The role of Rab46 in immune-mediated inflammatory diseases

Inflammation is an essential defense mechanism by which the body tries to protect us from infection and injury. However, inappropriate inflammation, where the immune system is active long after the injury has gone, plays a role in many chronic diseases, including rheumatoid arthritis, psoriasis and atherosclerosis. People, who are susceptible to chronic inflammation often, have more than one disease, which suggests there may be a common mechanism that triggers immune cells to act inappropriately.

Many immune cells contain special storage packages called granules, which rapidly release their contents upon stimulation (e.g. injury), in order to generate repair. Release of these contents is highly regulated but aberrant release is associated with many inflammatory-based diseases. Rab46 is a new protein that we know plays important roles in the release of granular contents in cells that, like mast cells, respond rapidly to injury. In addition, variations in the Rab46 gene have been associated with diseases characterized by inflammatory conditions. Therefore, in this study, we aim to investigate the contribution of Rab46 to the release of contents from immune cells and determine if impairment of this protein is common in chronic inflammation.

We will investigate Rab46 using three distinct approaches: First, there is a resource of genetic and health data from over 500,000 patients stored in the UK Biobank. Genetic analysis of thousands of patients, for example with rheumatoid arthritis, will inform us if variations in the Rab46 gene is common to inflammatory disease. Second, we have a special cohort of patients whose mast cells (a specific set of immune cells involved in allergies) are hypersensitive to stimulation and release their contents too readily (Mast Cell Activation Syndrome: MCAS). In approximately 50% of patients with MCAS, the underlying cause is unknown. We know Rab46 is expressed in these cells; thereby we will sequence the genetic material of MCAS patients and compare genetic variations in the Rab46 gene with control groups. Thirdly, we will determine how Rab46 regulates granule mobility in mast cells by using microscopy to compare granule dynamics in mast cells extracted from wild-type mice to those extracted from mice where the Rab46 gene has been deleted.

These studies will provide us with data to determine if Rab46 is an appropriate target for the development of novel therapeutics and/or could be utilized as a diagnostic tool to measure the probability of acquiring chronic inflammatory diseases.

Objective 1: To determine if nucleotide variations in the Rab46 gene (EFCAB4B) are associated with common IMIDs, we will analyse genomic data stored in the UK Biobank from patients with several candidate diseases. We will compare data from patients with rheumatoid arthritis, atherosclerosis and psoriasis to a non-diseased control group matched for age and sex. We will determine any associations between mutations/SNPs in EFCAB4B and susceptibility to one or all disease phenotypes.
Objective 2: To identify associations of Rab46 gene (EFCAB4B) with patient of non-clonal Mast Cell Activation Syndrome (ncMCAS) of unknown aetiology. We have a cohort of patients that are non-responders to omalizumab who exhibit hypersensitivity to mast cell degranulation. After collection of patient samples, we will prepare DNA libraries and whole genome sequencing will be performed using next generation sequencing facilities (NextSeq 500) based at the Leeds Institute of Molecular Medicine. Analysis will be performed by 'Leeds Omics' and focussed on Rab46 but the genomic sequencing data will be available for further GWA studies.
Objective 3: The overarching strategy of this study is to use super-resolution imaging techniques (iSIM and AiryScan) to quantify and measure the co-localisation of distinct populations of granules with Rab46 in human mast cells or mast cells extracted from mouse bone marrow. Cells will be stimulated with IgE, ATP, C3a and TLR ligands and granule dynamics observed in fixed and live cells. The role of Rab46 in mast cell degranulation will be assessed biochemically using cells extracted from a global Rab46 knockout mouse (currently validated and maintained in the McKeown lab) as compared to wild-type littermates.

The proposed grant aligns with the MRC strategic call of using a multidisciplinary team to push the frontiers of human knowledge into understanding common mechanisms underlying immune-mediated inflammatory diseases. The goal of this research is to produce both functional and mechanistic insights into the role of Rab46 in mast cells using patient data, physiologically relevant mast cells and specific knockout mice. Although focussed on mast cell-mediated disease, the use of the UK biobank genomic data will have the potential to present Rab46 as a fundamental player in immune-mediated inflammatory diseases.
For academic groups, determining the mechanisms by which Rab46 contributes to immune-mediated inflammatory diseases will represent a major breakthrough in this field that will not only impact on our understanding of how secretory granules are spatially controlled but also provide truly novel opportunities for future studies regarding the contribution of aberrant degranulation to chronic inflammatory diseases. First, the data on Rab46 as a regulator of signal-coupled secretion will foster further funding applications from immunologists, cellular biologists and researchers of many disease areas and result in the publication of several peer reviewed papers for international dissemination. In addition, the genomic data will encourage further analysis of both common and rare inflammatory diseases and inform the generation of more appropriate mouse models.
In the longer term there is potential to impact on patient health by therapeutically targeting Rab46 activity to manipulate secretory granule degranulation, thereby enhancing the treatment of mast cell activation syndrome. This would support further collaborations and investment with the pharmaceutical industry, clinicians and translational scientists and could involve the exploitation of intellectual property and patenting. As variants in Rab46 have been implicated in many inflammatory diseases, investigations that identify cell-specific mechanisms will ultimately impact patients with other pathologies, such as rheumatoid arthritis, atherosclerosis and psoriasis.
Benefits to the UK economy resulting directly from this research are unknown. However, since chronic inflammatory disorders are so prevalent in the population, a potential to develop a biomarker or a novel therapeutic target will relieve the economic burden associated with these disorders on the National Health Service.
Due to the prevalence of inflammatory disease within the population, these findings will be of interest to the public. Our research findings will be conveyed to the public through our Institute's 'Action group for public engagement' and by presence at the University's public events such as 'Be Curious'.