
==== Front
Bone Joint Res
Bone Joint Res
BJR
Bone & Joint Research
2046-3758
The British Editorial Society of Bone & Joint Surgery London

10.1302/2046-3758.139.BJR-2024-0122.R1
BJR-2024-0122.R1
Bone Biology
bj11388Orthopaedic diseasesbj11416Orthopaedic treatmentsbj2262Bone regenerationbj9337Lymphocytebj9357Macrophagebj9891Mesenchymal stem cellbj4173Cytokinesbj11453Ossificationbj2208Bone marrow aspirate concentratebj14659Secretionbj7463Inflammationbj11128NonunionThe interactions of macrophages, lymphocytes, and mesenchymal stem cells during bone regeneration
https://orcid.org/0009-0000-1228-482X
Murayama Masatoshi MD, PhD Visiting Instructor 1 muramasa@stanford.edu
muramasa218@gmail.com

https://orcid.org/0000-0003-1742-980X
Chow Simon K. PhD Senior Research Scientist 1 skhchow@stanford.edu
skhchow@ort.cuhk.edu.hk

https://orcid.org/0000-0003-3526-288X
Lee Max L. MA Medical Student 1 maxlee12@stanford.edu

Young Bill BS Medical Student 1 younbil@stanford.edu

Ergul Yasemin S. Visiting Student Researcher 1 yaseminsudeergul@gmail.com

https://orcid.org/0000-0002-4122-7119
Shinohara Issei MD, PhD Visiting Instructor 1 issei27@stanford.edu

https://orcid.org/0009-0002-1458-4361
Susuki Yosuke MD, PhD Postdoctoral Fellow 1 susuki10@stanford.edu

https://orcid.org/0000-0002-1244-8326
Toya Masakazu MD, PhD Postdoctoral Fellow 1 mtoyaorthop@gmail.com

https://orcid.org/0000-0003-0175-566X
Gao Qi PhD Postdoctoral Fellow 1 qigao7@stanford.edu

https://orcid.org/0000-0002-1919-3717
Goodman Stuart B. MD, PhD Professor 1 2 goodbone@stanford.edu

1 Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
2 Department of Bioengineering, Stanford University School of Medicine, Stanford, California, USA
Correspondence should be sent to Stuart B. Goodman. E-mail: goodbone@stanford.edu
S. B. Goodman reports partial funding from his position as Robert L. and Mary Ellenburg Professor of Surgery, Stanford University, related to this study.

6 9 2024
9 2024
13 9 462473
© 2024 Murayama et al.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ https://online.boneandjoint.org.uk/TDM This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (CC BY-NC-ND 4.0) licence, which permits the copying and redistribution of the work only, and provided the original author and source are credited. See https://creativecommons.org/licenses/by-nc-nd/4.0/

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Bone regeneration
Bone marrow aspirate concentrate therapy
Cell-based therapy
Immunomodulatory therapy
Stem cells
lymphocytes
macrophages
mesenchymal stem cells (MSCs)
cytokines
osteogenesis
bone marrow aspirate concentrate (BMAC)
secretion
inflammation
nonunion of fractures
odf2nlm-version2.0
price$2.00
principal-institutionStanford University School of Medicine, Stanford, California, USA
article-typeBone Biology
coi-statementS. B. Goodman reports partial funding from his position as Robert L. and Mary Ellenburg Professor of Surgery, Stanford University, related to this study.
==== Body
pmcArticle focus

Investigating the interactions of macrophages, lymphocytes, and mesenchymal stem cells (MSCs) in bone regeneration.

Exploring how the immune cells influence the osteogenic potential of MSCs and bone healing.

Evaluating potential strategies for optimizing cell-based therapies for bone regeneration by utilizing specific interactions among immune cell interactions with MSCs.

Key messages

Macrophages and lymphocytes play critical roles in bone regeneration, with different subtypes promoting either bone formation or resorption. Their interaction with MSCs is crucial for effective bone healing.

The balance between pro-inflammatory and anti-inflammatory immune cells significantly influences the osteogenic potential of MSCs, with M2 macrophages, helper T2 (Th2) and regulatory T (Treg) cells being particularly supportive of bone formation.

Enhancing the regenerative capacity of bone marrow aspirate concentrate (BMAC) by modulating immune cell composition and interactions offers a promising means to improve clinical outcomes in bone repair and regeneration.

Strengths and limitations

This study provides a comprehensive overview of the crosstalk between immune cells and MSCs, highlighting the potential for targeted immunomodulation to promote bone regeneration.

The manuscript identifies specific immune cell subtypes that can be targeted to improve the efficacy of BMAC-based therapies for bone healing, and provides practical insights for clinical applications.

The variability in outcomes due to differences in MSC concentration and immune cell composition in BMAC highlights the challenge of achieving consistent results, necessitating further research to standardize therapeutic approaches.

Introduction

Compromised bone healing often affects a patient’s function, potential for ambulation, and quality of life. Factors that could lead to undesirable clinical outcomes include: poor overall health; serious medical comorbidities such as diabetes and chronic renal disease; obesity; medications; and ageing.1,2 Chronic inflammation is present in many of these scenarios,3 and is associated with several commonly observed conditions including corticosteroid-induced osteonecrosis, fracture nonunions,4-6 and persistent bone defects.

Recently, bioengineering strategies have been developed for augmenting bone regeneration and repair. One approach includes grafting of reparative cells and a biocompatible scaffold. The most common source of cellular components is autologous bone marrow aspirate concentrate (BMAC), which fulfills the principle of “minimal manipulation” mandated by the USA Food and Drug Administration (FDA).7 Another strategy is the use of stem cell therapy, which is permissible by regulatory bodies in some countries despite these cells undergoing more than “minimal manipulation”.

BMAC is an autologous, safe, and reliable source of cells that has demonstrated a solid foundation with sufficient biological basis for bone regeneration. Some applications of BMAC include the enhancement of healing of osteonecrotic lesions during core decompression8,9 and healing of long bone fractures in pre-clinical animal studies, as well as in clinical trials.10

However, the outcomes of BMAC use are not uniformly positive. In a study by Cuomo et al,11 neither bone marrow aspirate (BMA) nor mesenchymal stem cell (MSC)-enriched BMA mixed with demineralized bone matrix resulted in reliable healing of a 6 mm critical-sized bone defect in the rat femur. The authors suggested that the number of MSCs, the presence of an enhanced osteoinductive signal (e.g. bone morphogenic protein-2 (BMP-2)), or the variability of the carrier are among many contributing factors to deficiency of bone formation in this model.

Clinical grafting of progenitor cells for fracture nonunion is safe and effective. In one study, the radiological outcome evaluated by preoperative and four-month postoperative CT scans, for the treatment of nonunions, was dependent on the percentage of progenitor cells present.12 BMAC contains a mean MSC concentration of 0.001% detected by flow cytometry; thus, the quality of the BMAC is an important factor when considering cell-based therapy.13 Furthermore, a recent meta-analysis of the use of BMAC for the treatment of small non-critical size fracture nonunions reported a healing rate of only 71% to 77%. Although there are reports that MSCs alone can promote bone formation,14,15 bone union was achieved in only 4% to 59% of cases using MSCs alone without immune cells.16 These healing rates in small defects suggest that substantially worse results are anticipated for larger critical-size bone defects. These results also highlight the importance of immune cells as a source of osteoinductive paracrine signals. Not only are immune cells involved, but the presence of endocrine/metabolic pathways and environmental factors also influence the process of bone formation.

Current clinical and pre-clinical reports together advocate for further opportunities for the optimization of BMAC as a cell-based therapy. The strategy could encompass specific targeting of the deficiencies of BMAC including the low progenitor cell number, suboptimal immune cell composition, or the variability of the delivery biomaterial. The purpose of this review was to summarize the roles of major immune cell components, including macrophages and lymphocytes (T cells) in BMAC, in terms of their effects on osteogenesis, and to identify the knowledge gap and potential opportunities to enhance osteogenesis by a minimal manipulative cytotherapeutic approach.

