Rhinovirus Induces Basolateral Release of IL-17C in Highly Differentiated Airway Epithelial Cells

Abstract

Human rhinovirus (HRV) is a major trigger of acute exacerbations of both asthma and chronic obstructive pulmonary disease. The airway epithelium is the primary site of HRV infection, and responds by releasing proinflammatory and antimicrobial cytokines. Epithelial cells release IL-17C in response to exposure to bacterial, viral, and fungal pathogens. We previously demonstrated a role for HRV in IL-17C production from undifferentiated epithelial cells, and showed that IL-17C could play a role in neutrophil recruitment. To extend these observations, highly differentiated human bronchial epithelial cells (HBE) were infected apically with HRV to assess the effect of dose, time, viral replication, and strain on the IL-17C response. Cellular lysates, and basolateral and apical secretions were analyzed for IL-17C and CXCL1 protein release following HRV or IL-17C stimulation. Upon HRV infection, IL-17C protein was exclusively released basolaterally in a dose-, time-, and viral replication-dependent manner. Several strains of rhinovirus were capable of inducing IL-17C release. Enriched columnar epithelial cell populations contained significantly higher viral titer, and expressed significantly more IL-17C mRNA than enriched basal cell populations. In addition, the kinetic profile of IL-17C release following HRV treatment closely mimics viral shedding kinetics, further implicating the role of rhinovirus replication in IL-17C production. Basolateral treatment of HBEs with IL-17C resulted in a dose-dependent increase in basolateral CXCL1 production. In summary, replicating rhinovirus drives basolateral IL-17C protein release from both apical and basal epithelial cells, which may then act in an autocrine/paracrine manner to promote basolateral CXCL1 protein release.

Keywords: IL-17C; air-liquid interface; airway epithelium; basolateral secretion; rhinovirus; well-differentiated.

Figure 1

IL-17C is released basolaterally in response to HRV-1A in a dose- and replication-dependent manner. Highly differentiated HBE were treated with control medium (Con), with increasing doses of HRV-1A (10⁶-10⁹ HRV copies) or 10⁹ copies of replication-deficient HRV-1A (UVHRV) for 4 h. (A) Apical wash and (B) basolateral medium were collected at the time of harvest and analyzed for IL-17C release (n = 4). Significant differences were assessed using a Kruskal-Wallis ANOVA with Dunn's multiple comparisons post-hoc test and indicated with asterisks. **p < 0.01.

Figure 2

The daily rate of basolateral IL-17C release peaks between 12 and 48 h following HRV-1A exposure. Highly differentiated HBE were treated apically with 10⁸ copy number HRV-1A for 12, 24, 48, 72, 96, or 120 h and apical wash and basolateral medium were collected every 24 h. Cellular RNA was collected at each time point and represents cumulative levels in the presence of daily apical washes and basolateral medium changes (n = 6–8). (A) IL-17C protein was measured via ELISA in the basolateral medium collected following indicated time periods. (B) Rhinovirus RNA was measured via real-time RT-PCR in the apical washes collected following indicated time periods. (C) Intracellular rhinovirus RNA was a cumulative measurement from t = 0 after daily washes and medium changes, and was measured via real-time RT-PCR in cellular lysates. Significant differences were assessed using a Two-way ANOVA with Holm-Sidak's multiple comparisons test at each time point and indicated with asterisks. *p < 0.05. The detection limit indicates the minimum viral copy number detected with real-time RT-PCR.

Figure 3

Cumulative basolateral IL-17C release remains significantly induced from 24 to at least 120 h following HRV-1A exposure. Highly differentiated HBE were treated apically with 10⁸ copy number HRV-1A for 12, 24, 48, 72, 96, or 120 h and apical wash, basolateral medium, and intracellular RNA was collected at time of harvest (n = 6–8). (A) IL-17C protein was measured via ELISA in the basolateral medium. (B) Apically shed RNA and (C) cellular RNA were isolated and analyzed for HRV-1A copy number. Significant differences were assessed using a Two-way ANOVA with Holm-Sidak's multiple comparisons test at each time point and indicated with asterisks. *p < 0.05. The detection limit indicates the minimum viral copy number detected with real-time RT-PCR.

Figure 4

Basolateral IL-17C release can be induced in response to multiple serotypes of human rhinovirus as can CXCL1. Highly differentiated HBE were treated apically with major group virus HRV-16 (10⁸ copies), minor group virus HRV-1A (10⁸ copies), or C clade virus HRV-C15 (10⁹ copies) for (A,C) 24 h or (B,D) 48 h and basolateral media were collected at the time of harvest (n = 4). Basolateral media were analyzed for release of IL-17C (A,B) and CXCL1 (C,D). Significant differences were assessed using a Kruskal-Wallis ANOVA with Dunnett's multiple comparisons post-hoc test and indicated with asterisks. *p < 0.05, **p < 0.01.

Figure 5

Characterization of trypsin-separated columnar and basal cell enriched populations from highly differentiated cultures of HBE. Highly differentiated HBE were treated with 0.025% trypsin in the apical and basolateral compartment for 20 min at 37°C. The columnar layer was jetted off the insert and collected for RNA isolation. The remaining basal layer was treated with 0.025% trypsin in the apical and basolateral compartments for 10 min at 37°C. Cells from the basal layer were collected separately (n = 7). (A,B) RNA from each subpopulation were isolated and analyzed for markers of columnar cells (FOXJ1/IFT140/MUC5B) and basal cells (KRT5/TP63). Horizontal dashed line represents a value of 1 where genes would be equally distributed between populations. (C,D) Cells were imaged before and after trypsin-separation and stained with alcian blue and haematoxylin counterstain.

Figure 6

Expression of mRNA for IL-17C, IL-17RA, and IL-17RE, and HRV1A genomic RNA in columnar and basal cell enriched populations. Highly differentiated HBE were infected with 10⁸ copy number of HRV-1A for 24 h. Cell fractions enriched for columnar and basal cell populations were isolated as described in the legend for Figure 5 (n = 7). RNA from each subpopulation was isolated and analyzed for (A) IL-17C, (B) HRV-1A, (C) IL-17RA, and (D) IL-17RE. Significant differences were assessed using a Two-way ANOVA with Holm-Sidak's multiple comparisons post-hoc test comparing HRV-1A between cell populations and indicated with asterisks. ****p < 0.0001.

Figure 7

IL-17C acts in a concentration-dependent manner at the basolateral surface to induce basolateral release of CXCL1. Highly differentiated HBE were treated (A) apically or (B) basolaterally with increasing doses of exogenous IL-17C (1–100 ng/ml) for 24 h (n = 6). Apical washes and basolateral media were collected at time of harvest and analyzed for CXCL1 release. Basolateral generation of CXCL1 in response to 100 ng/ml of IL-17C applied basolaterally was significantly different than control. *p < 0.05.