Administration of vitamin E attenuates airway inflammation through restoration of Nrf2 in a mouse model of asthma

Abstract Accumulating evidence reveals that ROS is one of the key mediators that contribute to the development of asthma. Studies on antioxidants have shown to have beneficial effects on asthma management. However, we still do not know the precise mechanism, and the effects depend on age. This study was conducted to assess the levels of ROS and the effect of antioxidants in younger and older mice using an eosinophilic asthma model. We analyzed airway hyperresponsiveness (AHR), cytokines in bronchoalveolar lavage fluid (BALF), inflammatory cell counts, and the expression levels of NFκB, Nrf2, EPx, and EDN in the lung tissue, as well as the level of ROS in the lung tissue and BALF. The degree of eosinophilia and the levels of IL‐5, ROS, and NFκB were significantly increased, whereas the endogenous levels of vitamin E and Nrf2 were decreased in the lung and BALF in the older mice compared to younger mice. The administration of vitamin E attenuated AHR, airway inflammation, and the level of IL‐13 and ROS and enhanced the Nrf2 level in the older mice compared to the younger mice. Taken together, vitamin E treatment may have the therapeutic potential through restoration of the Nrf2 level, especially in elderly asthma.

Immune cells, such as eosinophils and neutrophils, are the main source of ROS, and ROS levels are positively correlated with the severity of asthma and the age of onset of asthma. 2,11 In addition, recent studies have shown that increased ROS can contribute to tissue damage in the asthma airways by releasing eosinophil extracellular traps. 12,13 The antioxidant system consists of endogenous and exogenous parts. Endogenous enzymatic antioxidants, including superoxide dismutase (SOD), glutathione peroxidase, and catalase (CAT), have the neutralizing capacity on the degree of ROS production. 14 This system is controlled by nuclear factor erythroid 2-related factor 2 (Nrf2). Under normal conditions, Nrf2 was inactivated by binding natural inhibitors called Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. Nevertheless, stress-activated Nrf2 can then translocate to the nucleus and interact with HO-1 and antioxidant response element (ARE). 15 As a consequence, the Nrf2-HO1-Keap1 axis can regulate redox signaling, proteostasis, DNA repair, apoptosis prevention, and iron and heme metabolism. 14 Nrf2 is also known as an age-related factor. 16 Antioxidants are also supplied by reducing compounds, such as vitamin C, vitamin E, carotenoids, and polyphenols, which play integral roles in various antioxidant mechanisms. 17 Vitamin E is a group of potent, lipid-soluble, chain-breaking antioxidants that can prevent oxidative stress. 8 It consists of multiple natural and unsaturated isoforms, such as α-, β-, γ-, and δ-tocopherols, which differ in the number of methyl groups on a chromanol head. 8 The administration of vitamin E decreases C-reactive protein and pro-inflammatory cytokine release in coronary artery disease. 18 Additionally, vitamin E exerts anti-inflammatory and anticarcinogenic activities by reducing the inflammation index and the number of adenomas in the colon of azoxymethane-administered mice. 19 Previous studies reported that the α-tocopherol isoform has been measured to be low in asthmatic patients, and the treatment with this isoform has beneficial effects on the outcome of asthmatic patients. 20 The treatment of the α-tocopherol isoform attenuates eosinophil recruitment and blocks AHR through direct regulation of endothelial cell signals during leukocyte recruitment in an OVA asthma model. 21 However, mechanisms underlying effects of vitamin E in younger and older asthmatic patients have not been completely understood yet. Thus, this study was conducted to evaluate the antioxidant-oxidant defense mechanism in older asthmatic mice compared to younger asthmatic mice.

| Animals
Six weeks-old BALB/c mice purchased from Jackson Laboratory (Bar Harbor, ME) were used as the younger group. For the older group, 6 weeks-old mice were bred to the age of 24 weeks under specific pathogen-and ovalbumin (OVA)-free conditions and were housed in a 12 hour light-dark cycle with food and water ad libitum. All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC 2018-0041).

