**3. In vivo models for RA**

Autoimmunity is triggered when the response is persistent and leads to the development of uncontrolled cells that react aggressively against any component of the body [19]. These processes require the entry of effector cells into the target organ, whereby there are changes in blood vessels due to the inflammatory substances that are released into the blood [20]. There are different associated factors that trigger an autoimmune disease such as those shown in **Figure 1**. Recognition of the major histocompatibility complex (MHC) II by the T-lymphocyte receptor (TCR) will produce a clustering with other surface receptors that activate a signaling cascade, which in turn alters the T-lymphocyte transcriptional program. These events produce tissue destruction and loss of function of affected organs during the course of the autoimmune disease. There is evidence that CD4+ T cells are active in local inflammation and cell infiltration

RA is a chronic inflammatory disease characterized by synovitis with a symmetrical distribution that causes severe joint destruction [22]. RA is a common autoimmune systemic inflammatory disease that affects approximately 1% of the worldwide population and its incidence is 0.5–1% [3]. Cohort studies have shown that people with arthritis are 54% more likely to die than a healthy person, and there are data that indicate that this frequency is directly associated with the severity of the disease [23]. The process was reported in 1909 by Nichols and Richardson, and among the symptoms of RA are a prodromal period preceded by overt asthenia, general malaise, diffuse myalgia, fever, and anorexia, weight loss, pain, stiffness, and swelling in

RA initiates as an inflammation of synovial fluid, in which rheumatoid factor (RF), IgM and IgG, and anti-CCP antibodies are present in serum and joints. Complement is activated within the synovial fluid, with C3a and C5a being the most important components found [9]. The disease is perpetuated by the production of cytokines and the action of extracellular matrix

**Figure 1.** Factors associated with the development of autoimmunity. There are several important factors that are considered the trigger of an autoimmune disease, since the true etiology is not currently known. These include hormonal,

that result in inflammation [21].

affected joints [24].

metalloproteinases (MMP) [25].

immunological, environmental, or genetic factors (susceptibility).

**2.3. Rheumatoid arthritis, its causes and prognosis**

260 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

RA is a cosmopolitan disease that affects 60 million people, making it a big problem for the health sector [33]. The etiology and pathophysiology of RA remain poorly understood, but it is generally accepted that genetic, immunological, hormonal, and environmental factors could lead to chronic inflammatory infiltrates, the development of destructive arthropathy, and the manifestation of clinical symptoms [34].

Since 1806, RA has been associated with a certain degree of inheritance; its relationship with genetic and environmental components has not been neglected in greater proportion [35]. There are different experimental models that generate basic knowledge of the pathophysiology of RA for the development of diagnostic kits through the discovery of biological markers. These are grouped in genetic, immunological, hormonal and environmental characteristics; that is, these factors are always associated with most autoimmune diseases.

#### **3.1. Genetic models**

The first RA-associated gene appeared in 1978 with the elucidation of HLA association with the disease. Nowadays, it is a clear complex of predisposition to suffer RA [36].

The first set of studies aimed at demonstrating the genetic susceptibility to RA was performed in twins, in which the environment and the genome were similar. This led to the hypothesis that 60% of the changes are attributable to genetic components. One of the best strategies has been to link polymorphisms with RA; despite these efforts, only HLA and PTPN22 factors have been linked with accurate results, hence the importance of genetically modified animal models [3, 37].

The collagen-induced arthritis (CIA) model was developed through the induction of type II collagen although the usefulness of other types of collagen has been demonstrated [38]. This model has become the most effective genetic biomarker. Among the targets that have been evaluated are immunoglobulin-producing B cells which attack type II collagen. Additionally, TCR transgenic mice in which the development of arthritis has been generated by immunoglobulins have also been evaluated [39, 40].

The way in which the transgenic mice model is developed is by injecting a construct that weighs an expression promoter, the gene or molecule of interest, and, commonly, a reporter gene, which will facilitate the exact location of the construction generated by immunofluorescence techniques. Once this construction is obtained, it is introduced into a pronucleus of a fertilized egg. The mouse that will be obtained will be able to express the gene of interest and transmit it to its descendants [41].

