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Latent TGF-β1 protects against bleomycin-induced lung injury in mice Yong-Jiang Tang1, Jun Xiao1,2, Xiao Ru Huang1, Yang Zhang1, Chen Yang1, Xiao-Ming Meng1, Yu-Lin Feng2, Xiao-Jing Wang3, David S.C. Hui1, Cheuk-Man YU1, Hui Yao Lan1

1

Department of Medicine & Therapeutics, and Li Ka Shing Institute of Health Sciences, The Chinese

University of Hong Kong, Hong Kong, China. 2

Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu,

Sichuan, China, and 3Department of Pathology, University of Colorado Denver, Aurora, CO 80045, USA.

Correspondence and requests for reprints should be addressed to Professor Hui Y Lan, Department of Medicine &Therapeutics, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong; Tel: +852-37636061; Fax: +852-21457190; E-mail: [email protected]

Running title: Latent TGF-β1 protects against lung injury. Total word count for the manuscript without abstract: 3053. Author contributions: YJT conceived all experiments, data analysis, and drafted the manuscript. JX, XRH conceived animal model. YZ, CY performed flow cytometry.

XMM, YLF, DSCH, CMY conceived

experimental design, revising, and approval the article. XJW provided transgenic mice. HYL contributed to all aspects of the study including design, data interpretation, and writing up manuscript for publication. 1

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Sources of support: This work was supported by grants from the Research Grant Council of Hong Kong (GRF 419110, CUHK5/CRF/09, and CUHK3/CRF/12R) and Focused Investment Scheme A and B from the Chinese University of Hong Kong. Subject Category: 3.1 Animal Models of Pulmonary Fibrosis

AT A GLANCE COMMENTARY Scientific Knowledge of the Subject TGF-β1 is a potent mediator known to induce lung fibrosis. The present study explored the protective role of latent TGF-β1 against bleomycin-induced pulmonary inflammation and fibrosis in mice that overexpress human latent TGF-β1 in skin. What This Study Adds to the Field In contrast to active TGF-β1, latent TGF-β1 plays a protective role against both lung inflammation and fibrosis. Triggering the Smad7 negative feedback mechanism to block TGF-β/Smad3-mediated lung fibrosis and NF-κB-driven pulmonary inflammation and enhancing the Treg response to counter-regulate the Th17-mediated lung injury are potential mechanisms by which latent TGF-β1 protects against lung injury.

This article has an online data supplement, which is accessible from this issue’s table of content online at www.atsjournals.org

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Abstract Rationale: TGF-β1 is a potent mediator known to induce lung fibrosis. However, the role of latent TGF-β1 in lung inflammation and fibrosis is unclear. Objective: To investigate the role of circulating latent TGF-β1 in bleomycin-induced lung injury. Methods: Lung disease was induced in Keratin-5 promoter-driven TGF-β1wt transgenic mice by bleomycin. Role of latent TGF-β1 in pulmonary inflammation and fibrosis was examined at days 7 and 28 after administration of bleomycin. Results: Compared to littermate wild-type (WT) mice, TGF-β1wt transgenic mice had >2-fold higher levels of latent TGF-β1 in both plasma and lung tissue and were protected from bleomycin-induced pulmonary inflammation such as upregulation of IL-1β, TNF-α, and MCP-1, and infiltration of CD3+ T cells and F4/80+ macrophages. In addition, the severity of lung fibrosis with massive collagen matrix accumulation was markedly reduced in TGF-β1wt transgenic mice. These protective effects were associated with higher levels of Smad7, inactivation of both NF-κB and TGF-β/Smad3 signaling pathways, in addition to an increase in Foxp3-dependent regulatory T cells (Treg) but inhibition of Th17-mediated lung injury. Conclusions: Mice overexpressing latent TGF-β1 are protected from bleomycin-induced lung injury. Triggering the Smad7 negative feedback mechanism to inhibit both NF-κB and TGF-β/Smad signaling pathways and enhancing the Treg response to counter-regulate the Th17-mediated lung injury are potential mechanisms by which latent TGF-β1 protects against bleomycin-induced lung injury.

Key Words: latent TGF-β1, Th17, Treg, Smads, fibrosis, inflammation 3

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Introduction TGF-β1 has been shown to play a diverse role in pulmonary inflammation and fibrosis (1). In general, TGF-β1 plays a protective role in inflammation and autoimmune diseases. This is supported by the observation that targeted disruption of TGF-β1 gene in mice results in lethal inflammation in multiple organs including the lungs (2, 3). In contrast, deletion of TGF-β receptor II specifically from fibroblasts or epithelial cells protects against bleomycin-induced pulmonary fibrosis (4, 5). Recent studies have also shown that TGF-β1 plays a paradoxical role in immune homeostasis through the induction of immunosuppressive factor Foxp3 or by upregulation of Th17 response in a concentration-dependent manner (6). All these studies suggest the complexity of TGF-β1 in regulating the T cell immunity and the process of inflammation towards fibrosis under the influence by disease microenvironments.

