AhR agonist Ficz modulates T follicular helper cells and ameliorates skin fibrosis in a bleomycin-induced SSc mouse model

Abstract

Objectives

T follicular helper (Tfh) cells are involved in the pathogenesis of systemic sclerosis (SSc). Recent investigations have revealed aryl hydrocarbon receptor (AhR) regulated skin fibrosis in SSc. This study aims to explore the effect of AhR agonist Ficz on Tfh in a bleomycin (BLM)-induced SSc mouse model.

Methods

BALB/c mice were randomly assigned to the control group (Ctrl), BLM-induced SSc mouse group (BLM) and the Ficz treatment group (BLM+Ficz). BLM (1 mg/kg/day) and Ficz (1 µg/mouse/48 hours) were injected subcutaneously for 4 weeks. Skin lesions were harvested for HE staining, Masson’s trichrome (Masson) staining, double immunofluorescence as well as real-time quantitative PCR. Flow cytometry analysis was carried out to determine the proportion of peripheral Tfh cells.

Results

We found that Ficz treatment effectively attenuated dermal thickness, collagen content and the expression of fibrosis-related genes, including Col1a1, Col3a1, Fn1 and Tgfβ1 compared with the BLM-induced mice. Moreover, the percentage of peripheral Tfh cells (CD4+CXCR5+PD-1+) was significantly elevated in BLM-induced mice and Ficz appeared to reduce the percentage of peripheral Tfh cells. Double immunofluorescence staining verified the presence of Tfh-like cells (CXCR5+PD-1+) in skin lesions of BLM-induced mice. In addition, the messenger RNA level of a key Tfh cytokine, Il21 was decreased in skin of Ficz treated BLM-induced mice, with elevated expression of Cyp1a1, a marker of AhR signalling.

Conclusions

Ficz, an AhR agonist, can alleviate skin fibrosis in BLM-induced SSc mice, which may relate to the reduction of Tfh cells and its downstream cytokine interleukin-21 expression through the activation of AhR pathway.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• T follicular helper (Tfh) cells contribute to the pathogenesis of systemic sclerosis (SSc), and the aryl hydrocarbon receptor (AhR) has been implicated in regulating skin fibrosis. However, whether pharmacological activation of AhR can modulate Tfh cell responses to alleviate SSc-associated fibrosis remains unknown.

WHAT THIS STUDY ADDS

• Ficz, an AhR agonist, can alleviate skin fibrosis in bleomycin-induced SSc mice, which may relate to the reduction of peripheral Tfh cells and its downstream cytokine interleukin-21 expression through the activation of AhR pathway.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• These findings position AhR activation as a potential therapeutic strategy for SSc by concurrently suppressing Tfh-mediated immune pathways and dermal fibrosis.

Introduction

Systemic sclerosis (SSc) is a complex autoimmune disease marked by autoimmunity, fibrosis of the skin and internal organs, and vasculopathy with the highest mortality among all rheumatic diseases.¹ The pathogenesis of SSc involves dysfunction across multiple immune cell subsets, including Th1, Th2, Th17 and Treg cells.² T follicular helper (Tfh) cells are a special population of helper T cells essential for the biological progression of plasma cell development, formation of germinal centres as well as antibody production. Tfh cells express high levels of C-X-C chemokine receptor type 5 (CXCR5), programmed death-1 (PD-1) and inducible costimulatory molecule (ICOS). B-cell lymphoma 6 is considered a key transcription factor for Tfh cell differentiation.³ Emerging evidence has implicated Tfh cells in the pathogenesis of various autoimmune diseases, including SSc, systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren’s syndrome.⁴

The aryl hydrocarbon receptor (AhR) is a highly evolutionarily conserved ligand-activated transcription factor known as dioxin receptor to mediate environmental toxicity.⁵ AhR activation and subsequent gene transcription can involve a variety of physiological processes, such as cell proliferation and differentiation, immune function and tumourigenesis.^{6 7} Recently, AhR signalling in the pathogenesis of SSc has received increasing attention.⁸ The expression of AhR is particularly low in SSc skin lesions and dermal fibroblasts, and Ficz, an exogenous agonist of AhR, can ameliorate collagen production in SSc fibroblasts via AhR/TGF-β signalling activation.⁹ Esomeprazole alleviates fibrosis in SSc by modulating AhR/Smad2/3 signalling.¹⁰ These researches suggest that AhR signalling may be an effective intervention target for SSc. However, the precise mechanisms by which AhR activation ameliorates SSc require further elucidation.

