Oligosaccharide modification by N-acetylglucosaminyltransferase-V in macrophages are involved in pathogenesis of bleomycin-induced scleroderma Arisa Kato 1,2 , Mizuki Yutani 1,2 , Mika Terao 1 , Akihiro Kimura 1,2 , Saori Itoi 1 , Hiroyuki Murota 1 , Eiji Miyoshi 2 and Ichiro Katayama 1 1 Department of Dermatology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; 2 Department of Molecular Biochemistry and Clinical Investigation, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Correspondence: Mika Terao, MD, PhD, Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, Tel.: +81-6-6879-3031, Fax: +81-6-6879-3039, e-mail: mterao@derma.med.osaka-u.ac.jp Abstract: Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), which catalyses the formation of b1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is associated with various pathologies, such as cancer metastasis, multiple sclerosis and liver fibrosis. In this study, we demonstrated the involvement of GnT-V in the pathophysiology of scleroderma. High expression of GnT-V was observed in infiltrating cells in skin section samples from systemic and localized patients with scleroderma. Most of the infiltrating cells were T cells and macrophages, most of which were CD163 + M2 macrophages. To determine the role of GnT-V in scleroderma, we next investigated skin sclerosis in GnT-V knockout (MGAT5 / ) mice. Expression of GnT-V was also elevated in bleomycin (BLM)-injected sclerotic skin, and MGAT5 / mice were resistant to BLM-induced skin sclerosis with reduced collagen type 1 a1 content, suggesting the biological significance of GnT-V in skin sclerosis. Furthermore, the number of CD163 + M2 macrophages and CD3-positive T cells in BLM-induced skin sclerosis was significantly fewer in MGAT5 / mice. In bone marrow-derived macrophages (BMDMs), IL-4-induced expressions of Fizz1 and Ym1 were significantly reduced in MGAT5 / mice-derived BMDMs. Taken together, these results suggest the induction of GnT-V in skin sclerosis progression is possibly dependent on increased numbers of M2 macrophages in the skin, which are important for tissue fibrosis and remodelling. Abbreviations: BLM, bleomycin; BMDMs, bone marrow-derived macrophages; GnT-V, N-acetylglucosaminyltransferase-V; LPS, lipopolysaccharide; LS, localized scleroderma; MGAT5  / mouse, N- acetylglucosaminyltransferase-V knockout mouse; PBS, phosphate-buffered saline; SSc, systemic sclerosis. Key words: bleomycin – localized scleroderma – macrophages – N-acetylglucosaminyltransferase-V – systemic sclerosis Accepted for publication 8 April 2015 Introduction Glycans are oligosaccharides attached to proteins via two major linkage types, N-glycans and O-glycans. Changes in glycosylation are observed during differentiation and carcinogenesis (1) and are regulated by a series of glycosyltransferases. The enzyme N-acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase encoded by the MGAT5 gene, plays an important role in tumor growth and metastasis (2,3). Our previous study demonstrated that GnT-V expression is associated with poor disease prognosis as judged by immunohistochemical analysis (3,4). In addition, mammary tumor growth and metastasis induced by the polyo- mavirus middle T oncogene are considerably reduced in N-acet- ylglucosaminyltransferase-V knockout (MGAT5 / ) mice compared with littermate controls (5). We recently reported that GnT-V transgenic mouse keratinocytes exhibit an epithelial-to- mesenchymal transition-like phenotype and promote wound healing (6). Other than its promotion of tumor growth, GnT-V is associated with many physiological and pathological condi- tions, such as rat liver regeneration, steatohepatitis and epidermal hyperplasia (7–10). The enzyme also plays important roles in immune cell function and autoimmune diseases; specifically, GnT-V is involved in the negative regulation of T-cell activation by suppressing autoimmu- nity reactions (11). T-cell receptor signalling by GnT-V promotes the development of a Th2, rather than a Th1, response (12), and genetic changes in GnT-V are associated with multiple sclerosis (13–15). Recently, immune response during allergic inflammation is reported to be altered in MGAT5 / mice (16,17). Scleroderma, a subset of fibrosing disorders, is classified as either systemic sclerosis (SSc) or localized scleroderma (LS), also known as morphea. Systemic sclerosis is a generalized disease of connective tissue involving mainly the skin, gastrointestinal tract, lungs and the kidneys (18,19), while LS is limited to the skin, sub- cutaneous tissue, underlying bone and, rarely, the underlying cen- tral nervous system. Although autoimmunity is one factor associated with scleroderma formation, its underlying pathogenesis remains unknown. However, the disease is likely initiated by vas- cular injury, which increases collagen production and decreases collagen destruction. In our study, we found that GnT-V was expressed in infiltrating macrophages and T cells in the skin of patients with scleroderma. We further investigated the role of GnT-V in bleomycin (BLM)- induced scleroderma, especially focusing on macrophages using MGAT5 / mice. ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, Experimental Dermatology 1 DOI: 10.1111/exd.12730 www.wileyonlinelibrary.com/journal/EXD Original Article