IL-13 Mediates IL-33-dependent Mast Cell and Type 2 Innate Lymphoid Cell Effects on Bronchial Epithelial Cells.

Type 2 inflammation, characterized by increased expression of the cytokines IL-4, IL-5, and IL-13 and eosinophilic infiltration of the airways, is a feature of some patients with asthma and is prominent in patients with frequent exacerbations.1Lemanske Jr., R.F. Busse W.W. Asthma: clinical expression and molecular mechanisms.J Allergy Clin Immunol. 2010; 125: S95-S102Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar We and others have shown that “TH2 high” asthmatic patients exhibit distinct IL-13–induced gene expression in airway epithelial cells including eosinophil chemokine (C-C) ligand 26 (CCL26), periostin (POSTN), a biomarker of systemic eosinophilic airway inflammation and subepithelial fibrosis in asthmatic patients, and an increased ratio of mucin (MUC)5AC to MUC5B (MUC5AC:MUC5B), all of which may contribute to disease pathology.2Woodruff P.G. Boushey H.A. Dolganov G.M. Barker C.S. Yang Y.H. Donnelly S. et al.Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids.Proc Natl Acad Sci U S A. 2007; 104: 15858-15863Crossref PubMed Scopus (642) Google Scholar, 3Jia G. Erickson R.W. Choy D.F. Mosesova S. Wu L.C. Solberg O.D. et al.Periostin is a systemic biomarker of eosinophilic airway inflammation in asthmatic patients.J Allergy Clin Immunol. 2012; 130: 647-654.e10Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 4Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. et al.Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (130) Google ScholarMast cells are localized to the airway epithelium in “TH2 high” asthmatic patients, are associated with disease severity, and can be activated by epithelial cytokines including IL-33.5Deckers J. Branco Madeira F. Hammad H. Innate immune cells in asthma.Trends Immunol. 2013; 34: 540-547Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar IL-5 and IL-13–producing type 2 innate lymphoid cells (ILC2s) contribute significantly in animal models of type 2 inflammation and may potentially play a role in human asthma by responding to cytokines including IL-33, IL-25, and thymic stromal lymphopoietin.6Spits H. Artis D. Colonna M. Diefenbach A. Di Santo J.P. Eberl G. et al.Innate lymphoid cells–a proposal for uniform nomenclature.Nat Rev Immunol. 2013; 13: 145-149Crossref PubMed Scopus (1707) Google Scholar IL-33 is released after in vitro exposure to allergen extracts by human airway epithelial cells and in lungs of allergen-challenged mice, and has been detected in bronchoalveolar lavage fluid and airway smooth muscle in patients with asthma.7Cayrol C. Girard J.P. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy.Curr Opin Immunol. 2014; 31C: 31-37Crossref PubMed Scopus (443) Google Scholar However, specific effects of IL-33 on airway epithelium in the context of an inflammatory microenvironment have not been well characterized. As both mast cells and ILC2s have been found in human lungs, express the IL-33 receptor ST2, and produce IL-13, we sought to characterize the interactions of these cells with the airway epithelium in a proinflammatory context and determine the role of IL-13–dependent and IL-13–independent pathways in airway gene expression.We investigated global transcriptional patterns in IL-33–stimulated primary cultured in vitro differentiated human mast cells using gene expression microarrays, focusing on cytokines, chemokines, and growth factors (CCGFs),4Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. et al.Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (130) Google Scholar as they represent soluble mediators of intercellular communication (Table I). Detailed methods of culture and stimulation are given in this article's Methods section in the Online Repository at www.jacionline.org. Consistent with previous reports,8Allakhverdi Z. Smith D.E. Comeau M.R. Delespesse G. Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells.J Immunol. 