Abstract

Introduction

Osteoarthritis (OA), the most common musculoskeletal disease, is characterized by progressive degeneration of the articular cartilage with synovial inflammation (synovitis) and neovascular invasion into the articular surface¹. The gradual wear and tear of cartilage bring local inflammation of the joint lining and surrounding soft tissues. Rheumatoid arthritis (RA), another common chronic joint disease, affects 0.5–1.0% population and involves in autoimmune dysregulation¹. RA affects multiarticular cartilages, bones, synoviums, and sometimes internal organs. Severe OA and RA both are accompanied by progressively subchondral bone destruction, osteophyte formation, bone marrow remodeling, and meniscal lesions.

Accumulative evidence suggests that macrophage plays a crucial role in developing both OA and RA^2,3. Macrophages are the primary immune cells and the front-line responder to joint injury in the normal synovium^3,4. In the OA synovium, macrophages cause cartilage destruction by producing matrix metalloproteinases and inflammatory cytokines, such as IL-1β and TNF-α⁵. Increased macrophages in the affected synovium are correlated with the degree of synovial angiogenesis and synovitis^3,6,7. A recent clinical study by Kraus et al. showed that the numbers of activated macrophages are positively associated with the OA and RA severity and the disease progression⁸.

It is known that macrophages exhibit a spectrum of diverse functions and phenotypes. To elucidate the detailed role of the macrophage in arthritis, Bailey et al. locally depleted the intra-articular macrophages in genetically modified mice with joint injury. They surprisingly discovered that such depletion leads to enhanced synovial inflammation and M1 polarization after fracture⁹. This controversial result may be caused by the diverse functionally-distinct macrophage lineages, such as M1 and M2 macrophages. M1 macrophages are pro-inflammatory and can be polarized by granulocyte macrophage-colony stimulating factor (GM-CSF). In contrast, M2 macrophages are anti-inflammatory and can be polarized by macrophage colony-stimulating factor (M-CSF) to reduce synovitis. Depletion of macrophages in animal models of arthritis may lead to M2 macrophage reduction in the synovial membrane and subsequently trigger the synovitis progress⁹. These findings in genetically-edited mice suggest that polarized M1/M2 macrophages from monocytes are critical for arthritis progress and could be essential biomarkers in arthritis management.

The human type II collagen (Col II) is a crucial but non-renewable component of the adult joint cartilage^10,11. Providing exogenous Col II stimulates the secretion of cartilage glycosaminoglycans (GAGs) by chondrocytes and enhances cartilage repair in animal models^12,13. However, Col II shows very limited turnover during the life span of an adult, even in pathological arthritis condition^10,11. Current OA therapies include pain relief with oral NSAIDs or selective cyclooxygenase 2 (COX-2) inhibitors and intra-articular (IA) injections with corticosteroids or hyaluronan¹⁴. Partial or total joint replacement is the final solution for severe OA and RA patients. Although hyaluronan is used to lubricate damaged-joint, this treatment does not regenerate hyaline cartilage or modify disease progression¹⁴. Despite advances in understanding the biology of arthritis and other musculoskeletal diseases, developing disease-modifying arthritis drugs, such as biological molecules and novel cell therapy, is still an urgent unmet medical need for promoting the healing of damaged joints.

Results

Peripheral blood samples were collected from donors and subjected for isolating the peripheral blood mononuclear cells (PBMNCs) by Ficoll-Paque premium. After treating PBMNCs with growth factors for 3 days on fibronectin-coated dishes, we showed that 100 ng/mL M-CSF steered 46.7±6.0% adherent PBMNCs to express Col II in the cells, detected by a polyclonal antibody (Fig. 1A,B). Similar Induction efficacy of GM-CSF was observed (36.7±4.5%) (Fig. 1A,B). A Col II-specific monoclonal antibody further verified the Col II expression in adherent PBMNCs (Fig. 1C). Both antibodies showed positive and similar cytosolic expression patterns after fluorescent immunocytochemistry staining (Fig. 1C).

