TNF-α Expression In Macrophages Research Articles

γδ T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice.

γδ T cells are resident in AT and increase during diet-induced obesity. Their possible contribution to the inflammatory response that accompanies diet-induced obesity was investigated in mice after a 5 to 10 week milk HFD. The HFD resulted in significant increases in CD44(hi), CD62L(lo), and TNF-α(+) γδ T cells in eAT of WT mice. Mice deficient in all γδ T cells (TCRδ(-/-)) or only Vγ4 and Vγ6 subsets (Vγ4/6(-/-)) were compared with WT mice with regard to proinflammatory cytokine production and macrophage accumulation in eAT. Obesity among these mouse strains did not differ, but obese TCRδ(-/-) and Vγ4/6(-/-) mice had significantly reduced eAT expression of F4/80, a macrophage marker, and inflammatory mediators CCL2 and IL-6 compared with WT mice. Obese TCRδ(-/-) mice had significantly reduced CD11c(+) and TNF-α(+) macrophage accumulation in eAT after 5 and 10 weeks on the HFD, and obese Vγ4/6(-/-) mice had significantly increased CD206(+) macrophages in eAT after 5 weeks on the diet and significantly reduced macrophages after 10 weeks. Obese TCRδ(-/-) mice had significant reductions in systemic insulin resistance and inflammation in liver and skeletal muscle after longer-term HFD feeding (10 and 24 weeks). In vitro studies revealed that isolated γδ T cells directly stimulated RAW264.7 macrophage TNF-α expression but did not stimulate inflammatory mediator expression in 3T3-L1 adipocytes. These findings are consistent with a role for γδ T cells in the proinflammatory response that accompanies diet-induced obesity.

Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells

Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear. RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control. Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals. LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity. Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.

Abstract 1552: Cigarette smoke induces TNF-α secretion from macrophages through a pathway consisting of PPARγ, MUC1, ERK and JNK.

Abstract The glycoprotein Mucin 1 (MUC1 in human and Muc1 in animals) is expressed in the lung and has been shown to be associated with lung carcinogenesis. Recently we have found that cigarette smoke (CS) significantly increased MUC1 expression in mouse lung macrophages and that MUC1 is required for CS-induced tumor necrosis factor-alpha (TNF-α) secretion from macrophages. Because CS potently elicits chronic pulmonary inflammation that is important for the development of lung cancer, we investigated the cell signaling pathways for CS-induced MUC1 expression and MUC1-mediated TNF-α secretion in macrophages using an in vitro model. The results showed that: CS extract (CSE) strongly induced MUC1 expression at both the mRNA and protein levels; Suppression of PPARγ effectively blocked CSE-induced MUC1 expression in THP1 cells, suggesting CSE induces MUC1 expression through PPARγ-mediated transcription; The induction of TNF-α expression was effectively blocked when ERK and JNK were blocked, suggesting these pathways are involved in CSE-induced TNF-α expression in macrophages; Knockdown of MUC1 significantly suppressed CSE-induced ERK and JNK activation; and Suppression of PPARγ that effectively blocked CSE-induced MUC1 expression also inhibited CSE-induced TNF-α secretion from macrophages. Thus, our results suggest a possible involvement of a signaling pathway consisting of PPARγ, MUC1 and ERK/JNK for CSE-induced TNF-α secretion from macrophages that may contribute to inflammation-associated lung cancer development. Note: This work is supported by the NIH/NIEHS grant 1R01ES017328. Citation Format: Xiuling Xu, Mabel T. Padilla, Alexandria Wells, Kwang Chul Kim, yong Lin. Cigarette smoke induces TNF-α secretion from macrophages through a pathway consisting of PPARγ, MUC1, ERK and JNK. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1552. doi:10.1158/1538-7445.AM2013-1552

