There is an unmet need in severe asthma where approximately 40% of patients exhibit poor β-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated-Cl− channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ~580,000 compounds. The anthelmintics niclosamide, nitazoxanide and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting β-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the β-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

IL-33 is a mediator of allergic inflammation and is localized in mucosal tissues to respond rapidly to environmental insults. These include allergens themselves, which can directly activate IL-33 through their intrinsic proteolytic activity.

Atopic dermatitis (AD) often precedes asthma and food allergy, indicating that epicutaneous sensitization to allergens may be important in the induction of allergic responses at other barrier surfaces. Thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 are two cytokines that may drive type 2 responses in the skin; both are potential targets in the treatment of allergic diseases. We tested the functional role of IL-33 and the interplay between IL-33 and TSLP inmouse models of atopic march and gastrointestinal (GI) allergy. IL-33-driven allergic disease occurred in a TSLP-independent manner. In contrast, mice lacking IL-33 signaling were protected from onset of allergic diarrhea in TSLP-driven disease. Epithelial-derived IL-33 was important in this model, as specific loss of IL-33 expression in the epithelium attenuated cutaneous inflammation. Notably, the development of diarrhea following sensitization with TLSP plus antigen was ameliorated even when IL-33 was blocked after sensitization. Thus, IL-33 has an important role during early cutaneous inflammation and during challenge. These data reveal critical roles for IL-33 in the "atopic march'' that leads from AD to GI allergy.

BACKGROUND: Ozone increases IL-33 in the lungs, and obesity augments the pulmonary effects of acute ozone exposure.OBJECTIVES: We assessed the role of IL-33 in the augmented effects of ozone observed in obese mice.METHODS: Lean wildtype and obese db/db mice were pretreated with antibodies blocking the IL-33 receptor, ST2, and then exposed to ozone (2 ppm for 3 hr). Airway responsiveness was assessed, bronchoalveolar lavage (BAL) was performed, and lung cells harvested for flow cytometry 24 hr later. Effects of ozone were also assessed in obese and lean mice deficient in.gamma delta T cells and their wildtype controls.RESULTS AND DISCUSSION: Ozone caused greater increases in BAL IL-33, neutrophils, and airway responsiveness in obese than lean mice. Anti-ST2 reduced ozone-induced airway hyperresponsiveness and inflammation in obese mice but had no effect in lean mice. Obesity also augmented ozone-induced increases in BAL CXCL1 and IL-6, and in BAL type 2 cytokines, whereas anti-ST2 treatment reduced these cytokines. In obese mice, ozone increased lung IL-13(+) innate lymphoid cells type 2 (ILC2) and IL-13(+).gamma delta T cells. Ozone increased ST2(+) gamma delta T cells, indicating that these cells can be targets of IL-33, and.gamma delta T cell deficiency reduced obesity-related increases in the response to ozone, including increases in type 2 cytokines.CONCLUSIONS: Our data indicate that IL-33 contributes to augmented responses to ozone in obese mice. Obesity and ozone also interacted to promote type 2 cytokine production in.gamma delta T cells and ILC2 in the lungs, which may contribute to the observed effects of IL-33.

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (β = -0.21 SD, P = 2.5×10–16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10–4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Chronic rhinosinusitis (CRS) represents a heterogeneous disease comprising several different subtypes that are grouped together by common criteria lasting at least 12 weeks. The classification of CRS subtypes remains largely clinical and carries little prognostic value. At present, clinicians possess limited capability in predicting a patient's disease course and anticipating response to available therapies. More recently, efforts have focused on categorizing CRS subtypes into endotypes. Endotypes organize disease subtypes according to molecular patterns believed to underlie the expression of different clinical phenotypes.1Akdis C.A. Bachert C. Cingi C. Dykewicz M.S. Hellings P.W. Naclerio R.M. et al.Endotypes and phenotypes of chronic rhinosinusitis: a PRACTALL document of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma &Immunology.J Allergy Clin Immunol. 2013; 131: 1479-1490Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar The push for disease endotyping in CRS derives from the successful application of this method in asthma, a disease sharing many pathophysiologic features with CRS. Indeed, the presence of type 2 inflammation represents an important point of disease stratification, both in asthma and in CRS. In asthma, increased blood and tissue eosinophilia, IgE levels, and expression of type 2 inflammatory biomarkers, such as IL-4, IL-5, IL-13, and periostin, have been leveraged to endotype disease, develop novel biologic therapies, and monitor response to treatment.2Gauthier M. Ray A. Wenzel S.E. Evolving concepts of asthma.Am J Respir Crit Care Med. 2015; 192: 660-668Crossref PubMed Scopus (180) Google Scholar In this study, we characterized the expression patterns of several type 2 inflammatory cytokines of interest in different clinical subtypes of CRS. To this end, we use a top-down approach in the