Major cellular components of the bone marrow

MSCs have self-renewal and multipotent differentiation capabilities, and are able to differentiate into various cell types such as osteoblasts, adipocytes, chondrocytes, myotubes, fibroblasts, and more. Currently, MSCs find broad applications in cell-based therapies owing to their immunomodulatory properties and regenerative potential. In the bone marrow, MSCs constitute a very small fraction, ranging from 0.001% to 0.01% of nucleated cells, while macrophages (granulocytic lineage) account for 40% to 55%, and T cells account for up to 25%.17 However, in addition to the potential differentiation into osteoblasts, MSCs influence macrophages and T cells by secreting paracrine factors and extracellular vesicles (EVs), thereby contributing to bone formation. The relevance of macrophages and T cells interacting with MSCs in facilitating bone formation and remodelling is briefly outlined below. Nonetheless, other immune cells also play important roles in regulating bone generation, including neutrophils and mast cells in tandem with MSCs, macrophages, and T cells; these interactions will also be briefly discussed.

Macrophages

Macrophages are members of the monocyte/macrophage/foreign body giant cell/osteoclast/dendritic cell lineage. Macrophages are present in most tissues, where they play crucial immunomodulatory roles by recognizing, engulfing, and degrading cellular debris and pathogens. In the non-stromal cell population of the bone marrow, 50% of cells are white blood cells (WBCs), i.e. monocytes/macrophages, polymorphonuclear leucocytes, mast cells, and their precursors; 25% are in the erythropoietic lineage; and the balance, about 25%, are in the T cells lineage.18 Macrophages present antigens to T cells and induce the expression of co-stimulatory molecules on antigen-presenting cells. Macrophages can alter their polarization phenotype in response to local environmental cues.19 Activated macrophages are typically classified into two general phenotypes: the pro-inflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes.20,21 M1 macrophages are induced by inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) from helper T1 (Th1) cells, as well as inflammatory stimuli like bacterial lipopolysaccharide (LPS).22 M1 macrophages release high levels of pro-inflammatory cytokines such as interleukin-1-beta (IL-1β), IL-6, TNF-α, and IFN-γ.23 Conversely, M2 macrophages are induced by Th2 cytokines such as IL-4 and IL-13, and they secrete anti-inflammatory cytokines like IL-10 and transforming growth factor-beta (TGF-β).

Macrophages can differentiate into osteoclasts involved in bone resorption and metabolism.24 Furthermore, it has been reported that M1 macrophages are more prone to differentiate into osteoclasts rather than naïve or M2 macrophages.25 Osteoclasts are particularly involved in bone resorption and are promoted in inflammatory environments. Inflammatory cytokines such as IL-1 and TNF-α secreted by M1 macrophages enhance the production of receptor activator of nuclear factor kappa-B ligand (RANKL), which promotes osteoclast formation and activity. Furthermore, inflammatory cytokines IFN-γ and TNF-α induce apoptosis in bone marrow mesenchymal stem cells (BMMSCs), inhibit collagen and proteoglycan synthesis, and thereby impede bone formation.26,27 Consequently, during inflammation, the bone resorptive activity of osteoclasts can outpace the bone-forming ability of osteoblasts, greatly contributing to inflammatory bone loss. Therefore, the balance between M1 and M2 phenotypes plays a crucial role in various microenvironments including bone.28 This suggests a potential for immunomodulation at target sites by adjusting this macrophage balance, for the purpose of bone regeneration.

T cells

T cells comprise approximately 25% of the non-stromal cells in the bone marrow. They originate from haematopoietic stem cells in the bone marrow and mature in the thymus. T cells are further divided into αβ and γδ T cells; αβ T cells consist of CD4+ Th cells and CD8+ cytotoxic T cells. CD4+ Th cells are the most extensively studied subset. CD4+ Th cells interact with other immune cells via surface receptors and modulate activation states by secreting cytokines.29 CD8+ cytotoxic T cells play a crucial role in eliminating intracellular pathogens and emerging neoplasms.

CD4+ Th cells can be subdivided into different subsets based on their cytokine expression profiles, such as Th1, Th2, Th17, and regulatory T (Treg) cells. Th1 cells are polarized by IL-12, produce IFN-γ and TNF-α, induce cell-mediated immune responses, and regulate the activation of M1 pro-inflammatory macrophages and inflammatory reactions.30,31 Th2 cells, stimulated by IL-2 and IL-4, secrete IL-4, IL-5, IL-10, and IL-13 and regulate immune responses such as activation of B lymphocytes, eosinophils, and M2 macrophages.32,33 Th17 cells are stimulated by cytokines like IL-6 and IL-23, secrete IL-17 and IL-22, and participate in inflammatory and autoimmune responses.34,35 Treg cells, induced by cytokines such as TGF-β, IL-2, and IL-10, are involved in suppressing autoimmune responses and inflammation.

Generally, cytokines produced by Th1 and Th17 cells exhibit pro-inflammatory properties, while those from Th2 and Treg cells demonstrate anti-inflammatory effects. The inflammatory microenvironment has secondary effects on both osteoclasts and osteoblasts; thus, the balance between Th1/Th2 cells and Th17/Treg cells has great relevance to the homeostatic equilibrium between bone resorption and formation.

T cells are believed to play important roles in coordinating metabolism and assist in the process of tissue regeneration. For example, conditioned medium from human CD4+ T cells has been shown to statistically significantly upregulate the expression of Runt-related transcription factor 2 (Runx2), osteocalcin, alkaline phosphatase (ALP), and bone sialoprotein in allogenic MSCs, enhancing mineralization of bone in culture of MSCs.36 Therefore, it is important to delineate the roles of macrophages and T cells, and their crosstalk with stromal cells from BMA, in order to exploit their unique characteristics and enlist them as supporting factors for bone regeneration.

Neutrophils

Neutrophils are a subset of granulocytes derived from the haematopoietic stem cell lineages and are part of the innate immune system; neutrophils exhibit chemotaxis, phagocytosis, and bactericidal activity.37,38 Neutrophils are the first inflammatory cells to migrate to the injury site in response to chemotactic stimuli from resident macrophages, and participate in the clearance of bacteria, dead cells, and debris.39 Additionally, neutrophils secrete inflammatory and chemotactic mediators such as IL-6 and CCL2, which recruit monocytes/macrophages.40,41 These recruited monocytes/macrophages influence MSC migration and osteogenic differentiation.42 However, excessive and continued neutrophil-induced inflammation may contribute to impaired fracture healing, by heralding a state of chronic inflammation.3,43

Mast cells

Mast cells (MCs), derived from the haematopoietic stem cell lineage, are tissue-resident immune cells well known for promoting allergic reactions.44 Like neutrophils, MCs are part of the innate immune system and are capable of phagocytosis. They regulate vascular permeability and blood flow to initiate the rapid recruitment of effector cells such as neutrophils, eosinophils, and natural killer cells.45 MCs store and newly synthesize mediators, including cytokines and enzymes, which can be rapidly released in response to stimuli in acute inflammation or allergic reactions.46 These mediators include histamine, IL-6, and TNF-α, which promote osteoclast formation, and IL-1 and TNF-α, which inhibit osteoblast activity, thereby promoting bone resorption and inhibiting bone formation. Conversely, MCs can promote bone formation through TGF-β and potentially reduce osteoclast formation and bone resorption via IL-12. MCs also enhance MSC proliferation and migration.47 However, the effects on bone metabolism remain unclear, as studies using MC-deficient mice have shown contradictory results depending on the mouse model used.

