The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism.

Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex - a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype. Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.

Emerging concepts in alcohol, infection & immunity: A summary of the 2023 alcohol and immunology research interest group (AIRIG) meeting.

On December 8th 2023, the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The 2023 meeting focused broadly on how acute and chronic alcohol exposure leads to immune dysregulation, and how this contributes to damage in multiple tissues and organs. These include impaired lung immunity, intestinal dysfunction, autoimmunity, the gut-Central Nervous System (CNS) axis, and end-organ damage. In addition, diverse areas of alcohol research covered multiple pathways behind alcohol-induced cellular dysfunction, including inflammasome activation, changes in miRNA expression, mitochondrial metabolism, gene regulation, and transcriptomics. Finally, the work presented at this meeting highlighted novel biomarkers and therapeutic interventions for patients suffering from alcohol-induced organ damage.

Pioglitazone reverses alcohol-induced alterations in alveolar macrophage mitochondrial phenotype.

BACKGROUND: People with alcohol use disorder (AUD) have an increased risk of developing pneumonia and pulmonary diseases. Alveolar macrophages (AMs) are immune cells of the lower respiratory tract that are necessary for clearance of pathogens. However, alcohol causes AM oxidative stress, mitochondrial damage and dysfunction, and diminished phagocytic capacity, leading to lung injury and immune suppression. METHODS: AMs were isolated by bronchoalveolar lavage from people with AUD and male and female C57BL/6J mice given chronic ethanol (20% w/v, 12 weeks) in drinking water. The peroxisome proliferator-activated receptor γ ligand, pioglitazone, was used to treat human AMs ex vivo (10 µM, 24 h) and mice in vivo by oral gavage (10 mg/kg/day). Levels of AM mitochondrial superoxide and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA, a marker of oxidative stress, were measured by fluorescence microscopy and RT-qPCR, respectively. Mouse AM phagocytic ability was determined by internalized Staphylococcus aureus, and mitochondrial capacity, dependency, and flexibility for glucose, long-chain fatty acid, and glutamine oxidation were measured using an extracellular flux analyzer. In vitro studies used a murine AM cell line, MH-S (±0.08% ethanol, 72 h) to investigate mitochondrial fuel oxidation and ATP-linked respiration. RESULTS: Pioglitazone treatment decreased mitochondrial superoxide in AMs from people with AUD and ethanol-fed mice and HIF-1α mRNA in ethanol-fed mouse lungs. Pioglitazone also reversed mouse AM glutamine oxidation and glucose or long-chain fatty acid flexibility to meet basal oxidation needs. In vitro, ethanol decreased the rate of AM mitochondrial and total ATP production, and pioglitazone improved changes in glucose and glutamine oxidation. CONCLUSIONS: Pioglitazone reversed chronic alcohol-induced oxidative stress in human AM and mitochondrial substrate oxidation flexibility and superoxide levels in mouse AM. Decreased ethanol-induced AM HIF-1α mRNA with pioglitazone suggests that this pathway may be a focus for metabolic-targeted therapeutics to improve morbidity and mortality in people with AUD.

Alcohol and Immunology: Mechanisms of multi-organ damage. Summary of the 2022 alcohol and Immunology research interest group (AIRIG) meeting.

On October 26th, 2022 the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite symposium at the annual meeting of the Society for Leukocyte Biology in Hawaii. The 2022 meeting focused broadly on the immunological consequences of acute, chronic, and prenatal alcohol exposure and how these contribute to damage in multiple organs and tissues. These included alcohol-induced neuroinflammation, impaired lung immunity, intestinal dysfunction, and decreased anti-microbial and anti-viral responses. In addition, research presented covered multiple pathways behind alcohol-induced cellular dysfunction, including mitochondrial metabolism, cellular bioenergetics, gene regulation, and epigenetics. Finally, the work presented highlighted potential biomarkers and novel avenues of treatment for alcohol-induced organ damage.

RGS14 is neuroprotective against seizure-induced mitochondrial oxidative stress and pathology in hippocampus.

RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro . As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.

A critical review of recent knowledge of alcohol's effects on the immunological response in different tissues.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

3'UTR shortening of HAS2 promotes hyaluronan hyper-synthesis and bioenergetic dysfunction in pulmonary hypertension.

Pulmonary hypertension (PH) comprises a diverse group of disorders that share a common pathway of pulmonary vascular remodeling leading to right ventricular failure. Development of anti-remodeling strategies is an emerging frontier in PH therapeutics that requires a greater understanding of the interactions between vascular wall cells and their extracellular matrices. The ubiquitous matrix glycan, hyaluronan (HA), is markedly elevated in lungs from patients and experimental models with PH. Herein, we identified HA synthase-2 (HAS2) in the pulmonary artery smooth muscle cell (PASMC) layer as a predominant locus of HA dysregulation. HA upregulation involves depletion of NUDT21, a master regulator of alternative polyadenylation, resulting in 3'UTR shortening and hyper-expression of HAS2. The ensuing increase of HAS2 and hyper-synthesis of HA promoted bioenergetic dysfunction of PASMC characterized by impaired mitochondrial oxidative capacity and a glycolytic shift. The resulting HA accumulation stimulated pro-remodeling phenotypes such as cell proliferation, migration, apoptosis-resistance, and stimulated pulmonary artery contractility. Transgenic mice, mimicking HAS2 hyper-synthesis in smooth muscle cells, developed spontaneous PH, whereas targeted deletion of HAS2 prevented experimental PH. Pharmacological blockade of HAS2 restored normal bioenergetics in PASMC, ameliorated cell remodeling phenotypes, and reversed experimental PH in vivo. In summary, our results uncover a novel mechanism of HA hyper-synthesis and downstream effects on pulmonary vascular cell metabolism and remodeling.

