Inflammation resolution is an active process that functions to restore tissue homeostasis. Clearance of apoptotic leukocytes by efferocytosis at inflammatory sites plays an important role in inflammation resolution and induces remarkable macrophage phenotypic and functional changes. Here, we investigated the effects of deletion of either plasminogen (Plg) or the Plg receptor, Plg-R KT , on the resolution of inflammation. In a murine model of pleurisy, the numbers of total mononuclear cells recruited to the pleural cavity were significantly decreased in both Plg −/− and Plg-R KT −/− mice, a response associated with decreased levels of the chemokine CCL2 in pleural exudates. Increased percentages of M1-like macrophages were determined in pleural lavages of Plg −/− and Plg-R KT −/− mice without significant changes in M2-like macrophage percentages. In vitro , Plg and plasmin (Pla) increased CD206/Arginase-1 expression and the levels of IL-10/TGF-β (M2 markers) while decreasing IFN/LPS-induced M1 markers in murine bone-marrow-derived macrophages (BMDMs) and human macrophages. Furthermore, IL4-induced M2-like polarization was defective in BMDMs from both Plg −/− and Plg-R KT −/− mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg −/− and Plg-R KT −/− BMDMs after IL-4 or IL-10 stimulation. The extents of expression of CD206 and Annexin A1 (important for clearance of apoptotic cells) were reduced in Plg −/− and Plg-R KT −/− macrophage populations, which exhibited decreased phagocytosis of apoptotic neutrophils (efferocytosis) in vivo and in vitro . Taken together, these results suggest that Plg and its receptor, Plg-R KT , regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

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Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and BtcAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or BtcAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. studies showed that ROL and BtcAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these findings, H89 prevented ROL- and BtcAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using -Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and BtcAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or BtcAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in settings, ROL and BtcAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in a manner dependent on PKA and CCR2/CCL2 pathways. Furthermore, db-cAMP promoted reprogramming of bone-marrow-derived macrophages to a M2 phenotype as seen by increased Arg-1/CD206/Ym-1 expression and IL-10 levels (M2 markers). Db-cAMP also showed a synergistic effect with IL-4 in inducing STAT-3 phosphorylation and Arg-1 expression. Importantly, db-cAMP prevented IFN-γ/LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-α/IL-6) and p-STAT1. In vivo, db-cAMP reduced the number of M1 macrophages induced by LPS injection without changes in M2 and Mres numbers. Moreover, db-cAMP enhanced efferocytosis of apoptotic neutrophils in a PKA-dependent manner and increased the expression of Annexin A1 and CD36, two molecules associated with efferocytosis. Finally, inhibition of endogenous PKA during LPS-induced pleurisy impaired the physiological resolution of inflammation. Taken together, the results suggest that cAMP is involved in the major functions of macrophages, such as nonphlogistic recruitment, reprogramming and efferocytosis, all key processes for inflammation resolution.

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Nonphlogistic migration of macrophages contributes to the clearance of pathogens and apoptotic cells: critical steps for the resolution of inflammation and return to homeostasis. Angiotensin-(1-7) [Ang-(1-7)] is an heptapeptide of the Renin-Angiotensin system that acts through Mas receptor (MasR). Ang-(1-7) has recently emerged as a novel pro-resolving mediator, yet Ang-(1-7) resolution mechanisms are not fully determined. Herein, Ang-(1-7) stimulated migration of human and murine monocytes/macrophages in a MasR, CCR2 and MEK/ERK1/2-dependent manner. Pleural injection of Ang-(1-7) promoted nonphlogistic mononuclear cell influx alongside increased levels of CCL2, IL-10 and macrophage polarization towards a regulatory phenotype. Ang-(1-7) induction of CCL2 and mononuclear cell migration was also dependent on MasR and MEK/ERK. Noteworthy, MasR was upregulated during resolution phase of inflammation and their pharmacological inhibition or genetic deficiency impaired mononuclear cell recruitment during self-resolving models of LPS pleurisy and E. coli peritonitis. Inhibition/absence of MasR was associated with reduced CCL2 levels, impaired phagocytosis of bacteria, efferocytosis and delayed resolution of inflammation. In summary, we have uncovered a novel pro-resolving feature of Ang-(1-7), namely the recruitment of mononuclear cells favoring efferocytosis, phagocytosis and resolution of inflammation. Mechanistically, cell migration was dependent on MasR, CCR2 and the MEK/ERK pathway.

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