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. 2018 Jan 8:8:1958.
doi: 10.3389/fimmu.2017.01958. eCollection 2017.

M1 and M2 Monocytes in Rheumatoid Arthritis: A Contribution of Imbalance of M1/M2 Monocytes to Osteoclastogenesis

Shoichi Fukui  1   2 , Naoki Iwamoto  1 , Ayuko Takatani  1 , Takashi Igawa  1 , Toshimasa Shimizu  1 , Masataka Umeda  1   3 , Ayako Nishino  1   4 , Yoshiro Horai  1   5 , Yasuko Hirai  1 , Tomohiro Koga  1   6 , Shin-Ya Kawashiri  1   2 , Mami Tamai  1 , Kunihiro Ichinose  1 , Hideki Nakamura  1 , Tomoki Origuchi  1   7 , Ritsuko Masuyama  8 , Kosuke Kosai  9 , Katsunori Yanagihara  9 , Atsushi Kawakami  1
Affiliations

M1 and M2 Monocytes in Rheumatoid Arthritis: A Contribution of Imbalance of M1/M2 Monocytes to Osteoclastogenesis

Shoichi Fukui et al. Front Immunol. .

Abstract

Objectives: We investigated the relationships among M1 monocytes, M2 monocytes, osteoclast (OC) 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 also obtained and cultured CD14-positive cells from PBMCs from RA patients and healthy donors to investigate OC differentiation in vitro.

Results: Forty RA patients and 20 healthy donors were included. Twenty-two patients (55%) were anticitrullinated protein antibody (ACPA) positive. The median M1/M2 ratio was 0.59 (0.31-1.11, interquartile range). 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 OC number in vitro in RA patients (ρ = 0.81, p < 0.001). The ACPA-positive patients had significantly higher M1/M2 ratios in vivo (p = 0.028) and significantly greater numbers of OCs in vitro (p = 0.005) than the ACPA-negative patients. Multivariable regression analysis revealed that the M1/M2 ratio was the sole significant contribution factor to in vitro osteoclastogenesis. RA patients with M1/M2 ratios >1 (having relatively more M1 monocytes) had higher C-reactive protein and erythrocyte sedimentation rates than RA patients with M1/M2 ratios ≤1. M1-dominant monocytes in vitro produced higher concentrations of interleukin-6 upon stimulation with lipopolysaccharide than M2 monocytes.

Conclusion: M1/M2 monocytes imbalance strongly contributes to osteoclastogenesis of RA patients. Our findings cast M1 and M2 monocyte subsets in a new light as a new target of treatments for RA to prevent progression of osteoclastic bone destruction.

Keywords: anticitrullinated protein antibody; inflammation; monocytes; osteoclasts; rheumatoid arthritis.

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Figures

Figure 1
Figure 1
(A) M1 monocytes were defined as positive for CD14, CD68, and CCR2. (B) M2 monocytes were defined as positive for CD14, CX3CR1, and CD163 monocytes were defined as M2 monocytes. (C) We quantified three types of monocytes: classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16+ monocytes. There were no correlations between the M1/M2 ratio and the (D) classical, (E) intermediate, or (F) nonclassical monocytes.
Figure 2
Figure 2
(A) The significantly positive correlation between the M1/M2 ratio and the number of osteoclasts in the rheumatoid arthritis patients (ρ = 0.81, p < 0.001). (B) No correlation between the M1/M2 ratio and the number of osteoclasts in the healthy donors. (C) The significantly positive correlation between the number of osteoclasts and the area percentage of the pit formation area (ρ = 0.74, p = 0.001). (D) Patients who had lower M1/M2 ratios and fewer numbers of osteoclasts had smaller resorbed areas. (E) Patients who had higher M1/2 ratios and greater numbers of osteoclasts had larger resorbed areas.
Figure 3
Figure 3
There were no significant differences between the rheumatoid factor (RF)-positive patients and RF-negative patients regarding (A) the M1/M2 ratio and (B) the number of osteoclasts. Anticitrullinated protein antibody (ACPA)-positive patients had (C) significantly higher M1/M2 ratios (0.87 vs. 0.41, p = 0.028) and (D) greater numbers of osteoclasts (76 vs. 47 per well, p = 0.005). (E) ACPA-negative patients had fewer osteoclasts in vitro compared to the ACPA-positive patients (F).
Figure 4
Figure 4
There were no significant differences between erosive rheumatoid arthritis (RA) patients at entry (n = 13) and the non-erosive RA patients at entry (n = 27) regarding (A) the M1/M2 ratio and (B) the numbers of osteoclasts. There were no significant differences between RA patients treated with biologics (n = 17) and RA patients treated without biologics (n = 23) regarding (C) the M1/M2 ratio and (D) the numbers of osteoclasts.
Figure 5
Figure 5
(A) There were no significant differences of C reactive protein (CRP) levels between M1/M2 ratio >1 group and M1/M2 ratio ≤1 group in the whole rheumatoid arthritis (RA) patients. (B) Excluding patients treated with tocilizumab, the M1/M2 ratio >1 group had significantly higher CRP levels (0.45 vs. 0.08 mg/dL, p = 0.032). The M1/M2 ratio >1 group had significantly (C) higher erythrocyte sedimentation rate (29 vs. 9 mm/1 h, p = 0.011) and (D) greater numbers of osteoclasts (100 vs. 47 cells per well, p < 0.001) in the whole RA patients.
Figure 6
Figure 6
M1-dominant monocytes produced significantly more interleukin-6 than M2 monocytes did by stimulation of 100 ng/mL lipopolysaccharide (n = 5, healthy donors).
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