Cell and molecular markers of systemic vasculitis and autoantibodies against serum amyloid A as physiological regulators of the inflammatory responseKuret, Tadeja (Avtor) Sodin-Šemrl, Snežna (Mentor) Čučnik, Saša (Komentor) Božič, Borut (Član komisije za zagovor) Jeras, Matjaž (Član komisije za zagovor) Veranič, Peter (Član komisije za zagovor) systemic vasculitisautoantibodiescell and molecular markersGiant cell arteritis and immunoglobulin A vasculitis represent a group of chronic rheumatic inflammatory diseases that are categorized as primary systemic vasculitides. They are associated with increased morbidity and earlier mortality as a result of severe cardiovascular complications and long-term use of immunosuppressive therapy. Setting the appropriate diagnosis of both giant cell arteritis and immunoglobulin A vasculitis can be challenging due to diverse and unspecific clinical signs and symptoms and, in some cases, atypical clinical presentation. The therapeutic options for patients with both types of vasculitides are scarce, with glucocorticoids being the first choice. However, glucocorticoids can be associated with severe adverse effects and patients may still experience a relapse when glucocorticoid tapering regimen is applied. Both types of vasculitides are characterized by an acute inflammatory response that can be extended for several years due to impaired or inadequate resolution leading to chronic systemic inflammation. Persistently high levels of acute phase proteins (such as serum amyloid A) during the inflammatory process can lead to accelerated development of atherosclerosis, which is the most common underlying pathological process of cardiovascular diseases. Consequently, patients with giant cell arteritis and immunoglobulin A vasculitis have an increased risk of developing cardiovascular disease compared to the general population. Understanding the mechanisms involved in the resolution of inflammation may thus support the development of alternative, inflammation-blocking therapeutic options for these patients, whereas understanding the role of inflammation and serum amyloid A might unveil new insights into the acceleration of the pathogenic mechanism of atherosclerosis. The work outlined in this thesis aimed to: i) improve the diagnostic, prognostic and therapeutic options of patients with giant cell arteritis and immunoglobulin A vasculitis, ii) explore the role of autoantibodies against serum amyloid A as physiological regulators of the inflammatory response and iii) advance our understanding of the interplay between chronic inflammation, serum amyloid A, glucocorticoid treatment and possible development of atherosclerosis. Within the doctoral thesis, we first focused on reviewing the literature to identify potential serological and tissue markers of giant cell arteritis. We examined the strengths and limitations of existing methodology and identified missing gaps in the current literature. In order to foster the up-to-date scientific knowledge on giant cell arteritis biomarkers, we consequently designed and performed three different research studies. In the first study, we discovered that tissue micro RNA-125b, expressed in the temporal artery biopsies, could be used as a biomarker to identify giant cell arteritis patients, in whom the typical histopathological changes in the temporal arteries are not observed and thus improve their diagnostic options. However, to measure micro RNA expression, performing a temporal artery biopsy is required, which is an invasive procedure. We therefore performed a second, cross-sectional study on giant cell arteritis patients where we focused on measuring serum analytes that could be used to better characterize treatment-naïve patients with giant cell arteritis and to predict disease-associated complications. Serum samples are easy to obtain with a minimally invasive procedure and can be a valuable source of biomarkers. We found that therapy-naïve giant cell arteritis patients had significantly higher serum levels of pro-inflammatory cytokines (interleukins-1β, -6, -18, -23, -27 and -31), chemokines (e.g. interleukin-8), acute phase proteins (serum amyloid A, α1-acid glycoprotein), vascular cell adhesion molecule-1 and matrix metalloproteinases-1 and -9, while levels of interleukin-13, matrix metalloproteinase-2 and α-fetoprotein were significantly lower compared to healthy blood donors. The highest fold change elevations between patients with giant cell arteritis and healthy blood donors were observed for concentrations of serum amyloid A (83-fold change), interleukin-23 (58-fold), and interleukin-6 (11-fold). Giant cell arteritis patients with visual disturbances had lower levels of acute phase parameters serum amyloid A, C-reactive protein, haptoglobin and erythrocyte sedimentation rate, compared to patients without visual disturbances. In contrast, the levels of SAA, C-reactive protein, and erythrocyte sedimentation rate were increased in therapy-naïve giant cell arteritis patients with future relapse, compared to the patients without future relapse. Our third, longitudinal giant cell arteritis study, reported on an increased neutrophil surface expression of adhesion molecule L-selectin in peripheral blood of treatment-naïve patients with active disease. The expression of L-selectin rapidly decreased after initiation of glucocorticoid treatment, followed by a progressive increase to week 48, when patients received lower doses of glucocorticoid monotherapy. This progressive increase of L-selectin was not observed in patients under combinatory therapy with leflunomide, which indicates that the addition of leflunomide might have a beneficial long-term effect on the control of neutrophil adhesion. Since erythrocyte sedimentation rate and levels of C-reactive protein are usually used to monitor giant cell arteritis activity but are not always reliable markers, our study also focused on alternative serum biomarkers that might be useful in this regard. Prior to treatment, the relapsing patients had significantly higher median levels of interleukin-23, compared to patients in the responder group. Serum levels of interleukin-23 were also higher in all three relapsing patients, at the time closest to relapse, compared to the last time point before relapse. Levels of interleukin-23 decreased again after relapse and concurrent administration of leflunomide. Interleukin-23 could