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For three-quarters of a century, glucocorticoids (GCs) have been used to treat rheumatic and autoimmune diseases. Over these 75 years, our understanding of GCs binding to nuclear receptors, mainly the glucocorticoid receptor (GR) and their molecular mechanisms has changed dramatically. Initially, in the late 1950s, GCs were considered important regulators of energy metabolism. By the 1970s/1980s, they were characterised as ligands for hormone-inducible transcription factors that regulate many aspects of cell biology and physiology. More recently, their impact on cellular metabolism has been rediscovered. Our understanding of cell-type-specific GC actions and the crosstalk between various immune and stromal cells in arthritis models has evolved by investigating conditional GR mutant mice using the Cre/LoxP system. A major achievement in studying the complex, cell-type-specific interplay is the recent advent of omics technologies at single-cell resolution, which will provide further unprecedented insights into the cell types and factors mediating GC responses. Alongside gene-encoded factors, anti-inflammatory metabolites that participate in resolving inflammation by GCs during arthritis are just being uncovered. The translation of this knowledge into therapeutic concepts will help tackle inflammatory diseases and reduce side effects. In this review, we describe major milestones in preclinical research that led to our current understanding of GC and GR action 75 years after the first use of GCs in arthritis., Competing Interests: Competing interests: JPT is scientific cofounder of MetaImmune., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ on behalf of EULAR.)

Glucocorticoid (GC) signaling is essential for mounting a stress response, however, chronic stress or prolonged GC therapy downregulates the GC receptor (GR), leading to GC resistance. Regulatory mechanisms that refine this equilibrium are not well understood. Here, we identify seven lysine acetylation sites in the amino terminal domain of GR, with lysine 154 (Lys 154 ) in the AF-1 region being the dominant acetyl-acceptor. GR-Lys 154 acetylation is mediated by p300/CBP in the nucleus in an agonist-dependent manner and correlates with transcriptional activity. Deacetylation by NAD + -dependent SIRT1 facilitates dynamic regulation of this mark. Notably, agonist-binding to both wild-type GR and an acetylation-deficient mutant elicits similar short-term target gene expression. In contrast, upon extended treatment, the polyubiquitination of the acetylation-deficient GR mutant is impaired resulting in higher protein stability, increased chromatin association and prolonged transactivation. Taken together, reversible acetylation fine-tunes duration of the GC response by regulating proteasomal degradation of activated GR., Competing Interests: Authors declare that they have no competing interests., (© 2024 The Author(s).)

ChIP-qPCR offers the opportunity to identify interactions of DNA-binding proteins such as transcription factors and their respective DNA binding sites. Thereby, transcription factors can interfere with gene expression, resulting in up- or downregulation of their target genes. Utilizing ChIP, it is possible to identify specific DNA binding sites that are bound by the DNA-binding proteins in dependence on treatment or prevailing conditions. During ChIP, DNA-binding proteins are reversibly cross-linked to their DNA binding sites and the DNA itself is fragmented. Using bead-captured antibodies, the target proteins are isolated while still binding their respective DNA response element. Using quantitative PCR, these DNA fragments are amplified and quantified. In this protocol, DNA binding sites of the glucocorticoid receptor are identified by treatment with the synthetic glucocorticoid Dexamethasone in murine bone marrow-derived macrophages., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Male C57BL/6N mice exposed to the chronic subordinate colony housing (CSC; 19 days) paradigm, a preclinically validated model of chronic psychosocial stress, are characterized by unaffected basal morning plasma corticosterone (CORT) concentrations despite adrenal and pituitary hyperplasia and increased adrenocorticotropic hormone (ACTH) plasma concentrations, compared with single-housed control (SHC) mice. However, as CSC mice are still able to show an increased CORT secretion towards novel heterotypic stressors, these effects might reflect an adaptation rather than a functional breakdown of general hypothalamus-pituitary-adrenal (HPA) axis functionality. In the present study we used male mice of a genetically modified mouse line, to investigate whether genetically-driven ACTH overexpression compromises adaptational processes occurring at the level of the adrenals during CSC exposure. Experimental mice carried a point mutation in the DNA binding domain of the glucocorticoid (GC) receptor (GR), attenuating dimerization of GR (GR dim ), resulting in a congenially compromised negative feedback inhibition at the level of the pituitary. In line with previous studies, CSC mice in both the wild type (WT; GR +/+ ) and GR dim group developed adrenal enlargement. Moreover, compared with respective SHC and WT mice, CSC GR dim mice show increased basal morning plasma ACTH and CORT concentrations. Quantitative polymerase chain reaction (qPCR) analysis revealed neither a genotype effect, nor a CSC effect on pituitary mRNA expression of the ACTH precursor proopiomelanocortin (POMC). Finally, CSC increased anxiety-related behavior, active coping and splenocyte in vitro (re)activity in both WT and GR dim mice, while a CSC-induced increase in adrenal lipid vesicles and splenic GC resistance was detectable only in WT mice. Of note, lipopolysaccharide (LPS)-stimulated splenocytes of GR dim mice were resistant to the inhibitory effects of CORT. Together our findings support the hypothesis that pituitary ACTH protein concentration is negatively controlled by GR dimerization under conditions of chronic psychosocial stress, while POMC gene transcription is not dependent on intact GR dimerization under both basal and chronic stress conditions. Finally, our data suggest that adrenal adaptations during chronic psychosocial stress (i.e., ACTH desensitization), aiming at the prevention of prolonged hypercorticism, are protective only to a certain threshold of plasma ACTH levels., Competing Interests: DL, MK, SK, LG, LK, MT, MNH, SV, JT and SOR have nothing to declare and do not have any conflict of interest., (© 2023 The Authors.)

