Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages
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ABSTRACT:
SUBMITTER: Cao H
PROVIDER: S-EPMC10949775 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Bioactive plant extracts have been used for the prevention and treatment of various diseases. One of the major classes of bioactive compounds is plant polyphenols. Cottonseed ethanol extracts were determined by HPLC-MS analysis to be essentially free of toxic gossypol. The objective of this study was to investigate the effect of cottonseed ethanol extracts on the cytotoxicity and regulation of anti-inflammatory tristrataprolin (TTP) family gene expression in mouse cells. MTT, qPCR and immunoblotting assays tested the effects of cottonseed extracts in mouse RAW264.7 macrophages and 3T3-L1 adipocytes. No cytotoxicity effect was observed in macrophages treated with extracts from the coat or kernel of glanded and glandless cottonseed. Similarly, the viability of mouse adipocytes was not affected by cottonseed extracts. In contrast, gossypol and lipopolysaccharides were toxic to macrophages but not adipocytes under high concentration or long time treatment. Cottonseed extracts exhibited modest effect on TTP family gene expression in macrophages but glandless cottonseed coat extract significantly increased TTP mRNA and protein levels with a magnitude similar to cinnamon and green tea polyphenol extract and insulin. These results demonstrated that cottonseed extracts are harmless towards the mouse cells and that glandless cottonseed coat extract stimulates TTP gene expression. We propose that glandless cottonseed is a safe source of plant polyphenols with anti-inflammatory property.
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Project description:Nicotine inhibits the release of TNF-α from macrophage through activation of STAT3. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts containing AU-rich elements (ARE) in 3'-untranslated region (3'-UTR). Here we show that in LPS-stimulated human macrophages the anti-inflammatory action of nicotine is mediated by TTP. Nicotine induced activation of STAT3 enhanced STAT3 binding to the TTP promoter, increased TTP promoter activity, and increased TTP expression resulting in the suppression of LPS-stimulated TNF-α production. Overexpression of a dominant negative mutant of STAT3 (R382W) or down-regulation of STAT3 by siRNA abolished nicotine-induced TTP expression and suppression of LPS-stimulated TNF-α production. Nicotine enhanced the decay of TNF-α mRNA and decreased luciferase expression of a TNF-α 3'-UTR reporter plasmid in U937 cells. However, siRNA to TTP abrogated these effects of nicotine. In this experiment, we are reporting for the first time the involvement of TTP in the cholinergic anti-inflammatory cascade consisting of nicotine-STAT3-TTP-dampening inflammation.
| S-EPMC3137049 | biostudies-literature
Project description:Rapid initiation and timely resolution of inflammatory response in macrophages are synergistic events that are known to be equally critical to optimal host defense against pathogen infections. However, the regulation of these processes, in particular by a specific cellular metabolic program, has not been well understood. In this study, we found that IFN regulatory factor 2 (IRF2) underwent an early degradation in a proteasome-mediated pathway in LPS-treated mouse macrophages, followed by a later recovery of the expression via transactivation. We showed that IRF2 was anti-inflammatory in that knockdown of this protein promoted the production of LPS-induced proinflammatory mediators. Mechanistically, although IRF2 apparently did not target the proximal cytoplasmic signaling events upon LPS engagements, it inhibited HIF-1α-dependent expression of glycolytic genes and thereby cellular glycolysis, sequential events necessary for the IRF2 anti-inflammatory activity. We found that macrophages in endotoxin tolerant state demonstrated deficiency in LPS-augmented glycolysis, which was likely caused by failed downregulation of IRF2 and the ensuing upregulation of the glycolytic genes in these cells. In contrast to observations with LPS, knockdown of IRF2 decreased IL-4-induced macrophage alternative activation. The pro-IL-4 activity of IRF2 was dependent on KLF4, a key mediator of the alternative activation, which was transcriptionally induced by IRF2. In conclusion, our data suggest that IRF2 is an important regulator of the proinflammatory response in macrophages by controlling HIF-1α-dependent glycolytic gene expression and glycolysis. This study also indicates IRF2 as a novel therapeutic target to treat inflammatory disorders associated with dysregulations of macrophage activations.
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Project description:BackgroundThe tristetraprolin (TTP) family of mRNA-binding proteins contains three major members, Ttp, Zfp36l1, and Zfp36l2. Ttp down-regulates the stability of AU-rich element-containing mRNAs and functions as an anti-inflammation regulator.MethodsTo examine whether other TTP family proteins also play roles in the inflammatory response, their expression profiles and the possible mRNA targets were determined in the knockdown cells.ResultsTtp mRNA and protein were highly induced by lipopolysaccharide (LPS), whereas Zfp36l1 and Zfp36l2 mRNAs were down-regulated and their proteins were phosphorylated during early lipopolysaccharide stimulation. Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp. Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life. Increasing the expression of Mkp-1 inhibited the activation of p38 MAPK under lipopolysaccharide stimulation and down-regulated Tnfα, and Ttp mRNA. In addition, hyper-phosphorylation of Zfp36l1 might stabilize Mkp-1 expression by forming a complex with the adapter protein 14-3-3 and decreasing the interaction with deadenylase Caf1a.ConclusionsOur findings imply that the expression and phosphorylation of Zfp36l1 and Zfp36l2 may modulate the basal level of Mkp-1 mRNA to control p38 MAPK activity during lipopolysaccharide stimulation, which would affect the inflammatory mediators production. Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.
