Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

Background

Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.

Methods

Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.

Results

We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.

Conclusions

Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.     

An isopentenyl-substituted flavonoid norartocarpin activates Nrf2 signalling pathway and prevents oxidative insults in human lung epithelial cells

The nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in regulating the intracellular oxidative stress, and thus activation of Nrf2 by nature-derived molecules effectively alleviates the pathological process of oxidative stress-induced chronic diseases. The isopentenyl-substituted flavonoid norartocarpin (NOR) induced the activity of NAD(P)H: quinone reductase (QR), implying that it might be a potential Nrf2 activator. Further studies indicated that NOR upregulated the protein levels of Nrf2 and its downstream genes, NAD(P)H quinone oxidoreductase 1 (NQO1), and γ-glutamyl cysteine synthetase (GCLM) through facilitating the nuclear translocation of Nrf2 and enhancing Nrf2 protein stability. NOR-induced activation of Nrf2 pathway was associated with multiple upstream kinases, including mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase C (PKC), and protein kinase R-like endoplasmic reticulum kinase (PERK). Moreover, NOR protected human lung epithelial Beas-2B cells against sodium arsenite [As(III)]-induced cytotoxicity in an Nrf2-dependent manner. Collectively, NOR was firstly identified to be an Nrf2 activator, which demonstrated the capability of preventing oxidative insults in human lung epithelial cells.     

Nrf2-dependent repression of interleukin-12 expression in human dendritic cells exposed to inorganic arsenic

Inorganic arsenic, a well-known Nrf2 inducer, exerts immunosuppressive properties. In this context, we recently reported that the differentiation of human blood monocytes into immature dendritic cells (DCs), in the presence of low and noncytotoxic concentrations of arsenic, represses the ability of DCs to release key cytokines in response to different stimulating agents. Particularly, arsenic inhibits the expression of human interleukin-12 (IL-12, also named IL-12p70), a major proinflammatory cytokine that controls the differentiation of Th1 lymphocytes. In the present study, we determined if Nrf2 could contribute to these arsenic immunotoxic effects. To this goal, human monocyte-derived DCs were first differentiated in the absence of metalloid and then pretreated with arsenic just before DC stimulation with lipopolysaccharide (LPS). Under these experimental conditions, arsenic rapidly and stably activates Nrf2 and increases the expression of Nrf2 target genes. It also significantly inhibits IL-12 expression in activated DCs, at both mRNA and protein levels. Particularly, arsenic reduces mRNA levels of IL12A and IL12B genes which encodes the p35 and p40 subunits of IL-12p70, respectively. tert-Butylhydroquinone (tBHQ), a reference Nrf2 inducer, mimics arsenic effects and potently inhibits IL-12 expression. Genetic inhibition of Nrf2 expression markedly prevents the repression of both IL12 mRNA and IL-12 protein levels triggered by arsenic and tBHQ in human LPS-stimulated DCs. In addition, arsenic significantly reduces IL-12 mRNA levels in LPS-activated bone marrow-derived DCs from Nrf2^+/+ mice but not in DCs from Nrf2^−/− mice. Finally, we show that, besides IL-12, arsenic significantly reduces the expression of IL-23, another heterodimer containing the p40 subunit. In conclusion, our study demonstrated that arsenic represses IL-12 expression in human-activated DCs by specifically stimulating Nrf2 activity.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

Selenium deficiency alters epithelial cell morphology and responses to influenza

It is unknown whether nutritional deficiencies affect the morphology and function of structural cells, such as epithelial cells, and modify the susceptibility to viral infections. We developed an in vitro system of differentiated human bronchial epithelial cells (BEC) grown either under selenium-adequate (Se+) or selenium-deficient (Se–) conditions, to determine whether selenium deficiency impairs host defense responses at the level of the epithelium. Se– BECs had normal SOD activity, but decreased activity of the selenium-dependent enzyme GPX1. Interestingly, catalase activity was also decreased in Se– BECs. Both Se– and Se+ BECs differentiated into a mucociliary epithelium; however, Se– BEC demonstrated increased mucus production and increased Muc5AC mRNA levels. This effect was also seen in Se+ BEC treated with 3-aminotriazole, an inhibitor of catalase activity, suggesting an association between catalase activity and mucus production. Both Se– and Se+ were infected with influenza A/Bangkok/1/79 and examined 24 h postinfection. Influenza-induced IL-6 production was greater while influenza-induced IP-10 production was lower in Se– BECs. In addition, influenza-induced apoptosis was greater in Se– BEC as compared to the Se+ BECs. These data demonstrate that selenium deficiency has a significant impact on the morphology and influenza-induced host defense responses in human airway epithelial cells.     

Induction of claudins in passaged hTERT-transfected human nasal epithelial cells with an extended life span

The epithelial barrier of the upper respiratory tract, such as that of the nasal mucosa, plays a crucial role in host defense. The epithelial barrier is regulated in large part by the apical-most intercellular junctions, referred to as tight junctions. However, the mechanisms regulating of tight junction barrier in human nasal epithelial cells remain unclear because the proliferation and storage of epithelial cells in primary cultures are limited. In the present study, we introduced the catalytic component of telomerase, the hTERT gene, into primary cultured human nasal epithelial cells and examined the properties of the transfectants, including their expression of tight junctions, compared with primary cultures. The ectopic expression of hTERT in the epithelial cells resulted in adequate growth potential and a longer lifespan of the cells. The properties of the passaged hTERT-transfected cells including tight junctions were similar to those of the cells in primary cultures. The barrier function in the transfectants after treatment with 10% FBS was significantly enhanced with increases of integral tight junction proteins claudin-1 and -4. When the transfectants were treated with TGF-β, which is assosciated with nasal polyposis and chronic rhinosinusitis, upregulation of only claudin-4 was observed, without a change of barrier function. In human nasal epithelial cells, the claudins may be important for barrier function and a novel target for a drug-delivery system. Our results indicate that hTERT-transfected human nasal epithelial cells with an extended lifespan can be used as an indispensable and stable model for studying the regulation of claudins in human nasal epithelium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports Science, and Technology of Japan, the Ministry of Health, Labor, and Welfare of Japan, Japan Science and Technology Agency, the Akiyama Foundation, and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

