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

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.

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: rheumatoid arthritis, monocytes, anticitrullinated protein antibody, osteoclasts, inflammation inTrODUcTiOn Rheumatoid arthritis (RA) is the most common form of inflammatory arthritis and is characterized by inflammation and matrix destruction of bone and cartilage (1). RA affects approximately 1% of the world's population (2). Bone resorption (i.e., bone erosion, a hallmark of RA) was demonstrated histopathologically to be caused mainly by osteoclasts (OCs) (3). OCs are differentiated from monocytes and macrophages through the stimulation of receptor activator for nuclear factor kappa-B ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) (4). Increased in vitro osteoclastogenesis was observed in RA patients (5).
There is growing evidence that an imbalance of M1 macrophages and M2 macrophages is associated with many diseases such as asthma (11), chronic obstructive pulmonary disease (12), atherosclerosis (13) and tumors (14). Interestingly, in addition to macrophages, regarding monocyte subsets, M1 monocytes and M2 monocytes mirroring the M1/ M2 macrophage polarization concept were suggested, and they are reported to be associated with diabetes mellitus (15) and hypercholesterolemia and atherosclerosis (16). These M1/M2 monocyte subsets are different from the traditional monocyte subsets defined by CD14 and CD16 expression (17). However, the roles of this novel concept of monocytes in the etiology of various diseases have not been elucidated. Moreover, little is known regarding the relationships between osteoclastogenesis and M1/M2 monocyte subsets.
In this study, we investigated the relationships among M1/M2 monocyte subsets and the differentiation ability of OCs, which are derived from monocytes. We also sought to elucidate the relationships among M1 and M2 monocyte subsets, osteoclastogenesis, and the clinical backgrounds of RA patients.

Patients
We enrolled 40 patients with RA who were followed at Nagasaki University Hospital between January and July 2016. Their RA was diagnosed based on the ACR/EULAR 2010 RA classification criteria (18). We collected demographic, clinical, and laboratory characteristics of the RA patients and treatments for RA at the time of the monocyte subset analyses. We also examined the patients' clinical disease activities of RA as disease activity score (DAS) 28-erythrocyte sedimentation rate (ESR) and DAS 28-C reactive protein (CRP) at the time of the monocyte subset analyses. We defined the rheumatoid factor (RF)-positive status as >15.0 U/mL (upper normal value, measured by a latexenhanced immunonephelometric assay; Dade Behring, Marburg, Germany).
We evaluated the radiographic severity of peripheral joint damage at entry according to Steinbrocker stage (19). In addition, we defined Steinbrocker stage I or II as non-erosive disease and Steinbrocker stage III or IV as erosive disease.
The patients' characteristics are summarized in Table 1. We also enrolled 20 healthy donors to compare to the RA patients. This study was performed in accordance with the Declaration of Helsinki and was approved by the Investigation and Ethics Committee at Nagasaki University Hospital (approval number: 12072361). Patients and healthy donors gave their informed consent to be subjected to the protocol.
The relative frequency of these monocyte subsets is expressed as percent within the CD14-positive cell gate. Experiments were performed using a FACS Canto II Flow Cytometer and FACS Diva software (BD Biosciences).

Osteoclastogenesis
At the same time as the FACS analysis, we purified CD14-positive cells from whole peripheral blood from the RA patients and healthy donors using CD14 M-pluriBead (pluriSelect, Spring Valley, CA, USA). We cultured CD14-positive cells (1 × 10 5 cells per well) in a 96-well dish with Octeoclast Precursor BulletKit™ Medium containing l-glutamine, penicillin/streptomycin, M-CSF, and RANKL (Lonza, Walkersville, MD, USA) so that the cells would differentiate to OCs. We changed the medium three times a week. At day 12, the cells were fixed in 4% paraformaldehyde and AlexaFluor647-conjugated mouse IgG1 and APC-conjugated mouse IgG1 (all from BD Biosciences) were used isotype controls. stained with tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (Cosmo Bio). We defined OCs as TRAP staining-positive cells with >3 nuclei. We counted the OCs in the whole wells. Three wells were used to count OCs from one patient or one healthy donor, and we defined the average of the numbers of OCs of three wells as "the numbers of OCs. "

