Oxidative Stress Attenuates TLR3 Responsiveness and Impairs Anti-viral Mechanisms in Bronchial Epithelial Cells From COPD and Asthma Patients

COPD and asthma exacerbations are commonly triggered by rhinovirus infection. Potentially promoting exacerbations, impaired anti-viral signaling and attenuated viral clearance have been observed in diseased bronchial epithelium. Oxidative stress is a feature of inflammation in asthma and COPD and is prominent during exacerbations. It is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthmatic and COPD donors were infected with rhinovirus or treated with the oxidative stressor H2O2 followed by exposure to the synthetic viral replication intermediate poly(I:C). Poly(I:C) was used to ascertain a constant infection-like burden. Gene and protein levels of antioxidants as well as anti-viral responses were measured 3 and 24 h post poly(I:C) exposure. Rhinovirus infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. In asthmatic bronchial epithelial cells pre-treatment with H2O2 dose-dependently decreased the antioxidant response to poly(I:C), suggesting exaggerated oxidative stress. Further, poly(I:C)-induced IFNβ gene expression was reduced after pre-treatment with H2O2. This epithelial effect was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Pre-treatment with H2O2 did not alter antioxidant responses in COPD bronchial epithelial cells and, more modestly than in asthma, reduced poly(I:C)-induced IFNβ gene expression. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNβ gene expression by H2O2. We developed a method by which we could demonstrate that oxidative stress impairs anti-viral signaling in bronchial epithelial cells from asthmatic and COPD patients, most pronounced in asthma. The impairment apparently reflects reduced responsiveness of TLR3. These present findings shed light on molecular mechanisms potentially causing reduced interferon responses to rhinovirus infection at exacerbations in asthma and COPD. Together, our findings suggest a possible self-perpetuating vicious cycle underlying recurrent exacerbations, leading to an impaired anti-viral response, which in turn leads to viral-induced exacerbations, causing more airway inflammation.

COPD and asthma exacerbations are commonly triggered by rhinovirus infection. Potentially promoting exacerbations, impaired anti-viral signaling and attenuated viral clearance have been observed in diseased bronchial epithelium. Oxidative stress is a feature of inflammation in asthma and COPD and is prominent during exacerbations. It is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthmatic and COPD donors were infected with rhinovirus or treated with the oxidative stressor H 2 O 2 followed by exposure to the synthetic viral replication intermediate poly(I:C). Poly(I:C) was used to ascertain a constant infection-like burden. Gene and protein levels of antioxidants as well as anti-viral responses were measured 3 and 24 h post poly(I:C) exposure. Rhinovirus infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. In asthmatic bronchial epithelial cells pre-treatment with H 2 O 2 dose-dependently decreased the antioxidant response to poly(I:C), suggesting exaggerated oxidative stress. Further, poly(I:C)-induced IFNβ gene expression was reduced after pre-treatment with H 2 O 2 . This epithelial effect was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Pre-treatment with H 2 O 2 did not alter antioxidant responses in COPD bronchial epithelial cells and, more modestly than in asthma, reduced poly(I:C)-induced IFNβ gene expression. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNβ gene expression by H 2 O 2 . We developed a method by which we could demonstrate that oxidative stress impairs anti-viral signaling in bronchial epithelial cells from asthmatic and COPD patients, most pronounced in asthma. The impairment apparently reflects reduced responsiveness of TLR3. These present findings shed light on molecular mechanisms INTRODUCTION Asthma and COPD are chronic diseases of the airways, characterized by inflammation of the bronchi and airflow obstruction. Disease severity may be acutely increased at periods defined as exacerbations, which also may be involved in disease progression (1,2). Exacerbations are a major cause of morbidity and mortality in asthma, but at present, it is unclear why some patients have frequent exacerbations, while others rarely, or never experience exacerbations (3). Understanding the mechanisms driving recurrent exacerbations is crucial to the development of better management strategies. Infections with rhinoviruses are a main cause of exacerbations of asthma and COPD. The airway epithelium is the main target of rhinoviruses. The epithelial cells express a variety of pattern recognition receptors (PRRs), such as TLR3 and the RIG-I like helicases RIG-I and MDA5. PRRs recognize viruses and mount immunological responses increasing epithelial production of inflammatory cytokines, chemokines, and anti-viral interferons (4,5). Several studies have demonstrated a reduced anti-viral response toward rhinoviral infection, leading to a higher viral burden in asthmatics (6)(7)(8).
The airway inflammation at exacerbations of asthma and COPD likely involves oxidative stress (9,10). Inflammatory cells participating in inflammatory processes in these diseases, such as alveolar macrophages, eosinophils and neutrophils, are known sources of reactive oxygen species and free radicals (11). Oxidative stress is caused by an imbalance between the oxidant and the antioxidant system, in which free radicals overcome the cell's antioxidant defenses (12). Oxidative stress not only acts as an inflammatory trigger, likely by activation of the transcription factor NFκB (13), but might also lead to smooth muscle hyper-contractility (14). Both environmental and genetic factors contribute to oxidative stress. Polymorphisms in the gene coding for glutathione-S-transferase, which is involved in maintaining physiological oxidant levels in the cell, have been associated with asthma (15). Further, it has been demonstrated that proteases from allergens can induce production of reactive oxygen species (16) and oxidative stress produced by cigarette smoke is considered a major disease mechanism in COPD (17).
Infection with rhinovirus causes oxidative stress (18), probably through interaction with a NOD-like receptor (19,20). The dsRNA mimic poly(I:C) induces oxidative stress by the same mechanism (19,20) supporting the view that dsRNA is responsible for reactive oxygen production at rhinoviral infections. A previous study suggested that increased mitochondrial oxygen species could decrease early TLR9 mediated type I interferons while increasing a second wave of  type I interferons mediated via RIG-I signaling in plasmacytoid dendritic cells (21).
It is yet largely unknown how oxidative stress affects the antiviral signaling cascade in the major bronchial target for infection, the epithelium. Here we demonstrate that the oxidative stressor H 2 O 2 reduces dsRNA-induced expression of IFNβ and pattern recognition receptors in bronchial epithelial cells of asthmatics and to some extent also in COPD patients. Further, we provide evidence that the effect of H 2 O 2 on anti-viral responses might involve TLR3.

