Glucocorticoid Sensitivity Research Articles

PU.1 eviction at lymphocyte-specific chromatin domains mediates glucocorticoid response in acute lymphoblastic leukemia

The epigenetic landscape plays a critical role in cancer progression, yet its therapeutic potential remains underexplored. Glucocorticoids are essential components of treatments for lymphoid cancers, but resistance, driven in part by epigenetic changes at glucocorticoid-response elements, poses a major challenge to effective therapies. Here we show that glucocorticoid treatment induces distinct patterns of chromosomal organization in glucocorticoid-sensitive and resistant acute lymphoblastic leukemia xenograft models. These glucocorticoid-response elements are primed by the pioneer transcription factor PU.1, which interacts with the glucocorticoid receptor. Eviction of PU.1 promotes receptor binding, increasing the expression of genes involved in apoptosis and facilitating a stronger therapeutic response. Treatment with a PU.1 inhibitor enhances glucocorticoid sensitivity, demonstrating the clinical potential of targeting this pathway. This study uncovers a mechanism involving PU.1 and the glucocorticoid receptor, linking transcription factor activity with drug response, and suggesting potential therapeutic strategies for overcoming resistance.

Prediction of post-ESD esophageal stricture by a nomogram and risk factor analysis of ineffective oral steroids prophylaxis

Background and aimsSeveral risk models for esophageal stricture after endoscopic submucosal dissection have been developed. However, some of them did not include the use of steroids in the risk analysis. Glucocorticoid sensitivity mediated by glucocorticoid receptor expression has not been discussed in this condition.MethodsClinical and endoscopic characteristics were included in the logistic regression model to establish a nomogram for stenosis prediction. The score for each risk factor was estimated. Risk factors of ineffective oral steroid prophylaxis were analyzed and glucocorticoid receptor expressions were detected by immunohistochemistry.ResultsThree hundred fourteen patients of endoscopic submucosal dissection for esophageal superficial neoplasms were included to develop the nomogram. The circumferential range(≤ 3/4, 3/4–1 or the whole circumference), longitudinal diameter reached 4 cm (yes or not) and lesion location (the cervical and upper thoracic part, the middle thoracic part or the lower thoracic part) consisted of the nomogram. Patients have a high risk of esophageal stricture if they have a total point greater than 36. In the simplified risk score model, the corresponding cutoff score was 1. 92 patients with oral steroid prophylaxis were separately analyzed and the circumferential mucosal defect involving 7/8 or more was an independent risk factor of ineffective prevention (OR 12.2, 95%CI 5.27–28.11). The expression of glucocorticoid receptor β was higher in the stricture group (p = 0.042 for AOD; p = 0.016 for the scoring system).ConclusionsWe established a nomogram for esophageal stricture prediction. Depending on the characteristics of lesions, it is possible to estimate the risk of stricture under routine post-ESD treatments (no steroids or oral steroids). Alternative treatments should be considered if the risk is extremely high, especially for patients with mucosal defects involving 7/8 or more of circumference in which oral steroid treatment tends to be ineffective. The higher glucocorticoid receptor β may indicate potential glucocorticoid resistance.

Glucocorticoid receptor response and glucocorticoid-induced leucine zipper expression in neutrophils of critically ill patients with traumatic and non-traumatic brain injury.

Critically ill patients, including those with brain injuries (BI), are frequently hospitalized in an intensive care unit (ICU). As with other critical states, an adequate stress response is essential for survival. Research on the hypothalamic-pituitary-adrenal gland (HPA) axis function in BI has primarily focused on assessing ACTH and cortisol levels. However, the immunological, metabolic, and hemodynamic effects of glucocorticoids (GCs) are mediated through the glucocorticoid receptor (GCR), a ubiquitously distributed intracellular receptor protein. Data on GCR-α expression and its signaling in acute BI injury are lacking. We designed a prospective observational study, carried out in one academic multi-disciplinary ICU. Forty-two critically ill patients with acute (BI)were included. These patients suffered from traumatic BI (N= 20), subarachnoid hemorrhage (N= 12), intracranial hemorrhage (N= 7), or ischemic stroke (N= 3). All patients were steroid-free. Twenty-four age and sex-matched healthy controls were used for comparison. Expression of GCR-α and the glucocorticoid-inducible leucine zipper (GILZ), serum cortisol, interleukins (IL) 6, 8, 10 and TNF- α, and the BI biomarkers glial fibrillary acidic protein (GFAP) and total Tau were measured on ICU admission (within 48 hours) and 5-7 days from admission. Compared to healthy controls, in the critically ill patients with BI, GCR-α mRNA expression was significantly downregulated on admission, and after 5-7 days in the ICU (2.3-fold, p<0.05 and 2.6-fold, p<0.01, respectively). Even though GCR-α was downregulated, its downstream gene, GILZ, was expressed at the same levels as in normal controls on admission and was significantly upregulated 5-7 days following admission (2-fold, p<0.001). TNF-α levels were undetectable at both time-points. GCR-α expression levels inversely correlated with IL-6. The levels of cortisol and the BI biomarkers did not differ between the 2 time-points. We provide novel evidence on the downregulated expression and upregulated signaling of the ligand-binding and functionally active GCR-α isoform in the polymorphonuclear neutrophils (PMNs) of critically ill patients with BI. The increased GILZ expression indicates an increased GC sensitivity in the PMNs of BI critically ill patients.

