Animal models of rheumatoid arthritis - Wiley Online Library

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Autoimmune disease: Rheumatoid arthritis Animal models of rheumatoid arthritis Darren L. Asquith, Ashley M. Miller, Iain B. McInnes and Foo Y. Liew Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, UK

Animal models have been used extensively in studies of rheumatoid arthritis pathogenesis. Despite the inherent limitations of all animal models, several rodent models have significantly progressed our understanding of the fundamental mechanisms underpinning rheumatoid arthritis and contributed to several current major advances in treatment. These models include the induced arthritis models such as collagen-induced arthritis, collagenantibody-induced arthritis, zymosaninduced arthritis, and the methylated BSA model, and the genetically manipulated or spontaneous arthritis models such as the TNF-a-transgenic mouse, K/BxN mouse, and the Skg mouse. Here, we describe these animal models and discuss their advantages and limitations.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects primarily the joints manifesting as pain, stiffness, and synovitis (inflammation of the synovial membrane) leading in turn to articular destruction. The etiology of RA is multifactorial, with genetic and environmental components that together lead to early immune perturbation in both the innate and adaptive compartments and subsequent chronic inflammation. In particular, studies of sera from pre-disease onset human cohorts reveal the presence of autoantibodies against citrinullated selfproteins and rheumatoid factors that predate disease onset by up to 10 years. The trigger to articular disease onset is unknown and factors that define chronicity of the responses are similarly poorly

understood. By definition this phase of disease, although potentially crucial for future preventative therapeutics, is impossible to study at present in humans and thus provides powerful incentive for development of relevant animal models of disease, in which mechanisms and pathways can be modeled and explored. Moreover, in established RA synovitis there is ample evidence of innate and adaptive immune pathways that co-exist and interact to perpetuate tissue inflammation and destruction. Thus, germinal centers are found in a significant proportion of RA synovia, auto-antibody titers are characteristic of poor prognosis disease and recently therapeutics that target both innate cytokines, e.g. TNF-a, IL-6 and adaptive immune pathways, e.g. abatacept (co-stimulatory blockade) and rituximab (B-cell depletion) have proven to be efficacious. Whereas the studies in human tissues are helpful in defining such pathways, they are significantly limited in that true kinetic analyses are impossible, relevant tissues, e.g. lymph node, bone marrow, spleen, are not readily obtainable and even in synovial tissue, the target lesion is accessed only rarely and not normally on an ongoing basis. Therefore, animal models are essential to facilitate understanding of the mechanisms of disease in RA and development of new therapies.

Induced arthritis models Collagen-induced arthritis Collagen-induced arthritis (CIA) shares many similarities with human RA. Two characteristics of the CIA model – breach of tolerance and generation of autoantibodies toward self and collagen – make CIA the gold standard in vivo model

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DOI 10.1002/eji.200939578

for RA studies. CIA was first described in rats [1] and subsequently shown to be inducible in susceptible strains of mice [2], following inoculation with type II heterologous collagen in complete Freund’s adjuvant. Susceptibility has been linked to strains that have MHC Class II I-Aq haplotypes; however, it is clear that many mouse strains have variable degrees of susceptibility to CIA. Similarly restricted class II genotypes can be found in RA patients, for whom pathogenesis is associated with HLA-DR1 and HLA-DR4 (reviewed in [3]). CIA can also be initiated in non-human primates, making it a useful model in which to better assess efficacy of novel therapeutic targets and aid their transition through the primary stages of pre-clinical development. DBA/1 mice are most widely used in the CIA model. Clinical signs of disease typically develop 21–25 days after the initial inoculation and presents as a polyarthritis, which is most prominent in the limbs and characterized by synovial inflammatory infiltration, cartilage and bone erosion and synovial hyperplasia similar to human RA. The development of CIA is associated with both B- and T-lymphocyte responses with the production of anti-collagen type II antibodies and collagen-specific T cells. The auto-antibody response in CIA is predominated by the IgG2 subclass with high levels of both IgG2a and IgG2b present at the peak of arthritis. Disease severity is expected to peak at approximately day 35, after which DBA/1 mice enter remission, marked by increased concentrations of serum IL-10 and a subsequent decrease in pro-inflammatory Th1 cytokines [4]. In a further development of the model, inoculation with homologous type II collagen has been reported to cause chronic relapsing arthritis more akin to human RA, and has been suggested to be www.eji-journal.eu

