Glucocorticoid Resistance Research Articles

Synergy between interleukin-1β, interferon-γ and glucocorticoids to induce TLR2 expression involves NF-κB, STAT1 and GR.

Glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to downregulate inflammatory gene expression and are effective treatments for mild-moderate asthma. However, in severe asthma and virus-induced exacerbations, glucocorticoid therapies are less efficacious, possibly due to reduced repressive ability and/or the increased expression of pro-inflammatory genes. In human A549 epithelial and primary human bronchial epithelial cells (pHBECs), toll-like receptor (TLR) 2 mRNA and protein were supra-additively induced by interleukin-1β (IL1B) plus dexamethasone (IL1B+Dex), interferon-γ (IFNG) plus dexamethasone (IFNG+Dex) and IL1B plus IFNG plus dexamethasone (IL1B+IFNG+Dex). Indeed, ~34-2100-fold increases were apparent at 24 h for IL1B+IFNG+Dex and this was greater than for any single or dual treatment. Using the A549 cell model, TLR2 induction by IL1B+IFNG+Dex was antagonized by Org34517, a competitive GR antagonist. Further, when combined with IL1B, IFNG, or IL1B+IFNG, the enhancements by dexamethasone on TLR2 expression required GR. Likewise, inhibitor of κB kinase 2-inhibitors reduced IL1B+IFNG+Dex-induced TLR2 expression, and TLR2 expression induced by IL1B+Dex, with or without IFNG, required the nuclear factor (NF)-κB subunit, p65. Similarly, signal transducer and activator of transcription (STAT) 1 phosphorylation and γ-interferon-activated-sequence-dependent transcription were induced by IFNG. These, along with IL1B+IFNG+Dex-induced TLR2 expression, were inhibited by Janus kinase (JAK) inhibitors. As IL1B+IFNG+Dex-induced TLR2 expression also required STAT1, this study reveals cooperation between JAK-STAT1, NF-κB, and GR to upregulate TLR2 expression. Since TLR2 agonism elicits inflammatory responses, we propose that synergies involving TLR2 may occur within the cytokine milieu present in the immunopathology of glucocorticoid-resistant disease, and this could promote glucocorticoid resistance. Significance Statement This study highlights that in human pulmonary epithelial cells, glucocorticoids, when combined with the inflammatory cytokines, IL1B and IFNG, can synergistically induce the expression of inflammatory genes, such as TLR2. This effect involved positive combinatorial interactions between NF-κB/p65, GR and JAK-STAT1 signalling to synergistically upregulate TLR2 expression. Thus, synergies involving glucocorticoid-enhancement of TLR2 expression may occur in the immunopathology of glucocorticoid-resistant inflammatory diseases, including severe asthma.

JUN mediates glucocorticoid resistance by stabilizing HIF1a in T cell acute lymphoblastic leukemia

Dexamethasone (Dex) plays a critical role in T-ALL treatment, but the mechanisms of Dex resistance are poorly understood. Here, we demonstrated that the expression of JUN was regulated in Dex-resistant T-ALL cell lines and patient samples. JUN knockdown increased the sensitivity to Dex. Moreover, the survival data showed that high expression of JUN related to poor prognosis of T-ALL patients. Then, we generated dexamethasone-resistant clones and conducted RNA-seq and ATAC-seq. We demonstrated that the upregulation of JUN was most significant and regulated by JNK pathway in Dex-resistant cells. High-throughput screening showed that HIF1α inhibitors synergized with Dex could enhance Dex resistance cells death invitro and invivo. Additionally, JUN combined and stabilized HIF1α in Dex resistance cells. These results reveal a new mechanism of Dex resistance in T-ALL and provide experimental evidence for the potential therapeutic benefit of targeting the JNK-JUN-HIF1α axis for T-ALL treatment.

Predicting glucocorticoid resistance in multiple sclerosis relapse via a whole blood transcriptomic analysis.