TGF-β1 is secreted as a latent complex, consisting of active TGF-β1 and latency- associated peptide (LAP). LAP binds the NH2 terminal of active TGF-β1 to prevent the latter from binding to its receptors, whereas the latent TGF-β binding protein (LTBP) binds the LAP-TGF-β complex to prevent TGF-β from interacting with local matrix proteins (7). Latent TGF-β1 becomes active when an NH2-terminal LAP is cleaved from the mature TGF-β1 by pH, heat, proteases, thrombospondin-1 (TSP-1), reactive oxygen species, and integrin αvβ6 (8,9). It has been reported that latent TGF-β1 has a much longer plasma half-life than active TGF-β1 (>100 min versus 2-3 min) (10). Once released, active TGF-β1 binds its receptors to exert its biological and pathological activities via Smad-dependent and independent signaling pathways (11). In contrast, latent TGF-β1 may bind its own receptor, GARP, to exert its anti-inflammatory effects via the Foxp3-dependent mechanism (12). 4

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Moreover, LAP binds the N-terminal of active TGF-β1 to act as a potent inhibitor of the bioactive of TGF-β1 (13). CD4+ T cells that overexpress LAP have been shown to play an immunosuppressive role similar to Foxp3+ Trigs, thereby inhibiting airway allergic inflammation (14). Furthermore, LAP mediates Treg-induced inhibition of Th17 cells and regulates immune response independent of TGF-β1 (15, 16). Nevertheless, the roles and mechanisms of active versus latent TGF-β1 in inflammation and fibrosis under disease conditions remain largely unclear. In the present study, we sought to examine the regulatory role and mechanisms of latent TGF-β1 in bleomycin-induced lung injury in mice that overexpress human latent TGF-β1 by the skin keratinocytes under control of the keratin-5 promoter (17). In spite of the development of severe inflammatory skin phenotype mimicking human psoriasis, this phenotype of mice has several-fold higher circulating latent TGF-β1 levels without detectable abnormalities in other tissues/organs including lung and kidney (18) and, therefore, is suitable for the present study.

Materials and Methods A mouse model of bleomycin-induced lung injury K5.TGF-β1wt Tg mice were generated from ICR background mice and genotyped with primers as described previously (17). All Tg mice developed psoriasis-like skin lesions after the age of 2 months (17). Female mice (8-10 weeks) of both Tg and wild type (WT) mice were used in this study. Lung injury was induced by intratracheal instillation of 1.5U/kg bleomycin (Sigma, St. Louis, MO, USA). Control mice received the same volume of saline without bleomycin (n=6-8 mice per group). At day 7 or day 28 after bleomycin treatment, mice were killed by a lethal dose of ketamine and xylazine. Then, bronchoalveolar lavage (BAL) and lung tissues were collected for analysis by histology, 5

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immunohistochemistry, real-time PCR, Western blot, ELISA, and flow cytometry. In addition, groups of 6 normal mice (age-matched) were euthanized at day and day 28 as normal controls. The experimental procedures were approved by the Animal Experimental Ethics Committee at the Chinese University of Hong Kong.

Bronchoalveolar Lavage Bronchoalveolar lavage (BAL) was performed as previously described (19). Briefly, BAL was collected by slowly washing the lung through the tracheal tube with 800 µl sterilized saline three times. BAL samples (~2ml each) were centrifuged and the cell pellet was used to evaluate the number and cell phenotypes by Giemsa staining or flow cytometry. The total number of cells in the suspension was counted in a Neu-bauer chamber. Cells were cytospined and then stained with May-Grunewald-Giemsa. At least 500 cells per sample were differentiated into macrophages/monocytes, lymphocytes, and neutrophils and expressed as a percentage of total cells.

Histology and Immunohistochemistry Lung histology was evaluated in paraffin-embedded tissue sections (3 µm) stained with hematoxylin and eosin or Masson’s trichrome. The degree of lung fibrosis was determined by a numerical scale method (20). Immunohistochemical staining was performed in paraffin-embedded sections using a microwaved-based antigen retrieval protocol as previously described (21). The antibodies used in this study were as follows: rat anti-mouse monoclonal antibody to macrophages (F4/80) (Serotec, Ltd, Oxford, UK), rabbit polyclonal antibodies to CD3+ T cells (SP7) (Abcam, Cambridge, MA, USA), Goat anti-collagen I (Southern Biotech, Birmingham, AL,USA), and Rabbit anti- IL-17A 6

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(Santa Cruz Biotech Inc., Santa Cruz, CA, USA). Positive signals were quantitatively analyzed as described previously (18, 21).

Hydroxyproline assay Total amount of collagens in lung tissue was measured by hydroxyproline assay as described previously (22). Briefly, both normal and diseased lung tissues were homogenized in 0.5 ml of PBS. Then, 0.5 ml of 12 N HCl was added into the homogenate and hydrolyzed at 120°C for 24 hours and the samples were diluted with citrate/acetate buffer in 1:1 ration, followed by adding 100 µl of chloramine T solution and incubated for 30 minutes at room temperature. After addition of 100 µl of Ehrlich's reagent (2.5 g p-dimethylaminobenzaldehyde added to 9.3 ml of n-propanol and 3.9 ml of 70% perchloric acid), the samples were incubated at 65°C for 30 minutes. The absorbance of each sample was then measured at 550 nm. Standard curves for the experiment were generated using known concentrations (0-200 µg/ml) of reagent hydroxyproline (Sigma-Aldrich, St. Louis, MO, USA). Data were expressed as micrograms of hydroxyproline/mg dry weight of lung tissue.

Real-time PCR Total lung RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed using the Bio-Rad iQ SYBR Green supermix with Opticon2 (Bio-Rad, Hercules, CA, USA) as previously described (23). The primers used for detection mouse mRNA expression were described in Supplementary Table S1 or previously (23, 24). The ratio for the mRNA of interest was normalized to GAPDH mRNA expression.