Glucocorticoid Sensitivity Among Young Survivors of Childhood Acute Lymphoblastic Leukemia: What Does It Matter?

The aim of the study was to assess glucocorticoid sensitivity in survivors of childhood acute lymphoblastic leukemia using in vivo and in vitro tests. Thirty leukemia survivors of both sexes aged ≥18 years participated in the study and at least two years after therapy withdrawal. In vivo tests comprised: a) a very low dose intravenous dexamethasone suppression test for measurement of serum cortisol before, after, and % suppression, compared with 32 age-matched controls; and b) 0.25 mg overnight oral dexamethasone suppression test for assessment of salivary cortisol before, after, and % suppression. In vitro methods comprised: c) glucocorticoid receptor polymorphisms: BcI1-NR3C1 and A3669G; and d) splicing variant of glucocorticoid receptor GR-α mRNA by real-time quantitative polymerase chain reaction, compared with 32 controls. There was a reduction in salivary cortisol, and 73.3% of leukemia survivors showed high sensitivity according to % suppression after oral dexamethasone (p<0.05). Serum cortisol at baseline, after the test, % suppression after intravenous dexamethasone, and the percentage of high sensitivity were reduced in the leukemia group (%F=36.7; p<0.05). The BcI1-NR3C1 and A3669G polymorphisms were present in 11/30 (36.7%) and 5/30 (16.7%) patients, respectively. GR-α mRNA levels were lower in the leukemia group than in the controls (p<0.05). Survivors of acute lymphoblastic leukemia presented with reduced glucocorticoid sensitivity. Glucocorticoid sensitivity allows individualized treatment to avoid adverse effects and may be involved in cardiovascular disease risk among this particular group of cancer survivors.

Sex-specific associations between childhood trauma and adult systemic inflammation in daily life.

Childhood trauma may contribute to lifelong health through chronic systemic inflammation. However, associations between childhood trauma and inflammation are mixed, indicating that distinct types of childhood trauma may relate to inflammation differently. Moreover, most studies use a single assessment of inflammatory markers that may not reliably estimate stable interindividual differences. The current study is the first to examine relationships between childhood trauma and an ecologically valid measure of inflammation derived from repeated assessments of interleukin (IL)-6 in daily life. We also examine the possibility that glucocorticoid sensitivity and patterns of daily cortisol may contribute to observed associations. Finally, we explore whether biological sex moderates relationships between childhood trauma and IL-6. Participants were 283 healthy adults aged 40-64 (57% female, 23% Black, Indigenous, and People of Color) who completed the Childhood Trauma Questionnaire and self-collected dried blood spots at home on 4 days to measure IL-6. Measures of salivary cortisol and blood-based glucocorticoid sensitivity were also assessed. Childhood trauma was not associated with IL-6 in the sample as a whole. However, exploratory analyses showed that childhood trauma related to IL-6 differently for males and females, such that total trauma and emotional neglect predicted higher IL-6 for males but not females. Results persisted after adjustment for covariates. There was no evidence for indirect effects via cortisol or glucocorticoid sensitivity. Childhood trauma and, specifically, emotional neglect were associated with IL-6 in daily life among middle-aged males. Additional research is needed to elucidate biological and behavioral pathways underlying these associations. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Central role of the mTORC1 pathway in glucocorticoid activity against B-ALL cells

AbstractGlucocorticoids (GCs), such as dexamethasone and prednisone, are crucial components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapies. However, the molecular basis of GC-induced cell death remains elusive. Here, we show that GC suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling and that, conversely, oncogenic activation of mTORC1 confers resistance to GCs. Our genome-wide CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) dropout screens reveal that depletion of components of either the gap activity toward Rags 1 or tuberous sclerosis complexes, both negative regulators of mTORC1 signaling, significantly attenuates B-ALL cell sensitivity to dexamethasone. Dexamethasone primarily induces B-ALL cell death by downregulating mTORC1 activity, thus promoting autophagy and impairing protein synthesis. Dexamethasone treatment failed to suppress mTORC1 activity in B-ALL cells expressing mutant GC receptors lacking DNA-binding capacity, suggesting that dexamethasone transcriptionally represses mTORC1 activity. RNA-sequencing analysis identified multiple dexamethasone target genes that negatively regulate mTORC1 activity. Our findings suggest that GC sensitivity is significantly influenced by oncogenic stimuli and/or growth factors that activate the PI3K-AKT-mTORC1 pathway. This is consistent with the frequent GC resistance found in Ph and Ph-like ALLs.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GC), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-offunction experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GC-resistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GC against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

The Glucocorticoid Receptor: Isoforms, Functions, and Contribution to Glucocorticoid Sensitivity.

