Background/Purpose:  Increased expression of Notch signaling molecules has been reported in synovial samples of patients with rheumatoid arthritis (RA). However, the identity of the cell type(s) in RA joints with Notch activation or how they might contribute to RA pathogenesis is unknown. Here, we used GFP+ cells from Hes1-GFP/TNF transgenic (TNF-Tg) mice (a model of RA) to identify Notch-activated cells, i.e., those with high Hes1 expression, in the joints of these mice and to study the source and effect of the deletion of GFP+ cells on joint tissue damage.

Methods:  Hes1-GFP/TNF-Tg mice were generated by crossing Hes1-GFP reporter mice and TNF-Tg mice. Immunofluorescent (IF) staining, flow cytometry and bone marrow (BM) transplantation assays were used. BM macrophages (BMM) were generated by culturing BM cells with M-CSF. M1 inflammatory macrophages (M1s) and M2 anti-inflammatory cells (M2s) were induced from BMM by TNF and IL-4, respectively. Thapsigargin (Thap), a recently recognized Notch inhibitor, was used.

Results:  To determine the cell type(s) with high Hes1 expression in RA joints, we performed flow cytometry of BM and synovial cells from 6-m-old Hes1-GFP/TNF-Tg mice when they had developed severe RA joint damage. Compared to Hes1-GFP control (Ctl) mice, Hes1-GFP/TNF-Tg mice had significantly more GFP+ cells, especially in the synovial tissue (Ctl BM 2±0.6% vs. Hes1-GFP/TNF-Tg BM 5±0.7% vs. synovium 12±4%). Synovial GFP+ cells comprised 60% F4/80+, 2% B220+, 5% CD3+, 0.7% CD31+, and 3% CD45-Sca1+ cells. Significantly more of the F4/80+/GFP+ cells expressed the M1 marker, iNOS than the M2 marker CD206 (75+6% M1s vs. 25+6% M2s). IF staining showed that M1s were located mainly in the inflamed synovial tissue. To study if synovial M1s came from BM, we transferred Hes1-GFP/TNF-Tg BM cells into irradiated TNF-Tg and WT recipient mice. Chimeric mice were sacrificed 8 wks post-BM transfer. IF staining indicated numerous GFP+ cells in the synovium of TNF-Tg mice, but there were few GFP+ cells in the BM of the same mice. No GFP+ cells were detected in the synovium of WT recipient mice. To determine if Notch inhibition could reduce M1s and attenuate joint tissue damage in RA, we demonstrated that TNF treatment of Hes1-GFP BMMs promoted GFP+/iNOS+ M1s, while IL-4 promoted GFP-/CD206+ M2s. Thap reduced Hes1 expression and switched TNF-induced M1s to M2s (M1s: 32±18% vs. 79±11%; M2s: 63±14% vs. 23±11% in PBS). More importantly, the inhibitory effect of Thap on TNF-induced M1s was abolished in BMMs from mice carrying a constitutively active Notch intracellular domain. In vivo, Thap treatment of Hes1-GFP/TNF-Tg mice significantly reduced bone resorption (subchondral BV/TV: 30±3 vs. 20±4% in Ctl), inflammation area (0.7±0.3 vs. 1.3±0.2mm²in Ctl), synovial M1s and GFP+ cells (M1s: 35±28 vs. 78±16 % in Ctl; GFP+: 6±1 vs. 11±2% in Ctl).

Conclusion:  M1s derived from BM are the main cells with activated Notch signaling in the inflamed synovial tissues of TNF-Tg mice. Switching Notch-activated M1s to M2s by Thapsigargin attenuated joint inflammation and erosion; thus targeting Notch-activated M1s may represent a new therapeutic approach for patients with inflammatory arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-notch-activated-m1-macrophages-attenuates-joint-tissue-damage-in-a-mouse-model-of-inflammatory-arthritis/