form of large-scale microarray gene profiling techniques in 130 patients representing different clinical subtypes of CRS: aspirin-exacerbated respiratory disease (AERD), allergic fungal rhinosinusitis (AFRS), healthy control (HC), chronic rhinosinusitis without nasal polyps (CRSsNP), and chronic rhinosinusitis with nasal polyps (CRSwNP). We believe that a study of this design is well-suited to elucidate patterns of differential gene expression between subtypes that may serve to advance disease endotyping in CRS. To date, this is the largest microarray study of its type in CRS. Table E1 in this article's Online Repository at www.jacionline.org summarizes the different disease subtypes enrolled in each group and relevant clinical characteristics. We first performed hierarchical cluster analysis using microarray data from 130 patients with different subtypes of CRS. This resulted in the identification of 500 distinct gene expression clusters that are illustrated in a heat map (see Fig E1 in this article's Online Repository at www.jacionline.org). Many gene clusters had differential expression patterns characterized by either highly upregulated (red signal) or highly downregulated (green signal) gene expression. Polyp subtypes AERD, AFRS, and CRSwNP demonstrated similar patterns of increased or decreased cluster expression and segregated from CRSsNP and HC on the basis of their differential cluster expression, supporting a clear molecular delineation between polyp and nonpolyp CRS phenotypes. Clusters containing the most differentially expressed sequences between HC and disease, or those of particular interest, were examined for differential expression between subtypes. In addition, data were analyzed to identify correlative and reciprocal relationships between these clusters, and with other biological pathways of interest, especially those involved in type 2 inflammation. Sequences with the highest differential expression between groups were between HC and AFRS or between HC and AERD. The single highest differential signal was from a sequence specific to the soluble form of IgE (Fig 1, A). This expression was significantly upregulated in all subtypes when compared with HC, with AERD and AFRS demonstrating increased local IgE levels when compared with other disease subtypes. IgE expression was higher in patients with CRSwNP with asthma in comparison to patients with CRSwNP without asthma (P < .001), while other subtypes showed no difference in tissue IgE expression when comparing those with and without asthma. We next analyzed the expression of several canonical type 2 inflammatory cytokines (IL-4, IL-5, IL-13) and IFN-γ by digital droplet quantitative PCR (Fig 1, B). We also measured the expression of type 2 cytokine gene clusters that commonly act downstream of these cytokines (CCL13 + CCL18 clusters, CCL26 + periostin clusters, Fig 1, C and D). The cytokines CCL13, CCL18, CCL26, and periostin are of particular interest in CRS because they have been shown to mediate localized eosinophilic inflammation.3Kato A. Immunopathology of chronic rhinosinusitis.Allergol Int. 2015; 64: 121-130Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 4Wang M. Wang X. Zhang N. Wang N. Wang H. Li Y. et al.Association of periostin expression with eosinophilic inflammation in nasal polyps.J Allergy Clin Immunol. 2015; 136: 1700-1703Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar No subtypes demonstrated increased expression of IFN-γ, a canonical type 1 inflammatory cytokine, which is consistent with other studies showing lack of increased IFN-γ in CRS subtypes, including CRSsNP, in Japanese, Chinese, and American populations.3Kato A. Immunopathology of chronic rhinosinusitis.Allergol Int. 2015; 64: 121-130Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar All subtypes demonstrated relative increased expression of IL-13. Similarly, all subtypes exhibited increased expression of IL-5 when compared with HC. Only AFRS and CRSwNP subtypes demonstrated significant increases in IL-4 gene expression. Despite no differences detected between the mean copy of IL-13 and IL-4 among CRSwNP, AFRS, and CRSsNP samples, CRSwNP and AFRS had more samples expressing higher copies of IL-13 and IL-4 as compared with samples from patients with CRSsNP. Gene cluster expression analysis of CCL13 + CCL18 and CCL26 + periostin revealed overexpression in comparison to HC of these gene clusters in all CRS subtypes. Taken together, with the exception of IL-4, the expression of the above type 2 inflammatory markers did not separate clinical phenotypes but rather highlighted subgroups (possible endotypes) among the clinical phenotypes with different expression levels of these type 2 inflammatory markers when compared with HC. Because we could not readily delineate subtypes of CRS disease on the basis of relative expression of markers chosen in Fig 1, we explored additional components of type 2 inflammation that might be driving disease in CRS. The mast cell axis represents a central node of type 2 inflammation, capable of responding to both innate and adaptive immune arms to promote a type 2 response. Studies originating from the work of the senior author have found that mast cells are present at increased levels, independent of atopy, in polyp mucosa of patients with CRSwNP.5Shaw J.L. Ashoori F. Fakhri S. Citardi M.J. Luong A. Increased percentage of mast cells within sinonasal mucosa of chronic rhinosinusitis with nasal polyp patients independent of atopy.Int Forum Allergy Rhinol. 