Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice
© Atkinson et al.; licensee BioMed Central Ltd. 2011
Received: 13 December 2011
Accepted: 7 June 2012
Published: 7 June 2012
Rheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.
DTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.
We demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.
Our findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by severe synovitis leading to the destruction of articular cartilage and bone erosion. This ultimately results in joint destruction and severe disability and decreased quality of life for the affected patients . Although the precise etiology and pathogenesis of RA remain unknown, several therapeutic advances have been made in recent years, most notably through blockade of tumor necrosis factor (TNF) [2, 3].
To develop targeted therapeutic interventions, such as the TNF-blocking biologics currently on the market, investigators must attempt to dissect the multi-factorial nature of RA pathogenesis through the use of animal models mimicking different aspects of the disease. One animal model cannot stand alone, and in the pre-clinical screening of potential therapeutics, it is advantageous to use a range of different models that can supplement each other in the final evaluation of a drug candidate. Indeed, the predictive value of anti-rheumatic drug efficacy in pre-clinical animal models of RA is greatly enhanced if the pre-clinical efficacy testing is conducted using several different animal models [4, 5].
Several animal models of arthritis exist, but none of them truly represents the human condition. Most likely each model mimics certain aspects and thus can be used as tools to increase the understanding of specific pathways of the disease. In the murine collagen-induced arthritis (CIA) model, disease is induced by immunization with type II collagen (CII) in an adjuvant. This approach leads to activation of CII-specific T cells and generation of anti-CII-specific autoantibodies and results in polyarthritis characterized by severe synovitis, cartilage destruction, and bone erosion [6, 7]. Furthermore, it is possible to induce transient polyarthritis in mice on the basis of passive transfer of monoclonal anti-CII antibodies - anti-type II collagen antibody-induced arthritis (CAIA) [8, 9]. The disease pathogenesis differs in the two models. Arthritis in CIA mice is a combination of a delayed-type hypersensitivity (DTH) reaction and an immune complex disease of the joint, and the disease is dependent on T and B cells [10–12]. The arthritis that develops in CAIA is a combination of immune complex disease and innate immune cell-mediated tissue destruction of the joint, in which the administered anti-CII coats the cartilage surface and leads to complement activation and Fc receptor-mediated activation of cells of the innate immune system [8, 13–15]. Disease in CAIA arises independently of T and B cells. CIA susceptibility is dependent on major histocompatibility complex (MHC), and only strains carrying the H-2q or H-2r haplotype are susceptible. The DBA1 strain has the H-2q haplotype, and male mice of this strain are the most commonly used . C57BL/6 mice have the H-2b haplotype and thus are considered refractory to CIA under normal circumstances .
In 2007, Tanaka and colleagues , using male BALB/c mice, described a new arthritis model - termed DTH arthritis - in which arthritis can be induced in a single paw by immunization with methylated bovine serum albumin (mBSA) combined with injection of a low dose of an anti-CII antibody cocktail (anti-CII) followed by challenge with mBSA locally in the paw. The disease is characterized by severe paw swelling, infiltration of inflammatory cells, hyperplasia of the synovial membrane, cartilage destruction, and bone erosion, and it is thought that the local T cell-mediated DTH response to mBSA in conjunction with the presence of antibodies against collagen II gives rise to the arthritic phenotype.
In this study, we aim to establish the DTH arthritis model in female C57BL/6 mice and characterize the involved cell populations and cytokines. The motivation for the transfer to this strain is the prospect of genetically modified mouse studies, which are most often on a C57BL/6 background. A robust arthritis model on a C57BL/6 background thus would be of considerable value as a tool for understanding the disease process and evaluating novel therapeutics. We confirm and extend the previous publication  by demonstrating that arthritic disease can be induced in C57BL/6 mice. Disease induction is dependent on CD4+ cells, and macrophages, neutrophils. Osteoclasts are also heavily involved in the pathology. A clear profile of cytokines and chemokines associated with these cell subsets is induced locally in the diseased tissue. Furthermore, we demonstrate that disease can be both prevented and treated with compounds approved for the treatment of RA in the clinic.
Materials and methods
Female C57BL/6J mice (Taconic, Bomholtvej, Denmark) were used at 6 to 8 weeks of age. Animals were housed in a facility with a 12-hour light/dark cycle and with free access to water and standard rodent chow (Altromin®; Altromin Spezialfutter GmbH & Co. KG, Lage, Germany). All animal experiments have been conducted according to Danish legislation and have been approved by the Danish Animal Inspectorate and the local ethical review board.