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more useful for studying remission-inducing therapies [5, 6]. The ability to study the effect of genetic modification or targeted gene deletion has been problematic in the DBA/1 model as most transgenic mice are on the C57BL/6 background, which is generally considered resistant to CIA. However, with the development of a refined protocol for the induction of CIA in C57BL/6 mice [7, 8], the possibility to study arthritis in genetically modified mice is becoming more feasible. The C57BL/6 strain develops arthritis 4–7 days later compared with DBA/1 mice, eventually reaching severity at a level comparable to arthritis in DBA/1 mice. The arthritis was associated with sustained levels of serum anti-collagen antibody titers, higher levels of T-cell proliferation and IFN-g secretion in the late stage of disease. However, there are differences in the onset and progression of disease between the DBA/1 and C57BL/6, which may cause discrepancies when comparing studies between these two strains. The severity in the B6 model is sustained, but incidence of disease is lower than DBA/1 mice and more variable across different substrains of B6 mice. Therefore, while both strains of mice may be useful to study the pre-clinical development or prophylactic treatment of arthritis, the C57BL/6 model requires further characterization in different B6 substrains. As with most models of disease there is a requirement for uniformity in which disease severity is assessed and presented to provide clarity and direct comparability of results. It is generally recognized that variable incidence, severity and intergroup inconsistency is a feature of the CIA model, reflecting in part the model’s exquisite sensitivity to environment, maintenance conditions and general stress in the animals. Thus careful, independent, internal controls are mandatory for valid interpretation of data.

Collagen-antibody-induced arthritis RA is associated with auto-antibody production against self-type II collagen, citrullinated proteins (ACPA) and IgG

(rheumatoid factor). Similarly, in CIA, in which anti-type II collagen IgG antibodies are detectable, transfer of serum from an immunized mouse into a non-immunized recipient can induce arthritis [9]. This demonstrates a role for humoral immunity in the development of arthritis in which type II collagen is thought to be the predominant epitope (reviewed in [10]). Furthermore, anti-collagen antibody cocktails have been shown to induce the development of arthritis [11]. Similar protocols are now commonly used for the induction of collagen-antibodyinduced arthritis (CAIA). Identification of auto-antibody collagen epitopes allows the development of more arthritogenic antibody cocktails that may better represent the humoral auto-immunity in RA. This could also lead to the identification of conserved regions in type II collagen between species which may be central to driving disease pathology [12, 13]. Although the clinical development of arthritis is similar to that in CIA and RA, CAIA is characterized by macrophage and polymorphonuclear inflammatory cell infiltrate [14], but is not associated with a T- and B-cell response; although the administration of type II collagen reactive T cells has been shown to enhance disease severity [15, 16]. Therefore, CAIA can provide insight into the separate roles of innate and adaptive immune response in the development of arthritis. Furthermore, as disease develops within 48 h of antibody administration with 100% penetrance and is inducible regardless of the MHC class II haplotype, CAIA is wellsuited for studying the development of arthritis in genetically modified strains of mice.

Zymosan-induced arthritis Zymosan is a polysaccharide from the cell wall of Saccharomyces cerevisiae with repeating glucose units connected by b-1,3-glycosidic linkages. It binds to TLR2 in macrophages leading to the induction of proinflammatory cytokines, arachidonate mobilization, protein phosphorylation and also activates complement via the alternative pathway. Injection of zymosan intra-articularly into the knee joints of mice results in a


proliferative inflammatory arthritis with mononuclear cell infiltration, synovial hypertrophy and pannus formation with the peak of disease at about day 3 and inflammation subsiding by day 7 [17]. Recent data, however, demonstrate that the model is in fact biphasic, with both early (oday 7) and late phases (4day 25) [18]. The main limitation of this model is the monoarthritic nature of the disease and the technical skill required for an intra-articular injection in mice. Furthermore, an intra-articular injection model precludes analysis of the systemic component of the disease.