2012; 2: 233-240Crossref PubMed Scopus (32) Google Scholar We have also shown that inflamed mucosa of patients with CRSwNP harbor type 2 innate lymphoid cells expressing IL1RL1 (also known as ST2), a receptor for IL-33, and that these type 2 innate lymphoid cells secrete IL-13 in response to stimulation by IL-33.6Shaw J.L. Fakhri S. Citardi M.J. Porter P.C. Corry D.B. Kheradmand F. et al.IL-33-responsive innate lymphoid cells are an important source of IL-13 in chronic rhinosinusitis with nasal polyps.Am J Respir Crit Care Med. 2013; 188: 432-439Crossref PubMed Scopus (208) Google Scholar We wished to determine whether there existed a correlation between the expression of IL1RL1 and genes associated with mast cell activity, and, furthermore, assess whether these genes were exclusive to certain disease subtypes. We found that IL1RL1 transcripts were significantly overexpressed in polyp subtypes, AFRS and CRSwNP, when compared with CRSsNP and HC (Fig 2, A). In addition, the expression of IL1RL1 correlated with a cluster enriched for mast cell–related genes (tryptase cluster, Fig 2, B). We also found that IL1RL1 expression correlated with the expression of 21 genes associated with mast cell and eosinophil activity (see Table E2 in this article's Online Repository at www.jacionline.org). We performed an immunohistochemical analysis in a representative collection of surgically removed CRSwNP polyp tissue (Fig 2, C and D). IL-33 expression was evident in the epithelial cells and around vessels in all polyp samples (Fig 2, C). Immunohistochemistry indicated the presence of abundant eosinophils and mast cells, both known IL1RL1-expressing cells (Fig 2, D). Taken together, these results implicate an IL1RL1-mast cell signaling axis as a potential endotype marker and target for therapeutic intervention. Molecular pathways that drive disease in different CRS clinical phenotypes—so-called endotypes—need further clarification to facilitate the application of personalized therapies in CRS. Our top-down, microarray-based analysis allowed us to characterize the relative expression of several type 2 inflammatory gene clusters of interest in well-defined clinical subtypes of CRS, many of which take into account eosinophilic inflammation, comorbid asthma, aspirin sensitivity, fungal colonization, and atopy. Admittedly, we do not define specific endotypes in this study; however, we evaluated key type 2 inflammatory markers as a means of endotyping patients with CRS. We found that there were trends of elevated expression of certain type 2 inflammatory markers in clinical phenotypes but also found notable variation in expression within a given phenotype. One such marker, local IgE expression, was elevated in subgroups of patients with AERD, AFRS, and CRSwNP with asthma, clinical subtypes that characteristically exhibit increased local sinonasal eosinophilia. Our observations support previous studies that indicate a correlation between local sinonasal IgE expression and increased tissue eosinophilia.7Bachert C. Zhang N. Holtappels G. De Lobel L. van Cauwenberge P. Liu S. et al.Presence of IL-5 protein and IgE antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma.J Allergy Clin Immunol. 2010; 126: 962-968Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 8Tomassen P. Vandeplas G. Van Zele T. Cardell L.O. Arebro J. Olze H. et al.Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers.J Allergy Clin Immunol. 2016; 137: 1449-1456Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 9Bachert C. van Steen K. Zhang N. Holtappels G. Cattaert T. Maus B. et al.Specific IgE against Staphylococcus aureus enterotoxins: an independent risk factor for asthma.J Allergy Clin Immunol. 2012; 130: 376-381Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, E1De Schryver E. Devuyst L. Derycke L. Dullaers M. Van Zele T. Bachert C. et al.Local immunoglobulin E in the nasal mucosa: clinical implications.Allergy Asthma Immunol Res. 2015; 7: 321-331Crossref PubMed Scopus (69) Google Scholar Interestingly, with the exception of IL-4, all CRS subtypes demonstrated increased expression of several canonical type 2 inflammatory markers (Fig 1). The observed increase in these measured type 2 inflammatory markers in CRSsNP may in part be due to the high prevalence of comorbid allergic rhinitis in this patient population; however, multiple studies have suggested that atopic sinonasal inflammation represents a distinct process from that observed in CRS.E2Fritz S.B. Terrell J.E. Conner E.R. Kukowska-Latallo J.F. Baker J.R. Nasal mucosal gene expression in patients with allergic rhinitis with and without nasal polyps.J Allergy Clin Immunol. 2003; 112: 1057-1063Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, E3Wu J. Bing L. Jin H. Jingping F. Gene expression profiles of nasal polyps associated with allergic rhinitis.Am J Otolaryngol. 2009; 30: 24-32Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, E4Plager D.A. Kahl J.C. Asmann Y.W. Nilson A.E. Palanch J.F. Friedman O. et al.Gene transcription changes in asthmatic chronic rhinosinusitis with nasal polyps and comparison to those in atopic dermatitis.PLoS One. 2010; 5: e11450Crossref PubMed Scopus (61) Google Scholar Our findings complement those of Tomassen et al,8Tomassen P. Vandeplas G. Van Zele T. Cardell L.O. Arebro J. Olze H. et al.Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers.J Allergy Clin Immunol. 