Knock in p38g and knock out p38d effect on gene expression in TLR4 signalling in bone marrow derived macrophages using LPS.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE234776 | GEO | 2023/08/09
REPOSITORIES: GEO
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Project description:Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using p38γ/p38δ deficient (p38γ/δ-/-) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here we generated a p38γD171A/D171A/p38δ-/- (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to LPS-induced septic shock and Candida albicans infection than wild-type mice. Gene expression analyses in LPS-activated WT and p38γ/δKIKO macrophages revealed that p38γ/p38δ regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of iNOS and IL-1β mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.
2023-08-10 | PXD042626 | Pride
Project description:Studies in mice have indicated that TPL2 kinase plays important roles in immune responses to pathogens and in various models of inflammatory disease. We set out to determine how TPL2 (Map3k8) regulates transcription using an in vitro system of LPS-stimulated primary macrophages. TPL2 regulated genes are involved in a number of different processes including inflammation as well as reproduction, cell cycle and signal transduction. Differentially expressed genes were used in a K means analysis, grouping based on expression profile, which identified 12 different expression profiles. There was a strong correlation between rapidly-induced genes regulated by TPL2 kinase activity and their expression in cells treated with the MKK1/2 inhibitor (PD032590)1 or from cells where phosphorylation of P105 by IKK2 is blocked. Cluster 1 genes were found to be enriched for TCF family members. TPL2 was shown to regulate ELK1 phosphorylation and known TCF targets genes such as Egr1. TPL2 is known to regulate transcription of multiple cytokines and chemokines including IFNb. Like Map3k8[D270A] macrophages, expression of Ifnb1 was increased in Elk1-/-Elk4-/- macrophages compared to WT macrophages following LPS stimulation. In fact, this increase was also seen in MKK1/2 inhibitor treated macrophages. Expression of Fos was decreased in both Map3k8[D270A] and Elk1-/-Elk4-/- macrophages. Macrophages generated from mice with myeloid-specific deletion of Fos also had elevated levels of Ifnb1 compared to controls. Similar results were obtained following activation of TLR1/2, TLR9 and TNF-R in macrophages. Furthermore, LPS or TNF stimulation of MEFs lacking Elk1, Elk3 and Elk4 had reduced FOS protein levels and increased Ifnb1 mRNA suggesting a general role for ERK1/2 > TCF pathway regulating Ifnb1 levels through Fos expression.
2021-10-26 | GSE178921 | GEO
Project description:Studies in mice have indicated that TPL2 kinase plays important roles in immune responses to pathogens and in various models of inflammatory disease. We set out to determine how TPL2 (Map3k8) regulates transcription using an in vitro system of LPS-stimulated primary macrophages. TPL2 regulated genes are involved in a number of different processes including inflammation as well as reproduction, cell cycle and signal transduction. Differentially expressed genes were used in a K means analysis, grouping based on expression profile, which identified 12 different expression profiles. There was a strong correlation between rapidly-induced genes regulated by TPL2 kinase activity and their expression in cells treated with the MKK1/2 inhibitor (PD032590)1 or from cells where phosphorylation of P105 by IKK2 is blocked. Cluster 1 genes were found to be enriched for TCF family members. TPL2 was shown to regulate ELK1 phosphorylation and known TCF targets genes such as Egr1. TPL2 is known to regulate transcription of multiple cytokines and chemokines including IFNb. Like Map3k8[D270A] macrophages, expression of Ifnb1 was increased in Elk1-/-Elk4-/- macrophages compared to WT macrophages following LPS stimulation. In fact, this increase was also seen in MKK1/2 inhibitor treated macrophages. Expression of Fos was decreased in both Map3k8[D270A] and Elk1-/-Elk4-/- macrophages. Macrophages generated from mice with myeloid-specific deletion of Fos also had elevated levels of Ifnb1 compared to controls. Similar results were obtained following activation of TLR1/2, TLR9 and TNF-R in macrophages. Furthermore, LPS or TNF stimulation of MEFs lacking Elk1, Elk3 and Elk4 had reduced FOS protein levels and increased Ifnb1 mRNA suggesting a general role for ERK1/2 > TCF pathway regulating Ifnb1 levels through Fos expression.
2020-06-22 | GSE116220 | GEO
Project description:In autoimmune diseases, accumulation of activated leukocytes correlates with inflammation and disease progression, and therefore, disruption of leukocyte trafficking is an active area of research. The protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Herein, we addressed the contribution of Tpl2 to the regulation of macrophage chemokine and chemokine receptor expression and subsequent migration in vivo using a mouse model of Tpl2 ablation. We found that gene expression of the chemokine ligands CCL2, CCL7, CXCL2, and CXCL3 as well as the chemokine receptors CCR1 and CCR5 were reduced in macrophages from the bone marrow and peritoneal cavities of tpl2-/- mice following stimulation with LPS. LPS stimulation repressed chemokine receptor expression of CCR1, CCR2 and CCR5. Notably, LPS-induced repression of CCR1 and CCR5 was significantly enhanced in Tpl2-deficient macrophages and was observed to be dependent upon Erk activation and independent of PI3K and mTOR signaling. Consistent with alterations in chemokine and chemokine receptor expression, tpl2-/- macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of both chemokine receptors and their ligands and provides further insight into how Tpl2 inhibition may disrupt inflammatory networks in vivo. microarray was used to profile the genome-wide expression patterns in Tpl2 wild-type and deficient macrophage.