Silencing TNF-α in macrophages and dendritic cells for arthritis treatment

Objectives: Tumour necrosis factor (TNF)-α secreted by macrophages and dendritic cells (DCs) plays a predominant role in arthritis. Our previous studies suggest that a small peptide, RVG-9R (29-aa peptide derived from the rabies virus glycoprotein, fused to 9R residues), can deliver small interfering RNA (siRNA) to macrophages and DCs. We therefore tested whether knockdown of TNF-α expression in macrophages and DCs by RVG-9R/bound siRNA targeting TNF-α reduces the severity of collagen antibody-induced arthritis (CAIA) in mice.Method: Arthritis was induced in mice by injecting a combination of antibodies to collagen followed by lipopolysaccharide (LPS) treatment. Mice were also injected with TNF-α siRNA complexed with RVG-9R peptide or an irrelevant peptide RVMAT-9R on days 1, 3, 5, and 7. As a positive control, dexamethasone was injected intravenously. Paw thickness was measured every 2 days and the mice were killed on day 10 for testing synovial TNF-α levels and histological analysis of joints.Results: In control mice, arthritis developed on day 4 and reached its peak between day 7 and day 9. Treatment with siTNF-α bound to RVG-9R, but not to RVMAT-9R, resulted in reducing paw thickness scores to the same level as dexamethasone treatment, associated with reduced TNF-α level in synovial fluid. Histological analysis of joints in the control RVMAT-9R/TNF-α siRNA-treated mice showed marked pannus formation and destruction of cartilage and subchondrial bone, as well as severe infiltration of inflammatory cells into the synovium. By contrast, the joint pathology was markedly reduced in RVG-9R/TNF-α siRNA-treated mice resembling the dexamethasone-treated mice.Conclusions: Suppression of TNF-α expression in macrophages and DCs by RVG-9R-mediated siRNA delivery could potentially be a clinically viable strategy for treatment of arthritis.

Abstract 455: Decreased Experimental Hypertension and Inflammation in the Absence of Prolylendopeptidase Activity

Objective: Previously, we showed that inhibition of prolyloligopeptidase eliminated the differences in macrophage TNFα expression and BP response in mice lacking N-domain activity of ACE. Thus, functional studies of BP and inflammatory response in a model lacking prolylendopeptidase activity are performed. Results: After a 14 day high-dose AngII infusion (980 ng/kg-min), POP-KO(n=12) BP was 130±3 mmHg and 147±4 mmHg for POP-WT(n=12). After 14 days of low-dose AngII infusion(490 ng/kg-min), POP-KO(n=8) BP was 126±4 mmHg and 139±3 mmHg for POP-WT(n=8). To investigate differences in the inflammatory response, we examined peritoneal macrophage cytokine expression in response to AngII. The percentage of F4/80+/TNFαhigh cells vs. total F4/80+ was 17%±4.7% for POP-KO (n=8) and 49%±2.8% for POP-WT (n=8)[p<0.005]. The percentage of F4/80+/IL-6high cells vs. total F4/80+ was 15%±3.2% for POP-KO(n=8) and 39%±4.8% for POP-WT(n=8)[p<0.01]. To investigate whether there were baseline differences, TNFalpha secretion was measured in POP-KO(8%) vs. POP-WT(16%) without AngII stimulation. Baseline bone marrow analysis by flow shows 48% CD11b+Ly6ghi cells in POP-KO (n=6) vs. 39% in POP-WT (n=4). Moreover, F4/80hiLy6cintermediate cells were 1.19% in the POP-KO (n=6) vs. 1.75% in POP-WT(n=4). Conclusion: Inflammation is an emerging player in hypertension and prolylendopeptidase inhibition may play a role in reducing that inflammation. In the POP-KO mouse model, baseline differences in quantity and quality of inflammatory cells may contribute to the phenotypic differences found in vivo .

