The role of gutmicrobiota composition, epigenetics and autoimmunity in the development and treatment of vasculitis; the VASKIR biobank

Relevance: In the Netherlands, 100-300/100.000 people have some form of 
autoimmune systemic vasculitis which includes all types of vasculitis as 
defined by the Chapel Hill classification (1). Large-vessel vasculitides (LVV) 
include giant cell arteritis (GCA) and Takayasu arteriitis. Small-vessel 
vasculitides (SVV) are mainly associated with anti-neutrophil cytoplasmic 
antibodies (ANCA) and include granulomatosis with polyangiitis (GPA), 
microscopic polyangiitis (MPA) and eosinophilic GPA (EGPA). Other types 
affecting different vessel sizes (also termed variable vessel vasculitis) are 
Behçet*s disease, IgA vasculitis (formerly known as Henoch-Schönlein purpura), 
polyarteriitis nodosa (PAN), vasculitis associated with systemic lupus 
erythematosus (SLE), vasculitis due to systemic sclerosis and Sjögren's disease. Despite a clear 
improvement in outcome over the last decades and upcoming novel therapeutics 
(2), vasculitis is associated with considerable morbidity and mortality (3, 4). 
Improved survival coincides with an increased risk of side-effects from 
intensive long-term immunosuppression, resulting in a high incidence of 
infections. Better and structured follow-up of vasculitis patients is needed, 
to better categorize them on the road to patient tailored medical therapy.

Background: Autoimmunity is the hallmark of all types of vasculitis, and this process may 
originate from an immune response to gut microbiota. Indeed, the composition of 
gut microbiota was found to be altered in several studies including different 
types of vasculitis (5), but diagnostic and predictive values remain to be 
established. Nevertheless, molecular mimicry and microbiota driven antibodies 
are thought to play a role in vasculitis (6, 7). Activation of innate immunity 
by intestinal microbes may be critical for accelerating vasculitis by expanding 
both T-helper 1 (Th1) and T-helper 17 (Th17) cells in the small intestine 
(8-10). Another mechanism linking the microbiome to immunological tone are 
microbial metabolites (11) and subsequent epigenetic modification (12). While 
most microbiome research focused on bacteria, gut viruses (virome) and fungi 
(all present in fecal samples) are also implicated in development of vasculitis 
because of consequent T-cell activation and exhaustion (13). These parameters 
are influenced by the different types of treatment used (14). 
Thus the association between the gut microbiome/virome, T-cell exhaustion and immuno-tolerance in autoimmune vasculitis constitutes an important knowledge gap withholding a therapeutic target that will be addressed in this prospective cohort study. 

Primary Objective: Primary: Gut microbiota (oral and fecal) and nasal 
microbiota composition in relation to autoimmunity status (antibodies (ANA, 
ANCA) and HLA subtype) and inflammatory functional assays as well as disease 
activity parameters in patients with autoimmune diseases potentially leading to vasculitis.

Secondary Objective:
Gut microbiota (oral and fecal) and nasal microbiota composition in relation to:
• Questionnaires about abdominal complaints (to rule out intercurrent 
gastrointestinal infections), quality of life
• Efficacy of medication in relation to microbiota changes as well as on 
circulating immune cell panel (including T-cells, B-cells, neutrophils and 
monocytes measured by flow cytometry and functional assays) and plasma 
metabolites
• HLA type by high resolution sequencing 
• DNA buffycoat (whole genome sequencing and epigenetics)

This will be a cross-sectional observational cohort study. This study is 
performed in the outpatient setting. Individuals with autoimmune diseases potentially causing vasculitis >18 years old will be invited to participate and included if eligible and willing to participate. A cross-sectional design is sufficient to establish whether individuals with vasculitis carry a distinct microbiome and whether these 
individuals have altered circulating immune-cell and/or (epi)genetic signature. Moreover, we aim to study whether changes in gut, nasal and oral microbiota composition and plasma metabolite profiles are associated with outcome and treatment efficacy.

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On day 1, subjects will collect a urinary morning sample and a fecal sample. A nosal and oral swab will be collected. Furthermore, participants will fill in questionnaires and food diaries. The participants will be burdened by the discomfort associated with collecting and storing urine, fecal and nose and oral swab samples. Furthermore, the questionnaires inquire about the burden of complications, abdominal complaints and comorbidities. This may be distressing as the participant is reminded that they are at risk of developing these conditions. These data, however, also result in a thorough check-up of patients. On day 2 (visit to outpatient clinic for regular clinical care), participants will be asked for ablood sample taken during the blood drawn during usual care. Oral microbiome will be collected at different sites. Microbiome from tongue, hard palate and buccal mucosa will be collected with sterile sampling swabs. These sites will be rubbed for 5-10 seconds. If any bleeding occurs, the sample will be discharged. For nose swabs, after the patient has blown their nose, a cotton swab will be introduced in the nose 2cm and turned 2-3 times. Patients will undergo a fibroscan to measure extent of liver fibrosis, if any.
On day 3 (regular clinical care visit 1 month after any new treatment initiation (or dose increase because of relapse) fecal and plasma samples are collected again to study if treatment efficacy is related to changes in gut microbiota and plasma metabolites. We argue that the risk and discomfort associated with this study is similar to the yearly auto-immune disease check-up and justified in light of the potentially profound insights and novel treatments to be gained by studying the impact of the gut microbiome on autoimmunity status in (auto-immune diseases that could cause) vasculitis.
If explicitly agreed upon by the patient, after 1, 5 and 10 years after inclusion, research staff will ask participants about relapse rate, and whether cardiovascular events have occurred (i.e. myocardial infarction, hospitalization for unstable angina, hemorrhagic and ischemic cerebrovascular event, transient ischemic attacks and arterial revascularization procedures).