2007; 179: 2051-2054Crossref PubMed Scopus (409) Google Scholar we found that IL-33–stimulated mast cells upregulated multiple non–type 2 proinflammatory mediators compared with controls. However, IL-5 and IL-13 were among the top 5 most highly induced CCGF genes in mast cells on IL-33 stimulation, suggesting that a dominant response to IL-33 in mast cells is the increased expression of type 2 inflammatory cytokines. We not only detected significantly higher IL-5 (see Fig E1, A, in this article's Online Repository at www.jacionline.org) and IL-13 protein (Fig E1, B) in supernatants from IL-33–treated mast cells compared with controls but also observed increased production of non–type 2 mediators TNF-α and IL-8 in supernatants from IL-33–stimulated mast cells (Fig E1, C and D).Table ICCGF genes differentially expressed in mast cells after 24 hours of IL-33 stimulationAgilent gene IDEntrez gene IDFold changeAdjusted P valueGene nameA_24_P209047IL5352.12.6 × 10−08Interleukin 5A_23_P315364CXCL2114.62.5 × 10−08Chemokine (C-X-C motif) ligand 2A_23_P251031IL1373.01.3 × 10−08Interleukin 13A_32_P87013CXCL861.02.4 × 10−07Interleukin 8A_23_P49759CCL127.95.9 × 10−09Chemokine (C-C motif) ligand 1A_24_P183150CXCL317.11.3 × 10−13Chemokine (C-X-C motif) ligand 3A_23_P71037IL616.01.0 × 10−06Interleukin 6A_23_P72096IL1A8.83.7 × 10−08Interleukin 1, alphaA_23_P79518IL1B8.11.1 × 10−04Interleukin 1, betaA_23_P51936TNFRSF97.83.7 × 10−03TNF receptor superfamily, member 9A_24_P411121TNFRSF187.77.0 × 10−08TNF receptor superfamily, member 18A_23_P207564CCL47.62.5 × 10−05Chemokine (C-C motif) ligand 4A_24_P133253KITLG5.34.1 × 10−05KIT ligandA_23_P138352WNT2B4.31.3 × 10−05Wingless-type mouse mammary tumor virus integration site family, member 2BA_23_P76102GDF114.03.3 × 10−05Growth differentiation factor 11A_24_P228130CCL3L33.91.1 × 10−03Chemokine (C-C motif) ligand 3-like 3A_23_P376488TNF3.84.0 × 10−04TNFA_23_P152838CCL53.41.4 × 10−03Chemokine (C-C motif) ligand 5A_23_P72817GDF33.02.6 × 10−03Growth differentiation factor 3A_24_P151121A1BG2.01.1 × 10−03Alpha-1-B glycoprotein Open table in a new tab To assess IL-33's effects on ILC2s, we isolated lineage-negative human ILC2s from blood buffy coats (see Fig E2, A, in this article's Online Repository at www.jacionline.org) and expanded them in vitro in the presence of IL-2, IL-7, IL-25, IL-33, and thymic stromal lymphopoietin. We then assessed the production of TH2 cytokines in response to IL-33. IL-33 significantly enhanced IL-5 (Fig E2, B) and IL-13 (Fig E2, C) production from ILC2s, although substantial quantities of IL-5 and IL-13 were secreted from ILC2s even without IL-33 restimulation. IL-33 also significantly increased the production of TNF-α and IL-8 (see Fig E3, A and B, in this article's Online Repository at www.jacionline.org) in ILC2s. IL-4 was detectable albeit at much lower concentrations than IL-5 and IL-13, and trended toward an increase in ILC2s after IL-33 stimulation (Fig E3, C); however, this was not statistically significant.Because mast cells and ILC2s stimulated with IL-33 expressed both type 2 and non–type 2 CCGFs, we sought to determine which of these factors could activate primary mucociliary differentiated normal human bronchial epithelial NHBE cells. Mast cells required IL-33 for IL-13 production. However, because ILC2 differentiation is dependent on IL-33 and mature ILC2s produced abundant levels of type 2 cytokines without IL-33 restimulation, we assessed the effects of ILC2-NHBE cells coculture without additional IL-33. We cocultured NHBE cells with IL-33–stimulated mast cells or mature ILC2s and examined the expression of IL-13–regulated genes CCL26, POSTN, MUC5AC, and MUC5B. In NHBE cells alone, IL-33 did not change the expression of CCL26 (Fig 1, A), POSTN (Fig 1, B), or MUC5AC:MUC5B (Fig 1, C). In the presence of mast cells, IL-33 treatment significantly upregulated CCL26, POSTN, and MUC5AC:MUC5B compared with mast cell-NHBE cells medium control. Anti–IL-13 strongly and significantly suppressed differential expression of CCL26, MUC5AC:MUC5B, and POSTN compared with control antibody. ILC2 coculture led to significantly increased expression of CCL26 (Fig 1, D) and POSTN (Fig 1, E), which was blocked