Proinflammatory Gene Expression In Macrophages Research Articles

Abstract 190: Epigenetic Modifications of Pro-inflammatory Gene Expression in Macrophages by a Demethylase Enzyme, JMJD3, May Promote Chronic Inflammation in Type 2 Diabetic (T2D) Wounds

Introduction Type 2 diabetic(T2D) wounds are characterized by chronic inflammation, maintained by an exaggerated M1(pro-inflammatory) macrophage phenotype response. We seek to define a link between epigenetic modifications of bone marrow(BM) cells in T2D and dysregulated macrophages in wounds. We hypothesized that a chromatin modifying demethylase enzyme, JMJD3, is responsible for the decrease in H3K27me3 repressive methylation at the IL-12 gene promoter and thus drives an M1 macrophage phenotype in T2D wounds. Methods BM/adipose tissue(AT)/wounds were harvested from 30 diet-induced obese mice(DIO)(MG= 350g/DL) and 30 matched(WT) controls. For chromatin immunoprecipitation(ChIP) analysis, cells were isolated via ferromagnetic columns(CD34+,CD11b+). ChIP to detect histone methylation at the promoter regions of JMJD3 and IL-12(key M1 macrophage gene) was performed and RNA analysis was done with standard primers. Results JMJD3 mRNA in the BM is significantly increased in the DIO versus WT. ChIP showed increased H3K4me3(gene expression mark) in CD34+ progenitor cells and a corresponding decrease in H3K27me3(repressive mark) in monocytes at the promoter region of JMJD3. These changes correspond with the decrease in H3K27me3 seen at the IL-12 promoter in macrophages(CD11b+) from AT/T2D wounds. Conclusions Epigenetic changes initiated by JMJD3 in BM progenitor cells result in changes in histone methylation at the IL-12 promoter favoring an M1 phenotype in macrophages and thus contributes to the chronic inflammation seen in T2D wounds and AT. Whether manipulation of epigenetic enzymes could reduce chronic inflammation in T2D wounds requires further work.

Anti-inflammatory properties of histone deacetylase inhibitors

We have demonstrated that postshock administration of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, can significantly improve early survival in a highly lethal model of hemorrhagic shock. As the primary insult in hemorrhagic shock is cellular hypoxia, and transcription factor hypoxia-inducible factor-1α (HIF-1α) controls proinflammatory gene expression in macrophages, we hypothesized that SAHA would attenuate the HIF-1α associated proinflammatory pathway in a hypoxic macrophage model. Mouse macrophages were exposed to hypoxic conditions (0.5% O2, 10% CO2, and 89.5% N2) at 37°C in the presence or absence of SAHA (10 μmol/L). The cells and culture medium were harvested at 1 hour, 4 hours, and 8 hours. Sham (no hypoxia, no SAHA) served as a control. Western blots were performed to assess protein levels of prolyl hydroxylase 2 (PHD2), HIF-1α, and inducible nitric oxide synthase (iNOS) in the cells. Colorimetric biochemical assay and enzyme-linked immunosorbent assay were used to analyze the release of nitric oxide (NO) and secretion of tumor necrosis factor α (TNF-α), respectively, in the cell culture medium. Hypoxia significantly increased cellular level of HIF-1α (1 hour and 4 hours), gene transcription of iNOS (4 hours and 8 hours), iNOS protein (8 hours), NO production (8 hours), and TNF-α secretion (4 hours and 8 hours). SAHA treatment attenuated all of the above hypoxia-induced alterations in the macrophages. In addition, SAHA treatment significantly increased cellular level of PHD2, one of the upstream negative regulators of HIF-1α, at 1 hour. Treatment with SAHA attenuates hypoxia-HIF-1α-inflammatory pathway in macrophages and suppresses hypoxia-induced release of proinflammatory NO and TNF-α. SAHA also causes an early increase in cellular PHD2, which provides, at least in part, a new explanation for the decrease in the HIF-1α protein levels.

A Myeloid Hypoxia-inducible Factor 1α-Krüppel-like Factor 2 Pathway Regulates Gram-positive Endotoxin-mediated Sepsis

Although gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of gram-positive endotoxin-mediated sepsis.

Losartan inhibits LPS-induced inflammatory signaling through a PPARγ-dependent mechanism in human THP-1 macrophages

Macrophages have critical roles in the pathogenesis of atherosclerosis by activating the innate immune system and producing inflammatory cytokines. Accumulating evidence indicates that angiotensin type 1 receptor (AT1R) blockers exert anti-inflammatory effects in inflammatory diseases including atherosclerosis. In this study, we investigated the effect of losartan, an AT1R blocker, on the proinflammatory gene expression induced by bacterial lipopolysaccharide (LPS) in a well-defined in vitro human THP-1 macrophage system. We found that losartan significantly attenuated the LPS-induced expression of proinflammatory genes TNF-alpha, IL-8 and COX-2. However, exogenous angiotensin II (AngII) had no effect on LPS-induced inflammatory signaling despite the expression of AT1R. In addition, losartan did not block LPS-induced IkappaB phosphorylation, which is downstream of Toll-like receptor activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) antagonists, GW9662 and T0070907, reversed the inhibitory effects of losartan on LPS-induced TNF-alpha and IL-8 expression in THP-1 macrophages. These observations suggest that losartan inhibits LPS-induced proinflammatory gene expression in macrophages by activating the PPARgamma pathway rather than by the competitive inhibition of AT1R binding to AngII.

