Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis

Abstract

Rheumatoid arthritis is an autoimmune disease that is characterized by chronic inflammation and destruction of joints. Netrin-1, a chemorepulsant, laminin-like matrix protein, promotes inflammation by preventing macrophage egress from inflamed sites and is required for osteoclast differentiation. We asked whether blockade of Netrin-1 or its receptors [Unc5b and DCC (deleted in colorectal carcinoma)] may be useful therapeutic targets for treatment of inflammatory arthritis. Arthritis was induced in 8-wk-old C57Bl/6 mice by intraperitoneal injection of K/BxN serum. Murine monoclonal antibodies against Netrin-1, Unc5b, or DCC (10 µg/mouse) were injected weekly for 4 wk (n = 10). Paw swelling and thickness were assessed and following euthanasia 2–4 wk after serum transfer, paws were prepared for micro–computed tomography and histology. Paw inflammation was maximal 2 wk after injection. Anti–Netrin-1 or anti-Unc5b, but not anti-DCC, antibodies significantly reduced paw inflammation (clinical score: 9.8 ± 0.8, 10.4 ± 0.9, and 13.5 ± 0.5, respectively vs. 16 ± 0 for control; P < 0.001). Micro–computed tomography showed bony erosions in untreated or anti-DCC–treated mice, whereas there were no erosions in anti–Netrin-1/anti-Unc5b-treated-animals. Tartrate-resistant acid phosphatase staining demonstrated a marked decrease in osteoclasts in anti-Netrin-1/anti-Unc5b–treated animals. Immunofluorescence staining revealed a decrease in cathepsin K⁺ and CD68⁺ cells in anti–Netrin-1/anti-Unc5b–treated animals. Blockade of Netrin-1/Unc5b by monoclonal antibodies prevents bone destruction and reduces the severity of K/BxN serum transfer–induced arthritis. Netrin-1 may be a novel therapeutic target for treatment of inflammatory bone destruction.—Mediero, A., Wilder, T., Ramkhelawon, B., Moore, K. J., Cronstein, B. N. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic inflammation and destruction of the joints. It affects approximately 1% of the population worldwide, manifesting as pain, stiffness, and synovitis (inflammation of the synovial membrane), which, in turn, leads to articular destruction (1). Both genetic and environmental components contribute to the etiology of RA and together lead to an early immune alteration in both innate and adaptative compartments (1). Early cartilage and bone erosions are associated with accumulation of inflammatory cells in the synovial membrane, including macrophages, T and B lymphocytes, dendritic cells, and polymorphonuclear leukocytes, which mediate the destructive changes in the synovium (2). Despite marked improvements in the treatment of RA and other forms of inflammatory arthritis, the pathogenesis of inflammatory arthritis remains incompletely elucidated and, for many patients, novel approaches for antirheumatic therapies are needed.

Netrin-1 is a laminin-like matrix protein that belongs to the axonal guidance protein family. Netrin-1 acts as a chemorepulsant and inhibits migration of monocytes, neutrophils, and lymphocytes by activation of its receptors, Unc5b and adenosine A_2B receptor (3–5). Netrin-1 plays a pathogenic role in inflammation that leads to atherosclerosis and localization of macrophages to adipose tissue in diet-induced obesity by preventing macrophage egress from inflamed sites (6, 7). When localized to the vascular endothelium, Netrin-1 expression is regulated by infection and inflammatory cytokines and inhibits inflammatory cell migration into tissues, and its down-regulation at the onset of sepsis/inflammation may facilitate leukocyte recruitment (3). Of interest, administration of exogenous Netrin-1, acting via Unc5b receptor, reduces renal ischemia–reperfusion injury and its associated renal inflammation by preventing leukocyte recruitment to the inflamed site (8).