by the addition of anti–IL-13, suggesting a predominant role of IL-13 in this system. We observed a trend toward an increase in MUC5AC:MUC5B (Fig 1, F), which was reduced by blocking IL-13 although this did not reach nominal statistical significance. IL-33–treated NHBE cells in the presence of mast cells had higher levels of IL-13 and periostin in basolateral supernatants compared with control (see Fig E4, B and C, in this article's Online Repository at www.jacionline.org), suggesting that the effects in coculture were due to IL-13.On a genomewide transcriptional level, genes that were differentially expressed in NHBE cells when cocultured in the presence of IL-33–stimulated mast cells or ILC2s correlated positively and linearly with genes differentially expressed in NHBE cells directly stimulated with IL-13 alone (Fig 1, G, and Table E1, and Fig 1, H, and Table E2, respectively, in this article's Online Repository at www.jacionline.org). We did not observe differential expression of genes that were dependent on IL-33 nor dependent on ILC2 in the context of IL-13 blockade, suggesting that transcriptional effects in NHBE cells were predominantly dependent on IL-13.Although our studies focused on IL-13, we cannot completely rule out potential effects of IL-4. Although mast cells have been reported to express IL-4, we did not detect IL-4 in our mast cell cultures (data not shown). Non–type 2 mediators including IL-8 and TNF-α were also induced in IL-33–activated mast cells and ILC2s, and may be involved in severe asthma via the recruitment of neutrophils and smooth muscle activation.9Barnes P.J. New molecular targets for the treatment of neutrophilic diseases.J Allergy Clin Immunol. 2007; 119 (quiz 63-4): 1055-1062Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Furthermore, the presence of other immune cells in the airway in an IL-33 milieu may result in an abundance of non–type 2 mediators, which could affect differential gene expression not otherwise seen in the coculture system. Thus, further studies need to be conducted in vivo to examine the effects of anti–IL-13 on airway inflammation, mucus expression, and hyperreactivity in response to IL-33.In conclusion, we have shown that IL-33–activated mast cells and ILC2s drive predominantly IL-13–regulated gene expression when cocultured with NHBE cells, which initiates a transcriptional program that can subsequently exacerbate airway pathology and contribute to fibrosis, eosinophilia, and mucous metaplasia. Although mast cells and ILC2s produce a diverse set of cytokines and other mediators of intercellular communication in response to IL-33, the predominant effect on airway epithelium in this in vitro coculture system is mediated by IL-13. Type 2 inflammation, characterized by increased expression of the cytokines IL-4, IL-5, and IL-13 and eosinophilic infiltration of the airways, is a feature of some patients with asthma and is prominent in patients with frequent exacerbations.1Lemanske Jr., R.F. Busse W.W. Asthma: clinical expression and molecular mechanisms.J Allergy Clin Immunol. 2010; 125: S95-S102Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar We and others have shown that “TH2 high” asthmatic patients exhibit distinct IL-13–induced gene expression in airway epithelial cells including eosinophil chemokine (C-C) ligand 26 (CCL26), periostin (POSTN), a biomarker of systemic eosinophilic airway inflammation and subepithelial fibrosis in asthmatic patients, and an increased ratio of mucin (MUC)5AC to MUC5B (MUC5AC:MUC5B), all of which may contribute to disease pathology.2Woodruff P.G. Boushey H.A. Dolganov G.M. Barker C.S. Yang Y.H. Donnelly S. et al.Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids.Proc Natl Acad Sci U S A. 2007; 104: 15858-15863Crossref PubMed Scopus (642) Google Scholar, 3Jia G. Erickson R.W. Choy D.F. Mosesova S. Wu L.C. Solberg O.D. et al.Periostin is a systemic biomarker of eosinophilic airway inflammation in asthmatic patients.J Allergy Clin Immunol. 2012; 130: 647-654.e10Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 4Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. et al.Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (130) Google Scholar Mast cells are localized to the airway epithelium in “TH2 high” asthmatic patients, are associated with disease severity, and can be activated by epithelial cytokines including IL-33.5Deckers J. Branco Madeira F. Hammad H. Innate immune cells in asthma.Trends Immunol. 2013; 34: 540-547Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar IL-5 and IL-13–producing type 2 innate lymphoid cells (ILC2s) contribute significantly in animal models of type 2 inflammation and may potentially play a role in human asthma by responding to cytokines including IL-33, IL-25, and thymic stromal lymphopoietin.6Spits H. Artis D. Colonna M. Diefenbach A. Di Santo J.P. Eberl G. et al.Innate lymphoid cells–a proposal for uniform nomenclature.Nat Rev Immunol. 2013; 13: 145-149Crossref PubMed Scopus (1707) Google Scholar IL-33 is released after in vitro exposure to allergen extracts by human airway epithelial cells and in lungs of allergen-challenged mice, and has been detected in bronchoalveolar lavage fluid and airway smooth muscle in patients with asthma.7Cayrol C. Girard J.P. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy.Curr Opin Immunol. 2014; 31C: 31-37Crossref PubMed Scopus (443) Google Scholar However, specific effects of IL-33 on airway epithelium in the context of an inflammatory microenvironment have not been well characterized. As both mast cells and ILC2s have been found in human lungs, express the IL-33 receptor ST2, and produce IL-13, we sought to characterize the interactions of these cells with the airway epithelium in a proinflammatory context and determine the role of IL-13–dependent and IL-13–independent pathways in airway gene expression. We investigated global transcriptional patterns in IL-33–stimulated primary cultured in vitro differentiated human mast cells using gene expression microarrays, focusing on cytokines, chemokines, and growth factors (CCGFs),4Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. et al.Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (130) Google Scholar as they represent soluble mediators of intercellular communication (Table I). Detailed methods of culture and stimulation are given in this article's Methods section in the Online Repository at www.jacionline.org. Consistent with previous reports,8Allakhverdi Z. Smith D.E. Comeau M.R. Delespesse G. Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells.J Immunol. 2007; 179: 2051-2054Crossref PubMed Scopus (409) Google Scholar we found that IL-33–stimulated mast cells upregulated multiple non–type 2 proinflammatory mediators compared with controls. However, IL-5 and IL-13 were among the top 5 most highly induced CCGF genes in mast cells on IL-33 stimulation, suggesting that a dominant response to IL-33 in mast cells is the increased expression of type 2 inflammatory cytokines. We not only detected significantly higher IL-5 (see Fig E1, A, in this article's Online Repository at www.jacionline.org) and IL-13 protein (Fig E1, B) in supernatants from IL-33–treated mast cells compared with controls but also observed increased production of non–type 2 mediators TNF-α and IL-8 in supernatants from IL-33–stimulated mast cells (Fig E1, C and D). To assess IL-33's effects on ILC2s, we isolated lineage-negative human ILC2s from blood buffy coats (see Fig E2, A, in this article's Online Repository at www.jacionline.org) and expanded them in vitro in the presence of IL-2, IL-7, IL-25, IL-33, and thymic stromal lymphopoietin. We then assessed the production of TH2 cytokines in response to IL-33. IL-33 significantly enhanced IL-5 (Fig E2, B) and IL-13 (Fig E2, C) production from ILC2s, although substantial quantities of IL-5 and IL-13 were secreted from ILC2s even without IL-33 restimulation. IL-33 also significantly increased the production of TNF-α and IL-8 (see Fig E3, A and B, in this article's Online Repository at www.jacionline.org) in ILC2s. IL-4 was detectable albeit at much lower concentrations than IL-5 and IL-13, and trended toward