SAT0106\u2005NOVEL SUBCLASS OF INTRAVASCULAR NON-CLASSICAL SYNOVIAL MONOCYTES ARE CRITICAL FOR RHEUMATOID ARTHRITIS

Abstract

Background: There are at least three populations of circulating monocytes; classical, intermediate and non-classical. We demonstrated that circulating non-classical monocytes are required for the effector phase of arthritis and spontaneous models of arthritis in mice. While the vast majority of studies on monocytes have focused those in circulation, very little is known about the monocytes in the synovium. Objectives: The aim of this study was to examine the heterogeneity of tissue monocytes with those circulation and determine their involvement in inflammation. Methods: Female 8-10-week-old NR4A1-/-, CX3CR1ERCre.zsGFP, and C57Bl/6 mice were used in all studies. CX3CR1ERCre.zsGFP were utilized for cell tracking studies and joint shielded bone marrow chimeras via administration of tamoxifen (tam). Intravascular monocytes were identified using fluorescent anti CD45 antibody before perfusion. STIA was induced via I.V. KBxN sera. Monocyte populations were quantified by flow cytometry and FACS sorted for RNA-sequencing (RNA seq). Nonclassical tissue monocytes were identified CD45+CD11b+Ly6G-TIM4-CD64-Ly6clo and subdivided into intravascular (CD45-labeled, CD43+), trans-vascular (CD45-labeled CD43-) and extravascular (no CD45-label). Human synovium was obtained from ultrasound guided synovial biopsies and CD45+ cells were FACSorted for single cell RNA seq. Results: NR4A1-/- mice exhibit a 95% reduction in circulating Ly6clo monocytes but retain Ly6clo cells in the joint and develop STIA. The transcriptional profiling of bulk populations of Ly6clo cells in the synovium are distinct from those circulating in the blood. We then identified three populations of Ly6Clo monocytes in the joint; extra-vascular, trans-vascular cells, and intra-vascular cells using 18 color flow cytometry. Lineage tracing studies reveal that the origin of extra-vascular and trans-vascular synovial monocytes are from the embryo while the intravascular monocytes are derived post-natally. The intravascular monocytes are depleted with clodronate loaded liposomes while the extravascular and trans-vascular remain unaffected. Moreover, the intravascular monocytes rapidly expand during the first 1hour of STIA, increasing by 30x in population size. RA patients also display similar populations of non-classical monocytes using single cell RNA seq Conclusion: We have identified and described three previously uncharacterized populations of non-classical monocytes cells in the joint, an intra-vascular adherent, a trans-vascular population and an extra-vascular population. These cells have distinct origins and phenotype from both tissue resident macrophages and circulating non-classical monocytes. Further, the findings presented here strongly suggest intravascular are a key effector cells in inflammatory arthritis. Disclosure of Interests: None declared