Targeting pro-inflammatory cytokines following joint injury: acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis

Osteoarthritis (OA) is a debilitating disease characterized by degenerative changes in articular cartilage, bone, and other surrounding tissues. Of the nearly 27 million Americans with symptomatic OA, an estimated 12% have a post-traumatic etiology, making post-traumatic arthritis (PTA) one of the leading causes of joint disability [1, 2]. The financial burden of PTA is significant, as it is estimated to cost the US economy over $7 billion annually in work productivity and medical expenses [1]. Additionally, degenerative arthritis following injury is the most common reason for US service members not returning to active duty [3]. PTA can develop after a variety of joint injuries including soft tissue injuries such as ligament and meniscal tears [4‐6], articular impact [7, 8] or articular fracture [9]. Articular fractures are of particular interest, as they commonly and predictably cause accelerated joint degeneration [10]. The current standard of care for articular fractures is surgical reduction and fixation. Yet, surgical intervention alone does not prevent the development of PTA. Even with optimal treatment, displaced articular fractures of the lower extremity have exhibited a 10 to 20% incidence of clinically significant arthritic degeneration of joint tissues [11].

The pathogenesis of arthritis following joint trauma is not fully understood, and a variety of factors including chondrocyte death, altered joint mechanics, and inflammation have been implicated in the disease. Following joint injury, elevated synovial fluid levels of pro-inflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), have been reported with the highest levels observed acutely within the first 24 h after injury [12‐15]. However, levels remain elevated for weeks to months post-trauma [14, 16‐19]. Upregulation of IL-1 and TNF-α may play a significant role in the pathogenesis of PTA, similar to their role in chronic OA of joint tissue in patients without antecedent injury [20, 21]. Clinically, cartilage-derived biomarkers are significantly increased within the first month following knee injury [13, 22, 23], which suggests that significant cartilage damage is occurring within weeks of trauma and that early intervention may influence the long-term sequela of joint degeneration [24].

In order to further characterize arthritis development following joint trauma, we developed a murine model of closed articular fracture of the tibial plateau with progressive arthritic changes in the bone, articular cartilage, and other joint tissues [25] at 8 weeks post-injury in C57BL/6 mice. However, the MRL/MpJ strain of mice known as the superhealer strain was protected from PTA and did not develop degenerative joint changes following articular fracture [19], and exhibited lower levels of both local and systemic inflammation in MRL/MpJ mice compared to C57BL/6 mice [26]. This attenuated inflammatory response may help explain how MRL/MpJ mice are protected from the development of PTA after articular fracture [19]. These findings also suggest that the controlled inhibition of the inflammatory response, either systemically or locally, may represent a novel therapeutic approach for PTA after joint injury.

Targeted blocking of specific pro-inflammatory cytokines has been the focus of several therapies for rheumatic diseases such as rheumatoid arthritis. This approach has led to the development of specific inhibitors of IL-1, such as anakinra (Kineret^®, Biovitrum, Stockholm, Sweden), a recombinant form of human IL-1 receptor antagonist (IL-1Ra). Endogenous and recombinant IL-1Ra act similarly in competitively inhibiting the binding of both IL-1α and IL-1β to their active receptor [27]. Specific inhibitors of TNF-α have also been developed, such as etanercept (Enbrel^®, Amgen, Thousand Oaks, CA), a human soluble form of TNF-α receptor II (sTNFRII). sTNFRII binds directly to TNF to block its interaction with cell-surface TNF receptors and modulate the biological responses induced or regulated by TNF. Previous research has shown that administration of either IL-1Ra [28] or TNF inhibitors [29‐31] reduces inflammation and cartilage destruction in mouse models of collagen-induced arthritis. IL-1Ra was also shown to enhance meniscal repair in an in vitro model [32]. However, it is unclear how these inflammatory mediators if administered following joint injury would influence synovial inflammation and cartilage degeneration following intra-articular trauma. Although a role for IL-1 and TNF-α in the pathogenesis of arthritis has been suggested, the role of pro-inflammatory cytokines in the progression of PTA has not been elucidated. Using these cytokine inhibitors after joint injury may represent a novel therapeutic approach for PTA.

