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. 2021 Dec 3;12:767231. doi: 10.3389/fimmu.2021.767231

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Copyright © 2021 Flegar, Filipović, Šućur, Markotić, Lukač, Šisl, Ikić Matijašević, Jajić, Kelava, Katavić, Kovačić and Grčević

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Small-molecule inhibitor of CCR2 (SMI) alone or in combination with methotrexate (MTX) contributes to reduced osteoclast progenitor (OCP) frequency and their lower osteoclastogenic potential in DBA mice with collagen-induced arthritis (CIA). Starting approximately at day 19 (± 2 days) after the primary immunization, asymptomatic DBA mice were allocated to experimental groups [non-treated (NT) CIA, SMI CIA, MTX CIA, or combined SMI+MTX CIA] and treated every 48 h by intraperitoneal injections. Non-arthritic group was included as control (ctrl). (A) Flow cytometric analysis of the frequency of OCPs in periarticular bone marrow (PBM) bearing the phenotype CD45⁺CD3^-B220^-DX5^-Ly6G^-CD11b^-/loCD115⁺CCR2^hi and CD45⁺CD3^-B220^-DX5^-Ly6G^-CD11b^-/loCD115⁺CCR2^lo. (B) Flow cytometric analysis of the frequency of OCPs in spleen (SPL), bearing the phenotype CD45⁺CD3^-B220^-DX5^-Ly6G^-CD11b⁺CD115⁺CCR2^hi. (C) Sorted PBM OCPs (CD45⁺CD3^-B220^-DX5^-Ly6G^-CD11b^-/loCD115⁺) from in vivo treated mice were seeded to plates and stimulated by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Representative microphotographs of in vitro differentiated tartrate-resistant acid phosphatase (TRAP)⁺ osteoclast-like cells are presented (magnification 100×). (D) Quantification of the osteoclastogenic potential of PBM OCPs from treated mice. Culture-differentiated multinuclear TRAP⁺ osteoclast-like cells per well (Oc.N.) and percentage of TRAP⁺ area per well (Oc.Ar.) were calculated and normalized to the average value of NT group (NT avg). (E) qPCR analysis of c-Fos gene expression in cultures of OCPs from treated mice, stimulated by RANKL and M-CSF for 2 days, presented as relative quantity of mRNA. Dots represent technical replicates from single experiment. For (A, B, D) experiments were repeated three times and pooled data are shown (n = 10–12 mice per group). Values are presented as medians (middle horizontal lines), boxes represent the interquartile range (IQR), whiskers represent 1.5 times the IQR, and outliers are represented by circles. Statistically significant difference was determined at p < 0.05, Kruskal–Wallis test (p-value marked on the graph) with Conover post-hoc test for group-to-group comparisons; line denotes significant difference between groups.