Xian Fan

Alcohol misuse is twice as prevalent among people living with HIV (PWH), and this increases the risk of pulmonary complications even in those receiving antiretroviral therapy. Our prior work showed that the alcohol metabolite, acetaldehyde, activates nuclear factor kappa B p65 (p65), leading to HIV replication and interleukin (IL)-1β activation in alveolar macrophages (AMs). Since the aforementioned processes are energy-demanding, which conversely impair mitochondrial functions, we hypothesized that acetaldehyde-induced p65 drives AMs to a mitochondrial hyperactive state to promote HIV replication and IL-1β release and induces oxidative stress and mitochondrial dysfunction. Since we found pioglitazone (PIO) to be a negative regulator of p65, we postulate that PIO suppresses HIV replication and IL-1β activation in AMs by restricting p65-induced mitochondrial hyperactivation. Murine AMs were exposed to acetaldehyde via the acetaldehyde generating system (AGS) and infected in vitro with EcoHIV, a chimeric ecotropic HIV construct. AGS + EcoHIV activated p65, resulting in enhanced ATP-linked mitochondrial respiration, proton leak, non-mitochondrial respiration and the generation of reactive oxygen species (ROS) in AMs. Inhibition of mitochondrial ATP synthesis with low-dose oligomycin attenuated AGS-induced HIV replication and AGS + EcoHIV-induced IL-1β release from AMs. PIO treatment, which attenuated AGS-induced p65 activation, suppressed proton leak, non-mitochondrial oxygen consumption, ROS, and IL-1β and p24 release. While p65-induced mitochondrial hyperactivation represents AMs’ adaptive response to the energy demands imposed by HIV replication and proinflammatory activation when exposed to acetaldehyde, PIO treatment may offer a novel therapeutic strategy to restore adequate mitochondrial bioenergetics in the AMs of PWH who misuse alcohol.

Approximately 50% of people living with Human Immunodeficiency Virus (HIV) in the United States misuse alcohol, and they are at increased risk of chronic lung inflammation despite antiretroviral therapy. Acetaldehyde, a metabolite of alcohol, circulates systemically and directly impacts alveolar macrophages (AMs), the primary reservoir of HIV in the lungs. Acetaldehyde promotes AM HIV replication and triggers IL-1β release. We explored the mechanisms by which alcohol-derived acetaldehyde drives HIV replication and IL-1β release in AMs. Furthermore, we tested if the transcription factor peroxisome proliferator-activated receptor (PPAR)γ agonist, pioglitazone, attenuates AM HIV replication and IL-1β release. Primary mouse AMs, MH-S cells (an AM cell line), and THP-1 (human monocyte cell line)-derived macrophages were treated with alcohol-derived acetaldehyde (acetaldehyde-generating system [AGS]), HIV 1ADA, and EcoHIV, a chimeric HIV that infects murine cells. HIV expression was confirmed by HIV gag RNA (qRT-PCR) and p24 release (ELISA). IL-1β was measured by qRT-PCR and ELISA. Extracellular hydrogen peroxide (H2O2) release was quantified by Amplex Red assay. Furthermore, immunoblot analysis of ERK1/2, PPARγ, and NF-ĸB/p65 (p65) was used to identify how acetaldehyde potentiates HIV replication and IL-1β activation in AMs. AGS increased H2O2, leading to ERK1/2 phosphorylation, which deactivated PPARγ. AGS drove nuclear p65 translocation in HIV-infected cells, which enhanced HIV replication and IL-1β release. Treatment with pioglitazone decreased nuclear p65, attenuating AGS-induced HIV replication and IL-1β activation in AMs. We identified mechanisms underlying acetaldehyde-induced inflammatory activation and potentiation of HIV replication in AMs, which could be therapeutically targeted with pioglitazone to decrease HIV-related respiratory comorbidities among people living with HIV who misuse alcohol.

