Interleukin‐13 contributes to the occurrence of oral submucosal fibrosis

Abstract Oral submucous fibrosis (OSF) is a chronic progressive fibrosis disease that affects in oral mucosal tissues. Interleukin (IL)‐13 has been implicated in the development of fibrosis in multiple organs. Indeed, it contributes to diseases such as pulmonary fibrosis, liver cirrhosis among others. Currently, its expression in OSF and the specific mechanisms are not well understood. The aim of this study was to investigate the role of IL‐13 in OSF and further explore whether IL‐13 regulates—polarization of M2‐macrophages in OSF. Initially, in the tissues of patients with OSF, we observed a high expression of M2‐macrophages and IL‐13 protein. Additionally, we found a correlation between the expression of IL‐13 and the stage of OSF. Arecoline inhibited the proliferation of fibroblasts (FBs) and promoted IL‐13 production in vitro. Furthermore, our observations revealed that M2‐macrophages increased upon co‐culturing M0‐macrophages with supernatants containing the IL‐13 cytokine. In conclusion, our study demonstrated that arecoline stimulates FBs leading to increased secretion of IL‐13, which in turn IL‐13 leads to polarization of M2‐macrophages and promotes the occurrence of OSF. This suggests that IL‐13 may be a potential therapeutic target of OSF.


| INTRODUC TI ON
Oral submucous fibrosis (OSF) is a chronic progressive disease that primarily affects the oral mucosal tissues and has a predisposition towards malignancy. This disease, which was first described by Paymaster to be potentially malignant, 1 has been classified as a precancerous condition by the World Health Organization (WHO). OSF prevalence was reported to be 1.00%-3.03% in Hunan Province, 2 0.15%-14.40% in Vietnam 3 and 0.09%-17.60% in Taiwan. 4  whether the microenvironment develops persistent inflammation (M1), or fibrosis (M2) after regeneration and/or injury. 6,7 Studies have demonstrated that M2-macrophages directly affect development of fibrosis by producing a large number of pro-fibrotic factors, such as transforming growth factor-β1 (TGF-β1), among others, thereby promoting proliferation of myofibroblasts, and overproducing the extracellular matrix. 8,9 Interleukins (ILs) are cytokines that play an important role in the activation, differentiation, proliferation, maturation and migration of immune cells. 10 Studies have shown that IL-13 contributes to the pathogenesis of fibroproliferative diseases, such as pulmonary fibrosis, liver cirrhosis, myocardial fibrosis, progressive kidney disease and pathological skin scarring, among others. [11][12][13][14] Furthermore, IL-13 was associated with elevated production of total collagens in normal human skin and keloid fibroblasts (FBs), whereas FBs in various scarring diseases produced more fibrin, suggesting that IL-13 is associated with fibrosis. [15][16][17] Interestingly, a recent report demonstrated that in fibrosis, inhibition of both IL-13 and TGFβ signalling completely attenuated the fibrotic mechanism compared to TGFβ inhibition alone. 18 The Areca nut ranks fourth among the most commonly used addictive substances globally. 19 Some scholars believe that several components in the areca nut, especially arecoline and its thick fibres, can cause OSF. 20 In addition, arecoline has been shown to have exert cytotoxicity and genotoxicity, which not only causes DNA damage and oxidative stress, but also induces apoptosis of epithelial and vascular endothelial cells. 21 The active ingredients in the areca nut, such as arecoline and flavonoids, enter into the submucosal tissue, while the autologous tissue changes into autoantigen, a phenomenon that causes an autoimmune reaction that leads to infiltration of inflammatory cells including activated T cells and macrophages. 22 Studies have also shown that macrophages play a key role in fibrosis in the heart, lung, and kidney tissues, which occurs through inflammatory responses and overactivated damage repair. [23][24][25] In this study, we explored the possible mechanism underlying IL-13's action as an 'inducing factor' during stimulation of polarization of M2-type macrophages, and its role in pathogenesis of fibrosis.
Further, we investigated the effect of arecoline, the main pathogenic factor of OSF, on the function of FBs.

| Patients recruitment and selection criteria
The research protocol used in the current study was approved by

| Bioinformatics analysis
GSE64216, a dataset with mRNA expression profiles for OSF alongside normal samples, was downloaded from the Gene Expression Omnibus (GEO) database. Analysis of differentially expressed genes in the dataset was conducted at the following threshold: adjusted p (adj. p) <0.05, and log2 fold change (FC) >1.

