[Role of AHR-mediated histone acetylation at FOXP3 in Tfr cell differentiation in systemic lupus erythematosus]

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by abnormal autoantibody production, immune dysregulation, and multisystem involvement. This study aims to investigate the effects of aryl hydrocarbon receptor (AHR) activation on the differentiation of T follicular regulatory (Tfr) cells and the expression of forkhead box protein P3 (FOXP3) in patients with SLE, and to elucidate the underlying molecular mechanisms from an epigenetic perspective.

Eighteen patients with SLE who attended the Departments of Dermatology and Rheumatology and Immunology of The Third Xiangya Hospital, Central South University, between June and December 2025, and 18 healthy individuals undergoing routine health examinations were enrolled. In addition, 3 healthy volunteers were recruited for peripheral blood collection for mechanistic experiments. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation, and CD4⁺ T cells were sorted. Flow cytometry was used to determine the proportion of Tfr cells (Tfr%), as well as FOXP3 and AHR expression, and their correlations with disease activity were analyzed. CD4⁺ T cells isolated from healthy individuals were divided into a Control group [treated with dimethyl sulfoxide (DMSO)], an ITE group [treated with the AHR agonist 2-(1'H-indol-3'-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE)], an A-485 group (treated with the P300 inhibitor A-485), and an ITE+A-485 group (co-treated with ITE and A-485). Cell counting kit-8 (CCK-8) assay, flow cytometry, Western blotting and real-time reverse transcription polymerase chain reaction (PCR) were performed to assess cell proliferation activity, Tfr%, the protein expression of FOXP3 and AHR, and the mRNA expression levels of cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and interleukin-10 (IL-10) in the corresponding T cells. AHR and P300 overexpression plasmids were co-transfected into CD4⁺ T cells from healthy individuals, and co-immunoprecipitation (Co-IP) was conducted to verify the protein-protein interaction between AHR and P300. Chromatin immunoprecipitation (ChIP)-PCR was used to identify the binding sites of AHR and P300 within the FOXP3 promoter region, while ChIP-quantitative PCR (qPCR) was performed to quantify AHR and P300 binding to the FOXP3 promoter and histone acetylation levels in CD4⁺ T cells from healthy individuals in each group. Peripheral blood CD4⁺ T cells were also collected from patients with SLE, activated using CD3/CD28 T-cell activation beads, and treated with either DMSO or ITE. Flow cytometry was used to detect Tfr% and FOXP3 expression in CD4⁺ T cells from patients with SLE, and ChIP-qPCR was used to detect AHR and P300 binding to the FOXP3 promoter and histone acetylation levels.

Compared with healthy controls, patients with SLE had a significantly lower Tfr% among peripheral blood CD4⁺ T cells (P<0.001), which was negatively correlated with the systemic lupus erythematosus disease activity index (SLEDAI) (r=-0.803, P<0.001). FOXP3 and AHR protein expression levels in Tfr cells were also significantly downregulated (both P<0.01), and the two were positively correlated (r=0.596, P<0.05). CCK-8 assays showed that ITE at concentrations of 0 to 349.27 nmol/L and A-485 at concentrations of 0 to 93.20 nmol/L had no significant effects on CD4⁺ T-cell proliferation activity (both P>0.05). In vitro experiments showed that, compared with the Control group, the ITE group had significantly increased Tfr%, AHR and FOXP3 protein expression, and CTLA-4 and IL-10 mRNA expression levels (all P<0.05). Co-IP confirmed a protein-protein interaction between AHR and P300 in CD4⁺ T cells. ChIP-PCR showed that AHR had binding sites within the -78 to -263 bp and -431 to -596 bp regions of the FOXP3 promoter, whereas P300 bound to the -78 to -263 bp region of the FOXP3 promoter. ChIP-qPCR showed that, compared with the Control group, the ITE group exhibited significantly increased H3K9ac and H3K14ac acetylation within the FOXP3 promoter region, together with increased binding of AHR and P300 (all P<0.05). In contrast, the A-485 group showed significantly decreased H3K9ac and H3K14ac acetylation and P300 binding within the FOXP3 promoter region (all P<0.05), whereas AHR binding showed no significant change (P>0.05). In the functional rescue experiment, compared with the ITE group, the ITE+A-485 group showed significantly decreased FOXP3 protein expression and Tfr% (both P<0.05), as well as significantly decreased H3K9ac and H3K14ac acetylation and P300 binding within the FOXP3 promoter region (all P<0.05), while AHR binding showed no significant change (P>0.05). Compared with the A-485 group, the ITE+A-485 group showed significantly increased AHR binding to the FOXP3 promoter and increased FOXP3 protein expression (both P<0.05), although both remained lower than those in the ITE group. No significant differences were observed in H3K9ac and H3K14ac acetylation or P300 binding within the FOXP3 promoter region (all P>0.05). Validation experiments in CD4⁺ T cells from patients with SLE showed that ITE treatment increased Tfr%, FOXP3 expression, histone acetylation within the FOXP3 promoter region, and AHR and P300 binding (all P<0.05), consistent with the above findings.

The reduced Tfr% and decreased FOXP3 expression in peripheral blood CD4⁺ T cells from patients with SLE may be associated with insufficient AHR expression. AHR activation may promote FOXP3 expression and induce Tfr cell differentiation, potentially through recruitment of P300 to the FOXP3 promoter region and enhancement of local histone acetylation.