The therapeutic targets evaluated in CIA models or transgenic mice with adjuvants (from DBA/1 strain) are genes that code for T lymphocytes, within which the generation of a polymorphism has been associated with the development of RA. Finally, CMH, whose association with RA was reported in 1978, has been assigned to different loci such as HLA-DQ and HLA-DR, as well as to the H-2q region of HCM, demonstrating involvement in animal models with transgenic mice. Despite efforts to detect altered genes in this complex, to date, the exact pathway in which the pathogenesis is produced and the precise effect of this complex on the disease are unknown [42]. **Table 1** summarizes the animal models used for the genetic model of RA.

#### **3.2. Immunological models**

Among the immunological factors, commonly considered biomarkers are cytokines, chemokines, immune response cells, and adhesion molecules.

Cytokines often provide valuable information within the role of soluble factors that develop in RA. IL-1 β, TNF-α, IL-6, IL-15, and IL-18 are the most documented cytokines that play a regulatory role in RA, depending on the immune response or inflammatory processes.

CCP, cyclic citrullinated peptide; RF, rheumatoid factor; TNF, tumor necrosis factor; IL, interleukin; α, alpha; SKG,

**Model Inbred strain and mechanism**

IL-6R The genetic background is with strain C57BL / 6 and the model is generated due to a mutation generated in the amino acid tyrosine in the

IL-6 receptor.

IL-1Ra The strain used is C57BL / 6J and said mice are deficient in the gene that enconding for the IL-1Ra receptor antagonist.

K/BxN For this model, the strains of NOD and C57BL/6 are used, in which autoimmunity against the GPI is generated after its

crossing.

SKG A mutation occurs in

BALB / c

generated

**Table 1.** Genetic models for RA.

TNF-αtransgenic the SH2 region of the ZAP 70 protein, which is the result of an error in the selection of T cells, the mouse model is the

The animal strain used is C57BL / 6 in which an overexpression of the human TNF-α gene is

Sakaguchi; BL, black; ZAP 70, zeta-chain associated protein 70; SH<sup>2</sup>

receptor; Ra, receptor antagonist; NOD, nonobese diabetic; TCR, T cell receptor.

**Genotypic characteristics**

B6.129S-*IL1rntm1Dih/J*

B6.TCR. Cα−/−.H-2bxg7

gp130F759/F759 A high frequency of

response.

proteins.

arthritis

ZAP-70W163C -Do not develop

Tg197 Have impaired fertility

model.

and therefore it is very difficult to obtain offspring from this

small crosses.

B cells is generated, which could result in an increase in the humoral

Fail to respond to IL-1 and exhibit an altered immune response to many different target




lymphadenopathy or lupus-like diseases. -Arthritis is significantly reduced when the mice are rendered TNF-α, IL-1 or IL-6 deficient.

**Limitation Advantage Reference**

Mutation in GP130 enhances IL-6 expression.

Animal Models of Rheumatoid Arthritis http://dx.doi.org/10.5772/intechopen.72554

> Exhibit altered inflammatory responses.




, Src homology 2; GP130, glycoprotein 130; R,

[4, 43, 44]

263

[4, 43, 45]

[4, 43, 46, 47]

[4, 43, 48]

[4, 43, 49, 50]


CCP, cyclic citrullinated peptide; RF, rheumatoid factor; TNF, tumor necrosis factor; IL, interleukin; α, alpha; SKG, Sakaguchi; BL, black; ZAP 70, zeta-chain associated protein 70; SH<sup>2</sup> , Src homology 2; GP130, glycoprotein 130; R, receptor; Ra, receptor antagonist; NOD, nonobese diabetic; TCR, T cell receptor.