Glucocorticoids exert pleiotropic effects on all tissues to regulate cellular and metabolic homeostasis. Synthetic forms are used therapeutically in a wide range of conditions for their anti-inflammatory benefits, at the cost of dose and duration-dependent side effects. Significant variability occurs between tissues, disease states, and individuals with regard to both the beneficial and deleterious effects. The glucocorticoid receptor (GR) is the site of action for these hormones and a vast body of work has been conducted understanding its function. Traditionally, it was thought that the anti-inflammatory benefits of glucocorticoids were mediated by transrepression of pro-inflammatory transcription factors, while the adverse metabolic effects resulted from direct transactivation. This canonical understanding of the GR function has been brought into question over the past 2 decades with advances in the resolution of scientific techniques, and the discovery of multiple isoforms of the receptor present in most tissues. Here we review the structure and function of the GR, the nature of the receptor isoforms, and the contribution of the receptor to glucocorticoid sensitivity, or resistance in health and disease.

Implementation of a minimally invasive cell culture system to measure the regulation of inflammation in a school-based sample of adolescents.

Dysregulated inflammation underlies many human diseases, and measures of responsiveness to activation, and sensitivity to inhibition, provide important information beyond baseline assessments of chronic inflammation. This study implements a simplified cell culture protocol in a school-based setting, using finger stick capillary blood collected from 333 adolescents (age 11.4-15.6 years) incubated with lipopolysaccharide (LPS). Median cytokine responses for IL6, IL1β, and TNFα were 61.9, 26.2, and 11.2 pg/mL, respectively. Samples were also incubated with LPS and glucocorticoid (GC) to measure GC sensitivity. Median responses were reduced in the presence of GC inhibition for IL6 (20.3 pg/mL), IL1β (10.5 pg/mL), and TNFα (3.3 pg/mL). Minimally invasive cell culture protocols provide novel opportunities for measuring inflammatory phenotypes in a wide range of non-clinical settings.

Dysregulation of Glucocorticoid Receptor Homeostasis and Glucocorticoid-Associated Genes in Umbilical Cord Endothelial Cells of Diet-Induced Obese Pregnant Sheep.

Maternal obesity (MO) is associated with offspring cardiometabolic diseases that are hypothesized to be partly mediated by glucocorticoids. Therefore, we aimed to study fetal endothelial glucocorticoid sensitivity in an ovine model of MO. Rambouillet/Columbia ewes were fed either 100% (control) or 150% (MO) National Research Council recommendations from 60 d before mating until near-term (135 days gestation). Sheep umbilical vein and artery endothelial cells (ShUVECs and ShUAECs) were used to study glucocorticoid receptor (GR) expression and function in vitro. Dexamethasone dose-response studies of gene expression, activation of a glucocorticoid response element (GRE)-dependent luciferase reporter vector, and cytosolic/nuclear GR translocation were used to assess GR homeostasis. MO significantly increased basal GR protein levels in both ShUVECs and ShUAECs. Increased GR protein levels did not result in increased dexamethasone sensitivity in the regulation of key endothelial gene expression such as endothelial nitric oxide synthase, plasminogen activator inhibitor 1, vascular endothelial growth factor, or intercellular adhesion molecule 1. In ShUVECs, MO increased GRE-dependent transactivation and FKBP prolyl isomerase 5 (FKBP5) expression. ShUAECs showed generalized glucocorticoid resistance in both dietary groups. Finally, we found that ShUVECs were less sensitive to dexamethasone-induced activation of GR than human umbilical vein endothelial cells (HUVECs). These findings suggest that MO-mediated effects in the offspring endothelium could be further mediated by dysregulation of GR homeostasis in humans as compared with sheep.

Circulating cell-free mitochondrial DNA levels and glucocorticoid sensitivity in a cohort of male veterans with and without combat-related PTSD

