Exophilin-5 regulates allergic airway inflammation by controlling IL-33–mediated Th2 responses
Katsuhide Okunishi, … , Susumu Nakae, Tetsuro Izumi
Katsuhide Okunishi, … , Susumu Nakae, Tetsuro Izumi
Published April 2, 2020
Citation Information: J Clin Invest. 2020;130(7):3919-3935. https://doi.org/10.1172/JCI127839.
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Research Article Immunology

Exophilin-5 regulates allergic airway inflammation by controlling IL-33–mediated Th2 responses

Abstract

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.

Authors

Katsuhide Okunishi, Hao Wang, Maho Suzukawa, Ray Ishizaki, Eri Kobayashi, Miho Kihara, Takaya Abe, Jun-ichi Miyazaki, Masafumi Horie, Akira Saito, Hirohisa Saito, Susumu Nakae, Tetsuro Izumi

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Figure 9

Exophilin-5 deficiency inhibits intracellular trafficking of Nox2 upon stimulation in pathogenic Th2 cells.

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Exophilin-5 deficiency inhibits intracellular trafficking of Nox2 upon s...
(A and B) Levels of Nox2-encoding Cybb mRNA in the indicated T cells (A, n = 4–5 experiments), and in 4 fractions of CD44hiCD4+ T cells (see Figure 3A) obtained from OVA-sensitized mice (B, n = 3–5 experiments). #P < 0.05; ##P < 0.01 by 1-way ANOVA with Tukey’s post hoc test. Tpath2, pathogenic Th2 cells. L/L, CD62LloCXCR3lo; L/H, CD62LloCXCR3hi; H/H, CD62LhiCXCR3hi; H/L, CD62LhiCXCR3lo. (C) Binding of Rab27a, Nox2, and exophilin-5. Transfection of plasmids into HEK293A cells, immunoprecipitation of cell lysates, and detection of the indicated proteins in immunoprecipitates were conducted as described in Methods. We used 2 clones of FLAG-tagged Nox2-expressing plasmids (upper), and then subcloned Cybb cDNA from plasmid number 2 into the pEGFP-C1 vector (lower). An immunoblot representative of 3 different experiments is shown. (D) Immunostaining of Nox2 in sorted pathogenic Th2 cells before and after a 3-minute stimulation with PMA plus ionomycin (PMA/Iono). An image representative of 3 independent experiments is shown. The arrow in the WT cell indicates complete loss of cytoplasmic Nox2 staining. Scale bars: 5 μm. (E) Superoxide secretion by sorted pathogenic Th2 cells after PMA/Iono stimulation. Data were obtained from a total of 4 mice (n = 2 from both nontreated and OVA-sensitized mice). (F and G) Intracellular ROS production after stimulation. Pathogenic Th2 cells were sorted and cultured with and without PMA/Iono stimulation (F) or anti-CD3ε Ab (CD3) stimulation (G). (F) Percentages of ROShi population before stimulation (basal) and increases in them after 2.5 minutes of PMA/Iono stimulation (Δincrease) obtained from WT mice (n = 4) and Exph5-KO mice (n = 3) gathered from 2 independent experiments are shown. (G) Left: A dot plot representative of 3 individual experiments is shown. Right: Percentages of ROShi population without stimulation (basal) and increases in them with anti-CD3ε Ab stimulation compared with those at basal (Δincrease). Data were obtained from n = 4 mice gathered from 3 independent experiments. *P < 0.05 by unpaired t test.