Studies on pathogenesis, clinical features and comorbidities of idiopathic inflammatory myopathies

Sammanfattning: Idiopathic inflammatory myopathies (IIM), a group of rare chronic inflammatory disorders, are characterized by a broad spectrum of clinical manifestations with high morbidity and mortality. The pathogenesis of IIM is largely unknown but accumulating evidence suggests that autoantibodies promote the initiation and perpetuation of the disease. The aims of this thesis were 1) to increase the understanding of the role of anti-Jo1 antibodies and the histidylt- RNA synthetase (HisRS) autoantigen in the pathogenesis of the anti-synthetase syndrome (ASS), a distinct subgroup of IIM; 2) to study the incidence and the prevalence of a lifethreatening comorbidity of IIM, the arterial and venous thrombosis, and to assess the contribution of traditional risk factors, disease characteristics and biomarkers to its occurrence. Paper I: A sensitive electro-chemiluminescence immunoassay (ECLIA) was developed to detect HisRs in serum. For the first time, HisRS was found to circulate extracellularly in the serum of healthy individuals and, with higher concentrations, in the serum of anti-Jo1- patients. Serum levels of HisRS were, instead, undetectable in anti-Jo1+ patients and correlated negatively with anti-HisRS autoantibody levels in serum. A human muscle cell culture was set up showing that primary human myoblasts could release HisRS in the culture medium with increasing amount during differentiation into myotubes and upon stimulation with insulin growth factor 1. The tolerance to endogenous HisRS was efficiently disrupted in different strains of wild-type mice by immunizing mice with murine full-length (FL) HisRS and WHEP. No obvious muscle or lung inflammation was observed in immunized mice compared to control mice. However, upon external induction of tissue-specific damage, the degree of immune engagement with consequent muscle damage and lung injury was significantly exacerbated in immunized mice compared to controls. The administration of HisRS in mice previously lung or muscle-challenged resulted in a significant reduction of the inflammation in both lungs and muscle tissue. Moreover, in vitro, HisRS inhibited the activation of T cells isolated from fresh blood of human healthy donors. Paper II and III: Total IgG were isolated from anti-Jo1+ and anti-Jo1- patients as well as from healthy controls (HC) and glycans appended to the Fc region of the IgG were explored and compared between the three groups. The Fc-glycan profile of the anti-Jo1 IgG isolated from anti-Jo1+ patients was also investigated. Total IgG and specifically anti-Jo1 IgG from IIM/ASS patients displayed a pro-inflammatory Fc-glycan profile (i.e. agalactosylation) which was overrepresented in patients with interstitial lung disease (ILD). Anti-Jo1 IgG specifically presented lower abundance of bisected and afucosylated forms and Fc-glycan characteristics correlated positively with proteins involved in inflammatory processes. IgG and IgA were isolated from serum and matching bronchoalveolar lavage fluid (BALF), collected at time of diagnosis and longitudinally, of anti-Jo1+ and anti-Jo1- patients as well as from HC to determine the reactivity levels against the FL-HisRS protein and its different constructs and splice variants. Reactivity levels of IgG and IgA isolated from BALF and serum of anti-Jo1+ patients were found to be high already at the time of diagnosis and in some cases even before diagnosis, generally decreasing thereafter. Highest reactivity was registered against the HisRS-FL and the HisRS splice variants. Moreover, IgG against HisRS-FL displayed high affinity already at the time of diagnosis. Patients with high reactivity levels towards HisRS-FL were more likely to have ILD and arthritis, but less likely to have skin rash. Noteworthy, IgG anti-WHEP reactivity in BALF correlated with poor pulmonary function. In Paper IV, the incidence of venous thromboembolic events (VTE) was assessed in patients with IIM in comparison to the general population and patient categories at high risk and the timing of risk in relation to the diagnosis of IIM were identified. In Paper V, the prevalence of arterial and venous TE was retrospectively investigated in a large cohort of IIM patients and possible traditional and/or disease-related risk factors and biomarkers linked to arterial and venous TE in patients with IIM were explored. The incidence rate of VTE was significantly higher in IIM patients than in the general population, especially during the first year after diagnosis, and remained that high even after adjusting for education level, comorbidities, cancer, treatment at baseline and competing risk of death. Among IIM patients, the risk of VTE was even more elevated in those with a history of cancer, in patients with DM, and in those with age ≥72 years. In the retrospectively assessed cohort of IIM patients, one out of 5 patients had presented with an arterial and/or venous TE at the same time of or after the diagnosis of IIM. Even though a higher frequency of male gender and essential hypertension were observed in IIM patients with reported TE and of malignancy in those with history of exclusively venous TE, only older age was an independent risk factor for TE occurrence, while autoantibodies and clinical variables did not contribute. Interestingly, lower levels of e-selectin correlated with higher odds of getting TE in IIM patients. In conclusion, the discovery of HisRS extracellularly in both healthy individuals and IIM patients supports the hypothesis that HisRS exerts other physiological functions beyond the known intracellular protein synthesis. The inhibition of T cell activation by HisRS and the impact of HisRS in reducing the degree of inflammation in mice previously immunized against HisRS and with previously induced tissue damage suggest a possible immunosuppressive activity of this protein. This could open the path for a potential new therapeutically approach in anti-Jo1 positive patients. The inflammatory Fc-glycan profile as well as the high reactivity and affinity levels in serum and BALF of anti-Jo1 antibodies (conversely undetectable serum HisRS levels) already at the time of and even before diagnosis represent new evidence supporting the role of anti-Jo1 antibodies in the pathogenesis of IIM/ASS. The high incidence and prevalence of arterial and venous thrombotic events in patients with IIM, especially close to diagnosis and in those older, male patients with essential hypertension and history of malignancy, should indicate that a proper screening and preventive measures need to be recommended in this patient population. Eselectin levels could be used as biomarkers to identify IIM patients at higher risk of presenting with TE.