Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma.

Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single-nucleus transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as a driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis.

Osteoimmunology of Fracture Healing.

PURPOSE OF REVIEW: The purpose of this review is to summarize what is known in the literature about the role inflammation plays during bone fracture healing. Bone fracture healing progresses through four distinct yet overlapping phases: formation of the hematoma, development of the cartilaginous callus, development of the bony callus, and finally remodeling of the fracture callus. Throughout this process, inflammation plays a critical role in robust bone fracture healing. RECENT FINDINGS: At the onset of injury, vessel and matrix disruption lead to the generation of an inflammatory response: inflammatory cells are recruited to the injury site where they differentiate, activate, and/or polarize to secrete cytokines for the purposes of cell signaling and cell recruitment. This process is altered by age and by sex. Bone fracture healing is heavily influenced by the presence of inflammatory cells and cytokines within the healing tissue.

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Altered early immune response after fracture and traumatic brain injury.

Introduction: Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair. Methods: Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury. Results: We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers. Discussion: Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling.

Single-Cell RNA Sequencing Reveals B Cells Are Important Regulators in Fracture Healing.

The bone marrow microenvironment is composed primarily of immune and stromal cells that play important roles in fracture healing. Although immune cells have been identified in mouse bone marrow, variations in their numbers and type during the fracture healing process remain poorly defined. In this study, single-cell RNA sequencing was used to identify immune cells in fracture tissues, including neutrophils, monocytes, T cells, B cells, and plasma cells. The number of B cells decreased significantly in the early stage of fracture healing. Furthermore, B cells in mice fracture models decreased significantly during the epiphyseal phase and then gradually returned to normal during the epiphyseal transformation phase of fracture healing. The B-cell pattern was opposite to that of bone formation and resorption activities. Notably, B-cell-derived exosomes inhibited bone homeostasis in fracture healing. In humans, a decrease in the number of B cells during the epiphyseal phase stimulated fracture healing. Then, as the numbers of osteoblasts increased during the callus reconstruction stage, the number of B cells gradually recovered, which reduced additional bone regeneration. Thus, B cells are key regulators of fracture healing and inhibit excessive bone regeneration by producing multiple osteoblast inhibitors.

Fracture biomechanics influence local and systemic immune responses in a murine fracture-related infection model.

Biomechanical stability plays an important role in fracture healing, with unstable fixation being associated with healing disturbances. A lack of stability is also considered a risk factor for fracture-related infection (FRI), although confirmatory studies and an understanding of the underlying mechanisms are lacking. In the present study, we investigate whether biomechanical (in)stability can lead to altered immune responses in mice under sterile or experimentally inoculated conditions. In non-inoculated C57BL/6 mice, instability resulted in an early increase of inflammatory markers such as granulocyte-colony stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin (IL)-6 within the bone. When inoculated with Staphylococcus epidermidis, instability resulted in a further significant increase in G-CSF, IL-6 and KC in bone tissue. Staphylococcus aureus infection led to rapid osteolysis and instability in all animals and was not further studied. Gene expression measurements also showed significant upregulation in CCL2 and G-CSF in these mice. IL-17A was found to be upregulated in all S. epidermidis infected mice, with higher systemic IL-17A cell responses in mice that cleared the infection, which was found to be produced by CD4+ and γδ+ T cells in the bone marrow. IL-17A knock-out (KO) mice displayed a trend of delayed clearance of infection (P=0.22, Fisher's exact test) and an increase in interferon (IFN)-γ production. Biomechanical instability leads to a more pronounced local inflammatory response, which is exaggerated by bacterial infection. This study provides insights into long-held beliefs that biomechanics are crucial not only for fracture healing, but also for control of infection.

