Human Rhinovirus A16 Infection of Mast Cells Promotes T2 and Non‐ T2 Immune Responses That Are Modulated by the Airway Epithelium

Respiratory viral infection with major serotype human rhinoviruses (RVs) such as RV A16 (RV16) serves as a common trigger for acute asthma exacerbations and can generate a strong type-2 (T2) immune response in the lower airways of some individuals with asthma 1. Although much of this response is presumably coordinated by RV16 infection of airway epithelial cells (AECs), several immune cell populations that express the intracellular adhesion molecule-1 (ICAM-1) necessary for RV16 cell entry exist within the airway epithelial compartment of individuals with asthma and thus also have the capacity to become directly infected with RV16 2, 3, which may be an unrecognized mechanism of viral-mediated airway inflammation. We have been particularly interested in the role of mast cell (MC) populations in the airway epithelial compartment (intraepithelial MCs) in asthma as they are key sources of T2 cytokines in the airway, have been associated with defining physiologic and inflammatory features seen in asthma, and engage in considerable crosstalk with AECs 4-6. However, MC responses to viral infection are not well characterized, which prompted us to examine the effect of RV16 infection on MCs in isolation and in the context of interactions with AECs. We measured RV16 replication and gene expression of Laboratory of Allergic Diseases-2 (LAD2) MCs following RV16 infection (Multiplicity of Infection (MOI) 1.0). LAD2 MCs infected with RV16 were cocultured with primary AECs in organotypic culture from healthy donors (n = 5). MC gene expression was examined by qPCR and RNA-sequencing at multiple timepoints. Primary AECs were isolated from tracheal segments that were collected from a discarded segment of donor airway at the time of lung transplant, which is not considered human subjects research requiring informed consent per review by the University of Washington Human Subjects Review Committee. Materials and methods are further described in the Supporting Information. RV16 infection of MCs cultured in isolation resulted in increased expression of T2 genes (IL4, IL5, and IL13), which corresponded with RV16 viral replication (Figure S1A–D), as well as increased expression of IL33, IL1B1, and IFNB1 (Figure S2). MC IL13 expression was attenuated following RV16 infection in the presence of blocking antibodies against IL-33, IL-1β, and IFN-β (Figure S2C,E). RV16-infected MCs were cultured in the presence and absence of AECs obtained from healthy donors (Figure 1A). Coculture with AECs had no effect on RV16 viral counts in MCs (Figure 1B) but did result in attenuated T2 gene expression (Figure 1C–E). A more comprehensive analysis of MC transcriptional responses to RV16 infection over time was performed utilizing a weighted gene correlation network analysis (WGCNA) of differentially expressed genes, which generated 26 distinct gene expression modules (Figure 2, Table S1). RV16 infection promoted an anti-viral interferon response (Module 1) that did not change in the presence of AECs (FDR = 0.12). At 4 h and 8 h post-RV16 infection, MCs demonstrated decreased expression of genes related to MC function (Module 3) and increased expression of genes related to apoptosis and TGF-beta signaling (Module 4). At 28 h and 52 h post-RV16 infection, MCs exposed to AECs had higher expression of genes related to cell proliferation (Module 2) but lower expression of genes involved in protein synthesis (Module 9) and aerobic respiratory (Module 21). Overall, we demonstrate that MCs have a strong T2 response following direct infection with RV16, which may be a novel mechanism that contributes to rhinovirus-induced acute exacerbations of asthma. This response required infection with the virus and occurred at least in part through an autocrine loop involving IL-1 family members and IFN-β. Notably, the MC response to viral infection is modulated by the epithelium, with AECs having a dampening effect on T2 immune activation. However, a more comprehensive transcriptional analysis also revealed that culture with AECs in this context supported the expression of multiple factors that are implicated in the perpetuation of inflammation in asthma. These preliminary findings should prompt additional studies evaluating for differential effects of asthmatic AECs on MC responses to virus as well as modulation of MCs and classically defined epithelial-derived mediators (such as IL-33) in in vivo model systems of RV infection. R.C.M., T.S.H., and J.S.D. designed the studies. Y.L., R.C.M., M.L., and M.W.C. performed the experiments. R.C.M., D.G., K.A.D.