By using paired nasal and bronchial epithelial cells from subjects with various health conditions, we compared their responses to rhinovirus infection in the absence and presence of IL-13 treatment. Inclusion of human subjects with various disease states in the current study was aimed to broaden the implications of our research findings regarding the use of nasal epithelial cells in the research of various lung diseases. We found that both cell types have similar antiviral and pro-inflammatory responses. Specifically, nasal epithelial cells demonstrated similar IP-10, Mx1 and eotaxin 3 responses as bronchial cells to rhinovirus and/or IL-13 under ALI culture or submerged culture conditions.
Whether nasal epithelial cells can be used as a minimally invasive alternative to bronchial epithelial cells in the study of pathogenesis of asthma or other lung diseases remains uncertain. In our current study, we attempted to clarify this question by providing the following data. First, we utilized the cell culture model combining rhinovirus infection and IL-13 treatment, and found that viral infection in both cell types increased the expression of IP-10, although the increase was only significant in the ALI culture . In the literature, both submerged and ALI cultures have been used to study the mechanisms of rhinovirus infection. Their results are not always consistent. To clarify if the discrepancies of research data are related to the culture methods, it is important to perform both cultures in the same subjects. Second, the effect of IL-13 on viral load is similar in both cell types. Third, eotaxin 3 production following IL-13 treatment in both cell types is similar in the absence or presence of viral infection. Collectively, our data suggest that in the setting of airway epithelial exposure to rhinovirus 16 and type 2 cytokine IL-13, nasal epithelial cells may serve as an alternative to the invasive bronchial brushing procedure. However, future studies are necessary to further validate the use of nasal cells in studying other pathogenic conditions of lower airways such as exposure to additional cytokines, allergens and other pathogens including respiratory viruses.
Our current study is unique in several aspects. First, we performed both ALI and submerged cultures in order to clarify the controversial data in the literature regarding the use of nasal epithelial cells in the study of lower airway diseases. Comer et al.  used both submerged and ALI cultures to test the response of bronchial and nasal cells from COPD patients to LPS and cigarette smoke extract, and concluded that nasal cells could not be surrogates for bronchial cells based on their different levels of production of pro-inflammatory cytokines. However, our data support the use of nasal cells to indicate the responses of bronchial epithelial cells to rhinovirus infection. Thus, the types of treatments may affect the conclusions. Second, we sought to address the controversy about the effects of IL-13 on viral load and the antiviral genes such as Mx1. Some studies have found IL-13 increases viral load in airway epithelial cells in ALI cultures . Our findings in ALI cultures are contradictory to this publication in that a decrease in viral load was observed in both nasal and bronchial epithelial cells exposed to chronic, but not acute IL-13 treatments. However, in submerged culture, we found the opposite results because treatment with IL-13 trended to increase viral load. The decrease in viral load in ALI cultures could be due to the increased amount of mucous goblet cells in chronic IL-13-treated cells [17, 18]. These findings may not support the idea that asthmatic patients are more susceptible to viral infection because of a decreased interferon response [19, 20]. However, our data are consistent with those showing similar levels of viruses in airways of HRV-infected asthmatics with an exacerbation as compared to the normal subjects with HRV infection . Murine studies also showed similar effects of IL-13 on viral titer of other strains of respiratory viruses. Zhou W et al.  demonstrated that IL-13 transgenic (overexpressing) mice decreases respiratory syncytial virus (RSV) titer along with IFN-γ production in the lungs compared to the wild-type mice. Likewise, IL-13 knockout mice showed an increase in RSV titer.
There were heterogeneous responses of two cell types to HRV, even from the same subject. The heterogeneous responses were seen in both viral load and Mx1 expression. We speculate that these may be related to the degree of their susceptibility to HRV infection. Previous studies suggest that rhinoviruses replicate favorably at a lower temperature in the nasal cavity. Mechanistically, a study of mouse airway epithelial cell culture with HRV1B infection demonstrated that low temperature (i.e., 33°C) vs. warm temperature (i.e., 37 °C) allows more viral replication due to less induction of antiviral genes . In our current study, we initially infected both nasal and bronchial epithelial cells cultured at the ALI for 90 min at 34.5 °C, a temperature reflecting that in the nasal cavity. We then maintained the cell culture at 37 °C, a temperature best supporting the growth of epithelial cells. Temperature and differing antiviral gene levels could have resulted in slightly different responses from each cell type in some subjects. Variation in baseline Mx1 expression, as reported in the literature , between the two cell types could be another contributing factor in heterogeneous responses.
We determined the effect of IL-13 on the production of IP-10 which participates in the recruitment of inflammatory cells such as T cells and macrophages [7, 25]. As IP-10 is considered a biomarker for active rhinovirus infection in asthma [7, 25], it is important to uncover if IL-13 has any impact on IP-10 production in both injured (basal cells maintained in submerged cell culture) [13, 14] and intact (ALI cultured well-differentiated cells) airway epithelial cells. Although HRV16 significantly induced IP-10 production in ALI cultures, acute or chronic IL-13 treatment had no significant effect on IP-10 levels in both nasal and bronchial epithelial cells under submerged and ALI conditions. This finding is intriguing given the fact that rhinovirus alone increased IP-10, and IL-13 chronic treatment in virus-infected ALI cultured cells decreases viral load. We speculate that viral load is mainly controlled by the amount of goblet cells induced by IL-13, while IP-10 production may come from non-goblet cells (e.g., ciliated cells) that are more prone to viral infection with subsequent IP-10 production. Such a hypothesis could be tested in future studies using other experimental approaches such as single cell RNA sequencing to define the co-expression of the viral gene and IP-10 or other antiviral genes in different subtypes of nasal and bronchial epithelial cells under ALI cultures.
In addition, we determined if rhinoviruses had an impact on IL-13-induced eotaxin 3 production in paired nasal and bronchial epithelial cells. As eotaxin 3 is critical to airway eosinophilic inflammation , our data may unravel the role of viral infection in modulating eosinophilic inflammation. Although IL-13 clearly increased eotaxin 3 production, rhinoviruses had no significant effect on its levels in both nasal and bronchial epithelial cells, suggesting that viral infection may not further enhance eosinophilic inflammation in type 2 inflammation-high airways. We chose to measure eotaxin 3 in the current study because both eotaxin 2 and eotaxin 3 are similarly up-regulated by IL-13, and eotaxin 1 levels have not been shown to differ in normal subjects and asthmatics with the differing severity of the disease [27, 28]. Our finding of no up-regulation of eotaxin 3 by HRV infection is consistent with our recent publication . Although HRV infection alone has been reported to increase eotaxin and eotaxin 2 , this finding is still controversial, as cell culture studies by other research groups have not been able to show the similar data [31, 32].
Our study used the rhinovirus 16 and IL-13 treatment model to test whether nasal epithelial cells could be used as surrogates for bronchial epithelial cells in order to reduce the risks of bronchoscopy and the burden on patients. We are aware that other experimental models such as those related to airway remodeling could be tested in the future to compare these two cell types. Also, this study does not address the mechanisms by which nasal and bronchial epithelial cells have similar responses to viral infection and IL-13 treatment. For example, it is unclear whether there is similar cellular composition between the cell types that could affect their responses to HRV16 and IL-13. Finally, whether findings from our cell culture studies can be applied to the more complex in vivo models warrants further investigation.