Background/Purpose: Behcet’s disease (BD) is a chronic recurrent, multisystem inflammatory disorder. The phenotypic characteristics include oral aphtha, genital ulcers, uveitis and skin lesions. No specific laboratory tests have been known in BD. Reported in BD have been an increased number of γδ T cells in the peripheral blood and hyperactivity of neutrophils. Although a line of evidence has suggested genetic contributions to the disease, etiopathogenesis of BD is still unclear and non-genetic factors, like environment, infection or epigenetics may play pivotal roles in the pathogenesis.  Epigenetic mechanisms including posttranslational histone modifications are known to regulate gene expression without altering the genomic sequence. The association of specific histone modifications with gene expression is very well defined, such as tri-methylation of histone 3 lysine 27 (H3K4me27), which may be characteristic of repressed genes, or tri-methylation of histone 3 lysine 4 (H3K4me3), which may present in many active genes. Histone modifications in major rheumatic diseases, such as rheumatoid arthritis, have been investigated, while studies on histone modifications in BD are limited. From the functional point of view, it is important to analyze differences of histone modifications in each functional subclass of the entire peripheral blood nucleated cells. To examine the histone modifications of peripheral blood mononuclear cells (PBMCs) and neutrophils in BD, we have established a novel method analyzing histone methylation in each subset by fluorescence-activated cell sorting (FACS).

Methods: PBMCs and neutrophils were obtained from 13 patients with BD and 12 healthy controls (HC). Diagnosis of BD was made according to the criteria of Behcet’s Disease Research Committee of Japan, Ministry of Health, Labour and Welfare of Japan. Four immune cell types were stained surface epitopes: CD4+ T cells, CD8+ T cells, γδ T cells, and CD16+CD66b+ neutrophils. Flow cytometry for H3K4me3 and H3K4me27 were normalized using isotype controls. All samples were analyzed on a FACSCalibur cytometer. As a quantitative measure of H3K4me3 and H3K27me3, mean fluorescence intensity (MFI) was used.

Results:   H3K27me3 and H3K4me3 were detected in CD4+ T cells, CD8+ T cells, γδ T cells, and CD16+CD66b+ neutrophils. H3K27me3 MFI levels were not significantly different in those four immune cell types in BD and HC. In contrast, H3K4me3 MFI levels of BD in γδ T cells were significantly increased compared with HC. H3K4me3/H3K27me3 MFI ratio was significantly lower in neutrophils of BD and higher in γδ T cells of BD than that of HC. H3K4me3 MFI and H3K4me3/H3K27me3 MFI ratio of active BD were significantly increased in γδ T cells as compared to inactive BD.

Conclusion:   Difference in histone modifications could be detected by FACS in peripheral white blood cells in BD patients. Aberrant histone methylation may be associated with the pathogenesis of BD. It is suggested that histone methylation could be a new candidate-biomarker for BD.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/histone-methylation-profiling-in-peripheral-white-blood-cells-as-a-candidate-biomarker-for-behcets-disease/