Primary Bronchial Epithelial Cells
Primary human bronchial epithelial cells (HBECs) from asthmatic and COPD donors were obtained by bronchoscopy using a fiber optic bronchoscope with standard sterile-sheared nylon cytology brushes. Brushings were performed in accordance with standard guidelines and processed as described previously (22). Patients provided written informed consent and the regional ethical review board in Lund and the review board in Copenhagen approved the study. A characterization of patients can be found in Tables 1, 2.

Rhinovirus Infection
Rhinovirus RV16 was amplified in Ohio HeLa cells (European Collection of Cell Cultures) as described previously (24) and obtained from clarified cell lysates. HBECs were infected with RV16 at 1MOI (TCID 50 1.58 × 10 5 ) for 1 h at room temperature while shaking. Then virus was removed and fresh culture medium was added. After 48 h cell lysates were collected for subsequent protein expression analysis. A characterization of patients can be found in Table 3.

Stimulation With H 2 O 2 and Poly(I:C)
HBECs were cultured in bronchial epithelial growth medium (BEGM, Clonetics, San Diego, CA, USA or PneumaCult-Ex Medium, STEMCELL Technologies, Cambridge, UK) and subsequently seeded into 12-well plates (Nunc Technologies, Carlsbad, CA, USA). For experiments cells were exposed to H 2 O 2 (Sigma-Aldrich, Stockholm, Sweden) for 30 min before stimulation with 10 µg/ml poly(I:C) (InvivoGen, San Diego, CA, USA) for 3 and 24 h. Cell lysates were obtained for gene and protein expression analysis and cell-free supernatants were obtained for analysis of protein release.

Knockdown of Pattern Recognition Receptors
In subsequent experiments HBECs were transfected with 10 nM siRNA directed against MDA5, RIG-I or TLR3 or non-specific siRNA (scramble) using Lipofectamine RNAiMAX (Ambion, Thermo Scientific, Waltham, NA, USA) as a transfection agent prior to stimulation with H 2 O 2 and poly(I:C).