Potential role of myeloid-derived suppressor cells in transition from reaction to repair phase of bone healing process.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with immunosuppressive functions; these cells play a key role in infection, immunization, chronic inflammation, and cancer. Recent studies have reported that immunosuppression plays an important role in the healing process of tissues and that Treg play an important role in fracture healing. MDSCs suppress active T cell proliferation and reduce the severity of arthritis in mice and humans. Together, these findings suggest that MDSCs play a role in bone biotransformation. In the present study, we examined the role of MDSCs in the bone healing process by creating a bone injury at the tibial epiphysis in mice. MDSCs were identified by CD11b and GR1 immunohistochemistry and their role in new bone formation was observed by detection of Runx2 and osteocalcin expression. Significant numbers of MDSCs were observed in transitional areas from the reactionary to repair stages. Interestingly, MDSCs exhibited Runx2 and osteocalcin expression in the transitional area but not in the reactionary area. And at the same area, cllagene-1 and ALP expression level increased in osteoblast progenitor cells. These data is suggesting that MDSCs emerge to suppress inflammation and support new bone formation. Here, we report, for the first time (to our knowledge), the role of MDSCs in the initiation of bone formation. MDSC appeared at the transition from inflammation to bone making and regulates bone healing by suppressing inflammation.

Immune cell profiling in the joint following human and murine articular fracture.

OBJECTIVE: Human and in vivo animal research implicates inflammation following articular fracture as contributing to post-traumatic arthritis. However, relevant immune cell subsets present following injury are currently undefined. Immunophenotyping human and murine synovial fluid may help to identify immune cell populations that play key roles in the response to articular fracture. METHODS: Immunophenotyping by polychromatic flow cytometry was performed on human and mouse synovial fluid following articular fracture. Specimens were collected in patients with closed ankle fracture at the time of surgical fixation and from C57BL/6 mice with closed articular knee fracture. Immune cells were collected from injured and uninjured joints in mice via a novel cell isolation method. Whole blood samples were also collected. Immunohistochemistry (IHC) was performed on mouse synovial tissue to assess for macrophages and T cells. RESULTS: Following intra-articular fracture, the prominent human synovial fluid immune cell subset was CD3+ T cells, containing both CD4+ and CD8+ T cells. In mice, infiltration of CD45+ immune cells in synovial fluid of the fractured limb was dominated by CD19+ B cells and CD3+ T cells at 7 days after intra-articular fracture. We also detected adaptive immune cells, including macrophages, NK cells, dendritic cells and monocytes. Macrophage and T cell findings were supported by IHC of murine synovial tissue. CONCLUSIONS: Determining specific cell populations that mediate the immune response is essential to elucidating the chain of events initiated after injury and may be an important step in identifying potential immune signatures predictive of PTA susceptibility or potential therapeutic targets.

Age-related changes to macrophages are detrimental to fracture healing in mice.

The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age-related diseases. Therefore, we investigated age-related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA-seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up-regulation of M1/pro-inflammatory genes in macrophages from old mice as well as dysregulation of other immune-related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age-matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA-seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age-related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.

Does Allergy Break Bones? Osteoporosis and Its Connection to Allergy.

: Osteoporosis and allergic diseases are important causes of morbidity, and traditionally their coexistence has been attributed to causality, to independent processes, and they were considered unrelated. However, the increasing knowledge in the field of osteoimmunology and an increasing number of epidemiological and biological studies have provided support to a correlation between bone and allergy that share pathways, cells, cytokines and mediators. If the link between allergic pathology and bone alterations appears more subtle, there are conditions such as mastocytosis and hypereosinophilic or hyper-IgE syndromes characterized by the proliferation of cells or hyper-production of molecules that play a key role in allergies, in which this link is at least clinically more evident, and the diseases are accompanied by frank skeletal involvement, offering multiple speculation cues. The pathophysiological connection of allergy and osteoporosis is currently an intriguing area of research. The aim of this review is to summarize and bring together the current knowledge and pursue an opportunity to stimulate further investigation.

Effect of early restrictive fluid resuscitation on inflammatory and immune factors in patients with severe pelvic fracture.

