Pulmonary symptoms associated with heavy alcohol consumption among people living with HIV: an analysis of the NHANES 1999-2010.

AIM: This matched case-control study aimed to provide epidemiologic evidence of increased burden of respiratory symptoms and pulmonary function decline among people living with human immunodeficiency virus (HIV) and a history of heavy alcohol consumption. METHODS: Cases were participants with HIV (PWH; n = 75, 33%), and controls were participants without HIV (PWoH; n = 150, 67%). PWH were matched to PWoH by age and sex in the ratio of 1:2. Eligible participants responded to the respiratory health National Health and Nutrition Examination Survey questionnaire [prolonged coughs (≥3 months), bringing up of phlegm (≥3 months), and a history of wheezing or whistling in the chest (past year)]. The effects of both alcohol and HIV on participants' pulmonary function were determined using linear regression analysis. RESULTS: History of heavy alcohol consumption was more prevalent among PWH (40%) compared to PWoH (27%). PWH who had a history of heavy alcohol consumption had a higher prevalence of coughing most days (45% vs. 4%, P = .0010), bringing up phlegm most days (31% vs. 0%, P = .0012), and wheezing or whistling in the chest (40% vs. 20%, P = .058) compared to participants who did not heavily consume alcohol. Furthermore, a history of heavy alcohol consumption was associated with decreased forced expiratory volume (ml) in 1 s/forced vital capacity among PWH (ß = - 0.098 95% C.I. -0.16, -0.04, P = .03) after adjusting for having smoked at least 100 cigarettes in life. CONCLUSION: A history of heavy alcohol use increased respiratory symptoms and suppressed pulmonary function among people living with HIV. This study provides epidemiological evidence of the respiratory symptom burden of people living with HIV who have a history of heavy alcohol consumption.

Pioglitazone Reverses Alcohol-Induced Alveolar Macrophage Phagocytic Dysfunction.

Alcohol use disorders (AUD) increase susceptibility to respiratory infections by 2- to 4-fold in part because of impaired alveolar macrophage (AM) immune function. Alcohol causes AM oxidative stress, diminishing AM phagocytic capacity and clearance of microbes from the alveolar space. Alcohol increases AM NADPH oxidases (Noxes), primary sources of AM oxidative stress, and reduces peroxisome proliferator-activated receptor γ (PPARγ) expression, a critical regulator of AM immune function. To investigate the underlying mechanisms of these alcohol-induced AM derangements, we hypothesized that alcohol stimulates CCAAT/enhancer-binding protein ß (C/EBPß) to suppress Nox-related microRNAs (miRs), thereby enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. Furthermore, we postulated that pharmacologic PPARγ activation with pioglitazone would inhibit C/EBPß and attenuate alcohol-induced AM dysfunction. AM isolated from human AUD subjects or otherwise healthy control subjects were examined. Compared with control AM, alcohol activated AM C/EBPß, decreased Nox1-related miR-1264 and Nox2-related miR-107, and increased Nox1, Nox2, and Nox4 expression and activity. These alcohol-induced AM derangements were abrogated by inhibition of C/EBPß, overexpression of miR-1264 or miR-107, or pioglitazone treatment. These findings define novel molecular mechanisms of alcohol-induced AM dysfunction mediated by C/EBPß and Nox-related miRs that are amenable to therapeutic targeting with PPARγ ligands. These results demonstrate that PPARγ ligands provide a novel and rapidly translatable strategy to mitigate susceptibility to respiratory infections and related morbidity in individuals with AUD.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol.

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-ß (TGF-ß) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-ß, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-ß in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-ß was measured. Decreased sIgA and increased TGF-ß were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-ß release in both HBECs and mouse tracheal epithelial cells, but not in CD204 knockout mice. SPD-MAA stimulated TGF-ß in alveolar macrophage cells. These data show that MAA-adducted surfactant protein stimulates lung epithelial cell TGF-ß, down-regulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.

Glial Cell Line-Derived Neurotrophic Factor Enhances Autophagic Flux in Mouse and Rat Hepatocytes and Protects Against Palmitate Lipotoxicity.

