to +3000 region of a gene. Asterisks denote significance (p < 0.05). Data are expressed as mean ± standard error for the 12 replications.

Figure 5. Integration of CpG marker methylation pattern and gene expression profile

The CpG markers corresponding to TLR1, 2, 4, 6, 8, and 9 are cg22839308, cg03523945, cg13730105, cg14578677, cg07759587, and cg21578541, respectively. The methylation patterns of representative CpG markers and gene expression profiles of TLRs showed negative tendencies. Furthermore, they were found to be altered in the healthy and febrile control subjects, as well as in Kawasaki disease patients before and after IVIG treatment. For one TLR, the fold change values of gene expression in groups were determined by dividing them by the average of normal controls. The histogram and curve are presented as mean ± standard error.

Figure 6. Correlation analysis of gene expression and CpG marker methylation

The CpG markers corresponding to TLR1, 2, 4, 6, 8, and 9 are cg22839308, cg03523945, cg13730105, cg14578677, cg07759587, and cg21578541, respectively. We used scatter plots to demonstrate correlations between mRNA levels and DNA methylation. The results demonstrated that mRNA levels were negatively correlated with DNA methylation (Pearson's correlation coefficient around −0.4 and p < 0.001).