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Figure 1

The Col II expression in the differentiating PBMNCs. The cultured PBMNCs were adherent on fibronectin-coated plates and treated with 100 ng/mL cytokines, including the M-CSF, GM-CSF, or G-CSF for 3 days. The Col II expression (green) in the cultured PBMNCs was stained with an anti-human Col II polyclonal antibody (A), and the percentage of Col II expressing cells was summarized from triplicated experiments (B). The fidelity of Col II expression was further confirmed by a specific monoclonal antibody against human Col II (C). The nuclei were stained with DAPI as blue.

To quantify the Col II expression and examine the Col II under suspension culture, we examined the percentage of monocyte in the PBMNCs by flow cytometry using both anti-CD14 antibody and anti-Col II antibody. The population of monocytes could be determined by both CD14 expression or the size distribution in the plot of forward scatter (FSC), and side scatter (SSC) (Fig. 2A). To investigate the cell fate of Col II expressing cells, we co-stained the living PBMNCs with anti-CD14, anti-CD206, or anti-Col II antibodies on days 1, 3 and day 7 to identify the Col II expressing cells. CD206 is a specific M2 macrophage surface marker. With 100 ng/mL M-CSF treatment, CD14⁺ monocytes were polarized to become CD14⁺/CD206⁺ M2 macrophage at a ratio of 15.9%, 16.8% and 28.5% on day 1, day 3 and day 7, respectively (Fig. 2B). The flow cytometry further reveals a unique subgroup in the M2 macrophage population, named modified macrophage in this invention, which expressed Col II, CD14, and CD206 proteins on the cell membrane at day 3 (Fig. 2C). The results were further confirmed by the immunocytostaining of Col II and CD14 in the adherent PBMNCs (Fig. 2D).

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Figure 2

The Col II expression in M2 macrophages. The monocyte population of PBMNCs was identified either by flow cytometry of FSC/SSC or CD14-FITC histogram (A). The M2 population was verified by both CD14 (monocyte-specific marker) and CD206 (M2-specific marker) expressions at indicated days (B). The coexpression of CD206 and Col II in CD14⁺ monocytes was detected on Day 5 culture (C). The presence of CD206 indicates the cultured CD14⁺ monocytes have matured into modified macrophages. The M-CSF-treated PBMNCs on day 3 (D3) and day 7 (D7) were immunostained with Col II (green) and CD14 (red) (D). The nuclei were stained with DAPI as blue.

We next examine the dose–effect of M-CSF/GM-CSF on Col II expression. The PBMNCs were treated with 2, 10, 20, 50, and 100 ng/mL M-CSF (Fig. 3A) or GM-CSF (Fig. 3B) for 3 days. Cell populations were first gated based on the FSC-A and SSC-A to identify monocytes, and the monocyte population was further analyzed based on Col II expression. The result of Fig. 3 showed that 10 ng/mL M-CSF or 10 ng/mL GM-CSF, the typical concentrations for macrophage polarization, only activated about 5% Col II-expressing cells. Higher doses of the cytokines, such as 100 ng/mL M-CSF and GM-CSF, efficiently steered the modified macrophage induction in a dose-dependent manner (Fig. 3B and D).

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Figure 3

The dose effects of M-CSF (A, B) and GM-CSF (C, D) on Col II expression of modified macrophages were examined by flow cytometry on day 3. The fold induction results (B, D) were analyzed from triplicated experiments.

In contrast to the M2 polarizing factor of M-CSF, GM-CSF is a M1 polarizing factor and shows lower M2 induction efficacy. The treated PBMNCs with either control medium, M-CSF, GM-CSF, or G-CSF, were cultured in suspension for 3 days and then adhered on fibronectin-coated slides. Interestingly, immunocytochemical staining results with anti-CD206 antibody (red) and anti-Col II antibody (green) identified the robust induction of modified macrophage by either M-CSF or GM-CSF treatments (lane 2, 27.7±2.5% ; lane 3, 23.3±1.5%; Fig. 4A,B), but not the control medium (lane 1, 0% , Fig. 4A,B) and G-CSF (lane 4, 4.7±2.3%, Fig. 4A,B).

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Figure 4

The effect of 100 ng/mL M-CSF, GM-CSF, and G-CSF on the induction of Col II (green) and CD206 (red) coexpression in modified macrophages (A). The ratio of Col II expression in CD206⁺ cells was analyzed by Image J from three independent immunocytostaining results (B).

