Modulation of Macrophage Efferocytosis in Inflammation

A critical function of macrophages within the inflammatory milieu is the removal of dying cells by a specialized phagocytic process called efferocytosis (“to carry to the grave”). Through specific receptor engagement and induction of downstream signaling, efferocytosing macrophages promote resolution of inflammation by (i) efficiently engulfing dying cells, thus avoiding cellular disruption and release of inflammatory contents, and (ii) producing anti-inflammatory mediators such as IL-10 and TGF-β that dampen pro-inflammatory responses. Evidence suggests that plasticity in macrophage programming, in response to changing environmental cues, modulates efferocytic capability. Essential to programming for enhanced efferocytosis is activation of the nuclear receptors PPARγ, PPARδ, LXR, and possibly RXRα. Additionally, a number of signals in the inflammatory milieu, including those from dying cells themselves, can influence efferocytic efficacy either by acting as immediate inhibitors/enhancers or by altering macrophage programming for longer-term effects. Importantly, sustained inflammatory programming of macrophages can lead to defective apoptotic cell clearance and is associated with development of autoimmunity and other chronic inflammatory disorders. This review summarizes the current knowledge of the multiple factors that modulate macrophage efferocytic ability and highlights emerging therapeutic targets with significant potential for limiting chronic inflammation

Impaired phagocytosis of apoptotic cells by macrophages in chronic granulomatous disease is reversed by IFN-γ in a nitric oxide-dependent manner

Immunodeficiency in chronic granulomatous disease (CGD) is well characterized. Less understood are exaggerated sterile inflammation and autoimmunity associated with CGD. Impaired recognition and clearance of apoptotic cells resulting in their disintegration may contribute to CGD inflammation. We hypothesized that priming of macrophages (Ms) with IFN-γ would enhance impaired engulfment of apoptotic cells in CGD. Diverse M populations from CGD (gp91(phox)(-/-)) and wild-type mice, as well as human Ms differentiated from monocytes and promyelocytic leukemia PLB-985 cells (with and without mutation of the gp91(phox)), demonstrated enhanced engulfment of apoptotic cells in response to IFN-γ priming. Priming with IFN-γ was also associated with increased uptake of Ig-opsonized targets, latex beads, and fluid phase markers, and it was accompanied by activation of the Rho GTPase Rac. Enhanced Rac activation and phagocytosis following IFN-γ priming were dependent on NO production via inducible NO synthase and activation of protein kinase G. Notably, endogenous production of TNF-α in response to IFN-γ priming was critically required for inducible NO synthase upregulation, NO production, Rac activation, and enhanced phagocytosis. Treatment of CGD mice with IFN-γ also enhanced uptake of apoptotic cells by M in vivo via the signaling pathway. Importantly, during acute sterile peritonitis, IFN-γ treatment reduced excess accumulation of apoptotic neutrophils and enhanced phagocytosis by CGD Ms. These data support the hypothesis that in addition to correcting immunodeficiency in CGD, IFN-γ priming of Ms restores clearance of apoptotic cells and may thereby contribute to resolution of exaggerated CGD inflammation

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein–coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A2/2 mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A2/2 colons showed significantly more TNF-a+ and Ly6ChiMHCII2 proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A2/2 mice. G2A2/2 mice also had less IFN-g in inflamed colon tissues than wild-type mice. Fewer CD4+ lymphocytes were recruited to inflamed G2A2/2 colons, and fewer colonic lymphocytes produced IFN-g upon ex vivo stimulation. Administration of IFN-g to G2A2/2 mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-g reduced the numbers of TNF-a+ monocyte and enhanced their maturation from Ly6ChiMHCII2 to Ly6CintMHCII+ . Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-g, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

<div><p>Background/Objective</p><p>Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.</p> <p>Methodology/Principal Findings</p><p>In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated <i>via</i> activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.</p> <p>Conclusion/Significance</p><p>Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.</p> </div

Receptor CD36 is required for apoptotic cells induced TGF-β1 mRNA and protein expression.

<p>A, CD36 protein expression in RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h was analyzed by Western blotting with anti-CD36 (MF3) antibody, and band density was normalized to GAPDH. B, RAWTβRII cells transfected with CD36-target siRNA or control vehicle (Ctr siRNA) for 24 h were stimulated with apoptotic or viable Jurkat cells. C, Apoptotic Jurkat cells were pretreated with annexin V for 45 min and incubated with RAWTβRII transfected with CD36-target siRNA or control siRNA (Ctr siRNA). TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. D, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) were treated with either LPS (25 ng/ml) or PMA (50 nM). The expression of TGF-β1 mRNA was analyzed as above. Values represent means ± SD of five separate experiments. ns, no significant; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Both Lyn kinase and ERK1/2 MAPK are required for TGF-β1 synthesis induced by activating anti-CD36 mIgA.

<p>A, RAWTβRII cells were stimulated with activating anti-CD36 mIgA (2 µg/ml) for the times indicated. Total cell lysates were immunoblotted for phospho-Lyn kinase and the band density was normalized to total Lyn kinase. B, RAWTβRII cells were pretreated with the src-family kinase inhibitor PP2 (0.001 to 100 µM) for 2 h and then stimulated with anti-CD36 mIgA (2 µg/ml). After 6 h, TGF-β1 mRNA expression was analyzed by real-time PCR and normalized to GAPDH. Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h. C, A time course of ERK1/2 phosphorylation was assessed by Western blotting in RAWTβRII cells treated with anti-CD36 mIgA (2 µg/ml). Phospho-ERK1/2 band density was normalized to total ERK1/2. D, RAWTβRII cells were preincubated with MEK kinase inhibitor U0126 or inactive analogue U0124 for 2 h and then stimulated with anti-CD36 mIgA for 6 h to detect TGF-β1 mRNA expression or for 18 h to detect secreted TGF-β1 protein as in Figure 1. Values represent means ± SD of six separate experiments.</p