Induction and assessment of DTH arthritis
On day minus 7, the mice were immunized with mBSA (Sigma-Aldrich, St. Louis, MO, USA) emulsified in complete Freund's adjuvant (CFA) (Difco, Detroit, MI, USA) intradermally at the base of the tail. Four days later, they received 1,000 μg (approximately 50 mg/kg) CII mouse antibody 5-clone cocktail (Chondrex, Redmond, WA, USA) containing the clones A2-10 (IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G (IgG2b), and D2-112 (IgG2b) intravenously in 200 μL of phosphate-buffered saline (PBS). On day 0, the mice were challenged with 200 μg of mBSA subcutaneously in 20 μL of PBS in the right footpad. The left footpad was given 20 μL of PBS only and served as a control. Paw swelling was measured by using a dial thickness gage (Mitutoyo, Kanagawa, Japan) and was calculated as right paw thickness minus left paw thickness. Results are displayed as mean ± standard error of the mean (SEM). The clinical score was based on the following scoring system: 0 = normal, 1 = slight swelling of the footpad or digits, 2 = moderate swelling of the footpad or ankle or both, and 3 = severe swelling of the entire paw and ankle. Clinical score results are displayed as the median. The results are representative of at least five experiments.
Histopathological evaluation of DTH arthritis and immunohistochemistry
Hind paws were sampled and processed by standard histopathological procedures. Briefly, paws were fixed in 4% paraformaldehyde before decalcification for 7 days in formic acid bone decalcifier (Immunocal; Decal Chemical Corporation, Tallman, NY, USA). The tissue samples were dehydrated and embedded in paraffin before sections of 3 to 5 μm were prepared and deparaffinized. For histopathological evaluation, sections were stained by using a conventional hematoxylin and eosin (H&E) tissue stain. For detection of osteoclasts, a histochemical stain for the osteoclast enzyme tartrate-resistant acid phosphatase (TRAP) was performed. Deparaffinized sections were stained in a TRAP staining solution containing 1.35 mmol naphtol AS-MX phosphate (Sigma-Aldrich), 0.362 mmol N, N-dimethylformamide (Fluka; Sigma-Aldrich), 3.88 mmol Fast Red TR salt (Sigma-Aldrich), 0.5 mmol manganese chloride (Sigma-Aldrich), and 25 mmol sodium tartrate (Sigma-Aldrich) in 0.1 M Tris buffer and then were counterstained with hematoxylin. Cell nuclei appeared blue, and the osteoclast cytoplasm appeared red. For evaluation of cartilage destruction, the Safranin O staining protocol, which stains cartilage proteoglycan red, was used. Briefly, the deparaffinized sections were first counterstained with hematoxylin before staining in 0.1% Safranin O (BDH Chemicals, Poole, UK) in distilled water followed by staining in 0.1% Fast Green (Sigma-Aldrich) in distilled water to provide the green tissue contrast. For immunohistochemical detection of F4/80+, monoclonal rat anti-mouse F4/80 (Abcam, Cambridge, UK) was used, and rat IgG2b (BD Pharmingen, San Diego, CA, USA) was used as isotype control. These were diluted 1:250 and incubated with the tissue sections overnight at 4°C. Prior to incubation, antigen was retrieved by using Proteinase K (Roche, Basel, Switzerland). Detection was carried out with rabbit anti-rat antibodies (Dako, Glostrup, Denmark) followed by incubation in EnVision+ System horseradish peroxidase-labeled polymer anti-rabbit antibody (Dako). To visualize the target, sections were treated with 3-3'-diamino-benzidine-tetrahydrochloride (Sigma-Aldrich) for 5 minutes. The sections were all digitally scanned and studied by using a NanoZoomer Digital Pathology Virtual Slide Viewer (Hamamatsu Photonic, Shizuoka, Japan).
Histopathological scoring of DTH arthritis paws
Pathological changes in the paws were assessed on sections stained with H&E, TRAP, and Safranin O. The extra-articular infiltration of inflammatory cells (assessed on a scale of 0 to 3) and arthritic changes were assessed separately. Arthritic changes were assessed on metatarsal and tarsal joints, where synovitis, cartilage destruction, and bone erosion were scored separately on a scale of 0 to 3. For each of the three parameters of arthritic changes, an average between the two joint areas was calculated. In addition, new bone formation overall in the paw was scored on a scale of 0 to 3. The histology sum score was calculated by adding the five scores (extra-articular infiltration, synovitis, cartilage destruction, bone erosion, and bone formation), whereas the extra-articular infiltration score is left out in the arthritis score.