We have previously demonstrated that an acute and prolonged increase in the inflammatory response following joint injury has been associated with the development of arthritis in mice. To investigate the role of inflammation in the progression of arthritis following articular fracture, we targeted pro-inflammatory cytokines first, during the initial acute local response to joint injury, and secondly, systemically for a prolonged 4 week period following injury. Following closed intra-articular fracture in the C57BL/6 mouse knee, the anti-inflammatory agents IL-1Ra (anakinra) or sTNFRII (etanercept) were administered, either locally via a single intra-articular injection immediately following injury or systemically for 4 weeks following injury, and the severity of arthritis was assessed.

Increasing evidence from clinical and animal studies indicates that pro-inflammatory cytokines are elevated following joint injury, yet the specific roles of IL-1 and TNF-α in the development of post-traumatic arthritis are not fully understood. Here, we show that early local inhibition of IL-1 with a single intra-articular injection of IL-1Ra significantly reduced arthritic changes in cartilage, reduced synovitis, and did not alter bone morphology or bone healing after a closed joint fracture in mice. Local inhibition of TNF-α with a single intra-articular injection of sTNFRII had a moderate effect in reducing arthritic changes in the cartilage and synovitis, although not as effectively as IL-1Ra. However, degenerative changes in bone morphology were not reduced with sTNFRII. Systemic infusion of IL-1Ra for 4 weeks post injury, and both local and systemic inhibition of TNF-α with either a single intra-articular injection of sTNFRII or three times weekly injections of sTNFRII for 4 weeks did not reduce arthritic changes, and instead led to significant degenerative changes in bone morphology and evidence of fibrous fracture healing. These results show that intra-articular IL-1, rather than TNF-α, plays a critical role in the acute inflammatory phase following joint injury and can be inhibited locally to reduce post-traumatic arthritis following a closed articular fracture.

While the role of anti-cytokine therapy in PTA remains to be firmly established, its role in autoimmune mouse models of arthritis is relatively well-characterized. For example, continuous high doses of systemic IL-1Ra prevents collagen-induced arthritis (CIA) in DBA/1 mice [35]. Furthermore, chronic overexpression of TNF using a human TNF-transgenic mouse model of TNF-induced arthritis has been shown to be best reduced with a treatment of anti-TNF-antibody (infliximab) and recombinant human IL-1Ra (anakinra) [42]. When administered concurrently, combination therapy of infliximab and anakinra has been shown to block proteoglycan loss in a synergistic fashion [42]. However, systemic cytokine inhibition in the present study did not demonstrate any benefit in reducing PTA, suggesting that future efforts should target intra-articular methods of therapeutic administration. Given that IL-1Ra (anakinra) is commercially available and Food and Drug Administration (FDA)-approved for the treatment of rheumatoid arthritis, the results of the current investigation have broad therapeutic implication and support the extension of translational studies and potential clinical trials in humans. To date, there has been one pilot study of anakinra for acute joint injury in humans [53]; this trial showed that IL-1Ra, administered intra-articularly within the first month following severe knee injury, reduced knee pain and improved function over a 2-week interval. However, the ability of IL-1 inhibition to reduce the development of PTA was not addressed.