Crosstalk between major cell types

MSCs and macrophages

MSCs and macrophages mutually influence each other, promoting osteogenesis. Studies using human buffy coats showed that factors secreted by pro-inflammatory macrophages statistically significantly increased MSC adhesion and migration, whereas factors from anti-inflammatory macrophages enhanced MSC osteogenic activity and cell migration.48 However, research utilizing human inflammatory synovium revealed that only the conditioned medium from anti-inflammatory macrophages enhanced MSC migration, with no statistically significant impact observed with pro-inflammatory macrophage-conditioned medium.49

In studies involving direct co-culture of MSCs and macrophages, it has been reported that the initial inflammatory phase regulated by M1 macrophages promotes osteogenesis by MSCs via the COX-2-PGE2 pathway.50 Macrophages derived from human monocytic leukaemia THP-1 cell line secrete IL-23 in the inflammatory environment, which activates the signal transducer and activator of transcription 3 (STAT3) and β-catenin pathways, thereby enhancing expression of markers of bone formation and osteogenic differentiation by MSCs.51 Moreover, bone formation was enhanced by promoting the differentiation of inflammatory M1 macrophages into anti-inflammatory M2 macrophages 72 hours after the initial inflammatory phase,52,53 emphasizing the importance of M1 macrophages initially and the early inflammatory environment in bone formation.42 Furthermore, recent studies have revealed that EVs, containing proteins and microRNAs and other molecules, are endocytosed by target cells, where they exert their functional influence.54 Enrichment of miR-155 in the EVs of M1 macrophages decreased osteogenic differentiation of MSCs, while treatment of MSCs with miR-378a, enriched in the EVs of M2 macrophages, increased MSC osteoinductive gene expression.55 Additionally, polarized M2 macrophages release TGF-β, promoting osteogenesis by MSCs;56 BMP-2 secreted by M2 macrophages also enhances bone differentiation,57,58 suggesting that macrophages in the M2 polarized phenotype may have a greater impact on MSC osteogenesis compared to M1 macrophages. MSCs have also been shown to possess anti-inflammatory properties and immunomodulatory functions. MSCs were shown to regulate macrophage polarization, phagocytosis, and metabolism.59 In one study, naïve macrophages cultured with MSCs promoted the secretion of the proinflammatory cytokines TNF-α and IL-12. In contrast, M1 macrophages cultured with MSCs shifted towards an M2 macrophage phenotype.60 One of the MSC-derived EVs, miR-181c, has been found to inhibit the expression of Toll-like receptor 4 (TL4) and reduce the expression of inflammatory factors such as TNF-α and IL-1β.61 Furthermore, MSC-derived EVs promoted polarization towards the M2 phenotype, leading to enhanced expression of anti-inflammatory cytokines.62,63 Additionally, it has been observed that the polarization effect of MSC-derived EVs towards the M2 phenotype is more pronounced when MSCs are pre-treated with pro-inflammatory substances or cytokines, such as LPS or elevated reactive oxygen species (ROS).64-66 MSC-derived EVs regulate macrophage polarization toward anti-inflammatory M2 macrophage subtypes, especially when inflammatory cytokines are present. In summary, the interaction between MSCs and macrophages influences the process of osteogenesis (Figure 1). The presence of macrophages was generally shown to enhance osteogenesis of MSCs alone, with M2 macrophages showing a greater beneficial effect on bone formation compared to the M1 phenotype.

Fig. 1 Crosstalk between mesenchymal stem cells (MSCs) and macrophages. MSCs promote macrophage polarization into the anti-inflammatory M2 phenotype by the cytokines and extracellular vesicles (EVs) described. The cytokine interleukin-1 receptor antagonist (IL-1Ra) from MSCs inhibits polarization of macrophages into the pro-inflammatory M1 phenotype. Cytokines and EVs secreted by M2 macrophages promote osteogenesis of MSCs, while cytokines and EVs secreted by M1 macrophages inhibit MSC osteogenesis. BMP, bone morphogenic protein; COX-2, cyclooxygenase-2; HFG, hepatocyte growth factor; IFN-γ, interferon-gamma; miR, microRNA; OSM, oncostatin M; TGF-β, transforming growth factor beta; TNF-α, tumour necrosis factor-alpha.

MSCs and T cells

The absence of T cells in mice has been identified as a determinant for decreased differentiation and proliferation of MSCs, emphasizing the substantial crosstalk between T cells and MSCs.67 Focusing on the relationship between MSCs and cytokine-releasing CD4+ Th cells, the impact of T cells on MSCs is summarized below.

Activated T cells promoted the secretion of BMP-2 by MSCs, leading to enhanced bone formation.68 Additionally, conditioned media from human CD4+ T cells, but not CD8+ T cells, promoted bone formation in allogenic MSCs.36 CD4+ T cells consist of subsets with inflammatory Th1, Th17, and anti-inflammatory Th2, Treg characteristics. High levels of Th1 cytokines, such as IFN-γ and TNF-α, were correlated with decreased new bone formation.27 Furthermore, Th1 cells, which promote inflammation, inhibit osteoprotegerin (OPG) expression via IFN-γ production, leading to an increase in the RANKL/OPG ratio and promotion of osteoclast formation.69 Conversely, Th2 cytokines, such as IL-4 and IL-13, suppress RANKL expression by osteoblasts, enhance OPG expression, and decrease the overall RANKL/OPG ratio. These results suggest that Th2 cytokines decrease osteoclast formation and promote osteoblast activity.70-72

Treg cells can inhibit osteoclast formation through direct contact with high expression of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) by Treg cells and cytokine-dependent mechanisms.73,74 Treg cells promote the proliferation and differentiation of osteoblasts by activating intracellular effectors such as mitogen-activated protein kinases (MAPKs) and Smad-related proteins, which induce differentiation of MSCs to osteoblasts through the secretion of TGF-β.69,75,76 Furthermore, treatment of MSCs with dihydroepiandosterone (DHEA) in a mouse model increased the proportion of Tregs, and resulted in increased osteoblastogenesis and osteogenesis.77 Additionally, Tregs have been shown to enhance the immunomodulatory properties of MSCs through the secretion of anti-inflammatory factors such as IL-10.78-80

Unlike Treg cells, Th17 cells can promote osteoclast formation through both direct and indirect mechanisms. Th17 cells directly express RANKL on their surface, stimulating the proliferation and differentiation of osteoclast precursors.81,82 IL-17 secreted by Th17 cells indirectly induced the expression of macrophage colony-stimulating factor (M-CSF) and RANKL on the surface of MSCs, promoting osteoclast formation.81,83,84 Furthermore, many cytokines produced by Th17 cells induced the production of inflammatory factors, enhanced the expression of NF-κB, and further promoted RANKL expression. IL-17 is also associated with migration and motility of MSCs.4 Additionally, a study using mouse bone marrow MSCs found that IL-17 enhances MSCs’ immunosuppressive function by increasing the expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO).85 Similarly, Th1 cells have also been found to enhance the immunomodulatory functions of MSCs through the secretion of proinflammatory cytokines.86,87

In summary, anti-inflammatory Th2 and Treg cells are involved in bone formation, while pro-inflammatory Th1 and Th17 cells are implicated in osteoclast formation, thereby promoting bone resorption.

Regarding the impact of MSCs on T cells, co-culture of murine T cells and MSCs led to a decrease in the levels of TNF-alpha and IFN-gamma, suggesting that MSCs exert anti-inflammatory effects.88 One of the key mechanisms through which MSCs attenuate the immune response is through modulation of the Th1/Th2 cell and Th17/Treg cell balance.78,89 MSCs are involved in shifting the Th1/Th2 balance towards Th2 cells, demonstrating their anti-inflammatory effects.90-94

Treg cells have potent and well-established anti-inflammatory effects.95-97 Numerous studies have indicated that MSCs are involved in the proliferation and differentiation of Tregs, via different pathways including the Notch signalling pathway, the Fas/Fas ligand signalling pathway, and the mTOR signalling pathway.78,98-101

Th17 cells are pro-inflammatory cells that exert their effects through the secretion of pro-inflammatory cytokines including IL-17.102,103 Multiple studies suggest that MSCs are involved in the inhibition of Th17 cells and their ability to secrete IL-17. MSC-dependent suppression of Th17 cells involves many cells and pathways including the IL-10 signalling pathway, the prostaglandin E2 (PGE2) signalling pathway, the CCL2 signalling pathway, and the PD-1/PDL1 signalling pathway.104-110 These pathways have been shown to be context-dependent. For example, PGE2 signalling by MSCs can stimulate or downregulate Th17 cells under different conditions, depending on cell maturity and the local microenvironment.105,110,111

In summary, MSCs generally promote the differentiation of T cells to anti-inflammatory Th2 and Treg cell phenotype, particularly in inflammatory environments, leading to anti-inflammatory effects (Figure 2). Conversely, Th2 and Treg cells enhance osteoblastogenesis and bone formation. Therefore, it is suggested that the interactions between MSCs and T cells are mostly skewed towards the anti-inflammatory side where MSCs, Th2, and Treg are promoting one another toward resolving the acute inflammatory response, decreasing the secretion of pro-inflammatory cytokines, and increasing the secretion of anti-inflammatory cytokines, thus enhancing bone regeneration.

