Background: Over the past decade vital contributions has been made to the understanding of the role that subpopulations of monocytes and macrophages play in rheumatoid arthritis (RA) pathobiology. We are the first to identify a population of synovial cells that are transcriptionally distinct from circulating CM or NCM as well as synovial macrophages, distinguished by their failure to express canonical markers (RNA or protein) of monocytes and macrophages, which we call t issue- r esident m onocyte- l ineage c ells (TRMC). TRMC originate from the embryo, do not require CCR2, Nr4a1 or FLT3 for their development, are radio-resistant, capable of self-replenishment and rapidly expand following induction of inflammatory arthritis.

Objectives: Our goals are to determine whether is the TRMC population exists as a heterogenous population as well as the origin and function of each subpopulation. We will also examine whether TRMC population is associated with therapeutic response in patients with RA.

Methods: We wiill utilize murine models of RA as well as CCR2-/- and NR4A1-/- mice. We will also perfrom single cell analysis and spatial transcriptomics on murine cells and RA synovial biopsies.

Results: We identified three TRMC subpopulations, distinguished by expression of TIM4, CX3CR1, and MHCII. Clodronate-laden liposome (Clo-lip) reduces the number of TRMC but does not impact the proportions or transcriptional profiles of TRMC subpopulations at 7 days post-administration. TIM4 ⁺ CX3CR1 ⁺ and TIM4 ⁺ TRMC represent long-lived subpopulations, whereas MHCII ⁺ TRMC are BM-derived and dependent on Ccr2 during steady state. BM-derived TRMC expand and replenish TIM4 ⁺ CX3CR1 ⁺ and TIM4 ⁺ TRMC compartments throughout the peak and plateau of inflammatory arthritis. Genes were unique to TIM4 ⁺ TRMC from the peak and plateau stages of arthritis are associated with pathways such as leukocyte chemotaxis. and antigen processing and presentation of exogeneous antigen pathway. Since TRMC require LFA1 was sampling of the environment and since B6-->LFA1-/- mice have reduced arthritis, these data suggest that the TRMC may be crucial to prevent the initiation phase of arthritis. We furthered examined synovial biopsies from RA patients. These samples were collected through the infrastructure of the RhEumatoid Arthritis SynOvial tissue Network (REASON), which enables unprecedented access to RA samples and associated clinical data. In our Cell Reports manuscript, we identified the ortholog of human TR-MC using CITE seq as well as IF. Here, we examined 37,424 CD45+ synovial cells isolated from 17 patients who failed a DMARD or biologic DMARD and will change therapy for scRNA seq, CITE-seq analysis and spatial transcriptomic analysis. Our Preliminary data reveal 14 myeloid populations that consists of macrophages, DCs and TR-MC. Cluster 5, which is the TR-MC cluster correlates with response to biologic therapy. Expression of CD68, CD14 and CD163 are reproduced using spatial transcriptomics. Of note, Timd4 is not present in the 1K probe set. Currently, we designed myeloid probe set for Xenium and performed a study on 5 patients. Overall, these findings demonstrate that human TIM4 ⁺ TR-MC represent the human ortholog of TR-MCs and their proportion is associated with therapeutic response.

Conclusions: These findings underscore the importance of heterogeneity within TRMC and highlight their distinct responses to synovial disruption and potential roles in RA.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Harris Perlman Abbvie, Yidan Wang: None declared, Deborah Winter: None declared.