Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expression of IL-12 p40 and p35 mRNA, and the degradation of κBα induced by LPS or LPS/IFN-γ. EMSA showed that LPS could augment the NF-κB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-κB activity nor promote the degradation of kBa. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided wit     

Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages

Huntington's disease (HD) is associated with expansion of polyglutamine tract in a protein named huntingtin (htt) that is expressed in virtually all body tissues. Thus mutated htt (HD-htt) might affect all organs, although clinical manifestations of HD are associated with selective loss of corticostriatal neurons of the brain. In this work we studied how HD-htt affects mitochondria in human peripheral blood cells. We compared various functions of mitochondria isolated from cultured lymphoblastoid cells derived from three HD patients with juvenile onset of the disease (HD-LBM) and three age-matched control (C-LBM) individuals. Respiratory parameters in different metabolic states, with succinate and glutamate plus malate were the same for all control and HD cell lines. State 4 membrane potential in HD-LBM was slightly lower than in C-LBM. The calcium retention capacity (CRC) of mitochondria was estimated using simultaneously several methods to register permeability transition (PT). We found that LBM do not undergo swelling upon Ca2+-induced PT, and do not increase CRC in the presence of ADP + oligomycin. Although each cell line had different CRC values, qualitatively PT was different in C-LBM and HD-LBM. With C-LBM cyclosporin A (CsA) increased CRC significantly, while with HD-LBM CsA was ineffective. In C-LBM depolarization of mitochondria and a large pore opening (PT) always occurred simultaneously. In HD-LBM depolarization occurred at 20-50% lower Ca2+ loads than PT. We suggest that HD-htt promotes low H+ conductance of the mitochondria by interacting with proteins at the contacts sites without directly promoting PT or hampering mitochondrial oxidative phosphorylation.     

Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in Idiopathic Pulmonary Fibrosis

Background

Genes encoding cytokine mediators are prime candidates for genetic analysis in conditions with T-helper (Th) cell disease driven imbalance. Idiopathic Pulmonary Fibrosis (IPF) is a predominantly Th2 mediated disease associated with a paucity of interferon-gamma (IFN-γ). The paucity of IFN-γ may favor the development of progressive fibrosis in IPF. Interleukin-12 (IL-12) plays a key role in inducing IFN-γ production. The aim of the current study was to assess whether the 1188 (A/C) 3'UTR single nucleotide polymorphism (SNP) in the IL-12 p40 subunit gene which was recently found to be functional and the 5644 (G/A) 3' UTR SNP of the IFN-γ gene were associated with susceptibility to IPF.

Methods

We investigated the allelic distribution in these loci in UK white Caucasoid subjects comprising 73 patients with IPF and 157 healthy controls. The SNPs were determined using the polymerase chain reaction in association with sequence-specific primers incorporating mismatches at the 3'-end.

Results

Our results showed that these polymorphisms were distributed similarly in the IPF and control groups

Conclusion

We conclude that these two potentially important candidate gene single nucleotide polymorphisms are not associated with susceptibility to IPF.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Stimulation of IFN-γ production by garlic lectin in mouse spleen cells: Involvement of IL-12 via activation of p38 MAPK and ERK in macrophages

Several lectins, present in beans and edible plant products, have immuno-potentiating and anti-tumor activities. We here report the effects of garlic lectin purified from garlic bulbs on the production of cytokines such as interleukin-12 (IL-12) and interferon-γ (IFN-γ) in the mouse. Garlic lectin induced IFN-γ production in spleen cells in a bell-shaped time (24-60 h)- and concentration (0.25-2.0 mg/ml)-dependent manner. The maximal enhancement was observed at 36 h with 0.5 mg/ml of garlic lectin. The stimulatory effect of garlic lectin on IFN-γ production was completely inhibited by both actinomycin D and cycloheximide, an inhibitor of ribosomal protein synthesis and DNA-dependent RNA polymerase, respectively, and was associated with an increase in IFN-γ mRNA level. Garlic lectin also induced IL-12 production in mouse peritoneal macrophages in a concentration (0.25-1.0 mg/ml)- and bell-shaped time (3-24 h)-dependent manner. The lectin increased the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) in macrophages. Furthermore, specific pharmacological inhibitors of ERK kinase (U0126) and p38 MAPK (SB203580) also suppressed the production of IL-12 induced by garlic lectin. The present findings suggest that garlic lectin induces IL-12 production via activation of p38 MAPK and ERK in mouse macrophages, which, in turn, stimulates IFN-γ production through an increase in IFN-γ mRNA in the spleen cells.     