2016; 137: 1449-1456Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar which showed the presence of type 2 inflammation in both polyp and nonpolyp clinical subtypes, and highlight the complexity of the inflammatory milieu in CRS mucosa. Together, these findings prompt a need to investigate additional type 2 inflammatory pathways that better separate disease subtypes. Studies in asthma and CRS have highlighted multiple different mechanisms that can promote type 2 inflammation, including an adaptive immune component and a recently identified innate type 2 inflammatory component. Epithelial-derived cytokines, such as IL-25, IL-33, thymic stromal lymphopoietin, and mast cells, may play important roles in mediating type 2 inflammatory disease independent of adaptive immunity. Indeed, elevation of the IL-33 receptor, IL1RL1, in CRSwNP is not a novel finding; nonetheless, our results add credence to preceding studies that have shown the importance of this pathway in the pathogenesis of CRSwNP.E4Plager D.A. Kahl J.C. Asmann Y.W. Nilson A.E. Palanch J.F. Friedman O. et al.Gene transcription changes in asthmatic chronic rhinosinusitis with nasal polyps and comparison to those in atopic dermatitis.PLoS One. 2010; 5: e11450Crossref PubMed Scopus (61) Google Scholar, E5Baba S. Kondo K. Kanaya K. Suzukawa K. Ushio M. Urata S. et al.Expression of IL-33 and its receptor ST2 in chronic rhinosinusitis with nasal polyps.Laryngoscope. 2014; 124: E115-E122Crossref PubMed Scopus (52) Google Scholar, E6Endo Y. Hirahara K. Iinuma T. Shinoda K. Tumes D.J. Asou H.K. et al.The interleukin-33-p38 kinase axis confers memory T helper 2 cell pathogenicity in the airway.Immunity. 2015; 42: 294-308Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 6Shaw J.L. Fakhri S. Citardi M.J. Porter P.C. Corry D.B. Kheradmand F. et al.IL-33-responsive innate lymphoid cells are an important source of IL-13 in chronic rhinosinusitis with nasal polyps.Am J Respir Crit Care Med. 2013; 188: 432-439Crossref PubMed Scopus (208) Google Scholar In addition, to our knowledge, we present novel findings correlating IL1RL1 expression with mast cell activity in polyp subtypes and we extend these findings to the AFRS clinical subtype. We noted a trend toward increased IL1RL1 expression in the AERD clinical subtype (Fig 2, A); however, the small sample size of this group likely precluded achieving statistical significance. Taken together, our results implicate an IL1RL1-mast cell signaling axis as a potential disease marker, a mediator of type 2 inflammation and a potential target for therapeutic intervention. The study presented herein illustrates the complexity of the inflammatory makeup in CRS. Although features of type 2 inflammation appear active in many clinical subtypes of CRS, they may culminate in a type 2 inflammatory response via different mechanisms, including infection, barrier disruption, and allergy. In addition, the degree of type 2 inflammatory activation may be an important factor in differentiating disease endotypes. As clinical trials that test the efficacy of novel therapies in CRS progress, a better understanding of the specific molecular pathways that drive a patient's disease will aid in identifying those who will benefit most from these novel treatment strategies. We acknowledge Kim Merriam and Ken Ganley for their help with immunohistochemistry. Patients undergoing medically indicated functional endoscopic sinus surgery consented to having sinonasal tissue, which was removed as a standard of their surgery, collected and analyzed for gene expression. The Institutional Review Board at the University of Texas Health Science Center at Houston approved the study protocol. Patients were grouped into CRSwNP, CRSsNP, AERD, or AFRS according to criteria set forth in the European Position Paper on Rhinosinusitis and Nasal Polyps.E7Fokkens W.J. Lund V.J. Mullol J. Bachert C. Alobid I. Baroody F. et al.European Position Paper on Rhinosinusitis and Nasal Polyps 2012.Rhinol Suppl. 2012; : 1-298Google Scholar Patients were grouped into CRSwNP or CRSsNP on the basis of presence or absence of polyps on nasal endoscopy. Polyps documented by an ENT physician on nasal endoscopy at any time categorized a patient as CRSwNP subtype. Patients with CRSwNP were diagnosed with AFRS if there was evidence of eosinophil-rich mucus with noninvasive fungal hyphae, hypersensitivity to fungi, and characteristic radiographic findings.E8Bent J.P. Kuhn F.A. Diagnosis of allergic fungal sinusitis.Otolaryngol Head Neck Surg. 1994; 111: 580-588Crossref PubMed Scopus (586) Google Scholar AERD was characterized by the presence of asthma, the presence or documented history of nasal polyps, and history of intake of aspirin or nonsteroid anti-inflammatory drug inciting worsening respiratory symptoms. Patients classified as asthmatic had a previous diagnosis of asthma by a pulmonologist, allergist, and/or positive pulmonary function testing. In this study, CRSwNP excludes patients with AERD and AFRS. HCs were defined as patients with no history of atopy or asthma symptoms. These patients were undergoing sinus surgery as a component of surgical approach to the skull base for removal of benign pituitary lesion or repair of anterior skull base cerebrospinal fluid leak. A table delineating the number of patients enrolled according to each clinical subtype, along with relevant clinical factors, has been presented in Table E1. No clinical subtype was treated in an exclusive manner, in that they did not receive any specific medical or surgical therapy that deviated from the standard of care delivered to other subtypes. Per study protocol, all topical and systemic corticosteroids were withheld at least 4 weeks before surgery when tissue was harvested. A fragment of