Stelzig, Kimberly E.

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 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.

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and use of a simple method for small interfering RNA (siRNA) transfection of normal HBEs (NHBEs) in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology or morphology of NHBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.