Recent investigations in influenza virus infection and lupus revealed that AhR signalling can regulate Tfh cell differentiation.^{11 12} AhR agonist tapinarof ameliorated lupus autoimmunity by suppressing Tfh cell differentiation via regulation of the JAK2-STAT3 signalling pathway.¹³ AhR coordinated with AP-1 family member JUN to prevent CXCL13+Tph/Tfh cell differentiation.¹² Nevertheless, whether AhR activation influences Tfh cells to mediate therapeutic benefits in SSc remains unknown. In the current study, we used Ficz to treat a bleomycin (BLM)-induced SSc mouse model to observe the therapeutic effects of Ficz and further evaluated the consequences of AhR activation on Tfh cells.

Methods

Mice and treatments

Female BALB/c mice (8 weeks old, 18–22 g) were purchased from the Guangdong Animal Experiment. All mice were housed under specific pathogen‐free conditions at the Animal Laboratory Center of the Second Affiliated Hospital of Guangzhou Medical University. All animal experiments were approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou Medical University. Mice were randomly assigned to three groups, including the control group (Ctrl), BLM-induced SSc mouse group (BLM) and the Ficz treatment group (BLM+Ficz). BLM (Nippon Kayaku, Japan) and Ficz (MedChemExpress, USA) were individually dissolved in phosphate-buffered saline (PBS). Subcutaneous injection of BLM (1 mg/kg/day) into a 1 cm × 1 cm square area on the middle back for 4 weeks established a SSc mouse model. Ficz (1 µg/mouse, dissolved in PBS at a concentration of 10 µg/mL) was subcutaneously injected every 48 hours after BLM treatment for 4 weeks⁹. An equal amount of PBS was subcutaneously injected as a control. At the end of 4 weeks, skin lesions and peripheral blood were harvested for further analysis.

Histological analysis

Skin tissues of mice were fixed in 4% paraformaldehyde overnight, followed by dehydration, clearing, embedding and paraffin sectioning. H&E and Masson’s trichrome staining were administered. The collagen content was quantified using ImageJ (NIH, Bethesda, Maryland, USA).

Tissue section immunofluorescence

Skin tissue sections underwent standard deparaffinisation, antigen retrieval and blocking. Double immunofluorescence staining was processed as previously described.¹⁴ Primary antibodies included antibodies against the anti-CXCR5 antibody (1:200, Bioss, USA) and anti-PD-1 (1:100, Bioss, USA). After the second antibody was removed, the nuclei were counterstained with 4-amino-6-diamino-2-phenyl indole (Beyotime Biotechnology, China). Image analysis was performed with the EVOS M7000 Imaging System.

Flow cytometry analysis

Peripheral blood samples were collected, and mouse peripheral blood mononuclear cells (PBMCs) were isolated using the PBMC separation solution kit (Solarbio, China) to generate a single-cell suspension. 100 µL PBMC suspension was transferred to an microcentrifuge tube, followed by the addition of an equal volume of surface flow cytometry antibodies (CD4, CXCR5, PD-1). After gentle mixing, the samples were incubated at 4°C in the dark for 30 min, washed twice with PBS and then analysed by Navios Flow Cytometer (Beckman Coulter, USA). The following flow-cytometry antibodies were used in the present study: Ms CD4 FITC RM4-5, Ms CXCR5 PerCP-Cy5.5 2G8, Ms CD279 (PD-1) APC J43 (all from BD Biosciences, USA).

