Limper, Andrew H.

Abstract Alveolar macrophages (AMs) are critical for host defense and immunity against Pneumocystis. Through surface-associated receptors, AMs recognize, phagocytose, and kill Pneumocystis organisms, producing cytokines that promote pathogen clearance but also contribute to lung inflammation and injury. The EphA2 receptor, a member of the receptor tyrosine kinase (RTK) family, contains an extracellular region that binds activating ligands (Ephrins) and, more recently, has been shown to bind fungal β-glucans, leading to activation of its intracellular tyrosine kinase domain. The EphA2 signaling pathway has important roles in cancer, angiogenesis, and inflammatory diseases such as atherosclerosis. EphA2 is highly expressed in epithelial and endothelial cells, and emerging evidence indicates that it serves as a critical regulator of host defense against bacterial, viral, and fungal pathogens. In oropharyngeal candidiasis, EphA2 was identified as a receptor for fungal β-glucans in oral epithelial cells, mediating proinflammatory signaling through signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. Our laboratory recently demonstrated that EphA2 on lung epithelial cells binds Pneumocystis organisms, and that β-glucans from these organisms activate downstream signaling leading to IL-6 release. However, the role of EphA2 signaling in AM immune responses during Pneumocystis pneumonia (PCP) remains unknown. To address this, we examined EphA2-deficient (EphA2−/−) AMs stimulated with Pneumocystis β-glucans. Compared to wild-type AMs, EphA2−/− AMs exhibited significantly decreased production of proinflammatory cytokines, including IL-6 and TNF-α. In CD4-depleted mouse models of PCP, EphA2−/− lungs displayed markedly reduced levels of inflammatory cytokines and increased organism burdens, indicating impaired immune activation but preserved fungal persistence. Importantly, in CD4-depleted mice undergoing standard trimethoprim-sulfamethoxazole (TMP/SMX) therapy, concurrent administration of the EphA2 small-molecule inhibitor ALW-II-41-27 significantly reduced lung TNF-α responses without evidence of toxicity. These findings demonstrate that EphA2 receptor signaling plays a critical role in regulating macrophage-mediated inflammatory responses to Pneumocystis. While EphA2 deficiency dampens pulmonary inflammation, it also compromises fungal clearance, highlighting a delicate balance between host defense and immunopathology. Our data suggest that targeted modulation of EphA2 signaling during anti-Pneumocystis therapy may provide a novel adjunctive approach to reduce detrimental lung inflammation associated with PCP, potentially improving outcomes in affected patients. This abstract is funded by: NIH R01 HL-62150

Abstract Rationale Pneumocystis pneumonia (PJP) remains a major opportunistic infection in immunocompromised hosts, with limited therapeutic options. Previous studies identified a conserved cysteine residue (C353) in Pneumocystisphosphoglucomutase (PGM), analogous to that found in other pathogenic fungi but absent in mammalian PGMs, suggesting an exploitable antifungal target. We aimed to define the functional significance and therapeutic potential of targeting Pneumocystis PGM. Methods Full-length P. jirovecii and P. murina PGM cDNAs were cloned into S. cerevisiae pgm2Δ yeast (deficient in PGM activity) using the pYES2.1 TOPO vector for heterologous expression. Recombinant yeast lysates were tested for PGM activity in the presence of the thiol-reactive inhibitor ISFP10 (0-200 μM). Parallel assays were performed using recombinant human PGM lacking C353. Yeast growth inhibition in these strains in the presence of ISFP10 was also measured over 72 hours. For in vitro and ex vivo assays, P. murina organisms were treated with ISFP10, and viability was quantified via qPCR of the mitochondrial 16S ribosomal copy number. Precision-cut lung slice (PCLS) ex vivo cultures from a murine PCP model and in vivo CD4-depleted mice were used to assess ISFP10 efficacy and toxicity. Results Both P. jirovecii and P. murina PGMs showed potent, dose-dependent inhibition of enzymatic activity by ISFP10 up to 12.5 μM, with no inhibition of human PGM at concentrations up to 200 μM. Yeast complementation assays confirmed that ISFP10 significantly reduced the growth of pgm2Δ yeast expressing Pneumocystis spp. PGM. In vitro incubation of P. murina with ISFP10 resulted in significant reductions in organismal 16S mitochondrial copy number, comparable to pentamidine. In ex vivo PCLS models, ISFP10 decreased P. murina burden without affecting uninfected tissue viability. In vivo, ISFP10 administration for seven days significantly reduced fungal burden in CD4-depleted mice with an acceptable safety profile. Conclusions These findings demonstrate that Pneumocystis PGM activity depends on a conserved cysteine residue that is selectively targetable by ISFP10. Pharmacologic inhibition of PGM impairs P. murina viability in vitro, ex vivo, and in vivo with minimal host toxicity. Collectively, these data establish PGM as a promising, fungus-specific antifungal target and support the further development of small-molecule inhibitors targeting Pneumocystis PGM. This abstract is funded by: NIH R01 HL-62150, R21 AI-181542