Fig. 2 Crosstalk between mesenchymal stem cells (MSCs) and T cells. MSCs promote differentiation of CD4+ T cells into anti-inflammatory Th2 and regulatory T (Treg) cells, and inhibit differentiation of CD4+ T cells into inflammatory Th1 and Th17 cells. Th2 and Treg cells promote osteogenesis of MSCs, while Th1 cells inhibits MSC osteogenesis. Treg cells induce apoptosis of osteoclast precursors, while Th17 cells promote osteoclastogenesis by receptor activator of nuclear factor-kappa B ligand (RANKL) and cytokines. Th2 cytokines increase osteoprotegerin (OPG) and inhibit differentiation into osteoclasts, while Th1 cytokines decrease OPG and promote differentiation into osteoclasts. CTLA-4, cytotoxic T-lymphocyte associated protein 4; IFN-γ, interferon-gamma; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B; PTH, parathyroid hormone; TGF-β, transforming growth factor beta; Th, T helper; TNF-α, tumour necrosis factor-alpha.

Macrophages and T cells

When murine macrophages were co-cultured with Th1 cells to simulate inflammatory bowel disease (IBD), M1 polarization of macrophages was found to be promoted through STAT3 signalling.21 It was suggested that Th1 cells may play a causative role in the immune response and pathology in IBD patients, and perhaps other inflammatory disorders. The polarization pathway is dependent on the helper T cells’ phenotypes. Unlike Th1 cells, Th2 cells produce IL-4 to activate the M2 macrophage phenotype while simultaneously suppressing polarization of the M1 macrophage phenotype (Figure 3).112 Similarly, αβ T cells inhibit the inflammatory response by promoting M2 macrophage proliferation. One study found that knocking out αβ T cells led to increased polarization toward the M1 state, and decreased polarization toward the M2 state. This is a role that is also shared by Treg cells, which secrete cytokines to induce a shift towards M2 polarization, consistent with their anti-inflammatory function.67,113,114 This induction is associated with increased IL-10 production, and decreased major histocompatibility complex (MHC)-class II molecule expression. Subsequently, there is a decrease in MHC-related co-stimulation, which appears to control inflammatory processes such as Th17 cell expansion and promote immune tolerance.115 In tumours, Treg cells induce the release of IL-10 and IL-6 by macrophages, which subsequently promote tumour cell survival and Treg function.116 There is also modulation of the macrophage signalling pathway via microRNAs. Wu et al117 found that the release of IFN-γ by T cells primed the activation of macrophages, which was associated with a decrease in levels of miR-3473b. Indeed, restoration of miR-3473b levels reversed macrophage activation, suggesting a regulatory role of the microRNA in this inflammatory pathway.117 Understanding the downstream effects of these T cell/macrophage interactions, as well as potential modulators, can facilitate the identification of novel targets for immunotherapies and bone metabolism.

Fig. 3 Crosstalk between macrophages and T cells. Macrophages activate CD4+ T cells; M1 macrophages promote differentiation of CD4+ T cells into inflammatory Th1 and Th17 cells. Th1 and Th17 cells promote polarization of macrophages into M1 macrophages. M2 macrophages inhibit the differentiation of CD4+ T cells into inflammatory Th1 and Th17 cells, and promote the differentiation of CD4+ T cells into anti-inflammatory Th2 and regulatory T (Treg) cells. Th2 and Treg cells promote the polarization of macrophages into M2 macrophages. CTLA-4, cytotoxic T-lymphocyte associated protein 4; IFN-γ, interferon-gamma; MHC, major histocompatibility complex; RANKL, receptor activator of nuclear factor-kappa B ligand; TCR, T cell receptor; TGF-β, transforming growth factor beta; Th, T helper; TNF-α, tumour necrosis factor-alpha.

T cell activation requires antigen presentation via major MHC molecules. This is accomplished by different immune cells, including macrophages. Macrophages can present antigens via MHC class I or class II proteins. Macrophages also function as phagocytic cells and will digest foreign molecules and present subsequent antigens to activate T cells.118 Depending on the co-stimulatory molecules, these interactions can lead to variable T cell fates. The B7-1 ligand on macrophages can activate or inhibit T cell proliferation by binding to the CD28 receptor or cytotoxic T-lymphocyte associated protein 4 (CTLA-4) receptor on the T cell surface, respectively.119,120 One study found that the B7-CD28 interaction was synergistically acted upon by IL-12 secretion by macrophages, leading to enhanced T cell activation. These results suggest that IL-12 serves as a soluble signalling component in the T cell regulation pathway seen in macrophages.121 Another study found that administration of anti-B7 antibodies to macrophages diminished T cell responses, further underscoring the role of macrophages in early T cell activation.122

Cytokines released by macrophages also serve as a mechanism for regulating naïve T cell differentiation. Macrophages can secrete IL-12 and IFN-γ to induce Th1 proliferation. Acting in a cyclic manner, these Th1 effector cells will subsequently produce IFN-γ and TNF to upregulate macrophage phagocytosis. Another study found that synovial macrophages release IL-2 to induce Th1 differentiation. Th2 differentiation is induced by IL-4 secretion by macrophages, ultimately enhancing the anti-parasitic response. Egan et al123 showed that synovial macrophages release IL-1B, IL-6, and IL-23 to induce Th17 differentiation. Macrophages can also release TGF-β, which polarizes Treg cells and generates a phenotype characterized by immunosuppression.124 The delivery of TGF-β by M2 macrophages was found to induce the expression of CTLA-4 and other Treg-associated molecules on naïve CD4 T cells.125 MSCs secrete TGF-β, which can induce macrophages to secrete IL-10 and CCL-18. These factors subsequently stimulate Treg growth.126 Together, these findings suggest the potential of utilizing macrophages as a mechanism for controlling inflammation following surgery or as a therapy to target the immune disorders. For example, tumour-associated macrophages have been shown to suppress T cell proliferation through expression of programmed cell death ligand 1 (PD-L1) and secretion of IL-10, an anti-inflammatory cytokine. The immunosuppressive effect is also achieved by recruiting Treg cell migration to carcinomas via CCL22 signalling.127 However, this tumour-induced T cell proliferation can be reversed to target tumour growth. Macrophage-induced T cell stimulation in tumours was restored via inhibition of B7-H4 expression on tumour macrophages, highlighting a mechanism for how the macrophage–T cell axis can be regulated in various microenvironments.128 Given the key role that macrophages and T cells play in the immune and healing responses, manipulating immune cell subpopulations to optimize recovery in the clinical context should be explored. Future research should investigate how impacting the sub-composition of the immune microenvironment can improve the efficacy of immunotherapeutics and ultimately enhance clinical outcomes.

In summary, the results from crosstalk among various cell types (Figure 4) suggest opportunities to manipulate BMAC in terms of the composition or the proportions of macrophages, T cells, and MSCs so that bone regeneration can be optimized. MSCs have immunomodulatory effects. When they are co-cultured with macrophages, the macrophages would preferentially differentiate into the M2 phenotype, and these differentiated M2 macrophages promote osteoblast differentiation and bone formation. Additionally, when CD4+ T cells are added, MSCs and M2 macrophages will promote CD4+ T cells to differentiate into Th2 or Treg cells, the anti-inflammatory phenotypes. The increase in Th2 and Treg cells would further promote the polarization of macrophages into M2. Additionally, promoting anti-inflammatory M2 macrophages and Th2/Treg cells, rather than pro-inflammatory M1, Th1, and Th17 cells, reduces osteoclast formation. In other words, Th2 cells, Treg cells, and M2 macrophages are believed to not only enhance bone formation by MSCs, but also promote bone formation by inhibiting bone resorption. Therefore, the simultaneous presence of macrophages, CD4+ T cells, and MSCs is important for successful coordinated bone repair and regeneration. Understanding the optimal ratios of these cells could further enhance the bone-forming effects of BMAC, offering promising prospects for future treatments.

