The effects of the miR-21/SMAD7/TGF-β pathway on Th17 cell differentiation in COPD

Chronic obstructive pulmonary disease (COPD) is a complex disease with multiple etiologies, while smoking is the most established one. The present study investigated the modulation of T-helper 17 (Th17) cell differentiation by the miR-21/Smad7/TGF-β pathway, and their roles in COPD. Lung tissues were obtained from lung cancer patients with or without COPD who underwent lobotomy and the levels of miR-21, TGF-β/Smad signaling molecules, RORγT, and other Th17-related cytokines were detected. Mouse COPD models were built by exposing both wild-type (WT) and miR-21−/− mice to cigarette smoke (CS) and cigarette smoke extract (CSE) intraperitoneal injection. Isolated primary CD4+ T cells were treated with either CS extract, miR-21 mimics or inhibitors, followed by measuring Th17 cells markers and the expression of TGF-β/Smad signaling molecules and RORγT. Increased levels of miR-21, Smad7, phosphorylated (p)-Smad2, p-Smad3, TGF-β, and Th17-related cytokines was detected in the lungs of COPD patients. Lung function in modeled WT mice, but not miR-21−/− ones, deteriorated and the number of inflammatory cells in the lung tissues increased compared to the control WT-mice. Moreover, primary CD4+ lymphocytes tend to differentiate into Th17 cells after the treatment with CSE or miR-21 mimics, and the expression of RORγT and the TGF-β/Smad signaling were all increased, however miR-21 inhibitors worked reversely. Our findings demonstrated that Th17 cells increased under COPD pathogenesis and was partially modulated by the miR-21/Smad7/TGF-β pathway.


Materials and methods
Collection of human lung tissues. Lung tissues were collected from non-smoker patients both without COPD and with COPD who were undergoing pulmonary lobectomy due to lung cancer at the Xiangtan Central Hospital (Hunan, P.R. China). Healthy lung tissue at least 2 cm away from the tumor was also collected. Lung tissues of COPD patients were also collected from patients who underwent pulmonary lobectomy due to intractable pneumothorax or pneumatocele. COPD diagnoses were made according to GOLD2017 2 . Patients who had other chronic pulmonary diseases, such as bronchiectasia, asthma, tuberculosis, interstitial lung disease, or pneumosilicosis, were excluded. All collected lung tissues were stored at − 80 ℃ until further analysis.
The present study was approved by the Institutional Human and Animal Care Ethics Committee at the Third Xiangya Hospital of Central South University and Xiangtan Central Hospital, in accordance with the principles of the Helsinki Declaration II. Written informed consent was obtained from all patients prior to their enrollment into the study. All methods were carried out in accordance with relevant guidelines and regulations and the study was carried out in compliance with the ARRIVE guidelines.
Real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA from human lung tissue was isolated using the UNIQ-10 column Trizol RNA isolation kit (SangonBiotech, Shanghai, China). The Prime-Script RT reagent Kit (Takara Bio Inc., Shiga, Japan) was utilized for reverse transcription. Relative mRNA levels were quantified using two-step quantitative real-time polymerase chain reaction (PCR; Applied Biosystems, Carlsbad, CA, USA) with the SYBR Green PCR Master Mix (BioRad, Laboratories Inc., Hercules, CA, USA). U6 and GAPDH were utilized as internal controls. Primer for miR-21 is Bulge-Loop (RiboBio, Guangzhou, China). All other primers were designed by Primer Premier 5.0 (Premier Biosoft international, Palo Alto, CA, USA), and see Table 1.
Mouse COPD model building. Eight-week-old C57BL/6 wild-type (WT) mice (Vital River Laboratory Animal Technology Co., Ltd, Beijing, China, n = 5) and the miR-21 −/− mice (University of Texas, Southwestern Medical Center, TX, USA, n = 5) were enrolled in the present study. The WT mice were divided into a control group and a COPD model group, respectively. The COPD group was built as previously described by our group 4 . Briefly, mice were exposed to CS in a sealed box with a ventilation hole for two cycles per day and 28 days in total, except for days 1, 12, and 23, when mice were given the intraperitoneal injection of 100% cigarette smoke extract (CSE) solution (0.3 ml/20 g). The control group mice were maintained in fresh air with no special treatment and on days 1, 12, and 23 they were given an intraperitoneal injection of PBS (0.3 ml/20 g). IHC staining. Paraffin embedded lung tissue sections underwent IHC to detect RORγT (Abcam, UK, dilution 1:100). Primary antibodies were incubated overnight at 4 °C. Secondary antibodies are horseradish peroxidase-conjugated, being incubated for 15 min at 37 °C. The target proteins were visualized using diaminobenzidine (DAB, Sigma-Aldrich Co., USA). Five lung tissue sections from each mouse that underwent a complete lung function test were stained and five 400× fields from each section were randomly chosen for further analyses.