M2 macrophages can be initially induced by M-CSF from naïve monocytes and become mature by IL-4, IL-10, IL-13, or TGF-β. We next examined which mature factor can potentiate the M-CSF/GM-CSF effect on enforcing Col II expression. We isolated the PBMNCs and cultured the PBMNCs with control medium alone, IL-4, IL-10, IL-13, TGF-β at 20 ng/mL for 3 days. Cell populations were immunostained to identify modified macrophage populations based on Col II and CD206 expression. Our results indicated that among the tested cytokines, IL-4 showed the highest induction ratio of modified macrophage from the PBMNCs population (Fig. 5A,B).

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Figure 5

The effects of various cytokines on inducing Col II and CD206 expression of modified macrophages were examined by flow cytometry on day 3 (A, B). The ratio of Col II/CD206 modified macrophages of cultured PBMNCs was examined in the conditions of IL-4/M-CSF (C) and IL-4/GM-CSF (D), based on the results of the immunocytochemical staining on day 3. The Col II mRNA expression was analyzed by quantitative real-time RT-PCR at 0.5, 1, 2, 4, and 6 h after the addition of PBS (mock-treated) or M-CSF+IL-4 combination in the CD14 beads-enriched monocytes (E). The Col II mRNA expression was normalized to the housekeeping gene expression of glyceraldehyde 3-phosphate (GAPDH) (F) and shown as the fold difference at the indicated times (G). Statistic results are analyzed from three independent experiments. One-way ANOVA, *, p<0.05; ** p<0.01; ***, p<0.001.

We next examined the M-CSF/IL4 and GM-CSF/IL-4 combinations on Col II expression. The PBMNCs were cultured under four conditions, including control medium, 100 ng/mL IL-4, 100 ng/mL M-CSF or GM-CSF alone, 100 ng/mL M/CSF or GM-CSF with 100 ng/mL IL-4, for 3 days on fibronectin-coated slides. Compared to individual M-CSF (27.6±2.5%) or IL-4 groups (2.0±1.0%), M-CSF and IL-4 combinations showed synergistic induction (43.3±4.5%,>27.6±2.5%+2.0±1.0%) on modified macrophages from the cultured PBMNCs (Fig. 5C). A similar synergistic result was observed for the GM-CSF and IL-4 combination (Fig. 5D).

Based on this optimized Col II induction protocol, we determined the Col II RNA expression profile in isolated monocytes during the early induction. Treating the CD14-sorted PBMNCs (>90% purity, Fig. 5E) with M-CSF/IL-4 combination induced the Col II RNA expression in a time-dependent manner within the first 6 h culture (Fig. 5F,G). In the mock-treated cells, no significant difference in Col II RNA expression could be detected at the first 6 h of treatment.

CD206⁺ M2 macrophages are featured with anti-inflammatory activities, and CD206⁺/CD14⁺/Col II⁺ modified macrophages may exhibit similar potency for preventing inflammation. We investigated the therapeutic potential of the modified macrophages on arthritis. We established inflammatory arthritis in rats by administration of complete Freund’s adjuvant (CFA) in hindlimb joints. The RA model in both mice and rats was also established by injection of collagen into the tail vein. Significant joint and paw swelling were observed on day 7 and day 40 after the CFA and collagen treatment, respectively. The gross pathology persisted for 3 months if no intervention was introduced. On day 7 and day 50 after CFA and collagen induction, respectively, the rodents were intra-articularly injected with 0.2 mL PBS (mock) or with 2×10⁵ M-CSF/IL-4-treated PBMNCs (Figs. 6 and ​and7).7).

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Figure 6

The therapeutic effect of the modified macrophages on arthritic joints. The up panel A shows the hind knee joints of a rat without arthritis, treated with modified macrophages (cells) or without cells (PBS). The down panel A shows the hind knee joints of rats with adjuvant (CFA)-induced arthritis, treated with cells or without cells (PBS). The thickness of knee swelling in rats without arthritis (B) or with arthritis (C) was measured after the treatments with the modified macrophages or (PBS). Arrow, the time of cell transplantation. *, p<0.05, one-way ANOVA.

