Increased susceptibility to collagen-induced arthritis in female mice carrying congenic Cia40/Pregq2fragments
© Liljander et al.; licensee BioMed Central Ltd. 2008
Received: 20 February 2008
Accepted: 6 August 2008
Published: 6 August 2008
Collagen-induced arthritis (CIA) in mice is a commonly used experimental model for rheumatoid arthritis (RA). We have previously identified a significant quantitative trait locus denoted Cia40 on chromosome 11 that affects CIA in older female mice. This locus colocalizes with another locus, denoted Pregq2, known to affect reproductive success. The present study was performed to evaluate the role of the Cia40 locus in congenic B10.Q mice and to identify possible polymorphic candidate genes, which may also be relevant in the context of RA.
Congenic B10.Q mice carrying an NFR/N fragment surrounding the Cia40/Pregq2 loci were created by 10 generations of backcrossing (N10). The congenic mice were investigated in the CIA model, and the incidence and severity of arthritis as well as the serum levels of anti-collagen II (CII) antibodies were recorded.
Significant effects on onset, incidence, severity, and anti-CII antibody titers were observed in female mice carrying a heterozygous congenic Cia40/Pregq2 fragment of NFR/N origin, containing one or more polymorphic genes. Congenic male mice did not show increased incidence of CIA, but males carrying a heterozygous fragment showed a significant increase in severity in comparison with wildtype B10.Q males (littermates).
The Cia40/Pregq2 locus at chromosome 11 contains one or more polymorphic genes of NFR/N origin that significantly influence both incidence and severity of CIA in heterozygous congenic mice of the B10.Q strain. The major polymorphic candidate genes for the effects on CIA are Cd79b, Abca8a, and Map2k6. The congenic fragment also contains polymorphic genes that affect reproductive behavior and reproductive success. The Sox9 gene, known to influence sex reversal, is a candidate gene for the reproductive phenotype.
Collagen-induced arthritis (CIA) is a commonly used animal model for rheumatoid arthritis (RA). Although CIA shares several features with RA, there are some obvious differences between the mouse model and the human disease [1–3]. One such dissimilarity is the reversed sex susceptibility. A female predominance is characteristic for RA , whereas the opposite situation commonly is the case in mice developing CIA. Because of the male predominance of CIA in most strains of mice, including B10.Q, most published CIA experiments have been performed on males.
We have previously performed a genetic linkage analysis on multiparous female mice from an N2 cross between NFR/N and B10.Q, with the aim of finding CIA loci that are linked to disease development in females . We identified one novel significant CIA-associated locus on chromosome 11, which is now denoted Cia40. No other CIA loci/genes have previously been found in this region, but the central part of chromosome 11 is known to contain a number of inflammation loci, such as Eae22, Eae6b, Eae23, and Eae7 [6–8]. However, none of the experimental autoimmune encephalitis (EAE) loci is located close to the Cia40 linkage peak, indicating that other polymorphic genes might be of importance.
Interestingly, in an additional quantitative trait locus (QTL) analysis with females of the same cross (N2 generation of NFR/N and B10.Q), we detected a highly significant QTL close to Cia40 on chromosome 11 linked to the trait 'pregnancy frequency' . This locus is denoted Pregq2 and controls the frequency of successful pregnancies following successful copulation (successful coitus recorded by the detection of the 'vaginal plug'). In the initial QTL analysis, heterozygous mice carrying NFR/N genes at the Pregq2 locus suffered from an increased frequency of pregnancy failures . We hypothesized that the Cia40/Pregq2 region of chromosome 11 may contain polymorphic genes that influence both CIA incidence and breeding success.
Although our original QTL analysis was performed on (aged) female mice with the hope of finding CIA loci with female predominance, there would still be a possibility that the Cia40 locus is of equal importance in both sexes. In the present paper, we present results indicating that Cia40 congenic females are more affected by CIA than males are. We also show that the Cia40/Pregq2 locus is linked to a disturbed reproductive behavior and reduced breeding performance in females.
Materials and methods
Inbred NFR/N mice were originally obtained from the National Institutes of Health (Bethesda, MD, USA) and the B10.Q mice were originally from the animal colony of Professor Jan Klein (Tübingen University, Tübingen Germany). (B10.Q × NFR/N) × B10.Q N10 mice were bred in the animal house of the Department of Pathology of Lund University, Sweden. The animals were fed standard rodent chow and water in a photoperiod of light/dark 12:12. All mice used in the present study had clean health monitoring protocols according to the recommendations of the Federation of European Laboratory Animal Sciences Association. The ethical permission for reproduction and arthritis (M236-06,) was provided by the Swedish Board of Agriculture.