Adiponectin Inhibits Pro-inflammatory Signaling in Human Macrophages Independent of Interleukin-10

Macrophages participate pivotally in the pathogenesis of many chronic inflammatory diseases including atherosclerosis. Adiponectin, a vasculoprotective molecule with insulin-sensitizing and anti-atherogenic properties, suppresses pro-inflammatory gene expression in macrophages by mechanisms that remain incompletely understood. This study investigated the effects of adiponectin on major pro-inflammatory signaling pathways in human macrophages. We demonstrate that pretreatment of these cells with adiponectin inhibits phosphorylation of nuclear factor kappaB inhibitor (IkappaB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), induced by either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) alpha, as well as STAT3 phosphorylation induced by interleukin-6 (IL6). Antagonism of IL10 by either neutralizing antibodies or siRNA-mediated silencing did not abrogate the anti-inflammatory actions of adiponectin, indicating that the ability of adiponectin to render human macrophages tolerant to various pro-inflammatory stimuli does not require this cytokine. A systematic search for adiponectin-inducible genes with established anti-inflammatory properties revealed that adiponectin augmented the expression of A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, TNF receptor-associated factor (TRAF) 1, and TNFAIP3-interacting protein (TNIP) 3. These results suggest that adiponectin triggers a multifaceted response in human macrophages by inducing the expression of various anti-inflammatory proteins that act at different levels in concert to suppress macrophage activation.

MTOR‐dependent IL‐10 expression inhibits LPS induction of tissue factor and cytokines in macrophages.

Bacterial lipopolysaccharide (LPS) induces monocytes/macrophages to express proinflammatory cytokines and tissue factor (TF), the primary activator of the coagulation cascade. Anti‐inflammatory signaling pathways including the phosphatidylinositol‐3‐kinase (PI3K)‐Akt pathway inhibit proinflammatory gene expression in macrophages. We determined whether the mammalian target of rapamycin (mTOR) and induction of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) inhibited LPS‐induced proinflammatory and TF gene expression in peritoneal macrophages (PMs) in a PI3K‐Akt‐dependent manner. We used wild type (WT) peritoneal macrophages (PMs), and PMs from PTENflox/flox/LysMCre mice (PTEN−/− PMs), which have increased Akt activity. LPS induction of IL‐10 mRNA and protein was increased in PTEN−/− PMs compared to WT PMs. Pharmacologic inhibition of mTOR with rapamycin inhibited LPS induction of IL‐10 mRNA and protein, and enhanced the expression of TF and the proinflammatory cytokines interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNFα). Neutralization of endogenous IL‐10 also enhanced LPS induction of TNFα, IL‐6 and TF expression in WT and PTEN−/− PMs. The results indicate that mTOR‐dependent IL‐10 expression leads to inhibition of LPS induction of TF and the proinflammatory cytokines IL‐6 and TNFα in WT macrophages

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

Gliadin Stimulation of Murine Macrophage Inflammatory Gene Expression and Intestinal Permeability Are MyD88-Dependent: Role of the Innate Immune Response in Celiac Disease

Recent studies have demonstrated the importance of TLR signaling in intestinal homeostasis. Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. In this study, we sought to test the hypothesis that gliadin initiates this response by stimulating the innate immune response to increase intestinal permeability and by up-regulating macrophage proinflammatory gene expression and cytokine production. To this end, intestinal permeability and the release of zonulin (an endogenous mediator of gut permeability) in vitro, as well as proinflammatory gene expression and cytokine release by primary murine macrophage cultures, were measured. Gliadin and its peptide derivatives, 33-mer and p31-43, were found to be potent inducers of both a zonulin-dependent increase in intestinal permeability and macrophage proinflammatory gene expression and cytokine secretion. Gliadin-induced zonulin release, increased intestinal permeability, and cytokine production were dependent on myeloid differentiation factor 88 (MyD88), a key adapter molecule in the TLR/IL-1R signaling pathways, but were neither TLR2- nor TLR4-dependent. Our data support the following model for the innate immune response to gliadin in the initiation of CD. Gliadin interaction with the intestinal epithelium increases intestinal permeability through the MyD88-dependent release of zonulin that, in turn, enables paracellular translocation of gliadin and its subsequent interaction with macrophages within the intestinal submucosa. There, the interaction of gliadin with macrophages elicits a MyD88-dependent proinflammatory cytokine milieu that facilitates the interaction of T cells with APCs, leading ultimately to the Ag-specific adaptive immune response seen in patients with CD.

The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses.

A20 is a cytoplasmic protein required for the termination of tumor necrosis factor (TNF)-induced signals. We show here that mice doubly deficient in either A20 and TNF or A20 and TNF receptor 1 developed spontaneous inflammation, indicating that A20 is also critical for the regulation of TNF-independent signals in vivo. A20 was required for the termination of Toll-like receptor-induced activity of the transcription factor NF-kappaB and proinflammatory gene expression in macrophages, and this function protected mice from endotoxic shock. A20 accomplished this biochemically by directly removing ubiquitin moieties from the signaling molecule TRAF6. The critical function of this deubiquitinating enzyme in the restriction of TLR signals emphasizes the importance of the regulation of ubiquitin conjugation in innate immune cells.

The role and regulation of COX-2 during viral infection.

Prostaglandins are lipid mediators, generated by cyclooxygenase (COX), that have been shown to participate in the regulation of virus replication and the modulation of inflammatory responses following infection. A number of studies support a role for PGE2 in the modulation of virus replication and virulence in a cell type and virus selective manner. Virus infection also stimulates the expression of a number of proinflammatory gene products, including COX-2, inducible nitric oxide synthase (iNOS) as well as proinflammatory cytokines. This review will focus on the mechanisms by which proinflammatory prostaglandin production regulates virus replication and virulence. In addition, the signaling pathways that are activated during a virus infection, and that regulate proinflammatory gene expression in macrophages will be reviewed. Specific attention will be placed on the ability of virus infection to activate multiple signaling cascades (such as PKR, MAPK, iPLA2, NF-kappaB) and how these pathways are integrated in the regulation of individual target gene expression.