Immune Netw.  2011 Oct;11(5):299-306. 10.4110/in.2011.11.5.299.

Deficiency of Foxp3+ Regulatory T Cells Exacerbates Autoimmune Arthritis by Altering the Synovial Proportions of CD4+ T Cells and Dendritic Cells

• Jang E
• Cho ML
• Oh HJ
• Youn J

• KMID: 2168002
• DOI: http://doi.org/10.4110/in.2011.11.5.299

Abstract

BACKGROUND
CD4+Fop3+ regulatory T cells (Tregs) are needed to maintain peripheral tolerance, but their role in the development of autoimmune arthritis is still debated. The present study was undertaken to investigate the mechanism by which Tregs influence autoimmune arthritis, using a mouse model entitled K/BxN.
METHODS
We generated Treg-deficient K/BxNsf mice by congenically crossing K/BxN mice with Foxp3 mutant scurfy mice. The arthritic symptoms of the mice were clinically and histopathologically examined. The proportions and activation of CD4+ T cells and/or dendritic cells were assessed in the spleens, draining lymph nodes and synovial tissue of these mice.
RESULTS
K/BxNsf mice exhibited earlier onset and more aggressive progression of arthritis than their K/BxN littermates. In particular, bone destruction associated with the influx of numerous RANKL+ cells into synovia was very prominent. They also contained more memory phenotype CD4+ T cells, more Th1 and Th2 cells, and fewer Th17 cells than their control counterparts. Plasmacytoid dendritic cells expressing high levels of CD86 and CD40 were elevated in the K/BxNsf synovia.
CONCLUSION
We conclude that Tregs oppose the progression of arthritis by inhibiting the development of RANKL+ cells, homeostatically proliferating CD4+ T cells, Th1, Th2 and mature plasmacytoid dendritic cells, and by inhibiting their influx into joints.

Keyword

Regulatory T cells; Autoimmune arthritis; Synovium; Autoreactive T cells; Plasmacytoid dendritic cells

MeSH Terms

Animals
Arthritis
Dendritic Cells
Joints
Lymph Nodes
Memory
Mice
Peripheral Tolerance
Phenotype
Spleen
Synovial Fluid
Synovial Membrane
T-Lymphocytes
T-Lymphocytes, Regulatory
Th17 Cells
Th2 Cells

Figure

• 

  Figure 1 Accereration and exercerbation of arthritis in K/BxNsf mice. (A) Joint swelling in K/BxN and K/BxNsf mice was inspected blind. The ankle thickness of hind paws (left) and arthritic indices of all paws (right) are shown (n=6 mice/group). Data are means±SEM. *p<0.05, **p<0.01, and ***p<0.001 by Student's t-test. (B) Hind paws from BxN, K/BxN, and K/BxNsf mice were examined by histopathologic methods. Representative photographs of each group are shown. Original magnification, ×40. (C) Hind paw sections were stained with anti-RANK or RANKL Ab, followed by immunohistochemical processing. Original magnification, ×400.

• 

  Figure 2 Treg deficiency enhances autoreactive activation of CD4+ T cells. (A) CD4+ T cell numbers in spleens and dLNs of K/BxN and K/BxNsf mice. *p<0.05, **p<0.01, and ***p<0.001 by Student's t-test. (B) Splenocytes purified from 4 week-old mice were cultured with irradiated splenocytes from BxN mice and GPI peptide at the indicated concentrations for 72 hr. 3H-thymidine was added during the last 9 h, followed by 3H-thymidine incorporation assays. Values represent mean±SD (C) Percentage of CD4+CD44loCD62Lhi and CD4+CD44hiCD62Llo cells in the spleens and dLNs of 5-10-week-old BxN, K/BxN, and K/BXNsf mice. Histograms represent the percentage of CD25+ cells among CD4+CD44hiCD62Llo cells. (D) Absolute numbers of naïve, memory and activated CD4+ T cells in spleens and dLNs of K/BxN and K/BxNsf mice (n=7).

• 

  Figure 3 The profiles of Th1, Th2, and Th17 cells in the spleen and dLNs of K/BxNsf mice. (A and B) Splenocytes and dLN cells from 5 - 6 week-old BxN, K/BxN, and K/BxNsf mice were stimulated with PMA/ionomycin for 5 h and stained for CD4, IL-4, IFN-γ, and IL-17. FACS profiles gated on CD4+ cells are shown. A representative result of three independent experiments with the percentage of cells in each quadrant is shown. (C) Th1, Th2, and Th17 cell numbers in spleen and dLNs of K/BxN and K/BxNsf mice.

• 

  Figure 4 The profiles of synovial T cells from K/BxNsf mice. (A) Synovial cells extracted from ankle joints of 5 week-old BxN, K/BxN, and K/BxNsf mice were stained with mAbs to CD4 and Foxp3 and assayed by FACS. Representative FACS profiles gated on live lymphocytes are shown. (B) Synovial cells were stimulated with PMA/ionomycin for 5 h, followed by intracellular cytokine staining for CD4, IL-4, and IFN-γ. FACS profiles gated on CD4+ cells are shown. (C) Synovial cells were stimulated with PMA/ionomycin for 5 h and stained for IL-17, CD4, CD3, NK1.1, and γδ TCR. One representative result of three independent experiments with the percentages of cells in each gate is shown.

• 

  Figure 5 Enrichment of mature pDCs in the synovium of K/BxNsf mice. (A) Synovial cells extracted from ankle joints of 5 week-old BxN, K/BxN, and K/BxNsf mice were stained with mAbs to CD11b, CD11c, and B220. CD11c+CD11b+ cells (small circle) and CD11c+CD11b- cells (large circle) were gated for mDC and pDC, respectively. Histograms show the expression of B220 among each gate. Most CD11c+CD11b- pDC express B220 but most CD11c+CD11b+ mDC are B220 negative. The bar graphs on the right panel show the frequency of mDC or pDC on live lymphocyte gate. (B) The maturation status of CD11c+CD11b-B220+ pDCs was assessed by mean fluorescence intensity (MFI) of the co-stimulatory molecules CD40, CD80 and CD86. Bars and numbers indicate the mean of three animals. p-values in plots were calculated by Student's t-test.

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