-M., and M.C.A. performed the RNA-sequencing analysis. R.C.M. and T.S.H. wrote the manuscript. R.C.M., A.M.P., J.S.D., M.C.A., and T.S.H. edited and revised the manuscript. All authors reviewed and approved the manuscript prior to submission. This work was supported by the National Institute of Allergy and Infectious Diseases, U19AI175089, K24AI130263. National Heart, Lung, and Blood Institute, R01HL153979. A.M.P., J.S.D., M.C.A., and T.S.H. report funding from the NIH. K.A.D.-M. reports consulting fees from Seattle BioSoftware and EuropaDX related to computational tool development. T.S.H. reports receiving consulting fees from AstraZeneca. These relationships with private companies are not relevant to the current study. All other authors have no conflicts of interest or disclosures. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1: Materials And Methods. Figure S1: Human rhinovirus A16 (RV16) infection induces mast cell (MC) type-2 (T2) gene expression. (A-C) PCR analysis was performed on Laboratory of Allergic Diseases-2 (LAD2) MCs that were infected with RV16 (multiplicity of infection of 1.0), ultraviolet (UV)-inactivated RV16 (UV-IA RV16), or received a media control for 24 and 48 h. IL4 (A), IL5 (B), and IL13 (C) expression levels in comparison to the housekeeping gene HPRT1 (IL4 and IL5 n = 3 per condition, IL13 n = 4 per condition; each condition's PCR value represents the mean of three PCR reactions). Gene expression is normalized to baseline expression in LAD2 MCs. Mean values are shown with error bars representing the standard error of the mean. P values represent the result of two-way ANOVA analyses. *** indicates a P value < 0.001, and **** indicates a P value < 0.0001. (D) RV16 copy numbers between LAD2 MCs exposed to RV16, UV-IA RV16, or media control. In these experiments, no additional steps were taken to remove supernatant during the time course, which may have contributed to low-level viral copy numbers identified in MCs exposed to UV-IA RV16. P value represents results of a multiple comparisons analysis from a 2-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *** indicates a P value < 0.001, and **** indicates a P value < 0.0001. Figure S2: Human rhinovirus A16 (RV16) infection induces mast cell (MC) type-2 (T2) gene expression via autocrine signaling by IL-33, IL-1β, and IFN-β. (A, B, D) PCR analysis was performed on Laboratory of Allergic Diseases-2 (LAD2) MCs that were infected with RV16 (multiplicity of infection (MOI) 1.0) or received a media control for 4, 8, 12, and 24 h. IL33 (A), IL1B (B), and IFNB1 (D) expression levels in comparison to the housekeeping gene HPRT1 (n = 4 per condition, each data point represents the mean of three PCR reactions). Gene expression is normalized to baseline expression in LAD2 MCs. Mean values are shown with error bars representing the standard error of the mean. P value represents results of a multiple comparisons analysis from a 2-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (C, E) PCR analysis of IL13 expression relative to HPRT1 in LAD2 MCs following RV16 infection for 24 h in the presence of blocking antibodies against IL-33 and/or IL-1β (C) or IFN-β (E). P values represent the results of a multiple comparisons analysis from a 1-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. * indicates a P value < 0.05, ** indicates a P value < 0.01, *** indicates a P value < 0.001, and **** indicates a P value < 0.0001. Table S1: List of 5582 differentially expressed genes in mast cells (MCs) in our linear model (Timepoint*Coculture) included in the 26 modules from the weighted gene correlation network analysis. Pathway enrichment analysis of individual module gene sets were performed using the Hallmark pathways and the Gene Ontology (GO) C5 biological pathways and using Fisher's exact test in BIGprofiler from the R package SEARchways. Significant pathways were analyzed at an FDR < 0.25. Pairwise comparisons of individual modules from the weighted gene correlation network analysis at individual timepoints and based on the presence or absence of airway epithelial cells. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.