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Western blot analysis Protein from lung tissue was extracted using radioimmunoprecipitation assay (RIPA) buffer as previously described (21). The antibodies used in this study included: primary antibodies against phospho-IκBα(ser32), phospho-NF-κBp65(ser276), and phospho-Smad3 (Cell Signaling Technology, Beverly, MA, USA), GARP (Abgent, San Diego, CA, USA), IκBα (C-21), TβRI, CTGF, NF-κB p65, Smurf2, Smad7, Foxp3, ROR-γt, and Smad2/3 (Santa Cruz Biotech Inc.), and collagen I (Southern Biotech), and the LI-COR IRDye 800-labeled secondary antibodies (RocklandImmunochemicals, Gilbertsville, PA, USA). After being immunostained, the positive signals were visualized with Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA) and quantitated with Image J software (National Institutes of Health, Bethesda, MD, USA). The ratio for the protein examined was normalized against the GAPDH (Chemicon, Temecula, CA, USA).

ELISA To prepare lung tissue homogenate for ELISA analysis, frozen-lung tissues were homogenized at 50 mg/ml in HBSS (Invitrogen). After being centrifuged, the supernatant from the lung homogenate was collected for ELISA analysis. TGF-β1 levels in plasma and renal tissues including the active form, LAP, and total TGF-β1 were analyzed quantitatively by the commercial ELISA kits (R&D System Inc., Minneapolis, MN), as previously described (24). Briefly, samples were acidified with 1 N HCl and neutralized with 1.2 N NaOH/0.5 M HEPES to assay for the amount of total (the sum of latent and active) TGF-β1. The concentration of active TGF-β1 protein was analyzed on samples that were not acidified, whereas the levels of latent TGF-β1 protein were measured using a specific anti-LAP antibody. In addition, levels of IL-6 in plasma and lung tissue were measured by an ELISA kit 8

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(Abcam, Cambridge, MA, USA), according to the manufacturer’s protocol.

Two-color immunofluorescence staining Treg cells infiltrating the lung were identified in frozen sections (3 µm) by two-color immunofluorescence with FITC-anti-mouse Foxp3 and Dy550-anti-mouse CD4 (eBiosciences, San Diego, CA, USA). Then sections were counterstained with DAPI and examined under the Zeiss Axioplan2 imaging microscope (Carl Zeiss, Oberkoche, Germany).

Flow cytometry Flow cytometry was performed with a FACS Calibar using CellQuest Pro Analysis software (BD Biosciences, Franklin Lakes, NJ, USA). Cell suspensions from bronchoalveolar lavage fluid were restimulated with PMA/Ionomycin, and then harvested, fixed and permeabilized with IC Fixation buffer and permeabilization buffer (eBiosciences). Cells in the lymphocyte gate were used for analysis. Treg cells were labeled with the APC-anti-mouse Foxp3 and FITC-anti-mouse CD4 (eBiosciences) and Th17 cells were identified with the FITC-anti-mouse CD4 and PE-anti-mouse IL-17A (eBiosciences). PE-anti-mouse GARP (eBiosciences) was used to evaluate the change of GARP expression in CD4+ T cells.

Statistical analysis Data were expressed as mean ± stand error of mean (SEM). Statistical analyses were performed using one-way ANOVA and Post tests were performed by Bonferroni from GraphPad Prism 5.0 (Prism 5.0 GraphPad Software, San Diego, CA, USA). 9

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Results Latent TGF-β β 1 transgenic mice have higher levels of latent TGF-β β1 in both circulation and lung tissues and are protected from bleomycin-induced lung histological injury. First, we determined levels of latent, active, and total TGF-β1 in plasma and lung tissues by ELISA. As shown in Figure 1, in comparisons to WT mice, Tg mice presented higher levels of total and latent TGF-β1 in plasma (Fig. 1A, C) and in lung tissues (Fig. 1D, F) in both normal and bleomycin-treated mice. Interestingly, although active TGF-β1 in the plasma was no different between WT and Tg mice in either normal or disease conditions (Fig. 1B), it was significantly increased in bleomycin-treated disease lung tissues in WT mice (Fig.1E). Histologically, both hematoxylin/eosin and Masson’s trichrome staining showed that although Tg mice had higher levels of total and latent TGF-β1 in both circulation and lung tissues, no histological abnormalities were detected (Data not shown). Similarly, both Tg and WT mice that had received saline treatment exhibited normal lung histology without detectable inflammation and fibrosis (Fig.2). In contrast, WT mice that had been treated with bleomycin for 7 days developed a severe lung histological injury such as a profound inflammatory cell infiltration and subsequent severe lung fibrosis including an increase in hydroxyproline contents at day 28 (Fig.2). All these changes were largely inhibited in Tg mice (Fig.2).