Effect of Nrf2 signaling pathway on the improvement of intestinal epithelial barrier dysfunction by hyperbaric oxygen treatment after spinal cord injury

Disruption of the intestinal epithelial barrier following spinal cord injury (SCI) seriously affect long-term quality of life. Oxidative stress-induced epithelial cells’ injury contributes to the epithelial barrier dysfunction. Hyperbaric oxygen (HBO) treatment has been proved to alleviate SCI. However, it is unclear whether or not HBO treatment affects intestinal barrier function following SCI. In this study, our purpose was to explore the impact of HBO treatment on intestinal epithelial barrier function and underlying mechanisms following SCI. An SCI model was established in rats, and the rats received HBO treatment. Intestinal injury, mucosal permeability, intercellular junction proteins, and oxidative stress indicators were evaluated in our study. We found that HBO treatment significantly alleviated intestinal histological damage, reduced mucosal permeability, and markedly prevented bacterial translocation. Furthermore, HBO treatment significantly increased the expression of Claudin-1 and E-cadherin, inhibited intestinal tissue oxidative stress as demonstrated by upregulation of superoxide dismutase and glutathione, and HBO downregulated malondialdehyde. Mechanically, we demonstrated that HBO treatment ameliorated intestinal oxidative stress possibly through upregulating nuclear factor E2-related factor 2 (Nrf2) and its downstream targets, Heme oxygenase-1(HO-1), NADH-quinone oxidoreductase-1(NQO-1), and glutamate cysteine ligase catalytic subunit (GCLC). These results suggested that HBO treatment triggered antioxidative effects against intestinal epithelial barrier dysfunction by promoting Nrf2 signaling pathway after SCI.     

Biogenic nanoselenium particles activate Nrf2-ARE pathway by phosphorylating p38, ERK1/2, and AKT on IPEC-J2 cells

As the intestinal epithelium is vulnerable to oxidative stress because of frequent enterocyte renewal and continuous exposure to exogenous agents, it is meaningful to figure out how the epithelial cells exert antioxidant function. We previously synthesized a novel biogenic nanoselenium (BNS) particles and proved that BNS could effectively improve intestinal antioxidative function through activating Nrf2-ARE pathway. The objective of the present study was to investigate the mechanism by which BNS activate Nrf2-ARE pathway on the physiological function of intestinal epithelial cells. In the present study, we demonstrated that treatment of IPEC-J2 cells with BNS particles not only elevated the levels of downstream proteins of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) such as heme oxygenase-1 and NQO-1 in a time-dependent manner which started to weaken at 12 hr after treatment but also significantly activated Nrf2, mitogen-activated protein kinase (MAPK), and protein kinase B (AKT) pathway in a time-dependent manner within 24 hr. BNS particles significantly increased the content of phosphorylated-Nrf2, without evident influence on the level of Kelch-like ECH-associated protein 1 (Keap1). Moreover, BNS also induced the activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase, and AKT while phosphorylating Nrf2. Using specific protein kinase inhibitors, we found that the Nrf2-phosphorylating and antioxidative effects of BNS particles were abolished when p38, ERK1/2, and AKT were significantly inhibited. Overall, our data demonstrated that BNS particles activated Nrf2-ARE pathway through p38, ERK1/2, and AKT mediated-phosphorylation of Nrf2 to improve the antioxidant function of intestinal epithelial cells     

Regulation of uncoupling protein 2 expression by long-chain fatty acids and hormones in bovine mammary epithelial cells

Although mammary epithelial cells are known to synthesize and accumulate triacylglycerol (TAG) in order to produce milk lipid in the cytosol, lipid and energy metabolism is still not fully understood. In this study, we assessed the effects of long-chain fatty acid (LCFA) on the accumulation of cytosolic TAG and uncoupling protein (UCP) 2 in cloned bovine mammary epithelial cells (bMEC). LCFAs significantly raised the expression of UCP2 mRNA and the accumulation of TAG. We observed the rapid elevation in UCP2 shown at 6 h after LCFA treatment. Insulin (5-50 ng/ml) or dexamethasone (500 nM) significantly suppressed the expression of UCP2 mRNA. These results suggest that UCP2 play an important role of lipid and energy metabolism in mammary epithelial cells.     

不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H₂O₂刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h后测细胞活力,选择细胞H₂O₂刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21% O₂、12% O₂、8% O₂、5% O₂每组设8个复孔,12 h后,H₂O₂作用1 h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1 的mRNA表达量及细胞ROS水平。结果:选择H₂O₂作用时间相对较短的1 h和浓度0.5 mmol/L作为本实验的H₂O₂刺激条件。与21%O₂组相比,12%O₂组细胞Nrf2蛋白荧光增强,Nrf2 的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的 mRNA表达均显著增加(P＜0.05或P＜0.01),细胞 ROS水平明显降低(P＜0.01);8%O₂组仅GPX-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的 mRNA表达均明显降低(P＜0.05或P＜0.01),细胞 ROS水平则明显升高(P＜0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H₂O₂刺激反应不同,12 h的12% O₂浓度可促进C2C12细胞Nrf2的抗氧化作用,而5% O₂浓度的严重低氧则作用相反。     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.