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Bacterial pneumonia poses many challenges in ICU settings due to treatment resistance and the potential for lethal pulmonary injury and sepsis. Neutrophil recruitment is crucial for positive patient outcomes, but how to achieve the balance between effective microbial killing and restrained injurious effector functions remains elusive. Our study aims to investigate mechanisms underlying neutrophil effector functions, particularly the explosive release of chromatin via NETosis, a process associated with poor outcomes in various pathologies, including pneumonia, cardiovascular dysfunction, autoimmune disorders, and COVID-19. Using an LPS model of endotoxemia, we recently published that global nitric oxide synthase 1 (NOS1) knock-out mice are protected from injury and death. Notably, macrophages depleted of NOS1 activity also showed reduced transcription of a subset of pro-inflammatory genes. From this, we developed a CX3CR1-driven cre mouse model, in agreement with the guidelines and study protocols approved by the IACUC at MCW, to determine if selective deletion of NOS1 from macrophages conferred protection from injury during bacterial pneumonia. Preliminary data show protection from injury and death in NOS1ΔMac mice compared to controls post-infection, suggesting a link between macrophage NOS1 signaling and inflammatory injury. We hypothesize that NOS1-independent macrophage signaling programs neutrophils to suppress the release of reactive oxygen species (ROS) and explosive NETosis reducing tissue injury. To test this, we developed a novel in vitro system where primary neutrophils are exposed to cell-free, pre-conditioned media from either LPS-stimulated control (PCM) or macrophages depleted of NOS1 activity (PCMΔMac). After 30 minutes, injurious effector functions (ROS and NETosis) and surface marker expression of cd11b/Mac1 were evaluated. Using Amplex Red to detect extracellular H2O2, PCM treatment produced 10% more ROS after maximal NADPH oxidase stimulation with formyl peptide compared to PCMΔMac treatment. Next, NETosis was evaluated using long-duration, fluorescent microscopy with Hoechst and Sytox dyes to detect DNA intra- and extracellularly, respectively. We observed a 10% reduction of NETosis events with PCMΔMac treatment along with increased "vital NETosis". Finally, using flow cytometry, we observed increased surface expression of pro-inflammatory surface marker cd11b/Mac1 on neutrophils treated with PCM compared to PCMΔMac, suggesting suppression of the pro-inflammatory surface phenotype of neutrophils. These findings highlight the potential of macrophage NOS1-independent programming in suppressing injurious effector functions of neutrophils, holding promise for managing complex conditions such as sepsis without compromising antimicrobial control. We are grateful for funding provided by: NHLBI (1RO1HL 163820) Advancing a Healthier Wisconsin MCW Cancer Center MCW Research Affairs Committee Wisconsin Breast Cancer Showhouse.
LaFond et al. (Fri,) studied this question.