RNA Isolation and Quantification of Gene Expression
Total RNA was extracted using a RNA isolation kit (Nucleospin R RNA II, Macherey-Nagel, Düren, Germany) and 1 µg RNA was reverse transcribed to cDNA (Precision Nanoscript Reverse Transcription Kit, PrimerDesign, Southampton, UK). Quantitative real-time PCR was performed for real-time detection of PCR products on a Mx3005P qPCR system (Stratagene, La Jolla, CA, USA) using standard cycling parameters. Primers were obtained from Qiagen (Sollentuna, Sweden) or PrimerDesign (Southampton, UK). Target genes were normalized to UBC/GAPDH expression and related to unstimulated control using the Ct method (25).

Quantification of Protein Expression by Western Blot
For western blot quantification cells were lysed in a lysis buffer containing 1% TritonX-100, 10 mM Tris-HCl, 50

Statistical Analysis
Data is presented as mean with standard error of the mean. Comparison between different groups was performed by Kruskal-Wallis with Wilcoxon post-testing. P-values < 0.05 were regarded statistically significant. Statistical analysis was performed using R (26).

Rhinovirus Infection, as Well as Poly(I:C), Induces Superoxide Dismutase Protein Levels in Bronchial Epithelial Cells From Asthmatic Donors
As rhinovirus infection has been shown to act as an oxidative stressor (18) and thus induces antioxidant expression, we investigated expression of four well-known antioxidants after rhinovirus infection in bronchial epithelial cells of asthmatics. Infection with rhinovirus significantly induced protein levels of manganese-dependent superoxide dismutase (SOD2) and copper-zinc superoxide dismutase (SOD1) (p < 0.05; Figures 1A,B). There was only a tendential up-regulation of catalase and thioredoxin (TRX) protein levels after rhinovirus infection (Figures 1C,D). Next asthma bronchial epithelial cells were stimulated with the synthetic rhinovirus replication intermediate poly (I:C). Similarly to rhinovirus infection, stimulation with poly(I:C) induced gene and protein expression of SOD1 and SOD2 (Figure 2).

Poly(I:C)-Induced Superoxide Dismutase Expression Is Reduced in H 2 O 2 -Exposed Bronchial Epithelial Cells From Asthmatics
During exacerbations, frequently involving viral infections, an increase of levels of reactive oxygen species has been observed (27,28). It was thus of interest to develop an in vitro model that could mimic such biphasic effects. Hence, bronchial epithelial cells from asthmatic donors were first given increasing doses of the oxidative stressor H 2 O 2 which was then followed by stimulation with poly(I:C). Doses were chosen according to literature (29,30). We regarded the present employment of poly(I:C) a suitable mode for explorative studies as biological actions of rhinovirus infection are mimicked (31). Further, by using poly(I:C) one can avoid variations due to take off rates of infection, thus, supporting reproducibility of data. Pre-treatment with H 2 O 2 dose-dependently reduced poly(I:C)-induced SOD2 expression both on gene (Figure 2A) and protein level (Figure 2C), while there was no effect of H 2 O 2 on SOD1 gene expression (Figure 2B). Protein levels of poly(I:C)-induced SOD1 were reduced by H 2 O 2 pre-treatment ( Figure 2C). This data suggests that asthmatic epithelium

Poly(I:C)-Induced Expression of RIG-I Like Helicases Is Decreased Upon H 2 O 2 Pre-treatment in Asthma Bronchial Epithelial Cells
Observing an effect of H 2 O 2 pre-treatment on TLR3 gene and protein expression, we sought to study if other pattern recognition receptors involved in inducing IFNβ, such as RIG-I like helicases (4), are likewise affected. Pre-treatment with H 2 O 2 dose-dependently decreased poly(I:C)-induced expression of the RIG-I like helicases RIG-I and MDA5 on gene (Figures 4A,B) and protein level (Figure 4C). There was a significant correlation between RIG-I mRNA and IFNβ mRNA expression and between MDA5 mRNA and IFNβ mRNA expression (Supplementary Figure 2).