The Cia40congenic mice and the fragment
Genomic DNA was isolated from the tip of the tail according to a previously described protocol . Nine fluorescence-labeled polymorphic microsatellite markers (Interactiva, Biotechnologie GmbH, Ulm, Germany) were used to cover the heterozygous fragment derived from the NFR/N as previously described  (Figure 1). The polymerase chain reaction products were analyzed on a MegaBACE™ 1000 (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK) according to the manufacturer's protocol. Data were analyzed with Genetic Profiler 1.1. (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK).
Induction and evaluation of collagen-induced arthritis
To induce CIA, 8- to 12-week-old mice were immunized subcutaneously at the base of the tail with 100 μg rat collagen type II (CII) emulsified in 0.1 M acetic acid in complete Freund's adjuvant (Difco Laboratories, now part of Becton Dickinson and Company, Franklin Lakes, NJ, USA). After 30 days, a booster injection containing 50 μg CII emulsified in 0.1 M acetum in incomplete Freund's adjuvant (Becton Dickinson and Company) was given. The clinical scoring of arthritis was commenced 25 days after the first immunization. The scoring system is based on the number of inflamed joints, ranging from 1 to 15 for each affected paw. Each affected ankle/wrist was given a score of 5, and each inflamed knuckle and toe was given 1 point. The scores of the four paws were added, yielding a maximum total score of 60 points for each mouse. The severity trait is the maximum score observed in each individual female. Mice that did not develop CIA were given a score of 0 for the traits of severity, onset, and incidence. The onset is the number of days calculated from the first immunization to the first clinical signs of arthritis excluding unaffected animals.
Enzyme-linked immunosorbent assay
The mice were sacrificed at day 90 and sera were collected. Anti-CII antibody titers in sera were analyzed by a sandwich enzyme-linked immunosorbent assay technique . In short, immunosorbent plates were coated with CII (10 μg/mL) overnight at 4°C. Bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) was used for blocking, and thereafter different dilutions of control sera (purified mouse anti-collagen type II antibodies), test sera, and positive and negative controls were added. The presence of CII-specific IgG was visualized by peroxidase-conjugated goat anti-mouse IgG.
Statistical comparison between the different experimental groups was performed by using the Mann-Whitney U test.
Increased incidence, onset, and severity of collagen-induced arthritis in heterozygous Cia40congenic female mice
Incidence of collagen-induced arthritis in Cia40 congenic male and female mice
Onset of arthritis in Cia40 congenic male and female mice
55 (32, 82)
50 (29, 78)
45 (38, 84)
53 (32, 70)
38 (29, 59)b
42 (38, 72)
59 (35, 82)
56 (32, 78)
52 (47, 84)
Heterozygous Cia40/Pregq2congenic mice show increased anti-collagen type II antibody levels
Anti-collagen type II titers in Cia40 congenic male and female mice
Anti-collagen type II titers at day 90, mg/mL
0.68 ± 0.24
1.29 ± 0.31
0.86 ± 0.25
0.71 ± 0.32
1.57 ± 0.34a
0.96 ± 0.24
0.67 ± 0.38
0.75 ± 0.21
0.70 ± 0.17
Reduced breeding performance and disturbed breeding behavior in Cia40/Pregq2congenic mice
The results of the present study indicate that one or more polymorphic genes in the congenic Cia40/Pregq2 fragment affect severity, onset, and incidence of CIA as well as the reproductive performance of B10.Q mice. Interestingly, the increased incidence and severity are pronounced traits in heterozygous mice only, and the influence of the congenic fragment is particularly obvious in the heterozygous females, which actually show a much higher incidence than the males. This is striking since females of the strain B10.Q normally show a very low incidence of arthritis (around 15%). The female predominance in incidence of CIA makes polymorphic genes in the congenic fragment particularly interesting since female predominance is characteristic for RA in humans.