PURPOSE: To study the effect of early restrictive fluid resuscitation (EFR) on inflammatory and immune factors in patients with severe pelvic fracture (SPF). METHODS: A total of 174 SPF patients in the Department of Orthopaedics, the First Affiliated Hospital of Chengdu Medical College from July 2015 to June 2018 were involved in this study and divided into EFR group (n = 87) and control group (n = 87) using the random number table method. Conventional fluid resuscitation (CFR) was performed in control group, and EFR was performed in EFR group. The incidences of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) during rescue, successful rescue rate, blood transfusion volume, fluid input, and resuscitation time were compared between the two groups. The parameters including prothrombin time (PT), hematocrit (HCT), platelet (PLT) and blood lactate (BL) at the 4th hour after fluid resuscitation were recorded. The levels of inflammatory factors (TNF-α, IL-6, CRP) and immune factors (CD3+, CD4+, CD8+, CD4+/CD8+) were compared between the two groups before treatment and 7 days after treatment. The revised acute physiologic and chronic health evaluation system and the sequential organ failure assessment scores were adopted for evaluation before treatment and 7 days after treatment. RESULTS: The incidences of ARDS and MODS during rescue in EFR group were significantly lower than those in control group (p=0.015 and 0.010 respectively), and the successful rescue rate in EFR group was significantly higher than that in control group (p = 0.011). The blood transfusion volume, fluid input, resuscitation time in EFR group were significantly lower than those in control group (p = 0.016, 0.002 and 0.001 respectively). At the 4th hour after fluid resuscitation, PT and BL in EFR group were significantly lower than those in control group (p = 0.021 and 0.003 respectively), while HCT and PLT in EFR group were significantly higher than those in control group (p = 0.016 and 0.021 respectively). On day 7 after treatment, TNF-α, IL-6, CRP and CD8+ in EFR group were significantly lower than those in control group (p = 0.003, 0.004, 0.007 and 0.003 respectively), while CD3+, CD4+ and CD4+/CD8+ in EFR group were significantly higher than those in control group (p = 0.004, 0.000, 0.007 respectively). On day 7 after treatment, the revised acute physiologic and chronic health evaluation (APACHE) system and the sequential organ failure assessment (SOFA) scores in EFR group were significantly lower than those in control group. CONCLUSION: EFR can effectively eliminate inflammatory factors, improve immune function, maintain the stability of blood components, reduce the incidences of ARDS and MODS, and elevate the successful rescue rate in patients with SPF.

Compromised Bone Healing in Aged Rats Is Associated With Impaired M2 Macrophage Function.

Fracture repair is initiated by a multitude of immune cells and induction of an inflammatory cascade. Alterations in the early healing response due to an aged adaptive immune system leads to impaired bone repair, delayed healing or even formation of non-union. However, immuno-senescence is not limited to the adaptive immunity, but is also described for macrophages, main effector cells from the innate immune system. Beside regulation of pro- and anti-inflammatory signaling, macrophages contribute to angiogenesis and granulation tissue maturation. Thus, it seems likely that an altered macrophage function due to aging may affect bone repair at various stages and contribute to age related deficiencies in bone regeneration. To prove this hypothesis, we analyzed the expression of macrophage markers and angiogenic factors in the early bone hematoma derived from young and aged osteotomized Spraque Dawley rats. We detected an overall reduced expression of the monocyte/pan-macrophage markers CD14 and CD68 in aged rats. Furthermore, the analysis revealed an impaired expression of anti-inflammatory M2 macrophage markers in hematoma from aged animals that was connected to a diminished revascularization of the bone callus. To verify that the age related disturbed bone regeneration was due to a compromised macrophage function, CD14+ macrophage precursors were transplanted locally into the osteotomy gap of aged rats. Transplantation rescued bone regeneration partially after 6 weeks, demonstrated by a significantly induced deposition of new bone tissue, reduced fibrosis and significantly improved callus vascularization.

Immunomodulatory Effects of MSCs in Bone Healing.