The glucocorticoid receptor (GR) is a bona fide ligand-regulated transcription factor. Cloned in the 80s, the GR has become one of the best-studied and clinically most relevant members of the nuclear receptor superfamily. Cooperative activity of GR with other transcription factors and a plethora of coregulators contribute to the tissue- and context-specific response toward the endogenous and pharmacological glucocorticoids (GCs). Furthermore, nontranscriptional activities in the cytoplasm are emerging as an additional function of GR. Over the past 40 years, the concepts of GR mechanisms of action had been constantly changing. Different methodologies in the pregenomic and genomic era of molecular biological research and recent cutting-edge technology in single-cell and single-molecule analysis are steadily evolving the views, how the GR in particular and transcriptional regulation in general act in physiological and pathological processes. In addition to the development of technologies for GR analysis, the use of model organisms provides insights how the GR in vivo executes GC action in tissue homeostasis, inflammation, and energy metabolism. The model organisms, namely the mouse, but also rats, zebrafish, and recently fruit flies carrying mutations of the GR became a major driving force to analyze the molecular function of GR in disease models. This guide provides an overview of the exciting research and paradigm shifts in the GR field from past to present with a focus on GR transcription factor networks, GR DNA-binding and single-cell analysis, and model systems., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: We previously showed that attenuated glucocorticoid receptor (GR) function in mice (GR dim/dim ) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GR dim/dim ) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS., Methods: After 3 weeks of CS exposure, anesthetized and mechanically ventilated GR dim/dim and GR +/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid-base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range., Results: Nor CS exposure neither attenuated GR function affected survival. Non-CS GR dim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR +/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GR dim/dim and GR +/+ mice. Non-CS GR dim/dim mice presented with a lower pH and increased blood lactate levels compared to GR +/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GR dim/dim compared to GR +/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GR dim/dim compared to GR +/+ mice, but not in non-CS exposed animals., Conclusion: Whether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GR dim/dim mice warrants further investigation., Competing Interests: Authors CT, MK, and JB are employed by Boehringer Ingelheim Pharma GmbH & Co KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wepler, Preuss, Tilp, Keck, Blender, Wachter, Merz, Vogt, Kress, Gröger, Hoffmann, Fink, Calzia, Burret, Radermacher, Tuckermann and Vettorazzi.)

Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis, a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by siRNA, genetic deletion using the Cre-loxP system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing., (© 2022. The Author(s).)

In postmitotic neurons, several tumor suppressor genes (TSGs), including p53, Rb, and PTEN, modulate the axon regeneration success after injury. Particularly, PTEN inhibition is a key driver of successful CNS axon regeneration after optic nerve or spinal cord injury. In contrast, in peripheral neurons, TSG influence in neuronal morphology, physiology, and pathology has not been investigated to the same depth. In this study, we conditionally deleted PTEN from mouse facial motoneurons ( Chat-Cre/Pten loxP/loxP ) and analyzed neuronal responses in vivo with or without peripheral facial nerve injury in male and female mice. In uninjured motoneurons, PTEN loss induced somatic, axonal, and nerve hypertrophy, synaptic terminal enlargement and reduction in physiological whisker movement. Despite these morphologic and physiological changes, PTEN deletion positively regulated facial nerve regeneration and recovery of whisker movement after nerve injury. Regenerating PTEN-deficient motoneurons upregulated P-CREB and a signaling pathway involving P-Akt, P-PRAS40, P-mTOR, and P-4EBP1. In aged mice (12 months), PTEN deletion induced hair loss and facial hyperplasia of the epidermis. This suggests a time window in younger mice with PTEN loss stimulating axon growth after injury, however, at the risk of hyperplasia formation at later time points in the old animal. Overall, our data highlight a dual TSG function with PTEN loss impairing physiological neuron function but furthermore underscoring the positive effects of PTEN ablation in axon regeneration also for the PNS. SIGNIFICANCE STATEMENT Tumor suppressor genes (TSGs) restrict cell proliferation and growth. TSG inhibition, including p53 and PTEN, stimulates axon regeneration after CNS injury. In contrast, in PNS axon regeneration, TSGs have not been analyzed in great depth. Herein we show enhanced peripheral axon regeneration after PTEN deletion from facial motoneurons. This invokes a signaling cascade with novel PTEN partners, including CREB and PRAS40. In adult mice, PTEN loss induces hyperplasia of the skin epidermis, suggesting detrimental consequences when reaching adulthood in contrast to a beneficial TSG role for regeneration in young adult mice. Thus, our data highlight the double-edged sword nature of interfering with TSG function., (Copyright © 2022 the authors.)

