Innately activated TLR4 signal in the nucleus accumbens is sustained by CRF amplification loop and regulates impulsivity.

Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABAA receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.

Valproic acid induces neuronal cell death through a novel calpain-dependent necroptosis pathway.

A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase-related and -unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain-dependent necroptosis pathway that initiates with downstream activation of c-Jun N-terminal kinase 1 (JNK1) and increased expression of receptor-interacting protein 1 (RIP-1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis-inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis-inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis-associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3-K/Akt survival pathways, allowing them to resist VPA-induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain-dependent necroptosis pathway that includes JNK1 activation and RIP-1 expression. A growing body of evidence indicates that valproic acid (VPA) has neurotoxic activity, the mechanism of which is still poorly understood. We report, for the first time, that VPA activates a previously unrecognized calpain-dependent necroptosis cascade that initiates with JNK1 activation and involves AIF cleavage/nuclear translocation and H2AX phosphorylation as well as an altered Smac/DIABLO to XIAP balance.

Emerging evidence for endogenous neurosteroid modulation of pro-inflammatory and anti-inflammatory pathways that impact neuropsychiatric disease.

This mini-review presents emerging evidence that endogenous neurosteroids modulate both pro- and anti-inflammatory signaling by immune cells and brain cells that contribute to depression, alcohol use disorders, and other inflammatory conditions. We first review the literature on pregnenolone and allopregnanolone inhibition of proinflammatory neuroimmune pathways in the periphery and the brain - effects that are independent of GABAergic mechanisms. We follow with evidence for neurosteroid enhancement of anti-inflammatory and protective pathways in brain and immune cells. These studies draw clinical relevance from a large body of evidence that pro-inflammatory immune signaling is dysregulated in many brain disorders and the fact that neurosteroids inhibit the same inflammatory pathways that are activated in depression, alcohol use disorders and other inflammatory conditions. Thus, we describe evidence that neurosteroid levels are decreased and neurosteroid supplementation has therapeutic efficacy in these neuropsychiatric conditions. We conclude with a perspective that endogenous regulation of immune balance between pro- and anti-inflammatory pathways by neurosteroid signaling is essential to prevent the onset of disease. Deficits in neurosteroids may unleash excessive pro-inflammatory activation which progresses in a feed-forward manner to disrupt brain networks that regulate stress, emotion and motivation. Neurosteroids can block various inflammatory pathways in mouse and human macrophages, rat brain and human blood and therefore provide new hope for treatment of intractable conditions that involve excessive inflammatory signaling.

Novel neurosteroid therapeutics for post-partum depression: perspectives on clinical trials, program development, active research, and future directions.

This article reviews novel neurosteroid therapeutics for post-partum depression, with a focus on their development, clinical trial data, current practices, and future directions in this exciting field. We discuss the clinical impact of brexanolone and several other neurosteroids, particularly as they relate to the treatment of postpartum depression (PPD) and major depressive disorders outside of the perinatal period. There has been increasing interest in GABA signaling and modulation as it pertains to the development of altered circuity and depressive states. This scientific underpinning served as the rationale for the initial development of brexanolone. We review the clinical trials supporting its Food and Drug Administration (FDA) approval as the first rapidly acting antidepressant specific for PPD, and the subsequent development of a clinical brexanolone program at an academic medical center, highlighting new research and data from that site as well as the challenges with the delivery of this I.V. drug. In addition to the GABA signaling hypothesis, we discuss the new evidence demonstrating that brexanolone inhibits inflammatory signaling post-infusion, suggesting that inflammatory signaling may contribute to the etiology of PPD. Finally, we describe new and future directions in neurosteroid therapeutics, including the development of an oral agent, zuranolone, and the IV and oral formulations of ganaxolone. Ultimately, the hope is that these novel neurosteroid therapeutics will provide fast-acting treatment for these impairing disorders and improve our understanding of the underlying mechanisms of depressive disorders.