| Allergen sensitization, challenge, and drug treatment protocol
Mice were sensitized intraperitoneally with 10 µg of OVA (Fisher Scientific) in 1 mg of alum (Inject Alum; Pierce) on days 0 and 14, as previously described. 22 On days 28, 29, and 30, mice were subjected to airway allergen challenges by nebulization with 1% OVA for 30 minutes, using an ultrasonic nebulizer (NE-Y2, Omron). In some experiments, mice were given vitamin E (Sigma-Aldrich) (50 mg/kg) orally 30 minutes before OVA challenge, and the last doses were given from days 28 to 32. Control animals received PBS alone. On day 32, 30 minutes after the last vitamin E treatment, mice were euthanized for further analyses. The scheme of this study is shown in Figure S1.

| Evaluation of airway resistance, cytokines in bronchoalveolar lavage fluid (BALF), and lung histology
Mice were anesthetized with pentobarbital sodium. The trachea was exposed, and a cannula was inserted. It was connected to a FlexiVent system (Scireq). Then, ventilation was instituted with a tidal volume of 10 ml/kg at a frequency of 150 breaths/minutes. Airway hyperresponsiveness (AHR) to acetylβ-methyl choline chloride was recorded at methacholine doses of 0, 1.56, 3.12, 6.25, 12.5, and 25 mg/ml PBS. BALF was collected by washing with 1X phosphate-buffered saline (PBS) plus 1% bovine serum albumin (Sigma-Aldrich) through a cannula. Then, the BALF was centrifuged at 1200 revolutions per minute for 5 minutes at 4°C, and the supernatant was harvested and stored at −70°C until measurement of cytokine levels. Total and differential cell counts were determined by using a hemocytometer and analyzed by using ImageJ (National Institutes of Health, Bethesda). Consecutively, lung tissues were perfused as follows: the left lungs were fixed in 4% paraformaldehyde, while the right lungs were used to measure ROS levels and the expression of various proteins. The fixed tissues were embedded in paraffin and sectioned at 5 μm thickness. To detect inflammatory cells, the sections were stained with hematoxylin and eosin (H&E), and mucus-containing cells were stained with periodic acid-Schiff (PAS).

| Measurement of intracellular ROS
Intracellular ROS were measured as previously described. 23

| Measurement of inflammatory cytokines
The levels of inflammatory cytokines in the serum and BALF were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol. ELISA kits for IFNγ and IL-10 were purchased from R&D Systems. The IL-5 and IL-13 ELISA kits were purchased from Thermo Fisher Scientific. GSH was purchased from Cayman Chemical Co. For mouse serum, a superoxide dismutase (SOD) assay kit was purchased from Cayman Chemical Co, and the vitamin E ELISA kit was purchased from MyBioSource Inc.

| Detection of eosinophil activation and oxidative stress by Western blot
Proteins were isolated from the right lung tissues by lung homogenates, followed by incubation with lysis buffer. Aliquots of 50 μg of total protein per well were loaded onto 12% sodium dodecyl sulfatepolyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules). After blocking in 5% bovine serum albumin (BSA) or skim milk (Sigma Aldrich) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, the membranes were incubated with primary antibodies against EPx, p-NFκB, NFκB (Santa Cruz Biotechnology), Nrf2, and EDN (Abcam, Cambridge, UK) overnight at 4°C with gentle shaking. Then, the membranes were washed 3 times with TBS-T for 10 minutes each and incubated with horseradish peroxidase-conjugated with antigoat or anti-rabbit antibody for 1 hour at room temperature. Signals were detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare). The intensity of the bands was analyzed using a gel doc system (Bio-Rad Laboratories, Inc, Hercules).

