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Atherosclerosis is a cardiovascular disease caused mainly by dyslipidemia and is characterized by the formation of an atheroma plaque and chronic inflammation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that induces the degradation of the LDL receptor (LDLR), which contributes to increased levels of LDL cholesterol and the progress of atherosclerosis. Given that macrophages are relevant components of the lipidic and inflammatory environment of atherosclerosis, we studied the effects of PCSK9 treatment on human macrophages. Our data show that human macrophages do not express PCSK9 but rapidly incorporate the circulating protein through the LDLR and also activate the pro-inflammatory TLR4 pathway. Both LDLR and TLR4 are internalized after incubation of macrophages with exogenous PCSK9. PCSK9 uptake increases the production of reactive oxygen species and reduces the expression of genes involved in lipid metabolism and cholesterol efflux, while enhancing the production of pro-inflammatory cytokines through a TLR4-dependent mechanism. Under these conditions, the viability of macrophages is compromised, leading to increased cell death. These results provide novel insights into the role of PCSK9 in the crosstalk of lipids and cholesterol metabolism through the LDLR and on the pro-inflammatory activation of macrophages through TLR4 signaling. These pathways are relevant in the outcome of atherosclerosis and highlight the relevance of PCSK9 as a therapeutic target for the treatment of cardiovascular diseases.

The bioavailability and regulation of iron is essential for central biological functions in mammals. The role of this element in ferroptosis and the dysregulation of its metabolism contribute to diseases, ranging from anemia to infections, alterations in the immune system, inflammation and atherosclerosis. In this sense, monocytes and macrophages modulate iron metabolism and splenic function, while at the same time they can worsen the atherosclerotic process in pathological conditions. Since the nucleotide-binding oligomerization domain 1 (NOD1) has been linked to numerous disorders, including inflammatory and cardiovascular diseases, we investigated its role in iron homeostasis. The iron content was measured in various tissues of Apoe -/- and Apoe -/- Nod1 -/- mice fed a high-fat diet (HFD) for 4 weeks, under normal or reduced splenic function after ligation of the splenic artery. In the absence of NOD1 the iron levels decreased in spleen, heart and liver regardless the splenic function. This iron decrease was accompanied by an increase in the recruitment of F4/80 + -macrophages in the spleen through a CXCR2-dependent signaling, as deduced by the reduced recruitment after administration of a CXCR2 inhibitor. CXCR2 mediates monocyte/macrophage chemotaxis to areas of inflammation and accumulation of leukocytes in the atherosclerotic plaque. Moreover, in the absence of NOD1, inhibition of CXCR2 enhanced atheroma progression. NOD1 activation increased the levels of GPX4 and other iron and ferroptosis regulatory proteins in macrophages. Our findings highlight the preeminent role of NOD1 in iron homeostasis and ferroptosis. These results suggest promising avenues of investigation for the diagnosis and treatment of iron-related diseases directed by NOD1., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Protein tyrosine phosphatase 1B (PTP1B) is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs) from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Unlabelled: (18)F-FDG accumulates in glycolytically active tissues and is known to concentrate in tissues that are rich in activated macrophages. In this study, we tested the hypotheses that human granulocyte-macrophage colony-stimulating factor (GM-CSF), a clinically used cytokine, increases macrophage glycolysis and deoxyglucose uptake in vitro and acutely enhances (18)F-FDG uptake within inflamed tissues such as atherosclerotic plaques in vivo., Methods: In vitro experiments were conducted on human macrophages whereby inflammatory activation and uptake of radiolabeled 2-deoxyglucose was assessed before and after GM-CSF exposure. In vivo studies were performed on mice and New Zealand White rabbits to assess the effect of GM-CSF on (18)F-FDG uptake in normal versus inflamed arteries, using PET., Results: Incubation of human macrophages with GM-CSF resulted in increased glycolysis and increased 2-deoxyglucose uptake (P < 0.05). This effect was attenuated by neutralizing antibodies against tumor necrosis factor-α or after silencing or inhibition of 6-phosphofructo-2-kinase. In vivo, in mice and in rabbits, intravenous GM-CSF administration resulted in a 70% and 73% increase (P < 0.01 for both), respectively, in arterial (18)F-FDG uptake in atherosclerotic animals but not in nonatherosclerotic controls. Histopathologic analysis demonstrated a significant correlation between in vivo (18)F-FDG uptake and macrophage staining (R = 0.75, P < 0.01)., Conclusion: GM-CSF substantially augments glycolytic flux in vitro (via a mechanism dependent on ubiquitous type 6-phosphofructo-2-kinase and tumor necrosis factor-α) and increases (18)F-FDG uptake within inflamed atheroma in vivo. These findings demonstrate that GM-CSF can be used to enhance detection of inflammation. Further studies should explore the role of GM-CSF stimulation to enhance the detection of inflammatory foci in other disease states., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Macrophages are present in a large variety of locations, playing distinct functions that are determined by its developmental origin and by the nature of the activators of the microenvironment. Macrophage activation can be classified as pro-inflammatory (M1 polarization) or anti-inflammatory-pro-resolution-deactivation (M2), these profiles coexisting in the course of the immune response and playing a relevant functional role in the onset of inflammation (Figure 1). Several groups have analysed the metabolic aspects associated with macrophage activation to answer the question about what changes in the regulation of energy metabolism and biosynthesis of anabolic precursors accompany the different types of polarization and to what extent they are necessary for the expression of the activation phenotypes. The interest of these studies is to regulate macrophage function by altering their metabolic activity in a 'therapeutic way'., (© 2015 Authors; published by Portland Press Limited.)