Antigen-induced arthritis Various strains of mice develop inflammatory arthritis when primed with an antigen (e.g. methylated BSA in complete Freund’s adjuvant) and subsequently challenged by intra-articular injection of the same antigen [19, 20]. Such models are useful in that mice of several strains can be investigated to establish a hierarchical role for given factors in adaptive immunemediated articular damage. Subsequent pathology comprises immune complexmediated inflammation followed by articular T-cell-mediated responses. The model does not however recapitulate the endogenous breach of tolerance that is typical of RA pathogenesis and as such the model has limitations in applicability to RA. A recent development of this model comprises of prior adoptive transfer of transgenic ovalbumin-specific T cells followed by ovalbumin priming and later intra-articular challenge [21]. The recipient mice develop arthritis, which is followed by the emergence of auto-reactivity to collagen, and the presence over time of rheumatoid factors. This model has the advantage of facilitating imaging of the pathogenic T cells that in turn promote breach of self-tolerance to articular antigens [22].

Other induced models of arthritis A single subcutaneous injection of small amounts of pristane (a natural saturated terpenoid alkane) leads to the development of an acute severe inflammation

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followed by a chronic relapsing phase in rats and mice [23]. The model is largely T-cell dependent and the main pathological features include edema accompanied by an acute phase response, infiltration into the joint of mononuclear and polymorphonuclear cells, pannus formation, and the erosion of cartilage and bone. The proteoglycan-induced arthritis model involves immunization of genetically susceptible mouse strains, such as BALB/c, with human cartilage-derived proteoglycans [24]. These mice develop severe polyarthritis and spondylitis.

Genetically manipulated spontaneous arthritis models TNF-a transgenic mouse model of inflammatory arthritis A transgenic mouse over-expressing human TNF-a was developed by Kollias and co-workers in 1991 [25]. The mouse develops chronic inflammatory erosive polyarthritis and treatment with a monoclonal antibody against human TNF-a completely prevents the disease. In contrast to CIA and adjuvant-induced arthritis, which are acute and selflimiting, the chronic progressive nature of the arthritis in this model bears close resemblance to the human disease. Since then multiple lines of TNF transgenic mice have highlighted the importance of TNF-a in the cytokine hierarchy of RA, and given the success of antiTNF-a therapy in humans the TNF transgenic mice provide a useful tool for evaluating the efficacy of novel therapies in RA, particularly in which novel targets are considered to operate downstream of TNF. Moreover, the model has proven particularly useful in defining the distinct contribution of effector cytokines that regulate inflammation and those that regulate cartilage and bone destruction, e.g. RANKL.

K/BN model A variety of insights have emerged with the advent of sophisticated transgenic murine models. The K/BN spontaneous mouse model of arthritis was

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first described by Kouskoff et al. in 1996 [26]. These mice were generated by crossing the TCR transgenic KRN line with mice expressing the MHC class II molecule Ag7. K/BN mice develop severe and destructive inflammatory arthritis. They have high titers of autoantibodies recognizing glucose-6-phosphate isomerase, and serum from these mice induces arthritis in a wide range of normal recipient mouse strains (serum transfer model). The mechanism of action involves complement activation and mast cell degranulation, and is mediated not only by TNF but also by IL-1. The discovery of this model led to several studies investigating titers of pathogenic anti-glucose-6-phosphate isomerase antibodies in RA patients; however, to date the data remain controversial [27, 28]. Although this somewhat limits the utility of the model, it remains useful for the study of initial events involved in the induction of arthritis and in particular is invaluable in elucidating the contribution of discrete innate immune pathways in articular tissue damage.

the pathology of the human synovium with cartilage invasion and destruction mediated by the synovial fibroblast to be studied in an animal model.

