P083/O23 Novel subclass of non-classical monocytes are critical for inflammation
Authors: AB Montgomery, PJ Homan, D Winter, HR Perlman
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Career situation of first and presenting author</h3> Post-doctoral fellow. <h3>Introduction</h3> Monocytes in mice are distinguishable by expression of Ly6c. Ly6c<sup>hi</sup> (classical) monocytes are associated with pro-inflammatory responses, while Lyc6<sup>lo</sup> (non-classical) are involved in patrolling endothelial membranes. We have previously shown that depletion of monocytes prevents serum transfer induced arthritis (STIA) in mice, and that Lyc6<sup>lo</sup> monocytes are the critical population. <h3>Objectives</h3> We aim to contrast Ly6c<sup>lo</sup> monocytes from the circulation, lining vessels, and tissue to determine their involvement in inflammation. <h3>Methods</h3> Female 8–10 week old NR4A1<sup>-/-</sup>, CX3CR1<sup>ERCre.zsGFP</sup>, and C57Bl/6 mice were used in all studies. CX3CR1<sup>ERCre.zsGFP</sup> were utilized for cell tracking studies and joint shielded bone marrow chimeras via administration of tamoxifen (tam). Intravascular monocytes were identified by I.V. anti-CD45 labeling before perfusion. STIA was induced via I.V. KBxN sera. Cell populations were quantified by flow cytometry and FACS sorted for RNA-seq. Monocytes were identified CD45<sup>+</sup> CD11b<sup>+</sup> Ly6G<sup>-</sup> TIM4<sup>-</sup> CD64<sup>-</sup> Ly6c<sup>lo</sup> and subdivided into intravascular (labeled, CD43<sup>+</sup>), trans-vascular (labeled CD43<sup>-</sup>) and extravascular (no label). <h3>Results</h3> NR4A1<sup>-/-</sup> mice retain only 5% of circulating Ly6c<sup>lo</sup> monocytes but all joint Ly6c<sup>lo</sup> cells. STIA was comparable in NR4A<sup>-/-</sup> and C57Bl/6 mice suggesting circulating Ly6c<sup>lo</sup> are redundant. Transcriptional profiling of Ly6c<sup>lo</sup> cells identified distinct pathways enriched in upregulated genes between Ly6c<sup>lo</sup> from joint and blood. In the joint we identified three populations of Ly6C<sup>lo</sup> monocytes: extravascular unlabeled cells, labeled trans-vascular cells, and labeled intravascular cells adherent to endothelium. Mice given tam D8 of gestation had GFP<sup>+</sup> microglia only, whereas D15 tam induced GFP<sup>+</sup> synovial macrophages and unlabeled Ly6c<sup>lo</sup> monocytes. Both labeled Ly6c<sup>lo</sup> populations were GFP<sup>-</sup>, indicating unlabeled and unlabeled Ly6c<sup>lo</sup> arise from different progenitors. This was confirmed by bone marrow chimera studies showing labeled Ly6c<sup>lo</sup> cells are replenished from blood monocytes. Clodronate loaded liposomes depleted labeled CD43<sup>+</sup> cells but did not affect CD43<sup>-</sup> cells or unlabeled cells. With with our previous finding that clo-lip prevents STIA, these suggest adherent CD43<sup>+</sup> Ly6c<sup>lo</sup> cells are essential. This is supported by the finding that labeled Ly6c<sup>lo</sup> monocytes expand rapidly during the first 1 hour of STIA. Adherent CD43<sup>+</sup> cells expand especially rapidly, increasing in population size by 30x. <h3>Conclusions</h3> We have identified and described two previously uncharacterized populations of Ly6c<sup>lo</sup> cells in the joint- intra-vascular adherent and trans-vascular which have distinct origins and phenotype from both extravascular and circulating Ly6c<sup>lo</sup>. The findings presented here strongly suggest adherent Ly6c<sup>lo</sup> monocytes are a key effector cell in inflammatory arthritis. <h3>Disclosure of Interest</h3> None declared.