Sofie Vandevyver

New Insights into the Anti-inflammatory Mechanisms of Glucocorticoids: An Emerging Role for Glucocorticoid-Receptor-Mediated Transactivation

Glucocorticoids are anti-inflammatory drugs that are widely used for the treatment of numerous (autoimmune) inflammatory diseases. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor family of transcription factors. Upon ligand binding, the GR translocates to the nucleus, where it acts either as a homodimeric transcription factor that binds glucocorticoid response elements (GREs) in promoter regions of glucocorticoid (GC)-inducible genes, or as a monomeric protein that cooperates with other transcription factors to affect transcription. For decades, it has generally been believed that the undesirable side effects of GC therapy are induced by dimer-mediated transactivation, whereas its beneficial anti-inflammatory effects are mainly due to the monomer-mediated transrepressive actions of GR. Therefore, current research is focused on the development of dissociated compounds that exert only the GR monomer-dependent actions. However, many recent reports undermine this dogma by clearly showing that GR dimer-dependent transactivation is essential in the anti-inflammatory activities of GR. Many of these studies used GR(dim/dim) mutant mice, which show reduced GR dimerization and hence cannot control inflammation in several disease models. Here, we review the importance of GR dimers in the anti-inflammatory actions of GCs/GR, and hence we question the central dogma. We summarize the contribution of various GR dimer-inducible anti-inflammatory genes and question the use of selective GR agonists as therapeutic agents.

On the Trail of the Glucocorticoid Receptor: Into the Nucleus and Back

The glucocorticoid receptor (GR) belongs to the superfamily of steroid receptors and is an important regulator of physiological and metabolic processes. In its inactive state, GR is unbound by ligand and resides in the cytoplasm in a chaperone complex. When it binds glucocorticoids, it is activated and translocates to the nucleus, where it functions as a transcription factor. However, the subcellular localization of GR is determined by the balance between its rates of nuclear import and export. The mechanism of GR nuclear transport has been extensively studied. Originally, it was believed that nuclear import of GR is initiated by dissociation of the chaperone complex in the cytoplasm. However, several studies show that the chaperone machinery is required for nuclear transport of GR. In this review, we summarize the contribution of various chaperone components involved in the nuclear transport of GR and propose an updated model of its nuclear import and export. Moreover, we review the importance of ligand‐independent nuclear transport and compare the nuclear transport of GR with that of other steroid receptors.

Glucocorticoid receptor dimerization induces MKP1 to protect against TNF-induced inflammation.

Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflammation. Here, we demonstrate that GR dimerization plays a role in reducing TNF sensitivity. In mutant mice unable to dimerize GR, we found that TNF failed to induce MAPK phosphatase 1 (MKP1). We assessed TNF sensitivity in Mkp1(-/-) mice and found increased inflammatory gene induction in livers, increased circulating cytokines, cell death in intestinal epithelium, severe intestinal inflammation, hypothermia, and death. Mkp1(-/-) mice had increased levels of phosphorylated JNK, which promotes apoptosis, in liver tissue. We further examined JNK-deficient mice for their response to TNF. Although Jnk1(-/-) mice showed no change in sensitivity to TNF, Jnk2(-/-) mice were significantly protected against TNF, identifying JNK2 as an essential player in inflammation induced by TNF. Furthermore, we found that loss of Jnk2 partially rescued the increased sensitivity of Mkp1(-/-) and mutant GR mice to TNF. Our data show that GR dimerization inhibits JNK2 through MKP1 and protects from TNF-induced apoptosis and lethal inflammation.

Comprehensive Overview of the Structure and Regulation of the Glucocorticoid Receptor

Glucocorticoids are among the most prescribed drugs worldwide for the treatment of numerous immune and inflammatory disorders. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. There are several GR isoforms resulting from alternative RNA splicing and translation initiation of the GR transcript. Additionally, these isoforms are all subject to several transcriptional, post-transcriptional, and post-translational modifications, all of which affect the proteins stability and/or function. In this review, we summarize recent knowledge on the distinct GR isoforms and the processes that generate them. We also review the importance of all known transcriptional, post-transcriptional, and post-translational modifications, including the regulation of GR by microRNAs. Moreover, we discuss the crucial role of the putative GR-bound DNA sequence as an allosteric ligand influencing GR structure and activity. Finally, we describe how the differential composition and distinct regulation at multiple levels of different GR species could account for the wide and diverse effects of glucocorticoids.

Tumor Necrosis Factor Inhibits Glucocorticoid Receptor Function in Mice A STRONG SIGNAL TOWARD LETHAL SHOCK

As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved.

