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The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function.
Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively.
Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4–7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner.
These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.
Rheumatoid arthritis (RA) is a debilitating, chronic inflammatory disease of the distal joints characterized by hypertrophy and hyperplasia of the synovial epithelium, increased synovial fluid volume, radiographic evidence of bone erosion, and infiltration by inflammatory cells such as T lymphocytes, monocyte/macrophages, neutrophils, and dendritic cells. Resulting complement activation and accumulation of proinflammatory mediators in the synovial, subsynovial, and periarticular tissue adds to the cartilage damage and osteoclast-mediated bone resorption. Cytokines such as interleukin-6 (IL-6), IL-1, IL-15, tumor necrosis factor (TNF), and interferon-γ (IFNγ) have been implicated in the pathogenesis of RA (1), and their importance is underscored by the fact that several TNF inhibitors as well as an anti–IL-6 receptor (anti–IL-6R) antibody are currently marketed for the treatment of RA.
The nonreceptor protein tyrosine kinases of the JAK family, which includes JAK-1, JAK-2, JAK-3, and Tyk-2, transduce signals from multiple type I and type II cytokine receptors and mediate diverse inflammatory responses. Upon receptor activation, JAK kinases phosphorylate STAT proteins, which then translocate to the nucleus and regulate the expression of numerous genes that drive additional participation in the inflammatory response (2). Several small-molecule inhibitors of the JAK family are in various stages of clinical development. Tofacitinib is a potent and orally active inhibitor of the JAK family with a high degree of selectivity within the human kinome and greater potency for JAK-1 and JAK-3 compared to JAK-2 in models of inflammation and arthritis (3–6). Tofacitinib is currently in clinical development as an immunomodulatory agent and disease-modifying treatment for RA and other immune-mediated diseases (7–12); rapid clinical improvement in RA patients whose disease previously did not respond to other disease-modifying therapies, including TNF antagonists, has been observed (8, 13). Recently, findings from the ORAL Scan phase III trial (ClinicalTrials.gov identifier NCT00847613) demonstrated that tofacitinib in combination with methotrexate reduced the progression of structural damage in patients with moderate to severe RA (14).
In the clinical setting, the sequential downstream events following JAK inhibition and the impact of these events on structural damage to arthritic joints are not well understood. We therefore sought to better understand the role that JAK signaling plays at the cellular and molecular level in osteoclast-mediated bone resorption. An appropriate in vivo model for exploring early events in RA is rat adjuvant-induced arthritis (AIA) (15). Rat AIA shares several key pathophysiologic features with human RA, notably its dependence on TNFα (16, 17), and has a more robust presentation of osteoclast- mediated bone resorption than mouse collagen-induced arthritis (CIA) (18). Previous work has demonstrated both prophylactic and therapeutic efficacy of tofacitinib and other JAK inhibitors in rat AIA and mouse CIA (12, 19). However, these efforts did not address the mechanism by which this new class of antirheumatic drugs may impact structural joint damage in arthritis.
The objectives of this study were to evaluate the impact of tofacitinib treatment on osteoclast-mediated bone resorption and to investigate the immunologic mechanism of action underlying this activity. In doing so we evaluated the effect of oral JAK inhibition on established osteoclast bone resorption in rat AIA using histopathologic analysis and immunohistochemistry, as well as plasma and tissue cytokine/chemokine protein and gene expression end points. We also investigated whether JAK inhibition had a direct effect on human osteoclast differentiation or function. Last, we evaluated the impact of JAK inhibition on RANKL production by activated human CD4+ T lymphocytes. This study provides novel, comprehensive evidence supporting the notion that there is an immunopathophysiologic link between JAK signaling, RANKL production, and osteoclast-mediated bone resorption in RA.
MATERIALS AND METHODS
Animals and tofacitinib administration.
