Macrophage Proinflammatory Gene Research Articles

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

Biofilm of Klebsiella pneumoniae minimize phagocytosis and cytokine expression by macrophage cell line

Infectious bacteria in biofilm mode are involved in many persistent infections. Owing to its importance in clinical settings, many in vitro and in vivo studies are being conducted to study the structural and functional properties of biofilms, their drug resistant mechanism and the s urvival mechanism of planktonic and biofilm cells. In this regard, there is not sufficient information on the interaction between Klebsiella biofilm and macrophages. In this study, we have attempted to unravel the interaction between Klebsiella biofilm and macrophages in terms of phagocytic response and cytokine expression. In vitro phagocytosis assays were performed for heat inactivated and live biofilms of K. pneumoniae, together with the expression analysis of TLR2, iNOS, inflammatory cytokines such as IL-β1, IFN-γ, IL-6, IL-12, IL-4, TNF-α and anti-inflammatory cytokine, IL-10. A phagocytic rate of an average of 15% was observed against both heat inactivated and live biofilms when LPS + IFN-γ activated macrophages were used. This was significantly higher than non-activated macrophages when tested against heat inactivated and live biofilms (average 8%). Heat-inactivated and live biofilms induced similar phagocytic responses and up-regulation of pro-inflammatory genes in macrophages, indirectly conveying that macrophage responses are to some extent dependent on the biofilm matrix.

BHLHE40 promotes macrophage pro-inflammatory gene expression and functions.

Macrophages are the principal innate immune cells that populate all major organs and provide the first line of cellular defense against infections and/or injuries. The immediate and early-responding macrophages must mount a robust pro-inflammatory response to protect the host by eliminating deleterious agents. The effective pro-inflammatory macrophage response requires the activation of complex transcriptional programs that modulate the dynamic regulation of inflammatory and metabolic gene expression. Therefore, transcription factors that govern pro-inflammatory and metabolic gene expression play an essential role in shaping the macrophage inflammatory response. Herein, we identify the basic helix-loop-helix family member e40 (BHLHE40), as a critical transcription factor that promotes broad pro-inflammatory and glycolytic gene expression by elevating HIF1α levels in macrophages. Our in vivo studies revealed that myeloid-BHLHE40 deficiency significantly attenuates macrophage and neutrophil recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies show that BHLHE40 deficiency broadly curtails inflammatory signaling pathways, hypoxia response, and glycolytic gene expression in macrophages. Utilizing complementary gain- and loss-of-function studies, our analyses uncovered that BHLHE40 promotes LPS-induced HIF1α mRNA and protein expression in macrophages. More importantly, forced overexpression of oxygen stable form of HIF1α completely reversed attenuated pro-inflammatory and glycolytic gene expression in BHLHE40-deficient macrophages. Collectively, these results demonstrate that BHLHE40 promotes macrophage pro-inflammatory gene expression and functions by elevating HIF1α expression in macrophages.

LncRNA DRAIR is downregulated in diabetic monocytes and modulates the inflammatory phenotype via epigenetic mechanisms.

Long noncoding RNAs (lncRNAs) are increasingly implicated in the pathology of diabetic complications. Here, we examined the role of lncRNAs in monocyte dysfunction and inflammation associated with human type 2 diabetes mellitus (T2D). RNA sequencing analysis of CD14+ monocytes from patients with T2D versus healthy controls revealed downregulation of antiinflammatory and antiproliferative genes, along with several lncRNAs, including a potentially novel divergent lncRNA diabetes regulated antiinflammatory RNA (DRAIR) and its nearby gene CPEB2. High glucose and palmitic acid downregulated DRAIR in cultured CD14+ monocytes, whereas antiinflammatory cytokines and monocyte-to-macrophage differentiation upregulated DRAIR via KLF4 transcription factor. DRAIR overexpression increased antiinflammatory and macrophage differentiation genes but inhibited proinflammatory genes. Conversely, DRAIR knockdown attenuated antiinflammatory genes, promoted inflammatory responses, and inhibited phagocytosis. DRAIR regulated target gene expression through interaction with chromatin, as well as inhibition of the repressive epigenetic mark H3K9me2 and its corresponding methyltransferase G9a. Mouse orthologous Drair and Cpeb2 were also downregulated in peritoneal macrophages from T2D db/db mice, and Drair knockdown in nondiabetic mice enhanced proinflammatory genes in macrophages. Thus, DRAIR modulates the inflammatory phenotype of monocytes/macrophages via epigenetic mechanisms, and its downregulation in T2D may promote chronic inflammation. Augmentation of endogenous lncRNAs like DRAIR could serve as novel antiinflammatory therapies for diabetic complications.