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a biomarker of cellular injury or cellular stress and is a potential novel biomarker of psychological stress and of various brain, somatic, and psychiatric disorders. No studies have yet analyzed ccf-mtDNA levels in post-traumatic stress disorder (PTSD), despite evidence of mitochondrial dysfunction in this condition. In the current study, we compared plasma ccf-mtDNA levels in combat trauma-exposed male veterans with PTSD (n = 111) with those who did not develop PTSD (n = 121) and also investigated the relationship between ccf mt-DNA levels and glucocorticoid sensitivity. In unadjusted analyses, ccf-mtDNA levels did not differ significantly between the PTSD and non-PTSD groups (t = 1.312, p = 0.191, Cohen’s d = 0.172). In a sensitivity analysis excluding participants with diabetes and those using antidepressant medication and controlling for age, the PTSD group had lower ccf-mtDNA levels than did the non-PTSD group (F(1, 179) = 5.971, p = 0.016, partial η2 = 0.033). Across the entire sample, ccf-mtDNA levels were negatively correlated with post-dexamethasone adrenocorticotropic hormone (ACTH) decline (r = −0.171, p = 0.020) and cortisol decline (r = −0.149, p = 0.034) (viz., greater ACTH and cortisol suppression was associated with lower ccf-mtDNA levels) both with and without controlling for age, antidepressant status and diabetes status. Ccf-mtDNA levels were also significantly positively associated with IC50-DEX (the concentration of dexamethasone at which 50% of lysozyme activity is inhibited), a measure of lymphocyte glucocorticoid sensitivity, after controlling for age, antidepressant status, and diabetes status (β = 0.142, p = 0.038), suggesting that increased lymphocyte glucocorticoid sensitivity is associated with lower ccf-mtDNA levels. Although no overall group differences were found in unadjusted analyses, excluding subjects with diabetes and those taking antidepressants, which may affect ccf-mtDNA levels, as well as controlling for age, revealed decreased ccf-mtDNA levels in PTSD. In both adjusted and unadjusted analyses, low ccf-mtDNA levels were associated with relatively increased glucocorticoid sensitivity, often reported in PTSD, suggesting a link between mitochondrial and glucocorticoid-related abnormalities in PTSD.

Integrating Proteomics and Functional Genomics to Identify Targets for Improving Glucocorticoid-Based Treatment of B-Lymphoblastic Leukemia

Glucocorticoids are a critical component of successful combination chemotherapy regimens for B-lymphoblastic leukemia (B-ALL). Despite advances in immunotherapies, glucocorticoid-based chemotherapy continues to be the essential frontline treatment of patients with B-ALL. This approach has led to high cure rates but at the risk of both acute, potentially life-threatening complications and chronic toxicities. Additionally, glucocorticoids remain ineffective for some patients with B-ALL, which predicts poor outcomes. To achieve better outcomes and reduce the toxicity of treatment, we take a functional genomics approach to understand how glucocorticoids induce cell death and identify strategies to make them more effective. Glucocorticoids work through the glucocorticoid receptor (GR), a ligand-activated transcription factor, to induce cell death in B-ALL. By integrating gene expression and RNA interference screening, we have identified both effector genes (genes whose regulation by glucocorticoids contributes to B-ALL cell death) and sources of resistance that can be targeted to improve treatment (Kruth et al Blood 2017). This identified the network of BCL-2 family proteins (e.g., BCL2, BCL2L11, BMF), whose regulation contributes to apoptosis in B-ALL. Surprisingly, it also showed that suppression of B-cell development genes (e.g., EBF1, PAX5, IL7R) contributes to B-ALL cell death. However, only a fraction of the genes that affect the glucocorticoid sensitivity of B-ALL are effector genes. We therefore sought to identify functional interaction partners of GR, defined as proteins that physically interact with GR and influence glucocorticoid sensitivity. To identify proteins that physically interact with GR, we used proximity labeling. We fused an ascorbate peroxidase domain (APEX2) to the N-terminus of GR and expressed it REH cells, a B-ALL cell line that does not express GR protein. After verifying that APEX2-GR restored glucocorticoid sensitivity in the REH cells, we performed proximity labeling in the presence and absence of dexamethasone. After optimizing these reactions, cells were lysed and labeled proteins were isolated using magnetic streptavidin beads and identified by mass spectrometry. We were able to detect more than 4,000 proteins that interact with GR, including hundreds that are enriched upon addition of dexamethasone. To identify functional interaction, we intersected the proteins enriched upon addition of dexamethasone with proteins shown by us to modulate glucocorticoid sensitivity in B-ALL (Kruth et al Blood 2017). Supporting our working model, we found functional interaction partners that are involved in B-cell development and apoptosis. We found that GR interacts with EBF1 and MEF2C, B-cell development transcription factors that restrain glucocorticoid-induced cell death. This indicates that GR activity and glucocorticoid sensitivity is directly blunted by B-cell development, and further supports the model that glucocorticoids restrain B-cell development. The mechanism of how these interactions influence glucocorticoid sensitivity will guide rational targeting to improve treatment of B-ALL both in general and in different B-ALL subtypes. Additionally, the mitochondrial voltage dependent anion channel 2 (VDAC2) also interacts with GR in a dexamethasone-dependent manner and is integral to glucocorticoid-induced cell death. VDAC2 is important for nucleating and perhaps triggering pore formation through BAK and BAX. How GR influences this activity is not known but suggests a non-genomic role of GR in glucocorticoid-induced cell death. In summary, integration of functional genomics and proteomics provides rich data sets that will help understand the mechanism of glucocorticoid-induced B-ALL cell death. Targeting of non-canonical (B-cell development) and non-genomic (VDAC2) functions of GR will allow for optimization of glucocorticoid-based chemotherapy regimens.