Glial cell line-derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid ß-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid ß-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid-induced hepatic lipotoxicity.

TGF-ß1 Suppresses the Type I IFN Response and Induces Mitochondrial Dysfunction in Alveolar Macrophages.

TGF-ß1 is a pleiotropic cytokine with an established role in fibrosis; however, the immunosuppressive effects of TGF-ß1 are less characterized. Elevated levels of TGF-ß1 are found in patients with acute and chronic lung diseases, and the underlying disease processes are exacerbated by respiratory viral infections. The alveolar macrophage is the first line of cellular defense against respiratory viral infections, and its response to infections is dependent on environmental cues. Using the mouse alveolar macrophage line, MH-S, and human CD14+ monocyte-derived macrophages, we examined the effects of TGF-ß1 on the type I IFN antiviral response, macrophage polarization, and mitochondrial bioenergetics following a challenge with human respiratory syncytial virus (RSV). Our results showed that TGF-ß1 treatment of macrophages decreased the antiviral and proinflammatory response, and suppressed basal, maximal, spare mitochondrial respiration, and mitochondrial ATP production. Challenge with RSV following TGF-ß1 treatment further exacerbated mitochondrial dysfunction. The TGF-ß1 and TGF-ß1+RSV-treated macrophages had a higher frequency of apoptosis and diminished phagocytic capacity, potentially through mitochondrial stress. Disruption of TGF-ß1 signaling or rescue of mitochondrial respiration may be novel therapeutically targetable pathways to improve macrophage function and prevent secondary bacterial infections that complicate viral respiratory infections.

PPARγ Regulates Mitochondrial Structure and Function and Human Pulmonary Artery Smooth Muscle Cell Proliferation.

Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPARγ expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPARγ or PGC1α perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPARγ antagonist GW9662 or with siRNA against PPARγ or PGC1α for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT-PCR), target protein levels (Western blotting), mitochondria-derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPARγ or PGC1α increased HPASMC proliferation, enhanced mitochondria-derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPARγ diminishes PGC1α and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1α reversed hypoxia-induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPARγ as a novel therapeutic strategy in PH.

Peroxisome Proliferator-Activated Receptor γ Regulates Chronic Alcohol-Induced Alveolar Macrophage Dysfunction.

Peroxisome proliferator-activated receptor (PPAR) γ is critical for alveolar macrophage (AM) function. Chronic alcohol abuse causes AM phagocytic dysfunction and susceptibility to respiratory infections by stimulating nicotinamide adenine dinucleotide oxidases (Nox), transforming growth factor-ß1, and oxidative stress in the AM. Because PPARγ inhibits Nox expression, we hypothesized that alcohol reduces PPARγ, stimulating AM dysfunction. AMs were examined from: (1) patients with alcoholism or control patients; (2) a mouse model of chronic ethanol consumption; (3) PPARγ knockout mice; or (4) MH-S cells exposed to ethanol in vitro. Alcohol reduced AM PPARγ levels and increased Nox1, -2, and -4, transforming growth factor-ß1, oxidative stress, and phagocytic dysfunction. Genetic loss of PPARγ recapitulated, whereas stimulating PPARγ activity attenuated alcohol-mediated alterations in gene expression and phagocytic function, supporting the importance of PPARγ in alcohol-induced AM derangements. Similarly, PPARγ activation in vivo reduced alcohol-mediated impairments in lung bacterial clearance. Alcohol increased levels of microRNA-130a/-301a, which bind to the PPARγ 3' untranslated region to reduce PPARγ expression. MicroRNA-130a/-301a inhibition attenuated alcohol-mediated PPARγ reductions and derangements in AM gene expression and function. Alcohol-induced Toll-like receptor 4 endocytosis was reversed by PPARγ activation. These findings demonstrate that targeting PPARγ provides a novel therapeutic approach for mitigating alcohol-induced AM derangements and susceptibility to lung infection.

Peroxisome Proliferator-Activated Receptor γ and microRNA 98 in Hypoxia-Induced Endothelin-1 Signaling.

Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo. In PAECs isolated from patients with pulmonary arterial hypertension, microRNA (miR)-98 expression was reduced, and ET-1 protein levels and proliferation were increased. Similarly, hypoxia reduced miR-98 and increased ET-1 levels and PAEC proliferation in vitro. In vivo, hypoxia reduced miR-98 expression and increased ET-1 and proliferating cell nuclear antigen (PCNA) levels in mouse lung, derangements that were aggravated by treatment with the vascular endothelial growth factor receptor antagonist Sugen5416. Reporter assays confirmed that miR-98 binds directly to the ET-1 3'-untranslated region. Compared with littermate control mice, miR-98 levels were reduced and ET-1 and PCNA expression were increased in lungs from endothelial-targeted PPARγ knockout mice, whereas miR-98 levels were increased and ET-1 and PCNA expression was reduced in lungs from endothelial-targeted PPARγ-overexpression mice. Gain or loss of PPARγ function in PAECs in vitro confirmed that alterations in PPARγ were sufficient to regulate miR-98, ET-1, and PCNA expression. Finally, PPARγ activation with rosiglitazone regimens that attenuated hypoxia-induced PH in vivo and human PAEC proliferation in vitro restored miR-98 levels. The results of this study show that PPARγ regulates miR-98 to modulate ET-1 expression and PAEC proliferation. These results further clarify molecular mechanisms by which PPARγ participates in PH pathogenesis and therapy.

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases.

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 µM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.

Alcoholism causes alveolar macrophage zinc deficiency and immune dysfunction.

RATIONALE: Alcohol use disorders cause oxidative stress in the lower airways and increase susceptibility to pneumonia and lung injury. Currently, no therapeutic options exist to mitigate the pulmonary consequences of alcoholism. OBJECTIVES: We recently determined in an animal model that alcohol ingestion impairs pulmonary zinc metabolism and causes alveolar macrophage immune dysfunction. The objective of this research is to determine the effects of alcoholism on zinc bioavailability and alveolar macrophage function in human subjects. METHODS: We recruited otherwise healthy alcoholics (n = 17) and matched control subjects (n = 17) who underwent bronchoscopy for isolation of alveolar macrophages, which were analyzed for intracellular zinc, phagocytic function, and surface expression of granulocyte-macrophage colony-stimulating factor receptor; all three of these indices are decreased in experimental models. MEASUREMENTS AND MAIN RESULTS: Alcoholic subjects had normal serum zinc, but significantly decreased alveolar macrophage intracellular zinc levels (adjusted means [SE], 718 [41] vs. 948 [25] RFU/cell; P < 0.0001); bacterial phagocytosis (adjusted means [SE], 1,027 [48] vs. 1,509 [76] RFU/cell; P < 0.0001); and expression of granulocyte-macrophage colony-stimulating factor receptor ß subunit (adjusted means [SE], 1,471 [42] vs. 2,114 [35] RFU/cell; P < 0.0001]. Treating alveolar macrophages with zinc acetate and glutathione in vitro increased intracellular zinc levels and improved their phagocytic function. CONCLUSIONS: These novel clinical findings provide evidence that alcohol abuse is associated with significant zinc deficiency and immune dysfunction within the alveolar space and suggest that dietary supplementation with zinc and glutathione precursors could enhance airway innate immunity and decrease the risk for pneumonia or lung injury in these vulnerable individuals.

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.

HERPUD1 governs tumor cell mitochondrial function via inositol 1,4,5-trisphosphate receptor-mediated calcium signaling.