Summary of possible candidate genes on chromosome 11 for Cia40/Pregq2
Reproductive or inflammatory phenotypes of mutation
95.430132 – 95.449049
Nerve growth factor receptor
95.5528271 – 95.542087
Lethality before weaning
95.818477 – 95.867254
Insulin-like growth factor 2
96.565905 – 98.590749
Mediator complex subunit 24
Pups die prior to birth
98.490658 – 98.499540
Abnormal loss of skin and hair
101.631061 – 101.646685
Ets variant gene 4 (E1A enhancer-binding protein, E1AF)
Mammary gland abnormality, male infertility
106.172655 – 106.176076
109.510719 – 109.530970
Protein kinase, cAMP-dependent regulatory, type I, alpha
109.886948 – 109.957292
ATP-binding cassette, sub-family A (ABC1), member 8a
110.260436 – 110.386836
Mitogen-activated protein kinase kinase 6
Abnormal immune system
112.643538 – 112.649074
SRY-box containing gene 9
Perinatal lethality, cartilage formation, sex reversal
We have previously speculated that the same gene(s) might affect both arthritis incidence and pregnancy failure . This assumption is supported by the fact that the incidence of autoimmune CIA is elevated in females but not in males and that the elevated severity is particularly obvious in females. A modified gene that increases the risk of developing autoimmune inflammation in females can also be expected to interfere negatively with pregnancy success. Some types of early pregnancy failures could actually be caused by increased autoimmune reactivity. Again, it is possible that the MAP kinase is involved in the success of implantation. This assumption is strengthened by a recent observation that the MAP kinase cascade indeed affects preimplanted embryos . Still, it might be more likely that different mechanisms and genes are involved in the regulation of arthritic inflammation and the regulation of pregnancy success. If true, this would make it possible to separate Cia40 gene(s) from the breeding-suppressing Pregq2 gene(s), which would be of great advantage for the future characterization of the part of the Cia40 gene(s) that influence the outcome of arthritis.
The observation that the heterozygous Cia40 congenic mice show a quicker onset, and in the case of males, also develop a more severe disease, raises questions about the molecular mechanisms controlling arthritis. A polymorphism leading to an amino acid substitution in one allele could have strong effects on the function of a di- or multimeric protein and polymorphisms in noncoding regulatory regions could result in skewed transcription and altered protein levels. The observed phenotypic effects due to heterozygous alleles might be helpful in the identification of candidate genes. The heterozygous effect has previously been reported in a study of CIA development, in which mice with heterozygous alleles in a congenic fragment on mouse chromosome 15 were much more affected by the disease than homozygous littermates were .
We have found only a limited number of genes in the vicinity of the Cia40 and Pregq2 peaks, which show polymorphism between B10 and NMRI. In addition to Mapk6, we have focused some attention on the Abca8a gene and CD79b gene. The role of the Abca8a gene in the context of reproduction and immunity is largely unknown, whereas the CD79b gene is of importance primarily in the context of B-cell development . At present, it is not possible to speculate about the possible influence of these two genes for the phenotypes observed, but the function of these genes does not make them our main candidate genes.
The interesting reversal of sex susceptibility to arthritis and the observations that congenic males show impaired development of genital organs and that females are more aggressive and less caring mothers have made us pay attention to the Sox9 gene. The Sox9 gene has been reported to cause sex reversal , which is a highly relevant phenotype in the context of the Cia40/Pregq2 congenic mice. The possible presence of a Sox9 polymorphism/mutation on chromosome 11 in our congenic mice is under investigation.
The present results show that the Cia40 locus on chromosome 11 contains one or more polymorphic genes that particularly influence incidence and severity of CIA in female mice. These effects are significant in congenic B10.Q female mice carrying heterozygous Cia40 fragments of NFR/N origin. Congenic mice carrying heterozygous fragments also show quicker onset of the disease. The major polymorphic candidate genes in the congenic fragment are Cd79b, Abca8a, and Map2k6. The NFR/N fragment present in the congenic mice also contains a locus denoted Pregq2, which causes a change in reproductive behavior and reduces pregnancy success. This effect is significant in congenic B10.Q females carrying a homozygous NFR/N fragment. The Sox9 gene, known to influence sex reversal, is a candidate gene for the reproductive phenotype.
= collagen-induced arthritis
= collagen type II
= experimental autoimmune encephalitis
= mitogen-activated protein
= quantitative trait locus
= rheumatoid arthritis.
This study was supported by the Österlund's Fund, Kock's Fund, Crafoord's Fund, Gustav V 80-Year Foundation, the Royal Physiographic Society in Lund, and the Lars Hierta Memorial Foundation.
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