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- Bianchi, Matteo, et al.
(författare)
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Contribution of rare genetic variation to disease susceptibility in a large Scandinavian myositis cohort
- 2022
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Ingår i: Arthritis & Rheumatology. - : John Wiley & Sons. - 2326-5191 .- 2326-5205. ; 74:2, s. 342-352
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Tidskriftsartikel (refereegranskat)abstract
- Objective Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs.Methods Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune-related genes in a Scandinavian case–control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant– and gene-level enrichment analyses, was implemented to explore genotype–phenotype relations.Results Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle–specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants.Conclusion Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.
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| 3. |
- Galindo-Feria, Angeles S., et al.
(författare)
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Proinflammatory Histidyl-Transfer RNA Synthetase-Specific CD4+T Cells in the Blood and Lungs of Patients With Idiopathic Inflammatory Myopathies
- 2020
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Ingår i: Arthritis & Rheumatology. - : WILEY. - 2326-5191 .- 2326-5205. ; 72:1, s. 179-191
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Tidskriftsartikel (refereegranskat)abstract
- Objective Autoantibodies targeting histidyl-transfer RNA synthetase (HisRS; anti-Jo-1) are common in the idiopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome. Methods Bronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs) from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full-length HisRS protein or a HisRS-derived peptide (HisRS(11-23)). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12) were included as controls. The CD4+ T cell response was determined according to levels of CD40L up-regulation and cytokine expression using flow cytometry. Anti-Jo-1 autoantibody responses in the serum and BAL fluid were assessed by enzyme-linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syndrome (n = 14) were investigated by immunohistochemistry. Results In BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimulation with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7-14.7), and 2 of 3 patients with IIM/antisynthetase syndrome had the highest responses to HisRS(11-23) (median fold change 88, IQR 27-149)(.) In PBMCs, CD4+ T cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38, IQR 2.69-31.86; P < 0.001), whereas a HisRS(11-23) response was present in 11 of 14 patients with IIM/antisynthetase syndrome (median fold change 3.4, IQR 1.87-10.9; P < 0.001). In the control group, there was a HisRS(11-23) response in 3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45-3.29) and in 5 of 12 healthy controls (median fold change 2, IQR 1.89-2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1 phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon-gamma and interleukin-2 following stimulation. Anti-Jo-1 autoantibodies were detected in BAL fluid and germinal center (GC)-like structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome. Conclusion The results of this study demonstrate a pronounced presence of HisRS-reactive CD4+ T cells in PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoidosis and healthy controls. These findings, combined with the presence of anti-Jo-1 autoantibodies in BAL fluid and GC-like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of patients with IIM/antisynthetase syndrome.
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| 4. |
- Lundtoft, Christian, et al.
(författare)
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Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases
- 2022
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Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 74:8, s. 1440-1450
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Tidskriftsartikel (refereegranskat)abstract
- Objective Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjogrens syndrome (SS), or myositis. Methods Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. Results A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals capacity to deposit C4b on immune complexes. Conclusion We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.
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| 7. |
- Notarnicola, Antonella, et al.
(författare)
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Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome
- 2022
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Ingår i: Arthritis Research & Therapy. - : Springer Nature. - 1478-6362. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1(+) patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1(-) patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.
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| 8. |
- Notarnicola, Antonella
(författare)
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Studies on pathogenesis, clinical features and comorbidities of idiopathic inflammatory myopathies
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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 life-threatening 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.
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| 9. |
- Preger, Charlotta, et al.
(författare)
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Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies
- 2023
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Ingår i: Journal of Autoimmunity. - : Elsevier BV. - 0896-8411 .- 1095-9157. ; 134
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory my-opathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to known myositis-specific autoantibodies. We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM.Methods: Plasma samples from 217 patients with IIM according to 2017 EULAR/ACR criteria, including 50 pa-tients with ASSD, 165 without, and two with unknown ASSD status were identified retrospectively, as well as age and gender-matched sera from 156 population controls, and 219 disease controls. Patients with previously documented ASSD had to test positive for at least one of the five most common anti-aaRS autoantibodies (anti-Jo1,-PL7,-PL12,-EJ, and-OJ) and present with one or more of the following clinical manifestations: interstitial lung disease, myositis, arthritis, Raynaud's phenomenon, fever, or mechanic's hands. Demographics, laboratory, and clinical data of the IIM cohort (ASSD and non-ASSD) were compared. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 117 recombinant protein variants, representing 33 myositis-related proteins, including all nineteen cytoplasmic aaRS. Prospectively collected clinical data for the IIM cohort were retrieved and compared between groups within the IIM cohort and correlated with the results of the autoantibody screening. Principal component analysis was used to analyze clinical manifestations between ASSD, non-ASSD groups, and individuals with novel anti-aaRS autoantibodies. Results: We identified reactivity towards 16 aaRS in 72 of the 217 IIM patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four previously sero-negative patients for myositis-specific autoantibodies and eight with previously detected myositis-specific au-toantibodies. IIM individuals with novel anti-aaRS autoantibodies (n = 12) all had signs of myositis, and they had either muscle weakness and/or muscle enzyme elevation, 2/12 had mechanic's hands, 3/12 had interstitial lung disease, and 2/12 had arthritis. The individuals with novel anti-aaRS and a pathological muscle biopsy all presented widespread up-regulation of major histocompatibility complex class I. The reactivities against novel aaRS could be confirmed in ELISA and western blot. Using the multiplex bead array assay, we could confirm previously known reactivities to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n = 45)) and identified patients positive for anti-Zo,-KS, and-HA (n = 10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was also detected in controls.Conclusion: Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.
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