Introduction
Interstitial lung disease (ILD) is a frequent and serious complication in patients with connective tissue diseases (CTDs) and is associated with significant morbidity and mortality [
1,
2]. In addition to genetic factors, environmental factors are also thought to be important for triggering the disease progression of CTDs [
3,
4]. For example, occupational exposure to silica, asbestos, organic solvents such as trichloroethylene, and aromatic solvents are risk factors for systemic sclerosis, a prototype CTD with systemic fibrosis, including ILD [
5,
6]. Further, cigarette smoking is strongly associated with the disease progression of rheumatoid arthritis with ILD [
7]. However, the mechanisms by which these environmental factors cause immune dysregulation and ILD have not been well elucidated. The aryl hydrocarbon receptor (AhR) is a unique molecule with an emerging role that appears to directly connect environmental factors with the immune system [
8,
9]. AhR belongs to the basic helix-loop-helix PAS (PER-ARNT-SIM) domain family of transcription factors and was originally identified as a receptor targeted by xenobiotic toxins. However, subsequent studies have revealed the evolutionary conservation of the AhR and various endogenous AhR ligands such as tryptophan derivatives and indoles, suggesting physiological functions other than a xenobiotic receptor [
8,
10]. Of note, recent reports have revealed that the AhR is widely expressed in the immune system and that activation of AhR by its ligands has modulatory effects on the immune system, including balancing the differentiation of regulatory T cells (Tregs) and IL-17-producing helper T cells (Th17 cells), which may be associated with autoimmunity [
8,
9,
11,
12]. Furthermore, administration of AhR ligands has ameliorated the disease process of various animal models, such as experimental autoimmune encephalomyelitis and experimental colitis [
11,
13‐
15]. Thus, we hypothesized that AhR signaling may also be involved in the process of lung fibrosis via its modulatory effects on the immune system.
Materials and methods
Administration of FICZ to a BLM-induced pulmonary fibrosis mouse model
Female C57BL/6JJcl (8–10 weeks) mice were purchased from Clea Japan, Inc. (Tokyo, Japan). All experiments were performed in accordance with the guidelines for animal care and use approved by Keio University School of Medicine. For BLM-induced pulmonary fibrosis, mice were intratracheally administered BLM (Nippon Kayaku, Tokyo, Japan) dissolved in a total 50 μL of phosphate-buffered saline (PBS) at a dose of 0.06 U on day 0 [
16]. Control mice were administered 50 μL of PBS in the same manner. Either 1 μg of 5,11-dihydroindolo[3,2-b]carbazole-6-carboxaldehyde (FICZ) (Abcam, Cambridge, MA) dissolved in 200 μL corn oil (Sigma-Aldrich, St. Louis, MO) or vehicle was i.p. injected on day 0, 1, and 2 from BLM administration.
Histologic analysis and Sircol assay
Mice were sacrificed at week 3 after BLM administration for the evaluation of lung fibrosis. The left lung was fixed by infiltration with 4% paraformaldehyde for 48 h and embedded in paraffin. The lung sections were prepared at 5-μm thickness from paraffin-embedded blocks and stained with hematoxylin and eosin (H&E). Lung fibrosis was histopathologically quantified using the Ashcroft score as described previously [
17] at five points of each slide and the averaged score was used for the further analyses.
The deposition of soluble collagen in the right lung was analyzed using the Sircol assay according to the manufacturer’s protocol (Biocolor Ltd., Carrickfergus, UK). Briefly, the right lung was harvested and homogenized in PBS and incubated with acid-pepsin extraction media at 4 °C for 24 h. Lung extracts were incubated with Sircol dye, which binds to soluble collagen, and then centrifuged to form pellets. Pellets were solubilized in sodium hydroxide and the amount of eluted dye was measured using a microplate reader at 540 nm. Collagen standards supplied with the kit were used as controls.
Cell isolation for flow cytometry
Mice were sacrificed at week 1 after BLM administration for the analysis of infiltrating cells in the spleen and lungs. The spleen was minced with scissors and passed through a 40-μm cell strainer. Erythrocytes were lysed with Ammonium-Chloride-Potassium Lysing Buffer and spleen cells were re-suspended in a magnetic cell sorting buffer (PBS, 0.5% bovine serum albumin and 2 mM EDTA) after washing twice with PBS. Whole lungs were excised after transcardial perfusion with PBS. Excised lungs were digested with collagenase D (1 mg/ mL; Sigma-Aldrich) and DNase (1 mg/mL; Worthington, Columbus, OH) at 37 °C for 40 min. After filtering through a cell strainer, the extract was washed with a magnetic cell sorting buffer. Erythrocytes were lysed in the same manner as the spleen and lung cells including immune cells infiltrated in the tissue were re-suspended in magnetic cell sorting buffer.