| Analysis of NFκB p65 and EPx by immunofluorescence in lung tissues
Analysis of the immunocontent of NFκB p65 was performed in formalin-fixed lungs by immunofluorescence. First, the lungs were embedded in paraffin, and histological sections of 5 μm were made.
The slices were deparaffinated in xylene and rehydrated in ethanol in a concentration-dependent manner. The slides were boiled in a microwave in sodium citrate for 2 minutes for antigen retrieval. Then, the slices were incubated with 0.1% Triton X for 10 minutes and blocked with 5% BSA in 10% normal donkey serum at room temperature for 1 hour. The sections were incubated overnight with anti-NF-kB p65

| Eosinophil isolation
Eosinophils were isolated from healthy controls using the Eosinophil Isolation Kit (Miltenyi Biotec Inc). Briefly, blood was layered on the Lymphoprep (Axis-Shield), followed by density gradient centrifugation at 2000 rpm at 20°C for 25 minutes without brakes. The granulocyte fraction was lysed for red blood cells by using cold distilled water for 30 seconds. We collected unlabeled cells that pass through.
Cell purity (>5%) was evaluated by flow cytometry based on Siglec-8 and ECP expression for eosinophils, and cell viability evaluated after vitamin E treatment was more than 90% at 800 μmol/L (data not shown).

| H 2 O 2 measurement
Cells were pretreated with vitamin E (8, 80, 800 μmol/L) in serumfree media for 1 hour. Then, cells were primed with IL-5 for 30 minutes and LPS for 24 hours. An Amplex™ Red Hydrogen Peroxide/ Peroxidase assay kit was purchased from Thermo Fisher Scientific according to the manufacturer's protocols.

| ROS measurement for eosinophils
Cells were pretreated with vitamin E (8, 80, 800 μmol/L) in serumfree media for 1 hour. Eosinophils were pretreated with vitamin E (8, 80 μmol/L) in serum-free media for 1 hour. Then, cells were stimulated with H 2 O 2 in a time-and dose-dependent manner in order to measure the levels of ROS. Cells were labeled with H2DCDA within 30 minutes and analyzed by using FACS.

| Statistical analysis
Data are presented as mean ± SEM. The data obtained were analyzed using one-way ANOVA followed by Turkey's post hoc test for multiple comparisons of data. A nonparametric t test was done to examine the difference between 2 groups. Differences were considered significant at P < .05. Data analysis and graph preparation were performed by using the statistical software packages IBM SPSS 20.0 and GraphPad Prism 6 respectively.

| Different features in younger and older asthmatic mice
The OVA nebulization in OVA-sensitized mice led to significantly higher AHR and inflammatory cell numbers in BALF in both younger and older mice (P < .050 for all, Figure 1A-B). Although the degree of AHR was not significantly higher in the older mice than in the younger F I G U R E 1 Effects of age on airway hyperresponsiveness and inflammation. Mice were assessed for AHR to methacholine at different concentrations (0, 1.56, 3.12, 6.25, 12.5, and 25 mg/ml) (A). Total and differential cell counts in BALF (B). The level of cytokines released in BALF (C). Lung tissue histology with H&E and PAS staining (D). Data are presented as means ± SD. *P < .05, **P < .01, ***P < .001 obtained by Student's t test. BALF, bronchoalveolar lavage fluid; Eos, eosinophils; H&E, hematoxylin and eosin; IFNγ, interferon gamma; IL, interleukin; Mac, macrophages; NC, normal control; Neu, neutrophils; PAS, periodic acid-Schiff mice, the inflammatory cell counts, especially eosinophils and neutrophils, increased with age (P < .010). There was a significant increase in the level of IL-5, but not of IL-13, IFNγ, and IL-10 with age ( Figure 1C).
Lung histology revealed increased inflammatory cells and mucus secretion in the older mice compared to the younger mice ( Figure 1D).

| Oxidant stress and inflammatory markers in the younger and older asthmatic mice
Oxidative stress and endogenous antioxidant levels were measured in the younger and older asthmatic mice. There was a significant increase in the levels of ROS in BALF and lung tissues (P < .010, Figure 2A), whereas the level of vitamin E in serum (P < .010), GSH in BALF and lung tissues (P = .004, P < .050, respectively), and total SOD activity in serum (P < .001) were reduced in the older asthmatic mice compared to the younger asthmatic mice ( Figure 2B-D).
Next, pro-inflammatory markers including eosinophil activation were measured in lung homogenates. The expressions of NFκB and EPx were increased in the older asthmatic mice compared to the younger mice by Western blot ( Figure 3A). Signals for NFκB were enhanced in the younger and older asthmatic mice compared to the negative control mice in the immunofluorescence study. It was also showed that the expressions of EPx and NFκB were increased in the older mice compared to the younger mice ( Figure 3B). Interestingly, we also found the significant decrease level of Nrf2 in the older asthmatic mice. These findings suggest that there is a significant increase in oxidative stress, causing eosinophilic activation, especially in the older asthmatic mice.