Abstract Rationale Patients with idiopathic pulmonary fibrosis (IPF) living in disadvantaged neighborhoods have worse composite outcomes and pulmonary function. Although the current FDA approved drugs reduce lung function decline and may improve mortality for this progressive and fatal disease, equitable access to therapy remains a challenge, which may be contributing to low antifibrotic uptake. Existing understanding of social determinants of health in IPF is derived from local registries and special subpopulations, such as Veterans. The aim of our study is to investigate the association between neighborhood-level deprivation indicators and both the distribution of patients with IPF and outcomes in a large national database. Methods We performed a retrospective cohort analysis between October 1st, 2014, and December 31st 2022, using the Optum Labs Data Warehouse, a large de-identified administrative claims database. We used claims data from commercial, Medicare Advantage, and Medicare Fee-for-Service enrollees across the United States. Diagnosis of IPF was defined using ICD-9 and ICD-10 codes. Social deprivation index (SDI), assessed at the zip code tabulation area level, was determined based on 7 demographic characteristics from the 2015-2019 American Community Survey. Patients were grouped in quintiles based on SDI at diagnosis, where higher values reflect greater deprivation. Hazard ratios (HR) were calculated for all-cause mortality as primary and hospitalization as secondary outcome. Results A total of 96,143 patients (mean age 75.1 years ± 6.9 years) were included in the study, 23.5% received antifibrotics. As area deprivation increased, proportion of female, Black, and Hispanic patients increased, whereas, proportion of male, White, and Asian patients decreased. Similarly, antifibrotic use declined slightly, while prescription of steroids and oxygen increased with SDI. Residing in most disadvantaged neighborhoods increased risk of hospitalization (HR, 1.13 [95% CI 1.09 - 1.17] and all-cause mortality (HR, 1.15 [95% CI 1.11 - 1.19]) after adjusting for covariates over median follow-up of 301 days and 680 days, respectively. Conclusion Gender and racial disparities were evident among IPF patients who live in resource-limited areas. Our study shows that greater proportion of female, Black, and Hispanic IPF patients were disproportionately represented in more socioeconomically deprived areas. Furthermore, as area-level disadvantage increased, fewer patients received antifibrotics, while more patients were on steroids and oxygen therapy potentially indicating more advanced disease. Moreover, living in disadvantaged neighborhoods was associated with worse all-cause mortality and hospitalizations. Identifying individuals who live in socioeconomically disadvantaged areas is essential to provide equitable care and improve outcomes for this life-threatening disease. This abstract is funded by: None