Treatment of multiple sclerosis (MS) relapses consists of short-term administration of high-dose glucocorticoids (GCs). However, over 40% of patients show an insufficient response to GC treatment. We aimed to develop a predictive model for such GC resistance. We performed a receiver operating characteristic (ROC) curve analysis following the transcriptomic assay of whole blood samples from stable, relapsing GC-sensitive and relapsing GC-resistant patients with MS in two different European centers. We identified 12 genes being regulated during a relapse and differentially expressed between GC-sensitive and GC-resistant patients with MS. Using these genes, we defined a statistical model to predict GC resistance with an area under the curve (AUC) of the ROC analysis of 0.913. Furthermore, we observed that relapsing GC-resistant patients with MS have decreased GR, DUSP1, and TSC22D3 mRNA levels compared with relapsing GC-sensitive patients with MS. Finally, we showed that the transcriptome of relapsing GC-resistant patients with MS resembles those of stable patients with MS. Predicting GC resistance would allow patients to benefit from prompt initiation of an alternative relapse treatment leading to increased treatment efficacy. Thus, we think our model could contribute to reducing disability development in people with MS.

OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids

Abstract Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC50 4.55 nM [3.8;5.3]) compared to GR WT (EC50 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023

THU210 A Single-center Retrospective Study Of Brazilian Subjects With Differences In Sex Development (DSD) From Infancy To Adulthood

Abstract Disclosure: R.L. Batista: None. N.L. Gomes: None. T.A. Bachega: None. G. Madureira: None. M.C. Miranda: None. R.T. dallago: None. M.M. Ferrari: None. L.M. Lousada: None. F.L. Craveiro: None. J.P. Batatinha: None. R.D. Scalco: None. E.F. Costa: None. M.P. Sircili: None. F.T. Denes: None. M. Inacio: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. Context: DSD represent a wide range of conditions presenting at different ages to many health professionals with several backgrounds. Establishing a correct diagnosis is essential for appropriate management. Objective: To amplify the understanding of the first clinical presentation, prevalence, and gender change of Brazilian DSD subjects. Design: A retrospective, observational cohort study of all DSD subjects referred to a DSD multi-professional team over 41 years (from 1980 to 2021). Participants: 696 subjects.Outcome Measures: Data included DSD diagnosis, sex assignment, age at diagnosis, gender change, clinical presentation, and phenotypic features. Results: Subjects presented at prepubertal, post-pubertal, and adult age, usually with atypical genitalia, undescended testes, or primary amenorrhea. Amongst the three major DSD categories, sex chromosome DSD was diagnosed in 264 subjects (135 with 45,X karyotype); 101 are mosaics (45,X/46,Xi(Xq) and three chimerism (all ovotesticular DSD). Among the 4 ovotesticular DSD subjects, 3 were raised as females and 1 as male, no gender changes occurred in this group. Thirteen chromosome DSD patients with Y material were assigned as male and two female-assigned patients changed their gender. 258 subjects have 46,XY DSD (69 unknown DSD, 64 gonadal dysgenesis, 36 5-αRD2 deficiency, 18 17β-HSD3 deficiency, 11 17α-hydroxylase deficiency, 9 Leydig cell hypoplasia, 25 CAIS, 18 PAIS, 8 AMH defects). Among the 192 XY subjects with atypical genitalia, the sex of rearing was female in 89 (46%) and gender change from female to male occurred in 13%, most in 5 αRD2 (45%) followed by 17β-HSD3 (33%) deficiency. Among those reared as male, only 2.9% changed their gender. 46,XX DSD was diagnosed in 178 patients. Congenital adrenal hyperplasia-CAH (most 21-hydroxylase deficiency) was diagnosed in 123 (115 female-assigned). Among CAH, gender change from female to male occurred in 6 cases, of which most have VS form (5/6; p=.004), a late beginning of treatment (>2 ys old), and poor compliance. In the remaining 55 with 46,XX DSD, 24 have ovotesticular DSD (all with atypical genitalia), 16 have 46,XX testicular DSD (seven with atypical genitalia). Among the ovotesticular subjects, 14 were reared as male and ten as female, and gender change occurred in 4 cases (3 from female to male). The diagnosis was not established in twelve 46,XX DSD patients (all with atypical genitalia, three with syndromic features). One patient has aromatase deficiency, one POR deficiency, and one glucocorticoid resistance syndrome. Conclusion: gender change from female to male mainly occurred among subjects with 46,XY DSD, particularly in those with 5 αRD2 and 17β-HSD3 deficiency, suggesting that male sex assignment is desirable in patients with these diagnoses. Among 46,XX DSD, the VS form of CAH, a late start of treatment, and poor compliance were implicated in female-to-male gender change. Presentation: Thursday, June 15, 2023