The Tetraspanin CD9 Shapes Glucocorticoid Sensitivity in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Background: De novo or acquired resistance to glucocorticoids (GCs), the common therapeutic regimens for remission-induction in pediatric B-precursor acute lymphoblastic leukemia, confronts treatment success and is a strong predictor for disease relapse. Discovery of predictive markers and underlying mechanisms of GC resistance could lead to new treatment and prognostic modalities. Aims: This study aims: (1) to demonstrate the association between CD9 expression and GC sensitivity in childhood BCP-ALL; (2) to dissect the underlying mechanisms driving GC resistance; and (3) to discover the possible therapeutic modalities for restoring GC susceptibility. Methods: Ex vivo drug sensitivity profiling to prednisone (Pred) and dexamethasone (Dex) was performed on 6 BCP-ALL cell lines and 18 patient samples with differential CD9 expression. Association of CD9 expression with patient responses to Pred prophase was analyzed in a pediatric BCP-ALL cohort (n=182). Functional impact of CD9 on GC susceptibility was dissected by gain- and loss-of-function experiments in BCP-ALL cells. Mechanistic basis of CD9-driven GC response was investigated through genomics, transcriptomics, and proteomics. Reversal of GC resistance by pathway-targeted agents were explored on BCP-ALL cells and xenograft models. Results: Distinctive differences in the drug sensitivity pattern were found between CD9- and CD9+ BCP-ALL cell lines, with the former showing resistance to GCs but not other chemotherapeutic agents. Concurrently, CD9- primary lymphoblasts exhibited resistance to Dex (IC50s: 2,761 vs. 31 nM) and Pred (IC50s: 8,185 vs. 268 nM) compared with CD9+ samples (positivity defined by the presence of ≥ 20% CD9+ blasts). More Pred poor responders (defined by the presence of ≥ 1×10 9/L circulating lymphoblasts on day 8) were found in CD9- than CD9+ patients (19.4% vs. 6.2%, P=0.018). Subgroup analyses revealed more poor responders with CD9- phenotype were enriched in patients with older age (60% vs. 3.7%, P=0.008), male gender (21.7% vs. 6.7%, P=0.045), higher white blood cell count (66.7% vs. 15.4%, P=0.004), and in those with BCR-ABL1 translocation (100% vs. 12.5%, P=0.024), or not otherwise cytogenetically specified BCP-ALL (30.8% vs. 5.8%, P=0.019). Univariate and multivariate analyses further confirmed CD9 negativity as an independent predictor for GC resistance (OR=5.1, P=0.009). Overexpression of CD9 in CD9- cells (SEM, KOPN-8) by lentiviral transduction significantly enhanced GC sensitivity by 2.8-62.3-fold ( P&amp;lt;0.024), whereas CD9 knockout in CD9+ cells (697) using CRISPR-Cas9 decreased GC susceptibility ( P&amp;lt;0.003). No significant differences in mRNA or protein expression of the GC receptor NR3C1 were identified between CD9+ and CD9- cells, as well as its isoforms or mutants. The cytoplasm-to-nucleus translocation of GC receptor was consistent among cell lines regardless of CD9 expression status, but CD9 was physically interacted with NR3C1. Upon GC stimulation, the presence of CD9 increased the number of DEGs (110 vs. 82 DEGs) and magnitude of GC-responsive genes exclusively detected in the background of CD9 (1.3-18.6-fold; e.g. AKAP13, STAT5A, EZR). CD9- cells showed constitutive activation of the MAPK pathway and could be preferentially restored for GC susceptibility by the MEK1/2 inhibitor trametinib in vitro. BCP-ALL xenograft modeling revealed that, compared to single agent Dex treatment, combination with trametinib significantly reduced the leukemic load in animal transplanted with CD9- SEM but not CD9+ BV-173 cell lines. Ex vivo drug testing further showed that the resistance to GCs in some CD9+ cases could be partially reversed by combination of trametinib and the JAK inhibitor ruxolitinib due to the activation of STAT5. Conclusions: This study provides a definitive linkage of CD9 with GC sensitivity in childhood BCP-ALL mediated through a NR3C1-independent mechanism. CD9- cases could be prone to trametinib combination therapy, whereas CD9+ cases might need a third drug such as ruxolitinib to counteract GC resistance.

Association analysis of BclI with benign lymphoepithelial lesions of the lacrimal gland and glucocorticoids resistance.