Flow cytometry
For intracellular cytokine staining, cells were stimulated for 5 h in RPMI 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA) with PMA (1 μg/mL), ionomycin (1 μg/ mL), and Golgiplug (BD Biosciences, Franklin Lakes, NJ). Surface staining was performed for 20 min after incubating with FcBlock (BD Biosciences) for 5 min at 4 °C, and intracellular staining was subsequently performed using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. A single-cell suspension was stained with anti-mouse CD45 (BV510, BD Biosciences), CD3ε (PE-cy5, Thermo Fisher Scientific), CD4 (eFluor® 450, Thermo Fisher Scientific), γδTCR (FITC, Thermo Fisher Scientific), CD8a (PE-Cy™, BD Biosciences), B220 (PE, Thermo Fisher Scientific), and NK-1.1 (APC, BD Biosciences) for surface staining. For intracellular staining, a single-cell suspension was stained with anti-mouse INF-γ (PE-Cyanine7, Thermo Fisher Scientific), IL-17A (PE, Thermo Fisher Scientific), IL-22 (APC, Thermo Fisher Scientific), and Foxp3 (APC, Thermo Fisher Scientific). Cells were analyzed using MACS Quant Analyzer with Flowlogic software (Miltenyi Biotec, Gladbach, Germany).
Statistical analysis
Data were analyzed using Prism software version 6.05 (GraphPad Software, San Diego, CA). Mann-Whitney and Kaplan-Meier survival analyses were used to compare the data. Data were considered statistically significant at p < 0.05.
Discussion
We demonstrated that stimulation of AhR signals by FICZ attenuated lung fibrosis and improved the survival rate of mice with pulmonary fibrosis induced by BLM. In addition, we found that FICZ increased the number of infiltrating CD4+Foxp3+Tregs in the lungs and reduced the number of inflammatory subsets, such as IFNγ+CD4+ T cells and IL-17A+γδ+ T cells, during the inflammatory phase induced by BLM. These results suggest that modulation of AhR signaling might be one of candidate pathways to regulate the process of pulmonary fibrosis or remodeling in the diseases with fibrotic phenotype such as systemic sclerosis or idiopathic pulmonary fibrosis (IPF).
There has been a discussion whether inflammation is involved in the process of fibrosis, especially in IPF, but CD4
+ helper T cells, especially the shift to Th2, are also thought to be important for the patients with idiopathic pulmonary fibrosis [
19]. While AhR can modulate a wide variety of immune system components such as T cells, dendritic cells, and innate lymphoid cells [
8,
9], it was reported that AhR is important for the modulatory effects on the immune system including balancing the differentiation of regulatory T cells (Tregs) and IL-17-producing helper T cells (Th17 cells). Our study suggests that the effects of an AhR ligand, FICZ, on AhR led to increase the number of Tregs and reduced that of CD4
+IFNγ
+ T cells and contributed to alleviating lung fibrosis. Tregs are well known to play an important role in suppressing inflammation and excessive immune responses [
20,
21]. Moreover, previous reports have revealed that Tregs also alleviate lung inflammation in mice with lung fibrosis induced by lipopolysaccharide and ovalbumin [
22‐
24]. Given that a considerable portion of the inflammation phase also presents within the first week after the administration of BLM and leads to fibrosis in this animal model [
18], facilitating the accumulation of Tregs enhanced by FICZ in the lungs may suppress this inflammatory response and alleviate fibrosis induced by BLM. Previous report revealed latent TGF-β1 treatment caused more infiltration of CD4
+ Treg at inflamed lung lesion induced by BLM both with immunofluorescence staining and FACS analysis, suggesting infiltration of CD4
+ Treg at inflamed lung lesion also occurred in our model [
25]. We would like to conduct the immunohistochemistry or immunofluorescence in the future experiment. Several reports have also demonstrated that IFNγ is an important inflammatory mediator in the BLM-induced fibrosis. IFNγ
+CD4
+ T cells in the lungs and IFNγ in bronchoalveolar lavage fluid are increased following intratracheal administration of BLM, culminating in lung fibrosis. Furthermore, knocking
IFNγ out not only alleviates inflammation but also attenuates lung fibrosis [
26,
27]. Our study demonstrated decreased CD4
+IFNγ
+ T cells by the treatment with FICZ, both in the lungs and spleen at 1 week after BLM administration, suggesting that FICZ administration may also contribute to the alleviation of fibrosis through decrease of CD4
+IFNγ
+ T cells (Additional file
7). On the other hand, the increase of Tregs may directly contribute to reduce the number of CD4
+IFNγ
+ T cells, since previous reports have revealed that Tregs also contribute to the regulation of CD4
+ T helper type 1 (Th1) cells, which produce IFN-γ [
28,
29].
