Alcohol-induced lipid dysregulation impairs glycolytic responses to LPS in alveolar macrophages.

Several conditions are marked by increased susceptibility to, and enhanced severity of, bacterial infections. Alcohol use disorder, one of these conditions, is known to predispose to bacterial pneumonia by suppressing the lung's innate immune system, and more specifically by disrupting critical alveolar macrophage (AM) functions. Recently, we established that chronic ethanol consumption also perturbs surfactant lipid homeostasis in the lung and that elevated concentrations of free fatty acids contribute to blocking essential AM functions, such as agonist-induced cytokine expression. In this study, we extend these observations by showing that elevated free fatty acid levels impair metabolic responses to lipopolysaccharide (LPS) in AMs. In particular, we show that the glycolytic reprogramming characteristic of LPS-stimulated AMs is blunted by the saturated fatty acid palmitate, whereas oleate, an unsaturated fatty acid, or ethanol alone, had no effect on this adaptive metabolic response. Additionally, we found that elevated concentrations of palmitate induced mitochondrial oxidative stress and that glycolytic reprogramming and cytokine production to LPS could be partially restored in AMs by either pharmacologically blocking palmitate entry into mitochondria or administering a mitochondrial-specific antioxidant. Taken together, these findings suggest that alcohol and elevated levels of saturated fatty acids conspire to impair pulmonary innate immunity by altering metabolic responses in AMs. Additionally, our findings suggest that targeting the mechanisms involved in fatty acid metabolism can restore pulmonary immunity and possibly limit bacterial pneumonia in individuals with alcohol use disorder.

The involvement of GM-CSF deficiencies in parallel pathways of pulmonary alveolar proteinosis and the alcoholic lung.

Chronic alcohol consumption renders the lung more susceptible to infections by disrupting essential alveolar macrophage functions. Emerging evidence suggests that these functional deficits are due, in part, to a suppression of GM-CSF signaling, which is believed to compromise monocyte growth and maturation in the lung. However, in addition to controlling monocyte behaviors, GM-CSF also regulates surfactant homeostasis. For example, mice with targeted deletion of the gene for GM-CSF accumulate large amounts of surfactant phospholipids in their lungs. Moreover, decreased GM-CSF signaling in humans has been linked to the development of pulmonary alveolar proteinosis (PAP), a rare disorder in which surfactant lipids and proteins accumulate in alveolar macrophages and the lung exhibits enhanced susceptibility to infection. Consistent with parallel mechanisms in the PAP and alcoholic lung, we have recently reported that levels of intrapulmonary lipids, specifically triglycerides and free fatty acids, are increased in BAL fluid, whole lung digests and alveolar macrophages of chronically alcohol exposed rats. Additionally, we showed that uptake of saturated fatty acids alone could induce phenotypic and functional changes in alveolar macrophages that mimicked those in the alcohol-exposed rat and human lung. Herein, we discuss the role of GM-CSF in surfactant homeostasis and highlight the evidence that links decreased GM-CSF signaling to alveolar macrophage dysfunction in both the PAP and alcohol-exposed lung. Moreover, we discuss how lipid accumulation itself might contribute to altering alveolar macrophage function and propose how targeting these mechanisms could be employed for reducing the susceptibility to pulmonary infections in alcoholics.

Mitochondrial fusion and Bid-mediated mitochondrial apoptosis are perturbed by alcohol with distinct dependence on its metabolism.

Environmental stressors like ethanol (EtOH) commonly target mitochondria to influence the cell's fate. Recent literature supports that chronic EtOH exposure suppresses mitochondrial dynamics, central to quality control, and sensitizes mitochondrial permeability transition pore opening to promote cell death. EtOH-induced tissue injury is primarily attributed to its toxic metabolic products but alcoholism also impairs tissues that poorly metabolize EtOH. We embarked on studies to determine the respective roles of EtOH and its metabolites in mitochondrial fusion and tBid-induced mitochondrial apoptosis. We used HepG2 cells that do not metabolize EtOH and its engineered clone that expresses EtOH-metabolizing Cytochrome P450 E2 and alcohol dehydrogenase (VL-17A cells). We found that fusion impairment by prolonged EtOH exposure was prominent in VL-17A cells, probably owing to reactive oxygen species increase in the mitochondrial matrix. There was no change in fusion protein abundance, mitochondrial membrane potential or Ca2+ uptake. By contrast, prolonged EtOH exposure promoted tBid-induced outer mitochondrial membrane permeabilization and cell death only in HepG2 cells, owing to enhanced Bak oligomerization. Thus, mitochondrial fusion inhibition by EtOH is dependent on its metabolites, whereas sensitization to tBid-induced death is mediated by EtOH itself. This difference is of pathophysiological relevance because of the tissue-specific differences in EtOH metabolism.

Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity.

Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

Effect of millimeter waves and cyclophosphamide on cytokine regulation.

We have reported previously that millimeter waves (MMWs) protect T-cell functions from the toxic side effects of cyclophosphamide (CPA), an anticancer drug. Since the effect of MMWs has been reported to be mediated by endogenous opioids, the present study was undertaken to investigate the role of endogenous opioids in protection of T-cell functions by MMWs. The effect of MMWs (42.2 GHz, incident power density = 38 mW/cm²) was studied on CPA-induced suppression of cytokine release by T cells in the presence of selective opioid receptor antagonists (ORA). Production of cytokines was measured in CD4 T cells isolated from splenocytes. Treatment of mice with CPA suppressed the formation of Th1 cytokines (TNF-α, IFN-γ, and IL-2), shifting the overall balance toward Th2 (IL-4 and IL-5). MMW irradiation of CPA-treated groups up-regulated the production of Th1 cytokines suppressed by CPA. Treatment of the CPA+MMW group with selective kappa (κ) ORA further potentiated this effect of MMWs on Th1 cytokine production, whereas treatment with µ or δ ORA increased the imbalance of cytokine production in the Th2 direction. These results provide further evidence that endogenous opioids are involved in immunomodulation by MMWs.