| The effect of vitamin E treatment on AHR and airway inflammation in the younger and older asthmatic mice
To determine the effects of vitamin E treatment on OVA-induced AHR and airway inflammation, the mice were treated with 50 mg/kg vitamin E during the OVA challenge and continued 2 days more in both younger and older asthmatic mice. Vitamin E treatment significantly reduced AHR and the inflammatory cell counts, such as eosinophils and neutrophils, in the younger and older asthmatic mice (P < .050 for all, Figure 4A,B), which was more prominent in the older asthmatic mice than in the younger asthmatic mice. Vitamin E treatment also considerably decreased the release of IL-5 and IL-13 in BALF (P < .010 for all, Figure 4C) and the production of ROS in the lung tissues (P < .001, Figure 4D); it also restored the serum activity of SOD and GSH in serum and lung tissues, especially in the older asthmatic mice (P < .001, Figure 4E-F).

| The effect of vitamin E treatment on Nrf2 and NF-κB expressions and its cascade
The Nrf2-Keap1-HO1 pathway and NFκB-IkB pathway were evaluated after vitamin E treatment in the younger and older asthmatic  Figure 5A,B). In immunofluorescence, we finally found the increased expression of NFκB, and EPx was significantly reduced after vitamin E treatment ( Figure 5C).

| Ex vivo and in vitro effect of vitamin E in human eosinophils
Finally, we evaluated oxidant productions and cellular activation after vitamin E treatment on human peripheral eosinophils (PBEs).

| D ISCUSS I ON
It is well-known that aging causes a gradual loss of tissue and organ function processes through accumulation of damage caused by ROS. 24 ROS can be divided into 2 sources: one is the endogenous source of ROS mainly derived from immune cells, such as neutrophils, monocytes, and eosinophils, 2 and the other is the exogenous source of ROS, which is generated by exposure to the environmental factors that come inside the body and get metabolized into free radicals. 25 ROS plays a role as defense systems to remove invading pathogens and to maintain redox homeostasis and cellular signal transduction. 2,5 However, at high concentration, it has negative effects on the oxidative modification of major cellular macromolecules such as lipids, DNA, and proteins. 24 However, the exact mechanisms of oxidative stress-induced aging in asthma are still not clear.
Elderly asthma, one of the phenotypes of asthma, is difficult to treat because of steroid resistance, non-type 2 phenotype, and low atopy rate. 3,4 They could have significant alterations in pulmonary inflammation and structural changes during development of asthma with age. 26 Due to these clinical features, elderly asthma has become an important medical burden whose mechanism and novel therapeutic options according to its mechanism are warranted.
In the present study, we found different features of airway inflammation in younger and older asthmatic mice. First of all, the degree of airway inflammation in the BAL and lung tissues was significantly increased in the elderly asthmatic mice compared to the younger asthmatic mice, although there was no difference in AHR. These results