**Table 1.** Genetic models for RA.

Since 1806, RA has been associated with a certain degree of inheritance; its relationship with genetic and environmental components has not been neglected in greater proportion [35]. There are different experimental models that generate basic knowledge of the pathophysiology of RA for the development of diagnostic kits through the discovery of biological markers. These are grouped in genetic, immunological, hormonal and environmental characteristics;

The first RA-associated gene appeared in 1978 with the elucidation of HLA association with

The first set of studies aimed at demonstrating the genetic susceptibility to RA was performed in twins, in which the environment and the genome were similar. This led to the hypothesis that 60% of the changes are attributable to genetic components. One of the best strategies has been to link polymorphisms with RA; despite these efforts, only HLA and PTPN22 factors have been linked with accurate results, hence the importance of genetically modified animal

The collagen-induced arthritis (CIA) model was developed through the induction of type II collagen although the usefulness of other types of collagen has been demonstrated [38]. This model has become the most effective genetic biomarker. Among the targets that have been evaluated are immunoglobulin-producing B cells which attack type II collagen. Additionally, TCR transgenic mice in which the development of arthritis has been generated by immuno-

The way in which the transgenic mice model is developed is by injecting a construct that weighs an expression promoter, the gene or molecule of interest, and, commonly, a reporter gene, which will facilitate the exact location of the construction generated by immunofluorescence techniques. Once this construction is obtained, it is introduced into a pronucleus of a fertilized egg. The mouse that will be obtained will be able to express the gene of interest and

The therapeutic targets evaluated in CIA models or transgenic mice with adjuvants (from DBA/1 strain) are genes that code for T lymphocytes, within which the generation of a polymorphism has been associated with the development of RA. Finally, CMH, whose association with RA was reported in 1978, has been assigned to different loci such as HLA-DQ and HLA-DR, as well as to the H-2q region of HCM, demonstrating involvement in animal models with transgenic mice. Despite efforts to detect altered genes in this complex, to date, the exact pathway in which the pathogenesis is produced and the precise effect of this complex on the disease are unknown [42]. **Table 1** summarizes the animal models used for the genetic model of RA.

Among the immunological factors, commonly considered biomarkers are cytokines, chemo-

that is, these factors are always associated with most autoimmune diseases.

262 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

the disease. Nowadays, it is a clear complex of predisposition to suffer RA [36].

**3.1. Genetic models**

models [3, 37].

globulins have also been evaluated [39, 40].

transmit it to its descendants [41].

**3.2. Immunological models**

kines, immune response cells, and adhesion molecules.

Cytokines often provide valuable information within the role of soluble factors that develop in RA. IL-1 β, TNF-α, IL-6, IL-15, and IL-18 are the most documented cytokines that play a regulatory role in RA, depending on the immune response or inflammatory processes.

The mice strains that are used for evaluation of some cytokines or regions of the TCR are knockout mice or transgenic mice, which have a deficiency of the molecule to be studied. Typically, these strains develop the CIA model more easily than other strains such as C57BL/6 [51].

**Animal model Characteristics Species Limitations**

The induction is performed by injecting type II collagen. The generation of specific antibodies for type II collagen is well characterized. Mice used are B-cell deficient and resistant to developing CIA

mice and rats; it is the gold standard and causes polyarthritis. Animals are inoculated with collagen II and coadjuvant; a re-restriction of MHC II is generated. The most commonly used strain of mice is DBA 1/J

Is generated for passive transfer of anti-isozyme antibodies in mice, which are injected into the knee with

Mutations generated in the model are only generated in mice 4–5 weeks of age. Arthritis is mild and can be induced by serum transfer and induces changes in the MHC

RA, rheumatoid arthritis; CIA, collagen-induced arthritis; MHC, major histocompatibility complex.

poly-L-lysine-lysozyme

bacteria (*Streptococcus* sp., *Lactobacillus* sp.) are responsible for inducing the model. Biphasic arthritis is generated, and it persists for several months. The model is generated in Lewis rats

Rat The most important

evaluated

Mouse It does not generate B- or

infiltrates

acute

Rat, mouse The model does not comply

Mouse These models require specific

environmental stimulants, which are very sensitive to the changes; therefore they are usually models with considerably high costs. Another limitation is that antibodies to anti-glucose-6-phosphate isomerase are generated that mostly cause pathogenic effects

with endogenous factors that are generated, and it is very difficult to resemble certain models such as CIA. Reactivity is generated by the presence of type II collagen and increases the production of factors that trigger inflammation