Fig. 4 Upper panel: positive effects from the interactions between MSCs and macrophages. MSCs differentiate M0 into M2, and anti-inflammatory M2 macrophages promote osteogenesis of MSCs. Lower panel: positive effects from MSCs, macrophages, and CD4+ T cells. MSCs polarize M0 into M2 macrophages. MSCs and M2 macrophages differentiate CD4+ T cells into anti-inflammatory T helper (Th2) and regulatory T (Treg) cells. Th2 and Treg cells polarize M0 into M2 macrophages. M2 macrophages, Th2 cells, and Treg cells promote MSC osteogenesis. BMP, bone morphogenic protein; HFG, hepatocyte growth factor; miR, microRNA; MSCs, mesenchymal stem cells; M0, naïve macrophages; OSM, oncostatin M; PTH, parathyroid hormone; TGF-β, transforming growth factor beta.

However, in some patients, secretion of inflammatory cytokines due to senescense-associated secretory phenotypes (SASP) from senescent cells in ageing,129 decreased oestrogen levels in postmenopausal osteoporosis,130 sustained hyperglycaemia in diabetes,131,132 and chronic inflammatory conditions like rheumatoid arthritis may alter the functions of these cells.26

Conclusion

In the context of osteogenesis, co-culture of MSCs with macrophages has been demonstrated to enhance bone formation.50,57,133 This may be attributed to the potential of M2 EVs to promote MSC-associated osteogenesis and the capacity of MSCs to differentiate from an M1 to M2 phenotype. Elevated levels of inflammatory cytokines, including M1 cytokines such as IFN-γ and TNF-α, are associated not only with a deficiency in new bone formation but also with promoting osteoclast formation, thereby enhancing bone resorption.27 Co-culturing MSCs with T cells decreases the levels of TNF-α and IFN-γ in the co-culture medium, suggesting an anti-inflammatory effect of the MSCs,88 which could influence bone formation. Furthermore, activated CD4+ T cells produce soluble factors that contribute to osteoblastic differentiation of human MSCs.84 In macrophages and T cells, Th1 and Th17 promote the pro-inflammatory M1 phenotype,21 whereas Th2 and Treg cells promote the anti-inflammatory M2 phenotype.114,115 Additionally, M1 macrophages induce the proliferation of Th1 and Th17 cells, whereas M2 induces proliferation of Th2 and Treg cells. These findings suggest the potential for promoting bone formation by co-culturing MSCs, macrophages, and T cells together, highlighting the importance of understanding the roles of these cells. These findings have major implications for future therapies for immunomodulation of bone to enhance fracture healing and repair bone defects.

Data Availability

The data that support the findings for this study are available to other researchers from the corresponding author upon reasonable request.

Author contributions

M. Murayama: Conceptualization, Data curation, Resources, Visualization, Writing – original draft, Writing – review & editing

S. K. Chow: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing

M. L. Lee: Writing – original draft, Writing – review & editing

B. Young: Writing – original draft, Writing – review & editing

Y. S. Ergul: Writing – review & editing

I. Shinohara: Writing – review & editing

Y. Susuki: Writing – review & editing

M. Toya: Writing – review & editing

Q. Gao: Writing – review & editing

S. B. Goodman: Conceptualization, Formal analysis, Writing – review & editing

Funding statement

The authors disclose receipt of the following financial or material support for the research, authorship, and/or publication of this article: this project was partially funded by S. B. Goodman's position as the Robert L. and Mary Ellenburg Professor of Surgery, Stanford University.

ICMJE COI statement

S. B. Goodman reports partial funding from his position as Robert L. and Mary Ellenburg Professor of Surgery, Stanford University, related to this study.

Data sharing

The data that support the findings for this study are available to other researchers from the corresponding author upon reasonable request.

© 2024 Murayama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (CC BY-NC-ND 4.0) licence, which permits the copying and redistribution of the work only, and provided the original author and source are credited. See https://creativecommons.org/licenses/by-nc-nd/4.0/
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References