Dominance of the strongest: Inflammatory cytokines versus glucocorticoids

Pro-inflammatory cytokines are involved in the pathogenesis of many inflammatory diseases, and the excessive expression of many of them is normally counteracted by glucocorticoids (GCs), which are steroids that bind to the glucocorticoid receptor (GR). Hence, GCs are potent inhibitors of inflammation, and they are widely used to treat inflammatory diseases, such as asthma, rheumatoid arthritis and inflammatory bowel disease. However, despite the success of GC therapy, many patients show some degree of GC unresponsiveness, called GC resistance (GCR). This is a serious problem because it limits the full therapeutic exploitation of the anti-inflammatory power of GCs. Patients with reduced GC responses often have higher cytokine levels, and there is a complex interplay between GCs and cytokines: GCs downregulate pro-inflammatory cytokines while cytokines limit GC action. Treatment of inflammatory diseases with GCs is successful when GCs dominate. But when cytokines overrule the anti-inflammatory actions of GCs, patients become GC insensitive. New insights into the molecular mechanisms of GR-mediated actions and GCR are needed for the design of more effective GC-based therapies.

LPS resistance of SPRET/Ei mice is mediated by Gilz, encoded by the Tsc22d3 gene on the X chromosome

Natural variation for LPS‐induced lethal inflammation in mice is useful for identifying new genes that regulate sepsis, which could form the basis for novel therapies for systemic inflammation in humans. Here we report that LPS resistance of the inbred mouse strain SPRET/Ei, previously reported to depend on the glucocorticoid receptor (GR), maps to the distal region of the X‐chromosome. The GR‐inducible gene Tsc22d3, encoding the protein Gilz and located in the critical region on the X‐chromosome, showed a higher expressed SPRET/Ei allele, regulated in cis. Higher Gilz levels were causally related to reduced inflammation, as shown with knockdown and overexpression studies in macrophages. Transient overexpression of Gilz by hydrodynamic plasmid injection confirmed that Gilz protects mice against endotoxemia Our data strongly suggest that Gilz is responsible for the LPS resistance of SPRET/Ei mice and that it could become a treatment option for sepsis.

Neutralizing TNFα restores glucocorticoid sensitivity in a mouse model of neutrophilic airway inflammation

Asthma is a heterogeneous disorder, evidenced by distinct types of inflammation resulting in different responsiveness to therapy with glucocorticoids (GCs). Tumor necrosis factor α (TNFα) is involved in asthma pathogenesis, but anti-TNFα therapies have not proven broadly effective. The effects of anti-TNFα treatment on steroid resistance have never been assessed. We investigated the role of TNFα blockade using etanercept in the responsiveness to GCs in two ovalbumin-based mouse models of airway hyperinflammation. The first model is GC sensitive and T helper type 2 (Th2)/eosinophil driven, whereas the second reflects GC-insensitive, Th1/neutrophil-predominant asthma subphenotypes. We found that TNFα blockade restores the therapeutic effects of GCs in the GC-insensitive model. An adoptive transfer indicated that the TNFα-induced GC insensitivity occurs in the non-myeloid compartment. Early during airway hyperinflammation, mice are GC insensitive specifically at the level of thymic stromal lymphopoietin (Tslp) transcriptional repression, and this insensitivity is reverted when TNFα is neutralized. Interestingly, TSLP knockout mice displayed increased inflammation in the GC-insensitive model, suggesting a limited therapeutic application of TSLP-neutralizing antibodies in subsets of patients suffering from Th2-mediated asthma. In conclusion, we demonstrate that TNFα reduces the responsiveness to GCs in a mouse model of neutrophilic airway inflammation. Thus antagonizing TNFα may offer a new strategy for therapeutic intervention in GC-resistant asthma.

Pharmacological Inhibition of Type I Interferon Signaling Protects Mice against Lethal Sepsis

Current research on new therapeutic strategies for sepsis uses different animal models, such as the lipopolysaccharide-induced endotoxemia model and the cecal ligation and puncture (CLP) peritonitis model. By using genetic and pharmacologic inhibition of the type I interferon (IFN) receptor (IFNAR1), we show that type I IFN signaling plays a detrimental role in these sepsis models. Mortality after CLP was reduced even when type I IFN responses were blocked after the onset of sepsis. Our findings reveal that type I IFNs play an important detrimental role during sepsis by negatively regulating neutrophil recruitment. Reduced neutrophil influx likely occurs via the induction of the CXC motif chemokine 1. Moreover, human white blood cells exposed to heat-killed Pseudomonas aeruginosa secrete IFN-β and stimulate type I IFN signaling. We provide data that support pharmacologic inhibition of type I IFN signaling as a novel therapeutic treatment in severe sepsis.

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1

TNF is a central actor during inflammation and a well‐recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF‐induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1‐regulating miR‐511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR‐511 down‐regulates TNFR1 and protects against TNF, while anti‐miR‐511 up‐regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR‐511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti‐inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti‐inflammatory effects of glucocorti‐coids are mediated by induction of this miR, resulting in reduced TNFR1 expression.