Background/Purpose: 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. The aim of this study was to examine the heterogeneity of tissue monocytes with those circulation and determine their involvement in inflammation.

Methods: NR4A1^-/-, CX3CR1^ERCre.zsGFP, CCR2^-/-, LFA^-/- and C57Bl/6 mice were used in all studies. Intravascular and extravascular 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). Human synovium was obtained from ultrasound guided synovial biopsies and CD45+ cells were FACSorted for single cell RNA seq.

Results: Single cell profiling of CD45⁺NK1.1^–CD3^–CD19^–Ly6G^–CD64^–Tim4^–CD11b⁺ cells revealed 4 populations of non-classical monocytes that are distinct from circulating non-classical monocytes.  We then identified four populations of Ly6C^lo monocytes in the joint; 2 intravascular and 2 extravascular cells using 18 color flow cytometry. Similarly, RA patients also display populations of non-classical monocytes using single cell RNA seq of ultrasound guided synovial tissue biopsies.  Lineage tracing studies reveal that the origin of extravascular synovial monocytes are from the embryo while the intravascular monocytes are derived post-natally. The NR4A1-/- mice have fewer intravascular monocytes but still retain the extravascular monocytes, while CCR2-/- mice display similar numbers of intravascular non-classical monocytes yet lack the extravascular MHCII⁺ nonclassical monocytes compared to controls.  Nonetheless, NR4A1-/- and CCR2-/- mice develop rheumatoid arthritis (RA)-like disease.  Moreover, LFA-/- mice fail to have an expansion of the extravascular non-classical monocytes, which correlates with an ability to develop RA-like disease.  Clodronate loaded liposomes depletes the intravascular monocytes as well as the extravascular CD177+MHC- nonclassical monocytes.  These data suggest that only the loss of extravascular CD177+MHC- nonclassical monocytes but not the intravascular non-classical monocytes and the MHCII+ extravascular non-classical monocytes are essential for the development of RA.

Conclusion: We have identified and described four novel and uncharacterized populations of non-classical monocytes cells in the joint, an intravascular adherent and an extravascular populations.  These cells have distinct origins and phenotype from both tissue resident macrophages and circulating non-classical monocytes. Further, the findings presented here strongly suggest the extravascular CD177+ non-classical monocytes are a key effector cells in inflammatory arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-novel-subclass-of-intravascular-non-classical-tissue-resident-synovial-monocyte-is-critical-for-rheumatoid-arthritis-pathogenesis/