Latent TGF-β β 1 protects against bleomycin-induced lung inflammation and fibrosis possibly via Smad7-dependent inhibition of NF-κ κB-driven pulmonary inflammation and TGF-β β/Smad3-mediated lung fibrosis. 10

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As shown in Figure 3 (A-E), immunohistochemistry and real-time PCR detected a profound pulmonary inflammation in the diseased WT mice, such as a marked infiltration of CD3+ T cells and F4/80+ macrophages and upregulation of proinflammatory cytokines including IL-1β, TNF-α and MCP-1. However, all these inflammatory changes seen in the diseased WT mice were largely reduced in bleomycin-treated Tg mice. Similarly, bronchoalveolar lavage (BAL) analysis also showed that a significant increase in the number of total leukocytes, macrophages, lymphocytes, and polymorph neutrophils in WT mice was also largely reduced in Tg mice (Supplementary Fig.E1A-D). Interestingly, bleomycin-induced severe lung fibrosis in WT mice including a marked accumulation of interstitial collagen I and expression of connective tissue growth factor (CTGF) was also inhibited in Tg mice (Fig. 3F, G and Supplementary Fig. E2).

We then examined the activation of signaling pathways related to lung inflammation and fibrosis. As shown in Figure 4, bleomycin-induced pulmonary inflammation and fibrosis in WT mice were associated with low levels of Smad7 but higher levels of Smurf2. In addition, a marked activation of both NF-κB and TGF-β/Smad3 signaling pathways as demonstrated by higher levels of phosphorylated IκBα/p-65, phosphorylated Smad3, and upregulation of TGF-β receptor-I (TGFβRI) was also found in the diseased lung tissues of WT mice (Fig.4). In contrast, inhibition of lung inflammation and fibrosis in Tg mice was associated with higher levels of Smad7, lower levels of Smurf2, and inactivation of both NF-κB and TGF-β/Smad3 signaling (Fig.4). All these findings suggest that latent TGF-β1 may prevent Smurf2-mediated degradation of Smad7, thereby blocking NF-κB-driven pulmonary inflammation and TGF-β/Smad3-mediated lung fibrosis.

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Latent TGF-β β 1 protects against bleomycin-induced pulmonary inflammation by rebalancing the Treg/Th17 response. Increasing evidence shows that TGF-β1 is a key regulator of T cell immunity by rebalancing

the

Treg/Th17 response; therefore, we examined the Treg and Th17 responses in bleomycin-induced lung inflammation. First, we examined the Treg cell response by using two-color immunofluorescence and flow cytometry with anti-CD4 and Foxp3 antibodies. Interestingly, bleomycin treatment caused equal numbers of CD4+ T cells in both WT and Tg mice; however, Tg mice had much higher levels of Foxp3+ cells with a two-fold increase in CD4+Foxp3+ Treg cells when compared to the WT mice (Fig.5A, B and Fig. 6A). Further studies by real-time PCR and Western blot analysis also detected that both Foxp3 levels were significantly higher in the diseased lung tissues of Tg mice when compared with the WT mice (Fig.5C and D), which was associated with a two-fold increase in IL-10 mRNA expression (Fig.5E).

As latent TGF-β1 binds its own receptor, GARP, to upregulate Foxp3-dependent Treg response (12), we therefore examined whether an enhanced Treg response in Tg mice is associated with the upregulation of GARP on CD4+ T cells. As shown in Figure 6 (A and B), in comparisons to the WT mice, CD4+ T cells from the diseased lung of Tg mice highly co-expressed GARP, resulting in a three-fold increase in CD4+GARP+ cells. This was also confirmed by Western blot analysis with a higher level of GARP in the diseased lung tissues of Tg mice when compared to the WT mice (Fig.6C).

Then, we examined the Th17 cell response. Real-time PCR, Western blot, and ELISA analysis 12

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revealed that treatment with bleomycin markedly upregulated IL-6, IL-17A, and a Th17 transcriptional factor RORγt in WT mice, which was blocked in Tg mice (Fig.7 A-C, F). By using two-color flow cytometry, we also found that there was an 8-fold increase in CD4+IL-17A+ cells in the diseased lung tissues of WT mice. Again, this was virtually blocked in Tg mice (Fig.7D and E). Inhibition of Th17 cell response was further demonstrated by immunohistochemistry as shown in Supplementary Figure E3. The regulatory role of latent TGF-β1 in the Treg/Th17 response was further examined by comparing the ratio of Foxp3/ROR-γt and IL-10/IL-17A transcriptional activity. As shown in Supplementary Fig. E4 (A and B), Tg mice had a three-fold increase in the ratio of Foxp3/RORγt and IL-10/IL-17A mRNA when compared with the WT mice.

Discussion It is now well recognized that after being released, TGF-β1 becomes active and plays a diverse role in inflammation and fibrosis via Smad-dependent and independent mechanisms (1, 10). We reported here that K5.TGF-β1wt Tg mice overexpressing latent TGF-β1 in keratinocytes had higher levels of latent TGF-β1 in both plasma and lung tissues and therefore were protected from bleomycin-induced severe pulmonary inflammation and fibrosis, possibly via several potential mechanisms.