We have recently reported that Netrin-1 is an autocrine and paracrine regulator of osteoclast differentiation (9). Binding of Netrin-1 to its receptor Unc5b is essential for osteoclast differentiation and function and triggers the signaling cascade that is involved in the activation of the small GTPase RhoA via leukemia-associated guanine nucleotide exchange factor and repulsive guidance molecule A, which leads to cytoskeletal rearrangements required for osteoclast fusion and differentiation (9). Netrin-1 is also highly expressed by macrophages at sites of wear particle–induced osteolysis in the inflamed peri-implant soft tissue in patients who undergo implant revision and in macrophages and osteoclasts in a murine model of wear particle–induced bone destruction. Antibody-mediated blockade of Unc5b or Netrin-1 prevents both accumulation of inflammatory cells and bony destruction in this murine model (10). These results, both in mice and in humans, indicate that Netrin-1 plays an important role in inflammatory osteolysis.

Therefore, we asked whether blockade of Netrin-1 or its receptors Unc5b and DCC (deleted in colorectal carcinoma) may be useful therapeutic targets in the treatment of inflammatory arthritis. To answer this question, we employed the well-described K/BxN serum transfer–induced arthritis mouse model. This animal model shares features similar to human RA (11). The arthritis induced in mice by transfer of K/BxN serum is independent of the T- and B-cell–mediated autoimmune phase and has a predictable onset, as the same quantity of antibodies is injected into the affected mice. K/BxN serum transfer is a valuable tool for the investigation of factors that contribute to inflammation and bone and cartilage destruction during arthritis that develop independent of the autoimmune phase of the disease (11).

MATERIALS AND METHODS

K/BxN serum transfer–induced arthritis

Arthritic K/BxN mice were generated by crossing K/B mice with NOD/Lt mice. Adult arthritic K/BxN mice were bled and the sera were pooled. Age-matched, female recipient, 8-wk-old C57Bl/6 mice were injected with pooled serum (200 μl, i.p.) on d 0 and 2, and at the same time (d 0), murine monoclonal antibodies against Netrin-1 (LifeSpan Biosciences, Seattle, WA, USA), Unc5b (Abcam, Cambridge, MA, USA), DCC (AF5; Thermo Fisher Scientific, Waltham, MA USA), or IgG isotype control antibodies were intraperitoneally injected (10 µg/mouse; n = 10 mice in each group). Antibodies were administered weekly for up to 4 wk. Development of arthritis was assessed daily, and the severity of arthritis was assessed in each paw on a 4-point scale defined as follows: 0 = normal appearance, 1 = localized edema/erythema over one surface of the paw, 2 = edema/erythema involving more than one surface of the paw, and 3–4 = marked edema/erythema involving the whole paw. Scores of all 4 paws were added for a composite score. Mice were euthanized on d 14 and 28, and bones were prepared for micro-computed tomography (microCT) and histology.

MicroCT

After euthanasia, long bones were fixed in 70% ethanol and prepared for high-resolution microCT. Analyses were performed in Skyscan 1172 microCT (Bruker, Madison, WI, USA) by using the following imaging parameters: 60 kV, 167 uA, 9.7 μm pixel size, 2000 × 1332 matrix, 0.3° rotation steps, 6 averages, movement correction of 10, and 0.5 mm Al filter. Images were reconstructed by using the Skyscan NRecon software [histogram range 0–0.085, beam hardening correction of 40, gaussian smoothing (factor 1), and ring artifact correction of 8]. For qualitative analysis, 3-dimensional images of mice ankles were then reconstructed from cross-sectional slices by using CTNa and CTVox software provided by Skyscan.

Histologic studies

Long bones were removed and fixed in 4% paraformaldehyde for 48 h, followed by decalcification in 10% EDTA for 4 wk as well as paraffin embedding. Sections (5 µm) were cut and hematoxylin and eosin staining was performed. Photomicrographs were taken at an original magnification of ×400.