Pit Formation assay
Pit formation assays were performed using Dentin Slice (from Ivory, Thin Type for pit formation assay, Wako Pure Chemical Industries, Osaka, Japan.). CD14-positive monocytes were cultured with Octeoclast Precursor BulletKit™ Medium on the dentin slices for 17 days. Resorbed lacunae were visualized by Mayer's hemalum solution (Merck Millipore, Darmstadt, Germany) staining, and the relative resorbed area was measured under a microscope (BZ-9000, Keyence, Tokyo). Resorbing lacunae were quantified using Image J software (US National Institutes of Health, Bethesda, MD, USA). The relative area of resorbing lacunae is expressed as the percent of the area to the entire dentin slice.

stimulation assays and elisa
In another set of experiments, CD14+ CX3CR1+ CD163+ (M2 monocyte) cells were isolated from PBMCs derived from healthy donors by FACS. We used the rest of the CD14-positive cells as M1-dominant monocytes because M1 monocytes could not be isolated by FACS due to the intracellular expression of CD68. They were incubated in MEM Alpha medium with 10% fetal bovine serum, 1% penicillin/streptomycin (all from Gibco, Basel, Switzerland) for 24 h in a 12-well dish at 5 × 10 4 cells per well. Cells were stimulated for 24 h with LPS (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) or phosphate-buffered saline as a control, and supernatants were obtained. Proteins in the cultured supernatants were detected by ELISA using an IL-6-specific ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Absorption was measured at 450 nm.

statistical analysis
Variables are described using frequencies for categorical variables and with the median and interquartile range (IQR) for quantitative variables. We assessed the association between variables using Wilcoxon's rank sum test for quantitative variables. Correlations were analyzed using Spearman's correlation coefficient. Univariate and multivariable regression model analyses were performed to determine the factors that contribute to the number of OCs. All tests were two-sided, and a p-value < 0.05 was considered significant. All statistical analyses were performed using JMP Statistical Software, ver. 11 (SAS Institute, Cary, NC, USA) and GraphPad Prism ver. 7.0 (GraphPad Software, San Diego, CA, USA).

resUlTs
The Monocyte subsets (including the M1/M2 ratio) showed no Difference between the ra Patients and healthy Donors As shown in Table 2, we compared RA patients with healthy donors for each monocyte subset. The population of CD206-positive M2 monocytes was too small to examine (0-0.5% within CD14positive cells). Therefore, we redefined positive CD14, CX3CR1 and CD163 monocytes as M2 monocytes (Figure 1B). The median M1 and M2 monocyte percentages in CD14-positive cells were 16.7 and 40.0% in the RA group and 25.8 and 29.7% in the healthy donor group, respectively, with no significant differences between the RA and healthy groups. The RA and healthy groups had nearly equal M1/M2 ratios (0.60 vs. 0.62, respectively; p = 0.88). Likewise, the distribution of monocyte subtypes divided by CD14 and CD16 expression showed no significant differences between the RA patients and healthy donors. There was no correlation between the M1/M2 ratio and these subsets (Figures 1D-F).

Positive correlation between the M1/M2 ratio and greater number of Ocs
Monocytes are capable of differentiating into OCs. We next investigated the osteoclastogenic ability of M1/M2 monocytes. There was a significantly positive correlation between the M1/M2 ratio and the number of OCs in the RA patients (ρ = 0.81, p < 0.001) (Figure 2A). In contrast, there was no significant correlation between the M1/M2 ratio and the number of OCs in the healthy donors (ρ = 0.30, p = 0.196) (Figure 2B). The numbers of OCs in vitro were significantly correlated with the area percentage of the pit formation area (ρ = 0.74, p = 0.001) (Figure 2C), which demonstrated that the RA patients who had lower M1/M2 ratios and fewer OCs had smaller resorbed areas ( Figure 2D) compared to the RA patients who had higher M1/2 ratios and greater numbers of OCs ( Figure 2E).