Neurosteroid [3α,5α]3-hydroxypregnan-20-one inhibition of chemokine monocyte chemoattractant protein-1 in alcohol-preferring rat brain neurons, microglia, and astroglia.

BACKGROUND: Neuroimmune dysfunction in alcohol use disorder (AUD) is associated with activation of myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptors (TLR) resulting in overexpression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 overexpression in the brain is linked to anxiety, higher alcohol intake, neuronal death, and activation of microglia observed in AUD. The neurosteroid [3α,5α][3-hydroxypregnan-20-one (3α,5α-THP) has been reported as an inhibitor of MyD88-dependent TLR activation and MCP-1 overexpression in mouse and human macrophages and the brain of alcohol-preferring (P) rats. METHODS: We investigated how 3α,5α-THP regulates MCP-1 expression at the cellular level in P rat nucleus accumbens (NAc) and central amygdala (CeA). We focused on neurons, microglia, and astrocytes, examining the individual voxel density of MCP-1, neuronal marker NeuN, microglial marker IBA1, astrocytic marker GFAP, and their shared voxel density, defined as intersection. Ethanol-naïve male and female P rats were perfused 1 h after IP injections of 15 mg/kg of 3α,5α-THP, or vehicle. The NAc and CeA were imaged using confocal microscopy following double-immunofluorescence staining for MCP-1 with NeuN, IBA1, and GFAP, respectively. RESULTS: MCP-1 intersected with NeuN predominantly and IBA1/GFAP negligibly. 3α,5α-THP reduced MCP-1 expression in NeuN-labeled cells by 38.27 ± 28.09% in male and 56.11 ± 21.46% in female NAc, also 37.99 ± 19.53% in male and 54.96 ± 30.58% in female CeA. In females, 3α,5α-THP reduced the MCP-1 within IBA1 and GFAP-labeled voxels in the NAc and CeA. Conversely, in males, 3α,5α-THP did not significantly alter the MCP-1 within IBA1 in NAc or with GFAP in the CeA. Furthermore, 3α,5α-THP decreased levels of IBA1 in both regions and sexes with no impact on GFAP or NeuN levels. Secondary analysis performed on data normalized to % control values indicated that no significant sex differences were present. CONCLUSIONS: These data suggest that 3α,5α-THP inhibits neuronal MCP-1 expression and decreases the proliferation of microglia in P rats. These results increase our understanding of potential mechanisms for 3α,5α-THP modulation of ethanol consumption.

Neurosteroid [3α,5α]-3-Hydroxy-pregnan-20-one Enhances the CX3CL1-CX3CR1 Pathway in the Brain of Alcohol-Preferring Rats with Sex-Specificity.

This study investigates the impact of allopregnanolone ([3α,5α]3-hydroxypregnan-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP); 10 mg/kg, IP) on fractalkine/CX3-C motif chemokine ligand 1 (CX3CL1) levels, associated signaling components, and markers for microglial and astrocytic cells in the nucleus accumbens (NAc) of male and female alcohol-preferring (P) rats. Previous research suggested that 3α,5α-THP enhances anti-inflammatory interleukin-10 (IL-10) cytokine production in the brains of male P rats, with no similar effect observed in females. This study reveals that 3α,5α-THP elevates CX3CL1 levels by 16% in the NAc of female P rats, with no significant changes observed in males. The increase in CX3CL1 levels induced by 3α,5α-THP was observed in females across multiple brain regions, including the NAc, amygdala, hypothalamus, and midbrain, while no significant effect was noted in males. Additionally, female P rats treated with 3α,5α-THP exhibited notable increases in CX3CL1 receptor (CX3CR1; 48%) and transforming growth factor-beta 1 (TGF-ß1; 24%) levels, along with heightened activation (phosphorylation) of signal transducer and activator of transcription 1 (STAT1; 85%) in the NAc. Conversely, no similar alterations were observed in male P rats. Furthermore, 3α,5α-THP decreased glial fibrillary acidic protein (GFAP) levels by 19% in both female and male P rat NAc, without affecting microglial markers ionized calcium-binding adaptor molecule 1 (IBA1) and transmembrane protein 119 (TMEM119). These findings indicate that 3α,5α-THP enhances the CX3CL1/CX3CR1 pathway in the female P rat brain but not in males, primarily influencing astrocyte reactivity, with no observed effect on microglial activation.