Objectives: Glucocorticoids (GCs) are one of the most widely prescribed anti-inflammatory drugs. By acting through their cognate receptor, the glucocorticoid receptor (GR), GCs downregulate the expression of pro-inflammatory genes and upregulate the expression of anti-inflammatory genes. Metabolic pathways have recently been identified as key parts of both the inflammatory activation and anti-inflammatory polarization of macrophages, immune cells responsible for acute inflammation and tissue repair. It is currently unknown whether GCs control macrophage metabolism, and if so, to what extent metabolic regulation by GCs confers anti-inflammatory activity., Methods: Using transcriptomic and metabolomic profiling of macrophages, we identified GC-controlled pathways involved in metabolism, especially in mitochondrial function., Results: Metabolic analyses revealed that GCs repress glycolysis in inflammatory myeloid cells and promote tricarboxylic acid (TCA) cycle flux, promoting succinate metabolism and preventing intracellular accumulation of succinate. Inhibition of ATP synthase attenuated GC-induced transcriptional changes, likely through stalling of TCA cycle anaplerosis. We further identified a glycolytic regulatory transcription factor, HIF1α, as regulated by GCs, and as a key regulator of GC responsiveness during inflammatory challenge., Conclusions: Our findings link metabolism to gene regulation by GCs in macrophages., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, their long-term use leads to glucocorticoid-induced osteoporosis, increasing morbidity and mortality. Both anabolic and anti-resorptive drugs are used to counteract GC-induced bone loss, however, they are expensive and/or have major side effects. Therefore, identifying new targets for cost-effective, small-molecule inhibitors is essential. We recently identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation and showed that its inhibition with roscovitine promoted osteoblastogenesis, thus improving the skeletal bone mass and fracture healing. Here, we assessed whether Cdk5 knockdown or inhibition could also reverse the GC-mediated suppression of osteoblast differentiation, bone loss, and fracture healing. We first demonstrated that Cdk5 silencing abolished the dexamethasone (Dex)-induced downregulation of alkaline phosphatase (Alp) activity, osteoblast-specific marker gene expression ( Runx2 , Sp7 , Alpl , and Bglap ), and mineralization. Similarly, Cdk5 inhibition rescued Dex-induced suppression of Alp activity. We further demonstrated that Cdk5 inhibition reversed prednisolone (Pred)-induced bone loss in mice, due to reduced osteoclastogenesis rather than improved osteoblastogenesis. Moreover, we revealed that Cdk5 inhibition failed to improve Pred-mediated impaired fracture healing. Taken together, we demonstrated that Cdk5 inhibition with roscovitine ameliorated GC-mediated bone loss but did not reverse GC-induced compromised fracture healing in mice.

We previously showed that attenuated lung injury after hemorrhagic shock (HS) coincided with enhanced levels of the glucocorticoid (GC) receptor (GR) in lung tissue of swine. Here, we investigated the effects of impaired GR signaling on the lung during resuscitated HS using a dysfunctional GR mouse model (GR dim/dim ). In a mouse intensive care unit, HS led to impaired lung mechanics and aggravated lung inflammation in GR dim/dim mice compared to wildtype mice (GR +/+ ). After HS, high levels of the pro-inflammatory and pro-apoptotic transcription factor STAT1/pSTAT1 were found in lung samples from GR dim/dim mice. Lungs of GR dim/dim mice revealed apoptosis, most likely as consequence of reduced expression of the lung-protective Angpt1 compared to GR +/+ after HS. RNA-sequencing revealed increased expression of pro-apoptotic and cytokine-signaling associated genes in lung tissue of GR dim/dim mice. Furthermore, high levels of pro-inflammatory cytokines and iNOS were found in lungs of GR dim/dim mice. Our results indicate impaired repression of STAT1/pSTAT1 due to dysfunctional GR signaling in GR dim/dim mice, which leads to increased inflammation and apoptosis in the lungs. These data highlight the crucial role of functional GR signaling to attenuate HS-induced lung damage.

For more than 70 years, glucocorticoids (GCs) have been a powerful and affordable treatment option for inflammatory diseases. However, their benefits do not come without a cost, since GCs also cause side effects. Therefore, strong efforts are being made to improve their therapeutic index. In this review, we illustrate the mechanisms and target cells of GCs in the pathogenesis and treatment of some of the most frequent inflammatory disorders affecting the central nervous system, the gastrointestinal tract, the lung, and the joints, as well as graft-versus-host disease, which often develops after hematopoietic stem cell transplantation. In addition, an overview is provided of novel approaches aimed at improving GC therapy based on chemical modifications or GC delivery using nanoformulations. GCs remain a topic of highly active scientific research despite being one of the oldest class of drugs in medical use.

Immune response control is critical as excessive cytokine production can be detrimental and damage the host. Interleukin-10 (Il-10), an anti-inflammatory cytokine produced primarily by macrophages, is a key regulator that counteracts and controls excessive inflammatory response. Il-10 expression is regulated through the transcription factor c-Maf. Another regulator of Il-10 production is p35, an activator of the cyclin-dependent kinase 5 (Cdk5), which decreases Il-10 production in macrophages, thus increasing inflammation. However, Cdk5 regulation of c-Maf and the involvement of Il-10 production in macrophages has not yet been investigated. We used in vitro primary bone marrow-derived macrophages (BMDMs) lacking Cdk5 , stimulated them with lipopolysaccharid (LPS) and observed increased levels of c-Maf and Il-10. In an in vivo mouse model of LPS-induced endotoxemia, mice lacking Cdk5 in macrophages showed increased levels of c-Maf and elevated levels of Il-10 in lungs as well as in plasma, resulting in ameliorated survival. Taken together, we identified Cdk5 as a potential novel regulator of Il-10 production through c-Maf in macrophages under inflammatory conditions. Our results suggest that inhibition of Cdk5 enhances the c-Maf-Il-10 axis and thus potentiates improvement of anti-inflammatory therapy.