Firstly, latent TGF-β1 may protect against TGF-β/Smad3-mediated lung fibrosis by preventing TGF-β1 from activation. This may be associated with the known role of LAP which acts as an antagonist of active TGF-β1 by binding the N-terminal of active TGF-β1 to confer its latency, therefore preventing it from binding to the TGF-β receptors to exert its bioactivities (11, 25). It is 13

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also possible that the high levels of circulating latent TGF-β1 may act as a competitive inhibitor of endogenous matrix bound latent TGF-β1, thereby inhibiting integrin-mediated activation of TGF-β1 in the diseased lung tissues as previously reported (9, 26). These counter-regulating mechanisms between latent TGF-β1 and active TGF-β1 may well explain the notion that mice overexpressing latent TGF-β1 have much higher levels of latent TGF-β1 in both circulation and diseased lung tissues without an increase in the active form of TGF-β1 and activation of TGF-β/Smad signaling. This finding was also consistent with previous studies in kidney diseases in which mice overexpressing latent TGF-β1 developed much less kidney injury with lower levels of active TGF-β1 and TGF-β/Smad signaling (18, 24). In addition, because of a much longer plasma half-life of latent TGF-β1 (10), systemic administration of recombinant LAP can reverse active TGF-β1-induced proliferative suppression in a remnant liver model using transgenic mice overexpressing hepatic levels of bioactive TGF-β1(13). Similarly, administration of LAP can also prevent skin fibrosis by inhibiting bioactivities of active TGF-β1 (27). Thus, prevention of TGF-β1 from being active may be one mechanism by which latent TGF-β1 protects against bleomycin-induced lung injury.

Secondly, latent TGF-β1 may inhibit lung fibrosis and inflammation by triggering its negative feedback mechanism of Smad7. This was supported by the findings that Tg mice with higher levels of latent TGF-β1 had higher levels of Smad7 but lower levels of TGFβRI and phosphorylated Smad3. This finding was consistent with a known role for Smad7 as an inhibitory Smad to negatively regulate TGF-β/Smad signaling (11, 28). Indeed, Smad7 acts as an adaptor protein to recruit E3 ubiquitin ligase such as Smurf2 to the TGF-β receptor complex as well as Smads and then target their degradation (29). This interaction also causes degradation of Smad7 through the 14

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proteasomal-ubiquitin degradation pathway (29). Consistent with this known mechanism, bleomycin-induced degradation of lung Smad7 in WT mice may be attributed to higher levels of Smurf2, whereas, lower levels of Smurf2 in the diseased lung tissues of Tg mice may be associated with

higher levels of Smad7. Since Smad7 is an integrated molecule with inhibitory effect on

TGF-β/Smad signaling while inducing IκBα (an inhibitor of NF-κB) to block NF-κB activation (23, 24, 28), thus, once Smad7 is degraded, both NF-κB and TGF-β/Smad signaling pathways become activated. This may account for bleomycin-induced NF-κB-driven lung inflammation and TGF-β/Smad-mediated fibrosis in WT mice but not in Tg mice and may also explain the imbalance of TGF-β/Smad signaling as seen in this and other studies (30).

Importantly, we have also found that restoring the balance of Treg/Th17 response by upregulating Foxp3/IL-10 while downregulating RORγt/IL-17 was a key mechanism through which latent TGF-β1 might protect lung from bleomycin-induced inflammation. It is well known that TGF-β1 is an essential regulator in the development of both Treg and Th17 cells with opposing actions (3, 6). However, the lineage specification of anti- or pro-inflammatory T cells in a TGF-β1-rich microenvironment, such as the lung, remains to be fully determined. In the presence of proinflammatory cytokines such as IL-6 as well as IL-1β, TGF-β1 is capable of inducing the transcriptional factor RORγt to promote the differentiation of CD4+ T cells to the IL-17-producing Th17 phenotype, which contributes to the pathogenesis of many lung

diseases (31). On the other

hand, TGF-β1 is also capable of upregulating Foxp3 transcriptional factor to promote Treg cell differentiation, therefore counterbalancing RORγt-mediated Th17 response (6). In the present study, bleomycin-induced pro-inflammatory cytokines such as IL-1β and IL-6 in the WT mice may 15

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promote TGF-β1-induced RORγt-mediated Th17 cell differentiation while switching off TGF-β1-induced Foxp3-dependent Treg cell pathway, resulting in the imbalance of Treg/Th17 response. In contrast, inhibition of RORγt-mediated Th17-dependent lung injury in Tg mice may be associated with higher levels of latent TGF-β1 to antagonize the proinflammatory properties of active TGF-β1 in IL-6 expression. As IL-6 is capable of inhibiting TGF-β-induced Foxp3 expression (32), therefore, inhibition of IL-6 expression in Tg mice may further upregulate Foxp3-dependent Treg cell differentiation in this study. It is known that Foxp3 interacts with RORγt in the exon2-encoded sequence to inhibit RORγt-directed IL-17 expression (6), thus, Foxp3-mediated inhibition of RORγt-dependent Th17 response may be an essential mechanism by which latent TGF-β1 protects against bleomycin-induced lung injury through rebalancing Treg/Th17 response.

An interesting finding in the present study is that higher levels of Foxp3 expression in Tg mice was associated with higher levels of latent TGF-β1 both systemically and locally without significant activation of TGF-β signaling. This argues against the necessary role for TGF-β/Smad3 signaling in the induction of Foxp3 transcriptional factor as previously reported (33). One possible explanation is that latent TGF-β1 may exhibit its immunosuppressive effects independent of active TGF-β1 by binding to its own cell surface receptor GARP or LRRC32 to induce Foxp3 expression (12, 34). GARP or LRRC32 is a transmembrane protein containing leucine-rich repeats and is recognized as a receptor of latent TGF-β1 on the Tregs’ cells (12,34). Knockdown of GARP on activated Tregs prevents surface latent TGF-β1 expression (12). In the present study, we have found that GARP was highly expressed in the diseased lung tissue, particularly on CD4+ Treg cells in Tg mice. This suggests that latent TGF-β1 may bind GARP on the CD4+ T cells to upregulate Foxp3-dependent 16

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Treg response, although mechanisms for this action remain to be elucidated.

