Background/Purpose:

Populations of monocytes in mice are distinguishable by expression of Ly6c. Ly6c^hi monocytes are associated with pro-inflammatory responses, while Lyc6^lo are involved in lining and patrolling endothelial membranes. Both Ly6c^hi and Ly6c^lo monocytes can differentiate into macrophages and have been implicated in inflammatory arthritis and we have previously shown Lyc6^lo monocytes are essential for development of serum transfer induced arthritis (STIA). In the present study we show that NR4A1-deficient mice, which have a marked reduction of circulating Ly6c^lo monocytes but retain Ly6c^lo monocytes attached to endothelial vessels, also develop arthritis. Thus, we postulate that Ly6c^lo monocytes may be a heterogeneous population with distinct functions (circulating vs patrolling).

Methods:

Female NR4A1^-/- and C57Bl/6 mice were bred in house to 8-10 weeks old. STIA was induced by intravenous (IV) administration of KBxN sera at 85µl/20g. Arthritis was measured using clinical score, and mean clinical scores of N=4 per group were calculated. Monocytes were depleted using IV administration of clodronate-loaded liposomes (Clo-Lip). PBS-loaded liposomes were used as a negative control. Mice were euthanized by CO₂. Blood was collected by cardiac puncture and distal joints were collected following perfusion with PBS. Blood and joint monocytes were isolated by FACS and characterized by flow cytometry and RNA-sequencing (RNA-seq). Statistical analysis of disease and flow cytometry was carried out in GraphPad Prism. Statistical significance was considered at P ≤ 0.05.

Results:

NR4A1^-/- mice displayed a significant reduction in Ly6c^lo monocytes in the blood compared to C57Bl/6 controls, although joint Ly6c^lo monocytes in the joint were not significantly reduced. No significant differences were observed between arthritis severity between genotypes. In C57Bl/6 mice, Clo-lip administration resulted in over 90% depletion of CD115⁺ blood monocytes, however over 35% of synovial monocytes remained, suggesting they are not in the circulation. Of these, Ly6c^hi and Ly6c^int populations were significantly reduced (P<0.05), while Ly6c^lo monocyte numbers were unaffected. Further, flow cytometric analysis of blood and synovial macrophages identified a population of Ly6c^lo monocytes in the joint of C57Bl/6 mice which express endothelial cell surface marker CD105. 60% of Ly6clo monocytes in the joint were CD105⁺ compared to 14% and 16% of Ly6c^hi and Ly6c^int respectively. Finally, transcriptional profiling of Ly6c^lo monocytes reveals upregulated genes in blood Ly6c^lo cells are enriched for processes such as monocyte activation and differentiation processes, compared to processes involved in cell migration and localization enriched in Ly6c^lo cells isolated from the joint.

Conclusion:

These findings suggest a population of Ly6c^lo monocytes reside in the murine joint and have a distinct phenotype from blood Lyc6^lo monocytes measured by cell surface markers and transcriptional profile. Combined with the finding that NR4A1^-/- mice are susceptible to KBxN arthritis, this sub-population of Ly6c^lo monocytes is likely to have unique pro-inflammatory capabilities.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-distinct-profile-of-ly6clo-monocytes-in-the-murine-joint-compared-with-those-in-circulation-suggests-a-unique-role-in-inflammatory-arthritis/