an increase in ILC2s after IL-33 stimulation (Fig E3, C); however, this was not statistically significant. Because mast cells and ILC2s stimulated with IL-33 expressed both type 2 and non–type 2 CCGFs, we sought to determine which of these factors could activate primary mucociliary differentiated normal human bronchial epithelial NHBE cells. Mast cells required IL-33 for IL-13 production. However, because ILC2 differentiation is dependent on IL-33 and mature ILC2s produced abundant levels of type 2 cytokines without IL-33 restimulation, we assessed the effects of ILC2-NHBE cells coculture without additional IL-33. We cocultured NHBE cells with IL-33–stimulated mast cells or mature ILC2s and examined the expression of IL-13–regulated genes CCL26, POSTN, MUC5AC, and MUC5B. In NHBE cells alone, IL-33 did not change the expression of CCL26 (Fig 1, A), POSTN (Fig 1, B), or MUC5AC:MUC5B (Fig 1, C). In the presence of mast cells, IL-33 treatment significantly upregulated CCL26, POSTN, and MUC5AC:MUC5B compared with mast cell-NHBE cells medium control. Anti–IL-13 strongly and significantly suppressed differential expression of CCL26, MUC5AC:MUC5B, and POSTN compared with control antibody. ILC2 coculture led to significantly increased expression of CCL26 (Fig 1, D) and POSTN (Fig 1, E), which was blocked by the addition of anti–IL-13, suggesting a predominant role of IL-13 in this system. We observed a trend toward an increase in MUC5AC:MUC5B (Fig 1, F), which was reduced by blocking IL-13 although this did not reach nominal statistical significance. IL-33–treated NHBE cells in the presence of mast cells had higher levels of IL-13 and periostin in basolateral supernatants compared with control (see Fig E4, B and C, in this article's Online Repository at www.jacionline.org), suggesting that the effects in coculture were due to IL-13. On a genomewide transcriptional level, genes that were differentially expressed in NHBE cells when cocultured in the presence of IL-33–stimulated mast cells or ILC2s correlated positively and linearly with genes differentially expressed in NHBE cells directly stimulated with IL-13 alone (Fig 1, G, and Table E1, and Fig 1, H, and Table E2, respectively, in this article's Online Repository at www.jacionline.org). We did not observe differential expression of genes that were dependent on IL-33 nor dependent on ILC2 in the context of IL-13 blockade, suggesting that transcriptional effects in NHBE cells were predominantly dependent on IL-13. Although our studies focused on IL-13, we cannot completely rule out potential effects of IL-4. Although mast cells have been reported to express IL-4, we did not detect IL-4 in our mast cell cultures (data not shown). Non–type 2 mediators including IL-8 and TNF-α were also induced in IL-33–activated mast cells and ILC2s, and may be involved in severe asthma via the recruitment of neutrophils and smooth muscle activation.9Barnes P.J. New molecular targets for the treatment of neutrophilic diseases.J Allergy Clin Immunol. 2007; 119 (quiz 63-4): 1055-1062Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Furthermore, the presence of other immune cells in the airway in an IL-33 milieu may result in an abundance of non–type 2 mediators, which could affect differential gene expression not otherwise seen in the coculture system. Thus, further studies need to be conducted in vivo to examine the effects of anti–IL-13 on airway inflammation, mucus expression, and hyperreactivity in response to IL-33. In conclusion, we have shown that IL-33–activated mast cells and ILC2s drive predominantly IL-13–regulated gene expression when cocultured with NHBE cells, which initiates a transcriptional program that can subsequently exacerbate airway pathology and contribute to fibrosis, eosinophilia, and mucous metaplasia. Although mast cells and ILC2s produce a diverse set of cytokines and other mediators of intercellular communication in response to IL-33, the predominant effect on airway epithelium in this in vitro coculture system is mediated by IL-13. 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