Objective: Brown adipose tissue (BAT) displays a strong circadian rhythm in metabolic activity, but it is unclear how this rhythm is regulated. As circulating levels of corticosterone coincide with the rhythm of triglyceride-derived fatty acid (FA) uptake by BAT, we investigated whether corticosterone regulates BAT circadian rhythm., Methods: Corticosterone levels were flattened by implanting mice with subcutaneous corticosterone-releasing pellets, resulting in constant circulating corticosterone levels., Results: Flattened corticosterone rhythm caused a complete loss of circadian rhythm in triglyceride-derived fatty acid uptake by BAT. This effect was independent of glucocorticoid receptor expression in (brown) adipocytes and was not caused by deregulation of clock gene expression or overexposure to glucocorticoids, but rather seemed mediated by reduced sympathetic innervation of BAT. In a mouse model of hyperlipidemia and metabolic syndrome, long-term experimental flattening of corticosterone - and thus rhythm in BAT function - resulted in adiposity., Conclusions: This study highlights that a physiological rhythm in glucocorticoids is an important regulator of BAT function and essential for the maintenance of metabolic health., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The comprehensive analysis of serum cytokine levels can be challenging due to low sample volumes and time consuming when using single-target methods like enzyme-linked immunosorbent assay (ELISA). Bead-based detection systems allow the simultaneous detection of multiple analytes using minimal sample volumes. Here we describe the use of a multiplex cytokine, chemokine, and growth factor assay for mouse cytokines in a 96-well format. This assay is based on antibody-coupled fluorescent magnetic beads combined with biotinylated secondary detection antibody followed by fluorescent-tagged streptavidin in a sandwich-like composition. Final assay readout provides the concentrations of 23 different cytokines, chemokines, and growth factors in up to 76 samples.

Glucocorticoids (GCs) are stress hormones that regulate developmental and physiological processes and are among the most potent anti-inflammatory drugs to suppress chronic and acute inflammation. GCs act through the glucocorticoid receptor (GR), a ubiquitously expressed ligand-activated transcription factor, which translocates into the nucleus and can act via two different modes, as a GR monomer or as a GR dimer. These two modes of action are not clearly differentiated in practice and may lead to completely different therapeutic outcomes. Detailed aspects of GR mechanisms are often not taken into account when GCs are used in different clinical scenarios. Patients, with critical illness-related corticosteroid insufficiency, treated with natural or synthetic GCs are still missing a clearly defined therapeutic strategy. This review discusses the different modes of GR function and its importance on organ function in vivo.

Background: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear., Objectives: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation., Methods: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163 -/- mice., Results: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163 + BMRMs is strictly dependent on IFN regulatory factor-8. CD163 + BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163 -/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163 -/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well., Conclusions: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The pathophysiology of sepsis-induced myocardial dysfunction is not resolved to date and comprises inflammation, barrier dysfunction and oxidative stress. Disease-associated reduction of tissue cystathionine-γ-lyase (CSE) expression, an endogenous H 2 S-producing enzyme, is associated with oxidative stress, barrier dysfunction and organ injury. CSE-mediated cardio-protection has been suggested to be related the upregulation of oxytocin receptor (OTR). CSE can also mediate glucocorticoid receptor (GR) signaling, which is important for normal heart function. A sepsis-related loss of cardiac CSE expression associated with impaired organ function has been reported previously. The aim of this current post hoc study was to investigate the role of cardiac GR and OTR after polymicrobial sepsis in a clinically relevant, resuscitated, atherosclerotic porcine model. Anesthetized and instrumented FBM (Familial Hypercholesterolemia Bretoncelles Meishan) pigs with high fat diet-induced atherosclerosis underwent poly-microbial septic shock ( n = 8) or sham procedure ( n = 5), and subsequently received intensive care therapy with fluid and noradrenaline administration for 24 h. Cardiac protein expression and mRNA levels were analyzed. Systemic troponin, a marker of cardiac injury, was significantly increased in septic animals in contrast to sham, whereas OTR and GR expression in septic hearts were reduced, along with a down-regulation of anti-inflammatory GR target genes and the antioxidant transcription factor NRF2. These results suggest a potential interplay between GR, CSE, and OTR in sepsis-mediated oxidative stress, inflammation and cardiac dysfunction., (Copyright © 2020 Merz, Denoix, Wigger, Waller, Wepler, Vettorazzi, Tuckermann, Radermacher and McCook.)

