Collagen-Induced Arthritis Analysis in Rhbdf2 Knockout Mouse

Figures

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  Fig. 1.. Diagram describing collagen-induced arthritis induction in mice. The experiment was carried out for 13 weeks. Boosting was 3 weeks after the first immunization. Grip strength and rota-rod tests were carried out 3, 6 and 9 weeks after the first immunization. At the end of the experiment, all the mice were autopsied, and histological assessment for the severity of CIA was performed (n=14 for each group).

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  Fig. 2.. Confirmation of Rhbdf2 mutant allele in mutant mice. (A) Schematic representation of the target site on exon 5 in the Rhbdf2 gene. The target sequences and PAM sequences (gray letters) are indicated. (B) The mutated sequences were shown to be 8 bp deletions by sequencing analysis. (C) In the T7E1 cleavage assay, Rhbdf2 heterozygous mice showed two smaller bands that were cut by the enzyme and did not appear in homozygous mice. (D) PCR genotyping was performed with newly designed PCR primers to distinguish the wild allele (155 bp) from the mutant allele (147 bp). bp, base pair; M, DNA size maker; +/+, wild; +/−, heterozygous; −/−, homozygous. (E) Abolishment of full-length Rhbdf2 proteins production in Rhbdf2−/− mouse was confirmed by Western blot analysis. (F) TNF-α secretion assay was perfromed with macrophages derived from bone marrow cells. The TNF-α concentration in the culture medium of LPS-stimulated cells was determined by ELSIA. *p<0.5.

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  Fig. 3.. Collagen-induced arthritis in Rhbdf2 mice. Mice were immunized with type II collagen in complete Freund’s adjuvant, and paw diameter and clinical score were measured for 12 weeks (n=14 arthritic mice per group). Paw swelling peaked in RA-wild mice at week 10. In contrast, the mice in the RA-homo group did not show an increase in paw swelling from 4 weeks after the immunization through the end of the experiment. n=14 for each group. *p<0.05; **p<0.01.

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  Fig. 4.. The representative histopathologic observations in synovial membranes of Rhbdf2 mice. Representative H&E stained sagittal sections of the knee joints from vehicle-wild (A), RA-wild (B) and RA-homo (C) mice are shown. (D) The histopathological severity scores for RA were determined from the affected joints and expressed as the means ± SD (males: n=14 for each group). *p<0.05. The letters b, c and st indicate bone, cartilage, and synovial tissue, respectively.

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  Fig. 5.. Severity score determined using the results of the grip strength test with Rhbdf2−/− mice. (A) The grip strength was measured for mice in the RA-homo and RA-wild groups. The measurements were converted to the severity scores as described in Table 3. (B) The rota-rod test was measured for mice in RA-homo and RA-wild groups. The measurements were converted to the severity scores as described in Table 4. The severity scores were expressed as the means ± SD for each group. n=14 for each group. *p<0.05.

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  Fig. 6.. X-ray test in CIA mice. The skeletons of RA-homo mice and RA-wild mice at each week of age were analyzed by X-Ray system. Radiological examination was conducted 3, 6 and 9 weeks after immunization, and the same measurements were repeated with the same mice. The results are expressed as the means ± SD for 14 mice for each genotype (males: RA-homo, n=14; RA-wild, n=14). *p<0.5.