Inhibitory effect of paeoniflorin on the inflammatory vicious cycle between adipocytes and macrophages

Obesity is associated with a state of chronic, low-grade inflammation. It is considered that the paracrine loop involving free fatty acid (FFA) and tumor necrosis factor (TNF)α between adipocytes and macrophages establishes an inflammatory vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Paeoniflorin (PF), one of the major components of Paeony root, has been shown to have anti-inflammatory effects in vivo. We investigated the effect of PF on the production of FFA and TNFα in the interaction between adipocytes and macrophages. Coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages markedly enhanced the production of TNFα and FFA compared with the control cultures, however, treatment with PF dose-dependently inhibited the production. We further examined the effects of PF on TNFα-stimulated adipocyte lipolysis and on FFA-induced macrophage TNFα expression. PF inhibited TNFα-stimulated adipocyte lipolysis in a dose-dependent manner, which was compatible with suppressed phosphorylation of TNFα-activated ERK1/2 and preserved downregulation of perilipin. Palmitate, one of the most important saturated FFAs, induced macrophage TNFα upexpression, but PF partially attenuated the effect. These results indicate that PF exhibits anti-inflammatory properties by inhibiting the vicious cycle between adipocytes and macrophages. PF may be useful for ameliorating the inflammatory changes in obese adipose tissue.

Impact of Altered Methylation in Cytokine Signaling and Proteasome Function in Alcohol and Viral‐Mediated Diseases

Data from several laboratories have shown that ethanol (EtOH) feeding impairs many essential methylation reactions that contribute to alcoholic liver disease (ALD). EtOH is also a comorbid factor in the severity of hepatitis C virus-induced liver injury. The presence of viral proteins further exacerbates the methylation defects to disrupt multiple pathways that promote the pathogenesis of liver disease. This review is a compilation of presentations that linked the methylation reaction defects with proteasome inhibition, decreased antigen presentation, and impaired interferon (IFN) signaling in the hepatocytes and dysregulated TNFα expression in macrophages. Two therapeutic modalities, betaine and S-adenosylmethionine, can correct methylation defects to attenuate many EtOH-induced liver changes, as well as improve IFN signaling pathways, thereby overcoming viral treatment resistance.

PROLYL‐OLIGOPEPTIDASE INHIBITION AMELIORATES EXPERIMENTAL HYPERTENSION AND DECREASES INFLAMMATORY CYTOKINES IN MICE LACKING ANGIOTENSIN CONVERTING ENZYME N DOMAIN ACTIVITY

Using gene targeting, ACE domain‐specific knockout mouse lines N‐KO & C‐KO were created. After 14 day Ang II infusion, BP was 27 mm Hg (p<0.0001) higher in N‐KO vs. WT/C‐KO. N‐KO kidney Ang II levels were 1.5‐fold those of WT/C‐KO (p<0.015). Peritoneal macrophage cytokine expression in response to Ang II, measured as percentage of F4/80+/TNFαhigh cells vs total F4/80+, was 40.3% ± 4.7% for N‐KO and 19.8% ± 2.8% for WT (p<0.005). In response to LPS, N‐KO macrophages made 4‐fold the TNFα as C‐KO/WT cells (p<0.001). AcSDKP, a substrate specifically cleaved by the N‐domain, is made by the enzyme prolyl‐oligopeptidase (POP). Daily injections of the POP inhibitor S‐17092 were given to N‐KO and WT mice. This reduced the LPS‐mediated N‐KO macrophage production of TNFα to that of cells from WT. The hypertensive response to Ang II was also tested in N‐KO and WT mice treated with daily S‐17092. POP inhibition had no effect on the BP of WT, but reduced the BP of N‐KO to that of WT (p<0.01). Furthermore, FACS analysis of peritoneal macrophages showed a decreased percentage of F4/80+/TNFαhigh cells in N‐KO mice treated with daily S‐17092. The lack of N domain activity in ACE exhibited a pro‐inflammatory role in experimental hypertension via cytokine regulation, accumulation of renal Ang II, and increased BP response. Inhibition of POP eliminated the differences in macrophage TNFα expression and blood pressure response between N‐KO and WT mice.