Use of animals in these studies was reviewed and approved by the Pfizer Institutional Animal Care and Use Committee; the Pfizer Animal Care and Use Program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. AIA was induced in female Lewis rats as previously described (3). Rats were randomized according to hind paw volume and assigned to tofacitinib or vehicle treatment regimens. Groups of 7–8 rats per treatment group, and normal naive rats (n = 4 per group), were euthanized either 4 hours, 4 days, or 7 days after beginning treatment (days 16, 20, and 23 after immunization, respectively). Tofacitinib was prepared by Pfizer and suspended in 0.5% methylcellulose/0.025% Tween 20 (Sigma) for in vivo studies or in DMSO (Sigma) for in vitro use. Once-daily oral administration of vehicle or tofacitinib (6.2 mg/kg) was initiated on day 16 following immunization and continued through day 23. Paw volumes were reassessed 4 and 7 days after the beginning of treatment (days 20 and 23 after immunization, respectively). For micro–computed tomography (micro-CT) imaging, as well as tartrate-resistant acid phosphatase (TRAP) staining in paw tissue, AIA was induced in a separate cohort of Lewis rats.
Histopathologic analysis and immunohistochemistry.
The left hind paw was fixed and decalcified using previously described methods (20). Hematoxylin and eosin (H&E)–stained tissue sections were examined microscopically and peer-reviewed by 2 board-certified anatomic veterinary pathologists (TPL and ZAR), and scored using a semiquantitative system that included general inflammation and osteoclast bone resorption (i.e., numbers of osteoclasts present, amount of bone resorbed, and number of joints affected), according to previously published criteria (18). The use of microscopy-based semiquantitative scoring of bone resorption is an established end point for describing compound efficacy in rats with AIA (21), and microscopy-based outcomes in this study correlated with micro-CT–based bone volume/total volume (BV/TV) calculations (Figure 1). Detection of osteoclasts in paw tissue via H&E staining was confirmed by CD68/ED-1 IHC and TRAP staining, where TRAP-positive osteoclasts were identified using selected components of the Leukocyte Acid Phosphatase kit (Sigma). (Results are available from the corresponding author upon request.)
Microscopic scoring of monocyte/macrophage and T lymphocyte infiltrates detected by immunohistochemistry was performed using a semiquantitative scoring system as previously described (20). A biotinylated mouse anti-rat ED-1 primary monoclonal antibody (Serotec) was used to detect CD68/ED-1+ (panhistiocytic) cells. A rabbit anti-CD3 primary polyclonal antibody (Accurate Chemical) was used to detect CD3+ T lymphocytes, and a rabbit anti-RANKL polyclonal primary antibody (LSBio) was used to detect RANKL+ cells. To illustrate that the RANKL+ cells were also positive for CD3, dual immunohistochemistry analysis was performed using a rabbit anti-rat CD3 monoclonal antibody (Thermo Scientific) and the RANKL polyclonal antibody described above. Dual staining was accomplished sequentially using the Discovery XT system (Ventana Medical Systems). Multispectral images of the dual-stained tissue sections were acquired using the Vectra Multispectral Imaging system (CRi). Nuance and inForm software (CRi) were used to unmix the imaging data and perform colocalization, respectively.
Ex vivo samples (bilateral hind paws from 20 rats with AIA representing a wide array of disease severity) were scanned using a micro-CT imaging system (μCT40; Scanco Medical). Sequential transaxial images were acquired using an isotropic voxel resolution of 20 μm3, tube voltage of 45 kVp, current of 177 μA, and a 300 msec integration time. Total volume (TV) was identified as the medullary space of the distal tibia, extending 50 slices (1 mm) from the distal portion of the epiphysis proximally to the metaphysis. A Gaussian filter (σ = 0.1, support = 1, lower threshold = 575.6 mg HA/cc and upper threshold = 2,602 mg HA/cc) was used to quantify medullary trabecular bone (referred to as bone volume [BV]) (IPL version 5.15; Scanco Medical). The calculated bone volume fraction (BV/TV) represents the amount of trabecular bone within the medullary space of the distal tibia.