Glucocorticoids, their uses, sexual dimorphisms, and diseases: new concepts, mechanisms, and discoveries.

The normal stress response in humans is governed by the hypothalamic-pituitary-adrenal (HPA) axis through heightened mechanisms during stress, raising blood levels of the glucocorticoid hormone cortisol. Glucocorticoids are quintessential compounds that balance the proper functioning of numerous systems in the mammalian body. They are also generated synthetically and are the preeminent therapy for inflammatory diseases. They act by binding to the nuclear receptor transcription factor glucocorticoid receptor (GR), which has two main isoforms (GRα and GRβ). Our classical understanding of glucocorticoid signaling is from the GRα isoform, which binds the hormone, whereas GRβ has no known ligands. With glucocorticoids being involved in many physiological and cellular processes, even small disruptions in their release via the HPA axis, or changes in GR isoform expression, can have dire ramifications on health. Long-term chronic glucocorticoid therapy can lead to a glucocorticoid-resistant state, and we deliberate how this impacts disease treatment. Chronic glucocorticoid treatment can lead to noticeable side effects such as weight gain, adiposity, diabetes, and others that we discuss in detail. There are sexually dimorphic responses to glucocorticoids, and women tend to have a more hyperresponsive HPA axis than men. This review summarizes our understanding of glucocorticoids and critically analyzes the GR isoforms and their beneficial and deleterious mechanisms and the sexual differences that cause a dichotomy in responses. We also discuss the future of glucocorticoid therapy and propose a new concept of dual GR isoform agonist and postulate why activating both isoforms may prevent glucocorticoid resistance.

Suppression of mir-150–5p attenuates the anti-inflammatory effect of glucocorticoids in mice with ulcerative colitis

Glucocorticoids have been widely used in the treatment of ulcerative colitis, but not all patients benefit from this therapy due to hormone resistance. Mir-150–5p has been reported to enhance the efficacy of glucocorticoids, and low serum mir-150–5p expression has been linked to glucocorticoid resistance in ulcerative colitis patients. The aim of this study was to elucidate the mechanisms of mir-150–5p regulation on glucocorticoid resistance. An ulcerative colitis mouse model was used to evaluate changes in ulcerative colitis symptoms, inflammatory factors, and glucocorticoid resistance-related gene expression. The results showed that mir-150–5p suppression with antagomirs did not significantly interfere with or enhance the induction of ulcerative colitis symptoms by dextran sulfate sodium, but it did attenuate the inflammation inhibitory effect of dexamethasone by abnormally regulating the expression of IL-17a, IL-10, IL-2 and IL-6 levels and myeloperoxidase activity. Mir-150–5p inhibition also induced a glucocorticoid-resistant gene expression profile in colon tissues of ulcerative colitis mice, with upregulation of p-ERK, p-JNK, and HSP90 and downregulation of p-GRa, FKBP4, and HDAC2 expression. Our results indicate that mir-150–5p suppression attenuates the anti-inflammatory effect of glucocorticoids and may function as a driver element in ulcerative colitis glucocorticoid resistance. Availability of data and materialsAll data and figures analyzed in this study are available from the corresponding author by request.

CCL22 and Leptin associated with steroid resistance in childhood idiopathic nephrotic syndrome.