Background: Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGF beta 1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGF beta 1 and GM-CSF and thereby alter airway epithelial barrier function. Methods: Human or rat LF were cultured +/- ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGF beta 1 receptor type 1 (TGF beta R1) inhibitor (SB431542) or a TGF beta 1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGF beta 1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. Results: AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and a-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGF beta 1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGF beta 1 activity. Conclusions: Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGF beta 1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Chronic HIV infection causes redox stress and increases the risk of acute and chronic lung injury, even when individuals are adherent to antiretroviral therapy. HIV-1 transgene expression in rats inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which regulates antioxidant defenses and alveolar epithelial cell (AEC) barrier function, but the mechanism is unknown. In this study, we present novel evidence that these pathological effects of HIV are mediated by microRNA-144 (miR-144). HIV-1 transgene expression in vivo increases the expression of miR-144 in the alveolar epithelium, and this can be replicated by direct exposure of naïve primary AECs to either Tat or gp120 ex vivo. Further, treating naïve primary AECs with a miR-144 mimic decreased the expression and activity of Nrf2 and inhibited their barrier formation. In contrast, treatment with a miR-144 antagomir increased the expression and activity of Nrf2 and improved barrier function in primary AECs isolated from HIV-1 transgenic rats. Importantly, either delivering the miR-144 antagomir intratracheally, or directly activating Nrf2 by dietary treatment with PB123, increased Nrf2 expression and barrier formation in HIV-1 transgenic rat AECs. This study provides new experimental evidence that HIV-induced inhibition of Nrf2 and consequent AEC barrier dysfunction are mediated via miR-144, and that these pathophysiological effects can be mitigated in vivo by either directly antagonizing miR-144 or activating Nrf2. Our findings suggest that targeting the inhibition of Nrf2 in individuals living with HIV could enhance their lung health and decrease the lung-specific morbidity and mortality that persists despite antiretroviral therapy.

The master transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of antioxidant and phase II-metabolizing enzymes by activating the antioxidant response element (ARE) and thereby protects cells and tissues from oxidative stress. Pulmonary complications remain the leading cause of death in human immunodeficiency virus (HIV)-1-infected individuals, who display systemic oxidative stress and glutathione deficiency that can be modeled in transgenic rats where HIV-1-related viral proteins decrease glutathione levels and cause epithelial barrier dysfunction within the alveolar space by as yet unknown mechanisms. We hypothesized that HIV-1-related proteins inhibit Nrf2-mediated antioxidant defenses and thereby disrupt the normally tight alveolar epithelial barrier. Nrf2 RNA silencing dampened Nrf2/ARE activity, decreased the expression of the tight junction proteins zonula occludens-1, occludin, and claudin-18, increased paracellular permeability of alveolar epithelial monolayers derived from wild-type rats, and therefore reproduced the effects of HIV-1 transgene expression on the epithelial barrier that we had previously described. In contrast, upregulating Nrf2 activity, either by plasmid-mediated overexpression or treatment with the Nrf2 activator sulforaphane, increased the expression of ARE-dependent antioxidants, including NAD(P)H dehydrogenase, quinone 1 and glutathione, improved the expression of tight junction proteins, and restored the ability to form tight barriers in alveolar epithelial cells from HIV-1 transgenic rats. Taken together, these new findings argue that HIV-1-related proteins downregulate Nrf2 expression and/or activity within the alveolar epithelium, which in turn impairs antioxidant defenses and barrier function, thereby rendering the lung susceptible to oxidative stress and injury. Furthermore, this study suggests that activating the Nrf2/ARE pathway with the dietary supplement sulforaphane could augment antioxidant defenses and lung health in HIV-1-infected individuals.

Background: Alcohol abuse and HIV‐1 infection frequently coexist, and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV‐1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined. Methods and Results: In this study, we determined that chronic alcohol ingestion (12 weeks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV‐1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens‐1 localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of nuclear factor‐erythroid 2‐related factor 2 (Nrf2), the transcription factor required to activate the antioxidant‐response element, was decreased in primary alveolar epithelial cells isolated from HIV‐1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV‐related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol‐fed HIV‐1 transgenic rats. Conclusions: These results provide novel evidence that HIV‐related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of coexistent alcohol abuse and HIV infection.

Background: HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results: HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion: Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF‐κB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC‐4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature‐sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5–2%, 10 cycles per min, equibiaxial) caused magnitude‐dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT‐PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC‐4 cells require activation of ERK1/2 pathway. Therefore, the CIMC‐4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment. J. Cell. Physiol. 207: 454–460, 2006.