F I G U R E 4 Effect of vitamin E treatment on airway hyperresponsiveness and pulmonary inflammation with aging. Vitamin E decreased the AHR (A). The cell counts in BALF (B). Vitamin E decreased the level of Th2 cytokines (C) and ROS formation (D)
. Vitamin E restored the GSH level in serum and lung tissues (E) as well as the SOD level in serum (F). *** P < .001 at the respective weeks, between the positive asthma groups (young negative, old negative) vs. the treatment groups (young asthma, old asthma); ### P < .001 between the treatment groups at 6 and 24 w. Data were normalized in order to compare between the effects of antioxidant treatment on the asthma mouse model we showed reduced Nrf2 levels in the older asthmatic mice compared to the younger asthmatic mice, whereas the expression of p65 NFĸB was shown to be significantly increased in the lung tissues. Nrf2 is a transcriptional factor that controls more than 20 antioxidant genes in the human body. 34 Under oxidative stress, Nrf2 translocates to the nucleus, binds to ARE genes, and leads to reductions in the expression of pro-inflammatory cytokines. 34 Oppositely, NFĸB is another transcription factor that regulates the de novo synthesis of pro-inflammatory mediators; p65 NFĸB, one of the subunits of the NFĸB family, has a transactivation domain and is closely related to various diseases including asthma. 34 The Nrf2-HO-1-Keap1 pathway and IkB-NFĸB pathway have opposite effects in the control of inflammation, which suggests that they may play a crucial role in the treatment of NFĸB-related diseases via suppression or inactivation of Nrf2. 35 Third, we also found that eosinophilic activation was correlated with increased ROS production. H 2 O 2 -stimulated PBEs release ROS and EDN, which contributed to airway inflammation in asthma.
Eosinophil granulocyte proteins, such as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), were well-known activation markers for type 2 inflammatory diseases, 36 and the activation status of eosinophils plays an integral role in eosinophilic asthma. 37 ROS production is a key feature of activated eosinophils, and excessive ROS production can induce cellular death and tissue destruction to release cellular materials. 38,39 Taken together, increased eosinophilic activation and high IL-5 and ROS levels could be disease markers, especially in elderly eosinophilic asthma.
Finally, we evaluated the effect of vitamin E in an asthma model.
Antioxidants may act as scavengers of oxidants to maintain the biological redox steady state and to protect the body against aging and age-related diseases via different mechanisms. 40 In a normal physiological condition, there is a balance between ROS production and endogenous antioxidants including GSH, SOD, and CAT. 14,41 However, since endogenous antioxidants alone are not sufficient for neutralizing the high-levels of ROS, they require supplementation of exogenous antioxidants. 41 Vitamin E was used as one of the most effective antioxidants in inflammatory and age-related diseases, as previously reported. 40 For example, α-tocotrienols protect mouse hippocampal and cortical neuron cell death by regulating the neurodegenerative signaling cascade, 42 and administration of tocopherols and tocotrienols can also control age-related arthritis via reducing cytokine production. 43 In asthma, accumulating evidence reveals that AHR and airway inflammation are significantly decreased after treatment with vitamin E. 44    IgE-mediated inflammation in human atopic asthmatics in vivo. 46 We found different effects of vitamin E administration according to age, which stimulated downregulation of the IkB-NFκB pathway and EPx expression to reduce ROS production and eosinophil activation. In addition, vitamin E administration restored the dysregulation of the Nrf2-HO1-Keap1 pathway in the lung tissues from the older asthmatic mice. We showed that the exposure of H 2 O 2 for a long time will decrease the endogenous antioxidant defense, including SOD and GSH. Additionally, H 2 O 2 can decrease the level of Nrf2 by increasing the Keap1-Nrf2 interaction. As a result, the antioxidant defense will be disrupted, and overproduction of ROS will be induced.
Vitamin E can protect cell activation by enhancing endogenous antioxidant concentrations to suppress ROS levels, as well as decrease Keap1 levels to release active Nrf2. All of these effects of vitamin E have also been demonstrated in in vitro studies using PBEs.
There are some limitations to this study. Our model used 6-week-old and 24-week-old mice as the younger and older groups, respectively, so it could not completely reflect elderly asthmatic subjects. Moreover, since the majority of human asthma cases in the elderly are mainly neutrophilic asthma, our eosinophilic asthma model could not fully reflect the elderly asthma. These limitations can be overcome by using a neutrophilic asthma model and a much older mouse model in further study.
In conclusion, vitamin E may control airway hyperresponsiveness and inflammation in the elderly asthma cases through its antioxidant and inflammatory effects on NFκB and Nrf2 regulation. Further studies are needed to confirm our results.

CO N FLI C T O F I NTE R E S T
The authors confirm that there is no conflict of interest.