Antonio Castrillo

Reciprocal regulation of inflammation and lipid metabolism by liver X receptors

Macrophages have important roles in both lipid metabolism and inflammation and are central to the pathogenesis of atherosclerosis. The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression, but their role in inflammation and immunity is unknown. We demonstrate here that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Transcriptional profiling of lipopolysaccharide (LPS)-induced macrophages reveals reciprocal LXR-dependent regulation of genes involved in lipid metabolism and the innate immune response. In vitro, LXR ligands inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2 and interleukin-6 (IL-6) in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune functions in macrophages.

Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue

The control of lipid and glucose metabolism is closely linked. The nuclear receptors liver X receptor (LXR)α and LXRβ have been implicated in gene expression linked to lipid homeostasis; however, their role in glucose metabolism is not clear. We demonstrate here that the synthetic LXR agonist GW3965 improves glucose tolerance in a murine model of diet-induced obesity and insulin resistance. Analysis of gene expression in LXR agonist-treated mice reveals coordinate regulation of genes involved in glucose metabolism in liver and adipose tissue. In the liver, activation of LXR led to the suppression of the gluconeogenic program including down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase expression. Inhibition of gluconeogenic genes was accompanied by an induction in expression of glucokinase, which promotes hepatic glucose utilization. In adipose tissue, activation of LXR led to the transcriptional induction of the insulin-sensitive glucose transporter, GLUT4. We show that the GLUT4 promoter is a direct transcriptional target for the LXR/retinoid X receptor heterodimer and that the ability of LXR ligands to induce GLUT4 expression is abolished in LXR null cells and animals. Consistent with their effects on GLUT4 expression, LXR agonists promote glucose uptake in 3T3-L1 adipocytes in vitro. Thus, activation of LXR alters the expression of genes in liver and adipose tissue that collectively would be expected to limit hepatic glucose output and improve peripheral glucose uptake. These results outline a role for LXRs in the coordination of lipid and glucose metabolism.

LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

Crosstalk between LXR and Toll-like Receptor Signaling Mediates Bacterial and Viral Antagonism of Cholesterol Metabolism

The liver X receptors (LXR) alpha and beta are regulators of cholesterol metabolism and determinants of atherosclerosis susceptibility. Viral and bacterial pathogens have long been suspected to be modulators of atherogenesis; however, mechanisms linking innate immunity to cholesterol metabolism are poorly defined. We demonstrate here that pathogens interfere with macrophage cholesterol metabolism through inhibition of the LXR signaling pathway. Activation of Toll-like receptors (TLR) 3 and 4 by microbial ligands blocks the induction of LXR target genes including ABCA1 in cultured macrophages as well as in aortic tissue in vivo. As a consequence of these transcriptional effects, TLR3/4 ligands strongly inhibit cholesterol efflux from macrophages. Crosstalk between LXR and TLR signaling is mediated by IRF3, a specific effector of TLR3/4 that inhibits the transcriptional activity of LXR on its target promoters. These findings highlight a common mechanism whereby bacterial and viral pathogens may modulate macrophage cholesterol metabolism and cardiovascular disease.

Apoptotic Cells Promote Their Own Clearance and Immune Tolerance through Activation of the Nuclear Receptor LXR

Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.

Inhibition of IκB Kinase and IκB Phosphorylation by 15-Deoxy-Δ12,14-Prostaglandin J2 in Activated Murine Macrophages

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-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 IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Induction of NR4A Orphan Nuclear Receptor Expression in Macrophages in Response to Inflammatory Stimuli

Oxidized lipids and inflammatory cytokines are believed to play a causal role in atherosclerosis through the regulation of gene expression in macrophages and other cells. Previous work has implicated the nuclear receptors peroxisome proliferator-activated receptor and liver X receptor in the control of lipid-dependent gene expression and inflammation. Here we demonstrate that expression of a third group of nuclear receptors, the NR4A ligand-independent orphan receptors, is highly inducible in macrophages by diverse inflammatory stimuli. Treatment of macrophages with lipopolysaccharide (LPS), cytokines, or oxidized lipids triggers the transcriptional induction of Nur77 (NR4A1), Nurr1 (NR4A2), and NOR1 (NR4A3) expression. Several lines of evidence point to the NF-κB signaling pathway as a principal mediator of inducible NR4A expression in macrophages. Analysis of the murine and human Nur77 promoters revealed two highly conserved NF-κB response elements. Mutation of these elements inhibited LPS-dependent expression of the Nur77 promoter in transient transfection assays. Furthermore, induction of Nur77 expression by LPS was severely compromised in fibroblasts lacking the three NF-κB subunits, Nfkb1, c-Rel, and RelA. Consistent with its ability to be induced by oxidized lipids, Nur77 was expressed in macrophages within human atherosclerotic lesions. These results identified NR4A nuclear receptors as potential transcriptional mediators of inflammatory signals in activated macrophages.

The Increase in TNF-α Levels Is Implicated in NF-κB Activation and Inducible Nitric Oxide Synthase Expression in Brain Cortex after Immobilization Stress ☆

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex after three weeks of repeated stress and that its overexpression accounts for the neurodegenerative changes found in this situation. Now we have found that a short duration of stress (immobilization for 6 h) also induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in iNOS expression, we have studied the role of the cytokine tumor necrosis factor-α (TNF-α) released in brain during stress. We have shown that there is an increase in soluble TNF-α levels after 1 h of stress in cortex and that this is preceded by an increase in TNF-α-convertase (TACE) activity in brain cortex as soon as 30 min after immobilization. Stress-induced increase in both TACE activity and TNF-α levels seems to be mediated by excitatory amino acids since they can be blocked by MK-801 (dizocilpine) (0.2 mg/kg i.p.), an antagonist of the N-methyl-D-aspartate subtype of glutamate receptor. In order to study the role of TACE and TNF-α in iNOS induction, a group of animals were i.p. injected with the preferred TACE inhibitor BB1101 (2 and 10 mg/kg). Indeed, BB1101 inhibited iNOS expression induced by six hours of stress. In addition, we studied the role of the transcription factor nuclear factor κB (NF-κB), which is required for iNOS expression. We have found that the administration of the TACE inhibitor BB1101 inhibited the stress-stimulated translocation of NF-κB to the nucleus. Taken together, these findings indicate that glutamate receptor activation induces TACE up-regulation and subsequent increase in TNF-α levels, and this account for stress-induced iNOS expression via NF-κB activation, supporting a possible neuroprotective role for specific TACE inhibitors in this situation.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-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 IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Inducible nitric oxide synthase expression in brain cortex after acute restraint stress is regulated by nuclear factor κB-mediated mechanisms

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high‐output and long‐lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenerative changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium‐independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor κB (NF‐κB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF‐κB to the nucleus after 4 h and that the administration of the NF‐κB inhibitor pyrrolidine dithiocarbamate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress‐induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N‐methyl‐d‐aspartate) receptor activation has been recognized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF‐κB‐dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK‐801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress‐induced NF‐κB activation as well as the stress‐induced increase in iNOS expression. Taken together, these findings indicate that excitatory amino acids and subsequent activation of NF‐κB account for stress‐induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for specific inhibitors in this situation.