In contrast, for treatment of chronic OA, previous clinical trials have reported that neither systemic nor local inhibition of IL-1 were able to reduce clinical symptoms in patients with symptomatic OA of the knee. Systemic inhibition of IL-1 with human monoclonal antibody to IL-1 receptor 1 administered for 12 weeks was found to be well-tolerated, and showed a trend in pain reduction, but the effect was not significant, and the clinical benefit was small [54]. Local inhibition of IL-1Ra with a single intra-articular injection of human recombinant IL-1Ra (anakinra) was also shown to be well-tolerated but only showed a reduction of pain at 4 days and no improvement in OA symptoms compared to placebo at 4, 8, and 12 weeks [55]. Pharmacokinetic data indicate that the mean terminal half-life of IL-1Ra in serum after intra-articular injection is approximately 4 h and is undetectable at 24 h. With the acute local inhibition of IL-1 in this study, we observed less cartilage degeneration and less synovial inflammation. We were also able to detect IL-1Ra in serum up to three days after intra-articular injection, potentially because of decreased clearance due to joint swelling following injury. Our results support a direct role for IL-1 in the acute inflammatory phase of articular injury that can be inhibited at the injury site by exogenous administration of IL-1Ra to reduce PTA.

The systemic IL-1Ra group had the most severe arthritic changes demonstrated by the highest Mankin and synovitis scores. It is important to consider the role of IL-1 in the healing response to fracture. IL-1β was found to stimulate proliferation and differentiation of pre-osteoblasts in vitro, as MC3T3-E1 cells produced more mineralized bone matrix when IL-1β was introduced [56]. Moreover, IL-1β can alter the ratio of cartilage volume to callus volume in mice following a diaphyseal tibial fracture within two weeks after the injury [56]. The dosage and timing of administration of the 4-week continuous systemic infusion of IL-1Ra was selected based on previous data showing its effectiveness in ameliorating arthritis in mouse models of inflammatory arthritis [35‐37]. However, in our articular fracture model, this strategy of systemic IL-1Ra delivery appears to have altered the healing response. Given our findings, along with previous evidence suggesting a potential role of IL-1 in fracture repair, it could be speculated that IL-1 may transition from playing a negative role in the acute phase of trauma to a positive role in the healing and bone remodeling phase.

In this study, acute local inhibition of TNF-α or systemic inhibition of TNF-α for 4 weeks post injury did not prevent the progression of PTA. From histological evaluations, fibrous healing could be observed at the site of fracture 8 weeks post-injury with administration of sTNFRII. Likewise, bone morphology assessed with microCT indicated that bone fraction and bone density were significantly reduced with administration of sTNFRII. Our findings suggest that inhibiting TNF-α following articular fracture may inhibit fracture healing and bone remodeling. This is consistent with results in a model of simple closed fracture repair in wild-type and TNF-α receptor-deficient mice wherein the absence of TNF-α signaling led to impaired fracture healing [57]. We also found that detrimental changes in bone morphology were correlated to histologic measures of cartilage degeneration and synovial inflammation. This demonstrates the complex inter-relationship between the various joint tissues in the development of post-traumatic arthritis.

We have previously reported reduced bone fraction and bone mineral density following fracture [15, 19]. However, these degenerative bone changes appear to be reduced in the local saline group along with the local IL-1Ra group. The data suggest that intra-articular injections of saline may be altering the intra-articular environment in a manner which is beneficial to the periarticular bone. One hypothesis is that intra-articular injections may be diluting catabolic factors or washing out the joint. However, local saline provided no benefit in reducing cartilage degeneration or synovial inflammation. Normal fracture healing involves the upregulation of many inflammatory cytokines and growth factors, and the temporal profiles of these factors are different during the healing process [58, 59]. The cytokines IL-1β and TNF-α have also been shown to stimulate the production of active bone morphogenetic protein 2 (BMP-2) [60], which may be involved in the repair process. Understanding the role of such systemic factors found in the circulating serum following trauma may provide insight into articular fracture healing and the development of PTA. Although we saw differences in bone morphology between treatment groups, systemic measures of bone turnover were not significantly different among treatment groups following fracture. We found that markers of both osteoblast and osteoclast activity increased with increasing bone volume or bone fraction in the tibial plateau and metaphysis at 8 weeks post fracture. This time point would represent the remodeling phase of bone repair and has been characterized by high levels of bone resorption and formation markers [61]. Bone turnover markers vary throughout the fracture healing process, and although biochemical markers of bone-turnover have been helpful in understanding and clinical treatment of metabolic bone disease like osteoporosis, their usefulness in assessing fracture healing has not been established [62]. TGF-β1 may play a role in endochondral ossification [63], and is reported to be activated during osteoclast bone resorption [64, 65]. In this study, increasing total or free active TGF-β₁ was associated with decreased bone volume and bone fraction in the tibial plateau. TGF-β₁ promotes bone formation through chemotactic attraction of osteoblasts and the enhancement of osteoblast proliferation [66]. However, bone-resorbing osteoclasts may help to release free active TGF-β₁ via their acidic microenvironment [67, 68]. Longitudinal assessment of systemic markers of bone turnover and healing may provide more insight into our understanding of articular fracture healing and lead to new methods of assessing interventions that may prevent or mitigate the development of PTA.