Mesenchymal stem cells (MSCs) are capable of differentiating into multilineage cells, thus making them a significant prospect as a cell source for regenerative therapy; however, the differentiation capacity of MSCs into osteoblasts seems to not be the main mechanism responsible for the benefits associated with human mesenchymal stem cells hMSCs when used in cell therapy approaches. The process of bone fracture restoration starts with an instant inflammatory reaction, as the innate immune system responds with cytokines that enhance and activate many cell types, including MSCs, at the site of the injury. In this review, we address the influence of MSCs on the immune system in fracture repair and osteogenesis. This paradigm offers a means of distinguishing target bone diseases to be treated with MSC therapy to enhance bone repair by targeting the crosstalk between MSCs and the immune system.

Comparison of post-traumatic changes in circulating and bone marrow leukocytes between BALB/c and CD-1 mouse strains.

This manuscript emerged from a larger third-party funded project investigating a new poly-trauma model and its influence upon secondary sepsis. The present sub-study compared selected leukocyte subpopulations in the circulation and bone marrow after polytrauma in BALB/c versus CD-1 mice. Animals underwent unilateral femur fracture, splenectomy and hemorrhagic shock. We collected blood and bone marrow for flow cytometry analysis at 24h and 48h post-trauma. Circulating granulocytes (Ly6G+CD11+) increased in both strains after trauma. Only in BALB/c mice circulating CD8+ T-lymphocytes decreased within 48h by 30%. Regulatory T-cells (Tregs, CD4+CD25+CD127low) increased in both strains by approx. 32%. Circulating Tregs and lymphocytes (CD11b-Ly6G-MHC-2+) were always at least 1.5-fold higher in BALB/c, while the bone marrow MHC-2 expression decreased in CD-1 mice (p<0.05). Overall, immune responses to polytrauma were similar in both strains. Additionally, BALB/c expressed higher level of circulating regulatory T-cells and MHC-2-positive lymphocytes compared to CD-1 mice.

Fracture repair in the elderly: Clinical and experimental considerations.

Fractures in the elderly represent a significant and rising socioeconomic problem. Although aging has been associated with delays in healing, there is little direct clinical data isolating the effects of aging on bone healing from the associated comorbidities that are frequently present in elderly populations. Basic research has demonstrated that all of the components of fracture repair-cells, extracellular matrix, blood supply, and molecules and their receptors-are negatively impacted by the aging process, which likely explains poorer clinical outcomes. Improved understanding of age-related fracture healing should aid in the development of novel treatment strategies, technologies, and therapies to improve bone repair in elderly patients.

M2 macrophages are closely associated with accelerated clavicle fracture healing in patients with traumatic brain injury: a retrospective cohort study.

BACKGROUND: Mounting evidence indicate patients with traumatic brain injury (TBI) have an accelerated fracture healing. The healing process of bone fractures is greatly dependent on infiltrated macrophages. The macrophages are categorized into M1 or M2 phenotypes with different functions. This study is aimed to address the potential role of subtypes of macrophages in the process of fracture healing in patients with TBI. METHODS: Twenty-five cases of clavicle fracture alone (CF group) and 22 cases of clavicle fracture concomitant with TBI (CFT group) were retrospectively analyzed in this study. Callus tissues were harvested during operations. The expressions of COX-2, CD206, and CD68 were measured with immunohistochemistry. RESULTS: The percentages of M2 macrophages in total macrophages increased after bone fracture in both groups, while the percentages of M1-type macrophages are decreased. Interestingly, the increased percentages of M2 macrophages are significantly higher in CFT group than in CF group. Compared to CF group, the fracture callus volume was much larger (21.9 vs 8.5 cm3) and the fracture healing time was much shorter (82.2 vs 127.0 days) in CFT group. The percentage of M2 macrophages was negatively correlated with fracture healing time in patients (r = - 0.575, p < 0.01). CONCLUSIONS: The findings suggest that the percentages of M2 macrophages in callus tissues increased dramatically during the repairing stage in both CF and CFT group. Percentages of M2 macrophages are associated with accelerated fracture healing in patients with TBI. M2 macrophage polarization during the stage of bone regeneration may play a vital role in promoting bone fracture healing.

