Rhinovirus is a leading cause of acute respiratory infections and asthma attacks, but infections are also frequently cleared from the nasal mucosa without causing symptoms. We sought to better understand host defense against rhinovirus by investigating antiviral defense in primary human nasal and bronchial airway epithelial cells cultured ex vivo. Surprisingly, upon rhinovirus infection or RIG-I stimulation, nasal-derived epithelial cells exhibited much more robust antiviral responses than bronchial-derived cells. Conversely, RIG-I stimulation triggered more robust activation of the NRF2-dependent oxidative stress response in bronchial cells compared to nasal cells. NRF2 activation dampened epithelial antiviral responses, whereas NRF2 knockdown enhanced antiviral responses and was protective during rhinovirus infection. These findings demonstrate a tradeoff in epithelial defense against distinct types of airway damage, namely, viral versus oxidative, and reveal differential calibration of defense responses in cells derived from different airway microenvironments.

Here, we report fundamental differences in the responses of nasal and bronchial primary human epithelial cells to rhinovirus infection or direct stimulation of the viral RNA sensor RIG-I. We studied primary nasal- or bronchial-derived airway epithelial cells using a culture system that models basal cells, the regional progenitor cells of the airway epithelium central to epithelial defense and repair following mucosal injury (). In cells derived from both sites, RIG-I stimulation triggered activation of well-characterized signaling pathways, mediating protective responses against both viral infection and intracellular oxidative stress. Interestingly, however, nasal cells showed a more predominant interferon response, whereas bronchial cells exhibited a more predominant oxidative stress response. Further investigation revealed evidence for antagonism between activity of the NRF2-mediated oxidative stress response and RIG-I-dependent interferon and ISG defense in epithelial cells and a surprising cytoprotective effect of NRF2 knockdown during RV infection due to decreased viral replication. Based on these findings, we propose a model in which epithelial cell-intrinsic defense mechanisms are tailored for different airway microenvironments to optimize airway protection.

RV first enters the respiratory tract in the nasal passages, but cells and cell lines of bronchial origin are by far more commonly used as experimental tools. Here, we sought to compare antiviral responses of primary epithelial cells cultured from the nasal or bronchial airway mucosa of healthy donors. We were particularly interested in examining nasal epithelial cells because previous work showed that incubating primary airway cells at cool temperature, mimicking the conditions of the nasal passages, dampens antiviral responses triggered by cytoplasmic RNA (). This finding suggests that nasal and bronchial epithelial cells might require different calibration of innate responses to maintain effective antiviral defense in distinct in vivo anatomical microenvironments.

Multiple lines of evidence indicate that innate defenses of airway epithelial cells can efficiently block RV replication and clear infection at its earliest stages. Airway epithelial cells are the target cells within which RV replicates, but RV replication within these cells can trigger powerful innate defense responses, including induction of type I and type III interferons (IFNs) and interferon-stimulated genes (ISGs), programmed cell death, and RNaseL activity, all of which can block RV replication (). Furthermore, epithelial cell interferon responses triggered by RNA virus infection are attenuated in several patient groups susceptible to severe RV illness, including asthmatics and smokers, further supporting the idea that epithelial cell defenses are critically important for optimal control of RV infection ().

Respiratory virus infections cause an estimated 500 million colds per year in the US and contribute to the roughly 2 million annual hospitalizations for respiratory illness () However, recent evidence suggests that presence of respiratory viruses in the nasal passages is even more common but that viruses are often cleared without causing symptoms (). For example, in a recent family surveillance study, respiratory viruses were detected on average 7.3 weeks per year per person, but almost half of infections were asymptomatic (44%;). These data suggest that, in many cases, airway defense responses enable efficient local viral clearance without engaging defenses that lead to symptoms, such as excessive inflammation and mucus production. To better understand the molecular basis of antiviral defense responses in the airway, we have focused on interactions between epithelial cells and rhinovirus (RV), the most frequent cause of colds, asthma attacks, and exacerbations of chronic airway disease ().

