SUMMARY Alveolar macrophages (AMs), upon sensing pathogens, trigger host defense by activating toll-like receptor 4 (TLR4), but the counterbalancing mechanisms that deactivate AM inflammatory signaling and prevent lethal edema, the hallmark of acute lung injury (ALI), remain unknown. Here, we demonstrate the essential role of AM protease-activating receptor 2 (PAR2) in rapidly suppressing inflammation to prevent long-lasting injury. We show that thrombin, released during TLR4-induced lung injury, directly activates PAR2 to generate cAMP, which abolishes Ca2+ entry through the TRPV4 channel. Deletion of PAR2 and thus the accompanying cAMP generation augments Ca2+ entry via TRPV4, causing sustained activation of the transcription factor NFAT to produce long-lasting TLR4-mediated inflammatory lung injury. Rescuing thrombin-sensitive PAR2 expression or blocking TRPV4 activity in PAR2-null AMs restores their capacity to resolve inflammation and reverse lung injury. Thus, activation of the thrombin-induced PAR2-cAMP cascade in AMs suppresses TLR4 inflammatory signaling to reinstate tissue integrity.

Acute lung injury (ALI) is a lethal inflammatory lung disorder whose incidence is on the rise. Alveolar macrophages normally act to resolve inflammation, but when dysregulated they can provoke ALI. We demonstrate that monocyte-derived macrophages (CD11b + macrophages) recruited into the airspace upregulate the anti-inflammatory function of alveolar macrophages by suppressing their stimulator of type 1 interferon gene (STING) signaling. Depletion of CD11b + macrophages in mice (macrophage dep mice) after endotoxin or after Pseudomonas aeruginosa causes expansion of the inflammatory alveolar macrophage population, leading to neutrophil accumulation, irreversible loss of lung vascular barrier function, and lethality. We show that CD11b + macrophages suppress alveolar macrophage-STING signaling via sphingosine kinase-2 (SPHK2) generation of sphingosine-1-phosphate (S1P). Thus, adoptive transfer of wild-type (WT) or STING À/À , but not SPHK2 À/À , CD11b monocytes from murine bone marrow into injured macrophage dep mice rescue anti-inflammatory alveolar macrophages and reverse lung vascular injury. SPHK2-induced S1P generation in CD11b + macrophages has the potential to educate alveolar macrophages to resolve ALI.

Macrophages play a central role in dictating the tissue response to infection and orchestrating subsequent repair of the damage. In this context, macrophages residing in the lungs continuously sense and discriminate among a wide range of insults to initiate the immune responses important to host-defense. Inflammatory tissue injury also leads to activation of proteases, and thereby the coagulation pathway, to optimize injury and repair post-infection. However, long-lasting inflammatory triggers from macrophages can impair the lung's ability to recover from severe injury, leading to increased lung vascular permeability and neutrophilic injury, hallmarks of Acute Lung Injury (ALI). In this review, we discuss the roles of toll-like receptor 4 (TLR4) and protease activating receptor 2 (PAR2) expressed on the macrophage cell-surface in regulating lung vascular inflammatory signaling.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using i.t. rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.