Evaluation of mediators of inflammation in rat plasma and tissue.
Plasma samples were collected 4 hours after treatment on days 16, 20, and 23 and evaluated for IL-6, IL-17, and α2-macroglobulin (α2M) using an ultrasensitive rat IL-6 assay (Meso Scale Discovery), a rat IL-17 LincoPlex assay (Millipore), and a rat α2M enzyme-linked immunosorbent assay (Immunology Consultants Laboratory). Protein was extracted from frozen powdered paw tissue using T-PER tissue extraction buffer in the presence of protease inhibitors (Thermo Scientific). Soluble fractions of paw tissue extract were assayed for a panel of cytokines and chemokines using rat LincoPlex multiplex kits (Millipore). Cytokine/chemokine concentrations measured in each tissue extract were normalized to the total protein concentration in the same sample, as determined by a bicinchoninic acid total protein assay (Thermo Scientific).
Total RNA was prepared from powdered paw tissue using TRIzol (Invitrogen). Relative gene expression levels were determined by quantitative real-time PCR using TaqMan Gene Expression primer probe sets and an ABI Prism 7700 TaqMan system (Applied Biosystems). Cyclophilin was used for an internal reference standard, and data are expressed relative to cyclophilin values for each sample.
In vitro human osteoclast differentiation and function.
Primary human monocytes were obtained by negative selection of CD14+ cells from leukopaks (Biological Specialty Corporation) using magnetic-activated cell sorting (MACS) cell separation technology (Miltenyi Biotec). Cells were plated in 96-well black tissue culture plates at 1 × 105 cells/well in high-glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 5% fetal bovine serum (FBS; Hyclone) and 10 units/ml penicillin–streptomycin. Cultured cells were treated every other day for 14 days with either 25 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF) (R&D Systems) for macrophage differentiation, or M-CSF in the presence of 100 ng/ml of recombinant human RANKL (R&D Systems) for osteoclast differentiation. Cells were also treated with or without varying concentrations of tofacitinib in 0.2% DMSO at the same time as differentiating cytokines. TRAP activity was quantified using ELF-97 fluorescent phosphatase substrate (Invitrogen). Cells were then fixed and stained using a Leukocyte Acid Phosphatase kit according to the recommendations of the manufacturer (Sigma).
Functional bone resorptive activity of human osteoclasts was measured by OsteoLyse assay (Lonza) (22). Human osteoclast precursor cells (Lonza) were plated at 1 × 104 cells/well in medium containing 33 ng/ml M-CSF and 66 ng/ml RANKL on 96-well OsteoLyse cell culture plates precoated with europium-conjugated human type I collagen. During the differentiation phase (days 0–6), cells were treated with varying concentrations of tofacitinib or left untreated. After 6 days in culture, fresh medium containing M-CSF and RANKL was added, and tofacitinib was replaced at the same concentrations or added to previously untreated cells. Additionally, alendronate sodium (Cayman Chemical) was added to untreated cells as a positive control. Cells were cultured for an additional 4 days to allow collagen release by functionally active osteoclasts, and culture supernatants were assayed for europium fluorescence using OsteoLyse Fluorophore-Releasing Reagent (Lonza) with measurement of time-resolved fluorescence over a 400 μsec interval at 340 nm excitation and 615 nm emission.
In vitro human T lymphocyte RANKL production.
CD4+ T lymphocytes were negatively selected from a leukopak using MACS cell separation technology and cultured at 2.5 × 105 cells/well in round-bottomed 96-well tissue culture plates in RPMI 1640 medium containing glucose (Invitrogen), 10% FBS, and 10 units/ml penicillin–streptomycin. Cells were treated with or without varying concentrations of tofacitinib in 0.2% DMSO and activated for 5 days with 1 μg/ml anti-human CD3 and 0.1 μg/ml anti-human CD28 antibodies (BD Biosciences) together with 50 ng/ml recombinant human IL-2 (R&D Systems). RANKL secreted into culture medium was measured using a human LincoPlex assay (Millipore).
