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GSDMB Induces an Asthma Phenotype Characterized by Increased Airway Responsiveness and Remodeling Without Lung Inflammation

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GSDMB Induces an Asthma Phenotype Characterized by Increased Airway Responsiveness and Remodeling Without Lung Inflammation GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation Sudipta Dasa, Marina Millera, Andrew K. Beppua, James Muellerb, Matthew D. McGeoughb, Christine Vuonga, Maya R. Kartaa, Peter Rosenthala, Fazila Chouialic,d, Taylor A. Dohertya, Richard C. Kurtene, Qutayba Hamidc,d, Hal M. Hoffmanb, and David H. Broidea,1 aDepartment of Medicine, University of California, San Diego, La Jolla, CA 92093; bDepartment of Pediatrics, University of California, San Diego, La Jolla, CA 92093; cMeakins-Christie Laboratories of McGill University, McGill University, Montreal, QC, Canada H2X 2P2; dMcGill University Health Center Research Institute, Montreal, QC, Canada H2X 2P2; and eDepartment of Physiology and Biophysics, Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202 Edited by K. Frank Austen, Brigham and Women’s Hospital, Boston, MA, and approved September 27, 2016 (received for review June 28, 2016) Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic association studies. The human GSDMB gene located on genetic linkage to asthma, but its function in asthma is unknown. Here chromosome 17q21 belongs to the gasdermin (GSDM) protein we identified that GSDMB is highly expressed in lung bronchial epi- family (6, 7). The human GSDM family consists of four mem- thelium in human asthma. Overexpression of GSDMB in primary hu- bers: GSDMA, GSDMB, GSDMC, and GSDMD. The GSDMA man bronchial epithelium increased expression of genes important to and GSDMB genes are located at 17q21, and the GSDMC and both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway- GSDMD genes are located at 8q24. Several genetic linkage hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice studies have shown an association with GSDMB on chromosome expressing increased levels of the human GSDMB transgene showed 17q21 and asthma (2, 8), but none of the studies have linked a significant spontaneous increase in AHR and a significant spontane- asthma with chromosome 8q24 (where GSDMC and GSDMD ous increase in airway remodeling, with increased smooth muscle are located). This finding suggests that GSDMB (and perhaps mass and increased fibrosis in the absence of airway inflammation. Zp3-Cre GSDMA) are the only GSDM family members linked to asthma. In addition, hGSDMB mice showed increases in the same remod- The function of GSDMB (a 416-amino acid protein) is largely elingandAHRmediators(TGF-β1, 5-LO) observed in vitro in GSDMB- β unknown because GSDMB is a novel protein without charac- overexpressing epithelial cells. GSDMB induces TGF- 1 expression via teristic domains related to known function (6, 7). The single- induction of 5-LO, because knockdown of 5-LO in epithelial cells over- nucleotide polymorphism (SNP) linked to chromosome 17q21 expressing GSDMB inhibited TGF-β1 expression. These studies demon- and asthma is associated with increased expression of both strate that GSDMB, a gene highly linked to asthma but whose function GSDMB and ORMDL3 (genes located next to each other on in asthma is previously unknown, regulates AHR and airway remodel- chromosome 17q21) (2). Several studies indicate a strong linkage ing without airway inflammation through a previously unrecognized ORMDL3 GSDMB pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial disequilibrium between the SNPs of and (1, epithelium. 2), suggesting that these genes could be acting in concert toward asthma pathophysiology. However, at present the function of GSDMB | asthma | airway-hyperresponsiveness | remodeling | GSDMB in the lung and in asthma is largely unknown. inflammation Significance hromosome 17q21 was initially linked to asthma in genome- Cwide association studies (GWAS) in 2007, with confirmation in Because the SNP linking chromosome 17q21 to asthma is as- multiple GWAS and non-GWAS studies in populations of diverse sociated with increased gasdermin B (GSDMB) expression, we ethnic backgrounds (1, 2). Chromosome 17q21 contains a cluster of generated transgenic mice expressing increased levels of the Zp3-Cre four genes [ORMDL3, gasdermin B (GSDMB), IKZF3, and human GSDMB transgene (hGSDMB ), which develop an ZPBP2] (2), which may, either individually or in combination, be asthma phenotype characterized by a spontaneous increase in responsible for its genetic association to asthma. A precedent for airway responsiveness and airway remodeling (increased per- linkage of a cluster of genes in a single chromosomal region with ibronchial smooth muscle) in the absence of the development of asthma is evident from chromosome 5q31-33, where the genes IL-4, airway inflammation. These results challenge the current para- digm in asthma that airway inflammation induces smooth muscle IL-5, IL-9, and IL-13 are located (3). Although there are many Zp3-Cre genetic epidemiologic studies linking chromosome 17q21 with remodeling and airway responsiveness, as these hGSDMB asthma, there are limited