RNA isolation and quantitative real-time PCR

Total RNA was extracted from skin tissue using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Complementary DNA was synthesised with the PrimeScript RT Reagent Kit (Takara, Japan). The PCR reaction mixture was prepared according to the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA), and then detected using the QuantStudio 7 Flex. The primer sequences used in this study were shown in Supplementary material (online supplemental table).

Western blotting assay

Proteins were extracted from skins by RIPA lysis buffer (Beyotime, China) with 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, USA). Equal amounts of proteins from each sample were loaded onto 10% sodim dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). After blocking with 5% bovine serum albumin (Servicebio, China), blotted proteins were incubated with antibody anti-Collagen I (1:5000, Abcam, UK) at 4℃ overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary rabbit antibodies (Servicebio, China) for 2 hours. Finally, protein bands were rewashed with tris buffered saline with tween-20 (TBST) and visualised with an enhanced chemiluminescence system (Thermo Fisher Scientific, USA).

Statistical analysis

All statistical analyses were conducted using the SPSS V.25.0 statistical software. The data are presented as mean±SD. Differences between groups were analysed by using analysis of variance. p<0.05 was considered significant.

Results

Ficz improves BLM-induced skin fibrosis

We administered BLM as a subcutaneous injection in BALB/c mice for 28 consecutive days.

H&E and Masson staining revealed that the BLM-induced mice exhibited obvious dermal thickening and collagen deposition (figure 1A–D). To determine whether the AhR antagonist Ficz is effective in vivo, we treated the mice with Ficz after BLM treatment. Ficz treatment exhibited reduced dermal thickness and collagen content compared with the BLM-induced mice (figure 1A–D). These results indicate that the AhR agonist Ficz effectively alleviates skin fibrosis in BLM-induced SSc mice. Moreover, to further assess the fibrotic changes among the groups at the molecular level, we measured profibrotic markers, including Col1a1, Col3a1, Fn1 and Tgfβ1 in dorsal skin lesions by quantitative real-time PCR, the findings of which are in keeping with the results of H&E and Masson staining (figure 1E). Above results demonstrated that Ficz can improve BLM-induced skin fibrosis.

Figure 1. Ficz ameliorated skin fibrosis in a BLM-induced model mouse. (A–D). Sections were stained with HE and Masson staining of different groups. Fold changes in dermal thickness and collagen content in different groups were measured by ImageJ. (E). Expression of fibrosis-related genes, including Col1a1, Col3a1, Fn1 and Tgfb1 in different groups on 4 weeks. Data were presented as mean (SD) and compared with one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BLM, bleomycin; Col1a1, collagen type I alpha 1 chain; Col3a1, collagen type III alpha 1 chain; Ctrl, control group; Masson, Masson trichome; mRNA, messenger RNA; Tgfβ1, transforming growth factor beta 1.

Ficz decreased the Tfh cell population in BLM-induced mice

Increased Tfh cell lineage activity and exaggerated Tfh cell response are more likely to be associated with the pathogenesis of autoimmune diseases, including SSc. Flow cytometry demonstrated the percentage of peripheral Tfh cell was aberrantly elevated in BLM-induced mice. Compared with the BLM-induced mice, the Ficz-treated mice exhibited a decreased proportion of peripheral Tfh cells (CD4+CXCR5+PD-1+) (figure 2A,B). In a previous study, Tfh-like cell infiltrates were found in skin lesions of patients with SSc and the infiltrates were positively associated with modified Rodnan skin score (mRSS).¹⁴ We further investigated the Tfh-like cell infiltration in skin lesions of BLM-induced mice, and the effect of Ficz on Tfh-like cell infiltration. Double immunofluorescence confirmed the presence of Tfh-like cells (CXCR5+PD-1+) in skin lesions of BLM-induced mice (figure 2E), while absent in the control group and Ficz-treated group (data not shown). Tfh cell-derived interleukin (IL)-21 contributes to the development of SSc by promoting the expression of fibrosis-related genes and modulating the levels of CD4+ T cells. BLM-induced mice exhibited significantly higher Il21 mRNA levels compared with Control mice, which were reduced by Ficz treatment (figure 2C). These results supported that Ficz effectively reduced the Tfh cell response in BLM-induced mice.