Antigen-specific proliferation assay
Popliteal lymph nodes from the antigen- and PBS-challenged side from animals sacrificed on day 4 after DTH-arthritis induction were isolated and passed through a 70-μm cell strainer to produce single-cell suspensions and placed in complete medium containing RPMI 1640 (Invitrogen Corporation, Carlsbad, CA, USA) with 1.5% C57BL/6 mouse serum (Innovative Research, Novi, MI, USA), 1% penicillin/streptomycin (Invitrogen Corporation), and 50 μM 2-mercaptoethanol (Invitrogen Corporation). Lymph nodes draining the PBS-challenged side were pooled. Two hundred thousand cells per well were transferred to a 96-well plate, and 0, 5, 10, or 20 μg/mL mBSA (Sigma-Aldrich) was added. The plate was incubated at 37°C for 72 hours before the addition of 0.5 μCi/well 3H-thymidine (Amersham, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK) and incubation overnight. Results are displayed as mean ± SEM counts per minute with background subtracted. The experiment was performed once.
Enzyme-linked immunosorbent assay
Levels of serum amyloid P component (SAP) were measured in serum from mice with DTH arthritis by using sandwich enzyme-linked immunosorbent assay (ELISA) kits (GenWay Biotech, Inc., San Diego, CA, USA) in accordance with the instructions of the manufacturer. Levels of interleukin 6 (IL-6) and total (activated + proenzyme) matrix metalloproteinase 3 (MMP3) were measured in serum from mice with DTH arthritis by using sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with the instructions of the manufacturer. Results are displayed as mean ± SEM.
Analysis of cell subsets in the inflammatory exudates
Cells were isolated from antigen-challenged hind paws from mice sacrificed on days 2 and 7 after DTH-arthritis induction by incubating paw tissue for 1 hour in 50 U/mL collagenase (Sigma-Aldrich) and 50 U/mL DNAse (Roche) followed by washing in PBS and counting. Cells were stained with Fixable Near IR Vital dye (Invitrogen Corporation), CD45-E-fluor 450, clone 30.F11 (eBioscience, San Diego, CA, USA), TCRβ-PerCP Cy5.5, clone H57-597 (eBioscience), CD4-Qdot 605, clone RM4-5 (Invitrogen Corporation), CD19-PE-Cy7, clone 1D3 (BD Biosciences, San Jose, CA, USA), CD11c-APC, clone HL3 (BD Biosciences), CD11b-AF700, clone M1/70 (eBioscience), F4/80-FITC, BM8 (eBioscience), and Ly6G-PE, clone 1A8 (BD Biosciences) and analyzed by using the LSRII flow cytometer and FACSDiva software version 6.1.3 (BD Biosciences). Cells were gated on singlets, live cells, and CD45+ cells before individual subsets were defined. Results are displayed as mean ± SEM for fractions and as median for absolute cell counts and are representative of three experiments.
Depletion of CD4+ and CD8+ cells
A dose of 1,000 μg of depleting monoclonal antibody (mAb) against CD4 (clone GK1.5; BioXcell, West Lebanon, NH, USA) or CD8 (clone 53.6.72; BioXcell) in 200 μL of PBS or 200 μL of PBS alone was administered 24 hours prior to immunization (day minus 8) and 24 hours prior to challenge (day minus 1). Depletion was confirmed by flow cytometry analysis of whole-blood samples taken 24 hours after the first depletion treatment (day -7) and on day 9 after DTH-arthritis induction. The blood was stained with CD8-FITC (clone 2.43; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), CD4-PE (clone RM4-4; eBioscience), CD45-eFlour450 (eBioscience), and TcRβ-APC-eFluor780 (eBioscience). Samples were analyzed as described above. Results are displayed as mean ± SEM, and the experiment was performed once.