Objective: Although it is accepted that macrophage glycolysis is upregulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage proinflammatory activation and whether specific energy demands regulate cell viability in the atheromatous plaque., Approach and Results: We studied the interplay between macrophage energy metabolism, polarization, and viability in the context of atherosclerosis. Cultured human and murine macrophages and an in vivo murine model of atherosclerosis were used to evaluate the mechanisms underlying metabolic and inflammatory activity of macrophages in the different atherosclerotic conditions analyzed. We observed that macrophage energetics and inflammatory activation are closely and linearly related, resulting in dynamic calibration of glycolysis to keep pace with inflammatory activity. In addition, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase. As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages' ability to increase glycolysis through 6-phosphofructo-2-kinase is experimentally attenuated, cell viability is reduced if subjected to proinflammatory or hypoxic conditions, but unaffected under control conditions. In addition to this, granulocyte-macrophage colony-stimulating factor enhances anerobic glycolysis while exerting a mild proinflammatory activation., Conclusions: These findings, in human and murine cells and in an animal model, show that hypoxia potentiates macrophage glycolytic flux in concert with a proportional upregulation of proinflammatory activity, in a manner that is dependent on both hypoxia-inducible factor -1α and 6-phosphofructo-2-kinase., (© 2015 American Heart Association, Inc.)

The resolution of inflammation is an active process driven by specialized pro-resolving lipid mediators, such as 15-epi-LXA4 and resolvin D1 (RvD1), that promote tissue regeneration. Macrophages regulate the innate immune response being key players during the resolution phase to avoid chronic inflammatory pathologies. Their half-life is tightly regulated to accomplish its phagocytic function, allowing the complete cleaning of the affected area. The balance between apoptosis and autophagy appears to be essential to control the survival of these immune cells within the inflammatory context. In the present work, we demonstrate that 15-epi-LXA4 and RvD1 at nanomolar concentrations promote autophagy in murine and human macrophages. Both compounds induced the MAP1LC3-I to MAP1LC3-II processing and the degradation of SQSTM1 as well as the formation of MAP1LC3(+) autophagosomes, a typical signature of autophagy. Furthermore, 15-epi-LXA4 and RvD1 treatment favored the fusion of the autophagosomes with lysosomes, allowing the final processing of the autophagic vesicles. This autophagic response involves the activation of MAPK1 and NFE2L2 pathways, but by an MTOR-independent mechanism. Moreover, these pro-resolving lipids improved the phagocytic activity of macrophages via NFE2L2. Therefore, 15-epi-LXA4 and RvD1 improved both survival and functionality of macrophages, which likely supports the recovery of tissue homeostasis and avoiding chronic inflammatory diseases.

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.

Lipoxin A(4) (LXA(4)) is an endogenous lipid mediator that requires transcellular metabolic traffic for its synthesis. The targets of LXA(4) on neutrophils are well described, contributing to attenuation of inflammation. However, effects of lipoxins on macrophage are less known, particularly the action of LXA(4) on the regulation of apoptosis of these cells. Our data show that pretreatment of human or murine macrophages with LXA(4) at the concentrations prevailing in the course of resolution of inflammation (nanomolar range) significantly inhibits the apoptosis induced by staurosporine, etoposide and S-nitrosoglutathione or by more pathophysiological stimuli, such as LPS/IFNgamma challenge. The release of mitochondrial mediators of apoptosis and the activation of caspases was abrogated in the presence of LXA(4). In addition to this, the synthesis of reactive oxygen species induced by staurosporine was attenuated and antiapoptotic proteins of the Bcl-2 family accumulated in the presence of lipoxin. Analysis of the targets of LXA(4) identified an early activation of the PI3K/Akt and ERK/Nrf-2 pathways, which was required for the observation of the antiapoptotic effects of LXA(4). These data suggest that the LXA(4), released after the recruitment of neutrophils to sites of inflammation, exerts a protective effect on macrophage viability that might contribute to a better resolution of inflammation.