Neuroactive Steroids, Toll-like Receptors, and Neuroimmune Regulation: Insights into Their Impact on Neuropsychiatric Disorders.

Pregnane neuroactive steroids, notably allopregnanolone and pregnenolone, exhibit efficacy in mitigating inflammatory signals triggered by toll-like receptor (TLR) activation, thus attenuating the production of inflammatory factors. Clinical studies highlight their therapeutic potential, particularly in conditions like postpartum depression (PPD), where the FDA-approved compound brexanolone, an intravenous formulation of allopregnanolone, effectively suppresses TLR-mediated inflammatory pathways, predicting symptom improvement. Additionally, pregnane neurosteroids exhibit trophic and anti-inflammatory properties, stimulating the production of vital trophic proteins and anti-inflammatory factors. Androstane neuroactive steroids, including estrogens and androgens, along with dehydroepiandrosterone (DHEA), display diverse effects on TLR expression and activation. Notably, androstenediol (ADIOL), an androstane neurosteroid, emerges as a potent anti-inflammatory agent, promising for therapeutic interventions. The dysregulation of immune responses via TLR signaling alongside reduced levels of endogenous neurosteroids significantly contributes to symptom severity across various neuropsychiatric disorders. Neuroactive steroids, such as allopregnanolone, demonstrate efficacy in alleviating symptoms of various neuropsychiatric disorders and modulating neuroimmune responses, offering potential intervention avenues. This review emphasizes the significant therapeutic potential of neuroactive steroids in modulating TLR signaling pathways, particularly in addressing inflammatory processes associated with neuropsychiatric disorders. It advances our understanding of the complex interplay between neuroactive steroids and immune responses, paving the way for personalized treatment strategies tailored to individual needs and providing insights for future research aimed at unraveling the intricacies of neuropsychiatric disorders.

Sleep Deprivation in Middle Age May Increase Dementia Risk: A Review.

Neurodegenerative diseases present increasing interest in clinical practice for the aging population and involve dysregulation of sleep-wake behaviors. Approximately 5.8 million adults aged 65 and older were living with Alzheimer's disease (AD) in the United States in 2020 with increased mortality compared to the declining cardiovascular and cancer death rates. We conducted an extensive literature review to evaluate and synthesize evidence regarding the association between short sleep duration or sleep deprivation and the risk of developing all-cause dementia and Alzheimer's disease. There are multiple mechanisms describing brain damage, such as brain hypoxia, oxidative stress, or blood-brain barrier (BBB) impairment, induced by chronic sleep restriction (CSR) and the potential correlation with future cognitive decline and dementia. More studies are necessary to identify the specific factors involved in the sleep loss-cognitive decline association that could be taken into consideration while elaborating recommendations for dementia prevention measures.

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Background: Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. Methods: The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. Results: 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. Conclusion: The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Brexanolone therapeutics in post-partum depression involves inhibition of systemic inflammatory pathways.