To evaluate the relationship between gene polymorphism (BclI, ER22/23EK, N363S) and the occurrence, progression and sensitivity to glucocorticoid of lacrimal gland benign lymphoepithelial lesion (LGBLEL). Clinical peripheral blood samples of 52 LGBLEL patients and 10 normal volunteers were collected for DNA extraction and polymerase chain reaction sequencing to analyze single nucleotide polymorphism (SNP) genotypes. The lacrimal tissues of LGBLEL were surgically removed and made into paraffin sections for subsequent hematoxylin-eosin (HE) and Masson staining analysis. The duration of disease and hormone use of LGBLEL patients from diagnosis to surgery were also analyzed. The Meta-analysis follows PRISMA guidelines to conducted a systematic review of human studies investigating the relationship between the NR3C1 BclI polymorphism and glucocorticoids (GCs) sensitivity. There was no association between ER22/23EK or N363S and the occurrence of LGBLEL or GCs sensitivity (P>0.05); BclI GC genotype was closely related to GCs resistance (P=0.03) as is the minor allele C (P=0.0017). The HE staining and Masson staining showed that the GC genotype of BclI remarkably slowed down the disease progression and reduced fibrosis (P<0.05), especially for GCs-dependent patients (P<0.0001). Meta-analysis showed that BclI was not significantly associated with GCs responsiveness. The LGBLEL patients who carry the NR3C1 BclI allele C may be more sensitive to GCs and associated with lower fibrosis and slower disease progression. The results may guide the clinical treatment strategy for the LGBLEL patients.

The Transcription Factors NFATc1 and NFATc2 Control Glucocorticoid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia

Background: The overall survival of pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is significantly improved (˜75-80%) with the current therapeutic approaches. Nevertheless, for those patients that do not respond to conventional treatment and experience relapse, the prognosis is extremely poor. In this context, it is well defined that resistance to glucocorticoids (GCs), pillar drugs in the treatment protocol, can predispose pediatric T-ALL patients to a poor outcome. A pivotal role of LCK kinase has been elucidated in supporting GC resistance in T-ALL cells in our previous study and recently confirmed by other colleagues. However, to date, the biological processes modulated by LCK in this context are not yet defined. Aim: the identification of new mechanisms underlying GC resistance can lead to alternative therapeutic approaches to prevent or overcome GC resistance and ameliorate the outcome of this subgroup of patients. Methods and Results: we uncovered the involvement of NFATc1 and NFATc2 transcription factors, that act downstream LCK kinase, in guiding GC resistance in T-ALL cells. Of note, a high NFATc1 and NFATc2 transcriptional activity characterizes pediatric GC resistant T-ALL patients at diagnosis, that in turns show a low Glucocorticoid Receptor (GR) activity. In agreement, NFATc1 or NFATc2 specific gene silencing in 3 T-ALL GC resistant cell line models and primary cells increases dexamethasone response, by restoring GR canonical transcriptional activity through the increaseexpression of BIM GR targetgene (p value &amp;lt;0.05). Conversely, NFATc1 or NFATc2 overexpression in a murine T-ALL GC sensitive cell line confers resistance to dexamethasone treatment. Interestingly, by Gene Expression Profile (GEP) and Nuclear Magnetic Resonance (NMR) analysis, we observed that NFATc1 gene silencing in GC resistant cells significantly downregulates intracellular cholesterol abundance. Additionally, by Chromatin Immunoprecipitation (ChIP) we revealed that NFATc1 can directly control the transcription of HMGCS1, EBP and DHCR7, key enzymes of cholesterol biosynthesis process (n≥3, p value &amp;lt;0.05 for all the three genes). In agreement, exogenous cholesterol addition to NFATc1 knock-down cell lines rebuild dexamethasone resistance. Conversely, by GEP and flow cytometry analysis we observed that NFATc2 gene silencing in T-ALL GC resistant cells leads to a downregulation of the Wnt/β-catenin signaling pathway and to an increased T-cell differentiation. Furthermore, by ChIP analysis we revealed that in T-ALL GC resistant cells NFATc2 can directly affect the transcription of LRP6, a key Wnt/β-catenin signaling component (n=3, p value &amp;lt;0.05). In agreement, the Wnt/β-catenin signaling activation, by Wnt3a stimulation, restores dexamethasone resistance in NFATc2 knock-down T-ALL cells. Finally, we observed that the inhibition of cholesterol biosynthesis by simvastatin or of Wnt/β-catenin by PRI-724 increases GC sensitivity in T-ALL GC resistant cells by the High-Throughput drug synergism Screening (HTS) and the Highest Single Agent (HSA) approach. Conclusions: Overall, we revealed for the first time the involvement of NFATc1 and NFATc2 transcription factors in supporting GC resistance in T-ALL cells by the modulation of cholesterol biosynthesis and Wnt/β-catenin signaling, both processes well-described in sustaining chemotherapy resistance, paving the rationale to alternative therapeutic options for T-ALL GC resistant pediatric patients.