New insights into the mechanism of alcohol-mediated organ damage via its impact on immunity, metabolism, and repair pathways: A summary of the 2021 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On November 19th, 2021, the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Chicago Health Sciences Campus in Maywood, Illinois. The 2021 meeting focused on how alcohol misuse is linked to immune system derangements, leading to tissue and organ damage, and how this research can be translated into improving treatment of alcohol-related disease. This meeting was divided into three plenary sessions: the first session focused on how alcohol misuse affects different parts of the immune system, the second session presented research on mechanisms of organ damage from alcohol misuse, and the final session highlighted research on potential therapeutic targets for treating alcohol-mediated tissue damage. Diverse areas of alcohol research were covered during the meeting, from alcohol's effect on pulmonary systems and neuroinflammation to epigenetic changes, senescence markers, and microvesicle particles. These presentations yielded a thoughtful discussion on how the findings can lead to therapeutic treatments for people suffering from alcohol-related diseases.

Hyaladherins May be Implicated in Alcohol-Induced Susceptibility to Bacterial Pneumonia.

Although the epidemiology of bacterial pneumonia and excessive alcohol use is well established, the mechanisms by which alcohol induces risk of pneumonia are less clear. Patterns of alcohol misuse, termed alcohol use disorders (AUD), affect about 15 million people in the United States. Compared to otherwise healthy individuals, AUD increase the risk of respiratory infections and acute respiratory distress syndrome (ARDS) by 2-4-fold. Levels and fragmentation of hyaluronic acid (HA), an extracellular glycosaminoglycan of variable molecular weight, are increased in chronic respiratory diseases, including ARDS. HA is largely involved in immune-assisted wound repair and cell migration. Levels of fragmented, low molecular weight HA are increased during inflammation and decrease concomitant with leukocyte levels following injury. In chronic respiratory diseases, levels of fragmented HA and leukocytes remain elevated, inflammation persists, and respiratory infections are not cleared efficiently, suggesting a possible pathological mechanism for prolonged bacterial pneumonia. However, the role of HA in alcohol-induced immune dysfunction is largely unknown. This mini literature review provides insights into understanding the role of HA signaling in host immune defense following excessive alcohol use. Potential therapeutic strategies to mitigate alcohol-induced immune suppression in bacterial pneumonia and HA dysregulation are also discussed.

Alcohol-Induced Glycolytic Shift in Alveolar Macrophages Is Mediated by Hypoxia-Inducible Factor-1 Alpha.

Excessive alcohol use increases the risk of developing respiratory infections partially due to impaired alveolar macrophage (AM) phagocytic capacity. Previously, we showed that chronic ethanol (EtOH) exposure led to mitochondrial derangements and diminished oxidative phosphorylation in AM. Since oxidative phosphorylation is needed to meet the energy demands of phagocytosis, EtOH mediated decreases in oxidative phosphorylation likely contribute to impaired AM phagocytosis. Treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, pioglitazone (PIO), improved EtOH-mediated decreases in oxidative phosphorylation. In other models, hypoxia-inducible factor-1 alpha (HIF-1α) has been shown to mediate the switch from oxidative phosphorylation to glycolysis; however, the role of HIF-1α in chronic EtOH mediated derangements in AM has not been explored. We hypothesize that AM undergo a metabolic shift from oxidative phosphorylation to a glycolytic phenotype in response to chronic EtOH exposure. Further, we speculate that HIF-1α is a critical mediator of this metabolic switch. To test these hypotheses, primary mouse AM (mAM) were isolated from a mouse model of chronic EtOH consumption and a mouse AM cell line (MH-S) were exposed to EtOH in vitro. Expression of HIF-1α, glucose transporters (Glut1 and 4), and components of the glycolytic pathway (Pfkfb3 and PKM2), were measured by qRT-PCR and western blot. Lactate levels (lactate assay), cell energy phenotype (extracellular flux analyzer), glycolysis stress tests (extracellular flux analyzer), and phagocytic function (fluorescent microscopy) were conducted. EtOH exposure increased expression of HIF-1α, Glut1, Glut4, Pfkfb3, and PKM2 and shifted AM to a glycolytic phenotype. Pharmacological stabilization of HIF-1α via cobalt chloride treatment in vitro mimicked EtOH-induced AM derangements (increased glycolysis and diminished phagocytic capacity). Further, PIO treatment diminished HIF-1α levels and reversed glycolytic shift following EtOH exposure. These studies support a critical role for HIF-1α in mediating the glycolytic shift in energy metabolism of AM during excessive alcohol use.

D-serine mitigates cell loss associated with temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.