Mauger, D T

Abstract Background Prostaglandin E2 (PGE2) exerts paradoxical effects in asthma, acting as both a protective mediator and a contributor to pathology depending on context. Its role is shaped by type 2 (T2)-status, eosinophilia, and exacerbation frequency. In epithelial cell cultures from asthmatic and healthy individuals, interleukin (IL)-13 suppresses PGE2 synthesis genes (PTGS2 and PGES) while upregulating HPGD, which encodes for the PGE2 catabolic enzyme 15-prostaglandin dehydrogenase (15-PGDH) (PMID:16751012), suggesting diminished PGE2 availability during T2 inflammation. Similarly, in aspirin-exacerbated respiratory disease (AERD), impaired cyclooxygenase disrupts PGE2 biosynthesis, fueling unchecked leukotriene production and eosinophilic inflammation (PMID: 30516547), underscoring PGE2’s protective role in limiting disease progression. Conversely, in nasal polyps, tuft cells are activated by IL-13 to produce PGE2 (PMID: 35608904), highlighting PGE2’s potential pathogenicity. Objective To assess associations between PGE2-pathway gene expression in bronchial brushings and asthma severity, and to determine how nasal polyp history modifies these relationships. Methods We analyzed bronchial epithelial brushings from 109 adults with asthma (mean age 42.4±12.1, 64% female, 52% severe asthma, 18% nasal polyps) using normalized and batch-corrected RNA-seq data from the baseline visit in the Severe Asthma Research Program (SARP)3 (PMID: 36796454). Age-adjusted and nasal polyp-adjusted relationships were calculated between ratios of PGE2 synthesis and metabolism genes and clinical outcomes (ASSESS score, FEV1 %predicted). The composite PGE2 score (PMID: 35608904), derived from nasal polyp analysis, was calculated from the average scaled log-normalized expression of six PGE2-responsive genes. All data were analyzed using a linear regression mixed model with a random effect for site. Results Ratios of PGE2 synthesis genes (PTGS1/2, PTGES2) to HPGD were significantly associated with greater disease severity (change in ASSESS Score) (Fig.1A, solid lines) and lower lung function (change in FEV1 %predicted) (Fig.1B, solid lines). These relationships persisted after adjusting for nasal polyps (Fig.1, dashed lines). The composite PGE2 score was also related to disease severity (Fig.1A, black solid line) but attenuated when nasal polyps were accounted for (Fig.1A, black dashed lines). Interestingly, lower lung function remained associated with the PGE2 score even after nasal polyp adjustment (Fig.1B, dashed black lines). Conclusions Our analysis demonstrates that the balance between PGE2 synthesis and degradation in the bronchial epithelium is closely linked to asthma severity and lung function, independent of nasal polyp status. Future work will integrate transcriptomic and clinical data longitudinally to establish whether PGE2 could serve as a biomarker for asthma trajectory. This abstract is funded by: HL115118 (LCD), U10HL109152 (S.E.W.), U10HL109172 (to E.I. and B.D.L), U10HL109168 (N.N.J.), U10HL109250 (S.C.E), U10HL109164 (W.C.M), U10HL109257 (M.C), and U10HL109146 (to P.G.W. and S.A.C.)