| Immunohistochemistry (IHC)
Paraffin-embedded blocks were used to prepare OSF specimens, and 4μm sections were cut from them. These sections were then subjected to IHC by incubating them with specific primary antibod-

| Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
OSF lesion tissues were collected from patients and subjected to RNA TRIzol Reagent according to the manufacturer's instructions. The RNA was quantified then converted to cDNA using the

| Isolation and culture of primary FBs cells
Experimental specimens were obtained from healthy mucosal tissues, resected during oral and maxillofacial surgery. The tissues were first washed with PBS supplemented with 20% penicillin and streptomycin, chopped into small pieces then centrifuged at 1000 rpm for 5 min.
The tissue was digested with a mixture of Collagenase I (6 mg/mL) and Dispase (8 mg/mL) for 15 min, the cells counted, and transferred to a Petri dish. Culture medium was changed after every 3 days. FBs were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma), supplemented with 10% fetal bovine serum and 1% antibiotics, namely penicillin and streptomycin (100 μg/mL for each; Gibco). Cells at the 3rd to 6th passage three were used in subsequent experiments.

| Immunofluorescence
Fourth-generation FBs were seeded into 6-well plates for immunocytochemical staining. Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-x-100 at room temperature for 20 min, then blocked with 3% BSA at room temperature for 30 min. Next, they were incubated overnight with

| Cell proliferation assay
Proliferative activity of the FBs was assessed using the Cell Counting

| ELISA
Levels of IL-13 were quantified in the supernatant from FBs treated with arecoline using the ELISA kit (Zci Bio), following the manufacturer's instructions. Absorbance was measured at 450 nm wavelength using a microplate reader.

| Extraction and maturation of macrophages
Cubital venous blood was collected from normal people in an EDTA anticoagulation tube, and peripheral blood mononuclear cells (PBMC) obtained via density gradient centrifugation and cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (containing 10% FBS). The next day, the supernatant was discarded and anchorage-dependent cells were collected. The resulting monocytes were incubated with M-CSF (PeproTech) for 7 days to obtain differentiated mature macrophages (M0-phenotype).

| Flow cytometry
The supernatant of FBs stimulated by arecoline was used as the conditioned medium for the experimental group whereas the supernatant of FBs without arecoline stimulation was utilized as the

| Statistical analysis
All experiments were conducted in triplicates. All statistical analyses were performed using SPSS software (version 25.0), and data were presented as means ± SD. Comparisons between two groups were achieved using the t-test, whereas those among multiple groups were performed using one-way anova or two-way anova, followed by

| M2 macrophages are abundant in OSF tissue
Bioinformatics analysis results revealed ARG1 was significantly overexpressed in OSF tissues. (Figure 1A). ARG1 is a specific marker of M2-macrophages, and thus we hypothesized that M2-macrophages are highly expressed in OSF tissues. Similarly, immunohistochemistry results corroborated the hypothesis, as evidenced by the higher abundance of M2-macrophages in OSF relative to normal tissues ( Figure 1B-G).

| IL-13 is up-regulated in OSF tissues
Next, we performed immunohistochemistry staining on normal (n = 10 cases) and OSF tissues at different stages (25 cases each at the early, metaphasic, and advanced stages), and found that IL-13 was significantly upregulated in OSF relative to normal tissue across the stages (Figure 2A,B). Analysis of IL-13 expression in epithelial, and vascular endothelial cells, as well as FBs revealed higher cell numbers and the highest IL-13 expression in early OSF. In advanced OSF, the fibrotic tissue increased while the number of cells decreased, and these phenomena that were accompanied by low IL-13 expression, although it was still higher than that in normal mucosal tissue. qRT-PCR results showed that IL-13 was upregulated in OSF, relative to normal tissues ( Figure 2C).

| Profiles of primary FBs
The isolated cells were either spindle-shaped or irregularly triangular, with an oval nucleus in the center, as well as cytoplasmic protrusions and radial growth during growth ( Figure 3A). The immunofluorescence staining results indicated the presence of a cytoplasmic anti-vimentin antibody, which was later confirmed to be FBs ( Figure 3B).