Magnetic cell sorting (MACS) of CD4
Statistical analysis. All data were analyzed using the SPSS 22.0 statistical software (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 5 software (San Diego, CA, USA). Data are presented as the mean ± standard error of the mean (SEM). Comparisons between two groups of data were performed using the unpaired two-tailed Student's t test and ANOVA plus multiple comparison. All data were verified normal distribution. A P-value of less than 0.05 was considered statistically significant.

Results
Upregulation of miR-21 and Th17 cells level in COPD patients. 12 patients (6 non-smoker and 6 COPD patients) were enrolled. There were no significant differences in the distribution of age, gender, or BMI between the two groups. The smoking index and lung function parameters (FEV1 and FEV1/FVC%) in the COPD patients were significantly different compared to the non-smoking controls (P < 0.05, Table 2). We found that the relative miR-21 levels in the lung tissues of the COPD patients were significantly increased compared to the levels in the non-smokers by using RT-qPCR (Fig. 1A).The levels of Th17-related cytokines were quantified in the lung tissue homogenates of COPD patients and non-smokers using ELISA. IL-1β, IL-6, IL-17A, and RORγT protein expression, but IL-10 was significantly decreased and there were no changes in IL-21 www.nature.com/scientificreports/ and IL-22 between the COPD patients and non-smoking controls (4 out of 6 enrolled samples were randomly selected for western blot tests from control and COPD group respectively, Fig. 1B-G). ROR-γT is Th17 related key transcription factor, Compared with non-smokers, the COPD patients had higher expression of ROR-γT in their lung tissues, which we confirmed using both RT-qPCR (Fig. 1H) and western blot analyses (Fig. 1I). These results suggest that there had high expression of both miR-21 and activation of Th17 cells in COPD patients.
MiR-21 knockout alleviate COPD-related emphysema, lung functions and pulmonary inflammation changes. We utilized H&E staining and IHC to evaluate the pathological changes of lung and the number of several inflammatory cells in lungs of mice from different groups. We observed that modeled-WT mice exhibited lowest MAST and highest MLI and DI% compared to the control group ( Fig. 2A,B); and these changes in modeled miR-21 −/− mice were moderate, but still worse than control group (Fig. 2C). However, miR-21 −/− mice exposed to air showed no notable abnormal changes in lung tissue (Fig. 2D, Table 3). www.nature.com/scientificreports/ Moreover, modeled-WT mice showed the severest pathological changes as their RAW increased; Cdyn, PEF, and Ti/Te all decreased compared with the control. Modeled miR-21 −/− mice were also moderate, but still worse than the control, and meanwhile miR-21 −/− mice exposed to air showed no notable abnormal changes in lung function tests (Table 4).
Additionally, we analyzed the degree of inflammation in each group by using a histopathologic inflammatory scoring system as previously described 15 . As expected, the control mice scored the lowest, the modeled-WT mice scored the highest, and the modeled miR-21 −/− mice in the middle (Fig. 2E). The WT-COPD group exhibited enlarged alveolar space, thinner alveolar septum, and destroyed alveolar wall when compared with the control. Meanwhile, the miR-21 −/− COPD group had moderate pathological changes compared with the WT COPD group, but still worse than the control and the miR-21 −/− group have no notable changes when exposed to air. (E) The inflammation score of mice lung from different groups. The WT-COPD group had the highest inflammation score, followed by miR-21 −/− COPD group, and the miR-21 −/− control group was similar to WT control group. Magnification are all 400X, scale bar = 50 nm, The data are expressed as the mean ± SEM (n = 5 in each group). *: compared with the control group, P < 0.05; # : compared with the WT COPD group, t-test and ANOVA test were applied in this part, P < 0.05. Table 3. Morphological changes in the mouse lung following CS-exposure. Morphometric measurements of MAST (μm), MLI (μm) and DI (%) were performed in each group. *P < 0.05 versus WT control group; # P < 0.05 versusWT CSE + CS group (n = 5).

