Negative regulation of human IL-33 in endothelium during allergic airway inflammation
Maile K. Hollinger, Chanie L. Howard, Donna C. Decker, Kelly M. Blaine, Ivy Aneas, Emily M. Grayson, Tania E. Velez, Fernando A. Oliveira, Riley T. Hannan, Daniel F. Camacho, Philip A. Verhoef, Cara L. Hrusch, Rebecca S. Griffes, Jeffrey M. Sturek, Marcelo A. Nobrega, Nathan Schoettler, Anne I. Sperling
Maile K. Hollinger, Chanie L. Howard, Donna C. Decker, Kelly M. Blaine, Ivy Aneas, Emily M. Grayson, Tania E. Velez, Fernando A. Oliveira, Riley T. Hannan, Daniel F. Camacho, Philip A. Verhoef, Cara L. Hrusch, Rebecca S. Griffes, Jeffrey M. Sturek, Marcelo A. Nobrega, Nathan Schoettler, Anne I. Sperling
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Research Article Immunology Vascular biology

Negative regulation of human IL-33 in endothelium during allergic airway inflammation

Abstract

Lung IL-33 is involved in pathogen defense, barrier homeostasis, and development of allergic responses. We previously identified a 5 kb noncoding region within a GWAS-defined segment that regulates expression of human IL33 (hIL33) but is absent in the murine locus. To understand how this region affects IL-33 expression in vivo, we engineered 2 BAC-transgenic strains in which 166 kb of the human genome upstream of the hIL33 locus, along with a fluorescent reporter, was inserted into the murine genome, both with and without the 5 kb region. Comparison to a murine Il33 (mIl33) reporter strain revealed species-specific tropism; hIL33 reporter was mostly expressed in the endothelium, while mIl33 reporter was expressed in type 2 alveolar epithelium. hIL33 reporter expression in tracheal basal epithelium, submucosal glands, and lung microvasculature required the 5 kb region. Surprisingly, allergen and exogenous IL-33 downregulated hIL33 reporter in lung endothelium only when the 5 kb region was present. Similar IL-33–dependent downregulation of IL33 transcripts was observed in human endothelial cell lines, validating that our hIL33 reporter strain recapitulated human endothelial biology. Together, these data reveal the importance of the asthma-associated human 5 kb region in regulating human IL33 expression in a cell type– and context-dependent manner.

Authors

Maile K. Hollinger, Chanie L. Howard, Donna C. Decker, Kelly M. Blaine, Ivy Aneas, Emily M. Grayson, Tania E. Velez, Fernando A. Oliveira, Riley T. Hannan, Daniel F. Camacho, Philip A. Verhoef, Cara L. Hrusch, Rebecca S. Griffes, Jeffrey M. Sturek, Marcelo A. Nobrega, Nathan Schoettler, Anne I. Sperling

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Figure 1

Human and mouse IL33 reporters are expressed by distinct cell populations in the lungs.

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Human and mouse IL33 reporters are expressed by distinct cell population...
(A) Diagram of the BAC construct used to generate hIL33CrimBAC reporter mice. Black rectangles indicate LD blocks containing causal asthma-associated SNPs described previously (16). In the coding region of human IL33, we inserted an E2-Crimson coding sequence and stop codon to prevent expression of human IL33 in BAC-containing mice. (B) Confocal microscopy (40×) of lung from 6- to 8-week-old naive hIL33CrimBAC reporter mice (top row), mIL33GFP reporter mice (middle row), and hIL33CrimBAC x mIL33GFP coreporter mice. Red indicates Crimson, green indicates GFP, and white indicates Lyve-1, an endothelial cell marker. (C) Confocal microscopy (40×) of tracheas from naive hIL33CrimBAC reporter mice (top) or mIL33GFP reporter mice (bottom). Green indicates the endothelial cell marker CD31, cyan indicates the basal epithelial cell marker Krt5, and red indicates Crimson in hIL33CrimBAC or GFP in mIL33GFP mice, respectively. Yellow triangles: Krt5+ tracheal epithelium; white triangles: submucosal glands (SMG). (D) Quantification of Crimson human IL-33 reporter fluorescence in lung CD45– cells as gated in Supplemental Figure 2. (E) Quantification of GFP (murine IL-33 reporter) fluorescence in lung CD45– cells. (F) Expression of Il33 mRNA in sorted CD45– cells from 20-month-old human lung donor from the LGEA. (G) Expression of Il33 mRNA in sorted CD45– cells from mice postnatal day 28 from the LGEA. Quantified data are represented as mean ± first and third quartile. Data in D and E depict 2 of at least 3 independent experiments, with n ≥ 2 mice per group. ****P < 0.0001 by ordinary 1-way ANOVA with Šídák’s multiple-comparison test.