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Introduction: Interleukin 33 (IL33) and its receptor (IL1RL1/ST2) play important roles in asthma development. Multiple IL33 isoforms have been identified, while IL1RL1 contains coding region polymorphisms associated with asthma. Aim: To characterise the ability of IL33 isoforms to activate IL1RL1 signalling and determine the role of IL1RL1 TIR signalling domain haplotypes. Methods: A NFKB/AP1 promoter driven secretory alkaline phosphatase (SEAP) cell line was engineered to express asthma risk or protective IL1RL1 TIR domain haplotype receptors. Cells were treated with recombinant IL33 isoforms (95-270aa, 99-270aa, 102-270aa, 112-270aa, 113-270aa, Exon 3-4 deletion & oxidisation resistant) at 1, 10, 25 & 50ng/ml for 24 hours. Activity was measured by colorimetric assay (SEAP) & ELISA (IL8). Results: All IL33 variants activated IL1RL1, with elevated SEAP/IL8 responses observed in risk haplotype carriers. Only the 99-270aa & oxidisation resistant forms activated the protective haplotype. The two longest isoforms gave the greatest maximal response, while the Exon 3-4 deletion variant had limited activation. Oxidisation resistant IL33 had the largest SEAP signal, but limited IL8 response. Conclusion: The IL1RL1 asthma protective haplotype attenuates IL33 driven inflammation irrespective of isotype. The increased signalling observed for longer IL33 isoforms generated in vivo by proteolytic cleavage via e.g. allergens may be asthma-relevant. The limited activity of the Exon 4-5 deletion suggests a protective role for this variant as a competitive antagonist. Overall, we demonstrate that activation of the IL33/IL1RL1 axis is complex, with response governed by a combination of IL33 isoform and IL1RL1 receptor polymorphisms.

Introduction: No definite cure is available yet for asthma. A subset of asthmatics goes into spontaneous remission and becomes symptom free. Comparison of gene expression in patients with persistent asthma and asthma remission may provide better understanding of the biological mechanisms leading to the remission of asthma and the identification of novel therapeutic targets. Aim: To find differential gene expression in bronchial biopsies from patients with persistent asthma and subjects with well-documented asthma remission. Methods: We performed RNA sequencing in RNA isolated from bronchial biopsies from 25 patients with persistent asthma and 38 subjects with asthma remission, who did not use inhaled corticosteroids. Asthma remission was defined as no symptoms in the last 3 years and no medication in the past 3 months. Differences in raw read counts between persistent asthma and asthma remission were analysed using DESeq2 adjusting for age, gender and smoking status as possible confounders. The FDR was controlled at 10%. Results: We found that the expression of keratin 13 ( KRT13 ) was lower in subjects with complete asthma remission compared to persistent asthmatics (log2FC = 5.4, q-value = 0.078). KRT13 encodes a subunit of keratin intermediate filaments characteristic of squamous epithelium and (bronchial) basal cells. Conclusion: This is the first study on gene expression in bronchial biopsies from asthma remission subjects. We demonstrate that the expression of KRT13 is lower in bronchial biopsies from subjects in remission compared to persistent asthmatics, suggesting less squamous metaplasia and/or basal cell hyperplasia in the airways. Funded by GlaxoSmithKline

Genetic association of polymorphisms in the IL6R gene with severity of asthma, high levels of systemic sIL-6R and poor lung function has been reported (Hawkins et al. JACI 2012; 130:510-5). Higher levels of systemic IL-6 have been shown to be associated with asthma severity, elevated BMI, CRP and blood neutrophils (Peters et al. Lancet Respir Med 2016; 4:574-84). In this study, we investigated whether sputum IL-6 pathway activity is aberrant in a subgroup of asthma patients and is associated with particular clinical characteristics. We used a hypothesis driven analysis by investigating sputum transcriptomics (n=77), sputum and blood proteomics and clinical data from UBIOPRED consortium severe asthma cohort. As asthma is a heterogeneous disease, we used a clustering approach to identify subgroups of patients using genes correlated with IL6R sputum mRNA. The analysis identified two subgroups with high (n=33) and low (n=28) IL6R expression. Mean IL6R expression level in the high IL6R group was 2.8-fold (p This IL6R-high sub-population of severe asthma patients with high sputum neutrophilia has a high unmet need that could potentially benefit from targeting IL-6 pathway.

Cigarette smoke (CS) exposure is known to induce immunogenic cell death and release of Damage Associated Molecular Patterns (DAMPs) from airway epithelial cells. Previously, we have shown that CS exposure induces release of a specific pattern of DAMPs into BAL fluid of mice and serum of COPD patients. However, the role of DAMPs in CS-induced airway inflammation is still unknown. Here, we hypothesized that DAMPs increase pro-inflammatory activity of airway epithelial cells and induce airway inflammation. Airway epithelial cells isolated from COPD patients and healthy controls were stimulated with several DAMPs, including LL37 and mitochondrial DAMPs (MTDs). The levels of CXCL8 in supernatant were used as a marker for pro-inflammatory activity. Furthermore, mice previously determined as susceptible (BALB/cByJ) or non-susceptible (DBA/2J) for the development of CS-induced neutrophilic airway inflammation, were treated intranasally with recombinant LL37, isolated MTDs or BSA as a control, and inflammatory cell count and KC levels in BAL were analyzed. Both LL37 and MTDs were able to significantly induce CXCL8 secretion in epithelial cells from both COPD patients and healthy controls. Upon treatment with LL37 and MTDs, neutrophilic airway inflammation was induced in susceptible BALB/cByJ mice but not in non-susceptible DBA/2J mice. In conclusion, we show for the first time that LL37 and MTDs are potent inducers of pro-inflammatory responses, directly activating bronchial epithelial cells and inducing neutrophilic inflammation in mice. Thus, induction of immunogenic cell death by CS and subsequent release of LL37 and MTDs may contribute to airway inflammation in COPD.