inflamed ethmoid mucosa was removed during endoscopic sinus surgery, immediately wrapped in aluminum foil and flash frozen in liquid nitrogen. Frozen biopsies were placed in lysis buffer and homogenized with a Qiagen TissueRupter (Qiagen, Valencia, Calif). In instances in which a substantial amount of mucosa was harvested during surgery, tissue was separated and treated as independent samples. For microarray analysis, this was the case in a select group of patients: AFRS (45 patients yielded 51 samples), CRSwNP (38 patients, 45 samples), and HC (17 patients, 22 samples). For droplet digital PCR (described below), we included RNA from subsequent enrollees in addition to the index (n = 130, 147 samples) patient set: AERD (6 patients, 6 samples), AFRS (49 patients, 55 samples), HC (17 patients, 23 samples). RNA was prepared using the mirVana miRNA isolation kit (Applied Biosystems, Carlsbad, Calif), modified to include an on-column treatment with RNase-free DNase (Qiagen). RNA quality was assessed by RNA Integrity Number values from the BioAnalyzer 2100 (Agilent, Palo Alto, Calif). Fifty nanogram total RNA was amplified using the Ovation RNA Amplification System V2 and WB reagent (Nugen, Inc, San Carlos, Calif). Of the amplified cDNA, 4.4 μg was labeled using the FL Ovation cDNA Biotin Module V2 (Nugen, Inc) according to the manufacturer's recommendations. The labeled cDNA was hybridized onto Affymetrix human genome HG-U133_Plus_2 arrays (Affymetrix, Santa Clara, Calif) and processed according to Affymetrix technical protocols. The average intensity of each array was scaled to a target intensity of 500. Single genes were analyzed by droplet digital PCR on the BioRad (Hercules, Calif) QX100 system, in duplex mode. Target gene probe sets were labeled with FAM, and the RBM22 reference normalizer probe set was labeled with VIC. All probe sets were standard Applied Biosystems (Grand Island, NY) catalog probe sets (IL-4, Hs00174122_m1; IL-5, Hs01548712_g1; IL-13, Hs00174379_m1; IFNG, Hs00989291_m1). Raw Affymetrix. CEL files were preprocessed in Array Studio (OmicSoft, Cary, NC; http://www.omicsoft.com) using its default procedures including data normalization by Robust Multi-array Average approach. Microarray data were analyzed for different patterns of gene expression by hierarchical clustering analysis. To identify sets of genes with highly correlated expression patterns, a set of 32,719 sequences with coefficients of variation greater than 0.03 across all the samples was used. This method was used to identify (dis)similarities between data sets based on a measurement of Euclidean distance. These (dis)similarities between data sets were organized into discrete clusters that are visualized as a dendrogram (Fig E1). We used Ward's linkage method, which uses within-cluster sum of squares computations to group clusters of gene expression. Using these methods, we identified 500 clusters of differential gene expression and evaluated the separation of different subtypes qualitatively in Fig E1. Clusters containing the most differentially expressed sequences, or some of previously identified biological significance, were then examined for differential expression between the disease groups and their relationships with each other (Fig 1). Tissue sections from formalin-fixed, paraffin-embedded human nasal polyps from patients with CRS undergoing routine surgical removal (n = 9) were cut at 5 microns, baked at 65°C for 30 to 60 minutes, deparaffinized in xylene, and rehydrated through graded ethanol solutions. Serial sections were stained with hematoxylin and eosin following standard methodology and specific immunohistochemical staining was performed. Antigen retrieval for tissue sections stained for IL-33 and mast cell tryptase was accomplished by steaming the slides in DIVA buffer (Biocare Medical #DV2004, Concord, Calif) for 60 minutes. Endogenous protein was blocked with Background Sniper (Biocare Medical, #BS966), and then endogenous avidin and biotin were blocked (Biocare Medical #AB972). Antigen retrieval for tissue sections stained for eosinophil major basic protein was accomplished by incubating the sections with Pepsin for 30 minutes (Biocare Medical, Carezyme II # PEP956). Slides were then incubated with anti–IL-33 (Goat polyclonal, R&D Systems #AF3625), anti–mast cell tryptase (Rabbit polyclonal, Abcam #ab35118, Cambridge, Mass), or anti–eosinophil major basic protein (Mouse monoclonal, AbD Serotec #MCA 5751, Raleigh, NC) diluted in DaVinci Green diluent (Biocare Medical #PD900). Next, endogenous peroxidase was quenched (Biocare Medical #PX968). Slides stained for IL-33 were incubated with biotinylated anti-goat IgG (Vector PK6105, Vector Labs, Burlingame, Calif) followed by ABC-HRP (Vector PK6105, Vector Labs). Slides stained with mast cell tryptase were detected with goat anti-rabbit horseradish peroxidase–labeled polymer (Biocare Medical #RHRP520). Primary antibody against eosinophil major basic protein was detected using a goat anti-mouse horseradish peroxidase–labeled polymer (Biocare Medical #RMRP520). Slides were then developed with DAB chromogen (Biocare Medical #DCB859), rinsed in water, counterstained in hematoxylin, dehydrated, cleared, and mounted.Table E1Clinical phenotypes of patients with CRS and HCs enrolled in studyCRS subtype (n)HC (17)AERD (5)AFRS (45)CRSwNP (38)CRSsNP (25)Age (y), median5343265048Sex: Male/female10/71/425/2023/1515/10Asthma (%)0/17 (0)5/5 (100)12/45 (27)21/38 (55)5/25 (20)Inhalant allergy (%)0 (0)3 (60)45 (100)25 (66)12 (52)Aspirin sensitivity (%)0 (0)5 (100)0 (0)0 (0)0 (0) Open table in a new tab Table E2Mast cell– and eosinophil-related gene cluster and its correlation with IL1RL1 (ST2) expressionCorrelationRankGeneDescriptionProbe0.8852922IL1RL1ST2242809_PM_at0.7231863IL1RL1ST2207526_PM_s_at0.6858954IL10IL10207433_PM_at0.6760265SAMSN1(Nash1, mast cell nuclear adaptor protein)1555638_PM_a_at0.6709936SOCS1SOCS1213337_PM_s_at0.6625897CCL23CCL23 (eosinophil-enriched chemokine)210549_PM_s_at0.6542159LYVE1Lymphatic vessel endothelial hyaluronan receptor 1220037_PM_s_at0.65325110IL18R1IL18 receptor206618_PM_at0.64899411SRGNSerglycin (Mast cell protease-associated proteoglycan)201858_PM_s_at0.64887112ADORA3Adenosine A3 receptor (expressed by eosinophils and macrophages)206171_PM_at0.6376414IGHA1Immunoglobulin heavy locus217469_PM_at0.63023216ALOX5APArachidonate 5-lipoxygenase-activating protein (FLAP)204174_PM_at0.62687918C3AR1Complement component 3a receptor 1 (mast cells?)209906_PM_at0.62548319SOCS1SOCS1210001_PM_s_at0.61968820CCL18CCL18209924_PM_at0.61945621IL1R1IL1 receptor202948_PM_at0.61886422HPGDSProstaglandin D synthase (mast cells)206726_PM_at0.61683223PMCHPromelanin-concentrating hormone206942_PM_s_at0.61417524CCND2Cyclin D2200951_PM_s_at0.61363225NFIL3Nuclear factor, IL-3 regulated (IL4-induced, regulates IgE)203574_PM_at0.60820127SRGNSerglycin (Mast cell protease-associated proteoglycan)201859_PM_at0.60793528CTSGCathepsin G (Mast cell protease)205653_PM_at0.60694830PIM1Pim-1 oncogene209193_PM_at0.60225532PTGDR2Prostaglandin D2 receptor 2 (CRTH2) (expressed by eosinophils and type 2 innate lymphoid cells)206361_PM_atThe genes listed above were identified as highly correlated with one another as well as with the expression of IL1RL1. Data shown were generated from pooled expression values from all subtypes of CRS. Correlation coefficients (leftmost column) were generated using a Spearman's rank test and the IL1RL1 probe (234066_at). For all correlations, P < .001. Open table in a new tab The genes listed above were identified as highly correlated with one another as well as with the expression of IL1RL1. Data shown were generated from pooled expression values from all subtypes of CRS. Correlation coefficients (leftmost column) were generated using a Spearman's rank test and the IL1RL1 probe (234066_at). For all correlations, P < .001.

Thymic stromal lymphopoietin (TSLP), interleukin-25 (IL-25), and IL-33 are important initiators of type 2-associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 2 inflammation and fibrosis is much less clear. Using chronic models of helminth infection and allergic lung inflammation, we show that collective disruption of TSLP, IL-25, and IL-33 signaling suppresses chronic and progressive type 2 cytokine-driven inflammation and fibrosis. In a schistosome lung granuloma model or during chronic Schistosoma mansoni infection in the liver, individual ablation of TSLP, IL-25, or IL-33/ST2 had no impact on the development of IL-4/IL-13-dependent inflammation or fibrosis. However, significant reductions in granuloma-associated eosinophils, hepatic fibrosis, and IL-13-producing type 2 innate lymphoid cells (ILC2s) were observed when signaling of all three mediators was simultaneously disrupted. Combined blockade through monoclonal antibody (mAb) treatment also reduced IL-5 and IL-13 expression during primary and secondary granuloma formation in the lungs. In a model of chronic house dust mite-induced allergic lung inflammation, combined mAb treatment did not decrease established inflammation or fibrosis. TSLP/IL-33 double-knockout mice treated with anti-IL-25 mAb during priming, however, displayed decreased inflammation, mucus production, and lung remodeling in the chronic phase. Together, these studies reveal partially redundant roles for TSLP, IL-25, and IL-33 in the maintenance of type 2 pathology and suggest that in some settings, early combined targeting of these mediators is necessary to ameliorate progressive type 2-driven disease.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. Inhaled PM induces innate immune responses by airway epithelial cells that may lead to the exacerbation or de novo development of airway disease. We have previously shown that 10-μm PM (PM10) activates the nucleotide-binding domain, leucine-rich repeat protein (NLRP) 3 inflammasome in human airway epithelial cells. Our objective was to determine the innate and adaptive immune responses mediated by the airway epithelium NLRP3 inflammasome in response to PM10 exposure. Using in vitro cultures of human airway epithelial cells and in vivo studies with wild-type and Nlrp3(-/-) mice, we investigated the downstream consequences of PM10-induced NLPR3 inflammasome activation on cytokine production, cellular inflammation, dendritic cell activation, and PM10-facilitated allergic sensitization. PM10 activates an NLRP3 inflammasome/IL-1 receptor I (IL-1RI) axis in airway epithelial cells, resulting in IL-1β, CC chemokine ligand-20, and granulocyte/macrophage colony-stimulating factor production, which is associated with dendritic cell activation and lung neutrophilia. Despite these profound innate immune responses in the airway epithelium, the NLRP3 inflammasome/IL-1RI axis is dispensable for PM10-facilitated allergic sensitization. We demonstrate the importance of the lung NLRP3 inflammasome in mediating PM10 exposure-associated innate, but not adaptive, immune responses. Our study highlights a mechanism by which PM10 exposure can contribute to the exacerbation of airway disease, but not PM10-facilitated allergic sensitization.

