Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription

ABSTRACT: Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress response and also represses inflammation. However, the mechanisms how Nrf2 alleviates inflammation are still unclear. Here, we demonstrate that Nrf2 interferes with lipopolysaccharide-induced transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1Î². ChIP-seq and ChIP-qPCR analyses revealed that Nrf2 binds to the proximity of these genes in macrophages and inhibits RNA Pol II recruitment. Further, we found that Nrf2-mediated inhibition is independent of the Nrf2 binding motif and reactive oxygen species level. Murine inflammatory models further demonstrated that Nrf2 interferes with IL6 induction and inflammatory phenotypes in vivo. Thus, contrary to the widely accepted view that Nrf2 suppresses inflammation through redox control, we demonstrate here that Nrf2 opposes transcriptional upregulation of proinflammatory cytokine genes. This study identifies Nrf2 as the upstream regulator of cytokine production and establishes a molecular basis for an Nrf2-mediated anti-inflammation approach. Gene expression in BMDMs obtained from wild-type and Keap1-CKO mice. In Keap1-CKO (Keap1 flox/flox::LysM-Cre) BMDMs, Nrf2 transcription factor is activated due to Keap1-deficiency. BMDMs were obtained by a culture of bone marrow cells in the presence of M-CSF for7 days. M1-activated BMDMs were obtained by stimulation with LPS and IFNg for 6 hours, while M2-activated BMDMs were obtained by a stimulation with IL-4 for 6 hours. Two independent BMDM cultures were performed, and each experiment contains samples obtained from one wild-type and one Keap1-CKO mice, respectively.

ORGANISM(S): Mus musculus

SUBMITTER: Eri Kobayashi

PROVIDER: E-GEOD-71263 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BMDMs or iMACs were generated via differentiation of bone marrow cells or immortalized precursors, respectively, in culture media containing m-CSF for 7 day. Upon differentiation, BMDMs or iMacs were left untreated or stimulated as described below. 1) BMDM WT were stimulated with IFNa, LPS, PGE2, IL-4, IL-10, LPS+PGE2, LPS+IL-4, LPS+IL-10, IFNa+PGE2, IFNa+IL-10: each condition in duplicate. This experiment was performed to investigate transcriptional cross-antagonism in the concomitant presence of pro-inflammatory (LPS or IFNa) and anti-inflammatory stimuli (IL-4, IL-10, or PGE2). 2) BMDM WT were stimulated with LPS, in the presence or absence of PFI-1 (Brd2/4 inhibitor) or SGC-CBP30 (CBP/p300 inhibitor): each condition in triplicate. This experiment was performed to define pro-inflammatory genes dependent or not on chromatin remodeling for their induction. 3) BMDM Mef2C/D proficient (Vav-Cre) or BMDM Mef2C/D KO (obtained by crossing Mef2d-/- Mef2cfl/fl with Vav-Cre mice) were stimulated with LPS, PGE2, or LPS+PGE2: each condition in triplicate. This experiment was performed to investigate the role of MEF2C-D on LPS transcritptional response. 4) BMDM WT were pretreated for 40 minutes with an anti-IL10R antibody or the corresponding isotope control, and then left untreated or stimulated with LPS, PGE2 or LPS+PGE2: each condition in duplicate. This experiment was performed to investigate the crosstalk between IL-10 and PGE2. 5) BMDM WT were left untreated or stimulated with PGE2, LPS, LPS+PGE2, or LPS+PGE2+IFNb. In the latter condition, IFNb was added 1 hour after the beginning of the costimulation: each condition in triplicate. This experiment was performed to asses the impact of PGE2 into the LPS-mediated IFNb induction. 5) MEF2A-deficient iMac clones (D7, A7, A8, C7) and MEF2A-proficient (referred to as wild-type) iMac clones (NE, B3 and D10) were generated via CRISPR/Cas9 and stimulated or not with LPS: each condition in single. This experiment was performed to investigate the role of MEF2A on LPS transcritptional response.

Project description:Background & Aims: Inflammation in chronic liver diseases induces oxidative stress and thus may contribute to progression of liver injury, fibrosis, and carcinogenesis. The KEAP1/NRF2 axis is a major regulator of cellular redox balance. In the present study, we investigated whether the KEAP1/NRF2 system is involved in liver disease progression in human and mice. Methods: The clinical relevance of oxidative stress was investigated in a well-characterized cohort of NAFLD patients (n=63) by liver RNA sequencing and correlated with histological and clinical parameters. For functional analysis hepatocyte-specific NEMO knock-out (NEMO∆hepa) mice were crossed with hepatocyte-specific KEAP1 knock-out (KEAP1∆hepa) mice. Results: Immunohistochemical analysis of human liver sections showed increased oxidative stress and high NRF2 expression in patients with chronic liver disease. RNA sequencing of liver samples in a human pediatric NAFLD cohort revealed a significant increase of NRF2 activation correlating with the grade of inflammation, but not with the grade of steatosis, which could be confirmed in a second adult NASH cohort. In mice, microarray analysis revealed that KEAP1 deletion induces NRF2 target genes involved in glutathione metabolism and xenobiotic stress (e.g., Nqo1). Furthermore, deficiency of one of the most important antioxidants, glutathione (GSH), in NEMO∆hepa livers was rescued after deleting KEAP1. As a consequence, NEMO∆hepa/KEAP1∆hepa livers showed reduced apoptosis compared to NEMO∆hepa livers as well as a dramatic downregulation of genes involved in cell cycle regulation and DNA replication. Consequently, NEMO∆hepa/KEAP1∆hepa compared to NEMOΔhepa livers displayed decreased fibrogenesis, lower tumor incidence, reduced tumor number, and decreased tumor size. Conclusions: NRF2 activation in NASH patients correlates with the grade of inflammation, but not steatosis. Functional analysis in mice demonstrated that NRF2 activation in chronic liver disease is protective by ameliorating fibrogenesis, initiation and progression of hepatocellular carcinogenesis.

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Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription

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