Figure 2. Ficz modulated Tfh cells in a BLM-induced model mouse. (A–B). Representative FACS pseudo-colour plots showed peripheral Tfh cell populations (CD4+CXCR5+PD1+) in different groups and the result of the statistical analysis was shown. (C–D). Expression of Il21 (a key Tfh cytokine) and Cyp1a1 (a marker of AhR signalling) in the skin among different groups on 4 weeks. (E). Immunofluorescence of representative images from BLM-induced mouse skin sections stained with CXCR5, PD-1 and DAPI. Data were presented as mean (SD) and compared with one-way ANOVA. *p<0.05, **p<0.01. AhR, aryl hydrocarbon receptor; ANOVA, analysis of variance; BLM, bleomycin; Ctrl, control group; CXCR5, C-X-C chemokine receptor type 5, Cyp1a1, cytochrome P450 1a1; DAPI, 4-amino-6-diamino-2-phenyl indole; FACS, fluorescence-activated cell sorting; Il21, interleukin 21; mRNA, messenger RNA; ns, not significant; PD-1, programmed death-1; Tfh, T follicular helper.

Subsequently, we sought to investigate the role of AhR signalling in Ficz regulating Tfh cell, and examined the mRNA levels of AhR signalling marker Cyp1a1 in skin. Consistently, a significant elevated mRNA expression due to Ficz treatment was observed (figure 2D). These data suggested the involvement of AhR signalling on skin fibrosis improvement and Tfh cell inhibition mediated by Ficz. Nevertheless, further studies in vitro are warranted to confirm the effects and mechanism of AhR signalling in Ficz regulating Tfh cells.

Discussion

Tfh cells, a specialised subset of CD4+ T cells, are implicated in the pathogenesis of autoimmune diseases such as SSc, SLE, rheumatoid arthritis and Sjögren’s syndrome.^{15 16} Circulating Tfh cells were increased in SSc and correlate with SSc severity.¹⁷ Further analysis revealed elevated levels of Tfh cells were associated with lung involvement in SSc and there was a significant correlation between Tfh cell levels and the severity of lung disease.¹⁷ According to Morita et al, three subsets of cTfh were described regarding CXCR3 and CCR6 expression, cTfh1 (CXCR3+CCR6−), cTfh2 (CXCR3−CCR6−) and cTfh17 (CXCR3−CCR6+).¹⁸ Research focusing on Tfh cell subsets involved in SSc demonstrated both cTfh1 and cTfh17 cells appeared increased frequency in patients with SSc. Importantly, the frequency of cTfh 1 was correlated with cytokine concentrations of IL-21 and IL-6 that induced B cell differentiation in SSc. While cTfh17 secrete proinflammatory and profibrotic cytokines, cTfh 17 may be one of the potential cells that help to promote inflammatory response in SSc.¹⁹ There was also a correlation between Tfh cells and skin fibrosis in SSc, as patients with diffuse cutaneous SSc exhibited a higher proportion of circulating Tfh cells compared with patients with limited cutaneous SSc.¹⁷ Importantly, a research published in 2021 showed increased Tfh-like cell (CD4+ICOS+PD-1+) infiltration in SSc lesional skin by immunohistochemistry and multiplex immunofluorescence.¹⁹ In vitro, co-culture of Tfh cells with normal human dermal fibroblasts promoted myofibroblast differentiation, which demonstrated the role of Tfh cells in SSc fibrogenesis.¹⁴ IL-21, a critical cytokine among Tfh cell-mediated skin fibrosis in SSc, showed elevated expression in graft-versus-host disease (GVHD)-SSc mice skin.¹⁴ ICOS+Tfh like cells were found to be increased in the skin of GVHD-SSc mice and contributed to dermal fibrosis via an IL-21 and matrix metalloproteinase 12 dependent mechanism.¹⁴ Here, we established a BLM-induced SSc murine model, successfully mimicking higher percentages of cTfh cells observed in patients with SSc. The BLM-induced mouse also demonstrated CXCR5+PD-1+ Tfh like cells and elevated Il21 mRNA expression in the skin. It provides an important experimental means to investigate the molecular mechanisms of Tfh-mediated pathogenesis in SSc and their potential regulation approach like AhR signalling.

