Abstract Rationale Resolution of lung injury following infectious insult is a highly coordinated process and highly relevant to the 200,000 patients that develop acute respiratory distress syndrome (ARDS) each year in the USA. Over the course of lung injury, alveolar macrophages (AM) must transition from pro-inflammatory to pro-repair to promote lung healing, however the mechanisms that direct this shift are unclear. Pro-repair function in AM is also associated with metabolic changes, specifically a shift from glycolysis to fatty acid oxidation (FAO) and oxidative phosphorylation. To investigate signals upstream of pro-repair function and metabolic shifts in AM, we investigated calcium flux from these cells in a mouse model of ARDS, focusing on the cation channel transient receptor potential vanilloid-4 (TRPV4). Methods We utilized the S. pneumoniae murine model to emulate clinical ARDS (PNA-ARDS), with robust inflammation peaking predictably at days 2-5 and resolution beginning at day 7. Alveolar macrophages were isolated from BAL at days 4, 7, and 10 and calcium was measured ex vivo using live cell imaging. Cells isolated from BAL were characterized by flow cytometry and AM were characterized metabolically by enzyme expression and metabolite measurement. Results We found that basal intracellular calcium levels (Ca2+i) in AM increased on day 7 and 10 of lung injury compared to mock-infected mice. Basal Ca2+i levels in PNA-ARDS AMs decreased following treatment with TRPV4 inhibitor HC-067047 (1uM). Interestingly, basal Ca2+i from mock-infected mice did not change following HC treatment. TRPV4 activity (as measured by increase in Ca2+i following exposure to the TRPV4 agonist GSK1016790A (1.5uM) was increased in PNA-ARDS AMs at days 7 and 10. In addition to increased Ca2+i, we found that AM isolated on day 10 of injury had evidence enhanced FAO as evidenced by increased expression of several FAO enzymes and increased intracellular lipid droplets. Finally, PNA-ARDS AM expressed higher levels of proteins associated with reparative function on day 10 compared to day 4, such as CD36 and mannose receptor. Conclusion AM from mice infected with S. pneumoniae exhibited higher basal Ca2+I during the resolution phase of lung injury compared to mock-infected controls. Strikingly, basal Ca2+i in AM isolated from PNA-ARDS mice was dependent on TRPV4 as evidenced by marked attenuation of calcium flux TRPV4 inhibition. This TRPV4-dependent Ca2+i was temporally associated with increased FAO and expression of proteins with pro-reparative function of AM. The linkage between TRPV4 mediated increased Ca2+i and metabolic/phenotypic shifts in AM requires further investigation. This abstract is funded by: 5T32HL007534-43
Miller et al. (Fri,) studied this question.
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