The UK Refractory Asthma Stratification Programme (RASP-UK) will explore novel biomarker stratification strategies in severe asthma to improve clinical management and accelerate development of new therapies. Prior asthma mechanistic studies have not stratified on inflammatory phenotype and the understanding of pathophysiological mechanisms in asthma without Type 2 cytokine inflammation is limited. RASP-UK will objectively assess adherence to corticosteroids (CS) and examine a novel composite biomarker strategy to optimise CS dose; this will also address what proportion of patients with severe asthma have persistent symptoms without eosinophilic airways inflammation after progressive CS withdrawal. There will be interactive partnership with the pharmaceutical industry to facilitate access to stratified populations for novel therapeutic studies.

Background and objectives Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work demonstrated that the systemic administration of IL-33 reduces the development of atherosclerosis in the apolipoprotein E-deficient (ApoE-/-) mouse model of the disease by induction of a Th1-to-Th2 shift. However, the role of endogenous IL-33 in the atherogenesis remains elusive. Materials and methods Atherosclerosis was induced in 10 week-old ApoE-/-, IL-33-/-ApoE-/- and ST2-/-ApoE-/- mice by feeding a high-cholesterol diet (1.25%, no cholate) for 10 weeks. Additionally, a group of ApoE-/- mice were injected with a neutralising anti-ST2 antibody or an isotype control during the period of the diet. The atherosclerotic lesion development was measured with Oil Red O in the thoracic-abdominal aorta and in the aortic sinus. The mRNA levels of several cytokines, including IL-6, IFNγ, IL-17, IL-5 and IL-10 were assessed in the aorta and in in vitro-stimulated lymph node cells. Results We observed no differences in lipid-staining area in the aortas of IL-33-/-ApoE-/- mice (8.84 ± 0.97; mean ± SEM; n = 9–25), ST2-/-ApoE-/- mice (6.95 ± 0.78), ApoE-/- mice untreated (7.05 ± 0.78), ApoE-/- mice injected with either the neutralising anti-ST2 antibody (6.08 ± 0.79) or the isotype control (6.16 ± 0.86) after high-cholesterol diet feeding. Similar results were obtained in the aortic sinus compared to ApoE-/- controls. Total serum cholesterol and triglyceride levels were not different compared to ApoE-/- controls. IL-33 expression in aortic tissue was comparable in ApoE-/- and ST2-/-ApoE-/- mice and absent in IL-33-/-ApoE-/- mice. There was no difference in the transcript levels of inflammatory cytokines in the aorta and in in vitro-stimulated lymph node cells. Conclusions These data indicate that in contrast to the anti-atherosclerotic effect of systemically administered recombinant IL-33, the endogenously produced cytokine and its receptor do not significantly influence the severity of atherosclerosis in ApoE-deficient mice fed with a high-cholesterol diet.

Background: FEV1 correlates with symptoms and exacerbation risk in asthma. Although considered an index of airway resistance (R), FEV1 also varies with vital capacity (VC) as a function of airway recruitment. Aims: Quantify the contributions of airway narrowing vs. closure by analyzing airflow limitation and bronchodilator reversibility in U-BIOPRED patients with severe (S) vs. mild-moderate (MM) disease. Methods: Patients with S (n=305) and MM (n=95) underwent spirometry and plethysmography (PFTs) in a cross-sectional design. Employing a single compartment lung model, FEV1 = (TLC-RV){1-exp[-1/(RC)]}1. PFT values were entered into the model to assess airway narrowing and closure in each cohort. Results: Baseline % predicted FEV1 (67.4 vs. 88.4%; p Conclusion: Modeling of PFT data from U-BIOPRED reveals distinct patterns of baseline physiology and bronchodilator responsiveness in S and MM cohorts. S display lower FEV1 with airway collapse and gas trapping, partly reversible with bronchodilators. MM display minimal gas trapping, less bronchodilator %ΔFEV1, but larger changes in R suggesting greater b-agonist effect on airway tone in milder disease.