Next, we sought to test the effect of enhancing NRF2 activity in primary nasal epithelial cells by mimicking a physiological source of NRF2 activation in the airway, exposure to cigarette smoke. Airway epithelial cells from smokers show enhanced expression of NRF2 target genes compared to non-smokers (), and enrichment of NRF2 targets is also observed upon stimulation of cultured bronchial epithelial cells in vitro with cigarette smoke extract (). To test the effect of NRF2 activation in primary human nasal epithelial cells on RV1B replication, we exposed cells to cigarette smoke extract (CSE) and assessed RV amplification from a low MOI following incubation at 33°C for 40 hr. We observed a significant effect on RV1B replication, which doubled in cells exposed to CSE ( Figure 4 G). CSE exposure was toxic to NRF2 knockdown (KD) cells ( Figure S3 ), consistent with the known importance of NRF2-dependent responses for cell survival of oxidative stress and cigarette smoke exposure in particular (). Therefore, although we could not directly test the role of NRF2 in CSE-dependent increase in RV replication using knockdown, we explored the hypothesis that CSE exposure activates NRF2 and concomitantly suppresses antiviral interferon responses by testing the effect of CSE exposure on the basal and SLR14-induced expression of IFIT2 and NQO1 in these cells. We observed that CSE exposure increases expression of the NRF2 target NQO1 and decreases SLR14-dependent IFIT2 expression ( Figures 4 H and 4I). These findings are consistent with previous studies showing that CSE exposure leads to a decrease in ISG induction and a modest increase in viral replication following RV infection of human bronchial epithelial cells (). Together, these findings suggest that one mechanism whereby CSE exposure may promote RV replication is through antagonism of the interferon response by NRF2 activation. Overall, our findings support a model in which tissue-specific set points and environmental factors decrease or increase the level of NRF2 activation in RV host cells, which in turn promotes or antagonizes the antiviral response of airway epithelial cells ( Figure S4 ).

The observations reported here indicate that, within airway epithelial cells, NRF2 activity antagonizes RIG-I-mediated antiviral signaling. Previous work has shown that alteration in signaling by RIG-I-like receptors can have profound effects on the outcome of RV infection in host cells with robust interferon responses (). Therefore, we sought to probe the effect of modulating NRF2 activity within host cells on RV replication. First, we targeted NRF2 activity using small interfering RNA (siRNA) knockdown in nasal epithelial cells and then examined rhinovirus amplification from a low MOI. Strikingly, at 40 hr post-infection, viral titer was >10-fold higher in supernatants from control cells than NRF2 knockdown cells and virus-induced cytopathic effect was significantly more advanced in control cells than knockdown cells ( Figures 4 A–4C). NRF2 knockdown cells exhibited significantly higher expression of the ISG IFIT2 and significantly lower levels of mRNAs encoding NRF2 and the NRF2 target NQO1 compared to control cells ( Figures 4 D–4F). These findings indicate that NRF2 knockdown cells are protected from RV replication, consistent with the observed enhancement in ISG induction ( Figure 4 D).

In all panels, bars show mean and SEM of 3–6 replicate experimental wells per condition. Graphs show mRNA level relative to level in control, mock-treated cells. Significant differences by unpaired t test are indicated with asterisks:p < 0.05;p < 0.005; andp < 0.0001. The scale bar represents 50 μm. See also Figures S3 and S4

(H and I) Nasal epithelial cells were stimulated with SLR14 and then incubated for 3 hr with medium only or medium containing 2% or 4% CSE. RNA was isolated and levels of mRNA encoding IFIT2 (H) and NQO1 (I) were assessed by qRT-PCR.

(A–F) Primary human nasal epithelial cells (HNECs) were transfected with siRNA targeting NRF2 or RISC-free negative control siRNA and allowed to recover for 48 hr and then infected with RV-1B, MOI 0.05 at 33°C.

