Suppression of collagen-induced arthritis in growth arrest and DNA damage-inducible protein 45β-deficient mice

Abstract

Objective: Growth arrest and DNA damage-inducible protein 45β (GADD45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45β deficiency exacerbates K/BxN serum-induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45β plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45β regulates autoimmunity, we evaluated collagen-induced arthritis in GADD45β-/- mice.

Methods: Wild-type (WT) and GADD45β-/- DBA/1 mice were immunized with bovine type II collagen (CII). Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. Expression of cytokines and matrix metalloproteinases in the joint and spleen was determined by quantitative polymerase chain reaction. The in vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+ Treg cells and Th17 cells were quantified using flow cytometry.

Results: GADD45β-/- mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP-3 and MMP-13 expression was also markedly reduced in GADD45β-/- mice. However, serum anti-CII antibody levels were similar in both groups. FoxP3 and interleukin-10 (IL-10) expression was increased 2-3-fold in splenocytes from arthritic GADD45β-/- mice compared with those from WT mice. Flow cytometric analysis showed greater numbers of CD4+CD25+ Treg cells in the spleen of GADD45β-/- mice than in the spleen of WT mice. In vitro studies showed that interferon-γ and IL-17 production by T cells was significantly decreased in GADD45β-/- mice.

Conclusion: Unlike passive K/BxN arthritis and EAE, GADD45β deficiency in CIA was associated with lower arthritis severity, elevated IL-10 expression, decreased IL-17 production, and increased numbers of Treg cells. The data suggest that GADD45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.

Figure 1

Clinical arthritis scores in WT and Gadd45β−/− DBA/1 mice with collagen-induced arthritis (CIA). A. Arthritis was evaluated using a semiquantitative scoring system. Values are expressed as mean ± SEM. Clinical arthritis scores were significantly lower in Gadd45β−/− mice than WT mice (n=19 in each group, p < 0.05). B, Histology scores were significantly lower in Gadd45β−/− mice compared with WT (*p<0.05). C, Representative hematoxylin and eosin (H&E) stained sections of ankles obtained on day 55 from CIA mice.

Figure 2

JNK activation in Gadd45β−/− mice. Mice were sacrificed on day 55 and the ankle lysates were evaluated by Western blot analysis. Surprisingly, P-JNK and P-p38 in arthritic joints were similar in WT and Gadd45β−/− mice with CIA.

Figure 3

Anti-collagen antibodies in the CIA model. The level of anti-type II collagen antibody in serum was determined on day 20 and day 55. Values are the mean ± SEM μg/ml (n=5 mice at each time point). Differences were not statistically significant between the two groups.

Figure 4

Gene expression in CIA. Gene expression in joint and spleen extracts (day 20 and day 55) was determined by real-time qPCR and normalized to Hprt1. Data are expressed as average relative expression units ±SEM. A, Synovial gene expression (Wildtype =WT; −/− = Gadd45β−/−). Note effect on MMP mRNA levels that favor joint destruction in Gadd45β−/− mice. B, Gene expression in the spleen. Expression of IL-10 and Foxp3 was elevated in arthritic Gadd45β−/− mice compared with arthritic WT mice. n=3 for naive mice and n=5–10 for arthritic mice (*p < 0.05, *p < 0.01).

Figure 5

Cytokine production by regional lymph node cells from WT and Gadd45β−/− mice. A. Animals were immunized with bovine type II collagen (CII), and 10 days later, lymph node cells were incubated with CII for 72 hr. Cytokine levels in culture supernatants were measured by multiplex bead analysis. Values are the mean ±SEM. n=6 for each group. *p < 0.05 versus WT mice. B, Flow cytometric analysis of splenocytes and LN cells cultured in Th17 polarizing conditions for 72h and stained for CD4, IL-17 and IFNγ. The CD4+ population was analyzed for cytokine expression. The frequency of IL-17+ cells was similar in WT and Gadd45β−/− mice, indicating that Gadd45β is not required for Th17 differentiation.