B75-14 Adoptive Transfer of Monocytes by Airway Instillation Protects Against Lung Injury

Abstract Rationale Our goal is to develop a novel cell-based therapy for acute lung injury (ALI), which can lead to the high mortality of the acute respiratory distress syndrome (ARDS). We previously reported that sessile alveolar macrophages (AMs) suppress immunity and protect against ALI (PMID: 24463523). Since monocytes recruited to pulmonary alveoli differentiate into AMs, we considered the possibility that intra-airway adoptive transfer of bone marrow-derived monocytes (BMDMs) might be similarly immunosuppressive. Methods ALI was induced by airway instillation (a.i.) of LPS in anesthetized mice. This was followed 4 hours later by a.i. of BMDMs, or of PBS (control). After 24 hours, we carried out immunoblots in conjunction with knockdown and knockout strategies to detect Ca²+/calmodulin-dependent kinase kinase-2 (CaMKK2)-mediated phosphorylation, hence activation of AMP-activated kinase (AMPK), and IκB stabilization. For Ca2+ quantification, we viewed instilled BMDMs expressing the genetically encoded Ca²+ sensor GCaMP6. To assess ALI severity we quantified pulmonary edema, in terms of the extravascular lung water content (EVLW), and mouse survival. Results LPS-induced severe pulmonary edema and high mortality were markedly abrogated by a.i. of wild-type BMDMs, but not of BMDMs lacking the gap junctional protein connexin 43 (Cx43). PBS had no protective effect. In alveoli of LPS-treated mice, wild-type BMDMs evoked Ca2+ spikes every ∼2 minutes, which were blocked by intra-alveolar microinjection of the ATP hydrolyzer, apyrase. Further, wild-type BMDMs induced AMPK activation, stabilized IκBα and decreased pulmonary edema. These effects were blocked by silencing CAMKK2 in the alveolar epithelium (AE). In mice with CAMKK2 deletion in type 1 (AT1) cells of the AE, a.i. of wild-type BMDMs failed to protect against LPS-induced mortality. Conclusions Adoptive transfer of BMDMs by airway instillation markedly improved mouse survival in LPS-induced ALI. Mechanistically, we interpret that instilled BMDMs formed Cx43-containing gap junctions with AT1 cells, which released ATP to activate Ca2+ spikes in BMDMs. Ca2+ signaling thereby occurred between BMDMs and AT1 cells, leading sequentially to activation of CAMKK2 and AMPK, hence stabilization of IκBα. The resulting inhibition of proinflammatory NFkB signaling ameliorated injury to the air-blood barrier, decreasing pulmonary edema and improving survival. We conclude, airway delivery of BMDMs might be a potent therapeutic strategy for protecting against the high mortality in ALI and ARDS (Support: HL169515). This abstract is funded by: HL169515