Epithelial IL-33 appropriates exosome trafficking for secretion in chronic airway disease
Ella Katz-Kiriakos, … , Mark J. Miller, Jennifer Alexander-Brett
Ella Katz-Kiriakos, … , Mark J. Miller, Jennifer Alexander-Brett
Published January 28, 2021
Citation Information: JCI Insight. 2021;6(4):e136166. https://doi.org/10.1172/jci.insight.136166.
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Research Article Immunology Pulmonology

Epithelial IL-33 appropriates exosome trafficking for secretion in chronic airway disease

Abstract

IL-33 is a key mediator of chronic airway disease driven by type 2 immune pathways, yet the nonclassical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is cosecreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared with non-COPD specimens. Using an Alternaria-induced airway disease model, we found that the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a potentially novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type 2 inflammation.

Authors

Ella Katz-Kiriakos, Deborah F. Steinberg, Colin E. Kluender, Omar A. Osorio, Catie Newsom-Stewart, Arjun Baronia, Derek E. Byers, Michael J. Holtzman, Dawn Katafiasz, Kristina L. Bailey, Steven L. Brody, Mark J. Miller, Jennifer Alexander-Brett

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Figure 2

nSMase2 regulates tonic IL-33 and exosome secretion from epithelial cells.

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nSMase2 regulates tonic IL-33 and exosome secretion from epithelial cell...
(A) ELISA secretion assay for Flag-IL-33Δ34-His COPD and non-COPD airway basal cells treated with chemical inhibitors (concentrations in Methods), PBS, DMSO vehicle control, and Golgi (brefeldin and monensin); vesicle pathway inhibitors (GW4869, cambinol, spiroepoxide, and glutathione); and microautophagy inhibitors (3-methyladenine [3-MA]), showing marked blockade with GW4869 (n = 5). (B) COPD cells treated with DMSO or GW4869 (20 μM), demonstrating inhibition for all IL-33 variants (n = 3–5). (C) qPCR for SMPD3 mRNA in n = 12 non-COPD and n = 22 COPD cell specimens and nSMase2 enzyme activity for a subset (n = 6 non-COPD, n = 12 COPD), demonstrating increased expression in COPD. (D) shRNA knockdown in COPD cells and SMPD3–/– HBE-1 cells, showing decreased Flag-IL-33Δ34-His secretion with nSMase2 shRNA, which was further reduced in SMPD3–/– HBE-1 cells (n = 5). (E) COPD cells expressing Flag-IL33Δ34-His immunostained for VPS4A, LAMP2, or nSMase2 (green) and Flag (red). Coexpression of nSMase2 shRNA demonstrates loss of staining; Hoechst 33342 nuclear counterstain was also used. Scale bar: 10 μm. (F) Secreted exosomes (<150 nm) from shRNA knockdown cells quantified by tunable resistive pulse sensing (TRPS), demonstrating increased particles for VPS4A. #Undetectable for nSMase2 (detection limit 1 × 107 particles/ml). (G) Dynamic light scattering (DLS) particle size distribution on same samples with a larger peak shift for nSMase2 shRNA. (H) SMPD3 qPCR demonstrating increased relative expression in airway basal cells compared with HMC1.2 mast cell line (n = 3). (I) HMC1.2 cells expressing Flag-IL-33Δ34-His demonstrate no tonic secretion (ND) despite measurable total protein in lysate (n = 3). Data are shown as the mean ± SEM. Statistical analysis: 1-way ANOVA (A and COPD in D) and t test (B, C, H, and HBE-1 in D); *P < 0.05, **P < 0.01, ***P < 0.001.