Previous studies have indicated a decrease in T regulatory cells (Tregs) among patients with steroid-resistant nephrotic syndrome. CCL22 and Leptin influenced the immune function of Tregs through their respective pathways. This study aimed to compare patients with steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS) in terms of CCL22 and Leptin levels. This prospective study included 117 children diagnosed with idiopathic nephrotic syndrome (INS). Peripheral blood samples were collected before initiating steroid therapy, and serum levels of CCL22 and Leptin were measured. Patients were categorized into three groups based on their response to steroid treatment. Renal biopsies were recommended for all children diagnosed with INS, with higher acceptance rates in glucocorticoid resistance patients. Based on the response to steroid treatment, 117 children were divided as groups of SSNS (82 cases), frequent relapse nephrotic syndrome (FRNS) (10 cases), and SRNS (25 cases). A total of 41 patients underwent kidney biopsy, 11 cases (13.4%) in SSNS, 7 cases (70.0%) in FRNS and 24 cases (96.0%) in SRNS. 30 cases were minimal change disease (MCD), 9 cases were mesangial proliferative glomerulonephritis (MsPGN) and 3 cases were focal segmental glomerulosclerosis (FSGS). The levels of Leptin were significantly higher in SR patients (1208.1 ± 1044.1 pg/ml) compared to SS patients (515.4 ± 676.9 pg/ml) and controls (507.9 ± 479.8 pg/ml), regardless of the pathological type. CCL22 levels were significantly elevated in SRNS (92.2 ± 157.0 pg/ml), but the difference seemed to be attributed to the specific type of pathology, such as Minimal change disease (MCD) (127.4 ± 206.7 pg/ml) and focal segmental glomerulosclerosis (FSGS) (114.8 ± 22.0 pg/ml). For SRNS prediction, the AUC of Leptin, CCL22, and the joint prediction index were 0.764, 0.640, and 0.806, respectively. Serum levels of CCL22 and Leptin, detected prior to steroid therapy, were associated with steroid resistance in childhood INS.

Glucocorticoid receptor regulates protein chaperone, circadian clock and affective disorder genes in the zebrafish brain.

Glucocorticoid resistance is commonly observed in depression, and has been linked to reduced expression and/or function of the glucocorticoid receptor (NR3C1 in human, hereafter referred to as GR). Previous studies have shown that GR-mutant zebrafish exhibit behavioural abnormalities that are indicative of an affective disorder, suggesting that GR plays a role in brain function. We compared the brain methylomes and brain transcriptomes of adult wild-type and GR-mutant zebrafish, and identified 249 differentially methylated regions (DMRs) that are regulated by GR. These include a cluster of CpG sites within the first intron of fkbp5, the gene encoding the glucocorticoid-inducible heat shock protein co-chaperone Fkbp5. RNA-sequencing analysis revealed that genes associated with chaperone-mediated protein folding, the regulation of circadian rhythm and the regulation of metabolism are particularly sensitive to loss of GR function. In addition, we identified subsets of genes exhibiting GR-regulated transcription that are known to regulate behaviour, and are linked to unipolar depression and anxiety. Taken together, our results identify key biological processes and novel molecular mechanisms through which the GR is likely to mediate responses to stress in the adult zebrafish brain, and they provide further support for the zebrafish GR mutant as a model for the study of affective disorders.

Stress circuitry: mechanisms behind nervous and immune system communication that influence behavior.