All data are expressed as the mean ± SEM. Numerical data were statistically analyzed by Student's t-test or the Mann-Whitney test; multiple comparison procedures were not performed. P values less than or equal to 0.05 were considered significant. Statistical analyses were performed using either GraphPad Prism software or Excel software (Microsoft). The relationship between micro-CT–based and microscopy-based bone resorption scores was assessed by calculating Spearman's rank correlation coefficient (Figure 1). P values for correlation less than 0.05 were considered significant.
JAK inhibition suppresses inflammation and structural joint damage in rat AIA.
The rat AIA model of RA was used to characterize the in vivo effects of JAK inhibition on established arthritis and osteoclast-mediated bone resorption. Based on previous dose-response studies, a daily dose of tofacitinib of 6.2 mg/kg was selected to provide ∼80% inhibition of hind paw volume and plasma exposure capable of suppressing the JAK-1 and JAK-3 signaling pathways for >4 hours (3). Hind paw edema measured via plethysmography was reduced as early as 4 days after the beginning of treatment, with continued reduction to near normal levels by day 7 (Figure 2A). Plasma and paw tissue samples were collected from inhibitor- and vehicle-treated rats with AIA and compared with samples from normal rats after 4 hours, 4 days, and 7 days of treatment (days 16, 20, and 23 after adjuvant immunization, respectively).
Microscopic examination of the hind paw, which included the tibiotarsal/metatarsal (ankle) joints and the tarsal/interphalangeal joints (toes), revealed a destructive polyarthritis in 100% of the rats. This was characterized by synovial and periarticular edema, mixed inflammatory cell infiltration (monocyte/macrophages, neutrophils, and lymphocytes) within the joint space and synovial/periarticular tissue, and, lastly, significant osteoclast-mediated bone resorption of trabecular woven bone within the medullary space of the distal tibia, talus, and tarsal bones. After 7 days of treatment there were dramatic reductions in histopathologic severity scores for general inflammation (Figure 2B) and osteoclast-mediated bone resorption (Figure 2E). For instance, compared to a vehicle-treated rat (Figure 2C), edema and inflammatory cell infiltrates in a representative tofacitinib-treated rat were significantly reduced by day 7 (Figure 2D). Also, compared to vehicle (Figure 2F), tofacitinib significantly reduced osteoclast numbers within the medullary space of the distal tibia (Figure 2G).
Immunohistochemistry analysis was performed to semiquantitatively determine the inflammatory cell response to JAK inhibition. The CD68/ED-1 protein is widely used to detect panhistiocytic cells, such as monocytes, macrophages, and dendritic cells, in the rat (ED-1 is the rat homolog of CD68 on human histiocytic cells), and its expression on lysosomal membranes increases during phagocytic activity (23). The extent of macrophage infiltration and the macrophage activation state are known to correlate with joint pain and the general inflammatory status of the patient, and most therapies currently available for RA decrease the number of macrophages in the synovium (24, 25). In this study, a statistically significant decrease in tibiotarsal joint synovial CD68/ED-1+ cell numbers was observed 7 days after treatment was started (Figures 3A–C). CD3 is a marker of T cell lineage and, through its association with the T cell receptor, is involved in antigen recognition, T cell activation, and signal transduction (26). A statistically significant decrease in the number of CD3+ cells (coaggregated with osteoclasts at sites of bone resorption in vehicle-treated rats) was also detected 7 days after initiation of tofacitinib treatment (Figures 3D–F).
Both RANKL transcript levels (Figure 4A) and protein levels (Figure 4B) were significantly reduced in paw tissue sections after 7 days of treatment with tofacitinib. Many RANKL+ cells were noted at sites of bone resorption in vehicle-treated rats (Figure 4C), most of which stained positive for CD3 (Figure 4C, inset). (Additional results are available from the corresponding author upon request.) Consistent with the changes in transcript and protein levels, RANKL+ cell numbers were also dramatically reduced after 7 days of tofacitinib treatment (Figure 4D).