Background: Sepsis, that can be modeled by LPS injections, as an acute systemic inflammation syndrome is the most common cause for acute lung injury (ALI). ALI induces acute respiratory failure leading to hypoxemia, which is often associated with multiple organ failure (MOF). During systemic inflammation, the hypothalamus-pituitary-adrenal axis (HPA) is activated and anti-inflammatory acting glucocorticoids (GCs) are released to overcome the inflammation. GCs activate the GC receptor (GR), which mediates its effects via a GR monomer or GR dimer. The detailed molecular mechanism of the GR in different inflammatory models and target genes that might be crucial for resolving inflammation is not completely identified. We previously observed that mice with attenuated GR dimerization (GR dim/dim ) had a higher mortality in a non-resuscitated lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-induced inflammation model and are refractory to exogenous GCs to ameliorate ALI during inflammation. Therefore, we hypothesized that impaired murine GR dimerization (GR dim/dim ) would further impair organ function in LPS-induced systemic inflammation under human like intensive care management and investigated genes that are crucial for lung function in this setup. Methods: Anesthetized GR dim/dim and wildtype (GR +/+ ) mice were challenged with LPS (10 mg·kg -1 , intraperitoneal) and underwent intensive care management ("lung-protective" mechanical ventilation, crystalloids, and norepinephrine) for 6 h. Lung mechanics and gas exchange were assessed together with systemic hemodynamics, acid-base status, and mitochondrial oxygen consumption (JO 2 ). Western blots, immunohistochemistry, and real time quantitative polymerase chain reaction were performed to analyze lung tissue and inflammatory mediators were analyzed in plasma and lung tissue. Results: When animals were challenged with LPS and subsequently resuscitated under intensive care treatment, GR dim/dim mice had a higher mortality compared to GR +/+ mice, induced by an increased need of norepinephrine to achieve hemodynamic targets. After challenge with LPS, GR dim/dim mice also displayed an aggravated ALI shown by a more pronounced impairment of gas exchange, lung mechanics and increased osteopontin (Opn) expression in lung tissue. Conclusion: Impairment of GR dimerization aggravates systemic hypotension and impairs lung function during LPS-induced endotoxic shock in mice. We demonstrate that the GR dimer is an important mediator of hemodynamic stability and lung function, possibly through regulation of Opn, during LPS-induced systemic inflammation., (Copyright © 2020 Wepler, Preuss, Merz, Hartmann, Wachter, McCook, Vogt, Kress, Gröger, Fink, Scheuerle, Möller, Calzia, Burret, Radermacher, Tuckermann and Vettorazzi.)

Controversial data are available on hydrogen sulfide (H 2 S) during hemorrhage and resuscitation, depending on timing, dosing, mode of application, and the H 2 S donor used. Sodium thiosulfate (Na 2 S 2 O 3 ) is a recognized drug devoid of major side effects, which attenuated murine acute lung injury and cerebral ischemia/reperfusion injury. Therefore, we tested the hypothesis whether Na 2 S 2 O 3 would mitigate organ dysfunction in porcine hemorrhage-and-resuscitation. We studied animals with pre-existing coronary artery disease because of the reduced coronary arterial expression of the H 2 S producing enzyme cystathionine-γ-lyase (CSE) in this prospective, randomized, controlled, blinded experimental study. 20 anesthetized and instrumented pigs underwent 3 h of hemorrhage (removal of 30 % of the blood volume and subsequent titration of mean arterial pressure to 40 mmHg). Resuscitation (72 h) comprised re-transfusion of shed blood, crystalloids, and continuous i.v. norepinephrine. Animals randomly received vehicle or Na 2 S 2 O 3 (0.1 g·kg -1  h -1 ) for 24 h. Before, at the end of and every 24 h after shock, hemodynamics, metabolism, blood gases, lung, heart, kidney, and liver function and injury were evaluated together with cytokines and parameters of oxidative and nitrosative stress. Immediate post mortem lung, kidney, heart, and liver specimen were analyzed for marker proteins of inflammation and oxidative and nitrosative stress and mitochondrial respiratory activity in the heart, kidney, and liver. Immuno-histochemical analysis comprised lung extra-vascular albumin accumulation, nitrotyrosine formation, and CSE and glucocorticoid receptor (GCR) expression. Na 2 S 2 O 3 significantly attenuated shock-induced impairment of lung mechanics and gas exchange (plateau and positive end-expiratory pressure at 72 h p = 0.0006/p = 0.0264; Horovitz index at 48 h p = 0.0261), which coincided with a higher tissue GCR expression (p = 0.0415). During resuscitation from hemorrhagic shock Na 2 S 2 O 3 attenuated shock-induced acute lung injury in co-morbid swine, most likely due to a GCR expression related mechanism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

For many decades, glucocorticoids have been widely used as the gold standard treatment for inflammatory conditions. Unfortunately, their clinical use is limited by severe adverse effects such as insulin resistance, cardiometabolic diseases, muscle and skin atrophies, osteoporosis, and depression. Glucocorticoids exert their effects by binding to the Glucocorticoid Receptor (GR), a ligand-activated transcription factor which both positively, and negatively regulates gene expression. Extensive research during the past several years has uncovered novel mechanisms by which the GR activates and represses its target genes. Genome-wide studies and mouse models have provided valuable insight into the molecular mechanisms of inflammatory gene regulation by GR. This review focusses on newly identified target genes and GR co-regulators that are important for its anti-inflammatory effects in innate immune cells, as well as mutations within the GR itself that shed light on its transcriptional activity. This research progress will hopefully serve as the basis for the development of safer immune suppressants with reduced side effect profiles.