Tartrate-resistant acid phosphatase (TRAP) staining was carried out in paraffin sections with a homemade TRAP buffer [0.1 M acetate buffer, 0.3 M sodium tartrate, 10 mg/ml naphtol AS-MX phosphate, 0.1% Triton X-100, and 0.3 mg/ml Fast Red Violet LB (Sigma-Aldrich, St. Louis, MO, USA)]. After deparaffinization and acetate buffer washing processes, samples were incubated in TRAP buffer for 30 min and counterstained with Fast Green (Sigma-Aldrich).

Immunohistochemistry analysis for cathepsin K and CD68 was carried out as previously described (12). In brief, deparaffinized and hydrated sections were incubated with proteinase K solution (20 μg/ml in Tris EDTA buffer, pH 8.0) for 15 min in a water bath at 37°C for antigen retrieval. After blocking of nonspecific binding with PBS, 3% bovine serum albumin, and 0.1% Triton X-100 for 1 h, primary antibodies [rabbit polyclonal anti–cathepsin K (1:25) and rabbit polyclonal anti-CD68 (1:100); Santa Cruz Biotechnology, Santa Cruz, CA, USA] were incubated overnight at 4°C in a humidifying chamber. Secondary antibody goat anti-rabbit FITC (1:200) was incubated for 1 h in the dark. Slides were mounted with Fluoroshield with DAPI mounting medium (Sigma-Aldrich).

Statistical analysis

Statistical significance for differences between groups was determined by use of 1-way ANOVA and Bonferroni’s post hoc test. All statistics were calculated by using Prism software (GraphPad Software, La Jolla, CA, USA).

RESULTS

Blockade of Netrin-1 or Unc5b reduces K/BxN-induced signs of arthritis

To evaluate the effect of Netrin-1 blockade in inflammatory arthritis, K/BxN serum was transferred into C57Bl/6 mice that were monitored daily for inflammation and paw thickness for up to 4 wk. As previously described for this model, paw inflammation was maximal at 2 wk after injection (Fig. 1A, B) and weekly intraperitoneal injection of anti–Netrin-1 or anti-Unc5b antibodies significantly reduced paw inflammation (clinical score of 9.8 ± 0.8 and 10.4 ± 0.9, respectively vs. 16 ± 0 for control; P < 0.001; n = 10), whereas anti-DCC antibodies had no effect (13.5 ± 0.5 vs. 16 ± 0 for control; P = not significant; n = 10; Fig. 1A, B). The same results were observed for changes in paw thickness: weekly intraperitoneal injection of anti–Netrin-1 or anti-Unc5b antibodies significantly reduced paw thickness 2 wk after serum transfer (0.4 ± 0.05 mm and 0.3 ± 0.4 mm change from baseline, respectively vs. 0.7 ± 0.02 mm change from baseline for control; P < 0.001; n = 10) and anti-DCC antibodies had no effect (0.6 ± 0.04 mm change from baseline vs. 0.7 ± 0.02 mm change from baseline for control; P = not significant; n = 10; Fig. 1C).

Figure 1.

Blockade of Netrin-1 or Unc5b reduces K/BxN-induced arthritic signs. C57Bl/6 mice (8 wk old) were intraperitoneally injected with 0.2 ml K/BxN serum at d 0 and 2, and at the same time murine monoclonal antibodies against Netrin-1, Unc5b, or DCC (10 µg/mouse) were intraperitoneally injected for up to 4 wk (n = 10 per treatment). A) Representative images of inflammation at 2 and 4 wk after serum transfer. B) Clinical score (paw inflammation) among the 4 wk of experimentation (n = 10 per treatment). C) Paw thickness change in millimeters from basal during the 4 wk of experimentation (n = 10 per treatment). ***P < 0.001 vs. control mice (ANOVA).

