The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells

Macrophages take up oxidized low density lipoprotein (oxLDL) after being exposed to it in the blood vessels. oxLDL transforms macrophages into foam cells, which are a hallmark of atherosclerosis. The effects that oxLDL have on the inflammatory responses of foam cells are not clear. Here, we investigated how oxLDL modulates lipopolysaccharide (LPS)-induced inflammatory mediators in RAW 264.7 murine macrophages. Our results showed that oxLDL dramatically induced HO-1 expression, but did not increase pro-inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, iNOS, and monocyte chemoattractant protein (MCP)-1. In RAW 264.7 macrophages, oxLDL markedly inhibited LPS-induced inflammatory mediators such as inducible nitric oxide synthase (iNOS), IL-1β, IL-6, granulocyte macrophage colony-stimulating factor and stromal cell-derived factor-1. Interestingly, however, the down-regulation of HO-1 by siRNA did not recover the inhibition of LPS-induced expression and/or the secretion of inflammatory mediators. oxLDL blocked LPS-induced NF-κB nuclear translocation by inhibiting inhibitory κB (IκB) degradation. Taken together, our results suggest that oxLDL could modulate LPS-induced inflammatory responses by inhibiting NF-κB signaling independently of HO-1 expression.     

Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages

Proteinase-activated receptor 2 (PAR(2)), a 7-transmembrane G protein-coupled receptor, contributes to inflammation either positively or negatively in different experimental systems. Previously, we reported that concurrent activation of PAR(2) and TLRs in human lung and colonic epithelial cells resulted in a synergistic increase in NF-κB-mediated gene expression, but a down-regulation of IRF-3-mediated gene expression. In this study, the effect of PAR(2) activation on LPS-induced TLR4 signaling was examined in primary murine macrophages. The PAR(2) activation of wild-type macrophages enhanced LPS-induced expression of the anti-inflammatory cytokine, IL-10, while suppressing gene expression of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12. Similar PAR(2)-mediated effects on LPS-stimulated IL-10 and IL-12 mRNA were also observed in vivo. In contrast, PAR 2-/- macrophages exhibited diminished LPS-induced IL-10 mRNA and protein expression and downstream STAT3 activation, but increased KC mRNA and protein. PAR(2) activation also enhanced both rIL-4- and LPS-induced secretion of IL-4 and IL-13, and mRNA expression of alternatively activated macrophage (AA-M) markers, e.g. arginase-1, mannose receptor, Ym-1. Thus, in the context of a potent inflammatory stimulus like LPS, PAR(2) activation acts to re-establish tissue homeostasis by dampening the production of inflammatory mediators and causing the differentiation of macrophages that may contribute to the development of a Th2 response.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.     

Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens.     

Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression

The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.     

LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells

Bacterial inflammation in mucosa is accompanied by morphological and proliferative changes in goblet cells and mucin hypersecretion. Main stimulators of bacterial inflammation are bacterial lipopolysaccharides (LPS). In vitro investigation of the LPS effect on the molecular processes in goblet cells, using the human mucin-secreting goblet cell line HT29-MTX, showed the following results. LPS up-regulated mucin and cytokine mRNA expression and secretion in goblet cells in a concentration and time-dependent manner, with a maximum output at an LPS concentration of 100 ng/ml. LPS (100 ng/ml) increased mRNA expression of MUC5AC (2.4x), MUC5B (2.1x), and IL-8 (2.3x) and stimulated secretion of mucins (MUC5AC up to 39%, MUC5B up to 31%) and the inflammatory cytokine IL-8 (up to 10x). A significant correlation was found between the LPS-induced IL-8 secretion and secretion of mucins. These results suggest: (1) goblet cells, responding to the direct stimulation of bacterial LPS by two inflammatory-related processes such as production and secretion of the gel-forming mucins and the inflammatory cytokine IL-8, can be considered as an important part of mucosal immunity and (2) LPS- induced goblet cell mucin secretion can occur partly via IL-8-dependent pathway.     

Long noncoding RNA THRIL promotes foam cell formation and inflammation in macrophages

Tumor necrosis factor-α (TNF-α) and heterogenous nuclear ribonucleoprotein L (hnRNPL)-related immunoregulatory lincRNA (THRIL) is a long noncoding RNA (lncRNA) involved in various inflammatory diseases. However, its role in atherosclerosis is not known. In this study, we aimed to investigate the function of THRIL in mediating macrophage inflammation and foam cell formation. The expression of THRIL was quantified in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). The effect of THRIL overexpression and knockdown on oxLDL-induced inflammatory responses and lipid accumulation was determined. THRIL-associated protein partners were identified by RNA pull-down and RNA immunoprecipitation assays. We show that THRIL is upregulated in macrophages after oxLDL treatment. Knockdown of THRIL blocks oxLDL-induced expression of interleukin-1β (IL-1β), IL-6, and TNF-α and lipid accumulation. Conversely, ectopic expression of THRIL enhances inflammatory gene expression and lipid deposition in oxLDL-treated macrophages. Moreover, THRIL depletion increases cholesterol efflux from macrophages and the expression of ATP-binding cassette transporter (ABC) A1 and ABCG1. FOXO1 is identified as a protein partner of THRIL and promotes macrophage inflammation and lipid accumulation. Furthermore, overexpression of FOXO1 restores lipid accumulation and inflammatory cytokine production in THRIL-depleted macrophages. In conclusion, our data suggest a model where THRIL interacts with FOXO1 to promote macrophage inflammation and foam cell formation. THRIL may represent a therapeutic target for atherosclerosis.     