1. Chow SKH Chim YN Wang JY Wong RMY Choy VMH Cheung WH Inflammatory response in postmenopausal osteoporotic fracture healing Bone Joint Res 2020 9 7 368 385 10.1302/2046-3758.97.BJR-2019-0300.R2 32793332
2. Kushioka J Chow SK-H Toya M et al. Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy Inflamm Regen 2023 43 1 29 10.1186/s41232-023-00279-1 37231450
3. Bastian O Pillay J Alblas J Leenen L Koenderman L Blokhuis T Systemic inflammation and fracture healing J Leukoc Biol 2011 89 5 669 673 10.1189/jlb.0810446 21208896
4. Fong K Truong V Foote CJ et al. Predictors of nonunion and reoperation in patients with fractures of the tibia: an observational study BMC Musculoskelet Disord 2013 14 1 103 10.1186/1471-2474-14-103 23517574
5. Bishop JA Palanca AA Bellino MJ Lowenberg DW Assessment of compromised fracture healing J Am Acad Orthop Surg 2012 20 5 273 282 10.5435/JAAOS-20-05-273 22553099
6. Calori GM Albisetti W Agus A Iori S Tagliabue L Risk factors contributing to fracture non-unions Injury 2007 38 S11 8 10.1016/s0020-1383(07)80004-0 17920412
7. No authors listed Regulatory Considerations for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use: Guidance for Industry and Food and Drug Administration Staff Food and Drug Administration (FDA) 2020 https://www.fda.gov/media/109176/download#:~:text=Under%20the%20regulatory%20framework%20for,1271.3 date last accessed 21 August 2024
8. Goodman SB The biological basis for concentrated iliac crest aspirate to enhance core decompression in the treatment of osteonecrosis Int Orthop 2018 42 7 1705 1709 10.1007/s00264-018-3830-1 29435623
9. Maruyama M Lin T Kaminow NI et al. The efficacy of core decompression for steroid-associated osteonecrosis of the femoral head in rabbits J Orthop Res 2021 39 7 1441 1451 10.1002/jor.24888 33095462
10. Gianakos A Ni A Zambrana L Kennedy JG Lane JM Bone marrow aspirate concentrate in animal long bone healing: an analysis of basic science evidence J Orthop Trauma 2016 30 1 1 9 10.1097/BOT.0000000000000453 26371620
11. Cuomo AV Virk M Petrigliano F Morgan EF Lieberman JR Mesenchymal stem cell concentration and bone repair: potential pitfalls from bench to bedside J Bone Joint Surg Am 2009 91-A 5 1073 1083 10.2106/JBJS.H.00303 19411455
12. Hernigou P Poignard A Beaujean F Rouard H Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells J Bone Joint Surg Am 2005 87-A 7 1430 1437 10.2106/JBJS.D.02215 15995108
13. Brozovich A Sinicrope BJ Bauza G et al. High variability of mesenchymal stem cells obtained via bone marrow aspirate concentrate compared with traditional bone marrow aspiration technique Orthop J Sports Med 2021 9 12 23259671211058459 10.1177/23259671211058459 34901292
14. Xu C Liu Y Osteosarcoma cells/cell lines are not appropriate for studies on bone regeneration in vitro Bone Joint Res 2023 12 5 311 312 10.1302/2046-3758.125.BJR-2023-0088.R1 37144570
15. Gómez-Barrena E Padilla-Eguiluz N-G López-Marfil M Ruiz de la Reina R REBORNE Consortium Volume and location of bone regeneration after autologous expanded mesenchymal stromal cells in hip osteonecrosis: a pilot study Bone Joint Res 2022 11 12 881 889 10.1302/2046-3758.1112.BJR-2022-0152.R1 36464628
16. Palombella S Lopa S Gianola S Zagra L Moretti M Lovati AB Bone marrow-derived cell therapies to heal long-bone nonunions: a systematic review and meta-analysis - which is the best available treatment? Stem Cells Int 2019 2019 3715964 10.1155/2019/3715964 31949437
17. Hulme CH Perry J McCarthy HS et al. Cell therapy for cartilage repair Emerg Top Life Sci 2021 5 4 575 589 10.1042/ETLS20210015 34423830
18. Goodman SB Zwingenberger S Concentrated autologous bone marrow aspirate is not “stem cell” therapy in the repair of nonunions and bone defects Biomater Biosyst 2021 2 100017 10.1016/j.bbiosy.2021.100017 36824655
19. Ferrante CJ Leibovich SJ Regulation of macrophage polarization and wound healing Adv Wound Care (New Rochelle) 2012 1 1 10 16 10.1089/wound.2011.0307 24527272
20. Gordon S Martinez FO Alternative activation of macrophages: mechanism and functions Immunity 2010 32 5 593 604 10.1016/j.immuni.2010.05.007 20510870
21. Ruan S Xu L Sheng Y et al. Th1 promotes M1 polarization of intestinal macrophages to regulate colitis-related mucosal barrier damage Aging (Albany NY) 2023 15 14 6721 6735 10.18632/aging.204629 37494667
22. Awad F Assrawi E Jumeau C et al. Impact of human monocyte and macrophage polarization on NLR expression and NLRP3 inflammasome activation PLoS One 2017 12 4 e0175336 10.1371/journal.pone.0175336 28403163
23. Thomas L Rao Z Gerstmeier J et al. Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase Biochem Pharmacol 2017 130 71 82 10.1016/j.bcp.2017.02.004 28189727
24. Yao Y Cai X Ren F et al. The macrophage-osteoclast axis in osteoimmunity and osteo-related diseases Front Immunol 2021 12 664871 10.3389/fimmu.2021.664871 33868316
25. Liang B Wang H Wu D Wang Z Macrophage M1/M2 polarization dynamically adapts to changes in microenvironment and modulates alveolar bone remodeling after dental implantation J Leukoc Biol 2021 110 3 433 447 10.1002/JLB.1MA0121-001R 34254348
26. Goldring SR Pathogenesis of bone and cartilage destruction in rheumatoid arthritis Rheumatology (Oxford) 2003 42 90002 ii11 6 10.1093/rheumatology/keg327 12817090
27. Liu Y Wang L Kikuiri T et al. Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α Nat Med 2011 17 12 1594 1601 10.1038/nm.2542 22101767
28. Muñoz J Akhavan NS Mullins AP Arjmandi BH Macrophage polarization and osteoporosis: a review Nutrients 2020 12 10 2999 10.3390/nu12102999 33007863
29. Ziemkiewicz N Hilliard G Pullen NA Garg K The role of innate and adaptive immune cells in skeletal muscle regeneration Int J Mol Sci 2021 22 6 3265 10.3390/ijms22063265 33806895
30. Szabo SJ Sullivan BM Stemmann C Satoskar AR Sleckman BP Glimcher LH Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells Science 2002 295 5553 338 342 10.1126/science.1065543 11786644
31. Hölscher C The power of combinatorial immunology: IL-12 and IL-12-related dimeric cytokines in infectious diseases Med Microbiol Immunol 2004 193 1 1 17 10.1007/s00430-003-0186-x 12836019
32. Zhu J Min B Hu-Li J et al. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses Nat Immunol 2004 5 11 1157 1165 10.1038/ni1128 15475959
33. Gordon S Alternative activation of macrophages Nat Rev Immunol 2003 3 1 23 35 10.1038/nri978 12511873
34. Guo K Zhang X Cytokines that modulate the differentiation of Th17 cells in autoimmune uveitis J Immunol Res 2021 2021 6693542 10.1155/2021/6693542 33816637
35. Liang SC Tan X-Y Luxenberg DP et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides J Exp Med 2006 203 10 2271 2279 10.1084/jem.20061308 16982811
36. Grassi F Cattini L Gambari L et al. T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro J Tissue Eng Regen Med 2016 10 4 305 314 10.1002/term.1727 23653421
37. Deng Z Zhang Q Zhao Z et al. Crosstalk between immune cells and bone cells or chondrocytes Int Immunopharmacol 2021 101 Pt A 108179 10.1016/j.intimp.2021.108179 34601329
38. Teng T-S Ji A-L Ji X-Y Li Y-Z Neutrophils and immunity: from bactericidal action to being conquered 2017 J Immunol Res 2017 2017 9671604 10.1155/2017/9671604 28299345
39. Loi F Córdova LA Pajarinen J Lin T Yao Z Goodman SB Inflammation, fracture and bone repair Bone 2016 86 119 130 10.1016/j.bone.2016.02.020 26946132
40. Hurst SM Wilkinson TS McLoughlin RM et al. IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation Immunity 2001 14 6 705 714 10.1016/s1074-7613(01)00151-0 11420041
41. Xing Z Lu C Hu D et al. Multiple roles for CCR2 during fracture healing Dis Model Mech 2010 3 7–8 451 458 10.1242/dmm.003186 20354109
42. Omar OM Granéli C Ekström K et al. The stimulation of an osteogenic response by classical monocyte activation Biomaterials 2011 32 32 8190 8204 10.1016/j.biomaterials.2011.07.055 21835463
43. Gibon E Lu LY Nathan K Goodman SB Inflammation, ageing, and bone regeneration J Orthop Translat 2017 10 28 35 10.1016/j.jot.2017.04.002 29094003
44. Galli SJ Tsai M IgE and mast cells in allergic disease Nat Med 2012 18 5 693 704 10.1038/nm.2755 22561833
45. Ragipoglu D Dudeck A Haffner-Luntzer M et al. The role of mast cells in bone metabolism and bone disorders Front Immunol 2020 11 163 10.3389/fimmu.2020.00163 32117297
46. Wernersson S Pejler G Mast cell secretory granules: armed for battle Nat Rev Immunol 2014 14 7 478 494 10.1038/nri3690 24903914