Blockade of Netrin-1 or Unc5b reduces bone lesions and the inflammatory infiltrate produced by K/BxN serum transfer

Two weeks after serum transfer, microCT analysis showed bony erosions in ankles in untreated or anti-DCC–treated mice, whereas there were no erosions in anti–Netrin-1 and anti-Unc5b antibody–treated animals (Fig. 2). Hematoxylin and eosin staining revealed a decrease in the inflammatory infiltrate in anti–Netrin-1 and anti-Unc5b–treated animals (Fig. 3), with a nonsignificant decrease in TRAP-positive cells in anti–Netrin-1, anti-Unc5b, or anti-DCC antibody–treated animals compared with controls [5 ± 1, 6 ± 1, and 6 ± 1 cells/high-power field (hpf), respectively vs.7 ± 1 cells/hpf for control; P = not significant; n = 5; Fig. 3]. There was an increase in bony erosions detected by microCT in mice 4 wk after serum transfer (Fig. 4), and treatment with anti–Netrin-1 and anti-Unc5b antibodies diminished bone erosions. Histologic sections showed a marked decrease in inflammatory infiltrate and bone infiltration 4 wk after serum transfer (Fig. 5), and TRAP staining demonstrated a marked decrease in osteoclasts in anti–Netrin-1 and anti-Unc5b antibody–treated animals, but not in anti-DCC–treated or control mice 4 wk after serum transfer (4 ± 1, 3 ± 1, and 9 ± 2 cells/hpf, respectively vs.12 ± 1 cells/hpf for control; P < 0.001 and P = not significant; n = 5; Fig. 5).

Figure 2.

Blockade of Netrin-1 or Unc5b reduces bone lesions 2 wk after serum transfer. MicroCT analysis was performed 2 wk after serum transfer. Images show bony erosions (red arrows) in control and anti-DCC–treated mice, but not in anti–Netrin-1 or anti-Unc5b antibody–treated mice (n = 10 per treatment).

Figure 3.

Blockade of Netrin-1 or Unc5b reduces inflammation without affecting osteoclast number at 2 wk after serum transfer. Hematoxylin and eosin (H&E) and TRAP staining were performed on elbow sections 2 wk after serum transfer. H&E staining demonstrates a decrease in inflammatory infiltrate (black arrows). TRAP staining shows no decrease in osteoclast number at 2 wk after treatment with antibodies. P = not significant vs. control mice (ANOVA).

Figure 4.

Blockade of Netrin-1 or Unc5b reduces bone lesions at 4 wk after serum transfer. MicroCT analysis was performed 4 wk after serum transfer. Images show bony erosions (red arrows) in control and anti-DCC–treated mice, but not in anti–Netrin-1 or anti-Unc5b antibody–treated mice (n = 10 per treatment).

Figure 5.

Blockade of Netrin-1 or Unc5b reduces inflammation and osteoclast number at 4 wk after serum transfer. Hematoxylin and eosin (H&E) and TRAP staining were performed on elbow sections 4 wk after serum transfer. H&E staining demonstrates a decrease in inflammatory infiltrate (black arrows). TRAP staining shows a decrease in osteoclast number at 4 wk after treatment with Netrin-1 and Unc5b antibodies. ***P < 0.005 vs. control mice (ANOVA).

Immunofluorescence staining revealed diminished numbers of cathepsin K⁺ (osteoclast) and CD68⁺ cells (macrophage) in the bones and joints of anti–Netrin-1 and anti-Unc5b–treated animals, but not in anti-DCC–treated or control mice (Fig. 6).

Figure 6.

Blockade of Netrin-1 or Unc5b reduces cathepsin K⁺ osteoclast and CD68⁺ cells 4 wk after serum transfer. A) Immunofluorescence staining revealed a decrease in cathepsin K⁺ cells 4 wk after serum transfer in anti–Netrin-1 or anti-Unc5b antibody–treated mice (n = 10 per treatment). B) Immunofluorescence staining revealed a decrease in CD68⁺ cells 4 wk after serum transfer in anti–Netrin-1 or anti-Unc5b antibody–treated mice (n = 10 per treatment).