A glycosylphosphatidylinositol-based treatment alleviates trypanosomiasis-associated immunopathology

The GPI-anchored trypanosome variant surface glycoprotein (VSG) triggers macrophages to produce TNF, involved in trypanosomiasis-associated inflammation and the clinical manifestation of sleeping sickness. Aiming at inhibiting immunopathology during experimental Trypanosoma brucei infections, a VSG-derived GPI-based treatment approach was developed. To achieve this, mice were exposed to the GPI before an infectious trypanosome challenge. This GPI-based strategy resulted in a significant prolonged survival and a substantial protection against infection-associated weight loss, liver damage, acidosis, and anemia; the latter was shown to be Ab-independent and correlated with reduced macrophage-mediated RBC clearance. In addition, GPI-based treatment resulted in reduced circulating serum levels of the inflammatory cytokines TNF and IL-6, abrogation of infection-induced LPS hypersensitivity, and an increase in circulating IL-10. At the level of trypanosomiasis-associated macrophage activation, the GPI-based treatment resulted in an impaired secretion of TNF by VSG and LPS pulsed macrophages, a reduced expression of the inflammatory cytokine genes TNF, IL-6, and IL-12, and an increased expression of the anti-inflammatory cytokine gene IL-10. In addition, this change in cytokine pattern upon GPI-based treatment was associated with the expression of alternatively activated macrophage markers. Finally, the GPI-based treatment also reduced the infection-associated pathology in Trypanosoma congolense and Trypanosoma evansi model systems as well as in tsetse fly challenge experiments, indicating potential field applicability for this intervention strategy.     

Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus

Macrophage-derived foam cells in atherosclerotic lesions are generally thought to play a major role in the pathology of the disease. Because macrophages play a central role in the inflammatory response, and the atherosclerotic lesion has features associated with chronic inflammatory settings, we investigated foam cell inflammatory potential. THP-1-derived macrophages were treated with oxidized low density lipoprotein (OxLDL) for 3 days to lipid load the macrophages and establish a foam cell-like phenotype. The cells were then activated by treatment with lipopolysaccharide (LPS), and RNA was harvested at 0, 1, and 6 h after LPS addition. RNA from treated and control cells was hybridized to microarrays containing approximately 16,000 human cDNAs. Genes that exhibited a 4-fold or greater increase or decrease at either 1 or 6 h after LPS treatment were counted as LPS-responsive genes. Employing these criteria, 127 LPS-responsive genes were identified. Prior treatment of THP-1 macrophages with OxLDL affected the expression of 57 of these 127 genes. Among these 57 genes was a group of chemokine, cytokine, and signal transduction genes with pronounced expression changes. OxLDL pretreatment resulted in a significant perturbation of LPS-induced NF kappa B activation. Furthermore, some of the OxLDL effects appear to be mediated by the nuclear receptors retinoid X receptor and peroxisomal proliferator-activated receptor gamma because pretreatment of THP-1 macrophages with ligands for these receptors, followed by LPS treatment, recapitulates the OxLDL plus LPS results for several of the most significantly modulated genes.     

Phytosterols differentially influence ABC transporter expression, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells

Phytosterol supplements lower low-density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E (Apo E)-deficient mice, suggesting that the cholesterol-lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may be either beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high-density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect on cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL (agLDL) induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion.     

The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis

Interleukin-10 (IL-10) may have therapeutic potential in various inflammatory diseases, including atherosclerosis, as it can inhibit oxLDL-induced foam cell formation and apoptosis in macrophages. This study investigated the effect of IL-10 on mitogen-activated protein kinase (MAPK) activation, and apoptosis induced by oxidized low-density lipoprotein (oxLDL) in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that IL-10 significantly blocked the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and apoptosis induced by oxLDL. The inhibitory effect of IL-10 on oxLDL-induced apoptosis was partially dependent on reduced p38, but not JNK, phosphorylation. This study also discovered a linkage between IL-10 and p38 MAPK signaling in oxLDL-induced endothelial cell apoptosis. Interestingly, this study found that lectin-like oxidized LDL receptor-1 (LOX-1) was the only scavenger receptor, on the surface of HUVEC, that was upregulated by oxLDL and the increase in LOX-1 was not suppressed by IL-10. This study confirmed that IL-10 significantly upregulated the expression of suppressor of cytokine signaling-3 (SOCS3), whereas SOCS3 knockdown by siRNA effectively blocked the inhibitory effect of IL-10 on p38 MAPK-dependent apoptosis induced by oxLDL. These results showed for the first time, that IL-10 modulated oxLDL-induced apoptosis by upregulating SOCS3, which then interrupted p38 MAPK activation in endothelial cells. These findings support the essential role of p38 MAPK in the interplay of oxLDL and IL-10 in endothelial apoptosis.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase

IL-6 is a major inflammatory cytokine that plays a central role in coordinating the acute-phase response to trauma, injury, and infection in vivo. Although IL-6 is synthesized predominantly by macrophages and lymphocytes, skeletal muscle is a newly recognized source of this cytokine. IL-6 from muscle spills into the circulation, and blood-borne IL-6 can be elevated >100-fold due to exercise and injury. The purpose of the present study was to determine whether inflammatory stimuli, such as LPS, TNF-alpha, and IL-1beta, could increase IL-6 expression in skeletal muscle and C2C12 myoblasts. Second, we investigated the role of mitogen-activated protein (MAP) kinases, and the Jun NH2-terminal kinase (JNK) in particular, as a mediator of this response. Intraperitoneal injection of LPS in mice increased the circulating concentration of IL-6 from undetectable levels to 4 ng/ml. LPS also increased IL-6 mRNA 100-fold in mouse fast-twitch skeletal muscle. Addition of LPS, IL-1beta, or TNF-alpha directly to C2C12 myoblasts increased IL-6 protein (6- to 8-fold) and IL-6 mRNA (5- to 10-fold). The response to all three stimuli was completely blocked by the JNK inhibitor SP-600125 but not as effectively by other MAP kinase inhibitors. SP-600125 blocked LPS-stimulated IL-6 synthesis dose dependently at both the RNA and protein level. SP-600125 was as effective as the synthetic glucocorticoid dexamethasone at inhibiting IL-6 expression. SP-600125 inhibited IL-6 synthesis when added to cells up to 60 min after LPS stimulation, but its inhibitory effect waned with time. LPS stimulated IL-6 mRNA in both myoblasts and myotubes, but myoblasts showed a proportionally greater LPS-induced increase in IL-6 protein expression compared with myotubes. SP-600125 and the proteasomal inhibitor MG-132 blocked LPS-induced degradation of IkappaB-alpha/epsilon and LPS-stimulated expression of IkappaB-alpha mRNA. Yet, only SP-600125 and not MG-132 blocked LPS-induced IL-6 mRNA expression. This suggests that IL-6 gene expression is a downstream target of JNK in C2C12 myoblasts.     

Differential responses of hematopoietic and non-hematopoietic cells to anti-inflammatory cytokines: IL-4, IL-13 and IL-10.

Recombinant preparations of human anti-inflammatory cytokines: IL-4, IL-13 and IL-10, inhibited LPS-induced synthesis of TNFalpha and IL-6 in the whole human blood tested in vitro. These cytokines also inhibited LPS-induced IL-6 and TNF mRNA accumulation in isolated human blood monocytes/macrophages. On the other hand, similar concentrations of IL-4 and IL-13 (but not IL-10) enhanced synthesis of IL-6 in cultured human umbilical vein endothelial cells (HUVEC). In human hepatoma HepG2 cells IL-4 and IL-13 (but not IL-10) inhibited IL-6-induced synthesis of haptoglobin. These differential responses to the tested anti-inflammatory cytokines were observed at mRNA and protein levels and may reflect cell specificities in signalling pathways and gene expression. When HUVEC and HepG2 cells were cultured together and stimulated with LPS the addition of IL-4 or IL-13 resulted in the reduction of LPS-induced and IL-6-mediated haptoglobin synthesis. Thus in co-culture the inhibitory effects of IL-4 or IL-13 on HepG2 cells prevail over stimulation of IL-6 synthesis in HUVEC.     

Myeloid TAKI [corrected] acts as a negative regulator of the LPS response and mediates resistance to endotoxemia

TGFβ-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is considered a key intermediate in a multitude of innate immune signaling pathways. Yet, the specific role of TAK1 in the myeloid compartment during inflammatory challenges has not been revealed. To address this question, we generated myeloid-specific kinase-dead TAK1 mutant mice. TAK1 deficiency in macrophages results in impaired NF-κB and JNK activation upon stimulation with lipopolysaccharide (LPS). Moreover, TAK1-deficient macrophages and neutrophils show an enhanced inflammatory cytokine profile in response to LPS stimulation. Myeloid-specific TAK1 deficiency in mice leads to increased levels of circulating IL-1β, TNF and reduced IL-10 after LPS challenge and sensitizes them to LPS-induced endotoxemia. These results highlight an antiinflammatory role for myeloid TAK1, which is essential for balanced innate immune responses and host survival during endotoxemia.