Mechanosensitive Responses of Resident Alveolar

Background The extent of micromechanical coupling between resident alveolar macrophages (AMs) and the surrounding tissue niche remains unclear. Lung overexpansion due to therapeutic mechanical ventilation imposes distortional stresses on the alveolar epithelium (AE), causing acute lung injury (ALI). Sessile AMs, lying adjacent to the AE, are causally implicated in ALI. However, it is not clear whether micromechanical coupling with the AE induces proinflammatory distortional stresses in sessile AMs. We addressed this question in live mouse lungs by real‐time confocal microscopy (RCM). Methods For RCM, we inflated and blood‐perfused mouse lungs at physiological pressures. To detect cytosolic Ca 2+ (cCa 2+ ), we microinfused fluo‐4 by alveolar micropuncture. To detect sessile AMs, we microinfused fluorophore‐conjugated anti‐Siglec‐F and anti‐CD11c Abs. To induce transient lung overexpansion, we increased the alveolar pressure from 5 to 15 cmH 2 O for 15 seconds. We determined alveolar diameter in terms of fluo‐4 fluorescence, which marked the epithelial margins. We determined cell diameter of sessile AMs in terms of Siglec‐F fluorescence, which marked the cell boundary. We detected gap junctional communication (GJC) by fluorescence recovery after photobleaching (FRAP). Results Sessile AMs were exclusively located in non‐distending microniches of the alveolus and therefore, did not stretch during lung expansion. Nevertheless, a subset of AMs responded to lung overexpansion by transiently increasing cCa 2+ . The cCa 2+ increase initiated in 5 min, reaching peak at two‐times baseline in 20 min, then subsiding to baseline after a further 20 min. Concomitantly, in AE adjacent to AMs, there was marked increase in cCa 2+ oscillations, but no increase in mean cCa 2+ . In lungs with AM‐specific Cx43 deletion, the transient cCa 2+ response was blocked in AMs, but not in adjoining AE. FRAP revealed GJC only in cCa 2+ ‐responsive AMs. In paired comparisons, AMs lacking GJC failed to show expansion‐induced Ca 2+ responses (p<0.05). Conclusions We report the unexpected finding that sessile AMs were exclusively located in immobile microniches of alveoli and therefore did not stretch during lung expansion. Nevertheless, following expansion there was Cx43‐mediated Ca 2+ communication with the AE, causing cCa 2+ increases in sessile AMs. Although further studies are required to evaluate the potentially proinflammatory responses induced by this Ca 2+ response, we propose Cx43 of sessile AMs might be a therapeutic target to mitigate lung expansion‐induced ALI.