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease that continues to have high morbidity and mortality. There are only 3 approved medications, (nintedanib, pirfenidone and nerandomilast) which marginally slow FVC decline and only nerandomilast having some mortality benefit. All three have no effect on quality of life (exacerbations and hospitalizations). There is an urgent need to find more effective treatment options. Since IPF is predominantly a disease of the elderly with a peak prevalence in those 65 and older, it is common for these individuals to have other comorbidities such as Hyperlipidemia (HLD). Interestingly, in a prior small study of IPF patients, there is evidence to suggest clinical benefit from ezetimibe, a medication used to treat HLD. This study aims to further evaluate the clinical benefits of ezetimibe in patients with IPF in a large national cohort. Methods We conducted this study using the Optum Labs Data Warehouse (OLDW). The OLDW contains de-identified administrative claims data, including medical and pharmacy claims and enrollment records. This study used claims data from commercial, Medicare Advantage (MA), and Medicare Fee-for-service (FFS) enrollees between October 1st, 2014, and December 31st, 2021. Diagnosis of IPF was defined using ICD-9 and ICD-10 codes. Patients without a diagnosis of IPF were excluded. P < 0.05 was set to determine statistical significance. Two matched patient cohorts depending on ezetimibe use were defined. Primary outcome was all-cause mortality, and secondary outcome was hospitalizations. Results We identified 7724 patients with IPF. After adjusting for age, age group, gender, race, health plan, year of IPF diagnosis, smoking, diabetes, statin treatment, and antifibrotic treatment, two cohorts were identified and matched, patients treated with ezetimibe (n = 3862) and patients not treated with ezetimibe (n = 3862). Among the patients with IPF treated with ezetimibe, ezetimibe use was associated with a reduction in all-cause mortality (hazard ratio [95% CI]: 0.704 [0.660 - 0.752], p-value: <0.001) and hospitalizations(hazard ratio [95% CI]: 0.907 [0.857 - 0.959], p-value: 0.001) compared to patients not treated with ezetimibe. Conclusion In patients with IPF, treatment with ezetimibe may be associated with improved clinical outcomes. Further studies are necessary to confirm benefits, assess anti-fibrotic effects, and inform implementation in management of IPF. As IPF remains a disease with limited treatment options, repurposing drugs continues to offer an avenue to find affordable, safe and already approved medications for use. This abstract is funded by: None

Abstract Background Sputum color is a visual marker of airway inflammation and disease activity in bronchiectasis, ranging from clear or light gray (mucoid) to yellow-green (purulent), the latter largely reflecting neutrophil myeloperoxidase release. This study evaluated associations between sputum color and common clinical markers of disease activity to determine which parameters differed most between color categories. Methods Forty spontaneous sputum samples were analyzed from 80 consecutive adults with non-cystic fibrosis bronchiectasis (median age 70 years [IQR 12]; 74% female) enrolled between January 2024 and April 2025 in an observational study. Sputum color was graded using the Murray Sputum Color Chart and classified as mucoid (color #1) or purulent (colors #2-4). Clinical parameters, including blood cell counts, C-reactive protein (CRP), fractional exhaled nitric oxide (FeNO), and results of bacterial and acid-fast bacillus cultures (induced or bronchoscopic), were obtained from medical records if available within one month of enrollment. Categorical variables were analyzed using chi-square or Fisher’s exact tests and continuous variables with the Mann-Whitney U test. A secondary analysis compared patients who could versus could not produce spontaneous sputum. Results Of 40 samples, 20 (50%) were purulent. Blood neutrophil counts (median 4.5 vs. 3.9 x109/L), neutrophil-to-lymphocyte ratios (2.5 vs. 2.6), and eosinophil counts (1.7 vs. 1.7 x109/L) did not differ significantly between groups (n = 26). FeNO was normal in all 10 patients with available data and was not analyzed further. CRP was higher among patients with purulent sputum (4.6 vs. <3 mg/L; n = 20; p = 0.044). Bacterial growth was more frequent in purulent than mucoid samples (73% vs. 31%; n = 31; p = 0.032). Isolates in the purulent group included Klebsiella, Pseudomonas, Staphylococcus, and Streptococcus (n = 2 each), and single isolates of Acetobacter, Acinetobacter, Chryseobacterium, Delftia, Enterobacter, Escherichia, and Moraxella. In the mucoid group, Pseudomonas, Staphylococcus (n = 2 each), and Stenotrophomonas (n = 1) were identified. Mycobacterial growth did not differ significantly (20% vs. 12.5%; n = 31; p = 0.65). Patients unable to produce spontaneous sputum had a higher rate of mycobacterial isolation on induced/bronchoscopic samples (43% vs. 16%; n = 54; p = 0.027), though other parameters were similar. Over 90% of measurements were obtained within three days of enrollment, and freezing did not appear to alter sputum color. Conclusions Even within a small cohort, purulent sputum was associated with higher CRP and greater bacterial growth, supporting sputum color as a rapid, inexpensive marker of disease activity in bronchiectasis. Further research should clarify whether sputum color primarily reflects bacterial colonization, airway inflammation, or both. This abstract is funded by: None