DISCUSSION

Here, we report that blockade of Netrin-1 and its receptor Unc5b by treatment with murine monoclonal antibodies prevents bone destruction and inflammation in the K/BxN serum transfer–induced arthritis mice model. This model shares many features that are common to human RA and other types of inflammatory arthritis, such as leukocyte invasion, synoviocyte proliferation, pannus formation, and cartilage and bone erosion (13, 14). As the transfer of K/BxN sera induces robust and reproducible disease in multiple mouse strains, this model is ideal for the identification of the effector pathways that are involved in the disease process.

Antibody-mediated blockade of Netrin-1 and the receptor that mediates its effects in osteoclast differentiation and leukocyte migration, Unc5b, diminishes bone injury and inflammation in this arthritis model. In contrast, isotype control antibodies or anti-DCC antibodies, which also bind to inflammatory cells, have no effect on either inflammation or inflammatory bone destruction, which provides strong evidence that the molecules targeted are responsible for the effects seen in this model. Although study of this arthritis model in Netrin-1–deficient mice would provide further strong evidence of the role of Netrin-1 and its receptor in arthritis, for mice that lack Netrin-1, the defect is lethal in the embryonic stage. Moreover, synovial fibroblasts in radiation-induced bone marrow chimeras would still express Unc5b (15), and synoviocytes also participate in joint and bone destruction observed in inflammatory arthritis.

All cells that are involved in the inflammation and bone destruction that characterizes this model of arthritis—neutrophils, monocytes, macrophages, and synovial fibroblasts—express Unc5b and Netrin-1 (4, 5, 10, 16–20). In addition to its effects on leukocyte migration (3), Netrin-1 regulates the inflammatory response of neutrophils and macrophages via suppression of cyclooxygenase 2–mediated prostaglandin E2 production during ischemic acute kidney injury (19). Netrin-1, acting via the Unc5B receptor, induces activation of peroxisome proliferator-activated receptor-γ and other signaling pathways, which causes suppression of IκB degradation and inactivation of NF-κB transcription factor (21).

Netrin-1 contributes to the regulation of leukocyte migration and inflammation in peripheral tissues (22, 23) to reduce local injury and inflammatory responses (23, 24) and to stimulate resolution mechanisms and production of resolvins (25). In contrast to these generally anti-inflammatory actions, Netrin-1 plays a role in accumulation of macrophages at inflamed sites, such as atherosclerotic plaque (6, 16) and sites of wear particle deposition in prosthetic joints. Of note, Netrin-1 contributes to osteolysis associated with wear particle shedding by prosthetic joints as a result of its role both in maintaining the inflammatory infiltrate and in osteoclast differentiation (10, 20). Netrin-1 has also been reported to exert anti-inflammatory effects in such settings as pancreatitis, peritonitis, inflammatory bowel disease, and pulmonary inflammation (3, 4, 8, 15, 24, 26–30). During ischemia–reperfusion, Netrin-1 is significantly down-regulated, Unc5B mRNA expression is increased, and DCC is not altered significantly (26); these changes in Netrin-1 expression likely represent a compensatory change. Moreover, Netrin-1 administration regulates inflammation and infiltration of monocytes and ameliorates ischemia–reperfusion-induced kidney injury (8, 19) by inducing overexpression of a macrophage M2-like phenotype and suppression of expression of M1 markers (31), although the role of endogenous Netrin-1 in these phenomena has not been studied. In the setting of ischemia–reperfusion injury to the kidney, Netrin-1 also regulates the inflammatory response of neutrophils and macrophages and suppresses ischemic acute kidney injury by inhibiting cyclooxygenase 2–mediated prostaglandin E₂ and thromboxane A₂ production (19). Netrin-1 also reduces neutrophil influx and the levels of proinflammatory mediators and promotes the resolution of inflammation and hepatic repair and regeneration during liver ischemia–reperfusion injury (32). Up-regulation of Netrin-1 levels in blood–brain barrier endothelial cells has been observed during inflammatory conditions during which it prevents junctional breach and endothelial cell activation, which suggests that Netrin-1 protects the CNS against inflammatory conditions, such as multiple sclerosis and experimental autoimmune encephalomyelitis (33). Also, Netrin-1 acts as an intestinal epithelial-derived protein that is capable of limiting neutrophil recruitment to attenuate acute colitis and may have a beneficial function in chronic models of intestinal inflammation (26, 34).