Histone deacetylase 2 is essential for LPS‐induced inflammatory responses in macrophages

The role of specific histone deacetylase (HDAC) proteins in regulating the lipopolysaccharide (LPS)‐induced inflammatory response and its underlying mechanisms are unclear. Here, HDAC2, a class I HDAC family protein, is essential for the LPS‐triggered inflammatory response in macrophages. LPS stimulation increases HDAC2 expression in macrophages. Knockdown of HDAC2 decreases the expression of proinflammatory genes, such as IL‐12, TNF‐α and iNOS following stimulation with LPS. The adoptive transfer of HDAC2 knockdown macrophages attenuates the LPS‐triggered innate inflammatory response in vivo, and these mice are less sensitive to endotoxin shock and Escherichia coli‐induced sepsis. Mechanistically, the c‐Jun protein is the main target of HDAC2‐mediated LPS‐induced production of proinflammatory cytokines. Moreover, HDAC2 knockdown increases the expression of c‐Jun, which directly binds the promoters of proinflammatory genes and forms nuclear receptor corepressor complexes to inhibit the transcription of proinflammatory genes in macrophages. These effects are rescued by c‐Jun expression. According to the chromatin immunoprecipitation analysis, HDAC2 also selectively suppresses c‐Jun expression by directly binding to its promoter and modifying histone acetylation after LPS stimulation. Our findings define a new function and mechanism of the HDAC2/c‐Jun signaling network that regulates the LPS‐induced immune response in macrophages.

Effects of Three Thiazolidinediones on Metabolic Regulation and Cold-Induced Thermogenesis.

Insulin resistance is closely associated with metabolic diseases such as type 2 diabetes, dyslipidemia, hypertension and atherosclerosis. Thiazolidinediones (TZDs) have been developed to ameliorate insulin resistance by activation of peroxisome proliferator-activated receptor (PPAR) γ. Although TZDs are synthetic ligands for PPARγ, metabolic outcomes of each TZD are different. Moreover, there are lack of head-to-head comparative studies among TZDs in the aspect of metabolic outcomes. In this study, we analyzed the effects of three TZDs, including lobeglitazone (Lobe), rosiglitazone (Rosi), and pioglitazone (Pio) on metabolic and thermogenic regulation. In adipocytes, Lobe more potently stimulated adipogenesis and insulin-dependent glucose uptake than Rosi and Pio. In the presence of pro-inflammatory stimuli, Lobe efficiently suppressed expressions of pro-inflammatory genes in macrophages and adipocytes. In obese and diabetic db/db mice, Lobe effectively promoted insulin-stimulated glucose uptake and suppressed pro-inflammatory responses in epididymal white adipose tissue (EAT), leading to improve glucose intolerance. Compared to other two TZDs, Lobe enhanced beige adipocyte formation and thermogenic gene expression in inguinal white adipose tissue (IAT) of lean mice, which would be attributable to cold-induced thermogenesis. Collectively, these comparison data suggest that Lobe could relieve insulin resistance and enhance thermogenesis at low-concentration conditions where Rosi and Pio are less effective.

HADC5 deacetylates MKL1 to dampen TNF-α induced pro-inflammatory gene transcription in macrophages.