Multiplex analysis of cytokines in paw homogenate
Hind paws from mice with DTH arthritis were sampled at selected times after DTH-arthritis induction and each was placed in 1.25 mL of an ice-cold homogenization buffer containing 49.995 mL of 0.9% saline, one tablet of complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche), and 5 μL of Triton X-100 (Sigma-Aldrich). The paws were homogenized by using a T25 Ultraturrax homogeniser (IKA, Staufen, Germany) followed by centrifugation at 10,000g for 15 minutes. The supernatants were decanted and centrifuged once more at 10,000g for 15 minutes. The resulting supernatants were analyzed neat for levels of TNFα, IL-1β, IL-6, IL-17, IL-12(p40), CXCL10, CXCL2, CCL2, interferon-gamma (IFNγ), and receptor activator of nuclear factor kappa-B ligand (RANKL) by using bead-based Luminex® xMAP® technology with Milliplex kits from EMD Millipore Corporation (Billerica, MA, USA) in accordance with the instructions of the manufacturer. Results are displayed as the median, and difference from the control value was tested. In the analysis of cytokine production in untreated animals, left PBS-challenged hind paws removed on day 14 after DTH-arthritis induction were used as controls, and the results are representative of two experiments. In the analysis of cytokine production in anti-TNFα mAb-treated or isotype control antibody-treated animals, the contralateral left PBS-challenged hind paw was used as control, and the analysis was performed once. Any values below the detection limit were set to the detection limit for the analyte in question, and any values above the detection limit were set to the upper detection limit for the analyte in question.
TNFα blockade in vivo
Mice were treated with rat anti-mouse TNFα mAbs (clone XT3.11; BioXcell) or rat IgG1 (HRPN; BioXcell) in doses of 250 μg per mouse (approximately 12.5 mg/kg) in 200 μL of PBS intraperitoneally every 48 hours beginning at the time of immunization (prophylactic treatment) or the time of challenge (treatment at onset) and continuing until the end of the study (day 10 after DTH-arthritis induction). For treatment with etanercept (Enbrel™; Pfizer, Groton, CT, USA), the mice received doses of 500 μg per mouse (approximately 25 mg/kg) or 1,000 μg per mouse (approximately 50 mg/kg) in 200 μL of saline intraperitoneally every 48 hours from day 1 after DTH-arthritis induction until day 11 after DTH-arthritis induction, at which time the study was terminated. Control groups received humanized anti-trinitrophenol (TNP) IgG1 (anti-TNP hzIgG1; Novo Nordisk A/S, Måløv, Denmark) in accordance with the same protocol. An additional vehicle-treated group was included and received 200 μL of saline intraperitoneally every 48 hours from the time of DTH-arthritis induction. Results are displayed as mean ± SEM. The results of TNFα blockade are representative of several experiments, whereas the therapeutic etanercept treatment study was performed once.
Mice were treated daily with dexamethasone (Intervet, Milton Keynes, UK) in doses of 1 mg/kg in 200 μL of saline beginning at the time of DTH-arthritis onset (day 0) and continuing until day 11 after DTH-arthritis induction, at which time the study was terminated. Control groups received no treatment. Results are displayed as mean ± SEM, and the experiment was performed once.
Statistical analyses were conducted by using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). Non-parametric data or non-normal parametric data were analyzed by using the Mann-Whitney U test, and parametric data were analyzed by using a two-sided unpaired t test. For statistical analysis of the histological sum scores, an unpaired Student t test with Welch's correction was used. Differences between groups were considered significant when P values were not more than 0.05, and levels of significance were assigned as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
Establishment of DTH arthritis in C57BL/6J mice
These data, taken together, demonstrated that mice with DTH arthritis developed a semi-chronic arthritic disease limited to the mBSA-challenged paw and draining lymph node while still displaying a systemic component. The arthritis pathology observed included synovitis, hyperplasia of the synovial membrane and pannus formation, increased osteoclast activity, bone erosion and remodeling, and cartilage destruction.
