Investigating a role for activity-regulated cytoskeleton-associated protein in the mast cell response to hydrogen peroxide and rhinovirus infection

Abstract

Mast cells (MCs) are immune cells associated with mucosal surfaces where they are known as allergic and inflammatory effector cells. There is increased MC localisation to the airway epithelium of people with asthma, making them ideally located to respond to inhaled challenges that can trigger asthma exacerbations. A key example is rhinovirus (RV) infection, which is a major exacerbator of asthma. MCs are the only immune cell to support RV replication and release, and RV is the only respiratory virus known to replicate in MCs, but there is limited research showing the implications of this on RV-induced asthma exacerbations. Similarly, there is increased oxidative stress at the airway of people with asthma but the MC response to this oxidative stress is poorly understood. Activity-regulated cytoskeleton-associated protein (ARC) has a well-defined role in neuronal activity, but there is little research about a role for ARC outside of the brain. However, ARC is induced by oxidative stress and negatively regulates the heat shock response in HeLa cells, and ARC is associated with the neuronal and MC response to herpes simplex virus (HSV-1) and RV infection respectively. Therefore, ARC is a protein of interest in the investigation of the MC response to environmental challenges and I hypothesise that ARC plays a functional role in the MC response to both oxidative stress (H2O2 treatment) and RV infection.
LAD2 MCs and HeLa cells were treated with H2O2 (2 mM, 1h treatment, 3h or 9h recovery respectively) or infected with RV16 (MOI 1) or a UV-irradiated control prior to quantification of ARC gene and protein expression by RT-qPCR and western blotting. Samples from H2O2-treated and RV16-infected LAD2 MCs were sequenced, and a transcriptomic analysis was used to explore the global response of MCs to these stimuli. Additionally, stable plasmid transfection was used to overexpress ARC in HeLa cells prior to RV infection or H2O2 exposure, using ARC-overexpressing HeLa cells as an initial model to investigate a role for ARC by transcriptomic analysis.
To begin, conditions for H2O2 treatment and RV16 infection were optimised, and these data showed that ARC was induced by H2O2 treatment and RV16 infection of both LAD2 MCs and HeLa cells. Next, a HeLa cell line with stable ARC overexpression was produced and samples were generated for RNA-sequencing and subsequent investigation of a role for ARC in the cellular response to H2O2 and RV16. Transcriptomic data did not clearly indicate a role for ARC in the HeLa cells response to H2O2 treatment or RV16 infection, showing that future investigation of a role for ARC in MCs will require the use a MC model with altered ARC expression to gain further information. Transcriptomic analysis showed that H2O2 treatment upregulated the heat shock response and unfolded protein response in MCs, while ubiquitin proteasome system-mediated degradation was downregulated. This suggested that MCs favour protein refolding and autophagic degradation in their response to oxidative stress, contrasting the proteasomal degradation and upregulated the production of antioxidant systems that were associated with H2O2 treatment of HeLa cells. Transcriptomic data from RV16-infected MCs supported the hypothesis that RV is released from MCs via a non-lytic mechanism, in which RV assembly and maturation occurs at Golgi apparatus-derived membranes. Meanwhile, there was no enrichment of vesicle or Golgi apparatus-related pathways in RV16-infected HeLa cells where RV16 release is thought to occur by cell lysis.
During this project, I generated datasets which provided transcriptomic profiles of the MC response to both H2O2 treatment and RV16 infection. These give novel information about changes in mediator expression, metabolic pathways and general pathways associated with MC responses that can be further investigated in the future to understand how MCs contribute to oxidative stress- or RV16-induced inflammation in the asthmatic airway. Although these data did not elucidate a role for ARC in the cellular response to stimuli, a set of genes were identified that may be expressed downstream of ARC which could link to the role of ARC in non-neuronal cells.

More information

Identifiers

ORCID for Anna Willis: orcid.org/0000-0002-4675-9536

ORCID for Emily Swindle: orcid.org/0000-0003-3644-7747

ORCID for Cornelia Blume: orcid.org/0000-0001-6133-7318

ORCID for Tilman Sanchez-Elsner: orcid.org/0000-0003-1915-2410

Catalogue record

Export record