D101-17 Respiratory Pathogen DNA Is Recoverable From Circulating Red Blood Cells During Pseudomonas Aeruginosa Pneumonia

Abstract Rationale Pneumonia remains a leading cause of morbidity and mortality worldwide, yet definitive pathogen identification is often challenging. Current diagnostic methodologies, including lung-derived specimen collection and reliance on traditional culture techniques, are frequently invasive, time-consuming, and suffer from low sensitivity, delaying targeted antimicrobial therapy. This critical gap in rapid, non-invasive pathogen identification necessitates novel diagnostic strategies. We previously demonstrated that red blood cells (RBCs) express the nucleic acid sensing receptor Toll-like receptor 9 (TLR9) on their surface and can bind microbial DNA in a TLR9-dependent fashion. Thus, RBCs may play an underappreciated role in capturing and sequestering circulating pathogen DNA during pneumonia. Moreover, RBCs may offer an easily accessible target for blood-based detection of respiratory pathogens. We hypothesized that respiratory pathogen DNA could be recovered from RBCs in a murine model of pneumonia and that RBC-mediated DNA capture would depend on RBC-TLR9. Methods We subjected wild-type (WT) and erythrocyte-specific TLR9 (Erytlr9-/-) deleted mice to a Pseudomonas aeruginosa (Pa) pneumonia model. Animals were given 40µL of 1x109 CFU/mL Pa (ATCC 27583) or PBS intranasally and were euthanized after 24 hours. Whole blood was collected via sterile cardiac puncture and spun at 9300xg. RBCs were isolated by Ter119 antibody positive selection. DNA was extracted from RBCs, plasma, and bronchoalveolar lavage (BAL). qPCR was performed for the Pa-specific ECFX gene. Lung tissue was collected and plated on Brain-Heart Infusion agar plates with colony enumeration after 24 hours. Results Pa DNA was minimally detected in plasma and in a similar amount in the BAL between WT and Erytlr9-/- mice. In contrast, more Pa DNA was detected on circulating RBCs of WT pneumonia animals compared to PBS controls (n = 8-13, p = 0.005). Importantly, Erytlr9-/- animals had less Pa DNA on their RBCs (n = 12-13, p = 0.004) suggesting RBC-TLR9 dependent binding. Erytlr9-/- pneumonia animals had less lung bacterial growth compared to WT pneumonia animals (n = 8-12, p = 0.04). RBC Pa DNA copy number was positively correlated with lung colony forming units in WT mice (r2=0.4924, p = 0.002), but not in Erytlr9-/- mice (r2=0.1174, p 0.05). Conclusions Pneumonia pathogen DNA is recoverable from RBCs and positively correlates with lung bacterial burden. These findings suggest RBCs may serve as a platform for respiratory pathogen detection during pneumonia. Future studies are needed to determine if RBC pathogen detection will be a diagnostic tool for other respiratory pathogens. Moreover, microbial DNA is captured in a TLR9-dependent manner, warranting further investigation into RBC-TLR9's role in the host response to pneumonia. This abstract is funded by: NIH R21-AI 166813