Previous studies have reported that NRF2 activation during viral infection in macrophages and dendritic cells is associated with decreased inflammatory and antiviral responses (). This work, combined with the observed relatively lower antiviral response and greater NRF2-mediated response in bronchial cells compared to nasal cells ( Figure 2 ), suggested that NRF2 activation in airway epithelial cells might be antagonizing activation of RIG-I-dependent antiviral responses. To test this hypothesis, we asked whether NRF2 knockdown in bronchial epithelial cells increased RIG-I-dependent interferon and ISG induction. NRF2 knockdown enhanced induction of mRNA encoding IFNλ1 and the ISG IFIT2 following SLR14 exposure ( Figures 3 A–3C). As expected, knockdown of mitochondrial antiviral signaling protein (MAVS), an essential signaling adaptor downstream of RIG-I, abrogated SLR14-dependent antiviral responses ( Figures 3 A–3C). Next, we tested the effect of NRF2 activation on the robust SLR14-triggered interferon and ISG responses observed in nasal epithelial cells. To do this, we pretreated cells overnight with the well-characterized NRF2 activator sulforaphane prior to RIG-I stimulation and then stimulated cells with SLR14 and assessed gene expression after 5 hr. Sulforaphane (SULF) pretreatment significantly reduced interferon and ISG induction upon subsequent stimulation with SLR14 ( Figures 3 D and 3E). Sulforaphane pretreatment led to sustained NRF2 activation, as indicated by induction of an NRF2-regulated gene, glutamate-cysteine ligase catalytic subunit (GCLC), whereas SLR14 did not induce GCLC mRNA under these conditions ( Figure 3 F). These results indicate that NRF2 activity antagonizes interferon induction in both types of airway cells.

(D–F) Primary human nasal epithelial cells were pretreated with 10 μM sulforaphane (SULF) for 18 hr. After 3 hr recovery in medium only, cells were transfected with SLR14. After 5 hr incubation at 37°C, cells were collected for RNA isolation and qRT-PCR for mRNA encoding IFNλ1 (D), IFIT2 (E), or GCLC (F). Graph shows untreated cells with no stimulation (Ctrl) or SLR14 exposure (SLR14) or sulforaphane-pretreated cells with no stimulation (SULF/Ctrl) or SLR14 exposure (SULF/SLR14). Significant differences between control and SULF pretreated cells by unpaired t test are indicated (#p < 0.0001). Bars show mean and SD of 2–4 replicate experimental wells per condition. Graph titles indicate mRNA assessed by qRT-PCR. Results are representative of at least three independent experiments.

(A and B) Primary human bronchial epithelial cells were transfected with siRNA targeting NRF2 or MAVS or with control siRNA (RNA-induced silencing complex [RISC]-free). After recovery for four days, cells were transfected with the RIG-I ligand SLR14 and then incubated for 6 hr at 37°C, followed by RNA isolation and qRT-PCR for mRNA encoding IFNλ1 (A) and IFIT2 (B). Significant difference between transcript levels in control siRNA-treated and NRF2-siRNA-treated cells by unpaired t test is indicated with asterisks ( ∗ p = 0.02; ∗∗ p = 0.003).

Next, we compared the transcriptional changes observed in nasal or bronchial cells following stimulation with the RIG-I ligand SLR14 ( Figure 2 ). Consistent with the known role of RIG-I like receptors (RLRs), stimulation with SLR14 led to enrichment in transcripts associated with the antiviral response, including “interferon signaling,” “activation of IRF by cytosolic PRRs,” and “role of PRR in recognition of bacteria and viruses” ( Figures 2 A and 2B). Interestingly, the other top pathway enriched by RIG-I stimulation was the NRF2-mediated oxidative stress response ( Figures 2 A and 2B). NRF2 is a transcription factor that is activated by oxidative stress in the cytosol, leading to transcription of diverse targets involved in neutralizing reactive oxygen species and restoring homeostasis (). Notably, transcripts related to interferon signaling dominated the response to SLR14 in nasal cells, whereas transcripts related to the NRF2 pathway were more significantly enriched in bronchial cells ( Figures 2 B–2D). As shown on radar plots representing mean fragments per kilobase mapped (FPKM), overall mRNA levels of canonical ISGs were strongly induced and more highly expressed in nasal cells compared to bronchial cells ( Figure 2 C), whereas transcripts encoding antioxidant enzymes, a subset of NRF2 targets, were more highly expressed in bronchial cells compared to nasal cells following RIG-I stimulation ( Figure 2 D). A more complete list of enriched pathways and associated transcripts is shown in Table S1 . The effects of SLR14 treatment were not predictable from the resting transcriptomes, which showed a slight enrichment for both pathways in bronchial cells at rest compared to resting nasal cells ( Figure S2 E).