SKG model

Several other spontaneous models have also been reported. For example, mice with deficiency of IL-1 receptor antagonist similarly develop spontaneous arthritis that is dependent upon environmental stimuli and is mediated through a strong Th17-polarized response [33]. A homozygous mutation in the gp130 receptor results in enhanced STAT3 activation and the emergence of an inflammatory destructive arthritis [34]. The foregoing provides opportunities to explore the interface of T-cell mediated and innate immunity in inducing arthritis. A recent model further implicates DNA recognition in this process [35]. DNase II / IFN-IR / mice and mice with an induced deletion of the DNase II gene develop an inflammatory polyarthritis associated with high levels of anti-CCP antibody and rheumatoid factor. The model is in part TNF-a-dependent, suggesting that incomplete DNA disposal by macrophages may lead to the dysregulated cytokine release.

A point mutation in ZAP-70 induces inflammatory arthritis in part reflecting altered thymic T-cell selection [29]. This SKG model is dependent upon environmental stimuli and is absent in germ-free mice but can be induced by injection of zymosan in a dectin-1dependent manner.

Human/SCID chimeric mice Several investigators have exploited the ability of SCID mice to tolerate xenografts by implanting them with human synovial tissue. In the first model, human synovial tissue from RA patients and normal cartilage were implanted under the renal capsule of SCID mice [30]. After 35 days, focal erosions occurred at sites of synovial attachment to the cartilage. After 105 days, activated synovial fibroblast cells invaded the cartilage leading to cartilage destruction. Thus, this model allows


Human DR4-CD4 mice Genetic susceptibility to RA is associated with a group of HLA class II alleles, which all share a similar stretch of positively charged amino acids at the HLA-DRB1 locus. A mouse model that included four separate transgenes: HLA-DR0401 and human CD4 molecules, a RA-related human auto-antigenic protein (HCgp-39), and a TCR (TCR-ab) transgene specific for an important HCgp-39 epitope, allowed the analysis of strong Th1 responses in the context of HLA-DR0401 [31, 32]. This mouse has been particularly useful for the study of the mechanisms involved in the breach of self-tolerance that occurs in RA.

Other spontaneous transgenic models of arthritis

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Table 1. Characteristics of selected mouse models of arthritis

Model

Species

Characteristics

References

CIA

Mouse, rat, rabbit, non-human primate

Polyarthritis, only inducible in susceptible strains of rodents, antibody and T-cell response. Low incidence and variability of disease severity in C57BL/6 mice. Inoculation with homologous collagen induces relapsing/ remitting arthritis but otherwise is self-limiting.

[1, 2, 7, 39, 40]

CAIA

Mouse

Self-limiting polyarthritis in 100% animals, onset within 48 h, macrophage and polymorphonuclear cell involvement, no T- and B-cell involvement. Can be induced in most strains of mice.

[9, 11, 41]

Zymosan-induced arthritis

Mouse, rat

Monoarthritis, develops 3 days after inoculation and subsides by day 7, but has shown to relapse after day 25. Needs a high degree of technical ability to perform intra-articular injection in mice. TLR 2 dependent and can be induced in multiple strains of mice.

[17, 18]

Antigen-induced arthritis

Mouse, rat

Inoculation with antigen by intra-articular injection requires a high degree of technical ability and precludes analysis of the systemic component of disease.

[19, 20]

Spontaneous transgenic models of arthritis.

Mouse

Spontaneous chronic and progressive polyarthritis, onset of disease at 3–4 wk of age. This includes the KBxN, SKG and DNase II / IFN-IR / and human TNF-a transgenic mice. These mutations have so far only been identified in mice.

