Interleukin-17A

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Lung tissues of mice were excised, fixed in paraformaldehyde (4%), embedded in paraffin, and cut into sections (4 µm).The sections were stained with hematoxylin and eosin following standard protocols.The mean linear intercept was used to measure the alveolar size in the mouse tissue sections to evaluate the degree of emphysema, as described in the literature. 18iefly, a 10x objective slice image was imported into Image-ProPlus 6.0, and a cross line of length L was placed in the center of each picture and the number N of intersections between the alveolar septa and it was automatically identified by the software.We defined L/N as MLI.

Immunohistochemical staining
Immunohistochemical staining was performed using the avidin-biotin-peroxidase complex method.Briefly, 4% paraformaldehyde-fixed and paraffin-embedded murine lung tissue samples were cut into sections (4 µm).The sections were first heated at 56°C for 30 min.
Each slide was allowed to react with the 3,3'-diaminobenzidine (DAB) Substrate Kit (SK-4100, Vector Laboratories) for 10-20 s, and then, it was counterstained with hematoxylin for 5 min.For immunohistochemical scoring, two pathologists randomly selected five areas in each slice under a microscope (Axio Lab.A1; Zeiss AG) with a 10X objective according to the distribution (0 for < 5%, 1 for 5% to 25%, 2 for 26% to 50%, 3 for 51% to 75%, and 4 for 76% to 100%) and pigmentation depth (0 for colorless, 1 for yellowish, 2 for brownishyellow, and 3 for tan) of target protein staining.The score product of the distribution and coloration was the final fraction of the field.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.RNA was reverse-transcribed into complementary (c)DNA using oligo(dT) primers and a reverse transcriptase (TAKARA, Japan).cDNA was used for subsequent RT-qPCR analysis with the Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA).Each reaction was performed on a Mastercycler PCR machine (Applied Biosystems, Foster City, CA, USA).Primer sequences are listed in Table 1.The relative expression of the gene of interest was normalized to the housekeeping gene β-actin.Data were analyzed using the comparative CT method.Specificities of the resulting PCR products were confirmed using melting curves.

Western blotting
Fibroblasts and mouse lung tissues were lysed using radioimmunoprecipitation assay cell lysis buffer in the presence of a protease inhibitor (Beyotime, Shanghai, China) and a phosphatase inhibitor cocktail (Sangon Biotech, Shanghai, China).The total protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China).

Statistical analysis
The mean linear intercept of murine lung tissue was measured using Image-Pro Plus 6.0 (Media Cybernetics, USA).Data were expressed as mean ± standard deviation.All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA).The results were analyzed using one-way ANOVA and Tukey's test or Bonferroni's test for posthoc test.A p-value < 0.05 was considered statistically significant.

IL-17A activated primary mouse lung fibroblasts and increased CXCL12 secretion
Primary lung fibroblasts were successfully isolated from murine lung tissues and stably passaged three times.To confirm the isolated cell type, immunofluorescence staining was performed and the cells were found to express vimentin, consistent with lung fibroblasts (Fig.

1A).
To examine the effect of IL-17A on primary mouse lung fibroblasts, cells were treated with either 50, 100, or 200 ng/mL of rIL-17A, and then the markers of fibroblast activation were examined.RT-qPCR showed that the messenger (m)RNA expression levels of vimentin, α-SMA, and collagen I were increased after continuous stimulation with rIL-17A for 24 h compared to those in the control group.The largest differences in expression were seen following stimulation with 100 ng/mL of rIL-17A (Fig. 1B-D).Additionally, western blotting was performed to examine the protein expression levels of vimentin and α-SMA.Consistent with the RT-qPCR results, the protein expression levels of vimentin and α-SMA were increased in response to rIL-17A stimulation, and the most pronounced increase in levels was observed at a concentration of 100 ng/mL (Fig. 1E-G).To determine whether mouse lung fibroblasts secreted CXCL12 in response to rIL-17A stimulation, cells and supernatants were collected after 24 h of rIL-17A exposure.Immunofluorescence staining using a CXCL12specific antibody revealed that CXCL12 expression was significantly increased with rIL-17A treatment, particularly at a concentration of 100 ng/mL (Fig. 1H).RT-qPCR and immunofluorescence staining demonstrated upregulation of CXCL12 expression at the mRNA level (Fig. 1I) within cells, and an ELISA confirmed CXCL12 secretion (Fig. 1J).The optimal concentration of rIL-17A was 100 ng/mL.These data suggested that treatment with rIL-17A could activate mouse lung fibroblasts and enhance the secretion of CXCL12.