Brain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respond to the same glucocorticoid hormones but can have differential effects on cellular function. Several lines of evidence suggest that MR-specific target genes must exist and might underlie the distinct effects of the receptors. The present study aimed to identify MR-specific target genes in the hippocampus, a brain region where MR and GR are co-localised and play a role in the stress response. Using genome-wide binding of both receptor types, we previously identified MR-specific, MR-GR overlapping and GR-specific putative target genes. We now report altered gene expression levels of such genes in the hippocampus of forebrain MR knockout (fbMRKO) mice, killed at the time of their endogenous corticosterone peak. Of those genes associated with MR-specific binding, the most robust effect was a 50% reduction in Jun dimerization protein 2 (Jdp2) mRNA levels in fbMRKO mice. Down-regulation was also observed for the MR-specific Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1). Interestingly, the classical glucocorticoid target gene FK506 binding protein 5 (Fkbp5), which is associated with MR and GR chromatin binding, was expressed at substantially lower levels in fbMRKO mice. Subsequently, hippocampal Jdp2 was confirmed to be up-regulated in a restraint stress model, posing Jdp2 as a bona fide MR target that is also responsive in an acute stress condition. Thus, we show that MR-selective DNA binding can reveal functional regulation of genes and further identify distinct MR-specific effector pathways., (© 2019 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

The suppression of activated pro-inflammatory macrophages during immune response has a major impact on the outcome of many inflammatory diseases including sepsis and rheumatoid arthritis. The pro- and anti-inflammatory functions of macrophages have been widely studied, whereas their regulation under immunosuppressive treatments such as glucocorticoid (GC) therapy is less well-understood. GC-mediated glucocorticoid receptor (GR) activation is crucial to mediate anti-inflammatory effects. In addition, the anti-cancer drug roscovitine, that is currently being tested in clinical trials, was recently described to regulate inflammatory processes by inhibiting different Cdks such as cyclin-dependent kinase 5 ( Cdk5 ). Cdk5 was identified as a modulator of inflammatory processes in different immune cells and furthermore described to influence GR gene expression in the brain. Whether roscovitine can enhance the immunosuppressive effects of GCs and if the inhibition of Cdk5 affects GR gene regulatory function in innate immune cells, such as macrophages, has not yet been investigated. Here, we report that roscovitine enhances the immunosuppressive Dexamethasone (Dex) effect on the inducible nitric oxide synthase (iNos) expression, which is essential for immune regulation. Cdk5 deletion in macrophages prevented iNos protein and nitric oxide (NO) generation after a combinatory treatment with inflammatory stimuli and Dex. Cdk5 deletion in macrophages attenuated the GR phosphorylation on serine 211 after Dex treatment alone and in combination with inflammatory stimuli, but interestingly increased the GR-dependent anti-inflammatory target gene dual-specificity phosphatase 1 (Dusp1, Mkp1). Mkp1 phosphatase activity decreases the activation of its direct target p38Mapk, reduced iNos expression and NO production upon inflammatory stimuli and Dex treatment in the absence of Cdk5 . Taken together, we identified Cdk5 as a potential novel regulator of NO generation in inflammatory macrophages under GC treatment. Our data suggest that GC treatment in combination with specific Cdk5 inhibtior(s) provides a stronger suppression of inflammation and could thus replace high-dose GC therapy which has severe side effects in the treatment of inflammatory diseases.

Hepatic circadian gene transcription is tightly coupled to feeding behavior, which has a profound impact on metabolic disorders associated with diet-induced obesity. Here, we describe a genomics approach to uncover mechanisms controlling hepatic postprandial gene expression. Combined transcriptomic and cistromic analysis identified hundreds of circadian-regulated genes and enhancers controlled by feeding. Postprandial suppression of enhancer activity was associated with reduced glucocorticoid receptor (GR) and Forkhead box O1 (FOXO1) occupancy of chromatin correlating with reduced serum corticosterone levels and increased serum insulin levels. Despite substantial co-occupancy of feeding-regulated enhancers by GR and FOXO1, selective disruption of corticosteroid and/or insulin signaling resulted in dysregulation of specific postprandial regulated gene programs. In combination, these signaling pathways operate a major part of the genes suppressed by feeding. Importantly, the feeding response was disrupted in diet-induced obese animals, which was associated with dysregulation of several corticosteroid- and insulin-regulated genes, providing mechanistic insights to dysregulated circadian gene transcription associated with obesity., Competing Interests: The authors have declared that no competing interests exist.

