DISCUSSION

Our results show the number of CD4+CD25+T cells in BD-like mice were significantly lower than both BDN and normal mice. Moreover, frequency of Treg cells in BD-like mice was lower than BDN and normal mice. It has been reported that CD4+CD25+

Treg cells are lower in autoimmune and inflammatory diseases, such as Crohn’s Disease (Ricciardelli, et al., 2008), Multiple Sclerosis (MS)(Huan, et al., 2005), and Systemic Lupus Erythematosus (Lee, et al., 2008), than in healthy control and inactive patients. Also mouse models of autoimmune and autoinflammatory, such as collagen-induced arthritis (Morgan, et al., 2005) and experimental autoimmune encephalomyelitis (EAE)(Begum-Haque, et al.,

2008), were lower than healthy mice. But CD4+CD25+ Treg cells in rheumatoid arthritis was not different (Lee, et al., 2008) or higher than healthy controls (Han, et al., 2008).

Interestingly, Nanke et al. reported that the percentage of Treg cells among CD4+T cells from BD patients with an ocular attack were significantly decreased before ocular attack compared with those after ocular attack (Nanke, et al., 2008). Treg cells of BD-like mice with symptoms maintain lower levels than normal mice.

IL-2 and TGF-β are T cell growth factors and these can induce CD4+CD25+ Treg cells (Zheng, et al., 2007). In addition, these cytokines play important roles in the development, survival, and function of CD4+CD25+ Treg cells (Zheng, et al., 2007). Our experiments have shown these cytokines effectively expand CD4+CD25+ Treg cells.

Furthermore CD4+CD25+T and Treg cell levels by primary culture of normal splenocytes

were higher, compared to BD-like and BDN mice. Marguti et al. reported that bone marrow-derived DCs from BALB/c mice were able to promote expansion of CD4+CD25+ Treg cells in vitro. Both mature and immature DCs are capable of expanding the CD4+CD25+ Treg

cell population (Marguti, et al., 2008). Our results show the frequencies of CD4+CD25+T and Treg cells from the co-culture with CD4+T cells isolated from normal splenocytes and normal mDC was higher than CD4+T cells from splenocytes of BDN and BD-like mice. But, the frequencies of CD4+CD25+T and Treg cells from co-culture with normal CD4+T cells and each group of mDCs (normal, BDN, and BD-like) were not different among groups.

According to Fig. 2 and Fig. 3, we show the proliferation of CD4+CD25+T and Treg cells from normal healthy mice was higher than from BDN and BD-like mice. The proliferation of CD4+CD25+T and Treg cells was more dependent to CD4+T cell than DCs.

Increased regulatory T cell numbers or functions have been associated with improvement of inflammatory symptoms, such as MS, EAE, and diabetes in mice and humans (Baecher-Allan, et al., 2004; McGeachy, et al., 2005; Kukreja, et al., 2002;

Sakaguchi , 2004; Viglietta, et al., 2004). CD4+CD25+ Treg cells have a role in maintaining immunologic self-tolerance and lead to the decrease of spontaneous development of various autoimmune diseases (Takahashi, et al., 1998). Therefore we forecasted improvement of symptoms of BD-like mice by transfer with CD4+CD25+T cells on the basis of the foundation of our results. After transfer with CD4+CD25+T cells, the frequencies of

CD4+CD25+T cells. According to the amount of transferred CD4+CD25+T cells, frequencies of CD4+CD25+T and Treg cells were changed in splenocytes of BD-like mice.

We show CD4+CD25+T and Treg cells were up-regulated by transfer with CD4+CD25+T cells. After CD4+CD25+T cells transfer, we observed a change of symptoms in BD-like mice. Two weeks after transfer, the symptoms of BD-like mice showed improvement and also the disease severity score was decreased compared to transfer of CD4+CD25-T cells to BD-like mice. It has been reported that disease severity score decreased by transfer with CD4+CD25+T cell in EAE (Zhang, et al., 2004) and CIA (Morgan, et al., 2005). We will do further research at a later date to confirm direct effect or proliferation. The improvement of symptoms and increase of Treg in peripheral tissues occurred after CD4+CD25+T cells transfer. Therefore, adoptive transfer definitely affected the improvement of BD-like symptoms.

After transfer of CD4+CD25+T cells, cytokine expression profiles were compared with control groups. Our results show the expression of TGF-β and IL-10 was increased after transfer compared to not transferred BD-like mice. Zheng et al. reported that CD4+CD25+T cells cultured, with TGF-β and IL-2, changed cytokines levels of IL-10 and TGF-β. IL-10 and TGF-β were increased but IFN-γ was decreased (Zheng, et al., 2004).

According to a paper of Chai et al . Treg cells have up-regulated TGF-β and IL-10 (chai, et al., 2008). We show CD4+CD25+T cell transfer regulated the improvement of symptoms

through up-regulation of TGF-β and IL-10. TNF-α, and IFN-γ levels were decreased after transfer compared to not transferred BD-like mice. IFN-γ is a Th1 cytokine (Mosmann, et al., 1989). TNF-α is a mediator of inflammatory and immune functions. Th17 cells are highly

pro-inflammatory cells and emerging data suggest that they orchestrated tissue inflammation and organ-specific autoimmune diseases (Bettelli, et al., 2007). IL-17 is a Th17 cell mediated cytokine and IL-6 inhibits CD4+CD25+T cell regulatory functions (Wan, et al., 2007). IL-6 is highly elevated in the culture supernatants of peripheral blood mononuclear cells (PBMC) of patients with active Behcet’s disease (Yamakawa, et al., 1996). From our results, IL-17 and IL-6 proteins were decreased by transfer with CD4+CD25+T cells to BD-like mice and indicated similar levels of BDN mice. Foxp3 inhibits RORγt-mediated IL-17A mRNA transcription (Ichiyama, et al., 2008). mRNA levels of ROR-γt, transcription factor of Th17 cells, in 3 x 10⁵CD4+CD25+T cells transferred BD-like mice were lower than not transferred BD-like mice. The levels of IL-17A (IL-17) and IL-17F produced by Th-17 cells in 3 x 10⁵CD4+CD25+T cells transferred like mice were lower than not transferred BD-like mice. Also Th17 cell-mediated cytokines and transcription factors were inhibited by CD4+CD25+ Treg cells transfer.

In summary, frequency of CD4+CD25+ Treg cells in BD-like mice was lower than BDN and normal mice. In vitro, the proliferation of splenocytes from BD-like mice was doen-regulated compared to BDN. Adoptive transfer of CD4+CD25+T cells in BD-like mice improved symptoms and up-regulated CD4+CD25+T cells from splenocytes. After transfer of CD4+CD25+T cells, IL-10 and TGF-β expressions increased but IFN-γ, TNF-α, IL-6, and IL-17 expressions decreased compared with not transfer BD-like mice.