2014-06-03 | E-GEOD-48338 | biostudies-arrayexpress
Project description:In autoimmune diseases, accumulation of activated leukocytes correlates with inflammation and disease progression, and therefore, disruption of leukocyte trafficking is an active area of research. The protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Herein, we addressed the contribution of Tpl2 to the regulation of macrophage chemokine and chemokine receptor expression and subsequent migration in vivo using a mouse model of Tpl2 ablation. We found that gene expression of the chemokine ligands CCL2, CCL7, CXCL2, and CXCL3 as well as the chemokine receptors CCR1 and CCR5 were reduced in macrophages from the bone marrow and peritoneal cavities of tpl2-/- mice following stimulation with LPS. LPS stimulation repressed chemokine receptor expression of CCR1, CCR2 and CCR5. Notably, LPS-induced repression of CCR1 and CCR5 was significantly enhanced in Tpl2-deficient macrophages and was observed to be dependent upon Erk activation and independent of PI3K and mTOR signaling. Consistent with alterations in chemokine and chemokine receptor expression, tpl2-/- macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of both chemokine receptors and their ligands and provides further insight into how Tpl2 inhibition may disrupt inflammatory networks in vivo.
2014-06-03 | GSE48338 | GEO
Project description:Host defense against bacterial and fungal infections diminishes with age. In humans, this is thought to be caused in part by a decline in neutrophil responses. However, it remains unclear whether a similar decline in neutrophil function occurs in mice. Here, we show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment to the peritoneum was elevated during lipopolysaccharide (LPS)-induced septic peritonitis but not aseptic peritonitis. Neutrophils from old mice showed reduced chemoattractant-induced reactive oxygen species (ROS) production upon priming with LPS but not GM-CSF/TNF. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas NETosis was impaired independently of LPS. The chemoattractant-stimulated production of PIP3 was reduced upon priming with LPS but not GM-CSF/TNF, whereas PI(4,5)P2 levels were constitutively low. Unexpectedly, chemotaxis was normal regardless of priming pathway, as were the chemoattractant-stimulated activities of Rac1 and Rac2. The expression of 5% of neutrophil proteins was deregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as toll-like receptor (TLR) pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. Upregulation of Cd180 and downregulation of MyD88 may contribute to the impaired LPS priming and LPS-dependent PIP3 production. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS-TLR4 pathway dependence resolves some of the controversy regarding the effects of age on murine neutrophils and confirms mice are an appropriate model for the declining human neutrophil function.
2022-08-20 | PXD035577 | Pride
Project description:TPL2 (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates cytokine levels and activation states of microglia. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNAseq analysis indicates protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity promotes multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.
2023-08-10 | GSE196401 | GEO
Project description:Gene Expression profiling of the Arabidopsis thaliana MAP Kinase Kinases 1 (mkk1), MAP Kinase Kinases 2 (mkk2) knockout mutants and the double mutant mkk1/mkk2 before and 24 hours after treatment with the salicylic acid analog BTH, was measured by hybridisation to an Affymetrix ATH1 GeneChip. Experiment Overall Design: Whole Arabidopsis thaliana mkk1, mkk2 and mkk1/mkk2 knockout mutants and wild type plants (Col-0) were sampled in triplicates before and 24 hours after BTH treatment.
2008-06-11 | E-GEOD-10646 | biostudies-arrayexpress
Project description:Septic cardiac dysfunction is a key feature of severe sepsis and septic shock, contributing to multiorgan dysfunction syndrome and death. It has been established that persistent beta adrenergic stimulation is detrimental in sepsis, and that specific beta 1 blockade mitigates excessive systemic inflammation and improves myocardial function. The aim of this study was to investigate the effects of specific beta 1 blocker esmolol on septic mouse myocardium by genomic and proteomic techniques. We also evaluated survival of septic mice and systemic inflammation under esmolol treatment. C57BL/6 mice were rendered septic by 2 models: cecal ligature and perforation (CLP) and intraperitoneal injection of lipopolysaccharides (LPS). Effects of esmolol on myocardium were assessed by microarray technique. Total RNA were isolated and purified from a 30mg sample of the heart of 6 groups of 8 animals, depending on the sepsis model and the treatment. The labeled cDNA from the treated animals were hybridized against the labeled cDNA from the untreated animals with 2 dye-swaps done for each sepsis model.
2015-08-03 | E-GEOD-31175 | biostudies-arrayexpress
Project description:Carbon monoxide (CO) is an endogenous messenger that suppresses inflammation, modulates apoptosis and promotes vascular remodeling. Here, microarrays were employed to globally characterize the CO (250 ppm) suppression of early (1 h) LPS-induced inflammation in human monocytic THP-1 cells. CO suppressed 79 of 101 immediate-early genes induced by LPS; 19% (15/79) were transcription factors and most others were cytokines, chemokines and immune response genes. The prototypic effects of CO on transcription and protein production occurred early but decreased rapidly. CO activated p38 MAPK, ERK1/2 and Akt and caused an early and transitory delay in LPS-induced JNK activation. However, selective inhibitors of these kinases failed to block CO suppression of LPS-induced IL-1beta, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-kappaB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IkappaBalpha in human monocytes, thereby inhibiting NF-kappaB signal transduction. CO broadly suppresses the initial inflammatory response of human monocytes to LPS by reshaping proximal events in TLR4 signal transduction such as stress kinase responses and early NF-kappaB activation. These rapid, but transient effects of CO may have therapeutic applications in acute pulmonary and vascular injury.
2010-05-21 | GSE16193 | GEO