In summary, latent TGF-β1 plays a protective role against bleomycin-induced lung inflammation and fibrosis. The inhibitory effect of latent TGF-β1 on lung inflammation and fibrosis may be associated with the counter-regulatory mechanism between latent and active TGF-β1, the negative regulatory role of Smad7 in activation of both NF-κB and TGF-β/Smad signaling pathways, and importantly, the GARP-Foxp3 regulatory mechanism in rebalancing the Treg/Th17 response.

Acknowledgements This work was supported by grants from the Research Grant Council of Hong Kong (GRF 419110, CUHK5/CRF/09, and CUHK3/CRF/12R) and Focused Investment Scheme A and B from the Chinese University of Hong Kong.

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9. Jenkins RG, Su X, Su G, Scotton CJ, Camerer E, Laurent

GJ, Davis GE, Chambers RC, Matthay

MA, Sheppard D. Ligation of protease-activated receptor 1 enhances α vβ6 integrin-dependent TGF-β activation and promotes acute lung injury J Clin Invest. 2006;116(6):1606–1614. 10. Wakefield LM, Winokur TS, Hollands RS, Christopherson K, Levinson AD, Sporn MB. Recombinant latent transforming growth factor β1 has a longer plasma half-life in rats than active transforming growth factor β1, and a different tissue distribution. J Clin Invest 1990;86:1976-1984. 11. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 2003;425:577-584. 12. Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A 2009;106:13445-13450. 13.Bottinger EP, Factor VM, Tsang ML, Weatherbee JA, Kopp JB, Qian SW, Wakefield LM, Roberts AB, Thorgeirsson SS, Sporn MB. The recombinant proregion of transforming growth factor β1 (latency-associated peptide) inhibits active transforming growth factor β1 in transgenic mice. Proc Natl Acad Sci U S A 1996;93:5877-5882. 14. Duan W, So T, Mehta AK, Choi H, Croft M. Inducible CD4+LAP+Foxp3- regulatory T cells suppress allergic inflammation. J Immunol 2011;187:6499-6507. 15. Ali NA, Gaughan AA, Orosz CG, Baran CP, McMaken S, Wang Y, Eubank TD, Hunter M, Lichtenberger FJ, Flavahan NA, Lawler J, Marsh CB. Latency associated peptide has in vitro and in vivo immune effects independent of TGF-β1. PLoS One 2008;3:e1914. 16. Ye ZJ, Zhou Q, Zhang JC, Li X, Wu C, Qin SM, Xin JB, Shi HZ. CD39+ regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a 19

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LAP-dependent mechanism. Respir Res 2011;12:77. 17. Li AG, Wang D, Feng XH, Wang XJ. Latent TGFβ1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder. Embo J 2004;23:1770-1781. 18. Huang XR, Chung AC, Zhou L, Wang XJ, Lan HY. Latent TGF-β1 protects against crescentic glomerulonephritis. J Am Soc Nephrol 2008;19:233-242. 19. Bottoms SE, Howell JE, Reinhardt AK, Evans IC, McAnulty RJ. TGF-β isoform specific regulation of airway inflammation and remodelling in a murine model of asthma. PLoS One 2010;5:e9674. 20. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988;41:467-470. 21. Xiao J, Meng XM, Huang XR, Chung AC, Feng YL, Hui DS, Yu CM, Sung JJ, Lan HY. mIR-29 inhibits bleomycin-induced pulmonary fibrosis in mice. Mol Ther 2012;20:1251-1260. 22. Sisson TH, Mendez M, Choi K, Subbotina N, Courey A, Cunningham A, Dave A, Engelhardt JF, Liu X, White ES, et al. Targeted injury of type II alveolar epithelial cells induces pulmonary fibrosis. Am J Respir Crit Care Med 2010;181:254-263. 23. Chung AC, Huang XR, Zhou L, Heuchel R, Lai KN, Lan HY. Disruption of the Smad7 gene promotes renal fibrosis and inflammation in unilateral ureteral obstruction (uuo) in mice. Nephrol Dial Transplant 2009;24:1443-1454. 24. Wang W, Huang XR, Li AG, Liu F, Li JH, Truong LD, Wang XJ, Lan HY. Signaling mechanism of TGF-β1 in prevention of renal inflammation: Role of Smad7. J Am Soc Nephrol 2005;16:1371-1383. 25. Sheppard D. Transforming growth factor β: A central modulator of pulmonary and airway 20

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inflammation and fibrosis. Proc Am Thorac Soc 2006;3:413-417. 26. TatlerAL,JenkinsG.TGF-activation and lung fibrosis. Proc Am Thorac Soc 2012; 9:130-136. 27. Zhang Y, McCormick LL, Gilliam AC. Latency-associated peptide prevents skin fibrosis in murine sclerodermatous graft-versus-host disease, a model for human scleroderma. J Invest Dermatol 2003;121:713-719. 28. Lan HY. Smad7 as a therapeutic agent for chronic kidney diseases. Front Biosci 2008;13:4984-4992. 29. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 2000;6:1365-75. 30. Venkatesan N, Pini L, Ludwig MS. Changes in smad expression and subcellular localization in bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2004;287:L1342-1347. 31. Wong CK, Cao J, Yin YB, Lam CW. Interleukin-17A activation on bronchial epithelium and basophils: A novel inflammatory mechanism. The European respiratory journal 2010;35:883-893. 32. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature 2006;441:235-238. 33. Takimoto T, Wakabayashi Y, Sekiya T, Inoue N, Morita R, Ichiyama K, Takahashi R, Asakawa M, Muto G, Mori T, Hasegawa E, Saika S, Hara T, Nomura M, Yoshimura A. Smad2 and Smad3 are redundantly essential for the TGF-β-mediated regulation of regulatory T plasticity and Th1 development. J Immunol 2010;185:842-855. 34. Probst-Kepper M, Geffers R, Kroger A, Viegas N, Erck C, Hecht HJ, Lunsdorf H, Roubin R, 21

