Inflammatory biomarkers in ANCA-associated vasculitis

Background: Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a heterogenous group of relatively rare and serious small vessel vasculitides, characterized by the presence of proteinase 3 (PR3) ANCA or myeloperoxidase (MPO) ANCA. The disease manifestations vary widely, from a localized form to a serious disease with multi-organ failure. When untreated the mortality rate is high, around 80% in the first year from diagnosis. Improvements in treatment have resulted in better prognosis but disease relapses, comorbidities and treatment side effects remain a challenge.

Aims: the aims of this thesis were the following: 1) to identify potential non-invasive biomarkers that could aid in the evaluation of disease activity in AAV, and 2) to explore the role of these potential biomarkers in the pathogenesis of AAV.

Material and methods: Patients from a well characterized cohort of AAV patients were included in the studies. For comparison control samples from population-based cohorts were used. Biomarkers were measured in urine and/or plasma/serum. Analysis of extracellular vesicles (EVs) was performed with flow cytometry. Thrombin generation was evaluated with a modified Calibrated Automated Thrombogram (CAT) assay after addition of EV-enriched pellets. The disease activity was assessed with the Birmingham Vasculitis Activity Score (BVAS). Immunohistochemical staining was carried out on kidney biopsies and the well-established Berden score was used for histopathological classification.

Study I: MPO-positive extracellular vesicles (MPO+EVs) expressing pentraxin-3 (PTX3), high mobility group box 1 (HMGB1) protein, and tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were analysed in 46 AAV patient, (whereof 23 patients had an active disease) and 23 controls. Serum HMGB1, PTX3 and TWEAK levels were also measured. Levels of MPO+EVs expressing all investigated biomarkers were significantly higher in patients compared to controls. Levels of MPO+EVs expressing PTX3 and HMGB1 were significantly higher in active compared to inactive disease and correlated with BVAS. Moreover, serum PTX3 levels were higher in patients with active compared to inactive disease and correlated with BVAS but the same was not found for TWEAK or HMGB1.

Study II: Plasma PTX3 levels were measured in 79 patients with active AAV at baseline and 6-month follow-up as well as in 23 controls. Urinary PTX3 levels were measured in 34 of the patients. The plasma and urinary PTX3 levels were significantly higher at baseline compared to follow-up and correlated with BVAS at baseline. Both the plasma and urinary PTX3 levels correlated to albuminuria and the estimated glomerular filtration rate. Moreover, patients with kidney involvement had higher plasma and urinary PTX3 levels compared to those without.

Study III: Serum TWEAK levels were measured in 74 patients with active AAV at baseline and 6-month follow-up as well as in 20 controls. Urinary TWEAK levels were measured in 69 patients. Urinary TWEAK levels were higher at baseline compared to follow-up and correlated with BVAS at baseline. Patients with kidney involvement had higher urinary levels compared to those without and a correlation between urinary TWEAK and albuminuria was found. Serum TWEAK levels were higher in patients at inclusion compared to follow-up but did not correlate with BVAS. A significant difference in serum TWEAK levels in AAV patients compared to controls was not seen. Immunohistochemical staining of kidney tissue from AAV patients showed a clear expression of TWEAK and a weaker staining for fibroblast growth factor-inducible 14 (Fn14) (TWEAKs receptor).

Study IV: MPO-positive extracellular vesicles (MPO+EVs) expressing tissue factor (TF), citrullinated histone-3 (H3Cit) and plasminogen (Plg) were analysed in 46 patients with AAV (whereof 23 had active disease) and 23 controls. Concentrations of MPO+EVs expressing TF and H3Cit were significantly higher in patients with active compared to inactive disease and controls. Concentrations of MPO+EVs expressing Plg were higher in the total AAV group compared to controls but there was no difference between active and inactive patients. Concentrations of MPO+EVs expressing TF correlated with disease activity. Thrombin generation was augmented in AAV patients compared to controls and a correlation between MPO+EVs expressing TF and H3Cit and the parameters of thrombin generation was found.

Conclusions: Our findings indicate that circulating and urinary PTX3, urinary TWEAK as well as EVs expressing PTX3, HMGB1 and TF could be useful biomarkers in AAV. Thrombin generation is augmented in patients with AAV and is associated with concentrations of MPO+EVs expressing TF and H3Cit. These findings suggest that these biomarkers may have a role in the pathogenesis of the hypercoagulability seen in AAV.

List of scientific papers

I. Manojlovic M, Juto A, JONASDOTTIR A, Colic J, Vojinovic J, Nordin A, Bruchfeld A, Gunnarsson I, Mobarrez F, Antovic A. Microparticles expressing myeloperoxidase as potential biomarkers in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Journal of Molecular Medicine. 2020; 98:1279-1286.
https://doi.org/10.1007/s00109-020-01955-2

II. JONASDOTTIR AD, Antovic A, Qureshi AR, Nordin A, Malmström V, Gunnarsson I, Bruchfeld A. Pentraxin-3 – a potential biomarker in ANCA-associated vasculitis. Scandinavian Journal of Rheumatology. 2022; Apr 6:1-9. Epub ahead of print.
https://doi.org/10.1080/03009742.2022.2045790

III. JONASDOTTIR AD, Schwartz A, Söderberg M, Wernerson A, Qureshi AR, Antovic A, Gunnarsson I, Bruchfeld A. Urinary TWEAK reflects disease activity in ANCA-associated vasculitis. [Manuscript]

IV. JONASDOTTIR AD, Manojlovic M, Vojinovic J, Nordin A, Bruchfeld A, Gunnarsson I, Mobarrez F*, Antovic A*. Increased thrombin generation correlates with circulating extracelluar vesicles exposing tissue factor and citrullinated histone-3 in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. *Equal contribution. [Manuscript]