http://dx.doi.org/10.4110/in.2015.15.3.142 ORIGINAL ARTICLE pISSN 1598-2629 eISSN 2092-6685 Osteopontin Potentiates Pulmonary Inflammation and Fibrosis by Modulating IL-17/IFN-γ-secreting T-cell Ratios in Bleomycin-treated Mice Keunhee Oh1,2, Myung Won Seo1, Young Whan Kim3 and Dong-Sup Lee1,2* 1Laboratory of Immunology and Cancer Biology, Department of Biomedical Sciences, 2Transplantation Research Institute, 3Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-799, Korea Lung fibrosis is a life-threatening disease caused by overt and fibrosis by affecting the ratio of pathogenic or insidious inflammatory responses. However, the mecha- IL-17/protective IFN-γ T cells. nism of tissue injury-induced inflammation and sub- [Immune Network 2015;15(3):142-149] sequent fibrogenesis remains unclear. Recently, we and other groups reported that Th17 responses play a role in Keywords: Osteopontin, Pulmonary fibrosis, αβ T cells, amplification of the inflammatory phase in a murine model IL-17, Th17 differentiation induced by bleomycin (BLM). Osteopontin (OPN) is a cy- tokine and extracellular-matrix-associated signaling molecule. However, whether tissue injury causes inflammation and INTRODUCTION consequent fibrosis through OPN should be determined. In this study, we observed that BLM-induced lung inflam- Fibrosis is the result of inappropriate wound-healing proc- mation and subsequent fibrosis was ameliorated in OPN- esses, characterized as an excessive deposition of extra- deficient mice. OPN was expressed ubiquitously in the cellular matrix proteins in place of parenchymal cells (1,2). lung parenchymal and bone-marrow-derived components Fibrotic tissue remodeling causes the destruction of normal and OPN from both components contributed to patho- architecture, leading to organ malfunction. The persistent genesis following BLM intratracheal instillation. Th17 dif- activation of inflammatory pathways following tissue in- ferentiation of CD4+ αβ T cells and IL-17-producing γδ jury is associated with the development of fibrotic diseases. T cells was significantly reduced in OPN-deficient mice The mechanism involved in triggering the transition from compared to WT mice. In addition, Th1 differentiation of acute to chronic inflammation is under investigation (3). CD4+ αβ T cells and the percentage of IFN-γ-producing Recently, several groups have shown that Th17 differ- γδ T cells increased. T helper cell differentiation in vitro entiation in the lung connects the initial innate responses revealed that OPN was preferentially upregulated in CD4+ to the adaptive arms of immune responses in bleomy- T cells under Th17 differentiation conditions. OPN ex- cin-treated pulmonary fibrosis murine models (4-7). pressed in both parenchymal and bone marrow cell compo- Osteopontin (OPN) is a glycosylated protein expressed nents and contributed to BLM-induced lung inflammation in various tissues, and is implicated in many biological Received on March 27, 2015. Revised on May 28, 2015. Accepted on June 5, 2015. CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any me- dium, provided the original work is properly cited. *Corresponding Author. Dong-Sup Lee, Department of Biomedical Sciences, Seoul National University College of Medicine, 103 Daehak-ro Jongno-gu, Seoul, Korea. Tel: 82-2-3668-7625; Fax: 82-2-745-9528; E-mail: [email protected] Abbreviations: OPN, Osteopontin; BLM, Bleomycin; BALF, Bronchoalveolar lavage fluid; γδ T cell, gamma delta T cell 142 IMMUNE NETWORK Vol. 15, No. 3: 142-149, June, 2015 The Role of Osteopontin in Pulmonary Inflammation and Fibrosis Keunhee Oh, et al. processes including bone remodeling, stress response, in- wild-type (WT) mice. Moreover, OPN was preferentially flammation, and cancer progression (8-11). The OPN in- upregulated in CD4+ T cells under Th17 differentiation teraction with extracellular adhesion molecules has been conditions in vitro. Thus, OPN is expressed in both paren- well-characterized. Integrins including αVβ3, αVβ5, α9β1, chymal and bone marrow cell components, and contributed and CD44 variants (CD44v) are receptors for OPN (12-15), to BLM-induced lung inflammation and fibrosis by affect- and interactions between these receptors and OPN mediate ing the pathogenic IL-17/protective IFN-γ T cell ratios. the survival, migration, and adhesion of many cell types (16). In addition to its role in adhesion, OPN functions as MATERIALS AND METHODS a cytokine or intracellular signaling molecule during the development of various innate and adaptive immune cells Mice (17-19). C57BL/6 (B6) and OPN−/− mice were obtained from the Numerous studies have shown the important role of Jackson Laboratory (Bar Harbor, ME). Male, 8-12-week-old OPN in wound healing and fibrosis (20). OPN is strongly mice were used for experiments, and all mice, including expressed during fibrosis of the heart, lung, liver, and kid- wild-type B6, were bred and maintained at the animal fa- ney (21,22). In animal experiments, blockage of OPN ex- cility of Seoul