Casale, Thomas B.

Abstract Background Regulator of G protein signaling (RGS)2 terminates bronchoconstrictive Gaq signaling, and reductions in RGS2 expression associate with a hypercontractility phenotype. RGS2 promoter variants rs2746071 and rs2746072 are hypofunctional in gene reporter assays, and individuals with severe asthma homozygous for these two RGS2 variants experience higher asthma exacerbation rates. Whether these RGS2 promoter variants directly cause a hypercontractile human airway smooth muscle (HASM) phenotype is unknown. Methods We used the hTERT immortalized HASM cell line, derived from a human non-asthmatic donor. RGS2expression was reduced with siRNA (150nM anti-RGS2 vs. control siRNA) or through CRISPR transformation (Synthego©) to express the RGS2 promoter variants of interest, confirmed by Sanger sequencing. Gene and protein expression were quantified by qPCR and Western blotting. Intracellular Ca ++ flux and oscillation was measured with Fluo-8 (Abcam) with excitation/emission at 490/525 nm. HASM contractility to histamine treatment was measured using Traction Force Microscopy implemented in a 96-well plate format. Results Relative to cell lines with reference genotypes, HASM CRISPR transformed with the two RGS2 promoter variants exhibited a 41.8% (95% CI 28.6%--62.5%; p < 0.0001) reduction in RGS2 relative expression by qPCR, and a 35% (95% CI 8.6%—85.7%; p = 0.024) reduction in protein expression by Western blot. Methacholine-triggered calcium flux was nearly 50% greater for the cell lines with the RGS2 homozygous variants vs. those with reference genotypes (p = 0.043) as was the contractile response to histamine. HASM with reference genotypes exposed to anti-RGS2 siRNA showed the same phenotype, consistent with a causal relationship between the variants and expression (decreased) and receptor function (enhanced). Similarly, HASM CRISPR-transformed with the two RGS2 promoter variants exhibited an up to 3-fold increase in contraction magnitude (represented by strain energy, reported in pJ) in response to histamine >1uM (p < 0.05) relative to cell lines with reference genotypes. Conclusion Two promoter single nucleotide polymorphisms substitutions are sufficient to reduce RGS2 expression and induce a hypercontractile HASM phenotype. This abstract is funded by: AAAAI Bridge Award, NHLBI R21

Abstract RATIONALE: 4G4G/4G5G SERPINE1 genotypes produce high concentrations of plasminogen activator inhibitor-1 and are associated with worse asthma outcomes. A secondary analysis of the American Lung Association (ALA) SOYA trial showed that soy isoflavones significantly reduced asthma exacerbations versus placebo among patients with 4G4G/4G5G SERPINE1 genotypes. We hypothesized that baseline characteristics predict optimal responders to soy isoflavones among this subgroup. METHODS: ALA-SOYA was a randomized controlled trial that tested the efficacy of soy isoflavones vs. placebo for reducing asthma exacerbations among adolescents and adults with symptomatic asthma. In this responder analysis among participants with 4G4G/4G5G SERPINE1 genotypes, baseline demographic and clinical characteristics were tested for effect modification of the treatment assignment (soy/placebo) on the primary outcome of annualized number of asthma exacerbations using interaction terms in negative binomial regression models, with a significance threshold of p-interaction=0.15. RESULTS: Data were available for n=168 participants. Soy reduced the annualized number of asthma exacerbations relative to placebo among patients with 4G4G/4G5G SERPINE1 genotypes (incident rate ratio (IRR)=0.37, 95% confidence interval (CI): 0.15-0.90, p=0.028). Self-reported adherence to baseline controller therapy was the only significant effect modifier of this association (interaction p=0.115), with those using inhaled corticosteroid (ICS)/long-acting beta-agonist (LABA) controller therapy at least twice weekly experiencing reductions in asthma exacerbations with soy versus placebo (IRR = 0.25, 95% CI: 0.09-0.69; p= 0.007) but not among those using ICS/LABA controller therapy less frequently (IRR = 1.33, 95% CI: 0.22-8.02; p= 0.757). CONCLUSION: Soy isoflavones reduce asthma exacerbations among patients with the 4G4G/4G5G SERPINE1 genotypes and are most effective in those with greater adherence to asthma controller therapy regimens.

Asthma is a chronic inflammatory lung disease. Refractory asthma poses a significant challenge in management due to its resistance to standard therapies. Key molecular pathways of refractory asthma include T2 inflammation mediated by Th2 and ILC2 cells, eosinophils, and cytokines including IL-4, IL-5, and IL-13. Additionally, non-T2 mechanisms involving neutrophils, macrophages, IL-1, IL-6, and IL-17 mediate a corticosteroid resistant phenotype. Mediators including alarmins (IL-25, IL-33, TSLP) and OX40L have overlap between T2 and non-T2 inflammation and may signify unique pathways of asthma inflammation. Therapies that target these pathways and mediators have proven to be effective in reducing exacerbations and improving lung function in subsets of severe asthma patients. However, there are patients with severe asthma who do not respond to approved therapies. Small molecule inhibitors, such as JAK-inhibitors, and monoclonal antibodies targeting mast cells, IL-1, IL-6, IL-33, TNFα, and OX40L are under investigation for their potential to modulate inflammation involved in refractory asthma. Understanding refractory asthma heterogeneity and identifying mediators involved are essential in developing therapeutic interventions for patients unresponsive to currently approved biologics. Further investigation is needed to develop personalized treatments based on these molecular insights to potentially offer more effective treatments for this complex disease.

Abstract Background In the 5 years that have passed since the publication of the 2018 International Consensus Statement on Allergy and Rhinology: Allergic Rhinitis (ICAR‐Allergic Rhinitis 2018), the literature has expanded substantially. The ICAR‐Allergic Rhinitis 2023 update presents 144 individual topics on allergic rhinitis (AR), expanded by over 40 topics from the 2018 document. Originally presented topics from 2018 have also been reviewed and updated. The executive summary highlights key evidence‐based findings and recommendation from the full document. Methods ICAR‐Allergic Rhinitis 2023 employed established evidence‐based review with recommendation (EBRR) methodology to individually evaluate each topic. Stepwise iterative peer review and consensus was performed for each topic. The final document was then collated and includes the results of this work. Results ICAR‐Allergic Rhinitis 2023 includes 10 major content areas and 144 individual topics related to AR. For a substantial proportion of topics included, an aggregate grade of evidence is presented, which is determined by collating the levels of evidence for each available study identified in the literature. For topics in which a diagnostic or therapeutic intervention is considered, a recommendation summary is presented, which considers the aggregate grade of evidence, benefit, harm, and cost. Conclusion The ICAR‐Allergic Rhinitis 2023 update provides a comprehensive evaluation of AR and the currently available evidence. It is this evidence that contributes to our current knowledge base and recommendations for patient evaluation and treatment.