MUC1 attenuates neutrophilic airway inflammation in asthma by reducing NLRP3 inflammasome-mediated pyroptosis through the inhibition of the TLR4/MyD88/NF-κB pathway

BackgroundNeutrophilic airway inflammation is a challenge in asthma management and is associated with poor patient prognosis. Mucin 1 (MUC1), which contains a cytoplasmic tail (MUC1-CT), has been found to mediate glucocorticoid sensitivity in asthma; however, its role in modulating neutrophilic airway inflammation in asthma remains unknown.MethodsHuman-induced sputum cells were collected from healthy participants (n = 12), patients with mild-to-moderate asthma (n = 34), and those with severe asthma (n = 18). In vitro human lung bronchial 1 epithelial cell line (BEAS-2B) was transfected with small interfering RNA against MUC1 (MUC1-siRNA) and then stimulated by lipopolysaccharide (LPS), where some cells were pretreated with a TLR4 inhibitor (TAK-242). In vivo mouse model of asthmatic neutrophil airway inflammation was induced by ovalbumin (OVA)/LPS. Some groups were intraperitoneally injected with MUC1-CT inhibitor (GO-203) and/or TAK-242 .ResultsThe mRNA expression of MUC1 was downregulated in the induced sputum of patients with asthma and correlated with asthmatic neutrophilic airway inflammation. The mRNA expressions of TLR4, MyD88, nucleotide-binding oligomerization domain-like pyrin domain-containing protein 3 (NLRP3), caspase-1, interleukin (IL)-18, and IL-1β in induced sputum cells of patients with asthma were upregulated and related to the mRNA expression of MUC1. LPS activated the TLR4 pathway and NLRP3-mediated pyroptosis in BEAS-2B cells in vitro, which were significantly aggravated after MUC1-siRNA transfection. Furthermore, MUCl-CT interacted with TLR4, and the interaction between TLR4 and MyD88 was significantly increased after MUCl-siRNA transfection. Moreover, TAK-242 ameliorated TLR4/MyD88/nuclear factor kappa B (NF-κB) pathway activation, NLRP3 inflammasome-mediated pyroptosis, and neutrophilic inflammation exacerbated by MUC1 downregulation. GO-203 exacerbated TLR4/MyD88/NF-κB pathway activation in vivo, and NLRP3 inflammasome-mediated pyroptosis reduced in a mouse model of asthmatic neutrophil airway inflammation induced by OVA/LPS; these pathological changes were partially alleviated after TAK-242 application.ConclusionThis study revealed that MUC1 downregulation plays an important role in asthmatic neutrophilic airway inflammation. MUC1-CT reduces NLRP3 inflammasome-mediated pyroptosis by inhibiting the activation of the TLR4/MyD88/NF-κB pathway, thereby attenuating neutrophil airway inflammation in patients with asthma.

Preclinical Evidence for the Glucocorticoid-Sparing Potential of a Dual Toll-Like Receptor 7/8 Inhibitor in Autoimmune Diseases.

Toll-like receptor 7 (TLR7) and TLR8 are single-stranded RNA-sensing endosomal pattern recognition receptors that evolved to defend against viral infections. However, aberrant TLR7/8 activation by endogenous ligands has been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus. TLR activation and type I interferon (IFN) were shown recently to impart resistance to glucocorticoids (GC), which are part of the standard of care for multiple autoimmune diseases. While GCs are effective, a plethora of undesirable effects limit their use. New treatment approaches that allow for the use of lower and safer doses of GCs would be highly beneficial. Herein, we report that a dual TLR7/8 inhibitor (TLR7/8i) increases the effectiveness of GCs in inflammatory settings. Human peripheral blood mononuclear cell studies revealed increased GC sensitivity in the presence of TLR7/8i for reducing inflammatory cytokine production, a synergistic effect that was most pronounced in myeloid cells, particularly monocytes. Gene expression analysis by NanoString and single-cell RNA sequencing revealed that myeloid cells were substantially impacted by combining low-dose TLR7/8i and GC, as evidenced by the effects on nuclear factor-kappa B-regulated cytokines and GC-response genes, although IFNs were affected to a smaller degree. Low dose of TLR7/8i plus GC was more efficacious then either agent alone in the MRL/lpr mouse model of lupus, with improved proteinuria and survival. Overall, our findings indicate a GC-sparing potential for TLR7/8i compounds, suggesting TLR7/8i may offer a new strategy for the treatment of autoimmune diseases. SIGNIFICANCE STATEMENT: Some features of autoimmune diseases may be resistant to glucocorticoids, mediated at least in part by toll-like receptor (TLR) activation, necessitating higher doses that are associated with considerable toxicities. We demonstrate that TLR7/8 inhibition and glucocorticoids work synergistically to reduce inflammation in a cell-type specific manner and suppress disease in a mouse model of lupus. TLR7/8 inhibition is a promising strategy for the treatment of autoimmune diseases and has glucocorticoid-sparing potential.

Genetic glucocorticoid receptor variants differ between ethnic groups but do not explain variation in age of diabetes onset, metabolic and inflammation parameters in patients with type 2 diabetes.