National University College of Medicine. pression during wound healing decreases the formation of All animal experiments were performed with the approval granulation tissue and scarring (23). OPN is known to reg- of the Institutional Animal Care and Use Committee at ulate cell-extracellular matrix interactions and modulate Seoul National University (authorization no. SNU05050203). TGF-β1 and matrix metalloproteinase expression (24). For pulmonary fibrosis, OPN was found to distinguish idio- Induction of pulmonary fibrosis pathic pulmonary fibrosis (IPF) from normal lungs in hu- Pulmonary fibrosis was induced by intratracheal instilla- mans based on oligonucleotide arrays (25,26). OPN pro- tion with bleomycin (BLM, 1.5 mg/kg, Nippon Kayaku, duced by alveolar macrophages functions as a fibrogenic Japan) in 50 μl phosphate-buffered saline (PBS). cytokine that promotes migration, adhesion, and pro- liferation of fibroblasts during the development of BLM- Bone marrow chimera induced lung fibrosis (27). OPN expression is associated Recipient mice were lethally irradiated 950 rad whole with important fibrogenic signals in the lung and a sig- body irradiation in two split doses) and injected intra- nificant decrease in levels of active TGF-β1 and MMP2 venously with T cell-depleted bone marrow cells (3×106/ in OPN-deficient mice. The epithelium may be an im- mouse). Reconstituted mice were used in the experiments portant source of osteopontin during lung fibrosis (28). 8 weeks after bone marrow cell transfer. Since OPN is implicated in type I IFN production and Th17 differentiation (29,30), we evaluated its role in a Histopathology of lung tissue BLM-induced pulmonary fibrosis model focusing on fibro- Lung tissues were fixed in 4% paraformaldehyde, proc- genic T-helper cell differentiation following BLM adminis- essed, and embedded in paraffin. Sections were stained with tration. We also evaluated the effect of OPN expression H&E for histopathological analysis. in parenchymal cells (including lung epithelial cells) on pathogenesis using bone marrow chimeras (7). BLM-in- Collection of broncheoalveolar lavage fluid (BALF) duced pulmonary inflammation and subsequent fibrosis Broncheoalveolar lavage was performed with five 1.0-ml was ameliorated in OPN−/− mice, and OPN was expressed aliquots of PBS through a tracheal cannula. To evaluate in both lung parenchymal and bone-marrow-derived com- cytokine production, BALF cells were harvested and re- ponents, which contributed to pathogenesis following BLM stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin intratracheal instillation. Reduced Th17 differentiation of (Sigma-Aldrich) for 4 h. For intracellular staining, bre- CD4+ αβ T cells and IL-17-producing γδ T cells increased feldin A (BD-Pharmingen, San Diego, CA) was added dur- Th1 differentiation of CD4+ αβ T cells and increased the ing the final 2 h of stimulation. Cells were fixed with 4% percentage of IFN-γ-producing T cells; thus, the IFN-γ paraformaldehyde, permeabilized with 0.5% Triton X-100, /IL-17 ratio was increased in OPN−/− mice compared with and incubated with anti-CD4, anti-γδTCR, anti-IL-17, IMMUNE NETWORK Vol. 15, No. 3: 142-149, June, 2015 143 The Role of Osteopontin in Pulmonary Inflammation and Fibrosis Keunhee Oh, et al. and anti-IFN-γ antibodies (eBioscience). Intracellular cy- RESULTS tokine levels were assayed by flow cytometry. Osteopontin deficiency reduced BLM-induced pul- Flow cytometry monary inflammation and fibrosis BALF cells were incubated with mAbs to mouse CD4, To evaluate the effect of OPN on the pathogenesis of pul- CD8α, B220, CD11c, γδTCR, and βTCR that were con- monary fibrosis, a bleomycin (BLM)-induced lung fibrosis jugated to fluorescein, phycoerythrin, PerCP-Cy5.5, or al- model was induced in WT B6 and B6.OPN−/− (OPN−/−) lophycocyanin (eBioscience). Cells were analyzed using a mice. At day 21 of intratracheal BLM instillation, OPN−/− FACSCalibur (BD Biosciences, San Jose, CA) and FlowJo mice showed significantly reduced lung pathology in terms software (Tree Star, Ashland, OR). of cellular infiltration and collagen deposition compared to WT mice (Fig. 1A). Since OPN is expressed ubiquitously In vitro differentiation of effector T cells and its expression levels increased following BLM admin- Lymphocytes were isolated from lymph nodes and labeled istration (28), we evaluated OPN expression in various tis- with anti-CD4 or anti-CD8 antibodies. CD4+ or CD8+ T sues of normal adult untreated mice using semi-quantita- cells were purified using a FACS Aria cell sorter (BD). tive RT-PCR. High OPN expression was observed in pa- Purified T cells were stimulated with plate bound anti-CD3 renchymal organs such as the brain, lung, and liver, but and anti-CD28 (1 μg/ml each, BD-Pharmingen). For Th1 expression was lower in primary and secondary lymphoid cell differentiation, recombinant IL-12 (10 ng/ml) and an- organs such as the thymus, spleen, and lymph nodes (Fig. ti-IL-4 (10 μg/ml) were added during the stimulation.