Inflammatory processes are increased by stress and contribute to the pathology of mood disorders. Stress is thought to primarily induce inflammation through peripheral and central noradrenergic neurotransmission. In healthy individuals, these pro-inflammatory effects are countered by glucocorticoid signaling, which is also activated by stress. In chronically stressed individuals, the anti-inflammatory effects of glucocorticoids are impaired, allowing pro-inflammatory effects to go unchecked. Mechanisms underlying this glucocorticoid resistance are well understood, but the precise circuits and molecular mechanisms by which stress increases inflammation are not as well known. In this narrative review, we summarize the mechanisms by which chronic stress increases inflammation and contributes to the onset and development of stress-related mood disorders. We focus on the neural substrates and molecular mechanisms, especially those regulated by noradrenergic signaling, that increase inflammatory processes in stressed individuals. We also discuss key knowledge gaps in our understanding of the communication between nervous and immune systems during stress and considerations for future therapeutic strategies. Here we highlight the mechanisms by which noradrenergic signaling contributes to inflammatory processes during stress and how this inflammation can contribute to the pathology of stress-related mood disorders. Understanding the mechanisms underlying crosstalk between the nervous and immune systems may lead to novel therapeutic strategies for mood disorders and/or provide important considerations for treating immune-related diseases in individuals suffering from stress-related disorders.

The Hypothalamus-pituitary-adrenocortical Response to Critical Illness: A Concept in Need of Revision.

Based on insights obtained during the past decade, the classical concept of an activated hypothalamus-pituitary-adrenocortical axis in response to critical illness is in need of revision. After a brief central hypothalamus-pituitary-adrenocortical axis activation, the vital maintenance of increased systemic cortisol availability and action in response to critical illness is predominantly driven by peripheral adaptations rather than by an ongoing centrally activated several-fold increased production and secretion of cortisol. Besides the known reduction of cortisol-binding proteins that increases free cortisol, these peripheral responses comprise suppressed cortisol metabolism in liver and kidney, prolonging cortisol half-life, and local alterations in expression of 11βHSD1, glucocorticoid receptor-α (GRα), and FK506 binding protein 5 (FKBP51) that appear to titrate increased GRα action in vital organs and tissues while reducing GRα action in neutrophils, possibly preventing immune-suppressive off-target effects of increased systemic cortisol availability. Peripherally increased cortisol exerts negative feed-back inhibition at the pituitary level impairing processing of pro-opiomelanocortin into ACTH, thereby reducing ACTH-driven cortisol secretion, whereas ongoing central activation results in increased circulating pro-opiomelanocortin. These alterations seem adaptive and beneficial for the host in the short term. However, as a consequence, patients with prolonged critical illness who require intensive care for weeks or longer may develop a form of central adrenal insufficiency. The new findings supersede earlier concepts such as "relative," as opposed to "absolute," adrenal insufficiency and generalized systemic glucocorticoid resistance in the critically ill. The findings also question the scientific basis for broad implementation of stress dose hydrocortisone treatment of patients suffering from acute septic shock solely based on assumption of cortisol insufficiency.

Theory and Practice of Glucocorticoids in COVID-19: Getting to the Heart of the Matter-A Critical Review and Viewpoints.

Prolonged, low-dose glucocorticoids (GCs) have shown the highest efficacy among pharmacological and non-pharmacological treatments for COVID-19. Despite the World Health Organization's recommendation against their use at the beginning of the pandemic, GCs at a dose equivalent to dexamethasone 6 mg/day for 10 days are now indicated in all COVID-19 cases who require respiratory support. However, the efficacy of the intervention depends on the timing of initiation, the dose, and other individual factors. Indeed, patients treated with similar GC protocols often experience different outcomes, which do not always correlate with the presence of comorbidities or with the severity of respiratory involvement at baseline. This prompted us to critically review the literature on the rationale, pharmacological principles, and clinical evidence that should guide GC treatment. Based on these data, the best treatment protocol probably involves an initial bolus dose to saturate the glucocorticoid receptors, followed by a continuous infusion to maintain constant plasma levels, and eventually a slow tapering to interruption. Methylprednisolone has shown the highest efficacy among different GC molecules, most likely thanks to its higher ability to penetrate the lung. Decreased tissue sensitivity to glucocorticoids is thought to be the main mechanism accounting for the lower response to the treatment in some individuals. We do not have a readily available test to identify GC resistance; therefore, to address inter-individual variability, future research should aim at investigating clinical, physiological, and laboratory markers to guide a personalized GC treatment approach.