The effects of tofacitinib on inflammatory mediator expression were analyzed in both plasma and paw tissue from vehicle-treated and compound-treated rats with AIA and compared against normal controls. JAK inhibition significantly decreased the expression of messenger RNA (mRNA) for the cytokines IL-6 and IL-1β, the chemokine CXCL10, and the adhesion molecules P-selectin and E-selectin (Figure 5A). Interestingly, Il6, Cxcl10, Selp, and Sele gene expression levels were significantly reduced within 4 hours of initiation of treatment, whereas a statistically significant decrease in Il1b was not observed until 7 days after the beginning of treatment. Plasma IL-6 levels were significantly increased in vehicle-treated rats with AIA compared to normal rats; however, IL-6 was suppressed within 4 hours of the first dose of tofacitinib, and remained so through day 7 (Figure 5B). This result was consistent with the previously reported activity of the JAK inhibitor in a mouse model of established arthritis (6). Plasma IL-17 was also reduced within 4 hours of treatment initiation, with statistical significance achieved again after 7 days (Figure 5B). In contrast, 4 days of treatment was required before a significant reduction in the acute-phase protein α2M was observed (Figure 5B), closely paralleling the effect of JAK inhibition on edema (Figure 2A).
In paw tissue sections from rats with AIA, several inflammatory cytokines and chemokines were detectable. For instance, tissue levels of IL-6, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) were all significantly elevated compared to levels in normal animals. Similar to plasma, IL-6 levels in paw tissue were significantly suppressed within 4 hours of the first dose of tofacitinib, whereas MCP-1 and MIP-1α levels were not significantly decreased until after 4–7 days of treatment (Figure 5C). Anti-TNF therapy is effective in the rat AIA model (27), and elevated TNF levels have previously been demonstrated in the paws of rats with AIA (16); however, in the present study TNF levels were either below the level of detection or were not significantly elevated in rats with AIA compared to normal animals. These discrepancies are likely due to the relatively small increases that have been reported with this model, as well as differing sensitivities of the assays used to measure cytokine levels in the paw. IFNγ concentrations were also below the level of detection in both plasma and paw tissue in this study.
In summary, tofacitinib treatment mitigated active arthritic disease in vivo by reducing edema, CD68/ED-1+ and CD3+ inflammatory cell infiltration, tissue RANKL levels, and osteoclast-mediated bone resorption within 7 days. The rapid decrease in plasma and tissue IL-6 protein and transcript levels within 4 hours of tofacitinib administration was followed by a later reduction in RANKL (after 4–7 days of treatment) and correlated with a trend of decreasing infiltration of monocyte/macrophages and T lymphocytes in paw tissue. Taken together, these results suggest that tofacitinib had an immediate yet sustained impact on the lymphocyte-driven production of inflammatory mediators.
JAK inhibition does not impact in vitro human osteoclast differentiation or function, but does suppress human T lymphocyte RANKL production.
To determine whether reduced bone resorption was mediated through a direct effect on osteoclastogenesis, we evaluated the potential of tofacitinib to directly impact human macrophage and osteoclast differentiation in vitro. At concentrations up to 200 times greater than its cellular JAK-1 and JAK-3 50% inhibition concentration (IC50) (3), tofacitinib had no effect on either M-CSF–induced macrophage differentiation or M-CSF and RANKL–induced osteoclast differentiation, as demonstrated by in vitro cellular morphologic assessment and quantification of TRAP staining (Figure 6A). In contrast, the p38 MAPK inhibitor, SC-409, potently inhibited RANKL-induced osteoclast differentiation (data not shown) as previously described (28). Tofacitinib also had no effect on the resorptive capacity of differentiated human osteoclasts, as measured by their ability to digest human bone collagen (Figure 6B). By comparison, alendronate, a farnesyl diphosphate synthase inhibitor, inhibited collagen digestion in the same experiments at concentrations above 3 μM (data not shown).