OBJECTIVES: Pneumocystis jirovecii pneumonia (PCP) can cause severe hypoxemic respiratory failure in immunocompromised patients. Contemporary outcomes of venovenous extracorporeal membrane oxygenation (VV ECMO) support for PCP are poorly characterized, and existing reports are limited to small case series. DESIGN: Retrospective cohort study. SETTING: International registry study using the Extracorporeal Life Support Organization (ELSO) registry. PATIENTS/SUBJECTS: Adults with PCP-associated hypoxemic respiratory failure supported with VV ECMO between 2011 and 2024. INTERVENTIONS: VV ECMO. MEASUREMENTS AND MAIN RESULTS: A total of 209 patients with PCP-associated respiratory failure supported with VV ECMO were identified. The overall in-hospital mortality was 60.8%. Survivors were younger than nonsurvivors (median age 41.9 vs. 46.8 yr; p = 0.047). Duration of mechanical ventilation before VV ECMO was longer among nonsurvivors (median 4.7 vs. 1.6 d; p = 0.019). The proportion of patients with HIV infection was similar among survivors and nonsurvivors (19.5% vs. 22.1%; p = 0.73). Pre-ECMO vasopressor use, prone positioning, and renal replacement therapy were common and did not differ between groups. Clinical course was characterized by prolonged VV ECMO support (median 18.6 d) and frequent complications, including pneumothorax (21.1%), renal replacement therapy during VV ECMO (31.6%), intracranial hemorrhage or stroke (7.7%), and major pulmonary or gastrointestinal hemorrhage (16.8%). PCP accounted for a small proportion of VV ECMO runs reported to the ELSO registry throughout the study period at 0.36% of reported cases. No significant temporal trend in mortality was observed. CONCLUSIONS: PCP-associated respiratory failure supported with VV ECMO is associated with substantial mortality and prolonged VV ECMO support. These findings provide contemporary benchmarks that may inform VV ECMO candidacy discussions and expectations in this challenging patient population.

Abstract Background Alveolar macrophages (AMs) are central to host defense against Pneumocystis, mediating organism clearance while also contributing to lung inflammation. EphA2, a transmembrane receptor that binds fungal β-glucans, has emerged as a regulator of host–pathogen interactions, but its role in AM responses during Pneumocystis pneumonia (PCP) is unknown. Methods We evaluated AM inflammatory responses to Pneumocystis β-glucans in the presence and absence of EphA2. In vivo studies used a CD4-depleted mouse model of PCP comparing wild-type and EphA2-deficient (EphA2−/−) mice. Lung cytokine responses and organism burden were assessed. Additionally, EphA2 signaling was pharmacologically inhibited using ALW-II-41-27 during trimethoprim-sulfamethoxazole treatment. Results EphA2 deficiency significantly reduced AM proinflammatory cytokine responses to Pneumocystis β-glucans. In CD4-depleted PCP mice, EphA2−/− animals demonstrated decreased lung inflammatory cytokines, but increased organism burden compared to controls. Pharmacologic inhibition of EphA2 during antimicrobial therapy significantly suppressed lung inflammation. Conclusions EphA2 signaling plays a critical role in AM-mediated host defense against Pneumocystis by promoting inflammatory responses necessary for organism control. However, this pathway also contributes to lung inflammation. Targeting EphA2 may represent a novel strategy to modulate inflammation during severe PCP while preserving antimicrobial defense.

Summary Background Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. Methods Human lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. Findings RAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. Interpretation The inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in context Evidence before this study Pulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this study We demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGFβ-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidence Our findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.