Calpeptin may reverse glucocorticoid-resistance of allergic rhinitis associated with cigarette smoke exposure by down-regulating interferon regulatory factor 1

Cigarette smoke exposure is an important factor in chronic inflammation in patients with allergic rhinitis (AR); however, the relationship between cigarette smoke and AR-related glucocorticoid resistance requires further study. In mice, calpeptin significantly reduces inflammation of the lower respiratory tract caused by cigarette smoke, but whether it can treat glucocorticoid-resistant AR caused by cigarette smoke requires further research. In this study, we confirmed that cigarette smoke exposure can aggravate the Th2 inflammatory response in AR leading to glucocorticoid resistance. The underlying mechanism may be related to decreased expression of DNA methyltransferase 3a (Dnmt3a), and increased expression of interferon regulatory factor 1 (IRF1). In addition, we found that calpeptin can inhibit the expression of IRF1 and thus treat AR-associated glucocorticoid resistance in rats exposed to cigarette smoke. These data suggest that calpeptin may downregulate IRF1 and therefore treat glucocorticoid resistance in AR-associated with cigarette smoke exposure.

Long-term disease course, cost and prognosis of inflammatory bowel disease: epidemiological studies of a European and a Danish inception cohort.

Long-term disease course, cost and prognosis of inflammatory bowel disease: epidemiological studies of a European and a Danish inception cohort.

Inadequate therapeutic responses to glucocorticoid treatment in bronchial asthma.

Bronchial asthma (BA) is a heterogeneous disease. Some patients benefit greatly from glucocorticoid (GC) treatment, whereas others are non-responders. This could be attributable to differences in pathobiology. Thus, predicting the responses to GC treatment in patients with BA is necessary to increase the success rates of GC therapy and avoid adverse effects. The sustained inflammation in BA decreases glucocorticoid receptor (GR, NR3C1) function. Meanwhile, GRβ overexpression might contribute to GC resistance. Important factors in decreased GR function include p38 mitogen-activated protein kinase-dependent GR phosphorylated at Ser226, reduced expression of histone deacetylase 2 following activation of the phosphatidylinositol 3-kinase-δ signaling pathway, and increased nuclear factor-kappa B activity. MicroRNAs, which are involved in GC sensitivity, are considered biomarkers of the response to inhaled GCs. Some studies revealed that inflammatory phenotypes and disease-related modifiable factors, including infections, the airway microbiome, mental stress, smoking, and obesity, regulate individual sensitivity to GCs. Therefore, future investigations are warranted to improve treatment outcomes.

Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia

Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Beyond Corticoresistance, A Paradoxical Corticosensitivity Induced by Corticosteroid Therapy in Pediatric Acute Lymphoblastic Leukemias

Known as a key effector in relapse of acute lymphoblastic leukemia (ALL), resistance to drug-induced apoptosis, is tightly considered one of the main prognostic factors for the disease. ALL cells are constantly developing cellular strategies to survive and resist therapeutic drugs. Glucocorticoids (GCs) are one of the most important agents used in the treatment of ALL due to their ability to induce cell death. The mechanisms of GC resistance of ALL cells are largely unknown and intense research is currently focused on this topic. Such resistance can involve different cellular and molecular mechanisms, including the modulation of signaling pathways involved in the regulation of proliferation, apoptosis, autophagy, metabolism, epigenetic modifications and tumor suppressors. Recently, several studies point to the paradoxical role of GCs in many survival processes that may lead to therapy-induced resistance in ALL cells, which we called “paradoxical corticosensitivity”. In this review, we aim to summarize all findings on cell survival pathways paradoxically activated by GCs with an emphasis on previous and current knowledge on gene expression and signaling pathways.

Chronic stress, neuroinflammation, and depression: an overview of pathophysiological mechanisms and emerging anti-inflammatories.