BACKGROUND: Brexanolone has rapid, long-lasting, and remarkable efficacy in the treatment of post-partum depression (PPD). We test the hypothesis that brexanolone inhibits proinflammatory modulators and macrophage activation in PPD patients, which may promote clinical recovery. METHODS: PPD patients (N = 18) provided blood samples before and after brexanolone infusion according to the FDA-approved protocol. Patients were unresponsive to prior treatment before brexanolone therapy. Serum was collected to determine neurosteroid levels and whole blood cell lysates were examined for inflammatory markers and in vitro responses to the inflammatory activators lipopolysaccharide (LPS) and imiquimod (IMQ). FINDINGS: Brexanolone infusion altered multiple neuroactive steroid levels (N = 15-18), reduced levels of inflammatory mediators (N = 11) and inhibited their response to inflammatory immune activators (N = 9-11). Specifically, brexanolone infusion reduced whole blood cell tumor necrosis factor-α (TNF-α, p = 0.003), and interleukin-6 (IL-6, p = 0.04) and these effects were correlated with HAM-D score improvement (TNF-α, p = 0.049; IL-6, p = 0.02). Furthermore, brexanolone infusion prevented LPS and IMQ-induced elevation of TNF-α (LPS: p = 0.02; IMQ: p = 0.01), IL-1ß (LPS: p = 0.006; IMQ: p = 0.02) and IL-6 (LPS: p = 0.009; IMQ: p = 0.01), indicating inhibition of toll-like receptor (TLR)4 and TLR7 responses. Finally, inhibition of TNF-α, IL-1ß and IL-6 responses to both LPS and IMQ were correlated with HAM-D score improvements (p < 0.05). INTERPRETATION: Brexanolone actions involve inhibition of inflammatory mediator production and inhibition of inflammatory responses to TLR4 and TLR7 activators. The data suggest that inflammation plays a role in post-partum depression and that inhibition of inflammatory pathways contributes to the therapeutic efficacy of brexanolone. FUNDING: The Foundation of Hope, Raleigh, NC and UNC School of Medicine, Chapel Hill.

Outcomes of COVID-19 amongst patients with ongoing use of inhaled corticosteroids - a systematic review & meta-analysis.

Background: WHO quoted the numbers for the Coronavirus disease 2019 (COVID-19) pandemic as of August 2021 were 200 million cases with over 4 million deaths globally. COVID-19 is associated with several respiratory pathologies. Inhaled corticosteroids (ICS) are used to improve lung function by reducing inflammation, edema, mucus secretion, and inhibiting various cytokine activities. However, there is limited data on the effect of ICS usage in patients with COVID-19. In this study, we aim to evaluate the association between the use of ICS and the outcomes in COVID-19 patients compared to standard COVID-19 treatment. Methods: We followed PRISMA guidelines and MOOSE protocol for conducting the systematic review and meta-analysis comparing ICS and standard COVID-19 therapy. A search on PubMed is conducted yielding 270 articles of which 6 manuscripts are finalized for inclusion in the study. Patients with COVID-19 are identified from the studies based on confirmed positive RT-PCR tests. Hospitalization, ICU admission, and mortality are selected as the outcomes of our study. Using RevMan 5.3, we performed random-effects models to estimate the pooled effect size (pooled odds ratio), 95% confidence interval (95% CI), and heterogeneity (I2). Forest plots are obtained and p <0.05 is considered statistically significant. Results: Our study involves the comparison of ICS vs Non-ICS for mortality (N= 207,842 vs 166,217), ICU hospitalization (N= 1,084 vs 9,425), and the risk of hospitalization (N= 1,273 vs 1,676).Of the six studies, five reported mortality. We found a higher mortality rate in patients with asthma (60.88%, 107/160) and chronic obstructive pulmonary disease (COPD) (68.46%, 382/558) among ICS users. The overall mortality is 7.49% (107/1428). We found that ICS use was associated with higher odds of mortality (OR=1.45 95%CI: 1.10-1.91; p=0.009, I2= 68%) amongst COVID-19 patients. In subgroup analysis, higher odds of mortality among COPD patients using ICS was noted [pooled OR: 1.52 (1.24-1.86); p<0.0001; I2=0%]. However, no significant association between ICS and mortality was observed among asthma patients. Conclusion: ICS is associated with increased mortality and risk for hospitalization in patients with COVID-19 as compared to standard non-steroid-based COVID-19 therapy. It is crucial for healthcare providers to carefully evaluate the potential risks and benefits of ICS usage in the context of COVID-19 management to optimize patient outcomes and safety.

Prevalence of active and passive smoking among asthma and asthma-associated emergency admissions: a nationwide prevalence survey study.