Abstract Rationale Type-1 inflammation in asthma leads to significant airway level changes independent of Type-2 inflammation and may underlie the severity seen in combined Type1/2 high disease. As IFNs can cause airway epithelial injury and impaired repair, we applied a novel interpretable machine learning algorithm, SLIDE, to analyze the transcriptome of airway epithelial brushings of mild-moderate (MMA) and severe asthma (SA) participants in the Immune Mechanisms in Severe Asthma (IMSA) cohort to identify latent/hidden factors of disease that associate with disease severity. Methods The expression of genes identified by SLIDE as part of a co-expression program was assessed in the full IMSA cohort by severity and in an independent cohort of the NHLBI Severe Asthma Research Program (SARP). We also examined associations of these genes with clinical asthma outcomes in both cohorts. Results SLIDE identified aberrant increased expression of membrane-tethered mucin genes, MUC1 and MUC4. MUC1 appeared in each of 5 significant latent factors associated with greater severity and MUC4 in 2/5. Standard DGE analysis also identified the gene CEACAM5 as a predictor of severity. Pathway analysis of latent factor genes revealed airway keratinization which may contribute to airway remodeling and worsening obstruction over time. Expression of MUC1, MUC4, and CEACAM5 were increased in SA compared to HC in both the full IMSA and separate SARP cohorts (p < 0.001 for all genes in both cohorts). Expression in MMA was consistently lower than in SA in both cohorts, reaching significance in IMSA (p < 0.01) but not consistently in SARP (MUC1 p = 0.04, MUC4 p = 0.27, CEACAM5 p = 0.27). Protein analysis of endobronchial biopsies by immunofluorescence staining showed a trend towards increased MUC1 protein content with evidence of secretion. MUC1, MUC4 and CEACAM5 were negatively correlated with FEV1 % predicted in both cohorts (IMSA: MUC1 r=-0.47, p < 0.01, MUC4 r=-0.47, p < 0.01, CEACAM5 r=-0.62, p < 0.01; SARP: MUC1 r=-0.19, p = 0.05, MUC4 r=-0.29, p < 0.01, CEACAM5 r=-0.51, p < 0.01). All three genes positively correlated with prospective exacerbations in both cohorts (IMSA: MUC1 r = 0.345, p = 0.01, MUC4 r = 0.216, p = 0.08, CEACAM5 r = 0.368, p < 0.01; SARP: MUC1 r = 0.22, p = 0.02, MUC4 r = 0.32, p < 0.01, CEACAM5 r = 0.38, p < 0.01). Conclusions The expression of MUC1, MUC4 and CEACAM5 increased with asthma severity in the airway epithelium and correlated with both prospective exacerbations and obstruction. As pathway analysis of genes in the significant latent factors were associated with airway keratinization aberrant expression of these mucins (with known signaling functions) may contribute to maladaptive repair and airway remodeling that drives obstruction over time, contributing to worse asthma outcomes. This abstract is funded by: NIH

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 The activity of fucosyltransferase 2 (FUT2) determines if mucin glycan structures in epithelial mucus include histo-blood group antigens (HBGAs). People with active FUT2 express HBGAs in epithelial mucus secretions and are terms “secretors”, while non-secretors lack HBGAs in their secretions because they have a FUT2 genotype that inactivates the glycosyl transferase. Prior research suggests that secretors who are blood type O (type O secretors) are susceptible to asthma exacerbations1 and that FUT2 knockout mice (functional non-secretors) are protected from mucus plugging in allergen challenge models2,3. We set out here to determine if secretor status influences mucus plug prevalence in asthma and to confirm if type O secretors are susceptible to asthma exacerbations. Methods We determined secretor status in asthma participants from the Severe Asthma Research Program-3 (SARP-3) cohort (N = 507) in two ways, a specific fucose lectin binding assay in sputum and FUT2 genotyping. Blood group type was determined using ABO genotyping. We analyzed mucus plug segment scores, asthma exacerbations rates and other clinical and biological features in patients stratified by secretor status and blood type (non-secretors, type O secretors, and non-type O secretors). Results Secretor status determined by lectin binding in sputum was strongly concordant with genotypic analysis. Within the SARP-3 cohort, 26.2% were non-secretors, 32.3% type were O secretors, and 41.4% were non-type O secretors. Mucus plug segment scores, annualized asthma exacerbation rates, and other measures of asthma severity were similar in non-secretors, type O secretors, and non-type O secretors (Table). Although type O secretors had increased values for fractional exhaled nitric oxide (FeNO) and higher expression of type 2 gene network genes in their sputum cells, measures of sputum eosinophil inflammation were not higher in this subgroup (Table). Conclusion Secretor status does not influence the prevalence of mucus plugging in asthma patients, and we did not confirm a prior report that type O secretors have an increased risk of asthma exacerbation. Although type O secretors have modestly increased FeNO levels and increased expression of type 2 immune genes in their sputum, it is not clear if this is a biologically important signal. 1. Innes et al. AJRCCM; 2011. 2. Saku et al. JACI; 2019. 3. Hara et al. AJRCMB; 2025 This abstract is funded by: The study was supported by the National Institutes of Health grants R01 HL080414 (JVF), P01 HL107202 (JVF), R01 HL164787 (JVF), U10 HL109172 (EI), U10 HL109152 (SEW), U10 HL109257 (MC), U10 HL109250 (SCE), U10 HL109146 (JVF), U10 HL109164 (ERB), U01 HL146002 (BDL), U10 HL109086 (DTM) and Sandler Asthma Basic Research Center at UCSF (JVF). The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi–Genzyme Regeneron, and Teva. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.

