D101-15 Platelet Activation and Tnf Signaling Pathways Are Upregulated in Lung Megakaryocytes During Acute Lung Injury

Abstract Rationale Despite nearly sixty years of study, the mortality rate of the acute respiratory distress syndrome (ARDS) remains at 40-60% and the mechanisms facilitating resolution of lung injury remain unclear. Megakaryocytes (MK) are large multinucleated cells primarily residing in the bone marrow with the key function of platelet production. MKs also facilitate tissue repair, during radiation-induced bone marrow (BM) injury they release factors to drive proliferation of the BM stroma to recondition the niche for hematopoietic stem cell engraftment. Similarly, in a pneumonectomy model of lung regeneration, megakaryocyte-derived platelets secreted stromal derived factor 1 to support alveolar epithelial cell proliferation. MKs are also resident in the lung and are responsive to inflammation, having been demonstrated to release platelets under inflammatory conditions. Lung MKs detect multiple stimuli and upregulate immune receptors in response to pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), and cytokines, including IFN-γ, and IL-33. Considering lung MK responsiveness to inflammation and their role in tissue repair, we hypothesize that lung MKs actively respond to the inflammatory environment of the ARDS lung and facilitate resolution of ARDS. Methods We modeled ARDS with an LPS mouse model of acute lung injury (ALI) that replicated pulmonary edema and neutrophilic infiltration typical of ARDS. We injected 1.6 mg/kg LPS or vehicle i.t into the airway of wildtype C57BL6 mice and used flow cytometry to profile lung andBM MK quantity, proliferation, and apoptosis, while circulating platelets were quantified by hematological analyzer. We also used scRNA-seq to analyze the transcriptomes of lung and BM CD41+ cells. Results After ALI BM MKs remained stable over the course of 7 days, while the percentage of MKs in the lung decreased after 48 hours and increased after 7 days compared to vehicle, with no changes in proliferation nor apoptosis. Transcriptional analysis 48 hours after ALI revealed upregulation of platelet activation and tumor necrosis factor receptor 1 (TNFR1) signaling pathways in the lung compared to BM. Conclusions During early-stage ALI lung MKs are depleted from the lung in an apoptosis-independent whereas during late-stage ALI they repopulate the lung in a proliferation-independent manner.During early-stage ALI lung MKs upregulate transcription of genes associated with platelet activation, TNF and TNFR1 signaling, suggesting a responsiveness to inflammation and lung injury that leads to regulation of platelet activation. Further studies will directly investigate the role of lung MK TNF signaling on lung MK platelet production and activation during late-stage ALI. This abstract is funded by: NIH