therefore represent a viable biomarker to monitor giant cell arteritis activity. Similar to patients with giant cell arteritis, therapy-naïve adult patients with immunoglobulin A vasculitis also exhibited higher serum levels of serum amyloid A, interleukin-6 and interleukin-8 compared to healthy individuals, however these increases were not as high as those observed in giant cell arteritis. The inflammatory response, together with increased serum concentrations of immunoglobulin A and higher percentages of neutrophils determined in peripheral blood of immunoglobulin A vasculitis patients in our study, go in line with the theory of existing cross-talk between immunoglobulin A, endothelial cells and neutrophils in the pathogenesis of immunoglobulin A vasculitis that ultimately leads to substantial vascular damage. Our study confirmed the association of neutrophil to lymphocyte ratio with clinical manifestations involving gastrointestinal tract in patients with immunoglobulin A vasculitis. We also found that patients with gastrointestinal tract involvement had a lower expression of neutrophil adhesion molecule integrin αM, compared to patients with skin-limited presentation that would need further validation to be used as a marker of systemic involvement. Our results show that high- or medium-grade inflammatory response is present in therapy-naïve patients with giant cell arteritis and immunoglobulin A vasculitis, respectively, with upregulated levels of acute phase proteins, especially serum amyloid A. If the inflammatory response does not resolve, it can lead to chronic inflammation with significant organ damage and accelerated development of atherosclerosis. One of the potential mechanisms involved in the resolution of inflammation may include autoantibodies against acute phase proteins that could neutralize the pro-inflammatory activities of their respective antigens or promote their clearance. To check this hypothesis, we optimized an in house enzyme-linked immunosorbent assay for the detection of immunoglobulin G autoantibodies against human recombinant serum amyloid A1α (endogenous human protein) and human recombinant serum amyloid A (a hybrid between forms A1α and A2β). The presence of both autoantibodies was detected in serum samples of 300 healthy blood donors. The peak levels of autoantibodies against serum amyloid A and serum amyloid A1α were determined in individuals between 41 and 50 years. We also found that autoantibodies against both proteins are present in the therapeutic preparation of intravenous IgG and could be isolated using affinity chromatography. The isolated immunoglobulin G fractions of anti-serum amyloid A and anti-serum amyloid A1α autoantibodies decreased interleukin-6 release from serum amyloid A- or serum amyloid A1α-stimulated peripheral blood mononuclear cells in a dose-dependent manner. This could represent a novel endogenous mechanism that could regulate excessive pro-inflammatory functions of serum amyloid A. To confirm the presence of anti-serum amyloid A1α autoantibodies with another method, we developed a novel bead-based duplex immunoassay for simultaneous detection of anti-serum amyloid A1α and anti-α1-acid glycoprotein autoantibodies in human sera. We showed that autoantibodies against both serum amyoid A1α and α1-acid glycoprotein can be detected in serum samples of healthy blood donors and patients with systemic autoimmune diseases (giant cell arteritis, immunoglobulin A vasculitis, systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis and antiphospholipid syndrome) as well as intravenous immunoglobulin G. When tested for detection in human subjects the highest levels of both autoantibodies were determined in patients with giant cell arteritis. No significant differences were observed between healthy blood donors and any group of patients with systemic autoimmune diseases. Our method could be further optimized and developed to increase the number of autoantibodies against different acute phase proteins that can be simultaneously detected. Chronic inflammation is closely linked to the onset and development of atherosclerosis and cardiovascular disease with serum amyloid A playing a contributory role. Since atherosclerotic lesions typically develop in coronary arteries and the activation of endothelium represents one of the crucial early steps, we used an in vitro culture of human coronary endothelial cells to study endothelial activation and intracellular synthesis of SAA. We found upregulated messenger RNA expression of both serum amyloid A1 and serum amyloid A2 in interleukin-1β-stimulated coronary endothelial cells, while confirming the constitutive expression of serum amyloid A4, unchanged upon stimulation. No increase in serum amyloid A1 or serum amyloid A2 messenger RNA synthesis was observed when coronary endothelial cells were stimulated with interleukin-6 or glucocorticoids. We detected all three serum amyloid A proteins localized around the nuclei of interleukin-1β-stimulated, as well as unstimulated coronary endothelial cells, and in tunneling membrane nanotubes that connect the cells. The detection of serum amyloid A1 in tunneling membrane nanotubes led us to analyze potential serum amyloid A1 transport between cells. The intercellular exchange of serum amyloid A1 was more pronounced when coronary endothelial cells were stimulated with IL-1β. An inflammatory stress signal (e.g. interleukin-1β) can therefore increase intracellular serum amyloid A1 and serum amyloid A2 synthesis and promote serum amyloid A1 transport between cells. Taken together, we determined cell and molecular markers of primary systemic vasculitis, specifically giant cell arteritis and immunoglobulin A vasculitis, that could complement current diagnostics and might be used to monitor giant cell arteritis activity and response to treatment. We discovered that immunoglobulin G autoantibodies against serum amyloid A could function as physiological regulators of the inflammatory response by decreasing interleukin-6 release from serum amyloid A-stimulated peripheral blood mononuclear cells. Lastly, we showed that pro-inflammatory mediator interleukin-1β increased intracellular messenger RNA synthesis of serum amyloid A1 and serum amyloid A2 in human coronary artery endothelial cells, as well as promoted intercellular serum amyloid A1 transport. [T. Kuret]20202022-06-01 11:55:21Doktorska disertacija137107sl