Impairment of systemic DHA synthesis affects macrophage plasticity and polarization: implications for DHA supplementation during inflammation

Docosahexaenoic acid (DHA) is an omega-3 fatty acid obtained from the diet or synthesized from alpha-linolenic acid through the action of fatty acid elongases (ELOVL) and desaturases. DHA plays important roles in the central nervous system as well as in peripheral organs and is the precursor of several molecules that regulate resolution of inflammation. In the present study, we questioned whether impaired synthesis of DHA affected macrophage plasticity and polarization both in vitro and in vivo models. For this we investigated the activation status and inflammatory response of bone marrow-derived M1 and M2 macrophages obtained from mice deficient of Elovl2 (Elovl2^?/?), a key enzyme for DHA synthesis in mammals. Although both wild type and Elovl2^?/? mice were able to generate efficient M1 and M2 macrophages, M1 cells derived from Elovl2^?/? mice showed an increased expression of key markers (iNOS, CD86 and MARCO) and cytokines (IL-6, IL-12 and IL-23). However, M2 macrophages exhibited upregulated M1-like markers like CD80, CD86 and IL-6, concomitantly with a downregulation of their signature marker CD206. These effects were counteracted in cells obtained from DHA-supplemented animals. Finally, white adipose tissue of Elovl2^?/? mice presented an M1-like pro-inflammatory phenotype. Hence, impairment of systemic DHA synthesis delineates an alteration of M1/M2 macrophages both in vitro and in vivo, with M1 being hyperactive and more pro-inflammatory while M2 less protective, supporting the view that DHA has a key role in controlling the balance between pro- and anti-inflammatory processes.     

HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines

Activation of Kupffer cells (KCs) induced that inflammatory cytokine production plays a central role in the pathogenesis of HBV infection. The previous studies from our and other laboratory demonstrated miRNAs can regulate TLR-inducing inflammatory responses to macrophage. However, the involvement of miRNAs in HBV-associated antigen-induced macrophage activation is still not thoroughly understood. Here, we evaluated the effects and mechanisms of miR-155 in HBV-associated antigen-induced macrophage activation. First, co-culture assay of HepG2 or HepG2.2.15 cells and RAW264.7 macrophages showed that HepG2.2.15 cells could significantly promote macrophages to produce inflammatory cytokines. Furthermore, we, respectively, stimulated RAW264.7 macrophages, mouse primary peritoneal macrophages, or healthy human peripheral blood monocytes with HBV-associated antigens, including HBcAg, HBeAg, and HBsAg, and found that only HBeAg could steadily enhance the production of inflammatory cytokines in these cells. Subsequently, miRNAs sequencing presented the up- or down-regulated expression of multiple miRNAs in HBeAg-stimulated RAW264.7 cells. In addition, we verified the expression of miR-155 and its precursors BIC gene with q-PCR in the system of co-culture or HBeAg-stimulated macrophages. Meanwhile, the increased miR-155 expression was positively correlation with serum ALT, AST, and HBeAg levels in AHB patients. Although MAPK, PI3K, and NF-κB signal pathways were all activated during HBeAg treatment, only PI3K and NF-κB pathways were involved in miR-155 expression induced by HBeAg stimulation. Consistently, miR-155 over-expression inhibited production of inflammatory cytokines, which could be reversed by knocking down miR-155. Moreover, we demonstrated that miR-155 regulated HBeAg-induced cytokine production by targeting BCL-6, SHIP-1, and SOCS-1. In conclusion, our data revealed that HBeAg augments the expression of miR-155 in macrophages via PI3K and NF-κB signal pathway and the increased miR-155 promotes HBeAg-induced inflammatory cytokine production by inhibiting the expression of BCL-6, SHIP-1, and SOCS-1.     

Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis

Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.     