The acPa-Positive ra Patients had higher M1/M2 ratios and greater numbers of Ocs
To explore the factors that influence the M1/M2 subsets in RA, we analyzed the influence of RF/ACPA positivity on the M1/M2 subsets. There were no significant differences between the RF-positive RA patients and the RF-negative RA patients regarding the M1/M2 ratio and the number of OC (Figures 3A,B). In contrast, the ACPA-positive patients had significantly higher M1/M2 ratios (0.87 vs. 0.41, p = 0.028) ( Figure 3C) and greater numbers of OCs (76 vs. 47 per well, p = 0.005) (Figure 3D). We show an ACPA-negative patient's OCs in vitro of ( Figure 3E) and those of an ACPA-positive patient ( Figure 3F).
There were no significant differences between erosive RA patients at entry (n = 13) and the non-erosive RA patients at entry (n = 27) regarding the M1/M2 ratio and the number of OC (Figures 4A,B). In addition, there were no significant differences between RA patients treated with biologics (n = 17, 7 patients treated with TNF inhibitors, 5 patients treated with tocilizumab, and 5 patients treated with abatacept) and RA patients treated without biologics (n = 23) regarding the M1/M2 ratio and the number of OC (Figures 4C,D).

The M1/M2 imbalance is a strong contribution Factor for Osteoclastogenesis
Anticitrullinated protein antibody is a well-known bone erosion promoting factor in RA (20). To elucidate which factor is crucial for osteoclastogenesis, we investigated factors that may contribute to the number of OCs by performing univariate and multivariable analyses ( Table 3). In the univariate analysis, the positivities for RF and ACPA and the M1/M2 ratio significantly contributed to the numbers of OCs. However, the multivariable regression analysis revealed that the M1/M2 ratio was the sole significant factor contributing to the number of OCs (β-coefficient: 16.3, p < 0.0001).

high il-6 levels of culture supernatants of M1-Dominant Monocytes by lPs stimulation
To analyze the function of M1/M2 monocytes in inflammation, we extracted M2 monocytes from whole monocytes and  their cytokine production by measuring the IL-6 levels of the culture supernatants. The results demonstrated that the M1-domonant monocytes produced significantly more IL-6 than the M2 monocytes did (n = 5, p = 0.032, Figure 6).