AhR is a highly evolutionarily conserved ligand-activated transcription factor known as dioxin receptor to mediate environmental toxicity.²⁰ AhR activation and subsequent gene transcription participate in the pathogenesis of SSc.⁸ Shi et al had demonstrated the AhR expression is downregulated in SSc skin lesions and primary dermal fibroblasts.⁹ Ficz, an exogenous agonist of AhR signalling, can ameliorate collagen production in SSc fibroblasts via AhR/TGF-β signalling activation.⁹ Esomeprazole treatment can alleviate fibrosis in SSc via AhR/Smad2/3 signalling activation.¹⁰ In the current study, we treated the BLM-induced SSc mouse with Ficz, which effectively alleviated skin fibrosis with elevated expression of Cyp1a1, a typical AhR signalling molecule. However, the precise mechanisms by which AhR activation ameliorates SSc require further elucidation.

One of the important discoveries of our study was that activating AhR could modulate Tfh cell population and alleviate skin fibrosis in BLM-induced SSc mice. Indeed, the role of AhR signalling in immunoregulation had received more and more attention.^{6 21} Takei et al investigated the effects of AhR signals on the process of lung fibrosis and changes in immunological features using a BLM-induced lung fibrosis mouse model. After FICZ treatment, the number of Tregs (CD4+Foxp3+) cells was significantly increased and T cell subsets involved in inflammatory responses such as CD4+IFNγ+ and γδ+IL-17A+T cells were decreased in the lungs. Those results suggested that AhR signals attenuate lung fibrosis in the BLM-induced mouse model for pulmonary fibrosis through increase of regulatory T cells.²² There were some researches focusing on the regulatory effect of AhR for Tfh. In vitro model of Tfh cell differentiation, adding the AhR antagonist CH-223191 to the T cell culture further upregulated expression of a key Tfh marker, CXCR5, while AhR agonists downregulating CXCR5 expression.²³ Likely, AhR signalling participating in Tfh cell regulation was reported in recent researches on influenza virus infection and lupus.^{11 13} AhR agonist tapinarof ameliorates lupus autoimmunity by suppressing Tfh cell differentiation via regulation of the JAK2‐STAT3 signalling pathway.¹³ Consistently, Law et al revealed AhR coordinated with AP-1 family member JUN to prevent CXCL13+TPH/TFH cell differentiation in SLE.¹² Here, we also identified AhR as a negative regulator of Tfh cells in BLM-induced SSc mice, as the Ficz-treated mice exhibited a decreased proportion of peripheral Tfh cells (CD4+CXCR5+PD-1+) and expression of a key Tfh cytokine, IL-21 in skin. Interestingly, some researches had revealed the paradoxical dual role of AhR in Tfh cell differentiation. For instance, PM2.5 exposure promotes Tfh2 subset polarisation via the AhR/c-MAF axis, exacerbating allergic sensitisation and pulmonary inflammation.²⁴ The effect of AhR signalling on Tfh differentiation may exhibit ligand specificity.

There are some limitations in our study. Although we observed the regulation of Ficz on Tfh population, the precise mechanism should be further clarified in vitro and in vivo. Additionally, we cannot compare the Tfh cells in skin among three groups due to the difficulty of identification of Tfh cells in tissues. In the future, single-cell transcriptome may help to accurately measure the effect of AhR signalling on Tfh.

Conclusions

This study established a murine model partly mimicking Tfh cell dysregulation in patients with SSc and Ficz, an AhR agonist, can alleviate skin fibrosis in the BLM-induced SSc mice, which may relate to the reduction of Tfh cells and its downstream cytokine IL-21 expression via the activation of AhR pathway. These findings provide a novel therapeutic strategy targeting AhR and Tfh cells dysregulation for SSc treatment.

Data availability statement

Data are available upon reasonable request.