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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

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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 3

IL-33 cosecretion with exosomes as surface-bound cargo.

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IL-33 cosecretion with exosomes as surface-bound cargo.
(A) ELISA secret...
(A) ELISA secretion assay for Flag-IL-33Δ34-His expressing COPD airway basal cells treated with nSMase2 activator 4-methylumbelliferone (4-MU, 10 μM) and GW4869 (20 μM). 4-MU augmented secretion, which was blocked by GW4869 (n = 5). (B) Live-cell imaging for mCherry-IL33Δ34-GFP HBE-1 cells treated with 4-MU and GW4869 for 15 minutes, demonstrating foci (white arrows) of yellow merged signal. HBE-1 cells were also fixed and permeabilized after 15 minutes and imaged for GFP only (green) and immunostained for CD9 (red), demonstrating foci of colocalization (white arrows). DAPI nuclear counterstain was also used. Scale bar: 10 μm. (C) Secretion of Flag-IL-33full-His from COPD airway basal cells was augmented by disruption of nuclear entry (ivermectin, 1 μM) or by nSMase2 activation (4-MU, 10 μM), and both were inhibited by GW4869 (n = 5). (D) Live-cell imaging of mCherry-IL-33full-GFP HBE-1 cells treated with DMSO or ivermectin + 4-MU, showing accumulation of cytoplasmic IL-33full signal within 1 hour. Hoechst 33342 nuclear counterstain was also used. Scale bar: 10 μm. (E) Exosome and protein fractionation from Flag-IL33Δ34-His–expressing HBE-1 cell supernatant (sup). Secreted IL-33 was retained above centrifugal concentrator 100 kDa filter (conc sup) and detected by anti-Flag Western blot. Lysate (lys) was used for comparison. Exosomes were resolved from free proteins by a qEV size-exclusion column, and fractions were analyzed: nonfixed sup Flag-IL-33Δ34-His resolves into protein fractions and fixed sup protein migrates at a higher MW and resolves into both exosome and protein fractions. (F) Purified HBE-derived exosomes (108 particles) incubated for 15 minutes with recombinant site–specific biotinylated IL33Δ34 protein (1 μg) were resolved on the qEV column, showing that IL-33 coelutes in CD9-containing exosome fractions without fixation. Data are shown as the mean ± SEM. Statistical analysis: 1-way ANOVA (A and C); *P < 0.05, **P < 0.01, ***P < 0.001.

Copyright © 2021 American Society for Clinical Investigation
ISSN 2379-3708

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