In a subset of patients, chronic exposure to stress is an etiological risk factor for neuroinflammation and depression. Neuroinflammation affects up to 27% of patients with MDD and is associated with a more severe, chronic, and treatment-resistant trajectory. Inflammation is not unique to depression and has transdiagnostic effects suggesting a shared etiological risk factor underlying psychopathologies and metabolic disorders. Research supports an association but not necessarily a causation with depression. Putative mechanisms link chronic stress to dysregulation of the HPA axis and immune cell glucocorticoid resistance resulting in hyperactivation of the peripheral immune system. The chronic extracellular release of DAMPs and immune cell DAMP-PRR signaling creates a feed forward loop that accelerates peripheral and central inflammation. Higher plasma levels of inflammatory cytokines, most consistently interleukin IL-1β, IL-6, and TNF-α, are correlated with greater depressive symptomatology. Cytokines sensitize the HPA axis, disrupt the negative feedback loop, and further propagate inflammatory reactions. Peripheral inflammation exacerbates central inflammation (neuroinflammation) through several mechanisms including disruption of the blood-brain barrier, immune cellular trafficking, and activation of glial cells. Activated glial cells release cytokines, chemokines, and reactive oxygen and nitrogen species into the extra-synaptic space dysregulating neurotransmitter systems, imbalancing the excitatory to inhibitory ratio, and disrupting neural circuitry plasticity and adaptation. In particular, microglial activation and toxicity plays a central role in the pathophysiology of neuroinflammation. Magnetic resonance imaging (MRI) studies most consistently show reduced hippocampal volumes. Neural circuitry dysfunction such as hypoactivation between the ventral striatum and the ventromedial prefrontal cortex underlies the melancholic phenotype of depression. Chronic administration of monoamine-based antidepressants counters the inflammatory response, but with a delayed therapeutic onset. Therapeutics targeting cell mediated immunity, generalized and specific inflammatory signaling pathways, and nitro-oxidative stress have enormous potential to advance the treatment landscape. Future clinical trials will need to include immune system perturbations as biomarker outcome measures to facilitate novel antidepressant development. In this overview, we explore the inflammatory correlates of depression and elucidate pathomechanisms to facilitate the development of novel biomarkers and therapeutics.

Impact of the LPS from Cyanobacteria Brasilonema sp.on Lung Inflammation and Glucocorticoid Expression

Abstract Inhaled glucocorticoids (GCs) are the first-line treatment for asthma. However, patients with asthma characterized by recruitment of neutrophils show poor sensitivity to GC therapy. A potential etiology of this GC-resistant asthma is aerosolized cyanobacterial lipopolysaccharide (LPS) from cyanobacterial blooms. Previously, we demonstrated that inhalation of LPS from Geitlerinema sp. of cyanobacteria induces neutrophilic lung infiltration in mice. Our current data shows that the LPS from another cyanobacteria, Brasilonema sp., also induces lung inflammation after inhalation. Flow cytometric data demonstrate that Brasilonema sp. LPS induces neutrophil infiltration and ELISA identified increases in pro-inflammatory cytokine levels in bronchoalveolar lung fluid after Brasilonema sp. LPS exposure. Furthermore, IHC data demonstrate that mice exposed to Brasilonema sp. LPS show an increased level of lung epithelial GCRβ, a glucocorticoid receptor (GR) analog that inhibits GC activity. Conversely, expression of GCRα, the GR isoform responsible for glucocorticoid sensitivity, remains unchanged. These data help to elucidate the mechanisms of Brasilonema sp. LPS-induced lung-inflammation and potential glucocorticoid resistance, while highlighting the importance of the toxin in promoting human disease near cyanobacterial blooms. This work is supported by the Kenneth A. Suarez Fellowship and Intramural Funds at Midwestern University.

Intact GR dimerization is critical for restraining plasma ACTH levels during chronic psychosocial stress

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 (GRdim), 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 GRdim group developed adrenal enlargement. Moreover, compared with respective SHC and WT mice, CSC GRdim 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 GRdim 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 GRdim 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.