Neutrophils in Tissue Trauma of the Skin, Bone, and Lung: Two Sides of the Same Coin.

Following severe tissue injury, patients are exposed to various danger- and microbe-associated molecular patterns, which provoke a strong activation of the neutrophil defense system. Neutrophils trigger and modulate the initial posttraumatic inflammatory response and contribute critically to subsequent repair processes. However, severe trauma can affect central neutrophil functions, including circulation half-life, chemokinesis, phagocytosis, cytokine release, and respiratory burst. Alterations in neutrophil biology may contribute to trauma-associated complications, including immune suppression, sepsis, multiorgan dysfunction, and disturbed tissue regeneration. Furthermore, there is evidence that neutrophil actions depend on the quality of the initial stimulus, including trauma localization and severity, the micromilieu in the affected tissue, and the patient's overall inflammatory status. In the present review, we describe the effects of severe trauma on the neutrophil phenotype and dysfunction and the consequences for tissue repair. We particularly concentrate on the role of neutrophils in wound healing, lung injury, and bone fractures, because these are the most frequently affected tissues in severely injured patients.

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture.

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4+ T cells in patients with long bone fracture. We found that LAG-3+ cells represented approximately 13% of peripheral blood CD4+ T cells on average. Compared to LAG-3- CD4+ T cells, LAG-3+ CD4+ T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3+ CD4+ T cells expressed significantly higher levels of IL-10 and TGF-ß than LAG-3- CD4+ T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3+ CD4+ T cells. The frequency of LAG-3+ CD4+ T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4+ T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.

Anti-CCP-positive patients with RA have a higher 10-year probability of fracture evaluated by FRAX®: a registry study of RA with osteoporosis/fracture.

BACKGROUND: Positive anticyclic citrullinated peptide (anti-CCP+) is associated with bone loss in patients with rheumatoid arthritis (RA). However, whether overall positivity or specific levels of anti-CCP are associated with prevalent fracture or a 10-year probability of fracture remains unclear. METHODS: This interim analysis of an RA registry was conducted at Chang Gung Memorial Hospital in Kaohsiung (CGMHK) for RA-related osteoporosis/fracture. Consecutive patients with RA who had visited the rheumatology clinic at CGMHK since September 1, 2014, and fulfilled the classification criteria of RA were enrolled. The demographics, disease duration, Disease activity in 28 joints based on erythrocyte sedimentation rate (DAS28-ESR), lifestyle, evidence of previous fracture, risk factors of fracture in the Fracture Risk Assessment Tool (FRAX®), and FRAX® score of each participant were collected. Anti-CCP, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and bone mineral density (BMD) were measured at enrollment. The patients were grouped by positivity or quartiles of anti-CCP level (I-IV). RESULTS: Five hundred twenty-one patients with RA were enrolled through May 31, 2016. In total, 359 (68.9%) patients were anti-CCP+. Compared with anti-CCP- patients, anti-CCP+ patients had a significantly higher DAS28-ESR (p = 0.0001) and 10-year probability of major (15.0 [18.9] vs. 12.0 [15.3], p = 0.0461) or hip (5.0 [9.2] vs. 3.6 [8.2], p = 0.0118) fracture, but a significantly lower BMD of the FN (p = 0.0196). The rates of osteoporosis and previous fracture were comparable. There were 130, 127, 132, and 132 patients in groups I-IV, respectively. The DAS28-ESR was significantly different (p = 0.0001) among the groups and correlated to anti-CCP levels. The BMD and 10-year probability of major (p = 0.0067) and hip (p = 0.0013) fracture among the groups were also different. CONCLUSIONS: Anti-CCP+ RA patients had a higher 10-year probability of major or hip fracture, independent of anti-CCP levels, and a lower BMD of the FN than anti-CCP- patients.