functional studies of each of these four mice develop increased airway-hyperresponsiveness and smooth genes to understand how they contribute to the pathogenesis of muscle in the absence of airway inflammation. Furthermore, this asthma. We have previously demonstrated the importance of one study adds to our understanding of gene networks in asthma that we have identified can act in sequential pathways (i.e., GSDMB of the 17q21 localized genes (i.e., ORMDL3) to asthma, airway induces 5-lipoxygenase to induce TGF-β1). responsiveness, and airway remodeling (4, 5). Mice over- expressing human ORMDL3 regulated downstream pathways Author contributions: S.D., M.M., H.M.H., and D.H.B. designed research; S.D., A.K.B., J.M., (ATF6α, SERCA2b, metalloproteases) important in asthma. M.D.M., C.V., M.R.K., P.R., and F.C. performed research; R.C.K. and H.M.H. contributed Interestingly, mice overexpressing human ORMDL3 developed new reagents/analytic tools; S.D., M.M., T.A.D., Q.H., and D.H.B. analyzed data; and S.D. spontaneous increased airway responsiveness and airway and D.H.B. wrote the paper. remodeling in the absence of airway inflammation (5). The authors declare no conflict of interest. In this study, we are focusing on a second gene on chromo- This article is a PNAS Direct Submission. some 17q21, namely GSDMB, because little is known about its 1To whom correspondence should be addressed. Email: [email protected]. expression and function in the normal lung or asthmatic lung, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and published studies of GSDMB in asthma are limited to 1073/pnas.1610433113/-/DCSupplemental. 13132–13137 | PNAS | November 15, 2016 | vol. 113 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1610433113 Downloaded by guest on September 23, 2021 Because the SNP linking chromosome 17q21 to asthma is as- sociated with increased GSDMB expression (1, 2), we generated transgenic mice expressing increased levels of the human GSDMB transgene (hGSDMBZp3-Cre), which develop a significant sponta- neous increase in airway-hyperresponsiveness (AHR) and a significant spontaneous increase in airway remodeling (in- creased smooth muscle, increased fibrosis) in the absence of development of airway inflammation. This study, combined with our previous studies on ORMDL3 (4, 5), suggest that at least two genes localized to chromosome 17q21 (GSDMB and ORMDL3) contribute to airway remodeling in the absence of airway inflammation, and challenges the current paradigm that inflammation precedes lung remodeling and airway responsiveness in asthma. Results GSDMB Is Highly Expressed in Human Bronchial Epithelial Cells in Asthma. To determine which human lung cells express GSDMB, we exam- ined human lung sections from asthmatics and nonasthmatic controls. Immunohistochemistry analyses showed that levels of GSDMB were significantly increased in the bronchial epithelial cells in the lungs of asthmatics compared with control lungs (Fig. 1 A–C). We also examined the effect of asthma severity on GSDMB ex- + pression and found GSDMB cells to be significantly elevated in bronchial biopsy specimens from severe asthmatics com- pared with nonasthmatic controls (Fig. 1D). We further in- vestigated which human lung structural cells express GSDMB or its isoforms GSDMB-1, -2, -3,and-4 (Fig. S1A). We found that bronchial epithelial cells, but not alveolar epithelial cells, were a significant source of the GSDMB-1 isoform as quantitated by E F quantitative PCR (qPCR) (Fig. 1 and ). In addition, we did not Fig. 2. Nuclear localization of GSDMB activates TGF-β1, 5-LO, and MMP9 in detect significant GSDMB-1 expression in other human lung struc- human bronchial epithelial cells transfected with GSDMB. Primary bronchial tural cells (i.e., smooth muscle cells, fibroblasts), or in human lung epithelial cells were transfected with either empty, GSDMB-1, or GSDMB-1 macrophages. Moreover, GSDMB-1 was the most predominant vector lacking nuclear localization signal (GSDMB-1 ΔNLS) for 72 h and qPCR was isoform expressed in human bronchial epithelial cells (Fig. 1F). performed to measure (A)TGF-β1, (B)5-LO,and(C) MMP9 mRNA. β-Actin mRNA was used as normalization control. Fold-change is expressed as mean ± SEM from four independent experiments. ELISA was performed to measure (D)active TGF-β1, (E)5-LO,and(F) MMP9 levels in cells transfected with GSDMB-1 or empty vector. (G) Quantification of cellular localization of GSDMB in bronchial epithelial cells by Western blot. β-Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear extracts, respectively. The purity of these extracts was checked using HSP90, a cytoplasmic marker, and p53, a nuclear marker. Western blot image is a representative of three independent experi- ments. (H) Bronchial epithelial cells were transfectedwithRFP-taggedGSDMB-1, INFLAMMATION IMMUNOLOGY AND GSDMB-1 ΔNLS, or empty vector (red) and cells were mounted with DAPI (blue). (Magnification: 20×.) (I–L) Bronchial epithelial cells overexpressing GSDMB-1 or empty vector were transfected with scrambled (scr.) or 5-LO siRNA. (I) Efficiency of 5-LO mRNA knockdown was assessed by qPCR. β-Actin mRNA was used as normalization control.
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