Asthma affects 7% of children and 8% of adults in the United States. There is a paucity of studies examining the association between passive smoking and an increased risk of asthma exacerbations that led the authors to examine the association between various modes of smoking and rates of asthma exacerbations. A retrospective cross-sectional/case-control study was conducted using the National Health and Nutrition Examination Survey dataset (2013-2018). Out of 312,979 respondents, 35,758 (11.43%) had a history of asthma, 9083 (2.9%) had asthma attacks in the past year, and 4731 (1.51%) had asthma-related emergency room admissions in the past year. Prevalence of asthma-related emergency admissions were higher among active cigarette smoking (46.25 vs 35.46%), e-cigarette smoking (26.63 vs 16.07%), and passive smoking at home (37.53 vs 25.67%), workplace passive smoking (14.35 vs 12.11%), in bar (32.38 vs 26.16%), and car (26.21 vs 14.44%) (p < 0.0001). In multivariate regression analysis, we found regular cigarette smoking (OR 1.13, 95% confidence interval (CI) 1.009-1.260, p = 0.0252), e-cigarette (OR 2.13, 95% CI 1.92-2.36, p = 0.0043), cigar use (OR 1.21, 95% CI 1.1-1.33, p < 0.001), ultra-long cigarette length (OR 4.85, 95% CI 3.33-7.06, p < 0.0001), and passive smoking (OR 5.25, 95% CI 3.43-8.06, p < 0.0001) were associated with increased rates of asthma exacerbations over last 12 months. The study shows increased odds of asthma exacerbations among those using ultra-long cigarettes, e-cigarettes, and cigars. Consequently, passive inhalation from even a single smoker in the home, workplace, bars and cars is associated with worsening outcomes in asthma patients.

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Inhibition of human macrophage activation via pregnane neurosteroid interactions with toll-like receptors: Sex differences and structural requirements.

We recently discovered that (3α,5α)3-hydroxypregnan-20-one (allopregnanolone) inhibits pro-inflammatory toll-like receptor (TLR) activation and cytokine/chemokine production in mouse macrophage RAW264.7 cells. The present studies evaluate neurosteroid actions upon TLR activation in human macrophages from male and female healthy donors. Buffy coat leukocytes were obtained from donors at the New York Blood Center (http://nybloodcenter.org/), and peripheral blood mononuclear cells were isolated and cultured to achieve macrophage differentiation. TLR4 and TLR7 were activated by lipopolysaccharide (LPS) or imiquimod in the presence/absence of allopregnanolone or related neurosteroids and pro-inflammatory markers were detected by ELISA or western blotting. Cultured human monocyte-derived-macrophages exhibited typical morphology, a mixed immune profile of both inflammatory and anti-inflammatory markers, with no sex difference at baseline. Allopregnanolone inhibited TLR4 activation in male and female donors, preventing LPS-induced elevations of TNF-α, MCP-1, pCREB and pSTAT1. In contrast, 3α,5α-THDOC and SGE-516 inhibited the TLR4 pathway activation in female, but not male donors. Allopregnanolone completely inhibited TLR7 activation by imiquimod, blocking IL-1-ß, IL-6, pSTAT1 and pIRF7 elevations in females only. 3α,5α-THDOC and SGE-516 partially inhibited TLR7 activation, only in female donors. The results indicate that allopregnanolone inhibits TLR4 and TLR7 activation in cultured human macrophages resulting in diminished cytokine/chemokine production. Allopregnanolone inhibition of TLR4 activation was found in males and females, but inhibition of TLR7 signals exhibited specificity for female donors. 3α,5α-THDOC and SGE-516 inhibited TLR4 and TLR7 pathways only in females. These studies demonstrate anti-inflammatory effects of allopregnanolone in human macrophages for the first time and suggest that inhibition of pro-inflammatory cytokines/chemokines may contribute to its therapeutic actions.