| Arecoline treatment promotes IL-13 secretion by FBs
To verify the cytotoxic effect of arecoline on FBs, we treated FBs with different arecoline concentrations over time, and compared with the control group. The IC50 of arecoline on FBs was 33.25 μg/ mL after 3 days of treatment. Therefore, we selected 0-30 μg/mL, 0-3 days as the arecoline concentration for use in subsequent experiments. Results indicated that IL-13 was upregulated 1-3 days with increase in arecoline concentration ( Figure 4B,C). We then se-

| Differentiation of monocytes into mature M0 and their polarization into M2-phenotypes
Fractions of granulocytes and mononuclear cells were first isolated from human peripheral blood, centrifuged and the supernatant discarded to obtain anchorage-dependent cells (monocytes). Next, we cultured the monocytes in M-CSF-containing medium for 7 days to obtain target cells (M0 macrophages). Mature M0-macrophages were then polarized into M2-macrophages by induction with conditioned medium ( Figure 5A,B). Flow cytometry results showed that our method was effective in extracting macrophages ( Figure 5C,D).
These results were validated via flow cytometric analysis of cell markers.

| Conditioned media promotes M2 phenotype polarization of macrophages
To investigate whether IL-13 secreted by FBs under arecoline treatment can induce changes in polarization of macrophages, we first stimulated FBs with arecoline for 3 days, centrifuged the cells to obtain a supernatant for use as a conditioned medium. Next, we co-cultured this medium with M0-macrophages, with supernatant without arecoline used as a control group. Results revealed a slightly higher proportion of CD163 cells in the experimental (52.00%) than in the control group (46.77%). Similarly, more CD206 cells were recorded in the experimental (52.38%) than control group (46.94%). A similar trend was also observed with regard to CD209 cells, 51.92% and 46.78% in the experimental and control groups, respectively ( Figure 6A). These differences were statistically significant ( Figure 6B). Collectively, these results showed that arecoline promoted IL-13 secretion by FBs which could subsequently cause macrophages to undergo M2-phenotype polarization.

| DISCUS ION
The Results of the present study revealed that M2-macrophages and IL-13 were overexpressed in human OSF specimens relative to normal tissues. Notably, arecoline causes FBs to produce IL-13, which subsequently induces polarization of M0-macrophages into M2-macrophages, thereby affecting occurrence and development of OSF. Macrophages are immune cells with multiple functions.
For example, they are not only important targets for studying cellular phagocytosis, cellular immunity and molecular immunology, but are also involved in tissue inflammation and post-repair. 26 Notably, macrophages can be divided into pro-inflammatory M1 macrophages. Based on our results, we speculate that the high expression of M2-macrophages in OSF is due to upregulation of IL-13 cytokines. Previous studies have reported that IL-13 is highly expressed in lungs of patients with progressive fibrosing interstitial lung diseases. 13,32 Numerous studies have demonstrated IL-13's involvement in fibrosis, suggesting that it may be a target for treatment of fibrotic diseases in the future. Functionally, the IL-13 signalling pathway can activate FBs to produce extracellular matrix (ECM) and other constituent factors, which are required for collagen fibrogenesis. 33 Results from the present study showed that IL-13 was upregulated in FBs following arecoline intervention, which is consistent with previous studies. IL-13 expression is higher in OSF than in normal tissues, but its expression level is higher in the early OSF than in the middle and advanced stage. The reasons for these findings may be as fol- At present, most scholars believe that arecoline is the main cause of OSF. Arecoline stimulates the oral mucosa to induce secretion of numerous cytokines, ECM and collagen, thereby resulting in excessive collagen synthesis and reduced degradation. This imbalance is caused by fibrous key conditions for the transformation. 34 Furthermore, FBs are involved in degradation and synthesis of collagen, thus they play a key role in fibrosis formation. Our results showed that arecoline at a concentration of 5 μg/mL was cytotoxic to FBs, as evidenced by time-and dose-dependent inhibitory effects. Increasing the concentration and duration of action resulted in continued upregulation of IL-13 production by FBs. To avoid the influence on the subsequent experiments, we selected 0-30 μg/mL as the experimental concentration, and found that IL-13 cytokine level was highest at 30 μg/mL.
Next, we investigated whether IL-13 secreted by FBs would induce changes to polarization of macrophages under the arecoline.
To this end, we stimulated FBs with arecoline, then co-cultured the supernatant (at this time considered conditioned medium), with M0-macrophages. Results revealed that CD163, CD206 and CD209 were all upregulated, which is consistent with previous studies and is in line with our conjecture at the time. writing -review and editing (lead). Zhangui Tang: Funding acquisition (lead); resources (equal); supervision (lead).

ACK N O WLE D G E M ENTS
This study was supported by grants from the following institutions: The National Natural Science Foundation of China: 82170972; Human Provincial Science and Technology Department: 2022SK2052; Major Science and Technology Programs in Hainan Province: ZDKJ2021039.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors report no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.