0287: Comprehensive Profiling of Neutrophil Subsets Reveals Functional Signatures Linked to Vasculopathic Features in Patients with Inflammatory Myopathies

Background/Purpose: Vasculopathic manifestations of idiopathic inflammatory myopathies (IIM) such as interstitial lung disease (ILD), calcinosis, skin ulcers, mechanic’s hands, are characterized by immune-mediated vascular damage. However, operational definitions of activity/complete clinical response (CCR) underscore vasculopathy as a domain that impacts therapeutic decisions. Different subsets of neutrophils had been described as key regulators of activity in IIM. The “low-density granulocytes” (LDGs) in patients with vasculopathy, produce higher amount of neutrophil extracellular traps (NETs), correlate with IL18 and are found more frequently in different tissues from patients anti-MDA5 positive; a clinical subtype with predominance of vasculopathic features. A different unexplored subset are the neutrophils that migrate from sites of inflammation to the vascular system, known as reverse transendothelial migration (rTEM), characterized by the expression of CD54high, CXCR1low. The role of innate immune cells subsets in immune-mediated IM vasculopathy is unknown. The aim of this study was to address the role of neutrophil and monocyte subsets in the vasculopathic features of patients with IIM.

Methods: : A single-tertiary center cross-sectional study was performed. Fifty patients with definite IIM diagnosis according to ACR/EULAR 2017 criteria from the institutional (MYOTReCSZ) cohort were included, 25 patients with vasculopathy (7 with active IIM and 18 with CCR) who were compared with 25 patients without vasculopathy (7 with active IIM and 18 with CCR). Vasculopathy was defined as the presence of interstitial lung disease, calcinosis, ulcers, mechanic's hands or hiker’s feet. Disease activity was defined as MMT8 < 136 and/or CDASI >6. Immunophenotyping of neutrophils (LDGs: CD66b^posCD14^neg, rTEM: CD15^posCD16^hiCD54^hiCXCR1^lo) and monocytes (Classical monocytes: CD14^hiCD16^neg, Non-classical monocytes: CD14^posCD16^hi, Intermediate Monocyte: CD14^hiCD16^pos) was performed using flow cytometry, as well as phagocytosis and respiratory burst assays evaluation with pHRhodo and DHR123 assays, respectively.

Results: IIM patients with CCR and vasculopathy exhibit a significantly increased percentage of LDGs and rTEM compared with patients in CCR without vasculopathy (9.54% (2.9-29.18) vs 0.7% (0.48-2.32); p=0.0092) / (19.7% (9.85-23.8) vs 2.4% (0.99-2.88); furthermore, monocyte’s phagocytosis is increased in vasculopathy group compared with CCR patients (70% (45.35-94.08) vs 34.5% (20.3-50.7); p=0.0012). IIM patients with vasculopathy displayed a markedly greater reduction in respiratory burst activity compared with patients, IN CCR, indicating a distinct functional impairment in this subgroup. (1703 IMF (1547-2279) vs 2736 IMF (1946-3217); p=0.005).

Conclusion: The expansion of LDGs and rTEM, along with the increased monocyte phagocytosis and reduced respiratory burst in neutrophils, phenotypically and functionally differentiate the innate immune profiles of IIM patients with vasculopathy.