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Figure 7

The C57BL/6 mice (A, B) and SD rats (C, D) with collagen-induced arthritis were treated with (B, D) or not treated with (A, C) the modified macrophages. The thickness of knee swelling in the rodents was measured, and the days of cell transplantation were indicated as arrows. **, p<0.01; ***, p<0.001, one-way ANOVA.

We measured the diameter of each knee joint or paw every 2–3 days post-treatment. Treating the normal rats with PBS and modified macrophages showed no attenuation effect on joint swelling (Fig. 6B). Importantly, providing M-CSF/IL-4-treated PBMNCs significantly reduced the progression and the severity of the CFA-induced arthritis, compared to the joints on day 7 (p=0.016, p<0.05) and the PBS-treated joints on day 32 (p=0.017, p<0.05) (Fig. 6C). Similar results were observed in the collagen-induced arthritis model in mice (Fig. 7A,B) and rats (Fig. 7C,D). The inhibition of the arthritis could not be mediated by the soluble polarization factors due to the complete removal of the original medium by twice washes. Notably, the slight recurrence of tissue inflammation in modified macrophage-treated RA rodents was noticed on day 70 after the first cell administration on day 50 (Fig. 7B and D). A secondary cell transplantation boost on day 80 significantly attenuated the paw swelling till day 105, and no sign of recurrence was observed. These results suggest that the modified macrophages might be therapeutically effective for controlling OA or RA inflammation.

Discussion

Monocytes have multiple functions and can differentiate into several distinct lineages, such as macrophage and dendritic cells. Derivative M1 and M2 macrophages carry out pro- or anti-inflammatory responses, respectively. Dendritic cells in tissues are the primary antigen-presenting cells (APC) to trigger T cell recognition and activation. In addition to the roles of immune modulation, macrophages and local fibroblasts can promote tissue repair at inflammatory sites. In this study, we discovered that monocytes could become a novel population, featured with Col II expression, induced by high-dose M-CSF or GM-CSF. This unique cell might exhibit both anti-inflammatory and tissue-repairing properties to ameliorate the destructive progress of arthritis in patients.

In general, monocytes/macrophages are supposed to secret cytokines and promote collagen formation in the inflammatory tissues, including both Col I and Col III, for tissue repairing and immunomodulation^15,16. Notably, a recent study revealed that not only myofibroblast cells in the damaged tissues, but also blood monocyte-derived macrophages directly contribute to Col I deposit and scar formation during mouse heart repair¹⁷. This novel result highlights that monocytes/macrophages orchestrate the cellular properties of migration, invasion, and tissue reconstruction. In this study, treating the monocytes with both M-CSF and GM-CSF steered about 40–50% adherent PBMNCs (Fig. 1), and 10–20% suspended PBMNCs (Fig. 2) to express Col II. Higher Col II expression seems correlative to the M-CSF and adhesion-triggered maturation in monocyte-derived cells. Whether the down-regulation of Col II prevents the monocyte maturation or migration remains unclear. The maturation of the Col II and the extracellular seretion of the Col II need further investigation. It is also interesting to examine the role of Col II and its reciprocal relationship with Col I, Col III and other ECM in the cell migration or tissue repairment of modified macrophages.

Col II is specifically expressed by chondrocytes and is the major matrix protein in cartilage. In addition to the cartilage, Col II is also detected in the vitreous body of eyes, accounting for 60–75% of the total collagen proteins¹⁸. Non-cartilage expression of Col II, especially for the mRNA of IIA splicing form, is also found in developmental paraxial mesodermal cells, such as somites and skeletal primordial embryonic structure¹⁸. The spatial and temporal regulation of Col II in non-cartilage tissue is largely obscure. It is interesting to explore whether the non-cartilage Col II expression in modified macrophages and in embryonic paraxial mesoderm share similar core regulatory complexes and what novel regulators are required for the ex vivo enforced Col II expression.