Post-COVID-19 Pulmonary Fibrosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions worldwide with a high mortality rate due to a lack of definitive treatment. Despite having a wide range of clinical features, acute respiratory distress syndrome (ARDS) has emerged as the primary cause of mortality in these patients. Risk factors and comorbidities like advanced age with limited lung function, pre-existing diabetes, hypertension, cardiovascular diseases, and obesity have increased the risk for severe COVID-19 infection. Rise in inflammatory markers like transforming growth factor ß (TGF-ß), interleukin-6 (IL-6), and expression of matrix metalloproteinase 1 and 7 (MMP-1, MMP-7), along with collagen deposition at the site of lung injury, results in extensive lung scarring and fibrosis. Anti-fibrotic drugs, such as Pirfenidone and Nintedanib, have emerged as potential treatment options for post-COVID-19 pulmonary fibrosis. A lung transplant might be the only life-saving treatment. Despite the current advances in the management of COVID-19, there is still a considerable knowledge gap in the management of long-term sequelae in such patients, especially concerning pulmonary fibrosis. Follow up on the current clinical trials and research to test the efficacy of various anti-inflammatory drugs is needed to prevent long-term sequelae early mortality in these patients.

Cardiac Sarcoidosis Diagnostic Challenges and Management: A Case Report and Literature Review.

Sarcoidosis can be presented as cardiac sarcoidosis (CS), which is challenging to diagnose due to its clinical silence. Ventricular arrhythmias and atrioventricular blocks can be fatal and cause sudden death in patients with cardiac sarcoidosis. Five percent of sarcoidosis patients have clinically evident cardiac sarcoidosis. However, autopsy reports and imaging studies have shown a higher prevalence of cardiac involvement. Early recognition is important to prevent such detrimental consequences. Cardiac sarcoidosis is increasingly being diagnosed owing to increased awareness among physicians and new diagnostic tools like MRI and positron emission tomography (PET) scan replacing traditional endomyocardial biopsy. A definitive diagnosis of CS remains challenging due to the non-specific clinical findings that can present similar symptoms of common cardiac disease; therefore, the imaging and biopsies are substantial for diagnosis confirmation. Pharmacological and Implantable devices are two main therapeutic approaches in cardiac sarcoidosis, in which steroids and pacemaker therapy have shown better outcomes. This review summarizes the available data related to the prevalence, prognosis, diagnosis, and management of cardiac sarcoidosis.

Mitochondrial dysfunction and nicotinamide dinucleotide catabolism as mechanisms of cell death and promising targets for neuroprotection.

Both acute and chronic neurodegenerative diseases are frequently associated with mitochondrial dysfunction as an essential component of mechanisms leading to brain damage. Although loss of mitochondrial functions resulting from prolonged activation of the mitochondrial permeability transition (MPT) pore has been shown to play a significant role in perturbation of cellular bioenergetics and in cell death, the detailed mechanisms are still elusive. Enzymatic reactions linked to glycolysis, the tricarboxylic acid cycle, and mitochondrial respiration are dependent on the reduced or oxidized form of nicotinamide dinucleotide [NAD(H)] as a cofactor. Loss of mitochondrial NAD(+) resulting from MPT pore opening, although transient, allows detrimental depletion of mitochondrial and cellular NAD(+) pools by activated NAD(+) glycohydrolases. Poly(ADP-ribose) polymerase (PARP) is considered to be a major NAD(+) degrading enzyme, particularly under conditions of extensive DNA damage. We propose that CD38, a main cellular NAD(+) level regulator, can significantly contribute to NAD(+) catabolism. We discuss NAD(+) catabolic and NAD(+) synthesis pathways and their role in different strategies to prevent cellular NAD(+) degradation in brain, particularly following an ischemic insult. These therapeutic approaches are based on utilizing endogenous intermediates of NAD(+) metabolism that feed into the NAD(+) salvage pathway and also inhibit CD38 activity.

Overview of Sodium-Glucose Co-transporter 2 (SGLT2) Inhibitors for the Treatment of Non-diabetic Heart Failure Patients.