(C and D) Radar plots show average fragments per kilobase mapped (FPKM) for transcripts contributing to the “interferon signaling” ingenuity pathway (C) or for antioxidant enzymes associated with the NRF2-mediated oxidative stress response ingenuity pathway (D) in SLR14-stimulated (solid line) and unstimulated (dashed line) cells. Gridlines in (C) range from 0 to 500 FPKM (intervals of 100) and gridlines in (D) range from 0 to 200 FPKM (intervals of 50). Throughout, orange lines represent nasal cells and black lines represent bronchial cells.

(A) Dot plot depicts change in expression levels for all transcripts in stimulated versus unstimulated cells. Dots outside the gray box represent transcripts significantly increased (235 for bronchial; 533 for nasal) or decreased (47 for bronchial; 217 for nasal) in response to SLR14 stimulation (Log 2 FC > 1 or Log 2 FC < −1; p-adj < 0.05).

RNA-seq was performed on RNA isolated from two replicate wells of stimulated and unstimulated nasal or bronchial epithelial cells (total of eight samples) following SLR14 stimulation (for 1 hr) followed by incubation for 7 hr at 37°C. Libraries were prepared for paired-end RNA sequencing from two replicate samples per condition.

To assess whether the low-passage primary cells used in this study retained characteristics specific to the site of origin, we examined region-specific mRNA biomarkers. Previous work showed that regional progenitor cells derived from different airway regions retain gene expression patterns reflecting the site of origin within the respiratory tract, and furthermore that progenitor cells can proliferate and differentiate to form a differentiated airway epithelium with characteristics of the site of origin when reintroduced into a 3D matrix (). Using published microarray data (GSE32606), we identified the top 10 differentially expressed genes in nasal versus tracheal-derived airway progenitor cells and examined expression of these transcripts in cells used in this study. The differential expression pattern in our RNA-seq data comparing nasal and bronchial-derived cells largely mirrored the pattern seen previously in nasal versus tracheal-derived regional progenitor cells ( Figures S2 F and S2G). Next, we performed qRT-PCR on nasal and bronchial cells from different donors used in this study and found enrichment of the nasal-associated biomarker FOXG1 in nasal cells from different donors and higher expression of the tracheal and bronchial-enriched mRNA SERPINF1 in bronchial cells from different donors ( Figure S2 H). These results indicate that nasal and bronchial-derived cells cultured under the conditions used in this study retain gene expression patterns reflective of the site of origin within the respiratory tract.

To better understand the differences in the response to RIG-I stimulation in epithelial cells cultured from nasal or bronchial sites, we performed RNA sequencing (RNA-seq) to compare the transcriptomes of resting and SLR14-stimulated cells. First, we examined resting gene expression in both cell types ( Figure S2 ). We observed many commonalities and some differences in resting gene expression ( Figure S2 A). Both nasal and bronchial cells expressed lineage markers of airway basal cells, the self-renewing regional progenitor cells of the airway epithelium ( Figure S2 B). This finding is consistent with previous studies showing that the epithelial cells that proliferate from primary airway mucosa in conventional culture have a basal cell phenotype, with expression of basal cell lineage markers KRT5 and TP63 (). Examination of transcripts for RIG-I and other signaling molecules involved in innate immune recognition of RNA viruses revealed a trend toward equal or slightly higher expression levels in bronchial cells at rest compared to nasal cells ( Figures S2 C and S2D), therefore did not provide an explanation for the phenotype shown in Figure 1 . Ingenuity pathway analysis revealed some small differences in pathway activation at rest but did not reveal a clear-cut reason for the greater interferon response in nasal cells following response to RIG-I stimulation ( Figure S2 E).