Pre-treatment With H 2 O 2 Modestly Reduces Anti-viral Responses to Poly(I:C) in Bronchial Epithelial Cells of COPD Donors
Occurrence of oxidative stress is considered a hallmark of COPD (32), and viral infection is a major factor in exacerbations of COPD and asthma (1,33). Hence, it was thought of interest to examine whether oxidative stress reduced poly(I:C)-induced anti-viral responses also in bronchial epithelial cells from COPD patients. Similar to bronchial epithelial cells from asthmatics, pre-treatment with H 2 O 2 showed a trend toward dose-dependent reduction of poly(I:C)induced expression of IFNβ in COPD bronchial epithelium ( Figure 5A). The reduced IFNβ expression was associated with a decrease of RIG-I like helicases both on gene level and to some extent on protein level (Figures 5C-E). TLR3 gene expression was not affected by H 2 O 2 pre-treatment ( Figure 5B) but poly(I:C)-induced TLR3 protein expression was inhibited by H 2 O 2 ( Figure 5F). The more modest effects of oxidative stress on poly(I:C)-induced anti-viral responses in COPD patients was mirrored in their expression of SOD1 and SOD2 which were little affected by H 2 O 2 pre-treatment  Figure 3). Further, there was no difference in epithelial IFNβ gene expression at baseline or after poly(I:C) stimulation between the asthma and COPD donors (Supplementary Figure 4).

Knockdown of TLR3 Abrogates the Impairment of IFNβ Expression by H 2 O 2
As poly(I:C)-induced expression of pattern recognition receptors was affected by pre-treatment with H 2 O 2 we sought to investigate if the effect of H 2 O 2 on IFNβ expression might be due to its effects on pattern recognition receptor expression. Knockdown of pattern recognition receptors was confirmed (Supplementary Figure 5). Double knockdown of the RIG-I like helicases RIG-I and MDA5 did not affect H 2 O 2induced impairment of poly(I:C)-induced IFNβ expression and resulted in a similar expression pattern as knockdown with scramble siRNA (Figure 6A). In contrast, when TLR3 was knocked down pre-treatment with H 2 O 2 did not further reduce poly(I:C)-induced IFNβ expression (Figure 6A), suggesting involvement of TLR3 in the effects of H 2 O 2 on the anti-viral responses.

Pre-treatment With H 2 O 2 Reduces Expression of Poly(I:C)-Induced Cytokines in Asthma Bronchial Epithelial Cells
We next investigated if oxidative stress affected expression of other cytokines relevant to asthma. We observed that pre-treatment with H 2 O 2 decreased poly(I:C)-induced IFNβ protein levels (Figure 6B), confirming our findings on gene level ( Figure 3A). Further, oxidative stress reduced protein levels of poly(I:C)-induced IL-6, IL-8 and TNF, while increasing poly(I:C)-induced expression of IL-1β. There was no effect of oxidative stress on IL-33 and IL-17A protein levels ( Figure 6B) in asthma bronchial epithelial cells.