Macrophage-dependent inflammatory response on the one hand functions as a key line of defense in host immunity but on the other hand underlies the pathogenesis of a host of human pathologies when aberrantly activated. Our previous investigations have led to the identification of megakaryocytic leukemia 1 (MKL1) as a key co-factor of NF-κB/p65 participating in TNF-α induced pro-inflammatory transcription in macrophages. How post-translational modifications contribute to the modulation of MKL1 activity remains an underexplored subject matter. Here we report that the lysine deacetylase HDAC5 interacts with and deacetylates MKL1 in cells. TNF-α treatment down-regulates HDAC5 expression and expels HDAC5 from the promoters of pro-inflammatory genes in macrophages. In contrast, over-expression of HDAC5 attenuates TNF-α induced pro-inflammatory transcription. Mechanistically, HDAC5-mediated MKL1 deacetylation disrupts the interaction between MKL1 and p65. In addition, deacetylation of MKL1 by HDAC5 blocks its nuclear translocation in response to TNF-α treatment. In conclusion, our work has identified an important pathway that contributes to the regulation of pro-inflammatory response in macrophages.

MKL1 mediates TNF-α induced pro-inflammatory transcription by bridging the crosstalk between BRG1 and WDR5.

Tumor necrosis factor alpha (TNF-α) is a cytokine that can potently stimulate the synthesis of a range of pro-inflammatory mediators in macrophages. The underlying epigenetic mechanism, however, is underexplored. Here we report that the transcriptional modulator megakaryocytic leukemia 1 (MKL1) is associated with a histone H3K4 methyltransferase activity. Re-ChIP assay suggests that MKL1 interacts with and recruits WDR5, a component of the COMPASS complex responsible for H3K4 methylation, to the promoter regions of pro-inflammatory genes in macrophages treated with TNF-α. WDR5 enhances the ability of MKL1 to stimulate the promoter activities of pro-inflammatory genes. In contrast, silencing of WDR5 attenuates TNF-α induced production of pro-inflammatory mediators and erases the H3K4 methylation from the gene promoters. Of interest, the chromatin remodeling protein BRG1 also plays an essential role in maintaining H3K4 methylation on MKL1 target promoters by interacting with WDR5. MKL1 knockdown disrupts the interaction between BRG1 and WDR5. Together, our data illustrate a role for MKL1 in moderating the crosstalk between BRG1 and WDR5 to activate TNF-α induced pro-inflammatory transcription in macrophages.

Titanium dioxide nanoparticles prime a specific activation state of macrophages

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO2 NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO2 NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO2 NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO2 NPs (10 and 50 nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO2 NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO2 NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO2 NPs. These results demonstrate that TiO2 NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.

The role of transforming growth factor (TGF)-β in the infarcted myocardium.

The adult mammalian heart has negligible regenerative capacity. Following myocardial infarction, sudden necrosis of cardiomyocytes triggers an intense inflammatory reaction that clears the wound from dead cells and matrix debris, while activating a reparative program. A growing body of evidence suggests that members of the transforming growth factor (TGF)-β family critically regulate the inflammatory and reparative response following infarction. Although all three TGF-β isoforms (TGF-β1, -β2 and -β3) are markedly upregulated in the infarcted myocardium, information on isoform-specific actions is limited. Experimental studies have suggested that TGF-β exerts a wide range of actions on cardiomyocytes, fibroblasts, immune cells, and vascular cells. The findings are often conflicting, reflecting the context-dependence of TGF-β-mediated effects; conclusions are often based exclusively on in vitro studies and on associative evidence. TGF-β has been reported to modulate cardiomyocyte survival responses, promote monocyte recruitment, inhibit macrophage pro-inflammatory gene expression, suppress adhesion molecule synthesis by endothelial cells, promote myofibroblast conversion and extracellular matrix synthesis, and mediate both angiogenic and angiostatic effects. This review manuscript discusses our understanding of the cell biological effects of TGF-β in myocardial infarction. We discuss the relative significance of downstream TGF-β-mediated Smad-dependent and -independent pathways, and the risks and challenges of therapeutic TGF-β targeting. Considering the high significance of TGF-β-mediated actions in vivo, study of cell-specific effects and dissection of downstream signaling pathways are needed in order to design safe and effective therapeutic approaches.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells: A HOMEOSTATIC RESPONSE?