Because the decrease in hind paw RANKL mRNA and protein levels (Figures 4A and B, respectively) correlated with a decrease in CD3+ lymphocyte numbers (Figure 3D), it was initially unclear whether decreased RANKL levels resulted from JAK inhibitor reduction of T lymphocyte infiltration. To investigate this, we evaluated the impact of tofacitinib treatment on RANKL production from human T lymphocytes in vitro. Human CD4+ T lymphocytes were activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and peak RANKL production was observed after 5 days (174–241 pg/ml). Under those conditions, tofacitinib inhibited RANKL production in a concentration-dependent manner (Figure 6C) with an IC50 of ∼5 nM, which was consistent with the previously described potency against IL-2R signaling in cultured T cells (29).
The 4 members of the JAK tyrosine kinase family (JAK-1, JAK-2, JAK-3, and Tyk2) transduce signals from cytokine receptors during the immunopathogenesis of several inflammatory and autoimmune diseases, including RA (30). Tofacitinib is a potent and orally active small-molecule inhibitor of JAK with a high degree of human kinome selectivity (4) and specificity for JAK-1 and JAK-3 over JAK-2 at clinically relevant concentrations (3). Although the ability to prevent both inflammation and bone resorption in rat AIA has been described by others (12), the immunologic mechanism of action of tofacitinib on structural joint damage in active arthritis remained unclear.
In the present study, we investigated the mechanism(s) of action of tofacitinib suppression of osteoclast-mediated bone resorption in rat AIA. Treatment was initiated during peak disease and maintained for 7 days. In addition to decreased edema, we noted a time-dependent decrease in osteoclast-mediated bone resorption, as well as decreased CD68/ED-1+ monocyte/macrophage and CD3+ T lymphocyte infiltration in the joint and surrounding tissue. Increased expression of several inflammatory cytokines, chemokines, and adhesion molecules was observed in the plasma and/or paw tissue of rats with AIA, with significant inhibition of these biomarkers of inflammation following treatment. Notably, treatment resulted in significant and rapid decreases in both plasma and tissue levels of IL-6, a potent stimulator of innate immunity. Consistent with these findings, we have recently demonstrated the ability of tofacitinib to modulate innate responses to lipopolysaccharide in vivo through mechanisms likely involving inhibition of STAT-1 signaling (6). Further investigation is needed to determine the mechanism by which tofacitinib suppresses IL-6 production in such rapid order. It is possible that direct or indirect inhibition of cytokine-dependent IL-6 production by fibroblasts may play a role. The inhibition of plasma IL-17 in these studies could be explained by the potent effects of tofacitinib against IL-23R and retinoic acid receptor–related orphan nuclear receptor γ expression (6), whereas the rapid suppression of CXCL10, as well as E-selectin and P-selectin, likely involves the inhibition of IFNγ receptor signaling.
The process of osteoclast-mediated bone resorption (removal of mineralized bony matrix by osteoclasts) enables bones and joints to respond to ever-changing physical loads, yet this dogmatic view of bone turnover (first explained as Wolff's Law ) does not account for the influence of local, intense inflammation. Such interplay between biophysical and inflammatory stimuli is highly relevant in RA, particularly with respect to loss of joint function. Because of this, disease-modifying antirheumatic drugs, so-called because they are capable of reducing cartilage damage and bone resorption, are considered the standard of care for RA. At the center of bone resorption dynamics are osteoclasts, which are derived from the mononuclear phagocyte system and are formed by the fusion of mononuclear hematopoietic precursors (32). M-CSF is required for the growth and survival of osteoclast precursors, as underscored by the development of osteopetrosis in M-CSF–deficient mice (33); however, complete osteoclast differentiation also requires RANKL (34). RANKL is produced by osteoblasts under homeostatic conditions (35) and by lymphocytes and neutrophils under inflammatory conditions (36–39). The importance of RANKL in bone loss–associated disease is underscored by the efficacy of denosumab, a fully human monoclonal antibody against RANKL, for the treatment of postmenopausal women who have a high risk and/or a history of osteoporotic fractures (40).
