Role of IL-33 on Human Mast Cell Responses to Rhinovirus Infection in Asthma

Abstract

Mast cells (MCs) are classically associated with allergic inflammatory diseases, but they are important in parasitic and bacterial immunity. However, their role in viral immunity is less clear. Rhinoviruses (RV) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of RV replication is the airway epithelium and MCs localise to the bronchial epithelium with increasing asthma severity. RV-induced epithelial damage leads to IL-33 release, which promotes MC release of Th2 cytokines and inflammation in the airways. The majority of IL-33 studies have been focused on its role in allergic inflammation in the airways, making it an attractive target for asthma therapy. However, IL-33 may have an overlooked role in being protective, as its role in enhancing anti-viral responses is unknown. In chapter 3, IL-33 stimulated mouse MC transcriptome was analysed from publically available datasets using the limma package in R to identify genes and pathways involved in viral immunity. Several biological processes and pathways involved in both innate and adaptive anti-viral immunity were upregulated in mouse MC datasets. These include upregulation of genes involved in response to virus and upregulation of pathways involved in viral sensing, such as NOD-like receptor, TLR3 and RIG-1/MDA5 signalling. Therefore, IL-33 may have a role in viral immunity in mouse MCs. I hypothesise that IL-33 alters the global response of MCs, including innate immune responses to rhinovirus infection. In chapter 4, conditions for RNA-sequencing was optimised using the human MC line LAD2, which was treated with IL-33 (1-10 ng/ml) plus RV16 (multiplicity of infection (MOI): 1 or 5) or UV-irradiated RV16 as a control over a 24 hour time-course. Gene expression and viral copy number were quantified by RT-qPCR and protein release by Meso Scale Discovery (MSD). These results demonstrate that IL-33 induces anti-viral responses in MCs, which is further enhanced in the presence of RV infection that may be protective. However, RV also enhances IL-33-induced Th2 responses, which may contribute to inflammation and asthma exacerbations. For that reason, RNA-sequencing will elucidate the global response of human MCs to IL-33 and RV infection, which may have implications during RV-induced asthma exacerbations. These data were used to determine the optimal conditions for RNA-sequencing of human MCs which were stimulated with 10ng/ml of IL-33, RV at a MOI of 5 for 6 hours with a minimum sample size of 6. In chapters 5 and 6, the transcriptomic data was analysed using the EdgeR package in R to identify differentially expressed genes to IL-33 treatment, RV infection and co-treatment. Genes of interest were then validated at qPCR and protein level. In response to IL-33 treatment, MCs expectedly displayed a Th2 signature, but they also displayed an anti-viral signature. In response to co-treatment, MCs showed a set of genes not expressed in IL-33 or RV infection alone, which were involved in cell toxicity. These included GZMB1, PRF1 and CRTAM. In response to RV infection, MCs expectedly displayed an immune defense profile. In addition to that, we were also able to identify a potentially unique role for a neuronal gene, ARC. Expression of ARC was linked to RV copy numbers – it could be a novel role in MCs for viral replication and/or recruitment of T cells. Therefore, these results show the global response of human MCs to IL-33 treatment, RV infection and co-treatment. Wherein, MCs may be able to have a protective role during RVinduced asthma exacerbations, in addition to its role in Th2 inflammation. Most importantly, the identification of ARC as an RV-induced gene will enable us to better understand viral replication in MCs and could further add to the protective role of MCs during viral infections.

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