Th2-inducing cytokines IL-4 and IL-33 synergistically elicit the expression of transmembrane TNF-α on macrophages through the autocrine action of IL-6

Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine produced predominantly by activated macrophages, and plays a central role in the protective immunity against intracellular pathogens and the pathogenesis of autoimmune and inflammatory diseases. While both the soluble and transmembrane forms of TNF-α (sTNF-α and tmTNF-α) are biologically functional, the latter but not the former acts as a receptor besides as a ligand, and transmit a retrograde signal in a cell-to-cell contact manner. The production of TNF-α by macrophages under Th2-type (allergic) inflammatory conditions has been ill defined, compared to that under Th1-type inflammatory conditions. Here we examined the effect of representative Th2-inducing cytokines IL-4 and IL-33 on the TNF-α expression in macrophages. IL-4 induced the production of neither sTNF-α nor tmTNF-α while IL-33 promoted the production of sTNF-α with no detectable tmTNF-α. Notably, the combination of IL-4 and IL-33 elicited the tmTNF-α expression on macrophages, in addition to the enhanced production of sTNF-α and IL-6. The IL-4/IL-33-elicited tmTNF-α expression was not observed in IL-6-deficient macrophages, suggesting the involvement of macrophage-derived IL-6 in the tmTNF-α expression. Indeed, the stimulation of macrophages with the combination of IL-4 and IL-6 induced the tmTNF-α expression with no detectable production of sTNF-α. Thus, IL-4 and IL-33 synergistically elicit the tmTNF-α expression on macrophages through the autocrine action of IL-6.

Effects of S -adenosylmethionine and methylthioadenosine on inflammation-induced colon cancer in mice

Chronic inflammation is an underlying risk factor for colon cancer. Tumor necrosis factor alpha (TNF-α) plays a critical role in the development of inflammation-induced colon cancer in a mouse model. S-adenosylmethionine (SAMe) and its metabolite methylthioadenosine (MTA) can inhibit lipopolysaccharide-induced TNF-α expression in macrophages. The aim of this work was to examine whether SAMe and MTA are effective in preventing inflammation-induced colon cancer and if so identify signaling pathways affected. Balb/c mice were treated with azoxymethane (AOM) and dextran sulfate sodium to induce colon cancer. Two days after AOM treatment, mice were divided into three groups: vehicle control, SAMe or MTA. Tumor load, histology, immunohistochemistry, gene and protein expression were determined. SAMe and MTA treatment reduced tumor load by ∼40%. Both treatments raised SAMe and MTA levels but MTA also raised S-adenosylhomocysteine levels. MTA treatment prevented the induction of many genes known to play pathogenetic roles in this model except for TNF-α and inducible nitric oxide synthase (iNOS). SAMe also had no effect on TNF-α or iNOS and was less inhibitory than MTA on the other genes. In vivo, both treatments induced apoptosis but inhibited proliferation, β-catenin, nuclear factor kappa B activation and interleukin (IL) 6 signaling. Effect of SAMe and MTA on IL-6 signaling was examined using Colo 205 colon cancer cells. In these cells, SAMe and MTA inhibited IL-6-induced IL-10 expression. MTA also inhibited IL-10 transcription and signal transducer and activator of transcription 3 activation. In conclusion, SAMe and MTA reduced inflammation-induced colon cancer and inhibited several pathways important in colon carcinogenesis.

High TNF-alpha plasma levels and macrophages iNOS and TNF-alpha expression as risk factors for painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus. Recently it has become clear that nitric oxide (NO) and proinflammatory cytokines play an important role in the pathogenesis of PDN. We investigated whether the cytokine tumor necrosis factor alpha (TNF-α) and NO play a role in PDN pathogenesis by performing a cross-sectional and a case–control study in 110 type 2 diabetic patients. Of 110 subjects, 59 patients suffered from PDN (cases) and the remaining were painless DN (controls). Cross-sectionally, plasma TNF-α levels and immunoreactivity for inducible NO synthase (iNOS) and TNF-α were higher in patients with more severe pain on the visual analog scale. There were statistically significant differences between mild and severe pain for TNF-α levels, iNOS immunoreactivity, and TNF-α immunoreactivity. There were statistically significant differences between mild and severe pain for TNF-α levels (mean 15.24 pg/mL ± 5.42 vs 20.44 ± 10.34), iNOS immunoreactivity (9.76% ± 8.60% vs 15.48% ± 11.56%), and TNF-α immunoreactivity (13.0% ± 9.48% vs 20.44% ± 11.75%). The case–control study showed that TNF-α had an odds ratio of 5.053 (P < 0.001), TNF-α immunoreactivity of 4.125 (P < 0.001), and iNOS immunoreactivity of 3.546 (P = 0.002). DN patients with high TNF-α levels, and high iNOS and TNF-α expression in macrophages are at risk of suffering from pain. The higher the TNF-α level, and iNOS and TNF-α immunoreactivity, the more severe the pain. These findings could form the basis of further research into better management of PDN.