Background: Glucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation., Objectives: Define the role of the glucocorticoid receptor (GR) in stromal cells for suppression of inflammatory arthritis., Methods: Bone marrow chimeric mice lacking the GR in the hematopoietic or stromal compartment, respectively, and mice with impaired GR dimerisation (GR dim ) were analysed for their response to dexamethasone (DEX, 1 mg/kg) treatment in serum transfer-induced arthritis (STIA). Joint swelling, cell infiltration (histology), cytokines, cell composition (flow cytometry) and gene expression were analysed and RNASeq of wild type and GR dim primary murine fibroblast-like synoviocytes (FLS) was performed., Results: GR deficiency in immune cells did not impair GC-mediated suppression of STIA. In contrast, mice with GR-deficient or GR dimerisation-impaired stromal cells were resistant to GC treatment, despite efficient suppression of cytokines. Intriguingly, in mice with impaired GR function in the stromal compartment, GCs failed to stimulate non-classical, non-activated macrophages (Ly6C neg , MHCII neg ) and associated anti-inflammatory markers CD163, CD36, AnxA1, MerTK and Axl. Mice with GR deficiency in FLS were partially resistant to GC-induced suppression of STIA. Accordingly, RNASeq analysis of DEX-treated GR dim FLS revealed a distinct gene signature indicating enhanced activity and a failure to reduce macrophage inflammatory protein (Mip)-1α and Mip-1β., Conclusion: We report a novel anti-inflammatory mechanism of GC action that involves GR dimerisation-dependent gene regulation in non-immune stromal cells, presumably FLS. FLS control non-classical, anti-inflammatory polarisation of macrophages that contributes to suppression of inflammation in arthritis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Aims: Release of large amounts of free heme into circulation, overproduction of reactive oxygen species (ROS), and activation of toll-like receptor-4-dependent responses are considered critical for the ability of heme to promote oxidative stress and to initiate proinflammatory responses, posing a serious threat to the body. A deep understanding of the consequences of heme overload on the regulation of cellular and systemic iron homeostasis is, however, still lacking., Results: The effects of heme on iron metabolism were studied in primary macrophages and in mouse models of acute and chronic hemolysis. We demonstrated that hemolysis was associated with a significant depletion of intracellular iron levels and increased expression of the sole iron exporter protein, ferroportin. The pathophysiological relevance of this mechanism was further demonstrated in sickle cell anemia mice, which, despite chronic hemolysis, maintained high ferroportin expression and increased iron export. We identified a redox active iron species and superoxide as regulators for ferroportin induction by heme. Scavenging the ROS production, by use of a pharmacological antioxidant N-acetylcysteine, prevented ferroportin induction and normalized intracellular iron levels in macrophages and in experimentally induced hemolysis in mice., Innovation: Our data propose that scavenging ROS levels may be a novel therapeutic strategy to balance intracellular iron levels and systemic iron influx in conditions associated with heme overload., Conclusion: This study identifies that the pro-oxidant, and not the proinflammatory, actions of heme profoundly impact on iron homeostasis by critically regulating the expression of ferroportin and iron export in hemolytic conditions. Antioxid. Redox Signal. 29, 484-499.

Cell- and tissue-specific actions of glucocorticoids are mediated by the glucocorticoid receptor. Here, we demonstrate that the glucocorticoid receptor (GR) in macrophages is essential for cardiac healing after myocardial infarction. Compared with GR flox (wild-type controls), GR LysMCre mice that lacked GR in myeloid cells showed increased acute mortality as a result of cardiac rupture. Seven days after left coronary artery ligation, GR LysMCre mice exhibited worse cardiac function and adverse remodeling associated with impaired scar formation and angiogenic response to ischemic injury. Inactivation of GR altered the functional differentiation/maturation of monocyte-derived macrophages in the infarcted myocardium. Mechanistically, CD45 + /CD11b + /Ly6G - /F4/80 + macrophages isolated from GR LysMCre infarcts showed deregulation of factors that control inflammation, neovascularization, collagen degradation, and scar tissue formation. Moreover, we demonstrate that cardiac fibroblasts sorted from the ischemic myocardium of GR LysMCre mice compared with cells isolated from injured GR flox hearts displayed higher matrix metalloproteinase 2 expression, and we provide evidence that the macrophage GR regulates myofibroblast differentiation in the infarct microenvironment during the early phase of wound healing. In summary, GR signaling in macrophages, playing a crucial role in tissue-repairing mechanisms, could be a potential therapeutic target during wound healing after ischemic myocardial injury.-Galuppo, P., Vettorazzi, S., Hövelmann, J., Scholz, C.-J., Tuckermann, J. P., Bauersachs, J., Fraccarollo, D. The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling., (© The Author(s).)

Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although glucocorticoids (GCs) are a mainstay in the clinical management of asthma, the target cells that mediate their therapeutic effects are unknown. Contrary to our expectation, we found that GC receptor (GR) expression in immune cells was dispensable for successful therapy of allergic airway inflammation (AAI) with dexamethasone. Instead, GC treatment was compromised in mice expressing a defective GR in the nonhematopoietic compartment or selectively lacking the GR in airway epithelial cells. Further, we found that an intact GR dimerization interface was a prerequisite for the suppression of AAI and airway hyperresponsiveness by GCs. Our observation that the ability of dexamethasone to modulate gene expression in airway epithelial cells coincided with its potency to resolve AAI supports a crucial role for transcriptional regulation by the GR in this cell type. Taken together, we identified an unknown mode of GC action in the treatment of allergic asthma that might help to develop more specific therapies in the future., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Glucocorticoids (GCs) are important regulators of systemic energy metabolism, and aberrant GC action is linked to metabolic dysfunctions. Yet, the extent to which normal and pathophysiological energy metabolism depend on the GC receptor (GR) in adipocytes remains unclear. Here, we demonstrate that adipocyte GR deficiency in mice significantly impacts systemic metabolism in different energetic states. Plasma metabolomics and biochemical analyses revealed a marked global effect of GR deficiency on systemic metabolite abundance and, thus, substrate partitioning in fed and fasted states. This correlated with a decreased lipolytic capacity of GR-deficient adipocytes under postabsorptive and fasting conditions, resulting from impaired signal transduction from β-adrenergic receptors to adenylate cyclase. Upon prolonged fasting, the impaired lipolytic response resulted in abnormal substrate utilization and lean mass wasting. Conversely, GR deficiency attenuated aging-/diet-associated obesity, adipocyte hypertrophy, and liver steatosis. Systemic glucose tolerance was improved in obese GR-deficient mice, which was associated with increased insulin signaling in muscle and adipose tissue. We conclude that the GR in adipocytes exerts central but diverging roles in the regulation of metabolic homeostasis depending on the energetic state. The adipocyte GR is indispensable for the feeding-fasting transition but also promotes adiposity and associated metabolic disorders in fat-fed and aged mice., (© 2017 by the American Diabetes Association.)

