The Journal of Clinical Investigation RESEARCH ARTICLE Exophilin-5 regulates allergic airway inflammation by controlling IL-33–mediated Th2 responses Katsuhide Okunishi,1 Hao Wang,1 Maho Suzukawa,2,3 Ray Ishizaki,1 Eri Kobayashi,1 Miho Kihara,4 Takaya Abe,4,5 Jun-ichi Miyazaki,6 Masafumi Horie,7 Akira Saito,7 Hirohisa Saito,8 Susumu Nakae,9 and Tetsuro Izumi1 1Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan. 2National Hospital Organization Tokyo National Hospital, Tokyo, Japan. 3Division of Respiratory Medicine and Allergology, Department of Medicine, Teikyo University School of Medicine, Tokyo, Japan. 4Laboratory for Animal Resource Development and 5Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan. 6Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan. 7Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 8Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan. 9Laboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan. A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2- containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma. Introduction focused on the functions of such biologically active substances Allergic asthma is a chronic inflammatory airway disease charac- and their producers in the etiology of allergy. On the other hand, terized by airway eosinophilia, mucus hypersecretion, and airway although biological substances including cytokines are released hyperresponsiveness (1). Its incidence is steadily increasing, with from their producer cells extracellularly to engage their target an estimated 339 million people affected worldwide (2). The most cells, our understanding of the role of secretory machinery in common treatment is topical inhaled corticosteroid application, allergy has advanced little. which, while effective, is associated with corticosteroid nonspe- Secretory machinery is classified into 2 pathways: constitutive cific suppression of immune responses, resulting in increased and regulated. In the former, biologically active molecules are incidence of infections such as esophageal candidiasis. Further, released consecutively upon their synthesis. In the latter, mole- steroids are only temporarily effective and do not provide a cure cules are preformed, stored in vesicles, and rapidly released upon for asthma. To move toward a cure, we must better understand the stimulation, and comprises a highly developed regulatory pathway underlying pathophysiology of this process. in higher organisms that is essential for rapid intercellular com- A defining feature of allergic responses, such as in asthma, munication. Rab27 is a small GTPase belonging to the Rab super- is an antigen-specific Th2-dominant immune response, which family, and is well recognized as an essential player in regulated can develop through interactions among many different types of secretion (3). The role of Rab27a in insulin secretion by pancreatic cells. It is well established that biologically active substances, such β cells (4), as well as in melanosome transfer from melanocytes to as cytokines, play crucial roles, and allergy research has strongly keratinocytes (5), is well established. It is also known to positively regulate secretion of cytotoxic granules by cytotoxic cells such as NK cells and cytotoxic T cells (6, 7), and mutation of Rab27a in Related Commentary: p. 3419 humans results in type II Griscelli syndrome, one of the familial hemophagocytic syndromes accompanied by immune dysfunc- Authorship note: KO and HW contributed equally to this work. tion and albinism (8). In addition, Rab27a regulates cell functions Conflict of interest: The authors have declared that no conflict of interest exists. by controlling intracellular trafficking of NADPH oxidase–con- Copyright: © 2020, American Society for Clinical Investigation. taining vesicles and subsequent intracellular ROS production in Submitted: January 30, 2019; Accepted: March 31, 2020; Published: June 8, 2020. Reference information: J Clin Invest. 2020;130(7):3919–3935. macrophages and neutrophils (9, 10). On the other hand, although https://doi.org/10.1172/JCI127839. it is reported that Rab27 is expressed in other immune cells such jci.org Volume 130 Number 7 July 2020 3919 RESEARCH ARTICLE The Journal of Clinical Investigation as mast cells where it regulates exocytosis (11, 12), dendritic cells and mucus production (Figure 1F) were markedly enhanced in the (DCs) (13), Th cells (7), and lung epithelial cells (14), all of which lungs of Exph5-KO mice after 3 days of OVA challenges. Airway play essential roles in antigen-specific allergic immune responses hyperresponsiveness also tended to be enhanced in Exph5-KO in the lung, the roles of Rab27 in the net allergic immune respons- mice (Figure 1G). These results suggest that systemic exophilin-5 es in vivo are unknown. A recent study showed a link between deficiency enhances OVA-induced allergic airway inflammation, RAB27A single nucleotide polymorphism (SNP) and fractional possibly through upregulation of OVA-specific Th2 cytokine pro- exhaled nitric oxide (FeNO) levels (15), an indicator of allergic duction by immune cells. airway inflammation (16), suggesting the involvement of Rab27 in Exophilin-5 deficiency in hematopoietic cells is essential for the regulation of allergic immune responses. induction of enhanced allergic airway inflammation. Because exo- Eleven effectors have been shown to bind the active, GTP- philin-5 is reported to be expressed in both hematopoietic and bound form of Rab27 and regulate intracellular vesicle traffic at nonhematopoietic cells (17, 19, 20), we next used bone marrow– specific steps (3). Among these 11 Rab27 effectors, the present chimeric (BM-chimeric) mice to identify the cells responsible for study focused on exophilin-5, a large protein composed of 1960 the phenotypes observed in Exph5-KO mice in an OVA-induced and 1989 amino acids in mice and humans, respectively. Com- mouse model of asthma. Although exophilin-5 deficiency in pared with the other Rab27 effectors, its biological function BM cells did not affect the induction of OVA-specific Th2-type remains obscure, and few studies have investigated its biological immune responses, exophilin-5 deficiency in recipient mice tend- functions. For example, in humans, mutation of the exophilin-5– ed to enhance it (Figure 2A). However, exophilin-5 deficiency in encoding EXPH5 gene causes skin fragility, which results in the BM cells significantly exacerbated eosinophilic lung inflamma- development of epidermolysis bullosa (17). In cells, exophilin-5 tion to a level similar to that induced by exophilin-5 deficiency in is reported to positively regulate exosome secretion in HeLa cells the whole body (Figure 2B), along with enhanced mucus produc- (18) and intracellular trafficking of PI4-kinase type 2a–containing tion (Figure 2C). We confirmed that BM cell transplantation itself late endosomes in the human T cell leukemia cell line Jurkat (19). did not cause spontaneous development of eosinophilic lung Moreover, a recent study revealed that exophilin-5 is present in a inflammation in the absence of OVA antigen inhalation (Supple- human Th2-enriched memory-type CD4+ cell fraction (20). How- mental Figure 1F). These results indicate that lack of exophilin-5 ever, the role of exophilin-5 in asthmatic responses is unclear. In in hematopoietic cells is sufficient to phenocopy the exacerbated the present study, using newly developed exophilin-5–knockout allergic airway inflammation in Exph5-KO mice without affecting (Exph5-KO) mice and mouse models of asthma, we uncovered antigen sensitization induced by intraperitoneal antigen injec- a role of exophilin-5 as a regulator of IL-33 signaling in antigen- tions. The finding that exophilin-5 deficiency in recipient mice, induced allergic immune responses. regardless of types of transplanted BM, tended to affect