Challenge with CS induced emphysema in mouse lung tissues and increased CXCL12 expression
After 24 weeks of CS challenge and treatment with an anti-IL-17A antibody or AMD3100, lung tissues were harvested from mice.Hematoxylin and eosin staining showed that compared to the control group, lung tissues in the CS-attack group had significant alveolar space enlargement, severe alveolar wall thinning, rupture of the alveolar septa, and fusion of alveolar spaces (Fig. 2A).The mean linear intercept revealed that the alveolar cavities were larger in the COPD model group than in the control group (Fig. 2B, p < 0.001).These impairments were attenuated by both the anti-IL-17A antibody and AMD3100 treatments (Fig. 2A, B).To assess CXCL12 expression in the four experimental groups, BALF was collected at the end of the 24-week treatment period and an ELISA was performed to detect CXCL12.The levels of CXCL12 were significantly higher in the COPD mouse model group compared to the control group (Fig. 2E, p < 0.01).Additionally, treatment with either the anti-IL-17A antibody (Fig. 2E, p < 0.05) or AMD3100 (Fig. 2E, p < 0.05) downregulated CXCL12 secretion in the BALF compared to that in the COPD group.Immunohistochemical results of CXCL12 revealed significantly higher levels of CXCL12 in lung tissues of the COPD mouse model group compared to the control group (Fig. 2C, D, p < 0.01).CXCL12 levels in the COPD + anti-IL-17A antibody and COPD + AMD3100 groups were slightly higher compared to those in the control group, and they were significantly lower compared to those in the COPD mouse model group (Fig. 2C, D; p < 0.05 for all).The CXCL12 content in murine lung tissues was further assessed using RT-qPCR.The RNA expression of CXCL12 was higher in the COPD group than in the control group (Fig. 2F, p < 0.01).The RNA expression levels of CXCL12 in the COPD + anti-IL-17A antibody and COPD + AMD3100 groups were slightly higher compared to those in the control group, but they were significantly lower compared to those in the COPD group (Fig. 2F, p < 0.05 for all).

Anti-IL-17A or AMD3100 ameliorated EMT in mouse lung tissues under CS challenge
To investigate the effects of long-term CS challenge and anti-IL-17A antibody and AMD3100 treatments on EMT, mouse lung tissues were collected at the end of the 24-week treatment period.The protein expression levels of EMT-related markers, including E-cadherin, vimentin, collagen I, and α-SMA, were examined in each group using immunohistochemistry.
In the COPD mouse model, E-cadherin expression was decreased, (Figs.3A and a) while the expression of vimentin (Figs.3B and b), collagen I (Figs.3C and c), and α-SMA (Figs.3D  and d) was increased.However, treatment with either the anti-IL-17A antibody or AMD3100 reversed the CS-induced changes in EMT-related marker expression (Fig. 3A-D, Fig. 3a-d).
CS decreased the mRNA expression of E-cadherin (Fig. 3E), and it increased the mRNA expression of vimentin (Fig. 3F), collagen I (Fig. 3G), and α-SMA (Fig. 3H).Fig. 3I-L show the EMT-related markers in each group, and the trends were consistent with the immunohistochemistry and RT-qPCR results.Taken together, treatment with either the anti-IL-17A antibody or AMD3100 was able to reverse EMT in the mouse model of CS-induced COPD.

IL-17A stimulation caused activation of human lung primary fibroblasts and increased CXCL12 secretion
Primary human fibroblasts were isolated from the human lung tissue and were confirmed by immunofluorescence staining (Fig. 4A).Following stimulation with human rIL-17A, markers of fibroblast activation were measured by RT-qPCR and western blotting.Levels of human fibroblast activation were increased in a concentration-dependent manner (Fig. 4B, C).RT-qPCR and immunofluorescence staining showed a trend of higher CXCL12 expression in fibroblasts following rIL-17A treatment (Fig. 4D-E).An ELISA was used to measure the level of CXCL12 in the cell supernatant.Compared to that in the control group, human rIL-17A increased the production of CXCL12 in a concentration-dependent manner (Fig. 4F).
Therefore, among the concentrations tested, IL-17A had the greatest effect on human fibroblast activation at a concentration of 200 ng/mL.

Activated human lung fibroblasts promoted EMT in human bronchial epithelial (HBE) cells through CXCL12
After determining that the effective concentration of IL-17A for stimulation was 200 ng/mL, cell supernatants were collected from untreated human lung fibroblasts and fibroblasts treated with 200 ng/mL of rIL-17A.These cell supernatants were added to HBE cells for 48 h.Total RNA and protein of HBE cells were extracted to detect the markers of EMT.Following culture of HBE cells with the rIL-17A-stimulated fibroblast supernatant, the expression of Ecadherin (Fig. 5A, D, E) was decreased, and the expression of vimentin (Fig. 5B, D, F) and α-SMA (Fig. 5C, D, G) was increased, suggestive of EMT.When an anti-CXCL12 antibody was added to the fibroblast supernatant to neutralize CXCL12, the EMT process initiated in the HBE cells was significantly reversed.

IL-17A-induced EMT in COPD mouse lung tissues and 16HBE cells was mediated by CXCL12 through extracellular signal-regulated kinase (ERK) signaling
To explore the ERK-mediated signaling pathways during EMT in response to IL-17A stimulation, ERK phosphorylation (p) levels were measured in the lung tissues of BALB/c mice.Western blotting revealed that the ratio of p-ERK/ERK was increased in CS-challenged mice, and ERK phosphorylation was decreased following treatment with AMD3100 (Fig. 6A).Moreover, changes in ERK signaling were examined in 16HBE cells.The 16HBE cells cultured with the IL-17A-stimulated human fibroblast supernatant showed an increase in the p-ERK proportion, which was reduced upon CXCL12 neutralization (Fig. 6B).