To assess whether differences in nasal and bronchial cell responses to RIG-I ligand were due to differences in transfection efficiency, we created a modified SLR14 labeled with Alexa 488 (SLR14-488). Flow cytometric analysis of nasal and bronchial cells following transfection with SLR14-488 revealed an increase in fluorescence to an equivalent degree for both cell types, indicating equivalent transfection efficiency ( Figures S1 C and S1D). Consistent with results seen with unlabeled SLR14 ( Figure 1 ), SLR14-488 stimulated greater induction of mRNAs encoding IFNλ1 and the interferon stimulated gene IFIT2 in nasal cells compared to bronchial cells ( Figures S1 E and S1F). These findings indicate that differences between nasal and bronchial-derived cells in interferon and ISG induction are not due to differences in transfection efficiency.

Previous work showed that incubating primary airway epithelial cells at cool temperature (33°C), mimicking the conditions of the nasal passages, diminished antiviral responses triggered by cytoplasmic RNA, including induction of type I and type III interferons (). These findings suggest that nasal epithelial cells may require adaptations to maintain robust antiviral defense within their naturally cooler local microenvironment in vivo. To examine this hypothesis, we obtained primary nasal or bronchial epithelial cells from healthy donors (commercially; see STAR Methods ), cultured them on collagen under conditions that promote a basal cell phenotype, and then infected with rhinovirus 1B (RV1B). All experiments were performed at low passage number (P3 or fewer). Compared to bronchial epithelial cells, nasal-derived epithelial cells displayed much more robust secretion of the type III interferon, IFNλ1, following rhinovirus infection at 37°C, at a time point when viral load was equivalent in both cell types ( Figures 1 A and S1 ). Consistent with previous studies, when cells were incubated at 33°C, IFNλ1 secretion was greatly reduced ( Figure S1 ). Interestingly, the low but detectable level of IFNλ1 secreted by nasal cells at nasal temperature (33°C) was comparable to levels observed in bronchial epithelial cells at lung temperature (37°C; Figure S1 ). Because the levels of virus replication may contribute to the difference in IFNλ1 levels between the two cell types, we next performed experiments using a non-replicating RIG-I ligand, stem loop RNA (SLR), SLR14, and a short 5′-triphosphorylated RNA ligand of the cytoplasmic innate immune sensor RIG-I (). Following transfection of cells with SLR14, we observed more robust IFNλ1 secretion and induction of mRNA for IFNλ1, IFNβ, and the interferon-stimulated gene OAS1 in nasal cells compared to bronchial cells when both cell types were incubated at the same temperature ( Figures 1 B–1H).

Data are representative of at least 3 independent experiments with airway epithelial cells of each type from two or more different donors. Significant differences between nasal and bronchial cell levels by paired t test are shown with asterisks:p < 0.0005;p < 0.005; andp < 0.05. See also Figure S1

(F–H) Fold change in mRNA for IFNλ1 (F), IFNβ (G), and Oas1(H) at time points 0–8 hr post-stimulation. Bars represent mean and SD of 2 or 3 replicate experimental wells per condition. mRNA level is plotted relative to the level in resting bronchial cells (t = 0 hr).

(B–H) Primary nasal and bronchial epithelial cells were transfected with RIG-I ligand SLR14 for 1 hr and then medium was added and cells were incubated at 37°C. Supernatants were collected for ELISA, and cells were collected for RNA isolation and qRT-PCR at the time points shown.