Abstract Rationale Asthma contributes substantially to healthcare burden and morbidity through frequent exacerbations. Emerging evidence suggests that genetic factors play a crucial role in modulating susceptibility to asthma exacerbations. To better understand the genetic architecture of exacerbations, we conducted a genome-wide association study (GWAS) in the Tapestry cohort. Methods The Tapestry Study, a large-scale, decentralized and clinical grade genomic sequencing study, conducted Exome+® sequencing at Helix, Inc. on 98,075 Mayo Clinic participants. After excluding patients with ≥10 pack-years of smoking and/or emphysema, we performed a GWAS of asthma exacerbations in 3,960 adults with asthma based on ICD codes. Cases (n = 1,059) were defined as individuals who experienced at least two exacerbations between 2020 and 2025; the remaining 2,901 individuals served as controls. Asthma exacerbations were defined by oral or systemic corticosteroid prescriptions with durations ranging from 3 days up to 28 days. Logistic regression models adjusted for age, sex, BMI, and the first six principal components of ancestry. Replication was performed in 1,273 asthma patients (439 African Americans and 834 non-Hispanic Whites) from the NHLBI-sponsored Severe Asthma Research Program (SARP 1-3), with adjustments for age, sex, BMI, and the first 10 principal components of ancestry. Results Based on a minor allele frequency (MAF) threshold of > 0.05, we analyzed 5,256,135 genome-wide variants. Genome-wide significant associations for asthma exacerbations (p < 5.0 × 10⁻8) included loci at a pseudogene in SMPD4P1 (rs680548, MAF = 0.39, OR = 1.37, p = 9.10 × 10⁻9). Additional variants showing suggestive associations included loci within ENDOD1 (rs638776, OR = 0.69, p = 1.20 × 10⁻6), PCSK2 (rs2206454, OR = 1.32, p = 3.53 × 10⁻6), RBFOX1 (rs17667084, OR = 0.60, p = 5.11 × 10⁻6), and CDC42EP3 (rs721660, OR = 1.29, p = 5.68 × 10⁻6). Subthreshold associations in Tapestry including loci at CDC42EP3 (rs4670213, OR = 1.27, p = 1.41x10-5, MAF = 0.36), and SDR39U1 (rs12587663,OR = 0.83, p = 5.86x10-4, MAF = 0.46) were replicated in the SARP longitudinal visit 5 with p-values of 8.523x10-3 (rs4670213, OR = 1.78), and 2.88x10-4 (rs12587663,OR=0.43), respectively. Conclusion Our GWAS for asthma exacerbations in the Tapestry cohort identified variant loci relevant to mitophagy mechanisms (CDC42EP3, PMID: 40783597), immune signaling and RNA splicing regulation (RBFOX1, PMID: 31301374), and neuromodulatory pathways (PCSK2, PMID: 32794589), all previously implicated in asthma pathophysiology. Collectively, these findings highlight potential novel genetic contributors to asthma exacerbations and provide a foundation for improving risk stratification and therapeutic targeting in asthma management. This abstract is funded by: This abstract is funded by: Mayo Clinic Funding: X01 from the NHLBI for TOPMed whole-genome sequencing and NIH grants R01 HL142992, HL69116, HL69167, HL69170, HL69174, U10 HL109164, U10 HL109257, U10 HL109146, U10 HL109172, U10 HL109250, U10 HL109250, U10 HL109250, U10 HL109168, U10 HL109152, U10 HL109086, U10 HL109046.