Sox9, a member of the family of SRY (Sex determining region of the Y chromosome)-related high-mobility-group box (SOX) transcription factors, is a dominant factor for the Col II expression and cartilage formation¹⁹. Human SOX9 gene mutations contribute to campomelic dysplasia, a severe skeletal malformation syndrome with gonadal dysgenesis, indicating that SOX9 is a pivotal factor in controlling bone and testes development¹⁹. Whether Sox9 participates in the Col II expression in the modified macrophages remains unclear. Regarding the signaling pathway of M-CSF and Sox9 activation, M-CSF activation triggers classical receptor tyrosine kinases to activate intracellular MAPK, PI3K, and Stat3 pathway and to promote macrophage proliferation, differentiation, and survival²⁰. Stat3 signal is essential to activate Sox9 expression by binding to the proximal promoter region of Sox9 genome²¹. It is possible that after the treatment of extra-physiological M-CSF/GM-CSF stimuli on the cell membrane, intracellular phosphorylated Stat3 triggers the Sox9 induction and Sox9-downstream Col II gene activation. Interestingly, a recent finding indicated that RAW264.9 cells and mouse bone marrow-derived macrophages are competent to activate Sox9 genes after IL-4 treatment²². However, their Col II protein induction in the macrophages was not investigated.

Notably, Sox9 is a well-characterized chromatin modulator and usually binds to the super-enhancer to control the lineage status, cellular stemness, and differentiation¹⁹. Sox9 and its associated transcriptional regulators, such as Sox5 and Sox6, are master regulators for the spatiotemporal expression of Col II¹⁹ and might coordinately participate in the Col II expression in the modified macrophages. The genome-level regulatory elements of Col IIα1, such as the intron 1 enhancer and intron 6 enhancers, and their epigenetic modification on the ectopic Col II expression may also involve the Sox9-mediated Col II expression of the modified macrophages^23,24.

In OA and RA patients, the destructive cartilage and joint lining cells usually bring elevated inflammatory cytokines, polarized M1 macrophages, and synovitis during the disease progress. The current candidates of cell products for the disease-modifying OA drugs (DMOADs) include the mesenchymal stem cells, TGF-β expressing 293 cells and bone marrow-derived mononuclear cells to ameliorate the inflammation and promote tissue regeneration^25,26. Here, we provide a new candidate for DMOADs, modified macrophages, featured with both CD206 and Col II matrix expression to exhibit both anti-inflammatory and tissue repair activity. The Col II is crucial for the reconstruction of hyaline cartilage. A clinical trial demonstrated that intra-articular injection of bovine Col II showed pain-relief efficacy, promising immune suppression, and increased glycosaminoglycan production from the endogenous chondrocytes²⁷. Interestingly, a recent study also revealed that squid Col II could relieve OA symptoms by inhibiting the cell death of chondrocytes and promoting the M2 macrophage polarization²⁸. These findings emphasize the importance of Col II supplement in controlling arthritis progress. The discovery of the Col II-expressing modified macrophages, and the amelioration of arthritis diseases in rodents provide promising preclinical evidence bases for future clinical study of the cells in controlling arthritis.

Materials and methods

Acknowledgements

Author contributions

F.H.W., C.Y.H., and H.L.S. planned the study and designed all the in vitro, ex vivo, and in vivo experiments. F.H.W., C.Y.H., C.I.S., C.H.C., Y.H.C., and C.C.K. performed the in vitro, ex vivo, and in vivo experiments. F.H.W., C.Y.H., C.L.L., and H.L.S. performed the analysis of the statistical results. C.H.C, Y.H.C., C.C.K., K.D.L., and C.L.L. provided essential materials. C.L.L. and H.L.S. wrote the manuscript.

Funding

This work was supported by the Industry-Academic cooperation project (108-D-595, 109-D-525) between the Duogenic StemCells Corp. and National Chung Hsing University.

Data availability

The data that support the findings of this study are available from Hong-Lin Su upon request.

Competing interests

The authors have the following competing interests. The technology transfer office of National Chung Hsing University has received consultancy, speaker fees, and research grants on behalf of HLS from Duogenic StemCells Corp and Hualien Tzu-Chi Medical Center. FHW, CYH, and JIS are employees of Duogenic StemCells Corp. FHW, CYH, JIS, CHC, CCK, and CLL are shareholders of Duogenic StemCells Corp. There are patents, products in development, and marketed products associated with this research.

Footnotes

Contributor Information

Chih-Lung Lin, Email: moc.liamg@9250nilffej.

Hong-Lin Su, Email: wt.ude.uhcn@nilgnohus, Email: moc.liamg@nilgnohus.

References