Heart failure (HF) is a clinical syndrome that results from any structural or functional impairment of ventricular filling or ejection of blood, and results in low life quality and expectancy, creating a significant burden on the healthcare system. The pharmacological HF management has remained unchanged for a decade, however, several randomized clinical trials have demonstrated the potential clinical benefits of sodium-glucose cotransporter-2 inhibitors, an antidiabetic agent, by reducing the rate of hospitalizations for HF, cardiovascular death, and all-cause death. The cardioprotective effects are characterized by reduction of inflammatory, metabolic and ionic dyshomeostasis despite the diabetic status. Since the United States Food and Drug Administration (FDA) approval in May 2020, SGLT2 inhibitors have been used mostly in heart failure with reduced ejection fraction (HFrEF). In this review article, we provide a comprehensive overview of the potential benefits, effectiveness, and safety profile of SGLT2 inhibitors used in HF patients with no history of diabetes mellitus.

Neurosteroid allopregnanolone (3α,5α-THP) inhibits inflammatory signals induced by activated MyD88-dependent toll-like receptors.

We have shown that endogenous neurosteroids, including pregnenolone and 3α,5α-THP inhibit toll-like receptor 4 (TLR4) signal activation in mouse macrophages and the brain of alcohol-preferring (P) rat, which exhibits innate TLR4 signal activation. The current studies were designed to examine whether other activated TLR signals are similarly inhibited by 3α,5α-THP. We report that 3α,5α-THP inhibits selective agonist-mediated activation of TLR2 and TLR7, but not TLR3 signaling in the RAW246.7 macrophage cell line. The TLR4 and TLR7 signals are innately activated in the amygdala and NAc from P rat brains and inhibited by 3α,5α-THP. The TLR2 and TLR3 signals are not activated in P rat brain and they are not affected by 3α,5α-THP. Co-immunoprecipitation studies indicate that 3α,5α-THP inhibits the binding of MyD88 with TLR4 or TLR7 in P rat brain, but the levels of TLR4 co-precipitating with TRIF are not altered by 3α,5α-THP treatment. Collectively, the data indicate that 3α,5α-THP inhibits MyD88- but not TRIF-dependent TLR signal activation and the production of pro-inflammatory mediators through its ability to block TLR-MyD88 binding. These results have applicability to many conditions involving pro-inflammatory TLR activation of cytokines, chemokines, and interferons and support the use of 3α,5α-THP as a therapeutic for inflammatory disease.

(3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) regulation of hypothalamic and extrahypothalamic corticotropin releasing factor (CRF): Sexual dimorphism and brain region specificity in Sprague Dawley rats.

CRF is the main activator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF neurons are found mainly in the hypothalamus, but CRF positive cells and CRF1 receptors are also found in extrahypothalamic structures, including amygdala (CeA), hippocampus, NAc and VTA. CRF release in the hypothalamus is regulated by inhibitory GABAergic interneurons and extrahypothalamic glutamatergic inputs, and disruption of this balance is found in stress-related disorders and addiction. (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP), the most potent positive modulator of GABAA receptors, attenuates the stress response reducing hypothalamic CRF mRNA expression and ACTH and corticosterone serum levels. In this study, we explored 3α,5α-THP regulation of hypothalamic and extrahypothalamic CRF mRNA and peptide expression, in male and female Sprague Dawley rats, following vehicle or 3α,5α-THP administration (15 mg/kg). In the hypothalamus, we found sex differences in CRF mRNA expression (females +74%, p < 0.01) and CRF peptide levels (females -71%, p < 0.001). 3α,5α-THP administration reduced hypothalamic CRF mRNA expression only in males (-50%, p < 0.05) and did not alter CRF peptide expression in either sex. In hippocampus and CeA, 3α,5α-THP administration reduced CRF peptide concentrations only in the male (hippocampus -29%, p < 0.05; CeA -62%, p < 0.01). In contrast, 3α,5α-THP injection increased CRF peptide concentration in the VTA of both males (+32%, p < 0.01) and females (+26%, p < 0.01). The results show sex and region-specific regulation of CRF signals and the response to 3α,5α-THP administration. This data may be key to successful development of therapeutic approaches for stress-related disorders and addiction.