MiR-21 activate the differentiation of Th17 cells.
We collected MACS-sorted CD4 + lymphocytes cells from the spleens of mice. Moreover, CSE, miR-21 mimics and inhibitors were added to different groups respectively. IL-17A was detected using flow cytometry to confirm the proportion of Th17 cells. Compared with the control group, CSE and miR-21 mimics further increased the proportion of Th17 cells while the miR-21 inhibitors worked reversely (Fig. 3A-E). RORγT is a crucial transcription factor of Th17 cells. We further measured the effects of miR-21 on RORγT by IHC in all mice from different groups. The highest level of RORγT was observed in the lung tissues of modeled-WT mice (Fig. 4B), followed by modeled miR-21 −/− mice (Fig. 4C), and the control the last (Fig. 4A). The western blot results (Fig. 4D,E) of RORγT level was consistent with the IHC. Additionally, RORγT expression in MACS-sorted splenic CD4 + cells from WT mice could also be increased by either CSE or miR-21 mimics, while the miR-21 inhibitors reduced it (Fig. 4F,G).   www.nature.com/scientificreports/ MiR-21 partly activate Th17 cell differentiation partly through TGF-beta/Smad signaling pathway. To investigate the potential mechanism of how miR-21 affect the functions of Th17 cell, we observed a significant increase of Smad7 and TGF-β mRNA levels in the lung tissues of the COPD patients (Fig. 5A,B).
Western blot analysis showed that protein expression of Smad7, p-Smad2, p-Smad3, and TGF-β were increased in lung tissues of COPD patients compared to non-smokers (Fig. 5C,D). Furthermore, in Vitro, we found that the protein level of TGF-β, but not SMAD7, increased significantly in MACS-sorted CD4 + lymphocytes after the treatment of CSE, and moreover, TGF-β and Smad7 protein expressions were both significantly increased in Th17 cells following the treatment of CSE (Fig. 5E-G). Finally, we evaluated the roles of CSE and miR-21 on TGF-β/Smad signaling pathway by CSE, miR-21 mimics, or miR-21 inhibitors. CSE and miR-21 mimics increased the protein expression of p-Smad2, p-Smad3, Smad7, and TGF-β in Th17 cells, while the miR-21 inhibitors worked the other way around (Fig. 5H-L).