47. Nazari M Ni NC Lüdke A et al. Mast cells promote proliferation and migration and inhibit differentiation of mesenchymal stem cells through PDGF J Mol Cell Cardiol 2016 94 32 42 10.1016/j.yjmcc.2016.03.007 26996757
48. Vallés G Bensiamar F Maestro-Paramio L García-Rey E Vilaboa N Saldaña L Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells Stem Cell Res Ther 2020 11 1 57 10.1186/s13287-020-1578-1 32054534
49. Wesdorp MA Bastiaansen-Jenniskens YM Capar S Verhaar JAN Narcisi R Van Osch G Modulation of inflamed synovium improves migration of mesenchymal stromal cells in vitro through anti-inflammatory macrophages Van Cartilage 2022 13 1 19476035221085136 10.1177/19476035221085136 35306879
50. Lu LY Loi F Nathan K et al. Pro-inflammatory M1 macrophages promote osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway J Orthop Res 2017 35 11 2378 2385 10.1002/jor.23553 28248001
51. Tu B Liu S Liu G et al. Macrophages derived from THP-1 promote the osteogenic differentiation of mesenchymal stem cells through the IL-23/IL-23R/β-catenin pathway Exp Cell Res 2015 339 1 81 89 10.1016/j.yexcr.2015.10.015 26477825
52. Loi F Córdova LA Zhang R et al. The effects of immunomodulation by macrophage subsets on osteogenesis in vitro Stem Cell Res Ther 2016 7 15 10.1186/s13287-016-0276-5 26801095
53. Nathan K Lu LY Lin T et al. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex Bone Joint Res 2019 8 10 481 488 10.1302/2046-3758.810.BJR-2018-0231.R2 31728188
54. Kalluri R LeBleu VS The biology, function, and biomedical applications of exosomes Science 2020 367 6478 eaau6977 10.1126/science.aau6977 32029601
55. Kang M Huang C-C Lu Y et al. Bone regeneration is mediated by macrophage extracellular vesicles Bone 2020 141 115627 10.1016/j.bone.2020.115627 32891867
56. Liu H Wu Q Liu S et al. The role of integrin αvβ3 in biphasic calcium phosphate ceramics mediated M2 macrophage polarization and the resultant osteoinduction Biomaterials 2024 304 122406 10.1016/j.biomaterials.2023.122406 38096618
57. Zhang Y Böse T Unger RE Jansen JA Kirkpatrick CJ van den Beucken JJJP Macrophage type modulates osteogenic differentiation of adipose tissue MSCs Cell Tissue Res 2017 369 2 273 286 10.1007/s00441-017-2598-8 28361303
58. Jiang F Qi X Wu X et al. Regulating macrophage-MSC interaction to optimize BMP-2-induced osteogenesis in the local microenvironment Bioact Mater 2023 25 307 318 10.1016/j.bioactmat.2023.02.001 36844362
59. Lu D Xu Y Liu Q Zhang Q Mesenchymal stem cell-macrophage crosstalk and maintenance of inflammatory microenvironment homeostasis Front Cell Dev Biol 2021 9 681171 10.3389/fcell.2021.681171 34249933
60. Vasandan AB Jahnavi S Shashank C Prasad P Kumar A Prasanna SJ Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE2-dependent mechanism Sci Rep 2016 6 38308 10.1038/srep38308 27910911
61. Li X Liu L Yang J et al. Exosome derived from human umbilical cord mesenchymal stem cell mediates MiR-181c attenuating burn-induced excessive inflammation EBioMedicine 2016 8 72 82 10.1016/j.ebiom.2016.04.030 27428420
62. Li J Xue H Li T et al. Exosomes derived from mesenchymal stem cells attenuate the progression of atherosclerosis in ApoE−/- mice via miR-let7 mediated infiltration and polarization of M2 macrophage Biochem Biophys Res Commun 2019 510 4 565 572 10.1016/j.bbrc.2019.02.005 30739785
63. Li K Yan G Huang H et al. Anti-inflammatory and immunomodulatory effects of the extracellular vesicles derived from human umbilical cord mesenchymal stem cells on osteoarthritis via M2 macrophages J Nanobiotechnology 2022 20 1 38 10.1186/s12951-021-01236-1 35057811
64. An JH Li Q Bhang DH Song WJ Youn HY TNF-α and INF-γ primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis Sci Rep 2020 10 1 2115 10.1038/s41598-020-58909-4 32034203
65. Ti D Hao H Tong C et al. LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b J Transl Med 2015 13 308 10.1186/s12967-015-0642-6 26386558
66. Lo Sicco C Reverberi D Balbi C et al. Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: endorsement of macrophage polarization Stem Cells Transl Med 2017 6 3 1018 1028 10.1002/sctm.16-0363 28186708
67. Avery D Morandini L Gabriec M et al. Contribution of αβ T cells to macrophage polarization and MSC recruitment and proliferation on titanium implants Acta Biomater 2023 169 605 624 10.1016/j.actbio.2023.07.052 37532133
68. Rifas L T-cell cytokine induction of BMP-2 regulates human mesenchymal stromal cell differentiation and mineralization J Cell Biochem 2006 98 4 706 714 10.1002/jcb.20933 16619272
69. Tanaka Y Clinical immunity in bone and joints J Bone Miner Metab 2019 37 1 2 8 10.1007/s00774-018-0965-5 30324535
70. Young N Mikhalkevich N Yan Y Chen D Zheng W Differential regulation of osteoblast activity by Th cell subsets mediated by parathyroid hormone and IFN-gamma J Immunol 2005 175 12 8287 8295 10.4049/jimmunol.175.12.8287 16339569
71. Lubberts E Joosten LA Chabaud M et al. IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion J Clin Invest 2000 105 12 1697 1710 10.1172/JCI7739 10862785
72. Onoe Y Miyaura C Kaminakayashiki T et al. IL-13 and IL-4 inhibit bone resorption by suppressing cyclooxygenase-2-dependent prostaglandin synthesis in osteoblasts J Immunol 1996 156 2 758 764 10.4049/jimmunol.156.2.758 8543830
73. Zaiss MM Axmann R Zwerina J et al. Treg cells suppress osteoclast formation: a new link between the immune system and bone Arthritis Rheum 2007 56 12 4104 4112 10.1002/art.23138 18050211
74. Rossi M Rana I Buonuomo PS et al. Stimulation of Treg cells to inhibit osteoclastogenesis in Gorham-Stout disease Front Cell Dev Biol 2021 9 706596 10.3389/fcell.2021.706596 34513837
75. Runyan CE Liu Z Schnaper HW Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-β1 J Biol Chem 2012 287 43 35815 35824 10.1074/jbc.M112.380493 22942286
76. Zhu L Hua F Ding W Ding K Zhang Y Xu C The correlation between the Th17/Treg cell balance and bone health Immun Ageing 2020 17 1 30 10.1186/s12979-020-00202-z 33072163
77. Qiu X Gui Y Xu Y Li D Wang L DHEA promotes osteoblast differentiation by regulating the expression of osteoblast-related genes and Foxp3(+) regulatory T cells Biosci Trends 2015 9 5 307 314 10.5582/bst.2015.01073 26559023
78. Chen Q-H Wu F Liu L et al. Mesenchymal stem cells regulate the Th17/Treg cell balance partly through hepatocyte growth factor in vitro Stem Cell Res Ther 2020 11 1 91 10.1186/s13287-020-01612-y 32111238
79. Vasilev G Ivanova M Ivanova-Todorova E et al. Secretory factors produced by adipose mesenchymal stem cells downregulate Th17 and increase Treg cells in peripheral blood mononuclear cells from rheumatoid arthritis patients Rheumatol Int 2019 39 5 819 826 10.1007/s00296-019-04296-7 30944956
80. Lim J-Y Im K-I Lee E-S et al. Enhanced immunoregulation of mesenchymal stem cells by IL-10-producing type 1 regulatory T cells in collagen-induced arthritis Sci Rep 2016 6 26851 10.1038/srep26851 27246365
81. Huang H Kim HJ Chang E-J et al. IL-17 stimulates the proliferation and differentiation of human mesenchymal stem cells: implications for bone remodeling Cell Death Differ 2009 16 10 1332 1343 10.1038/cdd.2009.74 19543237
82. Sato K Suematsu A Okamoto K et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction J Exp Med 2006 203 12 2673 2682 10.1084/jem.20061775 17088434
83. Kotake S Udagawa N Takahashi N et al. IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis J Clin Invest 1999 103 9 1345 1352 10.1172/JCI5703 10225978
84. Croes M Öner FC van Neerven D et al. Proinflammatory T cells and IL-17 stimulate osteoblast differentiation Bone 2016 84 262 270 10.1016/j.bone.2016.01.010 26780388
85. Han X Yang Q Lin L et al. Interleukin-17 enhances immunosuppression by mesenchymal stem cells Cell Death Differ 2014 21 11 1758 1768 10.1038/cdd.2014.85 25034782
86. Cassano JM Schnabel LV Goodale MB Fortier LA Inflammatory licensed equine MSCs are chondroprotective and exhibit enhanced immunomodulation in an inflammatory environment Stem Cell Res Ther 2018 9 1 82 10.1186/s13287-018-0840-2 29615127
87. Han Y Yang J Fang J et al. The secretion profile of mesenchymal stem cells and potential applications in treating human diseases Signal Transduct Target Ther 2022 7 1 92 10.1038/s41392-022-00932-0 35314676
88. Sui B-D Chen J Zhang X-Y et al. Gender-independent efficacy of mesenchymal stem cell therapy in sex hormone-deficient bone loss via immunosuppression and resident stem cell recovery Exp Mol Med 2018 50 12 1 14 10.1038/s12276-018-0192-0 30559383