Inflammatory cell subset involvement
Effects of TNFα blockade and dexamethasone treatment in the DTH-arthritis model
In this study, we have established and characterized a novel arthritis model, the DTH-arthritis model, in female C57BL/6J mice. The model has been described in male BALB/c mice . We found that administering CII between the immunization and challenge steps of an mBSA-induced DTH reaction led to a robust T cell-dependent arthritis of the mBSA-challenged paw as reported by Tanaka and colleagues. We demonstrated that paw swelling persisted for at least 28 days. Pronounced involvement of neutrophils and macrophages was shown, findings supported by the observed local profile of inflammatory mediators. The increase in local IL-17 production suggested an involvement of T helper 17 (Th17) cells, a T-cell subset associated with strong inflammatory responses and autoimmunity [19, 20]. However, the very early peak production of this cytokine may also point toward the involvement of mast cells (a cell type also observed in the inflamed joint of patients with human RA ), where they have been shown to produce IL-17 . Mast cells have been found to play a role in other murine models of arthritis , and the presence and role of mast cells in DTH arthritis are currently under investigation. Histological evaluation of the inflamed paws in the present study showed synovitis, pannus formation, synovial hyperplasia, and bone and cartilage destruction, reminiscent of CIA [23, 24] and human RA [25, 26]. An increase in osteoclast number was also observed and this coincided with the onset of bone erosion. This finding was further supported by the increased production of RANKL locally in the arthritic paw, which coincided with the increase in TRAP-positive cells on day 4 after arthritis induction. RANKL is a TNF superfamily member expressed by osteoblasts, activated T cells, and synovial fibroblast-like cells. RANKL exists in both a soluble and a membrane-bound form and is an essential stimulatory factor for recruitment, differentiation, and activation of osteoclasts. RANKL binds to its receptor RANK, which is present on the surface of osteoclast precursors and mature osteoclasts , and the absence of RANKL prevents bone erosion in murine arthritis . In addition, the cytokines TNFα, IL-6, IL-1, and IL-17, which are elevated early after onset of the inflammatory response in the present study, are known to lead to an increase in RANKL expression , and these findings provide evidence that the increased osteoclast activation observed is a result of the inflammatory response in the affected joint. New bone formation was also observed, albeit in an aberrant pattern in the form of osteophytes adjacent to the affected joints. This finding suggests that there is a disturbance in the overall process of bone turnover in DTH arthritis; this disturbance may give an opportunity to study processes of new bone formation in inflammatory arthritis.
Onset and disease course of DTH arthritis were synchronized, incidence was 100% or close to it, and variation was low. These are very desirable features in an animal model, as all animals are exposed to disease and any treatment applied for an equal period of time. The fact that the arthritis affects only one paw allows the study of a more severe paw inflammation without severely compromising animal welfare; in other established models of arthritis, the humane endpoints set in place to limit animal suffering are more frequently reached, as the animals display severe inflammation in multiple paws. In addition, the phenotype of DTH arthritis makes the model very suitable for the application of in vivo imaging techniques to study inflammation and bone destruction because of the presence of an intra-animal contralateral control paw and the low intra-group variability. Although the arthritic phenotype is isolated to one paw, the model also displays a systemic component, illustrated by the increase in serum levels of SAP, IL-6, and total MMP3. MMP3 is notable in this situation, as studies have shown the serum levels of this enzyme to be a biomarker of disease severity in RA . The fact that the arthritic phenotype is induced in only one paw, however, also represents a limitation of the DTH-arthritis model by phenotypically removing the animal model from the human condition that it seeks to mimic: RA, which usually affects the joints of multiple extremities in a symmetrical pattern. Whether self-tolerance is broken in DTH arthritis was not investigated in the current study but remains an intriguing possibility. Breach of self-tolerance to joint antigens has been observed in a model of mono-arthritis, a model also induced by an inflammatory response to an antigen unrelated to the joint , the hypothesis being that joint auto-antigens are exposed to or released from the inflamed joint during the inflammatory response following antigen challenge. Thus, it remains a possibility that self-tolerance to joint-derived auto-antigens is broken in DTH arthritis.
The role played by anti-CII antibodies in disease induction is still not fully clear, but we hypothesize, on the basis of our results and available literature, that anti-CII antibodies play a role similar to what is observed in CAIA [13–15], namely that they activate complement and cells of the innate immune system through Fc receptors and that these events drive the joint pathology once the challenge with mBSA has initiated the T cell-dependent inflammatory response. The extent to which the anti-CII mAbs localize to the joint space and coat the cartilage surface immediately after administration remains unclear. It is clear, however, that there is no immediate effect of the systemic administration of anti-CII and that the inflammatory milieu of the DTH reaction is necessary to initiate the events leading to joint pathology in DTH arthritis. Anti-CII antibodies have been shown not to localize to the joints without the presence of an exogenous trigger . However, a different study demonstrated that anti-CII antibodies coated the cartilage surface of joints without any previous stimuli to increase vasopermeability . Thus, it is still unclear whether the anti-CII localizes to the joint space only after antigen challenge, due to the local increase in vasopermeability caused by inflammatory mediators and the effects of anti-antigen-antigen immune complexes on complement and FcR activation as some findings indicate , or whether it localizes there immediately following administration, at which time it is not pathogenic without further inflammatory stimuli. These questions are currently being addressed by means of in vivo imaging techniques. We did, however, measure equal levels of IL-1β in the mBSA-challenged and the PBS-challenged paws on days 1 and 3 after DTH-arthritis induction, and these measurements could point toward a direct effect of the anti-CII antibodies on the production of this cytokine.