Background: TH2 inflammation is considered important in asthma regardless of atopic status, but many asthmatics have TH2 biomarker levels similar to healthy controls. The clinical significance of TH2 biomarker elevation in asthma is unclear. Aims: Compare clinical features and lung function of severe (S) and mild-moderate (MM) asthmatics as a function of TH2 biomarker status. Methods: For this analysis, S and MM U-BIOPRED patients were classified as “TH2-Elevated” (TH2e) or “TH2-reduced” (TH2r) based on serum IgE, blood eosinophils, and serum periostin (> median for >2 = TH2e). Demographics, medications, exacerbation history, PFTs, and disease control were assessed vs biomarker status. Results: The proportion of TH2e patients in S and MM was 51% and 56% respectively. Among S, age (53.4 vs 50.2 yrs.), exacerbation rates (13.8% vs. 14.7%), disease control (ACQ5: 2.07 vs. 2.27U), medication use (inhaled/oral steroids), serum acute phase reactants (CRP: 6.7 vs 6.6 mg/dL), % pred FEV1 (63.4 vs. 69.4%) and FVC (84.8 vs. 88.7%; ), RV/TLC (0.44 vs. 0.42) and bronchodilator reversibility (19.9 vs 16.3%) were not significantly different (p>0.05) in TH2e and TH2r subgroups. MM clinical features, disease control, and PFTs were similar in TH2e vs. r patients, but TH2e MM patients displayed higher bronchodilator reversibility. Conclusion: TH2e vs.TH2r patients in S and MM U-BIOPRED cohorts displayed similar demographics, clinical features, and physiology despite distinct biomarker profiles. Increased IgE, eosinophils, and periostin were not associated with worse disease control or increased medication use in severe and mild-moderate asthma.

Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work demonstrated that the systemic administration of recombinant IL-33 reduces the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice by inducing a Th1-to-Th2 shift. The objective of our study was to examine the role of endogenous IL-33 and ST2 in atherosclerosis. ApoE(-/-), IL-33(-/-)ApoE(-/-), and ST2(-/-)ApoE(-/-) mice were fed with a cholesterol-rich diet for 10 weeks. Additionally, a group of ApoE(-/-) mice was injected with a neutralizing anti-ST2 or an isotype control antibody during the period of the cholesterol-rich diet. Atherosclerotic lesion development was measured by Oil Red O staining in the thoracic-abdominal aorta and the aortic sinus. There were no significant differences in the lipid-staining area of IL-33(-/-)ApoE(-/-), ST2(-/-)ApoE(-/-), or anti-ST2 antibody-treated ApoE(-/-) mice, compared to ApoE(-/-) controls. The absence of IL-33 signaling had no major and consistent impact on the Th1/Th2 cytokine responses in the supernatant of in vitro-stimulated lymph node cells. In summary, deficiency of the endogenously produced IL-33 and its receptor ST2 does not impact the development of atherosclerosis in ApoE-deficient mice.

Advanced vehicle control systems (AVCS) offer great potential to reduce traffic congestion, decrease travel times, and increase safety. Automated lateral control is an integral part of AVCS. To date, most research has focused on lateral control for lane and curve tracking, and lane merging. To implement an automated control system, it must be able to sense and safely react to emergency situations. An emergency lateral control strategy is presented that accounts for changing vehicle speed using continuous gain equations. A linear vehicle/tyre model is used in the development of a linear state model. A nonlinear vehicle/tyre model is used in the optimization of the feedback gains. Because the state model is velocity dependent, feedback gains are derived for discrete vehicle speeds. These data points are then used to derive continuous gain equations for the feedback gains. The performance using the gain equations is compared to that of using constant gains. Conclusions are drawn about the performance and robustness of the controller using the continuous gain equations. Vehicle response is improved with the use of continuous gain equations, for both single and double lane changes.

BACKGROUND:Exercise-induced bronchoconstriction (EIB) is a prototypical feature of indirect airway hyperresponsiveness. Mast cells are implicated in EIB, but the characteristics, regulation, and function of mast cells in patients with EIB are poorly understood.OBJECTIVES:We sought to examine mast cell infiltration of the airway epithelium in patients with EIB and the regulation of mast cell phenotype and function by epithelially derived cytokines.METHODS:Endobronchial biopsy specimens, epithelial brushings, and induced sputum were obtained from asthmatic patients with and without EIB and healthy control subjects. Mast cell proteases were quantified by using quantitative PCR, and mast cell density was quantified by using design-based stereology. Airway epithelial responses to wounding and osmotic stress were assessed in primary airway epithelial cells and ex vivo murine lung tissue. Mast cell granule development and function were examined in cord blood-derived mast cells.RESULTS:Tryptase and carboxypeptidase A3 expression in epithelial brushings and epithelial mast cell density were selectively increased in the asthma group with EIB. An in vitro scratch wound initiated the release of thymic stromal lymphopoietin, which was greater in epithelial cells derived from asthmatic patients. Osmotic stress induced the release of IL-33 from explanted murine lungs, which was increased in allergen-treated mice. Thymic stromal lymphopoietin combined with IL-33 increased tryptase and carboxypeptidase A3 immunostaining in mast cell precursors and selectively increased cysteinyl leukotriene formation by mast cells in a manner that was independent of in vitro sensitization.CONCLUSIONS:Mast cell infiltration of the epithelium is a critical determinant of indirect airway hyperresponsiveness, and the airway epithelium might serve as an important regulator of the development and function of this mast cell population.