89. Wang K Shi Y-J Song Z-L et al. Regulatory effect of rat bone marrow mesenchymal stem cells on Treg/Th17 immune balance invitro Mol Med Rep 2020 21 5 2123 2130 10.3892/mmr.2020.11019 32186771
90. Salek Farrokhi A Zarnani AH Moazzeni SM Mesenchymal stem cells therapy protects fetuses from resorption and induces Th2 type cytokines profile in abortion prone mouse model Transpl Immunol 2018 47 26 31 10.1016/j.trim.2018.01.002 29317300
91. Weiss ARR Dahlke MH Immunomodulation by mesenchymal stem cells (MSCs): mechanisms of action of living, apoptotic, and dead MSCs Front Immunol 2019 10 1191 10.3389/fimmu.2019.01191 31214172
92. Ge W Jiang J Arp J Liu W Garcia B Wang H Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression Transplantation 2010 90 12 1312 1320 10.1097/TP.0b013e3181fed001 21042238
93. Wang Q Sun B Wang D et al. Murine bone marrow mesenchymal stem cells cause mature dendritic cells to promote T-cell tolerance Scand J Immunol 2008 68 6 607 615 10.1111/j.1365-3083.2008.02180.x 18959624
94. Bai L Lennon DP Eaton V et al. Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis Glia 2009 57 11 1192 1203 10.1002/glia.20841 19191336
95. van der Veeken J Gonzalez AJ Cho H et al. Memory of inflammation in regulatory T cells Cell 2016 166 4 977 990 10.1016/j.cell.2016.07.006 27499023
96. Rocamora-Reverte L Melzer FL Würzner R Weinberger B The complex role of regulatory T cells in immunity and aging Front Immunol 2020 11 616949 10.3389/fimmu.2020.616949 33584708
97. Lei H Schmidt-Bleek K Dienelt A Reinke P Volk H-D Regulatory T cell-mediated anti-inflammatory effects promote successful tissue repair in both indirect and direct manners Front Pharmacol 2015 6 184 10.3389/fphar.2015.00184 26388774
98. Akiyama K Chen C Wang D et al. Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis Cell Stem Cell 2012 10 5 544 555 10.1016/j.stem.2012.03.007 22542159
99. Rashedi I Gómez-Aristizábal A Wang XH Viswanathan S Keating A TLR3 or TLR4 activation enhances mesenchymal stromal cell-mediated Treg induction via notch signaling Stem Cells 2017 35 1 265 275 10.1002/stem.2485 27571579
100. Abe Y Ochiai D Taguchi M et al. Human amniotic fluid stem cells ameliorate thioglycollate-induced peritonitis by increasing Tregs in mice Int J Mol Sci 2022 23 12 6433 10.3390/ijms23126433 35742877
101. Luo Y Guo J Zhang P et al. Mesenchymal stem cell protects injured renal tubular epithelial cells by regulating mTOR-mediated Th17/Treg axis Front Immunol 2021 12 684197 10.3389/fimmu.2021.684197 34122446
102. Tesmer LA Lundy SK Sarkar S Fox DA Th17 cells in human disease Immunol Rev 2008 223 87 113 10.1111/j.1600-065X.2008.00628.x 18613831
103. Singh RP Hasan S Sharma S et al. Th17 cells in inflammation and autoimmunity Autoimmun Rev 2014 13 12 1174 1181 10.1016/j.autrev.2014.08.019 25151974
104. Luz-Crawford P Hernandez J Djouad F et al. Mesenchymal stem cell repression of Th17 cells is triggered by mitochondrial transfer Stem Cell Res Ther 2019 10 1 232 10.1186/s13287-019-1307-9 31370879
105. Terraza-Aguirre C Campos-Mora M Elizondo-Vega R et al. Mechanisms behind the immunoregulatory dialogue between mesenchymal stem cells and Th17 cells Cells 2020 9 7 1660 10.3390/cells9071660 32664207
106. Rafei M Campeau PM Aguilar-Mahecha A et al. Mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4 Th17 T cells in a CC chemokine ligand 2-dependent manner J Immunol 2009 182 10 5994 6002 10.4049/jimmunol.0803962 19414750
107. Glenn JD Smith MD Kirby LA Baxi EG Whartenby KA Disparate effects of mesenchymal stem cells in experimental autoimmune encephalomyelitis and cuprizone-induced demyelination PLoS One 2015 10 9 e0139008 10.1371/journal.pone.0139008 26407166
108. Luz-Crawford P Noël D Fernandez X et al. Mesenchymal stem cells repress Th17 molecular program through the PD-1 pathway PLoS One 2012 7 9 e45272 10.1371/journal.pone.0045272 23028899
109. Qu X Liu X Cheng K Yang R Zhao RCH Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion Exp Hematol 2012 40 9 761 770 10.1016/j.exphem.2012.05.006 22634392
110. Wang D Huang S Yuan X et al. The regulation of the Treg/Th17 balance by mesenchymal stem cells in human systemic lupus erythematosus Cell Mol Immunol 2017 14 5 423 431 10.1038/cmi.2015.89 26435067
111. Rozenberg A Rezk A Boivin M-N et al. Human mesenchymal stem cells impact Th17 and Th1 responses through a prostaglandin E2 and myeloid-dependent mechanism Stem Cells Transl Med 2016 5 11 1506 1514 10.5966/sctm.2015-0243 27400792
112. DeNardo DG Barreto JB Andreu P et al. CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages Cancer Cell 2009 16 2 91 102 10.1016/j.ccr.2009.06.018 19647220
113. Collison LW Workman CJ Kuo TT et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function Nature 2007 450 7169 566 569 10.1038/nature06306 18033300
114. Tiemessen MM Jagger AL Evans HG van Herwijnen MJC John S Taams LS CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages Proc Natl Acad Sci U S A 2007 104 49 19446 19451 10.1073/pnas.0706832104 18042719
115. Romano M Fanelli G Tan N et al. Expanded regulatory T cells induce alternatively activated monocytes with a reduced capacity to expand T helper-17 cells Front Immunol 2018 9 1625 10.3389/fimmu.2018.01625 30079063
116. Kryczek I Wei S Zhu G et al. Relationship between B7-H4, regulatory T cells, and patient outcome in human ovarian carcinoma Cancer Res 2007 67 18 8900 8905 10.1158/0008-5472.CAN-07-1866 17875732
117. Wu C Xue Y Wang P et al. IFN-γ primes macrophage activation by increasing phosphatase and tensin homolog via downregulation of miR-3473b J Immunol 2014 193 6 3036 3044 10.4049/jimmunol.1302379 25092892
118. Unanue ER Antigen-presenting function of the macrophage Annu Rev Immunol 1984 2 395 428 10.1146/annurev.iy.02.040184.002143 6242349
119. Greenwald RJ Freeman GJ Sharpe AH The B7 family revisited Annu Rev Immunol 2005 23 515 548 10.1146/annurev.immunol.23.021704.115611 15771580
120. Sharpe AH Pauken KE The diverse functions of the PD1 inhibitory pathway Nat Rev Immunol 2018 18 3 153 167 10.1038/nri.2017.108 28990585
121. Kubin M Kamoun M Trinchieri G Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells J Exp Med 1994 180 1 211 222 10.1084/jem.180.1.211 7516408
122. Powers GD Faherty DA Connaughton SE et al. Expression and functional analysis of murine B7 delineated by a novel monoclonal antibody Cell Immunol 1994 153 2 298 311 10.1006/cimm.1994.1030 7509723
123. Egan PJ van Nieuwenhuijze A Campbell IK Wicks IP Promotion of the local differentiation of murine Th17 cells by synovial macrophages during acute inflammatory arthritis Arthritis Rheum 2008 58 12 3720 3729 10.1002/art.24075 19035489
124. Swain SL McKinstry KK Strutt TM Expanding roles for CD4 Nat Rev Immunol 2012 12 2 136 148 10.1038/nri3152 22266691
125. Schmidt A Zhang X-M Joshi RN et al. Human macrophages induce CD4(+)Foxp3(+) regulatory T cells via binding and re-release of TGF-β Immunol Cell Biol 2016 94 8 747 762 10.1038/icb.2016.34 27075967
126. Melief SM Schrama E Brugman MH et al. Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages Stem Cells 2013 31 9 1980 1991 10.1002/stem.1432 23712682
127. DeNardo DG Ruffell B Macrophages as regulators of tumour immunity and immunotherapy Nat Rev Immunol 2019 19 6 369 382 10.1038/s41577-019-0127-6 30718830
128. Kryczek I Zou L Rodriguez P et al. B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 2006 203 4 871 881 10.1084/jem.20050930 16606666
129. Franceschi C Bonafè M Valensin S et al. Inflamm-aging. An evolutionary perspective on immunosenescence Ann N Y Acad Sci 2000 908 244 254 10.1111/j.1749-6632.2000.tb06651.x 10911963
130. Fischer V Haffner-Luntzer M Interaction between bone and immune cells: Implications for postmenopausal osteoporosis Semin Cell Dev Biol 2022 123 14 21 10.1016/j.semcdb.2021.05.014 34024716
131. Li Z Zhang B Shang J et al. Diabetic and nondiabetic BMSC–derived exosomes affect bone regeneration via regulating miR-17-5p/SMAD7 axis Int Immunopharmacol 2023 125 111190 10.1016/j.intimp.2023.111190 37976598
132. Tang Y Zheng L Zhou J et al. miR‑203‑3p participates in the suppression of diabetes‑associated osteogenesis in the jaw bone through targeting Smad1 Int J Mol Med 2018 41 3 1595 1607 10.3892/ijmm.2018.3373 29328402
133. Romero-López M Li Z Rhee C et al. Macrophage effects on mesenchymal stem cell osteogenesis in a three-dimensional in vitro bone model Tissue Eng Part A 2020 26 19–20 1099 1111 10.1089/ten.TEA.2020.0041 32312178
