Role of Fas and Treg Cells in Fracture Healing as Characterized in the Fas-Deficient (lpr) Mouse Model of Lupus

Abstract

Previous studies showed that loss of tumor necrosis factor α (TNFα) signaling delayed fracture healing by delaying chondrocyte apoptosis and cartilage resorption. Mechanistic studies showed that TNFα induced Fas expression within chondrocytes; however, the degree to which chondrocyte apoptosis is mediated by TNFα alone or dependent on the induction of Fas is unclear. This question was addressed by assessing fracture healing in Fas-deficient B6.MRL/Faslpr/J mice. Loss of Fas delayed cartilage resorption but also lowered bone fraction in the calluses. The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Faslpr/J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis. Analysis of the apoptotic marker caspase 3 showed fewer positive chondrocytes and osteoclasts in calluses of B6.MRL/Faslpr/J mice. To determine if an active autoimmune state contributed to increased bone turnover, the levels of activated T cells and Treg cells were assessed. B6.MRL/Faslpr/J mice had elevated Treg cells in both spleens and bones of B6.MRL/Faslpr/J but decreased percentage of activated T cells in bone tissues. Fracture led to ∼30% to 60% systemic increase in Treg cells in both wild-type and B6.MRL/Faslpr/J bone tissues during the period of cartilage formation and resorption but either decreased (wild type) or left unchanged (B6.MRL/Faslpr/J) the numbers of activated T cells in bone. These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process. These data show that loss of Fas activity specifically in chondrocytes prolonged the life span of chondrocytes and that Fas synergized with TNFα signaling to mediate chondrocyte apoptosis. Conversely, loss of Fas systemically led to increased osteoclast numbers during later periods of fracture healing and increased osteogenesis. These findings suggest that retention of viable chondrocytes locally inhibits osteoclast activity or matrix proteolysis during cartilage resorption. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Fracture healing: mechanisms and interventions

“…41 Finally, in a model of SLE, in which mice have defective Fas receptors (TNF receptor superfamily member 6), the percentage of iT REG cells was increased in both callus and bone tissues during the period of active cartilage formation, while at the same time the number of activated T cells in these tissues decreased, despite increased numbers of activated T cells in the spleen, indicative of an autoimmune condition. 19 These results suggest that activated iT REG cells are chondroprotective and are consistent with studies showing that mesenchymal stem cells induce both the differentiation and immunosuppressive function of iT REG cells. 42,43 Pharmacological factors that alter immune function and inflammation might have negative and positive effects on fracture healing and are important to consider in the context of specific comorbidities that affect regeneration.…”

“…15–17 Interestingly, fracture leads to suppression of the immune system, 18 with a local increase in the number of induced T regulatory (iT REG ) cells that suppress active adaptive immune responses within the fracture callus. 19 Studies have further shown that mesenchymal stem cells actively maintain a hypoimmunogenic state 20,21 through the production of immunosuppressive paracrine factors, 22–24 or through the direct actions of these cells on immune-cell populations, including T cells. 25,26 Such effects suggest that these cells impart immune tolerance throughout the early stages of endochondral bone formation and provide protection to the developing tissues by suppressing allo-proliferation of T cells during stem-cell recruitment and cartilage formation.…”

“…25,26 Such effects suggest that these cells impart immune tolerance throughout the early stages of endochondral bone formation and provide protection to the developing tissues by suppressing allo-proliferation of T cells during stem-cell recruitment and cartilage formation. 19 At different stages of fracture healing, cytokines with inflammatory and immune functions, including IL-1β, 19 IL-6, 27,28 IL-17F, 19,29 IL-23 19 and TNF, 12,30 are variably expressed and have different effects. During the period of acute inflammation immediately after injury, TNF and IL-6 recruit cells needed for tissue regeneration and their complete absence has been shown to delay skeletogenic mesenchymal stem cell differentiation.…”

“…31 In the context of chronic inflammation, such as in streptozotocin-induced diabetes mellitus in mice, the cartilage callus is prematurely removed during fracture healing, 32,33 and in mouse models of systemic lupus erythematosus (SLE), osteoclast activity and bone turnover are high. 19 In both of these disease models, less primary and secondary bone accumulation occurs during fracture healing than in control mice. Anti-inflammatory biologic agents and antiresorptive agents might therefore have therapeutic value in fracture healing for patients with either SLE or diabetes mellitus.…”

Downregulation of regulatory T cell function in patients with delayed fracture healing

“…Multiple cytokines, including interleukin (IL)‐1β, IL‐6, IL‐17, IL‐23, and tumor necrosis factor alpha (TNF‐α), are expressed at various stages and present a role in healing . This initial infiltration of immune cells is followed by a suppression of the immune system, with increased differentiation of skeletogenic mesenchymal stem cells and regulatory T (Treg) cells, both of which are capable of inhibiting adaptive T cell responses and achieve a hypoimmunogenic state at the healing site . This suppression of continued inflammation is critical to proper healing, because in the presence of unresolved immune activation, such as when a bacterial infection is present or when the patient presents a pre‐existing inflammatory condition, the catabolic osteoclasts are also highly activated, resulting in reduced accumulation of mineralized bones and delayed union or nonunion …”