The effect of excess glucocorticoid receptor (GR) stimulation through glucocorticoid medication or cortisol on glucose metabolism is well established. There are genetic GR variants that result in increased or decreased GR stimulation. We aimed to determine the prevalence of genetic GR variants in different ethnic groups in a cohort of patients with type 2 diabetes, and we aimed to determine their association with age of diabetes onset and metabolic and inflammation parameters. A cross-sectional analysis was performed in a multiethnic cohort (n = 602) of patients with established type 2 diabetes. Polymorphisms in the GR gene that have previously been associated with altered glucocorticoid sensitivity (TthIIII, ER22/23EK N363S, BclI and 9β) were determined and combined into 6 haplotypes. Associations with age of diabetes onset, HbA1c, hs-CRP and lipid values were evaluated in multivariate regression models. The prevalence of the SNPs of N363S and BclI was higher in Dutch than in non-Dutch patients. We observed a lower prevalence of the SNP 9β in Dutch, South(East) Asian and Black African patients versus Turkish and Moroccan patients. We did not detect an association between SNPs and diabetes age of onset or metabolic parameters. We only found a trend for lower age of onset and higher HbA1c in patients with 1 or 2 copies of haplotype 3 (TthIIII + 9β). The prevalence of genetic GR variants differs between patients of different ethnic origins. We did not find a clear association between genetic GR variants and age of diabetes onset or metabolic and inflammation parameters. This indicates that the clinical relevance of GR variants in patients with established type 2 diabetes is limited.

Non-coding RNA-mediated epigenetic alterations in Grave's ophthalmopathy: A scoping systematic review.

It is becoming more and more apparent that Grave's Ophthalmopathy (GO) pathogenesis may be aided by epigenetic processes such as DNA methylation modifications, histone tail covalent modifications, and non-coding RNA (ncRNA)-based epigenetic processes. In the present study, we aimed to focus more on the miRNAs rather than lncRNAs due to lack of investigations on these non-coding RNAs and their role in GO's pathogenesis. A six-stage methodology framework and the PRISMA recommendation were used to conduct this scoping review. A comprehensive search was conducted across seven databases to discover relevant papers published until February 2022. The data extraction separately, and quantitative and qualitative analyses were conducted. A total of 20 articles were found to meet inclusion criteria. According to the results, ncRNA were involved in the regulation of inflammation (miR-146a, LPAL2/miR-1287-5p axis, LINC01820:13/hsa miR-27b-3p axis, and ENST00000499452/hsa-miR-27a-3p axis), regulation of T cell functions (miR-146a/miR-183/miR-96), regulation of glycosaminoglycan aggregation and fibrosis (miR-146a/miR-21), glucocorticoid sensitivity (miR-224-5p), lipid accumulation and adipogenesis (miR-27a/miR-27b/miR-130a), oxidative stress and angiogenesis (miR-199a), and orbital fibroblast proliferation (miR-21/miR-146a/miR-155). Eleven miRNAs (miR-146a/miR-224-5p/miR-Let7d-5p/miR-96-5p/miR-301a-3p/miR-21-5p) were also indicated to have the capacity to be used as biomarkers. Regardless of the fact that there is significant documentation of ncRNA-mediated epigenetic dysfunction in GO, additional study is needed to thoroughly comprehend the epigenetic connections concerned in disease pathogenesis, paving the way for novel diagnostic and prognostic tools for epigenetic therapies among the patients.

Circulating miR-146a predicts glucocorticoid response in thyroid eye disease.

Thyroid eye disease (TED) is an immune-mediated disorder of the eye. Intravenous glucocorticoid (GC) is the first-line treatment for patients with active moderate-to-severe TED. However, the response rate is between 50% and 80%. There are still no simple and reliable markers of responsiveness to GC therapy. We aimed to explore the possible role of miR-146a and miR-21 as predictors of responsiveness to GC treatment in TED. We carried out a prospective longitudinal study on 30 consecutive adult patients with active moderate-to-severe TED and eligible for GC therapy. All patients received the standard GC treatment with methylprednisolone i.v. In cases of progressive worsening of Gorman Score for diplopia or with duction restriction <30° in at least two consecutive controls, patients also underwent orbital radiotherapy. Response to GC treatment was defined as a decrease of two or more points in the clinical activity score (CAS) or CAS <4/10 at 24 weeks. Circulating miRNAs were extracted from patients' serum and quantified by real-time PCR. Twenty-three (77%) patients responded to GC. Thyroid surgery, higher CAS, greater proptosis and higher pre-treatment circulating levels of miR-146a emerged as predictive factors of responsiveness to GC. A ROC analysis revealed that miR-146a could predict responsiveness to GC with a positive predictive value of 100%. This is the first study investigating the role of pre-treatment circulating miR-21 and miR-146a to predict responsiveness to GC in TED. miR-146a emerged as a simple, objective, new marker of GC sensitivity that could be used to avoid ineffective administration of GC therapy to TED patients.