OP0155 ANTI-TOPOISOMERASE AUTOANTIBODIES ADDED IN ISOLATION OR AS IMMUNE COMPLEXES DIFFERENTIALLY REGULATE MACROPHAGE ACTIVATION IN SYSTEMIC SCLEROSIS
Authors: S. Lopez Garces, S. Zhang, H. Lopez, D. Abraham, C. P. Denton, T. Collins, J. Cross, B. Ahmed Abdi, R. Stratton
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Systemic sclerosis (SSc) is an autoimmune disease characterised by the production of autoantibodies, which leads to fibrosis and finally damage to various tissues and organs. Recent studies have demonstrated activation and aberrant persistence of M2-like macrophages in the disease tissues, implicating these cells as a source of pro-fibrotic and inflammatory mediators. However, the interplay between macrophages and autoantibodies remains largely unexplored. In this study, we aimed to investigate the role of disease-specific immunoglobulin G (IgG) autoantibodies, immune complexes, and plasma, on macrophage polarisation. We focused on the anti-topoisomerase (ATA) positive disease as a severe subgroup with strong M2 macrophage signature. <h3>Objectives:</h3> We aimed to test whether IgG purified from ATA positive SSc plasma, ATA:topoisomerase immune complexes or else unfractionated ATA SSc plasma affected the polarization state of healthy control or SSc disease macrophages. <h3>Methods:</h3> SSc and healthy control macrophages (both n=4) were obtained from blood monocytes following negative selection with RosetteSep and culture in X-vivo10 serum free media with the addition of M-CSF 4ng/ml for 7 days. IgG were isolated from ATA positive SSc plasma samples using Protein A IgG Purification Columns (Thermo Scientific) and added at 0.1, 10 and 100μg/ml to cultured macrophages. In other experiments the effects of ATA were compared with co-addition of human recombinant Topoisomerse I (Topo-I)(abcam, purity &gt; 95%)in 4 conditions, media only, ATA only, ATA+Topo-I, and Topo-I only. Final concentrations in each well for ATA and Topo-I were 100μg/ml and 20ng/ml, respectively. Recombinant Topo-I was mixed with ATA or media solution before the treatment and incubated at 37°C and 5% CO<sub>2</sub> for 2hrs to allow complex formation. qPCR assays for CD206 (mannose receptor), CD80 (co-stimulatory) and MerTK (regulatory/efferocytosis) were used to evaluate the polarization state and TBP used as the reference gene. In other experiments unfractionated ATA positive SSc plasma was added to media at 5% to stimulate the cells. <h3>Results:</h3> In healthy control macrophages ATA addition was found to decrease CD206 and CD86 but to enhance IL-10 and MerTK expression levels. Similar trends were seen in SSc macrophage lines, consistent with some reduction in M2a properties and enhancement of M2c by the effect of ATA. Different effects were seen when cells were stimulated by ATA:Topo-I immune complexes, leading to no change in CD206, but marked enhancement of CD86 (p&lt;0.031) and reduction of IL-10 (p&lt;0.019) and less MerTK (p&lt;0.067), consistent with M2b polarization. Finally, addition of 5% SSc plasma led to complex activation of healthy control macrophages as indicated by enhanced CD206 (p&lt;0.05), enhanced CD86 (p&lt;0.051) and greatly enhanced IL-10 (p&lt;0.016) and MerTK (p&lt;0.0049), with similar trends in SSc macrophages excepting that IL-10 was not induced. (SSc macrophage data Figure 1A). <h3>Conclusion:</h3> Taken together these results indicate that SSc ATA IgG autoantibodies are capable of inducing changes in macrophage activation state and exert differential effects when added in isolation or as immune complexes. ATA in isolation appeared to induce an M2c like phenotype normally associated with phagocytosis and resolution. When added as immune complexes the ATA:Topo-I induced CD86 consistent with an effector/inflammatory M2b phenotype. By contrast, adding unfractionated ATA positive SSc plasma, a known source of disease-related cytokines and growth factors, induced more complex activated M2-like changes. Failure to induce IL-10 was a major differences in SSc macrophages, as well as higher basal CD206. Taken together the results indicate a major influence of ATA on macrophage activation states, which are differentially induced dependent on presence of the Topoisomerase antigen. The formation of immune complexes with Topoisomerase released by damaged and apoptotic cells might have an important modulating effect on the induction of macrophages by ATA. <h3>REFERENCES:</h3> <b>NIL.</b> <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> Sandra Lopez Garces: None declared, Siyu Zhang: None declared, Henry Lopez CEO of Riptide Bioscience Inc, David Abraham Research grants received from UCB, Christopher P Denton: None declared, Teresa Collins: None declared, Jennifer Cross: None declared, Bahja Ahmed Abdi: None declared, Richard Stratton: None declared.