Alveolar macrophages undergo METosis in an inflammatory model of neonatal lung injury

Background: Activated macrophages can produce macrophage extracellular traps (METs) - DNA strands coated with cytotoxic components that are released to immobilize pathogens and control infection. However, METs can also contribute to the inflammatory process and lead to severe tissue damage. Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants initiated by inflammation and oxidative stress. Gram-negative bacteria and candida increase the risk of developing BPD and are strong inducers of extracellular traps. Macrophages are the most abundant immune cells in the neonatal lung tissue, responsible for controlling the inflammatory process. We aimed to determine whether inflammation triggers METosis in a neonatal model of inflammatory lung injury. Methods: Alveolar macrophages (AMs) from bronchoalveolar lavage fluid (BALF) were isolated from Sprague Dawley rat pups on postnatal day (PND) 8. In vitro - AMs were incubated with METosis agonists (lipopolysaccharide [LPS] and phorbol myristate acetate [PMA]) or control (media) for 4h. Cells were fixed and stained for markers of METosis (myeloperoxidase [MPO] and citrullinated histone H3 [CitH3]) and DNA (DAPI) for immunofluorescence imaging. In vivo - PND6-9 pups were treated with LPS or saline intrapharyngeally and DNase enzyme or saline intraperitoneally. BALF was collected on PND10. BALF was analysed using immunofluoresence imaging and picogreen assay. Results: Immunofluorescence imaging of neonate AMs showed increased expression of METosis markers MPO, CitH3, and DNA strands with in vitro exposure to METosis agonists PMA or LPS compared to control (n =3). In vivo LPS exposure also showed evidence of increased extracellular trap formation in the BALF collected from neonate rat lung compared to control (n=3; p<0.0001); further, rats exposed to both LPS and DNAse enzyme showed decreased extracellular trap formation (p = 0.002) in comparison to the rats exposed to LPS and saline. Conclusions: To the best of our knowledge, this study is first to establish (i) rat AMs undergo METosis, (ii) a plausible role for METosis in lung injury (iii), and the potential relevance of METosis in lung injury during early development. Novel therapies targeting METosis formation may reduce lung inflammation contributing to BPD in premature infants. This work was supported by the Women's Auxiliary Fund, The Hospital for Sick Children, Toronto, Ontario, Canada This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.