DiscUssiOn
This is the first study to show the possible relation of M1/M2 subsets with the pathogenesis of RA. We demonstrated that RA patients with positive ACPA had higher M1/M2 ratios and greater numbers of OCs. The patients with relatively more M1 monocytes in peripheral blood (M1/M2 ratio > 1) had higher ESR and CRP values and greater numbers of OCs.
To the best of our knowledge, a relationship between RA and M1/M2 monocyte subsets had not been reported prior to this study. However, research regarding CD68 and CD163 in inflammatory arthritis has been described. It was reported that CD68, one of the M1markers, positive synovial cells are mainly located in the sublining layer of rheumatoid synovial tissues, and the changes of the sublining of these cells were associated with clinical improvement by anti-rheumatic treatment (22). It was also reported that soluble CD163 in sera, one of the M2 markers, could be a diagnostic marker for macrophage activation syndrome in juvenile inflammatory arthritis (23). CD163 can bind and internalize hemoglobin-haptoglobin complexes, which leads to the strong anti-inflammatory effects by the release of IL-10 and carbon monoxide (24). CD163 may be produced to address overwhelming inflammatory reaction by macrophage activation syndrome in juvenile inflammatory arthritis.
There are two hypotheses regarding M1/M2 monocyte subsets and osteoclastogenesis. First, M1 monocytes may have higher osteoclastogenic differentiation ability than M2 monocytes. Second, higher M1/M2 ratios may create a proinflammatory environment that can induce more OC differentiation through some types of cytokines and chemokines, since M1-dominant monocytes produce excess amounts of IL-6, one of the most important osteoclastogenic cytokines in RA (25). An association between ACPA, which predicts erosive disease (20), and high M1/M2 ratios was observed in the present study, but it appears likely that varying pathogenic mechanisms may lead to a final common pathway, resulting in macrophage activation as well as OC differentiation. In this regard, our multivariable analysis which revealed that a high M1/M2 ratio, not positivity of ACPA, was the sole contributing factor to osteoclastogenesis in vitro suggests that there may be another mechanism underlying the increase in the M1/M2 ratio that does not involve ACPA. One of the possible ACPA-independent mechanisms contributing to in vitro osteoclastogenesis might be the intrinsic characteristic of M1 monocytes and/or the inflammatory condition derived by M1 monocytes.
In the meantime, polyclonal ACPA and some of the monoclonal ACPAs are reported to enhance OC differentiation from monocytes of RA patients in vitro (26). It is also reported that ACPA immune complex could induce the monocytes' production of inflammatory cytokines (27). Considering these results, one possible mechanism is that ACPA may bind to monocytes, creating an inflammatory environment and increasing the M1/ M2 ratio, resulting in enhanced OC activation. However, this hypothesis cannot be supported by our multivariable analysis. Accordingly, the associations among the M1/M2 ratio, ACPA and osteoclastogenesis must be elucidated.
Our present RA patients with M1/M2 ratios >1 (indicating more M1 monocytes than M2 monocytes) had higher levels of ESR and CRP compared to the RA patients with M1/M2 ratios ≤1. Monocytes from preeclampsia patients with surface receptors characteristic of the M1 monocytes were reported to produce higher levels of proinflammatory cytokines such as higher TNF-α and IL-12p40/70 (28). These results as well as our finding that M1 monocytes produce more IL-6 support the idea that RA patients with M1/M2 ratios >1 had higher ESR and higher CRP through inflammatory cytokine networks.  Our study has some limitations. First, we should have used M1 monocytes instead of M1-dominant monocytes. However, we could not sort M1 monocytes by fluorescence-activated cell sorting because CD68 expresses in cytoplasm. A new method to obtain M1 monocytes without deconstruction of the cell membrane is needed. Second, we did not evaluate time courses of changes of radiographic bone erosions. Our data finding that a higher M1/M2 ratio results in more osteoclastogenesis in vitro should be confirmed clinically by radiographic findings of bone erosions over time.
In conclusion, our present findings demonstrated that the M1/M2 ratio is strongly correlated with the in vitro differentiation of OCs in patients with RA. M1 and M2 monocyte subsets may become a new target of treatments for RA to prevent progression of bone erosions.

eThics sTaTeMenT
This study was performed in accordance with the Declaration of Helsinki and was approved by the Investigation and Ethics Committee at Nagasaki University Hospital (approval number: 12072361). Patients and healthy donors gave their informed consent to be subjected to the protocol.
aUThOr cOnTriBUTiOns SF drafted the manuscript and made substantial contributions to the concept and design with the assistance and supervision of NI. SF, NI, AT, TI, TS, MU, AN, YoH, YaH, TK, SYK, MT, KI, HN, TO, and AK treated the patients and collected the primary data. SF and NI performed the osteoclastogenesis in vitro and pit assays with the assistance of RM. SF and NI performed the ELISA. KK and KY performed the FACS analysis. SF performed the statistical analysis with the assistance of NI. NI, RM, KY, and AK critically revised the manuscript. AK supervised the entire study and gave final approval of the article. All authors read and approved the final manuscript.
acKnOWleDgMenTs We thank Mr. Kazuto Tsuruda and Ms. Mai Fuchigami for the FACS technical assistance, Ms. Kaori Furukawa for the ELISA assistance, and Dr. Shuntaro Sato, a biostatistician, for his advice.