POS1411 ANTI-PM/SCL75 AND ANTI-PM/SCL100 ANTIBODIES ARE PATHOGENIC IN PM/SSc-OVERLAP SYNDROMES

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Background: Anti-PM/SCL antibodies are significant autoantibodies found in patients with polymyositis (PM), systemic sclerosis (SSc), and overlap syndromes. Understanding the cellular function of the autoantibodies may provide insights into novel treatment approaches, addressing underlying pathogenic mechanisms and improving patient outcomes in these complex autoimmune disorders [1, 2]. Patients with overlap syndrome show a prevalence of 24% for being positive for one or both two autoantibodies. In contrast, patients with scleroderma or polymyositis exhibit the autoantibodies at rates of 2% and 6%, respectively [3]. The autoantibodies anti-PM/Scl-75 and -100 manifestation appears to be associated with interstitial lung disease, skin fibrosis, Raynaud's phenomenon, and arthritis [4]. Anti-PM/Scl-75 and anti-PM/Scl-100 autoantibodies target two proteins that are part of a nucleolar macromolecular exosome complex, consisting of more than 10 proteins. The core structure of the exosome comprises nine proteins and associates with various proteins in different subcellular locations. This complex is present in both the cytoplasm and nucleoplasm, but it is most abundant in the nucleolus [4]. The complex participates in the degradation and processing of RNA due to its 3' to 5' exoribonuclease activity function [5]. Objectives: It remains to be demonstrated if and how a functional interaction between anti-PM/SCL antibodies and the exosome complex leads to pathological changes associated with PM, SSc, and the PM/SSc overlap syndromes. In this study, we investigated the effects of these autoantibodies on a THP-1 cell line. Methods: Vectors for PM/SCL 75 and PM/SCL 100 were transfected into E. coli Rosetta™ (DE3), and the two proteins were subsequently purified. Total IgG was isolated from PM/SCL 75 and PM/SCL 100 positive patients (n=6) and healthy controls (HC) (n=3), see Tabel 1. The affinity of the antibodies towards the purified, recombinant proteins was analyzed using surface plasmon resonance (SPR). THP-1 cells were stimulated with anti-PM/SCL 75/100 IgG, followed by qPCR and ELISA, along with mesoscale multiplexing on supernatants for quantitation of type-1 IFN, IL-8, CXCL10, IL-6, and MCP-1. THP-1 cells with knockout (KO) of MAVS and STING served as controls, as did HEK-Blue cells for type I IFN. Results: Our findings support that patients who test positive for PM/SCL antibodies primarily exhibit positivity for anti-PM/SCL100 IgG in both an ELISA setup and through SPR techniques. As we had demonstrated that anti-PM/SCL IgG was present in the patients and absent in HC IgG, we further examined whether these antibodies had a pathogenic effect on cellular pathways. Stimulating THP-1 cultures with anti-PM/SCL IgG resulted in an increased secretion of IL-8 (p=0.01) and MCP-1 (p=0.01). CXCL10 (p=0.01), and IFNβ (p =0.01) in the THP-1 WT cell line, to a lesser extent, in the MAVS KO THP-1 cell line. The effect diminished to the level of non-stimulated controls when stimulating the STING KO THP-1 cell line. These results suggest that anti-PM/SCL antibodies impact the STING pathway pathogenic in THP-1 cells, whereas not the RIG-I/MDA-5/MAVS pathway, see figure 1. Conversely, the THP-1 cells did not exhibit an IL-6 response when stimulated with anti-PM/SCL IgG compared to HC IgG. Additionally, RT-PCR confirmed that IFNβ and CXCL10 synthesis were elevated. We used HEK-Blue™ IFN-α/β cells to confirm this, then quantified by reading their SEAP activity. Our results showed that IgG from anti-PM/SCL-positive patients significantly stimulated the production of bioactive type 1 IFN. No type 1 IFN production was observed in the STING KO THP-1 cell line. These data support the involvement of the STING pathway as an intracellular sensor for DNA or DNA/RNA complexes induced by anti-PM/SCL antibodies. Conclusion: Our research on IgG from PM/SSc overlap syndrome patients suggests that these antibodies promote chemokine secretion and are linked to STING, rather than the MAVS pathway, in THP-1 cells. REFERENCES: [1] Mahler, M. and R. Raijmakers, Novel aspects of autoantibodies to the PM/Scl complex: clinical, genetic and diagnostic insights. Autoimmun Rev, 2007. 6 (7): p. 432-7. [2] Reichlin, M., et al., Antibodies to a nuclear/nucleolar antigen in patients with polymyositis overlap syndromes. J Clin Immunol, 1984. 4 (1): p. 40-4. [3] Brouwer, R., et al., Autoantibody profiles in the sera of European patients with myositis. Ann Rheum Dis, 2001. 60 (2): p. 116-23. [4] Marguerie, C., et al., The clinical and immunogenetic features of patients with autoantibodies to the nucleolar antigen PM-Scl. Medicine (Baltimore), 1992. 71 (6): p. 327-36. [5] Brouwer, R., et al., Autoantibodies directed to novel components of the PM/Scl complex, the human exosome. Arthritis Res, 2002. 4 (2): p. 134-8. Figure 1IgG from PM/SCL 75/100 positive patients increases the secretion of bioactive type I IFN A) and CXCL10 B) in THP-1 cells. In THP-1 STING-KO cells, the effect diminished to the level of the non-treated control, whereas in the MAVS KO cells, the secretion did not change significantly to the wild-type cell line. This supports the idea that PM/SCL 75/100 antibodies are bioactive through the cGAMP/cGAS/STING pathway but not through the RIG-I/MAVS pathway. Table 1. Characteristics of the study population at the time of inclusion. Data are presented as median with range in brackets or as numbers with percentages in parentheses. Abbreviations: PM/SSc = Polymyositis/Systemic sclerosis, ILD=Interstitial lung disease, HRCT=High-resolution computed tomography scan of lungs, FVC=Forced Vital Capacity, DLCO=Diffusing Capacity of the Lungs for Carbon Monoxide, CRP=C-reactive protein, ESR=Erythrocyte Sedimentation Rate, DMARDs=disease-modifying anti-rheumatic drugs. Acknowledgements: The authors extend their gratitude to the individuals and patient associations who supported this project. Disclosure of Interests: None declared . © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.

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