While the role of JAK/STAT signaling in osteoclast bone resorption during RA is poorly understood, it has been demonstrated that RANKL action on osteoclast precursors can be modulated by cytokines that signal through JAK-dependent pathways. For instance, IL-15R signaling has been shown to be important for RANKL-dependent osteoclastogenesis (41). Blockade of IL-15R signaling in osteoclasts could reduce bone resorption by tempering the effect of RANKL, but RANKL levels were reduced in vitro and in vivo in our study. On the other hand, RANKL production by activated T cells can be mediated by cytokines such as IL-2, IL-6, IL-7, and IL-21 (42, 43), and signaling via the receptors for these cytokines is not only JAK dependent, but is also potently inhibited by tofacitinib (3, 6). It has also been proposed that T cells may actually modulate RANKL-mediated osteoclastogenesis via production of IFNγ (44, 45). Since IFNγ signals through a JAK-dependent mechanism (3, 6), one would anticipate that JAK inhibition would increase osteoclast activity; however, this was not the case in our in vivo study. Lastly, in addition to having a direct effect on T lymphocytes, JAK/STAT inhibition may modulate fibroblast RANKL expression by decreasing IL-6R signaling; however, this would only partially explain our observations, since TNF, IL-1, and IL-17 appear to drive RANKL expression in fibroblasts through JAK/STAT-independent mechanisms (46).
In this study, we showed a significant increase in both RANKL mRNA and protein levels in joint tissue from rats with AIA compared to normal rats, which correlated with an increase in systemic and paw inflammation. Although tofacitinib treatment decreased RANKL expression in joint tissue (both transcript and protein levels), we observed a corresponding decrease in CD3+ lymphocytes. Since it was unclear whether the decreased RANKL expression was simply due to the reduction in infiltrating T lymphocytes, and to better understand the clinical translatability of these findings, we evaluated the impact of tofacitinib in vitro on human CD4+ T lymphocyte RANKL production, as well as on human osteoclast differentiation/function. Tofacitinib did not affect either M-CSF–driven macrophage differentiation or RANKL-dependent osteoclastogenesis and osteoclast function, suggesting that JAK inhibition is unlikely to directly influence osteoclast numbers or activity in the arthritic joint. In contrast, there was a concentration-dependent inhibition of RANKL production in human CD4+ T lymphocytes activated in vitro, with an observed potency consistent with that previously reported for the inhibition of JAK-1 and JAK-3 signaling in T cells (29). Although T lymphocytes clearly appear to play a role in osteoclast activation by producing RANKL, it is possible that they do not represent the totality of RANKL production in the arthritic joint. Additional studies are needed to test the hypothesis that other cell types, taking direct cues from T lymphocytes, may produce the critical mass of RANKL.
In summary, the potent inhibition of RANKL production by T lymphocytes, taken together with the lack of an effect on osteoclast differentiation and function, suggests that osteoclast-mediated bone resorption in vivo is suppressed by tofacitinib through decreased RANKL production. These results support clinical radiographic evidence that tofacitinib reduces the progression of structural damage in patients with RA (14), and suggest an immunologic mechanism of action for this process.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. LaBranche had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. LaBranche, Jesson, Radi, Bonar, Thompson, Zhan, Hegen, Kishore, Mbalaviele, Meyer.
Analysis and interpretation of data. LaBranche, Jesson, Radi, Storer, Guzova, Bonar, Bansal, Wellen, Bailey, Symanowicz, Hegen, Head, Mbalaviele, Meyer.
All authors are employees of Pfizer Worldwide Research and Development.
The authors would like to acknowledge Drs. James D. Clark, Laurence O. Whiteley, Shawn P. O'Neil, and Birgitta Benda for reviewing this work, as well as Kimberly M. Shevlin, Denise M. Lay, and Matthew P. Lech for significant technical contributions.