Activating the Nrf2-mediated antioxidant response element restores barrier function in the alveolar epithelium of HIV-1 transgenic rats

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.

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Activation Of The NRF2/ARE Pathway Protects Against Oxidative Stress And Alveolar Epithelial Barrier Dysfunction In HIV-1 Transgenic Rats

10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a2805 ◽

2011 ◽

Author(s):

Xian Fan ◽

Pratibha C. Joshi ◽

Michael Koval ◽

David M. Guidot

Keyword(s):

Oxidative Stress ◽

Epithelial Barrier ◽

Barrier Dysfunction ◽

Transgenic Rats ◽

Alveolar Epithelial ◽

Epithelial Barrier Dysfunction ◽

Hiv 1

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MiR-144 mediates Nrf2 inhibition and alveolar epithelial dysfunction in HIV-1 transgenic rats

AJP Cell Physiology ◽

10.1152/ajpcell.00038.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. C390-C397 ◽

Cited By ~ 5

Author(s):

Abiodun T. Kukoyi ◽

Xian Fan ◽

Bashar S. Staitieh ◽

Brooks M. Hybertson ◽

Bifeng Gao ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Barrier Function ◽

Transgene Expression ◽

Antioxidant Defenses ◽

Transgenic Rats ◽

Alveolar Epithelial ◽

Barrier Formation ◽

Hiv 1 ◽

Expression And Activity

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.

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Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00299.2010 ◽

2011 ◽

Vol 301 (1) ◽

pp. L40-L49 ◽

Cited By ~ 63

Author(s):

Leslie A. Mitchell ◽

Christian E. Overgaard ◽

Christina Ward ◽

Susan S. Margulies ◽

Michael Koval

Keyword(s):

Epithelial Cells ◽

Tight Junction ◽

Barrier Function ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Epithelial Barrier ◽

Tight Junction Proteins ◽

Epithelial Barrier Function ◽

Alveolar Epithelial ◽

Junction Proteins

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.

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Zinc Supplementation Improves Alveolar Epithelial Barrier Dysfunction Induced By Chronic Alcohol Ingestion And/Or HIV-1 Viral Proteins By Activating The Nrf2/ARE Pathway

10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a1271 ◽

2012 ◽

Author(s):

Xian Fan ◽

Pratibha C. Joshi ◽

Michael Koval ◽

David M. Guidot

Keyword(s):

Zinc Supplementation ◽

Viral Proteins ◽

Alcohol Ingestion ◽

Epithelial Barrier ◽

Chronic Alcohol ◽

Barrier Dysfunction ◽

Alveolar Epithelial ◽

Epithelial Barrier Dysfunction ◽

Hiv 1 ◽

Chronic Alcohol Ingestion

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The role of zinc deficiency in alcohol-induced intestinal barrier dysfunction

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00350.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. G625-G633 ◽

Cited By ~ 115

Author(s):

Wei Zhong ◽

Craig J. McClain ◽

Matthew Cave ◽

Y. James Kang ◽

Zhanxiang Zhou

Keyword(s):

Oxidative Stress ◽

Tight Junction ◽

Zinc Deficiency ◽

Barrier Function ◽

Intestinal Barrier ◽

Alcohol Exposure ◽

Epithelial Barrier ◽

Tight Junction Proteins ◽

Barrier Dysfunction ◽

Junction Proteins

Disruption of the intestinal barrier is a causal factor in the development of alcoholic endotoxemia and hepatitis. This study was undertaken to determine whether zinc deficiency is related to the deleterious effects of alcohol on the intestinal barrier. Mice were pair fed an alcohol or isocaloric liquid diet for 4 wk, and hepatitis was detected in association with elevated blood endotoxin level. Alcohol exposure significantly increased the permeability of the ileum but did not affect the barrier function of the duodenum or jejunum. Reduction of tight-junction proteins at the ileal epithelium was detected in alcohol-fed mice although alcohol exposure did not cause apparent histopathological changes. Alcohol exposure significantly reduced the ileal zinc concentration in association with accumulation of reactive oxygen species. Caco-2 cell culture demonstrated that alcohol exposure increases the intracellular free zinc because of oxidative stress. Zinc deprivation caused epithelial barrier disruption in association with disassembling of tight junction proteins in the Caco-2 monolayer cells. Furthermore, minor zinc deprivation exaggerated the deleterious effect of alcohol on the epithelial barrier. In conclusion, epithelial barrier dysfunction in the distal small intestine plays an important role in alcohol-induced gut leakiness, and zinc deficiency attributable to oxidative stress may interfere with the intestinal barrier function by a direct action on tight junction proteins or by sensitizing to the effects of alcohol.

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Sodium-dependent lysine flux across bullfrog alveolar epithelium

Journal of Applied Physiology ◽

10.1152/jappl.1988.65.4.1655 ◽

1988 ◽

Vol 65 (4) ◽

pp. 1655-1661 ◽

Cited By ~ 20

Author(s):

K. J. Kim ◽

E. D. Crandall

Keyword(s):

