Background Monocytes can differentiate into either proinflammatory, microbicidal M1 macrophage or anti-inflammatory M2 macrophage subtypes. In addition to macrophages, regarding monocyte subsets, M1 monocytes and M2 monocytes mirroring the M1/M2 macrophage polarization concept were suggested.
Little is known regarding the relationships between osteoclastogenesis and M1/M2 monocyte subsets.
Objectives We investigated the relationships among M1 monocytes, M2 monocytes, osteoclast differentiation ability and clinical characteristics in patients with rheumatoid arthritis (RA).
Methods Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and healthy donors, and we then investigated the number of M1 monocytes or M2 monocytes by fluorescence-activated cell sorting. We defined positive CD14, CD68 and CCR2 monocytes as M1 monocytes, and in separate tubes, we defined positive CD14, CX3CR1 and CD163 or CD206 monocytes as M2 monocytes.
We also obtained and cultured CD14-positive cells from PBMCs from RA patients and healthy donors to investigate osteoclast differentiation in vitro. We defined osteoclasts as tartrate-resistant acid phosphatase (TRAP) staining-positive cells with >3 nuclei. We counted the osteoclasts in the whole wells of a 96-well dish. Pit formation assays were performed to evaluate function of osteoclasts.
Results Forty RA patients and 20 healthy donors were included. Twenty-two patients (55%) were ACPA-positive.
The median M1/M2 ratio was 0.59 (0.31–1.11, IQR). There were no significant differences between the RA patients and healthy donors.
There was a positive correlation between the M1/M2 ratio and the differentiated osteoclast number in vitro in RA patients (ρ=0.81, p<0.01) (A). The numbers of osteoclasts in vitro were significantly correlated with the area percentage of the pit formation area (ρ=0.74, p=0.001) (B).We demonstrated that the RA patients who had lower M1/M2 ratio and fewer osteoclasts (C) had smaller resorbed areas compared to the RA patients who had higher M1/2 ratio and greater numbers of osteoclasts (D).
The ACPA-positive patients had significantly higher M1/M2 ratio in vivo (p=0.028) (E) and significantly greater numbers of osteoclasts in vitro (p<0.01) (F) than the ACPA-negative patients. We show an ACPA-negative patient's osteoclasts in vitro (G) and those of an ACPA-positive patient (H).
Multivariable regression analysis revealed that the M1/M2 ratio was the sole significant contribution factor to in vitro osteoclastogenesis (β-coefficient 16.3, p<0.0001).
RA patients with M1/M2 ratio >1 (having relatively more M1 monocytes) had higher erythrocyte sedimentation rates (p=0.011) and C-reactive protein (p=0.032) than RA patients with M1/M2 ratio ≤1.
M1-dominant monocytes in vitro produced higher concentrations of IL-6 upon stimulation with lipopolysaccharide than M2 monocytes (p=0.032).
Conclusions The M1/M2 ratio is strongly correlated with the in vitro differentiation of osteoclasts in patients with RA. The RA patients with positive ACPA had higher M1/M2 ratio and higher numbers of osteoclasts.
M1 and M2 monocyte subsets may become a new target of treatments for RA.
Disclosure of Interest None declared