(A) Primary nasal and bronchial epithelial cells were inoculated with RV-1B, MOI 0.1, and incubated at 37°C for 48 hr, at which time supernatants were collected for ELISA. Bars show IFNλ1 protein in supernatant.

Discussion

Airway epithelial cells provide frontline defense against a variety of potentially harmful substances that enter the airway from the environment, including respiratory viruses and diverse substances that can cause oxidative damage. We found that stimulation of the innate immune sensor RIG-I within airway epithelial cells activates two central mechanisms that protect against these sources of damage: the antiviral interferon response (greater activation in nasal cells than in bronchial cells) and the NRF2-mediated response to oxidative stress (greater activation in bronchial cells compared to nasal cells). Here, we present evidence that NRF2 activation antagonizes the antiviral interferon response in the airway epithelium and evidence for cell intrinsic regulation (i.e., innate differences between nasal and bronchial cells) and environmental regulation (i.e., exposure to cigarette smoke that triggers NRF2 activation) of the balance between these two defense mechanisms in airway epithelial cells.

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Yamamoto M. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. The antagonism we observed between NRF2 activity and interferon and ISG induction in airway epithelial cells fits with antagonism of immune defense by NRF2 observed in other models. NRF2 activity was observed to suppress the interferon response and virus-induced apoptosis in a study of Dengue virus infection in dendritic cells (), and NRF2 knockout led to exaggerated IRF3 and nuclear factor κB (NF-κB) activation in mouse models of sepsis (). NRF2 activation has been shown to dampen production of NF-κB-dependent pro-inflammatory cytokines in diverse settings, and in fact, NRF2 activators are currently used clinically as anti-inflammatory agents (). Reactive oxygen species (ROS) promote cell-intrinsic innate antiviral signaling, including RIG-I signaling (), and neutralization of ROS has been proposed to be the mechanism whereby NRF2 activity suppresses antiviral and pro-inflammatory cytokine production (). Interestingly, ROS may enhance innate immune signaling in part by effects on nucleic acid ligands. For example, oxidation of cytosolic DNA was shown to enhance stimulator of interferon genes (STING)-dependent innate immune signaling due to decreased degradation of oxidized DNA by TREX1 (). It will be interesting to explore how oxidation of RNA ligands or other components of the RIG-I signaling pathway might influence innate immune signaling in future studies. In addition to effects on neutralization of ROS, an additional mechanism of NRF2-dependent suppression of innate immune signaling was proposed recently, in which chromatin immunoprecipitation (ChIP)-seq studies in lipopolysaccharide (LPS)-stimulated macrophages showed suppression of cytokine transcription by binding of NRF2 upstream of cytokine promoters (). The mechanism(s) underlying the antagonism between RIG-I signaling and NRF2 activation in airway epithelial cells will be an important avenue for future investigation. Our findings also emphasize the importance of considering how experimental conditions impact oxidative stress in studies of innate immune signaling.

Olagnier et al. (2014) Olagnier D.

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Hizawa N. Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice. Despite antagonism between antiviral innate immune defense and NRF2 activity, NRF2 activation can have either protective or deleterious effects for the host during viral infection. We observed a cytoprotective effect of NRF2 knockdown during rhinovirus infection in nasal epithelial cells, consistent with enhanced ISG induction and diminished viral replication ( Figure 4 ). However, NRF2 activity was essential for cell survival during CSE exposure ( Figure S3 ). Therefore, any suppressive effects of NRF2 activation on antiviral defense represent a necessary compromise in the presence of both CSE and viral infection. Likewise,observed a protective effect of NRF2 knockdown during Dengue virus infection of dendritic cells. In contrast, during influenza infection of cigarette-smoke-exposed mice, NRF2 knockout diminished host survival (). These results likely reflect the fact that host resistance and clearance of infection is the best pathway to health in some disease settings, whereas the antioxidant response takes precedence to promote host survival in others, particularly when multiple stressors are encountered at once.