The intricate relationship between calcium (Ca2+) homeostasis and mitochondrial function is crucial for cellular metabolic adaptation in tumor cells. Ca2+-initiated signaling maintains mitochondrial respiratory capacity and ATP synthesis, influencing critical cellular processes in cancer development. Previous studies by our group have shown that the homocysteine-inducible ER Protein with Ubiquitin-Like Domain 1 (HERPUD1) regulates inositol 1,4,5-trisphosphate receptor (ITPR3) levels and intracellular Ca2+ signals in tumor cells. This study explores the role of HERPUD1 in regulating mitochondrial function and tumor cell migration by controlling ITPR3-dependent Ca2+ signals. We found HERPUD1 levels correlated with mitochondrial function in tumor cells, with HERPUD1 deficiency leading to enhanced mitochondrial activity. HERPUD1 knockdown increased intracellular Ca2+ release and mitochondrial Ca2+ influx, which was prevented using the ITPR3 antagonist xestospongin C or the Ca2+ chelator BAPTA-AM. Furthermore, HERPUD1 expression reduced tumor cell migration by controlling ITPR3-mediated Ca2+ signals. HERPUD1-deficient cells exhibited increased migratory capacity, which was attenuated by treatment with xestospongin C or BAPTA-AM. Additionally, HERPUD1 deficiency led to reactive oxygen species-dependent activation of paxillin and FAK proteins, which are associated with enhanced cell migration. Our findings highlight the pivotal role of HERPUD1 in regulating mitochondrial function and cell migration by controlling intracellular Ca2+ signals mediated by ITPR3. Understanding the interplay between HERPUD1 and mitochondrial Ca2+ regulation provides insights into potential therapeutic targets for cancer treatment and other pathologies involving altered energy metabolism.

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.

Paired ATAC- and RNA-seq offer insight into the impact of HIV on alveolar macrophages: a pilot study.

People with HIV remain at greater risk for both infectious and non-infectious pulmonary diseases even after antiretroviral therapy initiation and CD4 cell count recovery. These clinical risks reflect persistent HIV-mediated defects in innate and adaptive immunity, including in the alveolar macrophage, a key innate immune effector in the lungs. In this proof-of-concept pilot study, we leveraged paired RNA-seq and ATAC-seq analyses of human alveolar macrophages obtained with research bronchoscopy from people with and without HIV to highlight the potential for recent methodologic advances to generate novel hypotheses about biological pathways that may contribute to impaired pulmonary immune function in people with HIV. In addition to 35 genes that were differentially expressed in macrophages from people with HIV, gene set enrichment analysis identified six gene sets that were differentially regulated. ATAC-seq analysis revealed 115 genes that were differentially accessible for people with HIV. Data-driven integration of the findings from these complementary, high-throughput techniques using xMWAS identified distinct clusters involving lipoprotein lipase and inflammatory pathways. By bringing together transcriptional and epigenetic data, this analytic approach points to several mechanisms, including previously unreported pathways, that warrant further exploration as potential mediators of the increased risk of pulmonary disease in people with HIV.

Pharmacological reversal of post-transcriptional alterations implicated in alcohol-induced alveolar macrophage dysfunction.

Alcohol use disorders (AUD) cause alveolar macrophage (AM) immune dysfunction and increase risk of lung infections. Excessive alcohol use causes AM oxidative stress, which impairs AM phagocytosis and pathogen clearance from the alveolar space. Alcohol induces expression of NADPH oxidases (Noxes), primary sources of oxidative stress in AM. In contrast, alcohol decreases AM peroxisome proliferator-activated receptor gamma (PPARγ), a critical regulator of AM immune function. To explore the underlying molecular mechanisms for these effects of alcohol, we hypothesized that ethanol promotes CCAAT/enhancer-binding protein beta (C/EBPß)-mediated suppression of Nox-related microRNAs (miRs), in turn enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. We also hypothesized that PPARγ activation with pioglitazone (PIO) would reverse alcohol-induced C/EBPß expression and attenuate AM oxidative stress and phagocytic dysfunction. Cells from the mouse AM cell line (MH-S) were exposed to ethanol in vitro or primary AM were isolated from mice fed ethanol in vivo. Ethanol enhanced C/EBPß expression, decreased Nox 1-related miR-1264 and Nox 2-related miR-107 levels, and increased Nox1, Nox2, and Nox 4 expression in MH-S cells in vitro and mouse AM in vivo. These alcohol-induced AM derangements were abrogated by loss of C/EBPß, overexpression of miRs-1264 or -107, or PIO treatment. These findings identify C/EBPß and Nox-related miRs as novel therapeutic targets for PPARγ ligands, which could provide a translatable strategy to mitigate susceptibility to lung infections in people with a history of AUD. These studies further clarify the molecular underpinnings for a previous clinical trial using short-term PIO treatment to improve AM immunity in AUD individuals.

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.