We chose to qualify the model with rat anti-mouse TNFα and etanercept (TNFRIg), which is approved for use in the clinic to treat patients with RA. Both agents are efficacious in several other experimental arthritis models [33–35]. Taken together, the results from the two treatment studies demonstrated that DTH arthritis can be suppressed by systemic administration of TNFα-blocking biologics whether administration was prophylactic, at the time of DTH-arthritis induction, or therapeutic from day 1 after DTH-arthritis induction, when paw swelling had reached peak level. In addition, blocking TNFα leads to effects on multiple readouts, both systemic and local, including serum biomarkers also useful as biomarkers in RA: MMP3 and acute-phase proteins. Also, most of the arthritic changes observed histopathologically were prevented when TNFα was blocked in a prophylactic manner. Together, these results suggest that the DTH-arthritis model of experimental arthritis could have some degree of predictive validity with regard to the therapeutic efficacy of biologics for the treatment of RA. In addition, the efficacy of blocking TNFα suggests that DTH arthritis indeed shares immunological pathways with human RA.
In this study, we have shown that it is possible to elicit a robust arthritis in C57BL/6 mice with synchronized onset and progression and high incidence displaying severe bone and cartilage destruction, something that presents difficulties in the CAIA  and CIA [36, 37] models when using the C57BL/6 strain. In these models, the disease responses and incidence frequencies observable in C57BL/6 mice are much lower and more variable. We have verified that DTH-arthritis induction is dependent on CD4+ T cells and anti-CII antibody-mediated tissue damage of the joint. Moreover, we have shown the arthritic pathology by histology and found that it is characterized by synovitis, hyperplasia of the synovial membrane, pannus formation, enhanced osteoclast activity, bone erosion and remodeling, and cartilage destruction, features also observed in human RA [25, 26]. In addition, inflammation of the affected joints is governed by a massive neutrophil and macrophage infiltration, verified by flow cytometry, histopathology, and analysis of the involved cytokines and chemokines, which also demonstrates the involvement of T cells. We have shown that disease can be reduced by both preventive and therapeutic treatment with TNFα-blocking biologics. Importantly, therapeutic intervention was effective when initiated at peak of disease (day 1 after the challenge). The possibility of robust arthritis induction in C57BL/6 mice allows studies that use genetically modified mice on a C57BL/6 background, permitting accurate comparison of disease in wild-type and genetically modified mice, and this represents a major advantage. Importantly, because disease onset is pre-defined and simultaneous, the start of a treatment plan can be standardized both within and across treatment groups. In addition, all animals are exposed to disease and treatment for exactly the same amounts of time. When seen from a pharmacological perspective, this represents an improvement compared with the CIA model, the current gold-standard arthritis model in the mouse, which has a lower and more variable frequency of disease incidence and a non-synchronized onset . In addition, the DTH-arthritis model is run in female mice, which are much less prone to grouping stress than male mice and thus are easier to house. In summary, we believe that the DTH-arthritis model may be highly useful in the pre-clinical screening of potential protein-based drugs targeting RA, since our data demonstrate that drugs approved for RA treatment have an effect in the model. In addition, the model is highly suited for the application of in vivo imaging techniques for studying inflammation and bone remodeling and yields reliable and reproducible data, and this strengthens its potential. Still, additional work is needed to further characterize the immunological mechanisms underlying DTH arthritis and the degree to which they can be aligned to RA pathogenesis.
anti-type II collagen monoclonal antibodies
collagen antibody-induced arthritis
complete Freund's adjuvant
type II collagen
chemokine (C-X-C motif) ligand
hematoxylin and eosin
methylated bovine serum albumin
matrix metalloproteinase 3
receptor activator of nuclear factor kappa-B ligand
serum amyloid P component
standard error of the mean
tumor necrosis factor
tumor necrosis factor receptor
tartrate-resistant acid phosphatase.
The authors sincerely thank Julie Jensen, Mie Berndorff, Kristine Smedenfors, Liv Svarrer Hesselholdt, Malik Nygaard Nielsen, Jeanette Juul, and Lise-Lotte Kruse for invaluable technical assistance and the staff of Animal Unit Maaloev, Novo Nordisk A/S, for taking care of the animals and assisting with blood sampling. AN, CH, HM, PAU, and PHK are employees of Novo Nordisk A/S. SMA is an employee of the University of Copenhagen. Apart from the salary of SMA, the study was funded by Novo Nordisk A/S.
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