Particulate β-glucan induces TNF-α production in wound macrophages via a redox-sensitive NF-κβ-dependent pathway.

Glucans are known to promote wound repair. Noncellulosic β-glucans are recognized as potent immunological activators. β-Glucans are generally safe and are known to attenuate the rate of postoperative infection. Glyc101 is a particulate β-glucan isolated from Saccharomyces cerevisiae. In this study, the hypothesis that Glyc101 regulates wound macrophage function was tested. Glyc101 induced tumor necrosis factor (TNF) α transcription in macrophages isolated from murine wound site. Multiplex assay identified interleukin (IL)-10 and TNFα as two cytokines that are induced by Glyc101 in human blood monocyte-derived macrophages. Glyc101-induced TNFα production was observed to be mediated via the TLR-2 and dectin-1 receptors, receptor tyrosine kinases and NFκB activation. In murine wound macrophages, Glyc101 potentiated phorbol 12-myristate 13-acetate-induced respiratory burst. In vivo, implantation of Glyc101-enriched polyvinyl alcohol-sponges at the wound-site induced TNFα expression in macrophages. Consistently, Glyc101 induced TNFα expression in wound-site macrophages isolated from two patients with chronic wounds. These observations establish the translational significance of the net findings of this study. Activation of wound macrophages by Glyc101 represents one of the potential mechanisms by which this β-glucan may benefit chronic wounds where inefficient inflammatory response is one of the underlying causes of impaired healing.

Sauchinone, a lignan from Saururus chinensis, reduces tumor necrosis factor-α production through the inhibition of c-raf/MEK1/2/ERK 1/2 pathway activation

We investigated the anti-inflammatory effects of sauchinone, a lignan isolated from Saururus chinensis, and the underlying mechanism in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. To assess the effects of sauchinone on LPS-induced macrophages activation, we measured the production of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, and activation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK) 1/2, c-Jun amino terminal kinases and p38 mitogen-activated protein kinase, and NF-κB activation in RAW264.7 cells. Sauchinone decreased the production of TNF-α, but not MIP-2 production in RAW264.7 cells stimulated with LPS. Sauchinone also decreased c-Raf-MEK1/2-ERK1/2 phosphorylation and NF-κB activation in RAW264.7 cells stimulated with LPS. Our results show that sauchinone inhibits LPS-induced TNF-α expression in macrophages by suppression of NF-κB activation via ERK1/2 pathway, which may constitute anti-inflammatory effects of sauchinone.

Sex hormone modulation of proinflammatory cytokine and C-reactive protein expression in macrophages from older men and postmenopausal women

Inflammation plays a central role in the development and progression of coronary heart disease (CHD). The sex hormones estrogen and testosterone have been shown to modify the inflammatory response by influencing cytokine expression in human macrophages obtained from younger individuals. The effect of these hormones on the expression of proinflammatory markers in macrophages obtained from a CHD age-relevant population has not been studied. Human monocyte-derived macrophages (HMDMs) were obtained from healthy normolipidemic men and postmenopausal women (age 50-70 years), and cultured in autologous serum along with both physiological and supraphysiological concentrations of estrogen or testosterone. HMDMs were stimulated with oxidized low-density lipoproteins, and the expression of the cytokines tumor necrosis factor alpha (TNF-alpha or TNF), interleukin (IL)6, and IL-1 beta (IL1B) and of the acute-phase protein C-reactive protein (CRP) was measured. Both physiological and supraphysiological concentrations of testosterone reduced the expression and secretion of TNF-alpha and reduced the expression of IL-1 beta, but did not affect the expression of IL6 or CRP. Estrogen did not modify the expression of TNF-alpha, IL6, and IL-1 beta. Estrogen caused a variable response in CRP expression that was positively associated with the plasma small dense LDL-cholesterol concentration of the donors. There were no gender differences in any of the observed effects. Our results indicate that testosterone may exert anti-inflammatory effects by reducing macrophage TNF-alpha expression, while the effects of estrogen on macrophage CRP expression may depend upon the extracellular lipid environment.