[25, 26, 29, 35]

Concluding remarks In this Viewpoint we have attempted to describe some of the advantages and limitations associated with the most commonly used murine models of arthritis and compare them with human pathology (summarized in Table 1). Although none of these models entirely recapitulate clinical pathology, there is no doubt that they have relevance to processes thought to be involved in disease development and progression. As knowledge of the etiology of human RA expands, it is important to adapt and modify animal models to better represent human disease. It should be emphasized that RA is a systemic disease in which there is multiple associated comorbidities. Alterations in serum lipids, and changes in vascular [36] and neurological functions [37] are all recognized as potential contributors to disease pathology with yet unknown mechanisms [38]. Nevertheless, despite these limitations it is clear that animal models have provided valuable information relating to the pathogenesis of RA. Acknowledgements: The authors acknowledge funding support from the Medical

Research Council, Arthritis Research Council (UK) and the Wellcome Trust. A. M. Miller is supported by a BHF Intermediate Basic Science Research Fellowship (FS/08/035/25309). Conflict of interest: The authors declare no financial or commercial conflict of interest. 1 Trentham, D. E. et al., J. Exp. Med. 1977. 146: 857–868. 2 Courtenay, J. S. et al., Nature 1980. 283: 666–668. 3 Brand, D. D. et al., Springer Semin. Immunopathol. 2003. 25: 3–18. 4 Mauri, C. et al., Eur. J. Immunol. 1996. 26: 1511–1518. 5 Holmdahl, R. et al., Arthritis Rheum. 1986. 29: 106–113. 6 Malfait, A. M. et al., Arthritis Rheum. 2001. 44: 1215–1224. 7 Inglis, J. J. et al., Nat. Protoc. 2008. 3: 612–618. 8 Campbell, I. K. et al., Eur. J. Immunol. 2000. 30: 1568–1575. 9 Stuart, J. M. and Dixon, F. J., J. Exp. Med. 1983. 158: 378–392. 10 Rowley, M. J. et al., Mod. Rheumatol. 2008. 18: 429–441. 11 Holmdahl, R. et al., Arthritis Rheum. 1986. 29: 400–410. 12 Hutamekalin, P. et al., J. Immunol. Methods 2009. 343: 49–55. 13 Nandakumar, K. S. and Holmdahl, R., J. Immunol. Methods 2005. 304: 126–136.


14 Santos, L. L. et al., Clin. Exp. Immunol. 1997. 107: 248–253. 15 Nandakumar, K. S. et al., Arthritis Res. Ther. 2004. 6: R544–R550. 16 Yoshino, S. et al., Eur. J. Immunol. 1990. 20: 2805–2808. 17 Keystone, E. C. et al., Arthritis Rheum. 1977. 20: 1396–1401. 18 Frasnelli, M. E. et al., Arthritis Res. Ther. 2005. 7: R370–R379. 19 Brackertz, D. et al., Arthritis Rheum. 1977. 20: 841–850. 20 Brackertz, D. et al., J. Immunol. 1977. 118: 1639–1644. 21 Maffia, P. et al., J. Immunol. 2004. 173: 151–156. 22 Nickdel, M. B. et al., Ann. Rheum. Dis. 2009. 68: 1059–1066. 23 Wooley, P. H. et al., Arthritis Rheum. 1989. 32: 1022–1030. 24 Glant, T. T. et al., Crit. Rev. Immunol. 2003. 23: 199–250. 25 Keffer, J. et al., EMBO J. 1991. 10: 4025–4031. 26 Kouskoff, V. et al., Cell 1996. 87: 811–822. 27 van Gaalen, F. A. et al., Arthritis Rheum. 2004. 50: 395–399. 28 Matsumoto, I. et al., Arthritis Rheum. 2003. 48: 944–954. 29 Sakaguchi, N. et al., Nature 2003. 426: 454–460. 30 Geiler, T. et al., Arthritis Rheum. 1994. 37: 1664–1671.

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31 Fugger, L. et al., Proc. Natl. Acad. Sci. USA 1994. 91: 6151–6155. 32 Eming, R. et al., Arthritis Res. 2002. 4: S133–S140. 33 Koenders, M. I. et al., Arthritis Rheum. 2008. 58: 3461–3470. 34 Sawa, S. et al., J. Exp. Med. 2006. 203: 1459–1470. 35 Kawane, K. et al., Nature 2006. 443: 998–1002. 36 Choy, E. and Sattar, N., Ann. Rheum. Dis. 2009. 68: 460–469. 37 Zolcinski, M. et al., Rheumatol. Int. 2008. 28: 281–283. 38 Kelly, S. et al., Eur. J. Neurosci. 2007. 26: 935–942.