The effect of rofecoxib, a selective cyclooxygenase-2 inhibitor, on inflammatory signaling has been investigated in elicited murine peritoneal macrophages. Macrophages treated with 10 microM rofecoxib exhibited an important inhibition in the early activation of nuclear factor kappa B (NF-kappa B) and the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase. Moreover, this drug decreased the protein levels of nitric-oxide synthase-2 and cyclooxygenase-2 in lipopolysaccharide (LPS)-treated macrophages. Rofecoxib delayed and attenuated NF-kappa B activation, which impaired significantly the expression of kappa B-dependent genes. This drug and related coxibs did not affect cell viability and protected against LPS-induced apoptosis through the impairment of the inflammatory response. These data show an additional anti-inflammatory mechanism of selective cyclooxygenase-2 inhibitors through the attenuation of macrophage activation.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) transiently activates protein kinase C zeta (PKC zeta) and Jun N-terminal kinase (JNK) through a phosphoinositide-3-kinase (PI3-kinase)-dependent pathway. Incubation of LPS-treated cells with the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) promoted a sustained activation of PKC zeta and JNK and inhibited I kappa B kinase (IKK) and NF-kappa B activity. Accordingly, 15dPGJ(2) induced an imbalance between JNK and IKK activities by increasing the former signaling pathway and inhibiting the latter signaling pathway. Under these conditions, apoptosis was significantly enhanced; this response was very dependent on PKC zeta and JNK activation. The effect of 15dPGJ(2) on PKC zeta activity observed in LPS-activated macrophages was not dependent on a direct action of this prostaglandin on the enzyme but was due to the activation of a step upstream of PI3-kinase. Moreover, LPS promoted the redistribution of activated PKC zeta from the cytosol to the nucleus, a process that was enhanced by treatment of the cells with 15dPGJ(2) that favored a persistent and broader distribution of PKC zeta in the nucleus. These results indicate that 15dPGJ(2) and other cyclopentenone prostaglandins, through the sustained activation of PKC zeta, might contribute significantly to the process of resolution of inflammation by promoting apoptosis of activated macrophages.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) inhibited the LPS- and IFN-gamma-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J(2), prostaglandin E(2), or rosiglitazone, a peroxisomal proliferator-activated receptor gamma ligand. Incubation of activated macrophages with 15dPGJ(2) inhibited the degradation of IkappaBalpha and IkappaBbeta and increased their levels in the nuclei. NF-kappaB activity, as well as the transcription of NF-kappaB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IkappaB phosphorylation showed an inhibition of IkappaB kinase by 15dPGJ(2) in cells treated with LPS and IFN-gamma, resulting in an impaired phosphorylation of IkappaBalpha, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IkappaB kinase with 2 microM 15dPGJ(2) reduced by 83% the phosphorylation in serine 32 of IkappaBalpha, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IkappaB kinase complex. These results show rapid actions of 15dPGJ(2), independent of peroxisomal proliferator receptor gamma activation, in macrophages challenged with low doses of LPS and IFN-gamma.

Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolonged activation of NF-kappaB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experiments using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Cotransfection of cells with a catalytically active p110 subunit of PI3-kinase impaired the responsiveness of the iNOS promoter to LPS stimulation, whereas transfection with a kinase-deficient mutant of p110 maintained the up-regulation in response to wortmannin. These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflammatory cytokines.

The effect of acanthoic acid analogs on the response to proinflammatory challenge was investigated. Some pimarane diterpenes are known activators of the LXRαβ nuclear receptors, but we show here that they also exert a rapid, potent, and selective activation of the p110γ and p110δ subunits of PI3K. Combination of these effects results in an important attenuation of the global transcriptional response to LPS in macrophages. PI3K/Akt activation leads to inhibition of the LPS-dependent stimulation of IKK/NF-κB and p38 and ERK MAPKs. Macrophages from LXRαβ-deficient mice exhibited an inhibition of these pathways similar to the corresponding wild-type cells. Silencing or inhibition of p110γ/δ suppressed the effect of these diterpenes (DTPs) on IKK/NF-κB and MAPKs signaling. Taken together, these data show a multitarget anti-inflammatory mechanism by these DTPs including a selective activation of PI3K isoenzymes.