Serum IL-6 concentrations were significantly lower in mice that received local IL-1Ra following articular fracture compared to those that received local sTNFRII, and no differences in synovial fluid concentrations of IL-6 were found between fractured and contralateral limbs among any groups. IL-6 is reported to be elevated in synovial fluid following meniscal and ligamentous tears [12, 69]. However, IL-6 serum concentrations significantly decrease during fracture healing [70], which may explain the minimal differences among treatment groups at 8 weeks post fracture. Interestingly, serum and synovial fluid IL-6 concentrations increased with increasing cartilage degenerative changes in the medial tibia following articular fracture. In vitro studies have demonstrated that IL-6 influences cartilage catabolism with mechanical trauma resulting in increased proteoglycan loss [71]. IL-6 with the soluble IL-6 receptor triggered osteoclast formation and has also been associated with osteoclast-like cell formation in rheumatoid arthritis patients and may contribute to bone resorption [72, 73], which supports our observation of an association between increased synovial fluid IL-6 concentrations and decreasing bone fraction in the medial tibial plateau. Serum IL-6 also correlated with increased synovitis scores following articular fracture. The interaction between inflammatory cytokines like IL-1, TNF-α and IL-6 following joint trauma is not well-understood. However, in this mouse model of joint injury, the data suggest a complex relationship between systemic and local biochemical factors and joint pathology of the cartilage, synovium and adjacent bone.

The progression of PTA following joint injury is not well-characterized, and current clinical measures are unable to predict which patients may develop PTA following injury. Identifying serum or synovial fluid molecular biomarkers of degenerative joint changes following injury would provide insight into the early stages of the disease and be a useful and relatively noninvasive diagnostic tool. In this study, synovial fluid COMP was able to distinguish between fractured and contralateral control limbs at 8 weeks post fracture. COMP is an extracellular matrix protein found predominantly in cartilage, but also in synovium, meniscus, ligaments, tendons, and associated with osteoblasts [74‐76]. COMP has been suggested as a candidate biochemical molecular marker of arthritis because of its relative specificity to joint tissues. Interestingly, synovial fluid COMP correlated with synovitis and decreased bone fraction but not cartilage degenerative changes. Although COMP has been used as a marker of cartilage turnover [76], COMP has also been reported to be associated with clinical synovitis in patients with knee OA [77], elevated in injured tendon sheath synovial fluid [78], and expressed by adult osteoblasts and may be indicative of metabolic bone activity [74]. In this study, synovial fluid COMP was not significantly different among treatment groups. However, COMP was only measured at a single time point of 8 weeks post fracture. Longitudinal analysis of COMP may provide more insight in future studies.

This study indicates that acute treatment of an articular fracture with local IL-1Ra therapy can prevent cartilage degeneration and synovial inflammation in the mouse knee. Our investigation supports a direct role for IL-1 in the acute phase of the inflammatory process that follows articular injury. These results further our understanding of the biological mechanisms governing PTA and provide evidence to support the therapeutic benefit of a novel method of treating acute joint injuries that may be used as adjunctive therapy to surgical stabilization.