Dendritic cells (DC) initiate the adaptive immune response. Glucocorticoids (GCs) down-modulate the function of DC. Compound A (CpdA, (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride) is a plant-derived GR-ligand with marked dissociative properties. We investigated the effects of CpdA on in vitro generated GM-CSF-conditioned bone marrow-derived DC (BMDC). CpdA-exposed BMDC exhibited low expression of cell-surface molecules and diminution of the release of proinflammatory cytokines upon LPS stimulation; processes associated with BMDC maturation and activation. CpdA-treated BMDC were inefficient at Ag capture via mannose receptor-mediated endocytosis and displayed reduced T-cell priming. CpdA prevented the LPS-induced rise in pErk1/2 and pP38, kinases involved in TLR4 signaling. CpdA fully inhibited LPS-induced pAkt Ser473 , a marker associated with the generation of tolerogenic DC. We used pharmacological blockade and selective genetic loss-of-function tools and demonstrated GR-independent inhibitory effects of CpdA in BMDC. Mechanistically, CpdA-mediated inactivation of the NF-κB intracellular signaling pathway was associated with a short-circuiting of pErk1/2 and pP38 upstream signaling. Assessment of the in vivo function of CpdA-treated BMDC pulsed with the hapten trinitrobenzenesulfonic acid showed impaired cell-mediated contact hypersensitivity. Collectively, we provide evidence that CpdA is an effective BMDC modulator that might have a benefit for immune disorders, even when GR is not directly targeted.

Background & Aims: Kupffer cells (KC) play a key role in the onset of inflammation in non-alcoholic steatohepatitis (NASH). The glucocorticoid receptor (GR) induces glucocorticoid-induced leucine zipper (GILZ) expression in monocytes/macrophages and is involved in several inflammatory processes. We hypothesized that the GR-GILZ axis in KC may contribute to the pathophysiology of obesity-induced liver inflammation., Methods: By using a combination of primary cell culture, pharmacological experiments, mice deficient for the Gr specifically in macrophages and transgenic mice overexpressing Gilz in macrophages, we explored the involvement of the Gr-Gilz axis in KC in the pathophysiology of obesity-induced liver inflammation., Results: Obesity was associated with a downregulation of the Gr and Gilz, and an impairment of Gilz induction by lipopolysaccharide (LPS) and dexamethasone (DEX) in KC. Inhibition of Gilz expression in isolated KC transfected with Gilz siRNA demonstrated that Gilz downregulation was sufficient to sensitize KC to LPS. Conversely, liver inflammation was decreased in obese transgenic mice specifically overexpressing Gilz in macrophages. Pharmacological inhibition of the Gr showed that impairment of Gilz induction in KC by LPS and DEX in obesity was driven by a downregulation of the Gr. In mice specifically deficient for Gr in macrophages, Gilz expression was low, leading to an exacerbation of obesity-induced liver inflammation., Conclusions: Obesity is associated with a downregulation of the Gr-Gilz axis in KC, which promotes liver inflammation. The Gr-Gilz axis in KC is an important target for the regulation of liver inflammation in obesity., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Acute lung injury (ALI) is a severe inflammatory disease for which no specific treatment exists. As glucocorticoids have potent immunosuppressive effects, their application in ALI is currently being tested in clinical trials. However, the benefits of this type of regimen remain unclear. Here we identify a mechanism of glucocorticoid action that challenges the long-standing dogma of cytokine repression by the glucocorticoid receptor. Contrarily, synergistic gene induction of sphingosine kinase 1 (SphK1) by glucocorticoids and pro-inflammatory stimuli via the glucocorticoid receptor in macrophages increases circulating sphingosine 1-phosphate levels, which proves essential for the inhibition of inflammation. Chemical or genetic inhibition of SphK1 abrogates the therapeutic effects of glucocorticoids. Inflammatory p38 MAPK- and mitogen- and stress-activated protein kinase 1 (MSK1)-dependent pathways cooperate with glucocorticoids to upregulate SphK1 expression. Our findings support a critical role for SphK1 induction in the suppression of lung inflammation by glucocorticoids, and therefore provide rationales for effective anti-inflammatory therapies.