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Molony R.D. Early local immune defences in the respiratory tract. We propose a model in which airway epithelial cells’ responses to airway injury may be differentially regulated to optimize defense responses in distinct microenvironments and can vary depending on the recent exposures of the cell. As shown in Figure S4 , in our model, virus-induced RIG-I signaling leads to activation of both the antiviral interferon response and the NRF2-dependent oxidative stress response. In nasal epithelial cells, there is an inherent bias toward less NRF2 activation and greater activation of the interferon and ISG response compared to bronchial cells ( Figure S4 A). This bias may serve to enable nasal epithelial cells to maintain robust antiviral defense in the relatively cool temperatures of the nasal passages ( Figure S1 ). This bias may also reflect the fact that the nasal epithelium is also the initial site of entry for many respiratory viruses and a site in which the consequences of local epithelial damage and inflammation are relatively low. In contrast, in the bronchi, maintaining tissue integrity and suppressing inflammatory responses to keep the large airways open is of paramount importance, fitting with the observed difference in set point favoring the NRF2 response in bronchial epithelial cells. It is also important to note that this study models defense responses of airway basal cells from different airway microenvironments, but in vivo, the relative proportions of differentiated epithelial cell types likely also play an important role in airway region-specific defense, such as the increased proportion of mucus-producing cells in the nasal passages compared to the bronchi ().

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et al. Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes. In our model, in addition to inherent region-specific adaptations of progenitor cells from different airway sites, recent environmental exposures can further influence the balance between defense responses in the airway epithelium. For example, the NRF2 response would be expected to be activated in cells exposed to sources of oxidative stress in the airway, which could include environmental pollutants, such as diesel exhaust, cigarette smoke, microbial metabolites, or the byproducts of oxidative metabolism by resident cells or infiltrating leukocytes. Although NRF2 activation would move cells to a new state of adaptation that enhances cell survival during oxidative stress, such cells would be maladapted to viral infection due to dampening of the antiviral interferon response ( Figure S4 B). We modeled this scenario in this study by exposing nasal epithelial cells to cigarette smoke extract, resulting in increased NRF2 activity and decreased RIG-I signaling ( Figures 4 G–4I). Previous studies have shown diminished antiviral responses in bronchial epithelial cells following cigarette smoke exposure (); our results indicate that a possible underlying mechanism for this finding is antagonism between NRF2 activation and antiviral signaling.

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Noah T.L. Reduced expression of IRF7 in nasal epithelial cells from smokers after infection with influenza. Importantly, there is considerable evidence that epithelial cells from patient groups susceptible to severe rhinovirus infection have a depressed antiviral interferon and ISG response. Our findings suggest that activation of the NRF2-mediated oxidative stress response in these cells could be the mechanistic basis for this phenotype. There is evidence supporting this model in one such patient group: smokers. Published data from smokers reveal a striking transcriptional signature of NRF2 activation (). Chronic obstructive pulmonary disease (COPD) and asthma patients are also highly susceptible to serious illness following RV infection, and in fact, RV is the top trigger of childhood asthma exacerbations (). Ex vivo experiments have shown evidence for defects in cell-intrinsic innate immunity in airway epithelial cells from asthmatics and smokers compared to healthy controls (). Our findings suggest that it will be important to investigate whether an aberrant shift toward NRF2-mediated antioxidant defense could underlie the observed defect in epithelial antiviral defense, leading to increased RV susceptibility in these patients.

In summary, the findings reported here demonstrate antagonism between two key defense mechanisms in airway epithelial cells and demonstrate how the activity levels of these responses are tailored to different set points in cells derived from different airway regions (nasal versus bronchial). We also demonstrate that NRF2 activation by an environmental oxidative stress can shift this balance and create vulnerability to rhinovirus infection. These results compel further investigation of the role of NRF2 activation in RV-susceptible patient groups and indicate that finding ways to protect the airway epithelium from intracellular oxidative stress, and thereby avert NRF2 activation, may lead to effective strategies to enhance natural defense against rhinovirus infection.