Arginase expression in peritoneal macrophages and increase in circulating polyamine levels in mice infected with Schistosoma mansoni

We investigated the nitric oxide (NO) synthase and arginase pathways in resident peritoneal macrophages of mice infected with the tropical parasite Schistosoma mansoni. The two enzymes may have opposite effects, insofar as NO may be involved in the killing of the parasite whereas arginase may stimulate parasite growth via polyamine synthesis. We determined the effects of the infection on the expression and activity of the two enzymes in macrophages, before and after cytokine activation. Cells from infected mice expressed the hepatic type I arginase, whereas in control cells, the enzyme was expressed only after cytokine activation, as were NO synthase II and type II arginase in both groups of cells. Moreover, we found that in infected mice, arginase expression in macrophages was associated with a ten fold increase in the concentration of circulating ornithine-derived polyamines. This may be of pathological importance, since parasitic helminths are though to be dependent on their hosts for the uptake and interconversion of polyamines. Received 13 March 2001; received after revision 4 May 2001; accepted 7 June 2001     

Role of hsp70 in cytokine production

Interleukin-1 and tumor necrosis factor-alpha are potent, multifunctional cytokine mediators of inflammation and immune responses that are produced primarily by activated monocytes and macrophages. Three published papers by different groups have shown that heat shock and chemical stress with heavy metal salts or sulfhydryl reagents, all of which induce the expression of heat shock protein 70 (hsp70), concomitantly inhibit the production of these cytokines in human monocytes and mouse macrophages activated by lipopolysaccharide. These papers are reviewed and discussed in some detail. Other studies suggest that various anti-inflammatory drugs, including acetylsalicyclic acid, auranofin and dexamethasone, can also facilitate HSP expression in macrophages. However, while these studies are interesting, it is clear that not a great deal of work has been done and/or published in this area. Since many pharmaceutical companies are developing cytokine synthesis inhibitors as potential anti-inflammatory drugs, one aim of this article is to emphasize that understanding the molecular mechanism(s) that lead to increased HSP expression and decreased cytokine biosynthesis may assist in achieving this goal.     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Blocking of melatonin synthesis and MT1 receptor impairs the activation of Jurkat T cells

Melatonin has been proposed as regulating the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. To further investigate the melatonin’s role in IL-2/IL-2R system, we established an inducible T-REx expression system in Jurkat cells in which the protein levels of HIOMT enzyme or MT₁ receptor were significantly down-regulated upon tetracycline incubation. We found that T-REx Jurkat cells with lower levels of HIOMT activity, and consequently lower content of endogenous melatonin, showed IL-2 production decrease after activation with lectin. Likewise, tetracycline-inducible stable cell line expressing MT₁ antisense produced decreased amounts of IL-2 (mRNA and protein levels) after stimulation. Moreover, in T-Rex-MT₁ cells incubated with tetracycline, a sub-optimal PHA dose failed to induce the early activation marker CD25 on the cell surface. The results shown here support the relevance of endogenous melatonin and its signaling in T cell activation.     

The pathogenesis of cardiac fibrosis

Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. This review discusses the cellular effectors and molecular pathways implicated in the pathogenesis of cardiac fibrosis. Although activated myofibroblasts are the main effector cells in the fibrotic heart, monocytes/macrophages, lymphocytes, mast cells, vascular cells and cardiomyocytes may also contribute to the fibrotic response by secreting key fibrogenic mediators. Inflammatory cytokines and chemokines, reactive oxygen species, mast cell-derived proteases, endothelin-1, the renin/angiotensin/aldosterone system, matricellular proteins, and growth factors (such as TGF-β and PDGF) are some of the best-studied mediators implicated in cardiac fibrosis. Both experimental and clinical evidence suggests that cardiac fibrotic alterations may be reversible. Understanding the mechanisms responsible for initiation, progression, and resolution of cardiac fibrosis is crucial to design anti-fibrotic treatment strategies for patients with heart disease.     

Regulation of macrophage activation

IFN- rapidly primes the macrophage via JAK1/2-STAT1 pathway so that it can subsequently undergo a slower classical type 1 activation upon exposure to T helper (Th)1 cytokines such as IFN or other activators, including tumor necrosis factor and lipopolysaccharide, e.g. in intracellular killing of phagocytosed Mycobacterium tuberculosis. If instead it is driven by Th2 cytokines interleukin (IL)-4 and IL-13, it undergoes alternate type 2 activation, which enhances endocytotic antigen uptake and presentation, mast cell and eosinophil involvement and type 2 granuloma formation, e.g. in response to parasitic and extracellular pathogens. Particle-induced macrophage activation was shown to differ from classical and alternate activation, showing in DNA microarray experiments (complete linkage/ Euclidean distance metric analysis) upregulation of nonsecreted structural/signaling molecules and lack of secreted proin-flammatory cyto- and chemokines. The switch-off (deactivation) of already activated macrophages is an active, controlled process in which IL-10 and corticosteroids play important roles and to which15dPGJ2, PGA1/2 and vasoactive intestinal peptide often contribute.Received 16 January 2003; received after revision 14 March 2003; accepted 2 April 2003     