Ameliorating effect of lipo-ATRA treatment on the expression of TIG3 and its suppressing effect on PPARγ gene expression in lung cancer animal model

Vitamin D₃ deficiency was found to be tightly linked to many health problems including metabolic syndrome, cancer, cardiovascular diseases, and type 2 diabetes mellitus. In our study, we tested the possible antidiabetic effects of one of vitamin D₃ analogs, alfacalcidol, solely or in a combination with metformin on type 2 diabetic rats. Type 2 diabetic model rats were induced by feeding high-fat diet for 4 weeks followed by intraperitoneal injection of streptozotocin. In addition to the control group, the diabetic rats were divided into four groups: untreated, metformin-treated, alfacalcidol-treated, and combination-treated group (metformin?+?alfacalcidol) for 4 weeks. The level of fasting blood glucose, fasting serum insulin, homeostatic model of insulin resistance, serum lipid profile, liver enzymes, calcium, phosphorus, and 25-hydroxyvitamin D₃ were also determined. Besides, sterol regulatory element binding protein-1c (SREBP-1c) and vitamin D receptors (VDR) gene expression at mRNA and protein levels were evaluated. The level of significance was fixed at P?≤?0.05 for all statistical tests. Alfacalcidol, solely or combined with metformin, significantly ameliorated glucose homeostasis and lipid profile parameters (P?<?0.001) with a neutral effect on calcium and phosphorus levels. Significant downregulation of mRNA expression of SREBP-1c in the liver, white as well as brown adipose tissues (P?<?0.001) and different patterns of mRNA expression of VDR gene in pancreas and white adipose tissue were observed in rats treated with alfacalcidol solely or in combination with metformin. Vitamin D₃ analogs can modulate glucose parameters and lipid metabolism in a diabetic rat model and it provides additional protective effects when combined with metformin.

     

oxHDL decreases the expression of CD36 on human macrophages through PPARγ and p38 MAP kinase dependent mechanisms

CD36, belongs to class B scavenger receptor family, is a macrophage receptor for oxidized low-density lipoprotein (oxLDL) and has been proven to play a critical role in atherosclerotic foam cell formation. In addition, CD36 expression is regulated by many factors including oxLDL and HDL. A recent study suggests that CD36 can also bind with oxidized high-density lipoprotein (oxHDL). However, the direct role of oxHDL in atherosclerosis is still not clear and it is not known whether oxHDL has any influence on the expression of CD36 in macrophages. Here, we performed experiments to investigate the effect of oxHDL on the expression of CD36 on human peripheral blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by CD36 on macrophages accelerates foam cell formation. In addition, oxHDL can down-regulate both the mRNA and surface protein expression of CD36 on human peripheral macrophages in vitro. oxHDL increased the mRNA expression and protein phosphorylation of peroxisome proliferators-activated receptor-γ (PPARγ). Using different mitogen-activated protein kinase (MAPK) inhibitors, we demonstrated that oxHDL regulated CD36 and PPARγ expression in a p38-MAP kinase dependent mechanism.     

The influence of drinking-water pollution with heavy metal on the expression of IL-4 and IFN-γ in mice by real-time polymerase chain reaction

In recent years, water pollution has been converted to a challenging discussion in health area of human being. Heavy elements are one of the most important water pollutants and their negative adverse effects on body systems have been confirmed. In this study, investigation of effects of two heavy elements including lead (Pb) and copper (Cu) on expression of interlukin-4 (IL-4) and interferon-gamma (IFN-γ) as humoral and cellular immunity biomarkers, respectively, was aimed and PCR, real-time PCR and electrophoresis techniques were used. In this study, BALB/c mice were studied that had free access to drinking water which contained Cu or Pb salts. After 2 weeks, spleens of mice were removed, RNA extracted, and cDNA was prepared for RT-PCR. Then the expression of IL-4 and IFN-γ genes were assessed by real-time PCR. The expression of IFN-γ was up-regulated in both treated groups and the expression of IL-4 was only up-regulated in the group treated with Cu and down-regulated in the group treated with Pb. This study shows that the presence of heavy elements as drinking-water pollutants results in a disproportion of natural cytokines balances, and thus may result in a negative effect on immune system.     

Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages

Background

PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear.

Methodology and Principal Findings

In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively.

Conclusions and Significance

In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.