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Project description:A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs) for complex traits are sex specific, but the reason for this is not well understood.RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F(2) mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs) were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only approximately 1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing.These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific.
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Project description:Lipopolysaccharide (LPS) is ubiquitous in the environment. Inhalation of LPS has been implicated in the pathogenesis and/or severity of several lung diseases, including pneumonia, chronic obstructive pulmonary disease and asthma. Alveolar macrophages are the main resident leukocytes exposed to inhaled antigens. To obtain insight into which innate immune pathways become activated within human alveolar macrophages upon exposure to LPS in vivo, we conducted a study in eight healthy humans, in which we instilled sterile saline into a lung segment by bronchoscope, followed by instillation of LPS into the contralateral lung. Six hours later, a bilateral bronchoalveolar lavage was performed and whole-genome transcriptional profiling was done on purified alveolar macrophages, comparing cells exposed to saline or LPS from the same individuals. LPS induced differential expression of 2,932 genes in alveolar macrophages; 1,520 genes were upregulated, whereas 1,440 genes were downregulated. A total of 26 biological functions were overrepresented in LPS-exposed macrophages; 44 canonical pathways affected by LPS were identified, among which the genes associated with the role of pattern recognition receptors in recognition of bacteria and viruses represented the top pathway. Other pathways included cellular immune response, signaling by tumor necrosis factor (receptor) family members, cytokine signaling and glucocorticoid receptor signaling. These results reveal for the first time a large number of functional pathways influenced by the biologically relevant challenge provided by LPS administered into the airways. These data can assist in identifying novel targets for therapeutic intervention in pulmonary diseases associated with LPS exposure, including pneumonia, asthma and chronic obstructive pulmonary disease.
| S-EPMC3521791 | biostudies-literature
Project description:We have isolated an 18-kDa peptide (designated sp18, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified sp18 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that sp18 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of sp18 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse sp18 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that sp18/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions.
| S-EPMC48898 | biostudies-other
Project description:Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor alpha mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 microM Zn(2+). The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex.
| S-EPMC1351390 | biostudies-literature
Project description:Metallothionein (MT) is a low-molecular-mass, cysteine-rich metal binding protein thought to be involved in the detoxification of heavy metals and scavenging of free radicals. MT is directly induced not only by heavy metals, but also by hormones and cytokines. The present study, which uses mice with genetic deletions of the MT proteins (MT(-/-) mice), was designed to evaluate the effects of MT on the expression of pro-inflammatory cytokines in macrophages. We found that the production of tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS) in peritoneal macrophages is up-regulated by MT via the modulation of nuclear factor kappaB (NF-kappaB) activity. This conclusion is supported by the following observations: (1) LPS stimulated the secretion of less TNF activity from MT(-/-) peritoneal exudate macrophages (PEMs) than from wild-type controls (MT(+/+) mice) without a difference in the pattern of kinetics; (2) LPS-stimulated expression of TNF-alpha mRNA was decreased in MT(-/-) PEMs; (3) LPS-stimulated activation of NF-kappaB was decreased in MT(-/-) PEMs; and (4) production of TNF in PEMs of MT(-/-) mice after LPS treatment in vivo was decreased (compared with MT(+/+) PEMs). Expression of other inflammatory cytokines, interleukin (IL)-1alpha and IL-6 mRNA, which were modulated by NF-kappaB, were also down-regulated in MT(-/-) PEMs. Thus MT plays a key role in the LPS-induced activation of PEMs via the modulation of NF-kappaB activity.
| S-EPMC1222316 | biostudies-other
Project description:PURPOSE:Lipopolysaccharide (LPS), a bacterial endotoxin, is known to stimulate leuokotriene B4 (LTB4) secretion by human corneal (HCECs), conjunctival (HConjECs) and meibomian gland (HMGECs) epithelial cells. We hypothesize that this LTB4 effect represents an overall induction of proinflammatory gene expression in these cells. Our objective was to test this hypothesis. METHODS:Immortalized HCECs, HConjECs and HMGECs were cultured in the presence or absence of LPS (15 μg/ml) and ligand binding protein (LBP; 150 ng/ml). Cells were then processed for RNA isolation and the analysis of gene expression by using Illumina BeadChips, background subtraction, cubic spline normalization and GeneSifter software. RESULTS:Our findings show that LPS induces a striking increase in proinflammatory gene expression in HCECs and HConjECs. These cellular reactions are associated with a significant up-regulation of genes associated with inflammatory and immune responses (e.g. IL-1β, IL-8, and tumor necrosis factor), including those related to chemokine and Toll-like receptor signaling pathways, cytokine-cytokine receptor interactions, and chemotaxis. In contrast, with the exception of Toll-like signaling and associated innate immunity pathways, almost no proinflammatory ontologies were upregulated by LPS in HMGECs. CONCLUSIONS:Our results support our hypothesis that LPS stimulates proinflammatory gene expression in HCECs and HConjECs. However, our findings also show that LPS does not elicit such proinflammatory responses in HMGECs.
| S-EPMC6026076 | biostudies-literature