Although blockade of Netrin-1 and Unc5b does not completely prevent inflammation in this model of arthritis, the magnitude of the effect is similar to that observed with other therapeutic maneuvers or knockout of molecules that are thought to play a role in the pathogenesis of inflammatory arthritis (11). More importantly, antibody-mediated blockade of Netrin-1 and Unc5b nearly completely blocked the development of bony destruction in arthritic mice. The disparity between the effect on inflammation and bony destruction most likely results from the antibody-mediated inhibition of osteoclast differentiation and function and the inflammatory cytokine-mediated increase in osteoclast function (35).

This work is a continuation of previous work in which we demonstrated that Netrin-1 is a critical autocrine/paracrine regulator of osteoclast differentiation (9) via activation of its receptor, Unc5b, and the downstream activation of RhoA via leukemia-associated guanine nucleotide exchange factor and repulsive guidance molecule A, which leads to cytoskeletal rearrangements and osteoclast differentiation. In the same work, we observed that treatment with antibodies to either Netrin-1 or Unc5b blocks in vitro osteoclast differentiation. Here, we further extend the observation that Netrin-1 is highly expressed by macrophages and osteoclasts in a murine model of wear particle–induced osteolysis and by macrophages at sites of osteolysis in the inflamed peri-implant soft tissue from patients who undergo implant revision (10). When studied in vivo, blockade of Netrin-1 or its receptor, Unc5b, by monoclonal antibodies prevents wear particle–induced bone destruction, which is consistent with the hypothesis that Netrin-1 plays a critical role in inflammatory osteolysis (10).

Members of the axon guidance molecule family have been found to play critical roles in RA. On one hand, high levels of semaphoring (Sema) 4D have been directly associated with RA. Thus, serum levels of Sema4D correlate with known clinical and biologic markers of RA, which suggests that Sema4D is a potentially useful biomarker for RA disease activity (36). On the other hand, protein expression of Sema3A in synovial lining cells was decreased in RA tissues, with a negative correlation between disease activity score in patients with RA and Sema3A expression (37). Migration of inflammatory cells to the site of injury is a critical cellular response to initiate the removal of dead cells and induce a regeneration response (21). In different settings, both up- or down-regulation of chemorepellent factors, such as Netrin-1, during organ injury may exacerbate inflammation (3, 5). It has been also observed that ephrins (EFNB1 and EFNB2) in T cells are essential for pathogenic antibody production and for T-cell migration to inflamed paws in mice with collagen-induced arthritis; T cells from patients with RA expressed higher EFNB1 mRNA levels, which correlate with RA symptoms and biomarkers (38).

In summary, Netrin-1 may be a novel therapeutic target for the treatment and prevention of inflammatory bone destruction and other forms of osteoclast-mediated bone resorption.

Glossary

DCC

deleted in colorectal carcinoma

EFN

ephrin

hpf

high-power field

microCT

micro-computed tomography

RA

rheumatoid arthritis

Sema

semaphoring

TRAP

tartrate-resistant acid phosphatase

AUTHOR CONTRIBUTIONS

K. J. Moore and B. N. Cronstein designed the experiments and wrote and revised the manuscript; A. Mediero designed the experiments, carried out the experimental procedures, and wrote the manuscript; T. Wilder performed clinical score measurements and revised the manuscript; and B. Ramkhelawon performed immunohistochemical staining and revised the manuscript.