Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.

BACKGROUND: Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder. OBJECTIVES: We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense. METHODS: Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated. RESULTS: As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo. CONCLUSIONS: Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.

Neutrophils regulate tissue Neutrophilia in inflammation via the oxidant-modified lipid lysophosphatidylserine.

Resolution of neutrophilia characteristic of acute inflammation requires cessation of neutrophil recruitment and removal of tissue neutrophils. Based on in vitro studies, a role in these events was hypothesized for oxidant-generated lysophosphatidylserine (lyso-PS) on recruited neutrophils signaling via the G2A receptor on macrophages. Peritoneal exudate neutrophils harvested from wild type (WT) mice had 5-fold more lyso-PS (lyso-PS(high)) than those of gp91(phox)(-/-) (lyso-PS(low)) mice. Ex vivo engulfment of lyso-PS(high) neutrophils (95% viable) by WT peritoneal macrophages was quantitatively similar to UV-irradiated apoptotic blood neutrophils, although the signaling pathway for the former was uniquely dependent on macrophage G2A. In contrast, lyso-PS(low) neutrophils were poorly engulfed unless presented with exogenous lyso-PS. Enhanced clearance of lyso-PS(high) neutrophils was also seen in vivo following their adoptive transfer into inflamed peritonea of WT but not G2A(-/-) mice, further supporting a requirement for signaling via G2A. To investigate downstream effects of lyso-PS/G2A signaling, antibody blockade of G2A in WT mice reduced macrophage CD206 expression and efferocytosis during peritonitis. Conversely, adoptive transfer of lyso-PS(high) neutrophils early in inflammation in gp91(phox)(-/-) mice led to accelerated development of efferocytic(high) and CD206(high) macrophages. This macrophage reprogramming was associated with suppressed production of pro-inflammatory mediators and reduced neutrophilia. These effects were not seen if G2A was blocked or lyso-PS(low) neutrophils were transferred. Taken together, the results demonstrate that oxidant-generated lyso-PS made by viable tissue neutrophils is an endogenous anti-inflammatory mediator working in vivo to orchestrate the "early" and rapid clearance of recruited neutrophils as well as the reprogramming of "resolving" macrophages.

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity.

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA.

The adipocyte-specific protein FSP27, also known as CIDEC, is one of three cell death-inducing DFF45-like effector (CIDE) proteins. The first known function for CIDEs was promotion of apoptosis upon ectopic expression in mammalian cells. Recent studies in endogenous settings demonstrated key roles for CIDEs in energy metabolism. FSP27 is a lipid droplet-associated protein whose heterologous expression enhances formation of enlarged lipid droplets and is required for unilocular lipid droplets typical of white adipocytes in vivo. Here, we delineate relationships between apoptotic function and lipid droplet localization of FSP27. We demonstrate that ectopic expression of FSP27 induces enlarged lipid droplets in multiple human cell lines, which is indicative that its mechanism involves ubiquitously present, rather than adipocyte-specific, cellular machinery. Furthermore, promotion of lipid droplet formation in HeLa cells via culture in exogenous oleic acid offsets FSP27-mediated apoptosis. Using transient cotransfections and analysis of lipid droplets in HeLa cells stably expressing FSP27, we show that FSP27 does not protect lipid droplets from action of ATGL lipase. Domain mapping with eGFP-FSP27 deletion constructs indicates that lipid droplet localization of FSP27 requires amino acids 174-192 of its CIDE C domain. The apoptotic mechanism of FSP27, which we show involves caspase-9 and mitochondrial cytochrome c, also requires this 19-amino acid region. Interaction assays determine the FSP27 CIDE C domain complexes with CIDEA, and Western blot reveals that FSP27 protein levels are reduced by coexpression of CIDEA. Overall, our findings demonstrate the function of the FSP27 CIDE C domain and/or regions thereof for apoptosis, lipid droplet localization, and CIDEA interaction.

Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4.

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.