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Moharregh-Khiabani D, Wagner K, Ocklenburg F, Jeron A, Garritsen H, Arstila TP, Kekäläinen E, Balling R, Hauser H, Buer J, Weiss S. Garp: A key receptor controlling Foxp3 in human regulatory T cells. J Cell Mol Med 2009;13:3343-3357.

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Figure legends Figure 1. Levels of LAP, active TGF-β β 1 and total TGF-β β1 in plasma and lung tissues of normal and bleomycin-treated mice detected by ELISA. (A, D) Levels of LAP in plasma and lung tissues; (B, E) levels of active TGF-β1 in plasma and lung tissues; and (C, F) levels of total TGF-β1 in plasma and lung tissues. Each bar represents the mean± SEM for groups of six to eight mice. *P< 0.05, **P<0.01,*** P﹤0.001 compared with the age-matched normal mice; #P< 0.05, ##P<0.01, ###P﹤0.001 compared with the WT mice.

Figure 2. Mice overexpressing latent TGF-β β1 are protected against bleomycin-induced lung injury. (A) Hematoxylin & eosin-stained lung tissue sections from K5.TGF-β1wt Tg and WT mice at day 7 after bleomycin or saline instillation. Scale bar =100 µm. (B) A high-power field (×40) from a selected area in (A). Scale bar = 50 µm. (C) Masson’s trichrome-stained lung tissue section at day 28 after saline or bleomycin instillation. Scale bar = 100 µm. (D) Fibrosis score from Masson’s Trichrome-stained sections. (E) Total lung collagens by hydroxyproline assay. Each bar represents the mean± SEM for groups of six to eight mice. *P﹤0.05, ***P﹤0.001 vs. saline-treated group; ###P﹤0.001 vs. bleomycin-treated WT group.

Figure 3. Bleomycin-induced lung inflammation and fibrosis are attenuated in mice overexpressing latent TGF-β β 1. At day 7 after bleomycin or saline instillation, (A) Immunohistochemical staining of CD3+ T cells and

(B) F4/80+ macrophages, scale bar =100 µm.

(C-E) Real-time PCR for il-1β, tnf-α, and mcp-1 expression. Each bar represents the mean± SEM for 23

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groups of six to eight mice. At day 28 after bleomycin or saline instillation, (F-G) Western blot and quantitation analysis of collagen I and CTGF protein expression. Each bar represents the mean± SEM for groups of six to eight mice. *P﹤0.05, **P﹤0.01, ***P﹤0.001 vs. saline-treated group; # P﹤0.05, ##P﹤0.01, ###P﹤0.001 vs. bleomycin-treated WT mice.

Figure 4. Western blot analysis shows that blockade of TGF-β/Smad3 and NF-κB signaling by triggering the Smad7 negative feedback loop may be a key mechanism by which mice overexpressing latent TGF-β β 1 inhibit bleomycin-induced lung inflammation and fibrosis. (A) Smad7 expression.(B) Smurf2 expression,and (C, D) Phosphorylation of IκBα and NF-κB-p65 at day 7 after bleomycin treatment. (E) TGF-β receptor I (TGFβRI) expression and (F) Phosphorylation of Smad3 at day 28 after bleomycin treatment. Each bar represents the mean± SEM for groups of six to eight mice. *P﹤0.05, **P﹤0.01, ***P﹤0.001 vs. saline group; # P﹤0.05, ##P﹤0.01, ###P﹤ 0.001 vs. bleomycin-treated WT mice.

Figure 5. Mice overexpressing latent TGF-β β1 show an enhanced Treg response after bleomycin treatment at day 7. (A) Treg cells infiltrating the diseased lung tissues at day 7 after bleomycin treatment are identified by two-color immunofluorescence with Foxp3+ (green) and CD4+ (red). The cell nuclei are labeled with

DAPI (blue) and examples of CD4+Foxp3+ cells are indicated by

arrows. Scale bar = 50 µm. (B) Quantitative analysis of CD4+, Foxp3+, and Treg cells. (C, D) Real-time PCR and Western blot analysis of Foxp3 mRNA and protein expression. (E) Real-time PCR analysis of il-10 mRNA expression. Each bar represents the mean± SEM for groups of six to eight mice. **P<0.01,*** P﹤0.001 compared with saline-treated mice; #P<0.05, ##P<0.01, 24

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compared with WT group.

