lavage fluid of patients with sarcoidosis Proinflammatory exosomes in bronchoalveolar

Background Sarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon g (IFNg)-producing T cells that cause inflammation and tissue damage in multiple organs, especially the lung. Exosomes are nano-sized immunomodulatory vesicles of endosomal origin released from a diverse range of cells and are also found in physiological fluids including bronchoalveolar lavage fluid (BALF) from healthy individuals. Objective To investigate whether exosomes are enriched in the lungs of patients with sarcoidosis compared with healthy individuals and whether they could contribute to pathogenesis. Design BALF exosomes from patients with sarcoidosis (n1⁄436) and healthy controls (n1⁄414) were compared by electron microscopy, flow cytometry, western blot analysis and mass spectrometry. BALF exosomes were incubated with autologous peripheral blood mononuclear cells (PBMCs) or the human bronchial epithelial cell line 16HBE14o-. Cytokines were measured by ELISPOT and ELISA. Results BALF from patients with sarcoidosis showed increased levels of exosomes compared with healthy individuals. Exosomes from patients showed significantly higher expression of MHC class I and II, tetraspanins CD9, CD63 and CD81 as well as neuregulin-1, known to be associated with cancer progression. Furthermore, BALF exosomes from patients induced significantly higher IFNg and interleukin (IL)-13 production in autologous PBMCs compared with healthy individuals and could also stimulate IL-8 production from epithelial cells. Conclusion The results indicate for the first time a role for exosomes in human lung disease with possible contributions to the initiation and progression of inflammation in sarcoidosis. This suggests that exosomes may be a new potential target for the clinical treatment of lung diseases. INTRODUCTION Sarcoidosis is a systemic granulomatous disease with diverse organ system manifestations that predominantly affects the lung. Common symptoms include dyspnoea, cough and fatigue. Spontaneous remission occurs in most patients but some develop chronic disease leading to death in 1e4% of patients. Although the aetiology of sarcoidosis is not known, many observations including the presence of oligoclonal Th1-like CD4+ T cells and macrophages in bronchoalveolar lavage fluid (BALF) and blood, as well as granuloma formation in the lungs, suggest an antigen-driven autoimmune disease. 2 Autoantigens such as vimentin and ATP-synthase have been identified as targets for expanded T cell clones in the lung. Data suggest contributing roles of Mycobacterium tuberculosis and Proprionibacterium spp. in the pathology of sarcoidosis. Exosomes are nano-sized vesicles (30e100 nm in diameter) produced by inward budding of multivesicular bodies. Fusion of multivesicular bodies with the plasma membrane leads to the extracellular release of intraluminal vesicles now called exosomes. The term exosome was first coined in 1987 by Johnstone who isolated exosome vesicles from culture supernatants and were discovered as a waste product in erythrocyte development. However, a wide variety of other cell types have since been shown to release exosomes, including B cells, dendritic cells (DC), macrophages, mast cells, Tcells, epithelial cells, platelets and tumour cells. 7 The in vivo role of exosomes still remains elusive, but their presence in several body fluids such as breast milk and plasma suggest a role in vivo. The analysis of exosomes is complicated because of their small size and the lack of exosome-specific markers, hence several methods including electron microscopy are needed to identify them. Exosomes have received great attention lately owing to their potential to induce immune responses or tolerance, depending on their cellular origin. Exosomes loaded with tumour antigens are considered as promising vaccine candidates in cancer 12 and infectious diseases such as toxoplasmosis. In 2003 our group isolated exosomes from BALF (BALF exosomes) which had phenotypic similarities to DC-derived exosomes. Studies by Prado et al showed that BALF exosomes from tolerised mice can be given intranasally to prevent allergic sensitisation. Furthermore, it was recently shown that BALF exosomes from Mycobacterium bovis BCGinfected mice have the capacity to stimulate tumour necrosis factor a (TNFa) production in naïve macrophages. Even though the role of exosomes in the lungs or other compartments in humans is not known, they could either participate in immune surveillance or in pathogenesisdfor example, by potentiating inflammation. In this study we found major differences between BALF exosomes from patients with sarcoidosis and healthy individuals. Patients showed increased numbers of exosomes in their lung compared with healthy individuals and had higher levels of MHC class I and II, tetraspanins and heat shock protein 70 (HSP70). The novel finding that BALF exosomes contain neuregulin-1 (NRG1), previously connected to cancer progression, suggests a pathological role for exosomes in sarcoidosis. Furthermore, exosomes Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden Department of Medicine, Division of Respiratory Medicine, Lung Research Laboratory, Karolinska Hospital, Stockholm, Sweden Correspondence to Associate Professor Susanne Gabrielsson, Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet and University Hospital, KS L2:04, SE-171 76 Stockholm, Sweden; [email protected] KRQ and PTP share first authorship of the paper. Received 25 November 2009 Accepted 29 July 2010 Published Online First 29 September 201