Amino Acid ◽

Opposite Direction ◽

Ussing Chamber ◽

Alveolar Epithelium ◽

Epithelial Barrier ◽

Alveolar Epithelial ◽

Control Value ◽

Electrical Gradient ◽

Upstream Reservoir ◽

Net Flux

Amino acid transport across the alveolar epithelial barrier was studied by measuring radiolabeled lysine fluxes across bullfrog lungs in an Ussing chamber. In the absence of a transmural electrical gradient, L-[14C]lysine was instilled into the upstream reservoir and the rate of appearance of the radiolabel in the downstream reservoir was determined. Two lungs from the same animal were used simultaneously to determine tracer fluxes both into and out of the alveolar bath. Results showed that the radiolabel flux measured in the alveolar to the pleural direction was greater than that measured in the opposite direction in the presence of sodium in the bathing fluids. The net flux of L-[14C]lysine was saturable with [Na+], with an apparent transport coefficient (Kt) of 28 mM for Na+. Hill analysis of [14C]lysine flux vs. [Na+] indicated a coupling ratio of 1:1 between sodium and radiolabeled L-lysine. Total L-lysine flux as a function of [L-lysine] was also saturable, with Kt of 7.3 mM for L-lysine. Ouabain significantly decreased absorptive (alveolar-to-pleural) radiolabel flux, while slightly increasing the flux observed in the opposite direction. L-leucine completely inhibited absorptive net flux of L-[14C]lysine. alpha-Methylaminoisobutyric acid (MeAIB), on the other hand, only slightly reduced net flux of L-[14C]lysine from the control value. The presence of a net absorptive, Na+-dependent amino acid flux across the alveolar epithelial barrier indicates that the tissue is capable of removing amino acids and sodium from the alveolar fluid by a coupled cotransport mechanism, which may be important for both protein metabolism and fluid balance by alveolar epithelium.

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Angiotensin II mediates glutathione depletion, transforming growth factor-β1 expression, and epithelial barrier dysfunction in the alcoholic rat lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00141.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. L363-L370 ◽

Cited By ~ 42

Author(s):

Rabih I. Bechara ◽

Andres Pelaez ◽

Andres Palacio ◽

Pratibha C. Joshi ◽

C. Michael Hart ◽

...

Keyword(s):

Oxidative Stress ◽

Angiotensin Ii ◽

Transforming Growth Factor ◽

Glutathione Depletion ◽

Ethanol Ingestion ◽

Epithelial Barrier ◽

Barrier Dysfunction ◽

Alveolar Epithelial ◽

Epithelial Barrier Dysfunction ◽

Tgf Β1

Alcohol abuse markedly increases the risk of sepsis-mediated acute lung injury. In a rat model, ethanol ingestion alone (in the absence of any other stress) causes pulmonary glutathione depletion, increased expression of transforming growth factor-β1 (TGF-β1), and alveolar epithelial barrier dysfunction, even though the lung appears grossly normal. However, during endotoxemia, ethanol-fed rats release more activated TGF-β1 into the alveolar space where it can exacerbate epithelial barrier dysfunction and lung edema. Ethanol ingestion activates the renin-angiotensin system, and angiotensin II is capable of inducing oxidative stress and TGF-β1 expression. We determined that lisinopril, an angiotensin-converting enzyme inhibitor that decreases angiotensin II formation, limited lung glutathione depletion, and treatment with either lisinopril or losartan, a selective angiotensin II type 1 receptor blocker, normalized TGF-β1 expression. The glutathione precursor procysteine also prevented TGF-β1 expression, suggesting that TGF-β1 may be induced indirectly by angiotensin II-mediated oxidative stress and glutathione depletion. Importantly, lisinopril treatment normalized barrier function in alveolar epithelial cell monolayers from ethanol-fed rats, and treatment with either lisinopril or losartan normalized alveolar epithelial barrier function in ethanol-fed rats in vivo, as reflected by lung liquid clearance of an intratracheal saline challenge, even during endotoxemia. In parallel, lisinopril treatment limited TGF-β1 protein release into the alveolar space during endotoxemia. Together, these results suggest that angiotensin II mediates oxidative stress and the consequent TGF-β1 expression and alveolar epithelial barrier dysfunction that characterize the alcoholic lung.

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Asymmetric [14C]albumin transport across bullfrog alveolar epithelium

Journal of Applied Physiology ◽

10.1152/jappl.1985.59.4.1290 ◽

1985 ◽

Vol 59 (4) ◽

pp. 1290-1297 ◽

Cited By ~ 30

Author(s):

K. J. Kim ◽

T. R. LeBon ◽

J. S. Shinbane ◽

E. D. Crandall

Keyword(s):

Alveolar Epithelium ◽

Ringer Solution ◽

The Other ◽

Epithelial Barrier ◽

Apparent Permeability ◽

Simple Diffusion ◽

Ussing Chambers ◽

Alveolar Epithelial ◽

Other Hand ◽

Apparent Permeability Coefficient

Bullfrog lungs were prepared as planar sheets and bathed with Ringer solution in Ussing chambers. In the presence of a constant electrical gradient (20, 0, or -20 mV) across the tissue, 14C-labeled bovine serum albumin or inulin was instilled into the upstream reservoir and the rate of appearance of the tracer in the downstream reservoir was monitored. Two lungs from the same animal were used to determine any directional difference in tracer fluxes. An apparent permeability coefficient was estimated from a relationship between normalized downstream radioactivities and time. Results showed that the apparent permeability of albumin in the alveolar to pleural direction across the alveolar epithelial barrier is 2.3 X 10(-7) cm/s, significantly greater (P less than 0.0005) than that in the pleural to alveolar direction (5.3 X 10(-8) cm/s) when the tissue was short circuited. Permeability of inulin, on the other hand, did not show any directional dependence and averaged 3.1 X 10(-8) cm/s in both directions. There was no effect on radiotracer fluxes permeabilities of different electrical gradients across the tissue. Gel electrophoretograms and corresponding radiochromatograms suggest that the large and asymmetric isotope fluxes are not primarily due to digestion or degradation of labeled molecules. Inulin appears to traverse the alveolar epithelial barrier by simple diffusion through hydrated paracellular pathways. On the other hand, [14C]albumin crosses the alveolar epithelium more rapidly than would be expected by simple diffusion. These asymmetric and large tracer fluxes suggest that a specialized mechanism is present in alveolar epithelium that may be capable of helping to remove albumin from the alveolar space.

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