DISCUSSION
Levels of reactive oxygen species are enhanced in asthmatic airways as a result of inflammation (11) and are inversely correlated with lung function (34). Activity of the antioxidant enzymes is also reduced in asthmatic bronchoalveolar lavage fluid and airway epithelial cells (35,36). The disequilibrium in oxidation state of the lungs is associated with asthma severity (37). During exacerbations, frequently involving viral infections, levels of reactive oxygen species are further increased (27,28). In COPD oxidative stress is considered a major disease mechanism (17) and oxidative stress markers in sputum are further increased at exacerbation (38). Our results of impaired viral stimulus-induced expression of antioxidant enzymes in diseased epithelium is consistent with these clinical observations. Further, we demonstrate here that oxidative stress in epithelial cells from diseased donors reduces anti-viral responses possibly by reducing responsiveness to TLR3.
It has been previously shown that experimental rhinovirus infection induces oxidative stress in mild asthmatics, particularly in those with high eosinophil counts. Depletion of eosinophils resulted in reduced oxidative stress after rhinovirus infection in this group, demonstrating the role of oxidative stress at exacerbations (39). In this study, both live rhinovirus infection and stimulation with the synthetic viral replication intermediate poly(I:C) induces antioxidant expression in bronchial epithelial cells obtained from diseased individuals. Considering the need for a model that mimics both the initial impact of viral infection, which produces an oxidative burden, but also the ensuing infectious state we pre-treated bronchial epithelial cells from asthmatics and COPD patients with H 2 O 2 to produce a low level oxidative stress followed by stimulation with poly(I:C) as an infection-like burden. These interaction experiments did not stimulate cell death and release of cellular material as suggested by LDH measurements. The H 2 O 2 -poly(I:C) co-stimulation dose-dependently reduced gene and protein levels of SOD2 and partially SOD1, thus mimicking clinical findings of antioxidant deficiency at asthma exacerbation (40). This data supports the potential relevance of our in vitro model.
Deficiency of anti-viral interferons has been observed in many cohorts of asthmatics (6)(7)(8). However, mechanisms causing interferon deficiency are largely unknown. We have previously shown that house dust mite impaired anti-viral responses at experimental asthma exacerbations in vitro and in vivo (41). Proteolytic allergens, such as house dust mite, are a known source of reactive oxygen species (16,42), suggesting that oxidative stress could have been involved in the reduced antiviral responses that we observed (41). Here we show that asthmatic bronchial epithelial cells pre-treated with H 2 O 2 have reduced IFNβ expression upon poly(I:C) stimulation. Reduction Further, oxidative stress induced protein levels of IL-1β upon poly(I:C) stimulation, a cytokine which is involved in driving inflammation and a predictor for future asthma exacerbations (46). In contrast poly(I:C)induced protein expression of IL-8 and TNF was reduced by pre-treatment with H 2 O 2 , thus predisposing toward a reduced immune response against infectious agents (47).
We further investigated the effect of H 2 O 2 on anti-viral responses in bronchial epithelial cells obtained from COPD patients. Similarly to asthma the COPD epithelium responded with reduced expression of IFNβ and pattern recognition receptors to oxidative stress, however, the effects were more modest compared to the asthmatic epithelium included in this study. Further, the oxidative stress produced in our model did not affect expression of the antioxidants SOD1 and SOD2 in the COPD epithelium. As oxidative stress is considered a major player in the pathogenesis of COPD (32) it is possible that a high oxidant burden in COPD (48) may make epithelial cells less responsive to further oxidative stress.
We demonstrate that pre-treatment with H 2 O 2 reduced poly(I:C)-induced pattern recognition receptor expression, which is upstream of IFNβ. Hence, we hypothesized that H 2 O 2 might directly affect PRR expression. Employing knockdown studies using siRNA we demonstrated that knockdown of TLR3 completely abrogated poly(I:C)-induced IFNβ expression, while knockdown of RIG-I/MDA5 had no effect on poly(I:C)-induced IFNβ, thus demonstrating the importance of TLR3 for IFNβ signaling. Further, absence of RIG-I/MDA5 did not result in alteration of H 2 O 2 -induced impairment of poly(I:C)-induced IFNβ expression. While these findings need further exploration, they hint at a possible involvement of TLR3 in H 2 O 2 -induced FIGURE 7 | Self-perpetuating circle of inflammation, impaired anti-viral defense, exacerbations, and inflammation in asthma and COPD.
impairment of poly(I:C)-induced IFNβ expression. The present reduction of poly(I:C)-induced RIG-I like helicase expression by H 2 O 2 might thus be due to a IFNβ-mediated feedback mechanism (49).
To our knowledge this is the first study investigating innate immune responses in an in vitro model of H 2 O 2 -poly(I:C) costimulation using primary bronchial epithelial cells. However, as the number of included patients was low further studies are needed to elucidate if the presented findings can be replicated in a larger patient cohort. Further, thorough quantification of the effects of H 2 O 2 -poly(I:C) co-stimulation on protein expression of anti-viral responses is warranted.
In conclusion, we developed a method by which we could show that oxidative stress attenuates anti-viral responses in primary bronchial epithelial cells of asthmatic and COPD patients. These findings could contribute to a better understanding of reduced anti-viral signaling observed at viral-induced exacerbations. Together, our findings suggest a possible self-perpetuating vicious cycle underlying recurrent exacerbations, leading to an impaired anti-viral response, which in turn leads to viral-induced exacerbations, causing more airway inflammation (Figure 7). This might provide part of the explanation for the increased risk of recurrent exacerbations and exacerbation relapses observed in patients with eosinophilic airway inflammation (50). They may further suggest that in order to stabilize asthma patients with recurrent exacerbations, treatment approaches that reduce oxidative stress may be relevant.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the regional ethical review board at Lund University and the review board in Copenhagen. The patients/participants provided their written informed consent to participate in this study.

FUNDING
This study was supported by financial grants from the Swedish Research Council, the Swedish Heart-Lung Foundation and VINNOVA.