Sputum transcriptomics reveal upregulation of IL-1 receptor family members in patients with severe asthma

Abstract

Background: Sputum analysis in asthma is used to define airway inflammatory processes and may guide therapy. Objective: To determine differential gene and protein expression in sputum samples from patients with severe asthma (SA) compared to mildmoderate non-smoking asthmatics (MMA). Methods: Induced sputum was obtained from non-smoking SA (SAn), smokers/ex-smokers with SA (SAsm), MMA and healthy non-smoking controls. Differential cell counts, microarray analysis of cell pellets and SOMAscan analysis of sputum analytes was performed. CRID3 was used to inhibit the inflammasome in a mouse model of severe asthma. Results: Eosinophilic and mixed neutrophilic/eosinophilic inflammation were more prevalent in SA compared to MMA. 42 genes probes were upregulated (>2-fold) in SAn compared to MMA including IL-1R family and NRLP3 inflammasome members (FDR<0.05). The inflammasome proteins NLRP1, NLRP3 and NLRC4 were associated with neutrophilic asthma and with sputum IL--13-induced Th2 signature and IL1RL1 mRNA expression. These differences were sputum-specific since no activation of NLRP3 or enrichment of IL-1R family genes in bronchial brushings or biopsies in SA was observed. Expression of NLRP3 and of the IL-1R family genes was validated in the Airway Disease Endotyping for Personalized Therapeutics (ADEPT) cohort. Inflammasome inhibition using CRID3 prevented airway hyperresponsiveness and airway inflammation (both neutrophilia and eosinophilia) in a mouse model of severe allergic asthma.Conclusion: IL1RL1 gene expression is associated with eosinophilic SA whilst NLRP3 inflammasome expression is highest in neutrophilic SA. Th2-driven eosinophilic inflammation and neutrophil-associated inflammasome activation may represent interacting pathways in SA.Imperial College of Science, Technology and Medicine We enclose a revised version of the above manscript entitled 'Sputum transcriptomics reveal upregulation of IL-1 receptor family members in severe asthma' by Rossios and collagues.We have responded to the Reviewer's comments in a point by point manner below and have incorporated the changes requested.We hope that the manuscript is now acceptable for publication. Responses to CommentsImperial College of Science, Technology and Medicine EDITOR'S SPECIFIC COMMENTS: Thank you for your thoughtful revision of this manuscript. However, I agree with Reviewer 2 in that adjusting for cell composition will allow you to determine whether your results are driven largely by differences in cellular composition or by true differences in gene expression. This will affect the interpretation of your results and provide important biological insight. Response: we have added this detail as detailed in response to Reviewers 1 and 2 below. COMMENTS FROM REVIEWER #1:The authors have addressed most of my original comments and have rewritten some sections of the manuscript to increase overall clarity. Response: We thank the Reviewer for their helpful comments which have improved the paper considerably.The one issue they did not address is...

Complex association patterns for inflammatory mediators in induced sputum from subjects with asthma

“…As indicated in the introduction, other recent studies in severe asthma have reported mainly transcriptomic gene expression data from various cell sources, including sputum, epithelial brush and biopsy, in addition to more limited information for protein analysis. Proteins identified by Somascan in sputum supernatants of U‐BIOPRED subjects presented only mild/moderate asthma, or severe asthma data (either smoking or not) compared to healthy controls subjects, without comparison between mild/moderate and severe asthma groups . They noted upregulation of IL‐1 receptor family members by gene expression in those with severe asthma not only compared to healthy controls but also to the mild/moderate asthma group.…”

“…Moreover, this approach avoids reliance on measures of mRNA which lack linear association with actual protein synthesized . Although several recent reports have examined gene expression patterns in sputum and bronchial epithelial samples from subjects with severe asthma, these have included more limited information on corresponding protein analyses . One report from U‐BIOPRED, examining protein differences between 4 clinically identified cluster groups, found 9 differentially abundant sputum proteins in more severe clusters compared to moderate, well‐controlled asthma with normal FEV1 .…”

“…23 Although several recent reports have examined gene expression patterns in sputum and bronchial epithelial samples from subjects with severe asthma, these have included more limited information on corresponding protein analyses. [24][25][26][27] One report from U-BIOPRED, examining protein differences between 4 clinically identified cluster groups, found 9 differentially abundant sputum proteins in more severe clusters compared to moderate, well-controlled asthma with normal FEV1. 28 However, the 1 cluster group with greatest number of differentially abundant proteins was characterized by 16-packyear smoking history which may influence and contribute to these elevated proteins.…”

The Role of Lung and Gut Microbiota in the Pathology of Asthma

“…Non-type 2 asthma, observed more often in obesity-related asthma, neutrophilic asthma, and paucigranulocytic asthma (Kuo et al, 2017;Cosío et al, 2017;Ray and Kolls, 2017), is usually connected with poor responsiveness to steroids and more severe phenotypes, even in childhood (Ramratnam et al, 2017). Nontype 2 inflammation is characterized by infiltration of Th1 and Th17 cells and neutrophils, the presence of type I interferons, NLRP3 inflammasome activation, and an IL-1b and IL-17 signature (Rossios et al, 2018;Kim et al, 2017;Tan et al, 2019). The effect of microbiota dysbiosis on asthma heterogeneity seems intuitive, but, until recently (Taylor et al, 2018;Boutin et al, 2017), lung and gut microbiome composition have not been addressed in (Turnbaugh et al, 2007), lungs were not sampled, partially due to the existing dogma at the time that lungs are sterile.…”

Six gene and TH2 signature expression in endobronchial biopsies of participants with asthma

“…It has also been reported that the inhibition of NLRP3 prevents neutrophilia and decreases airway hyper-responsiveness. 34 CPA3 is a metalloexopeptidase specifically expressed in a particular subtype of mast cells in combination with tryptase. 35 Expression of CPA3 has been associated with TH2-high asthma in sputum and epithelial brushings of steroid naïve asthma.…”