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39 Bakker, N. P. et al., Rheumatol. Int. 1990. 10: 21–29. 40 Terato, K. et al., Arthritis Rheum. 1989. 32: 748–758. 41 Stuart, J. M. et al., J. Exp. Med. 1982. 155: 1–16.

Correspondence: Prof. Foo Y. Liew, Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, Scotland, UK Fax: 144-141-330-4297 e-mail: [email protected]

Received: 5/5/2009 Revised: 22/5/2009 Accepted: 22/5/2009 Key words: Animal Arthritis Collagen Model Rheumatoid Abbreviations: CAIA: collagen-antibodyinduced arthritis CIA: collagen-induced arthritis RA: rheumatoid arthritis See accompanying article http://dx.doi.org/10.1002/eji.200939514

A clinical perspective of rheumatoid arthritis Hans Ulrich Scherer1,2,3 and Gerd R. Burmester1,2,3 1

Department of Rheumatology and Clinical Immunology, Charite´ – University Medicine Berlin, Berlin, Germany

2 3

Free University, Berlin, Germany Humboldt University of Berlin, Berlin, Germany

In recent years, factors potentially involved in pathogenesis of rheumatoid arthritis have mostly been identified by studying well-defined patient cohorts. Characterization of antibodies from sera of affected patients, family-based heritability studies, genome-wide association scans, the analysis of environmental factors and data from clinical trials have contributed largely to a thorough reassembly of our perception of rheumatoid arthritis. Modified animal models are now crucial to obtain experimental evidence for suggested pathogenetic pathways based on these observations and are currently being developed and explored. Some of the novel pathogenetic aspects, however, already influence decision making in the clinic.

Clinically relevant issues of disease pathogenesis Based on genetic background and autoantibody status, rheumatoid arthritis (RA) can be sub-classified in at least

two distinct entities, which in clinical practice are characterized by the presence or absence of anti-citrullinated protein antibodies (ACPA) [1]. This recent recognition is of relevance, as studies indicate that sub-classes of RA with distinct pathogenetic mechanisms might necessitate different treatment modalities. So far, ACPA exhibit the highest known specificity for RA (up to 98%). Citrulline-specific reactivity has been identified against a number of proteins (e.g. fibrinogen, collagen, vimentin), and recent data suggest that antibodies targeting citrullinated vimentin may most closely reflect the course of disease [2, 3]. ACPA are present (sometimes for years) before the onset of clinical symptoms [4, 5]; ACPA titres are initially low in preclinical disease, and rise steadily towards disease onset [6]; active citrullination occurs in rheumatoid synovium (i.e. the site of inflammation) [7], and the presence of ACPA predicts progression from undifferentiated arthritis (UA) to overt RA. Moreover, ACPA identify patients at high risk for erosive and extra-articular disease


DOI 10.1002/eji.200939514

manifestations, and are thus associated with poor disease outcome [8]. ACPA develop preferentially (yet not exclusively) in the presence of sharedepitope alleles [9]. This set of HLA-DRB1 alleles represents the strongest genetic risk factor for ACPA-positive RA. Several additional genetic risk factors (e.g. TRAF1-C5, TNFAIP3, STAT4) and at least one environmental factor (smoking) have been identified that associate with ACPApositive disease only, indicating that genetic background determines the type of immune response that eventually leads to clinical features of RA [10–13]. However, the exact mechanism by which any of these genetic polymorphisms contributes to disease pathogenesis has not yet been elucidated. In contrast to ACPA-positive RA, only few genetic risk factors have been identified for ACPA-negative disease. However, as the degree of heritability is comparable between ACPA-positive and -negative disease, still unidentified genetic factors have been postulated that predispose individuals to ACPAnegative RA [14]. www.eji-journal.eu