Aberrant histone modification and inflammatory cytokine production of peripheral CD4+ T cells in patients with oral lichen planus

Backgrounds To investigate alterations in histone modification and histone deacetylases (HDACs) in patients with oral lichen planus (OLP), and to evaluate correlations with inflammatory cytokine production. Methods Global histone H3/H4 acetylation and HDAC activity in CD4+ T cells from 23 patients with OLP and 10 healthy control subjects were examined using spectrophotometry. The mRNA levels of eight members of four classes of HDAC genes were measured by real‐time quantitative polymerase chain reaction. Forty cytokines involved in inflammation were examined with a cytokine array. The correlation between histone modification and cytokine production was analyzed. Results Global histone H3 hypo‐acetylation was observed in OLP patients. Patients with OLP had significantly higher HDACs activity,and higher HDAC6 and HDAC7 mRNA level compared with the controls. Of the 40 cytokines in the cytokine array, eight were significantly increased in OLP patients: interleukin (IL)‐4, IL‐8, IL‐1ra, tumor necrosis factor receptor II (TNFR II), macrophage inflammatory protein 1b (MIP‐1b), fibrosis‐associated tissue inhibitors of metalloproteinase 1 (TIMP)‐1, monocyte chemotactic protein 1 (MCP‐1), and eotaxin‐2. In the OLP group, the acetylation level of histone H3 was negatively correlated with IL‐4 and MCP‐1 production, and the expression of HDAC6 mRNA was positively correlated with MCP‐1 production. In the non‐erosive subgroup, acetylation of histone H3 was negatively correlated with IL‐4, IL‐16, and TIMP‐2 production. In the erosive OLP subgroup, the expression of HDAC7 mRNA was positively correlated with MIP‐1a production. Conclusion Aberrant histone modification of CD4+ T cells in peripheral blood could occur in OLP patients, and possibly affects inflammatory cytokine production.

of CD4+ T cells and various inflammatory cytokines. 5,6 These cytokines are produced by a variety of cells under chronic inflammatory conditions, which in turn leads to the development of immune-mediated inflammatory diseases. In OLP, several inflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, have been shown to have abnormal expression patterns. [7][8][9] However, the production and regulation of cytokines is complex. As such, studies that have focused on single or several inflammatory cytokines have produced contradictory results, and the mechanisms of cytokine regulation and their role in the chronic inflammatory environment of OLP are not understood.
In recent years, epigenetic mechanisms have been shown to play fundamental roles in a variety of biological processes including cell growth, development, differentiation, and genomic stability. Epigenetic changes can result in gene dysregulation, leading to various pathological conditions such as cancer or autoimmune diseases. 10,11 Histone acetylation, a primary aspect of epigenetic mechanisms, plays key roles in remodeling chromatin conformation. The level of histone acetylation is highly dynamic and regulated by the opposing action of two enzyme families, histone deacetylases (HDACs) and histone acetyltransferases (HATs). HDACs remove acetyl groups from target histones, resulting in histone hypo-acetylation that restores the positive charge of histones leading to a closed chromatin configuration, which may potentially induce further epigenetic changes and altered gene expression. 12 There is increasing evidence that HDACs and their inhibitors may mediate the development of chronic inflammation by modulating the expression of many inflammatory cytokines and mediators such as IFN-γ, TNF-α, and interleukins. 13,14 These studies have highlighted the involvement of histone modification in the pathogenesis of various autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). [15][16][17] To date, the involvement of epigenetic histone modification in the pathogenesis of OLP has not been examined. In this study, we investigated whether the histone modification pattern is altered in patients with OLP and examined the expression of eight members of four classes of HDACs in OLP CD4+ T cells. In addition, we also examined the relation of acetylation modification and peripheral blood cytokine levels in OLP. The statistical significance was set at P ≤ 0.05.

| Aberrant global histone acetylation in OLP patients and controls
Compared with the control group, the histone H3 acetylation level in the OLP group was significantly lower (P = 0.0116). There was a significant difference in the histone H3 acetylation level among EOLP, NEOLP group and controls (P = 0.025). No significant difference was found in histone H3 acetylation level between EOLP and NEOLP groups (P > 0.05). No significant difference in the acetylation level of histone H4 was found between different groups (P > 0.05) (Figure 1).

| HDAC activity and related modifier HDAC genes
Compared with the control, the HDAC activity in the OLP group was significantly higher (P = 0.023). The HDAC activity in the EOLP been reported that histone modification is associated with several critical events in T cells, such as T cell development, activation, differentiation, and cytokine production. 18 The different results may be due to the different methods of detection, or the influence of differences in genetic background, lesion site, sex, and age between study populations.
We eventually identified five cytokines, IL-4, IL-16, MCP-1, TIMP-2, and MIP-1a that may be associated with histone modification. The level of HDAC6 mRNA expression positively correlated with MCP-1 production in the OLP group and the level of HDAC7 mRNA expression positively correlated with MIP-1a production in the EOLP group. HDAC6 and HDAC7 are both class II HDACs that have been found to be correlated with pathologic grade and tumor stage in several human cancers. 25,26 They also appear to have crucial roles in T cell development and function. HDAC6 overexpression results in increased T cell migration and chemotaxis, 27 and nuclear export of HDAC7 regulates the expression of cytokines, cytokine receptors, and adhesion molecules. 28 A selective class II HDAC inhibitor has been found to decrease inflammatory cytokine levels in different cell lines. 29,30 We can infer that the inhibition of HDAC activation, and the subsequent modification of chromatin status, change the molecular signature of mediators, leading to changes in the inflammatory environment.
In summary, our results revealed that histone modifications are apparently altered in peripheral blood of CD4+ T cells and are correlated with cytokine production in OLP patients. These findings provide novel insights into the pathogenesis of OLP. It is possible that the reversal of epigenetic changes could be an effective therapy for OLP. Further research is necessary to confirm our results, and to determine the causes leading to the deregulation of epigenetic mechanisms in patients with OLP.

ACKNOWLEDG EMENTS
This work was supported by the grants from Guangdong Medical Science and Technology Research Fund (No. A2016207).

CONFLI CT OF INTEREST
The authors declared that they have no conflict of interest.