Reduced transcript stabilization restricts TNF-α expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan

Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

Retinoic Acid-Inducible Gene-I Mediates Late Phase Induction of TNF-α by Lipopolysaccharide

LPS is the known component of bacterial pathogens that stimulates a number of proinflammatory factors. However, the mechanism of the induction of these factors by LPS has not been fully elucidated. We show here that LPS induces retinoic acid-inducible gene-I (RIG-I) in vitro and in vivo as a result from autocrine secretion of IFN-beta in macrophages. TIR-domain-containing adapter-inducing IFN-beta-deficient mouse embryo fibroblast (trif(-/)(-)) fail to show expression of RIG-I following LPS stimulation. Interference of RIG-I expression short interfering RNA represses the expression of LPS-induced TNF-alpha, whereas over-expression of RIG-I leads to the activation of TNF-alpha promoter and the induction of TNF-alpha expression. LPS- and IFN-beta-induced TNF-alpha are suppressed in RIG-I-deficient mouse embryo fibroblasts (rig(-/)(-)). Thus, RIG-I plays a key role in the expression of TNF-alpha in macrophages in response to LPS stimulation, mainly for the late phase LPS-induced expression of TNF-alpha.

Induction of murine macrophage TNF-α synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid

We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-α expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-α levels. Blockage of the CR4 iC3b site yielded increases in TNF-α for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-α only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-α expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways.

Virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus.

Macrophages respond to virus infections by rapidly secreting proinflammatory cytokines, which play an important role in the first line of defense. Tumor necrosis factor alpha (TNF-alpha) is one of the major macrophage-produced cytokines. In this study we have investigated the virus-cell interactions responsible for induction of TNF-alpha expression in herpes simplex virus (HSV)-infected macrophages. Both HSV type 1 (HSV-1) and HSV-2 induced TNF-alpha expression in macrophages activated with gamma interferon (IFN-gamma). This induction was to some extent sensitive to UV treatment of the virus. Virus particles unable to enter the cells displayed reduced capacity to stimulate TNF-alpha expression but retained a significant portion which was abolished by HSV-specific antibodies. Recombinant HSV-1 glycoprotein D was able to trigger TNF-alpha secretion in concert with IFN-gamma. Sugar moieties of HSV glycoproteins have been reported to be involved in induction of IFN-alpha but did not contribute to TNF-alpha expression in macrophages. Moreover, the entry-dependent portion of the TNF-alpha induction was investigated with HSV-1 mutants and found to be independent of the tegument proteins VP16 and UL13 and partly dependent on nuclear translocation of the viral DNA. Finally, we found that macrophages expressing an inactive mutant of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) produced less TNF-alpha in response to infectious HSV infection than the empty-vector control cell line but displayed the same responsiveness to UV-inactivated virus. These results indicate that HSV induces TNF-alpha expression in macrophages through mechanisms involving (i) viral glycoproteins, (ii) early postentry events occurring prior to nuclear translocation of viral DNA, and (iii) viral dsRNA-PKR.

Expression of TNF-alpha by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by NF-kappa B and activating transcription factor 2/Jun and translational regulation through the AU-rich region of the 3' untranslated region.

Here we have investigated the regulation of TNF-alpha expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-alpha mRNA present in resting macrophages, no TNF-alpha protein is detectable. HSV-2 infection marginally increases the level of TNF-alpha mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-gamma-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-alpha promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-alpha protein, indicating that the infection lifts a block on translation of TNF-alpha mRNA. EMSA analysis showed that specific binding to the kappaB#3 site of the murine TNF-alpha promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-alpha expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-alpha promoter. Inhibitors against either NF-kappaB activation or the activating transcription factor 2 kinase p38 abrogated TNF-alpha expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-alpha, the AU-rich sequence in the 3' untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-alpha production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.