Targeting microglia-mediated neurotoxicity: the potential of NOX2 inhibitors

Microglia are key sentinels of central nervous system health, and their dysfunction has been widely implicated in the progressive nature of neurodegenerative diseases. While microglia can produce a host of factors that are toxic to neighboring neurons, NOX2 has been implicated as a common and essential mechanism of microglia-mediated neurotoxicity. Accumulating evidence indicates that activation of the NOX2 enzyme complex in microglia is neurotoxic, both through the production of extracellular reactive oxygen species that damage neighboring neurons as well as the initiation of redox signaling in microglia that amplifies the pro-inflammatory response. More specifically, evidence supports that NOX2 redox signaling enhances microglial sensitivity to pro-inflammatory stimuli, and amplifies the production of neurotoxic cytokines, to promote chronic and neurotoxic microglial activation. Here, we describe the evidence denoting the role of NOX2 in microglia-mediated neurotoxicity with an emphasis on Alzheimer's and Parkinson's disease, describe available inhibitors that have been tested, and detail evidence of the neuroprotective and therapeutic potential of targeting this enzyme complex to regulate microglia.     

Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constititive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetricLimulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.     

The role of the thioredoxin/thioredoxin reductase system in the metabolic syndrome: towards a possible prognostic marker?

Mammalian thioredoxin reductase (TrxR) is a selenoprotein with three existing isoenzymes (TrxR1, TrxR2, and TrxR3), which is found primarily intracellularly but also in extracellular fluids. The main substrate thioredoxin (Trx) is similarly found (as Trx1 and Trx2) in various intracellular compartments, in blood plasma, and is the cell’s major disulfide reductase. Thioredoxin reductase is necessary as a NADPH-dependent reducing agent in biochemical reactions involving Trx. Genetic and environmental factors like selenium status influence the activity of TrxR. Research shows that the Trx/TrxR system plays a significant role in the physiology of the adipose tissue, in carbohydrate metabolism, insulin production and sensitivity, blood pressure regulation, inflammation, chemotactic activity of macrophages, and atherogenesis. Based on recent research, it has been reported that the modulation of the Trx/TrxR system may be considered as a new target in the management of the metabolic syndrome, insulin resistance, and type 2 diabetes, as well as in the treatment of hypertension and atherosclerosis. In this review evidence about a possible role of this system as a marker of the metabolic syndrome is reported.     

Immunoregulatory properties of the cytokine IL-34

Interleukin-34 is a cytokine with only partially understood functions, described for the first time in 2008. Although IL-34 shares very little homology with CSF-1 (CSF1, M-CSF), they share a common receptor CSF-1R (CSF-1R) and IL-34 has also two distinct receptors (PTP-ζ) and CD138 (syndecan-1). To make the situation more complex, IL-34 has also been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that this cytokine is released by some tissues that differ to those where CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Our recent work has demonstrated a new and possible therapeutic role for IL-34 as a Foxp3⁺ Treg-secreted cytokine mediator of transplant tolerance. In this review, we recapitulate most recent findings on IL-34 and its controversial effects on immune responses and address its immunoregulatory properties and the potential of targeting this cytokine in human.     

Macropinocytosis,mTORC1 and cellular growth control

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.     

The plasminogen activation system in tumor growth, invasion, and metastasis

Generation of the serine proteinase plasmin from the extracellular zymogen plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type plasminogen activators (uPA and tPA). The plasminogen activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the plasminogen activation system may allow utilization of its components as targets for anti-invasive therapy.     

Molecular aspects of pathogenesis in osteoarthritis: the role of inflammation

Arthritic diseases cause enormous burdens in terms of pain, crippling, and disability. Osteoarthritis (OA), the most common form of arthritis, is characterized by a slow progressive degeneration of articular cartilage. The exact etiology of OA is not known, but the degradation of cartilage matrix components is generally agreed to be due to an increased synthesis and activation of extracellular proteinases, mainly matrix metalloproteinases. Insufficient synthesis of new matrix macromolecules is also thought to be involved, possibly as a consequence of deficient stimulation by growth factors. Although OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of nitric oxide and prostaglandin E₂, two factors that have been shown to induce a number of the cellular changes associated with OA. The generation of these key signal molecules depends on inducible enzymes and can be suppressed by pharmacological inhibitors.