Discussion
Airway remodeling is a structural change characterized by thickening of airway walls, subepithelial collagen deposition, and excessive mucus secretion. 19Airway epithelial cells are the primary targets of inhaled toxic gases and particles, including CS. Chronic exposure to a repetitive environmental injury may lead to the persistent activation of pathways involved in airway epithelial repair, such as EMT, which can initiate airway remodeling. 20The interaction between constituent cells of lung tissues, particularly fibroblasts, and HBE cells plays a key role in EMT in patients with COPD.Culture supernatants derived from lung fibroblasts of patients with COPD promote EMT in healthy adult HBE cells.Consistently, it has been demonstrated that activated primary lung fibroblasts promote EMT in 16HBE cells. 15 is a common etiologic factor for COPD, and it is associated with a series of pathophysiological changes.IL-17A is elevated in patients with COPD as well as in smokers with normal lung function.In the current study, IL-17A expression was elevated in lung tissues of mice chronically exposed to CS, particularly at 24 weeks.In a bleomycin-induced murine model of fibrosis, IL-17A significantly increased the pulmonary fibroblasts, type I collagen, and transforming growth factor-beta in vitro, which were attenuated by an anti-IL-17A antibody. 21In asthma, IL-17A can cause lung fibroblasts to overexpress α-SMA, collagen 1, and other activation markers. 22Thus, IL-17A is directly associated with fibroblast activation during chronic airway inflammation.In this study, primary lung fibroblasts were cultured from fresh lung tissues and treated with IL-17A to mimic a COPD-related inflammatory response in vitro.Our results demonstrated that fibroblasts are indeed activated by IL-17A; however, the most appropriate IL-17A stimulation concentrations are based on the After binding to CXCR4, CXCL12 can initiate various signaling pathways, including mitogen-activated protein kinase (MAPK) and its downstream p38 and ERK1/2 protein kinases (including Akt, protein kinase, and Rac), phosphoinositide 3-kinase, Ras, and c-Jun N-terminal kinase. 27,28 -ERK is a downstream signaling protein that contributes to cascade amplification of the MAPK signaling pathway.Binding of CXCL12 to CXCR4 has been shown to stimulate the proliferation of various tumor cell lines and their migration and adhesion to extracellular matrix components by activating these downstream signal transduction pathways.For example, Lin et al. demonstrated that CXCL12, which acts through CXCR4 and activates the Rac/ERK signaling pathways, could induce the expression of connective tissue growth factor in human lung fibroblasts, and potentiate their transdifferentiation into myofibroblasts.29 Wang et al. demonstrated that the autocrine CXCL12/CXCR4 axis could mediate the metastatic properties of esophageal cancer stem cells and it was dependent on ERK1/2 signaling.30 Pulmonary fibrosis and metastatic progression of tumors are similar to airway remodeling and inseparable from EMT.The current study investigated ERK signaling downstream of airway remodeling in COPD.In lung tissues of the COPD mouse model, the ERK pathway was clearly activated and was partially inhibited following treatment with an anti-IL-17A antibody or AMD3100.In vitro experiments revealed a markedly activated ERK pathway in 16HBE cells cultured with the CXCL12-containing fibroblast supernatant, followed by its partial inhibition upon CXCL12 neutralization.AMD3100, which has been approved for use by the United States Food and Drug Administration, plays an important role in mobilizing hematopoietic stem/progenitor cells and treating autoimmune diseases and asthma.31 In recent years, studies on AMD3100 have been limited to malignant tumors, such as cholangiocarcinoma, pancreatic cancer, and ovarian cancer, and they have confirmed its anti-pulmonary fibrosis effect.32 The results of the current study support the known role of AMD3100.Treatment of mice with AMD3100 was able to decrease the production of CXCR4/CXCL12 and it attenuated CS-induced COPD, despite incomplete inhibition.Barwinska et al. proposed that the protective effects of AMD3100 on CS-induced chronic lung injury may be due to bone marrow mobilization that increases the availability of hematopoietic progenitor cells (HPCs) for lung cell maintenance or repair.This is particularly necessary since both the number and proliferative potential of bone marrow HPCs have been shown to drop with CS exposure.33 However, the blood or sputum levels of CXCR4-positive HPCs in humans or mice with COPD were not measured, while altered CXCL12 levels in HPCs derived from blood samples of subjects with COPD are an observational marker.
In this study, IL-17A promoted EMT progression in a mouse model of CS-induced COPD through CXCL12 derived from activated lung fibroblasts.EMT progression was associated with an interaction between epithelial cells and fibroblasts, mediated by ERK activation.

Figure 2 .
Figure 2. CS challenge induced emphysema in mouse lung tissues and increased CXCL12

Figure 3 .
Figure 3. Anti-IL-17A or AMD3100 ameliorated EMT in mouse lung tissues under CS

Figure 5 .
Figure 5. Activated human lung fibroblasts promote EMT in HBE cells through CXCL12.