Discussion
In the present study, we demonstrated that levels of Th17-related cytokines (IL-1β, IL-6, IL-17A, and TGF-α) and RORγT were increased in the lung tissues of COPD patients. We further found an increased Th17 cells proportion in the COPD mouse model. These results support previous findings from Wang and colleagues who demonstrated that the number of Th17 cells was increased in the peripheral blood of moderate-to-severe COPD patients, along with the levels of ROR-γT (an important transcription factor of Th17 cells), and many other inflammatory mediators, including IL-17A, IL-6, IL-21, IL-22, and IL-23. The levels of these inflammatory mediators have been shown to be negatively correlated with lung function 16 . Additionally, Imani and colleagues found that the levels of ROR-γT, IL-17A, and TGF-β1 were all increased in lung biopsies of COPD patients 17 .
These studies were consistent with our present results, demonstrating a crucial role of Th17 cells and Th17-related cytokines in COPD pathogenesis. Some studies suggested that miRNAs also play indispensable roles in the occurrences of many pulmonary diseases, such as lung cancer, COPD, asthma, and IPF 18 . In addition, Smigielska-Czepiel and colleagues found that miR-21 could interfere with the growth of activated memory T lymphocytes leading to apoptosis, indicating that miR-21 affects the activity of memory T lymphocytes 19 . Ando and colleagues confirmed that transfection of miR-21 into T lymphocytes could increase expressions of both TNF-α and IFN-γ 20 . By knocking down miR-21 in murine COPD models, we found that the levels of COPD-related lung pathological changes ameliorated, and this knockdown process would not affect the lung tissue structure when exposed to air. Further, we confirmed, using miR-21 mimics and inhibitors, that miR-21 could regulate Th17 cell differentiation and expression of the Th17 related transcription factor, ROR-γT. These results suggest that miR-21 plays an important role in COPD pathogenesis by regulating Th17 cell differentiation and ROR-γT expression.
TGF-β plays a crucial role in Th17 cell differentiation 21 . Specifically, overexpression of TGF-β1 has been shown to promote Th17 cell differentiation 13 . It has been hypothesized that miRNAs are co-regulators of this differentiation process. A previous study using a luciferase assay showed that miR-21 regulates the Smad/TGF-β pathway by suppressing Smad7 22 , and this discrepancy might be resulted from some other co-regulatory mechanisms by other pathways. Our results are similar to those in a study of Syed and colleagues, which showed a correlation between increases in the levels of Smad proteins and increased miR-21 expression in children with inflammatory bowel diseases 23 . Understanding exactly how different miRNAs co-regulate the Smad/TGF-β pathway, affecting Th17 cell differentiation and function, requires further investigations.
Smad proteins are important signal transduction molecules in the TGF-β superfamily 24 . Abnormal expression of molecules in the TGF-β/SMAD signaling pathway has been previously demonstrated in lung tissues from COPD patients 25 , and the changes in TGF-β precedes visible changes in lung pathology and function. The present study confirmed the abnormal expression of several key molecules in this pathway, under COPD pathogenesis. Moreover, we found the TGF-β/Smad pathway and the level of Th17 cells were both elevated under the stimulation of CSE, and these suggested that TGF-β/Smad pathway could at least contribute to the level of Th17 cells to some extents. And miR-21, which activates TGF-β/Smad pathway, could indirectly contribute to the Th17 cells differentiation. A further study of how miR-21 directly promote Th17 cells differentiation is necessary.
With the present study, there are some limitations merit consideration. Initially, we did not collect lung tissue from heavy smokers without COPD, for the reason that we just planned to make comparison between health The western blot of p-Smad2 and p-Smad3 in Th17 cells after the treatment of CSE, miR-21 mimics or miR-21 inhibitors. (L) p-Smad2 and p-Smad3 are both increased after the treatment of either CSE or miR-21 mimics but decreased by miR-21 inhibitors. In western blot, different samples of the same marker are from the same blot. The data are expressed as the mean ± SEM (n = 6 in each human lung sample group and n = 5 in each mice sample), t-test was applied in this part, *P < 0.05. www.nature.com/scientificreports/ people between confirmed COPD patient. However, with the data of heavy smokers could make this study more convincing as smoking is the leading cause of COPD. Further, we collected 6 human samples in each group but only showed 4 of them as representatives western blot results. Moreover, a further TGF-β/Smad pathway inhibitor experiment, comparing with the effects of miR-21 mimics/inhibitors, could contribute to the persuasion of the whole study.

Scientific
To summarize, the present study, for the first time, demonstrated that CSE could give rise to miR-21 expression and promote Th17 cell differentiation, partly through the Smad/TGF-β pathway. Furthermore, an increase in Th17 cells could release numerous inflammatory cytokines which could further contribute to the developments of COPD. Additionally, inhibition of miR-21, either by genetic knockdown or miR-21 inhibitors, could be of great therapeutic value to COPD (Supplementary information).