Glucocorticoid receptor dimers control intestinal STAT1 and TNF-induced inflammation in mice
Marlies Ballegeer, Kelly Van Looveren, Steven Timmermans, Melanie Eggermont, Sofie Vandevyver, Fabien Thery, Karen Dendoncker, Jolien Souffriau, Jolien Vandewalle, Lise Van Wyngene, Riet De Rycke, Nozomi Takahashi, Peter Vandenabeele, Jan Tuckermann, Holger M. Reichardt, Francis Impens, Rudi Beyaert, Karolien De Bosscher, Roosmarijn E. Vandenbroucke, Claude Libert
Marlies Ballegeer, Kelly Van Looveren, Steven Timmermans, Melanie Eggermont, Sofie Vandevyver, Fabien Thery, Karen Dendoncker, Jolien Souffriau, Jolien Vandewalle, Lise Van Wyngene, Riet De Rycke, Nozomi Takahashi, Peter Vandenabeele, Jan Tuckermann, Holger M. Reichardt, Francis Impens, Rudi Beyaert, Karolien De Bosscher, Roosmarijn E. Vandenbroucke, Claude Libert
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Research Article Endocrinology Immunology

Glucocorticoid receptor dimers control intestinal STAT1 and TNF-induced inflammation in mice

Abstract

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer–dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.

Authors

Marlies Ballegeer, Kelly Van Looveren, Steven Timmermans, Melanie Eggermont, Sofie Vandevyver, Fabien Thery, Karen Dendoncker, Jolien Souffriau, Jolien Vandewalle, Lise Van Wyngene, Riet De Rycke, Nozomi Takahashi, Peter Vandenabeele, Jan Tuckermann, Holger M. Reichardt, Francis Impens, Rudi Beyaert, Karolien De Bosscher, Roosmarijn E. Vandenbroucke, Claude Libert

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Figure 2

ISGs are expressed in the IECs of naive GRdim/dim mice.

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ISGs are expressed in the IECs of naive GRdim/dim mice. (A–C) RNA-seq of...
(A–C) RNA-seq of IECs of GRWT/WT (black) and GRdim/dim (white) mice (n = 3 per group). (A) HOMER motif analysis of DE genes in GRdim/dim compared with GRWT/WT mice. Motifs with highest rank and their P values and q values are displayed. (B) Heat map of DE genes containing an ISRE and/or IRF-1 element. (C) Confirmation of RNA-seq data with qPCR on independent new samples (n = 3 per group). Ifit1, Irf8, Irf1, and Stat1 mRNA expression are shown as mean ± SEM. P values were calculated using Student’s t test. (D–F) GRWT/WT and GRdim/dim mice received drinking water with or without antibiotics for 3 weeks, after which IEC samples were taken (n = 3 per group). (D) STAT1 and p-STAT1 protein levels were analyzed via Western blot using actin as a loading control. (E) Relative STAT1 and p-STAT1 signal intensities were quantified and normalized to ACTIN and STAT1 levels respectively. P values were calculated using 2-way ANOVA. (F) Stat1 and Ifit1 mRNA expression were determined in IECs of GRfl/fl (black) and GRVillinKO mice (white) via qPCR and are shown as mean ± SEM (n = 5 per group). P values were calculated using Student’s t test. (G) GR recruitment to 2 IR-nGRE sites in the STAT1 promoter (IR-nGRE1 and IR-nGRE2). GRWT/WT (black) and GRdim/dim (white) mice were treated with PBS or 10 mg/kg DEX for 2 hours (n = 5 per group; combined data of 3 independent experiments). ChIP on IEC samples was performed against GR using an H300 antibody. Data were normalized to input for each sample and expressed as fold change of H300 to IgG control. P values were calculated using 2-way ANOVA. ****P < 0.0001; ***P < 0.001; **P ≤ 0.01; *P ≤ 0.05.