Rat, mouse Prior experience is required

T-cell responses, which are important factors to evaluate in RA despite having macrophages and polymorphonuclear cell

to perform the injection of the agents to evaluate intra-articularly. Arthritis characterized by polyarthritic does not meet all the characteristics, and results generated are variable. The arthritis that is triggered is

limitation is that the arthritis that is generated is monoarticular, and because the inflammation is generated by injecting intra-articularly, the systemic effects cannot be effectively

Animal Models of Rheumatoid Arthritis http://dx.doi.org/10.5772/intechopen.72554 265

Streptococcal cell wall arthritis Peptidoglycans in the cell walls of

Collagen-induced arthritis This model is inducible in susceptible

Passive transfer of CIA

Immune complex-induced

Spontaneous arthritis in knockout or transgenic mice

**Table 2.** In vivo models for the study of RA.

arthritis

antibodies

In addition, different therapeutic targets such as chemokines, signaling molecules, and cellular trafficking can be evaluated in arthritis models like collagen-induced arthritis (CIA), streptococcal cell wall (SCW), adjuvant-induced arthritis (AIA), and models in chemokinedeficient knockout mice. On the other hand, immune response cells (NK, monocytes, etc.) are evaluated in K/BxN mice with CIA, which must be pre-stimulated with LPS to inhibit Fc receptors. Adhesion molecules, whose function resides in leukocyte trafficking, have been referred to be evaluated in SCW models with BALB/c mice and among the markers found are P-selectin, ICAM-1, and VCAM-1 [52].

For this model, K/BxN mice, which will jointly express the cell receptor T and the CMH II allele, are usually used. These mice typically develop no problem with a severe form of arthritis, when they are inoculated with serum antibodies due to high levels of GPI. Two mice are required to generate the model: one from the C57BL/6 strain that has the KRN and NOD/Lt transgene carrying the CMH II allele [53].

#### **3.3. Environmental models**

One marker that has been associated with RA is a low socioeconomic level, from which unhealthy diets are derived. It has recently been found that the consumption of certain vitamins and minerals from healthy food provides protection and reduces the effects of RA in patients [54, 55].

In animal models, the first link in which the association of the environment in arthritis was observed was when the HLA-B27 complex was evaluated in transgenic rats with spondyloarthritis. The rats did not develop the disease, so it was deduced that normal intestinal flora in B27 plays an important role [56]. Among the main environmental links with RA are smoking, the presence of infections, antibodies to rheumatoid factor, and anti-cyclic citrullinated peptides [57].

Cigarette smoke is strongly associated with RA, and during a study in 2011 in which mice of the DBA/1 J strain were in contact with condensed smoke, an increase in the induction of RA mediated in the CIA model. This strongly suggests that smoking may be an etiologic cause of this pathology [58].

### **4. Implication of hormones in animal models**

Sex hormones are linked to RA because they function normally as inhibitors or suppressors of the immune response. Over the last few years, it has been speculated that the appearance


RA, rheumatoid arthritis; CIA, collagen-induced arthritis; MHC, major histocompatibility complex.

**Table 2.** In vivo models for the study of RA.

The mice strains that are used for evaluation of some cytokines or regions of the TCR are knockout mice or transgenic mice, which have a deficiency of the molecule to be studied. Typically, these strains develop the CIA model more easily than other strains such as

264 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

In addition, different therapeutic targets such as chemokines, signaling molecules, and cellular trafficking can be evaluated in arthritis models like collagen-induced arthritis (CIA), streptococcal cell wall (SCW), adjuvant-induced arthritis (AIA), and models in chemokinedeficient knockout mice. On the other hand, immune response cells (NK, monocytes, etc.) are evaluated in K/BxN mice with CIA, which must be pre-stimulated with LPS to inhibit Fc receptors. Adhesion molecules, whose function resides in leukocyte trafficking, have been referred to be evaluated in SCW models with BALB/c mice and among the markers found are