According to the previous reports, Lehmann et al. revealed that 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE), one of AhR ligands, inhibits myofibroblast differentiation in vitro, suggesting that AhR signals directly alleviate fibrosis via inhibiting activation of fibroblast [
30]. Moreover, regarding anti-inflammatory role, Beamer et al. showed that 2,3,7,8-Tetrachlorodibenzodioxin (TCDD), which is also one of AhR ligands, attenuated the lung inflammation induced by silica via inhibiting IL-1β production in macrophages in vivo [
31]. Besides, Simonian et al. revealed that IL-22 production via AhR signaling protected lung fibrosis induced by
Bacillus subtilis [
32], whereas IL-22 was not induced by FICZ in CD4
+ and γδ
+ T cells in our model (Fig.
5). Taken together, we hypothesize that FICZ attenuated lung fibrosis induced by BLM via AhR and at least one of the mechanisms was expanding CD4
+ Tregs.
The role of Tregs in pulmonary fibrosis remains controversial. Kotsianidis et al. and Shimizu et al. showed that a decrease in the number of Tregs was associated with the progression of lung fibrosis in IPF [
33,
34]. In contrast, Reilkoff et al. showed that the number of Sema 7a
+ Tregs was increased in the peripheral blood of IPF patients and that Sema 7a
+ Tregs induced lung fibrosis in TGF-β1 transgenic mice [
35]. Lo Re et al. showed that Tregs promoted lung fibrosis in a silica-induced murine lung fibrosis model [
36]. Conflicting results have also been observed for Tregs in BLM-induced pulmonary fibrosis models. While some reports have shown that Tregs alleviated fibrosis [
37,
38], Birjandi et al. showed that Tregs exacerbated fibrosis [
39]. Birjandi et al.’s study elegantly demonstrated that the increase in the number of Tregs with administration of IL-2 complex or adoptive transfer of Tregs at BLM administration exacerbated lung fibrosis. The same study showed that this was caused by changes in Tregs from a protective to damaging phenotype during the inflammatory phase. Given that FICZ enhanced the accumulation of CD4
+Foxp3
+ Tregs in the lungs of BLM-treated mice during the inflammatory phase only and that BLM induced the accumulation of CD4
+Foxp3
+ Tregs in this lung fibrosis model (Fig.
6c), increasing the number of protective Tregs during the inflammatory phase with FICZ as shown in the present study may lead to weaken the inflammation and result in the alleviation of pulmonary fibrosis. Furthermore, because AhR signaling affects the plasticity of T cells [
11,
14], modulating AhR signaling with its ligands may be an effective strategy for increasing the protective Treg population. Later administration of FICZ after week 1 can also increase Tregs at week 3; however, considering inflammation is provoked within the first week and subsided thereafter in BLM-induced mouse model [
18], expanding Tregs at week 3 may not be beneficial.
Our study has several limitations. First, the BLM-induced mouse model does not completely recapitulate IPF and/or connective tissue disease-associated
interstitial lung diseases (CTD-ILD). Although it is still controversial whether inflammation plays a substantial role in the pathogenesis, especially in IPF, as reflected by a lack of efficacy of anti-inflammatory therapy on IPF prognosis, this model has been used as a model of IPF [
40] and inflammation is still thought to have a potential role and also a trigger of the process. Furthermore, anti-inflammatory therapies such as glucocorticoids, cyclophosphamide, and mycophenolate are effective against CTDs with ILD [
1,
2]. Thus, we believe that our finding will be a benefit for these fibrotic lung diseases but may be more suitable for CTD-ILD than IPF. Second, while we showed that FICZ increased the number of CD4
+Foxp3
+ Tregs and reduced inflammatory T cell subsets such as CD4
+IFNγ
+ T cells and γδ
+IL-17A
+ T cells, we could not perform the functional analysis of these cell subsets directly, especially induction of fibrosis, due to difficulties with their isolation of infiltrating cells in the lungs whose number was quite limited.
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