Figure 6. Two-color flow cytometry shows that mice overexpressing latent TGF-β β 1 are promoting Treg differentiation by upregulating GARP on CD4+ T cells in bleomycin-induced pulmonary inflammation at day 7. (A) The percentage of Foxp3+CD4+ Treg cells in total CD4+ T cells. (B) The percentage of CD4+GARP+ T cells in total CD4+ T cells. (C) Western blot analysis of GARP in lung tissues. Each bar represents the mean± SEM for groups of six to eight mice. *P<0.05 compared with saline-treated mice; #P<0.05, ##P<0.01 compared with bleomycin-treated WT mice.

Figure 7. Mice overexpressing latent TGF-β β 1 are inhibited Th17 differentiation in bleomycin-induced pulmonary inflammation at day 7. (A) IL-6 mRNA levels by real-time PCR. (B) IL-6 protein levels from lung homogenates by ELISA. (C) ROR-γt (encoded by RORC) levels determined by Western blot and real-time PCR. (D, E) Two-color flow cytometry analysis of Th-17 cells from BAL cells. (F) Real-time PCR analysis of IL-17A mRNA expression. Each bar represents the mean± SEM for groups of six to eight mice. *P<0.05, **P<0.01,*** P﹤0.001 compared with saline-treated group; #P< 0.05, ##P<0.01, ###P﹤0.001 compared with bleomycin-treated WT mice.

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Online Supporting Information

Latent TGF-β1 Protects Against Bleomycin-Induced Lung Injury in Mice

Yongjiang Tang, Jun Xiao, Xiao Ru Huang, Yang Zhang, Chen Yang, XiaoMing Meng, Yulin Feng, Xiao-Jing Wang, David SC. Hui, Cheuk-Man Yu, Hui Yao Lan

Supplementary Table E1. Primers used for real-time PCR Gene

Forward primer

Reverse primer

IL-6

5’-AGGATACCACTCCCAACAGACCT-3’

5’-CAAGTGCATCATCGTTGTTCATAC-3’

IL-17A

5’-GATCAGGACGCGCAAACATG-3’

5’-AGTTTGCTGAGAAACGTGGG-3’

RORC

5’-CCGCTGAGAGGGCTTCAC-3’

5’-TGCAGGAGTAGGCCACATTACA-3’

Foxp3

5’-CCCAGGAAAGACAGCAACCTT-3’

5’-TTCTCACAACCAGGCCACTTG-3’

IL-10

5’-GGTTGCCAAGCCTTATCGGA-3’

5’-ACCTGCTCCACTGCCTTGCT-3’

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A

B 100

40

***

75 ### ***

50

*** ### ***

25

20 10

0

0

Saline 40

WT Tg

Bleomycin

Saline

D

12

***

WT Tg

Bleomycin ***

9

30 ### ***

20 10

PMN (X104)

Lymphocyes (x104)

WT Tg

30 M (X104)

Total cells (x104)

WT Tg

C

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6 ### ***

3 0

0

Saline

Bleomycin

Saline

Bleomycin

Supplementary Figure E1. Cell recruitment in the airways after saline or bleomycin treatment in both WT and Tg mice. At day 7 after bleomycin or saline instillation, number of total cells (A), macrophages (B), lymphocytes (C), and neutrophils (D) in BAL fluid as determined by Giemsa staining. Each bar represents the mean± SEM for groups of six to eight mice. *** P﹤0.001 compared with saline-treated group, ###P﹤0.001 vs.. bleomycin-treated WT mice.

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A

Tg

Bleomycin

Saline

WT

B Ratio(col-I/GAPDH)

0.02 0.015

** WT Tg

0.01 ###

0.005 0

Saline

Bleomycin

Supplementary Figure E2. Collagen I expression in saline or bleomycin-treated lung tissues of both WT and Tg mice. (A) Representative immunohistochemically-stained lung tissue

sections for collagen I expression, scale bar =100 μm. (B) Realtime PCR analysis of collagen I mRNA expression. Each bar represents the mean± SEM for groups of six to eight mice. **P﹤0.01 vs. saline-treated control; ###P﹤0.001 vs. bleomycintreated WT mice. Copyright © 2014 by the American Thoracic Society

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Tg

Bleomycin

Saline

WT

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IL-17A+ cells /mm2

100 *** 75 50 ### ***

25 0

Saline

Bleomycin

Supplementary Figure E3. Mice overexpressing latent TGF-1 are protected against IL-17A+ cells infiltration in bleomycin-induced lung tissues. Immunohistochemistry shows that mice with higher latent TGF-1 are protected against IL-17A+ cell infiltration in bleomycin-treated lung tissues. Data represent groups of 6 mice. Scale bar = 100 m.

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A ##

3

Ratio (Foxp3/ROR-γt)

WT Tg 2

1

0

Saline

Bleomycin

B 1.5

WT

Ratio ( IL-10/IL-17A)

Tg ## 1.0

0.5

*** 0

Saline

Bleomycin

Supplementary Figure E4. The ratio of Foxp3/RORγt and IL-10/IL-17A between WT and Tg mice treated with saline or bleomycin. (A) The ratio of Foxp3/RORγt protein as measured by Western blot. (B) The ratio of il-10/il-17a mRNA as measured by real-time

PCR. Each bar represents the mean± SEM for groups of six to eight mice. *** P﹤0.001 compared with salinetreated group; ##P<0.01 compared with bleomycintreated WT group.

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Latent transforming growth factor-β1 protects against bleomycin-induced lung injury in mice.

Transforming growth factor (TGF)-β1 is a potent mediator known to induce lung fibrosis. However, the role of latent TGF-β1 in lung inflammation and fi...
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