For this model, K/BxN mice, which will jointly express the cell receptor T and the CMH II allele, are usually used. These mice typically develop no problem with a severe form of arthritis, when they are inoculated with serum antibodies due to high levels of GPI. Two mice are required to generate the model: one from the C57BL/6 strain that has the KRN and NOD/Lt

One marker that has been associated with RA is a low socioeconomic level, from which unhealthy diets are derived. It has recently been found that the consumption of certain vitamins and minerals from healthy food provides protection and reduces the effects of RA in patients

In animal models, the first link in which the association of the environment in arthritis was observed was when the HLA-B27 complex was evaluated in transgenic rats with spondyloarthritis. The rats did not develop the disease, so it was deduced that normal intestinal flora in B27 plays an important role [56]. Among the main environmental links with RA are smoking, the presence of infections, antibodies to rheumatoid factor, and anti-cyclic citrullinated peptides [57].

Cigarette smoke is strongly associated with RA, and during a study in 2011 in which mice of the DBA/1 J strain were in contact with condensed smoke, an increase in the induction of RA mediated in the CIA model. This strongly suggests that smoking may be an etiologic cause of

Sex hormones are linked to RA because they function normally as inhibitors or suppressors of the immune response. Over the last few years, it has been speculated that the appearance

C57BL/6 [51].

P-selectin, ICAM-1, and VCAM-1 [52].

transgene carrying the CMH II allele [53].

**4. Implication of hormones in animal models**

**3.3. Environmental models**

[54, 55].

this pathology [58].

of polymorphisms in genes encoding testosterone, progesterone, and androgens is what trigger an imbalance in the hormone complex with immune system and thus be associated with RA [59].

**5. Therapeutic strategies**

Over the years, different RA therapies have been developed; one of the most representatives is TNF blockers, which, although effective, generate some notable side effects. The importance of animal models serves to generate the knowledge and evaluation of new therapies [52]. All these models are intended to induce inflammation and subsequent destruction of one or more peripheral joints in different animal species, with rodent species, such as mice and rats, being the most frequently used. The most common administration routes for evaluation of compounds in murine RA models are two, as shown in **Figure 2**. The most common administration route used for molecules including viral vectors such as lentivirus, retrovirus, and adeno-associated virus is intra-articular, which consists of administering a vector that contains a gene with regulatory effects on some mechanism of RA in the joint of the animal model being evaluated. Recently, murine models have been evaluated in which the viral vector is administered systemically (intravenously), and these have been able to reduce the inflammation generated by the disease as well as other clinical symptoms, without decreasing

Animal Models of Rheumatoid Arthritis http://dx.doi.org/10.5772/intechopen.72554 267

Some of the therapeutical goals of drugs, irrespective of their route of administration, include regularizing cytokine levels, since there is an overproduction of these, adjusting the expression levels of transcriptional products that cause inflammation and bone degradation, and decreasing chemokines and adhesion molecules, all of which are determinants of RA pathogenesis [52]. The models most used for the evaluation of exogenous substances that help reduce the dis-

**Figure 2.** Animal models in RA. The models that are currently used for the generation of RA in mice and rats are varied and depend on the purpose. This can be generated by adjuvants with susceptible animals or spontaneously with genetically modified animals. There are several routes of administration which depend on the vehicle to be administered;

for gene therapy the most used are the intra-articular and systemic routes.

ease are varied [65]; besides they depend on the animal model, as thus illustrated.

the effectiveness associated with the route of administration [52].

A higher prevalence of arthritis is present in females, but in murine models, it has been shown that for CIA and antibody-induced arthritis models, male mice and rats (CIA only) exhibit a higher prevalence. Castration of male mice produces a high prevalence of arthritis in the SCW model [60].

Another hormone that can also be a marker is cortisol secreted during periods of stress. In mice it has been shown to reduce the sensitivity to be induced in the CIA model; therefore, it usually confers some protection [61]. **Tables 2** and **3** summarize the in vivo models for the study of RA and the information of the genetic, immunological, and environmental animal models used in RA, respectively.


RA, rheumatoid arthritis; CIA, collagen induced arthritis.

**Table 3.** Comparison of animal models for the study of RA.