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.

Kruppel-like Factor 6 Promotes Macrophage-mediated Inflammation by Suppressing B Cell Leukemia/Lymphoma 6 Expression

Macrophages are the predominant innate immune cells recruited to tissues following injury or infection. These early-responding, pro-inflammatory macrophages play an essential role in the amplification of inflammation. However, macrophage pro-inflammatory gene expression should be tightly regulated to avert host tissue damage. In this study, we identify the Kruppel-like transcription factor 6 (KLF6)-B cell leukemia/lymphoma 6 (BCL6) signaling axis as a novel regulator of macrophage inflammatory gene expression and function. Utilizing complementary gain- and loss-of-function studies, we observed that KLF6 is essential for macrophage motility under ex vivo and in vivo conditions. Concordant with these observations, myeloid-specific deficiency of KLF6 significantly attenuates macrophage pro-inflammatory gene expression, recruitment, and progression of inflammation. At the molecular level, KLF6 suppresses BCL6 mRNA and protein expression by elevating PR domain-containing 1 with ZNF domain (PRDM1) levels in macrophages. Interestingly, pharmacological or genetic inhibition of BCL6 in KLF6-deficient macrophages completely abrogated the attenuation of pro-inflammatory cytokine/chemokine expression and cellular motility. Collectively, our observations reveal that KLF6 repress BCL6 to enhance macrophage inflammatory gene expression and function.

Abstract IA10: Regulating transcription elongation at stimulus responsive genes

Abstract The majority of mammalian protein-coding genes display transcription in both sense and anti-sense directions. At such bidirectional promoters, Pol II has been shown to initiate transcription and undergo promoter-proximal pausing near both the sense-strand mRNA transcription start site (TSS) and from within the upstream region in the anti-sense direction. This enigmatic, dual promoter structure raises fundamental questions about the specificity, purpose and regulation of upstream anti-sense transcription. Interestingly, transcription in the anti-sense direction is generally non-processive and creates short, unstable non-coding RNAs (ncRNAs). Intriguingly, many distal enhancer regions display bidirectional transcription and generate short unstable ncRNAs, raising parallels between enhancers and anti-sense promoters near protein-coding genes. Importantly, enhancers and upstream anti-sense promoters show exquisite cell type specificity in their location and activity, suggesting key roles in establishing cell state and behavior. Further, perturbation of either transcription or chromatin structure at such non-coding loci has been implicated in the development of cancer and disease, with a profound impact on maintenance of cell state and responsiveness to stimuli. These recent findings raise a number of intriguing questions about the communication between promoters and non-coding loci. Our work has recently defined a mechanism through which upstream anti-sense transcription impacts stimulus-responsive gene expression. Specifically, we found that the distance between sense and anti-sense TSSs at bidirectional promoters strongly influences activation of pro-inflammatory genes in macrophages. We discovered that anti-sense TSSs organize chromatin structure, creating a region of open chromatin between bidirectional TSSs that is optimal for recruitment of the transcription machinery and signal-dependent transcription factors like NF-kB. Further, we discovered that key inflammatory genes possess enhancer-like histone modifications around the anti-sense TSS, and that this characteristic selectively marks genes that are highly activated upon immune stimulation. Citation Format: Karen Adelman, Benjamin S. Scruggs, Telmo Henriques, Adam Burkholder, David C. Fargo. Regulating transcription elongation at stimulus responsive genes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA10.

Chemical epigenetics to assess the role of HDAC1–3 inhibition in macrophage pro-inflammatory gene expression

Azobenzene ortho-aminoanilides inhibit HDACs 1–3 and possess anti-inflammatory properties in murine macrophages.

Axonally derived matrilin-2 induces proinflammatory responses that exacerbate autoimmune neuroinflammation.

In patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE), inflammatory axonal injury is a major determinant of disability; however, the drivers of this injury are incompletely understood. Here, we used the EAE model and determined that the extracellular matrix protein matrilin-2 (MATN2) is an endogenous neuronal molecule that is regulated in association with inflammatory axonal injury. Compared with WT mice, mice harboring a deletion of Matn2 exhibited reduced disease severity and axon damage following induction of EAE. Evaluation of neuron-macrophage cocultures revealed that exogenous MATN2 specifically signals through TLR4 and directly induces expression of proinflammatory genes in macrophages, promoting axonal damage. Moreover, the MATN2-induced proinflammatory response was attenuated greatly in macrophages from Myd88 KO mice. Examination of brain sections from patients with MS revealed that MATN2 is expressed in lesions but not in normal-appearing white matter. Together, our results indicate that MATN2 is a deleterious endogenous neuroaxonal injury response signal that activates innate immune cells and could contribute to early axonal damage in CNS inflammatory diseases like MS.

Pathogenic Role of Basic Calcium Phosphate Crystals in Destructive Arthropathies

Backgroundbasic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals.Methodology/ Principal Findingssynovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages.Conclusions/ Significanceintra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

Reader's block

Protein factors can regulate gene expression by binding to specifically modified DNA-associated proteins. Small molecules that selectively interfere with such interaction may be of therapeutic value. See Article p.1067 & Letter p.1119 Small molecules that perturb chromatin proteins are an emerging focus of current biomedical research. Two groups reporting in this issue have targeted bromodomain-containing BET proteins that bind acetylated lysine residues during gene activation, arriving at cell-permeable small molecule compounds with similar structures based on fused triazole-diazepine rings. James Bradner and colleagues report the development of a compound named JQ1. The BET protein BRD4, with two bromodomains, is implicated in human squamous cell carcinoma. JQ1 inhibits the growth of BRD4-dependent tumours in mouse models. Alexander Tarakhovsky and colleagues' inhibitor, I-BET, is shown to interfere with the binding of certain BET family members to acetylated histones. It inhibits activation of pro-inflammatory genes in macrophages and has immunomodulatory activity in a mouse model of inflammatory disease.

Matricellular Protein CCN1 Activates a Proinflammatory Genetic Program in Murine Macrophages

CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NF-kappaB activity and enhancing the apoptotic activity of Fas ligand and TRAIL. In this paper, we show that CCN1 supports the adhesion of macrophages through integrin alpha(M)beta(2) and syndecan-4, activates NFkappaB-mediated transcription, and induces a proinflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-12b), chemokines (MIP-1alpha; MCP-3; growth-related oncogenes 1 and 2; and inflammatory protein 10), and regulators of oxidative stress and complement (inducible NO synthase and C3) and downregulation of specific receptors (TLR4 and IL-10Rbeta) and anti-inflammatory factors (TGF-beta1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NF-kappaB by CCN1, leading to the synthesis of cytokines including TNF-alpha and inflammatory protein 10; and a delayed response resulting from CCN1-induced TNF-alpha, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1beta and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates proinflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation.

Gambogic acid covalently modifies IκB kinase-β subunit to mediate suppression of lipopolysaccharide-induced activation of NF-κB in macrophages

GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.

The human myeloperoxidase gene is regulated by LXR and PPARα ligands

Myeloperoxidase (MPO) is an oxidant-generating enzyme expressed in macrophages and implicated in atherosclerosis and cholesterol homeostasis. LXRα and PPARα regulate genes involved in cholesterol metabolism and the inflammatory response in macrophages. Here, we examine the effect of LXR and PPARα ligands on MPO expression. LXR and PPARα, as heterodimers with RXR, are shown to bind overlapping sites in an Alu receptor response element (AluRRE) in the MPO promoter. The LXR ligand T0901317 suppresses MPO mRNA expression in primary human macrophages, and in bone marrow cells and macrophages from huMPO transgenic mice. The PPARα ligand GW9578 downregulates MPO expression in GMCSF-macrophages, while upregulating in MCSF-macrophages. In contrast, the mouse MPO gene, which lacks the primate-specific AluRRE, is not regulated by LXR or PPARα ligands. These findings identify human MPO as a novel LXR and PPARα target gene, consistent with the role of these receptors in regulation of proinflammatory genes in macrophages.