The frequencies of IL-15Rα expressing cells in BD mice compared with BDN

The frequencies of IL-15Rα were analyzed in PBMC, splenocytes, and lymph node (LN) cells of BD and BDN mice (BD normal, HSV was inoculated but no symptomatic mice) by FACS analysis. The frequencies of IL-15Rα-expressing cells in PBMC from BD mice (n=11) were lower than BDN mice (n=10)(10.19 ± 3.22 vs. 13.58 ± 7.23%, p=0.03) (Fig.

1A). The expression pattern of frequencies was similar in splenocytes (n=3)(4.63 ± 0.12 vs.

5.7 ± 0.61%, p=0.04) (Fig. 1B). Whereas in lymph node cells, the frequencies were not significantly different between BD (n=8) and BDN mice (n=10)(11.55 ± 5.84 vs. 11.09 ± 5.66%, p=0.9) (Fig. 1C). The frequencies of IL-15Rα-expressing cells in PBMC and splenocytes from BD mice were significantly lower than BDN mice.

Fig. 1. The expression of IL-15R

and BDN mice. The data show the frequencies of IL cytometry in cells isolated from

BDN mice.

15Rα in PBMC, splenocytes and lymph node cells

The data show the frequencies of IL-15Rα were analyzed by Flow PBMC (A), spleens (B), and lymph nodes (C) from

lymph node cells from BD were analyzed by Flow (C) from BD and

B. Poly I:C up-regulates expression of IL-15Rα in normal mice

To determine whether the expression of IL-15Rα can be regulated by Poly I:C, Poly I:C or PBS (control) was injected into normal mice. At 2 days after the second injection of Poly I:C or PBS, the cells were isolated from blood of mice. Then, the frequencies of IL-15Rα were measured by flow cytometry (Fig. 2A). The frequencies of IL-IL-15Rα expression on PBMC from normal mice following Poly I:C 0.05 µg/g (n=2)(6.9 ±0.85), Poly I:C 0.2 µg/g (n=4)(8.3 ±2.45), Poly I:C 1 µg/g (n=2)(7.7 ±2.83), and PBS (n=3)(4.3 ±0.79) (Fig. 2B).

The frequencies of IL-15Rα on PBMC from 0.2 µg/g of Poly I:C treated mice were significantly increased than that found in PBMC from PBS administered control mice (p=0.04). 0.2 µg/g of Poly I:C most efficiently up-regulated the expression of IL-15Rα.

Therefore, we chose to apply 0.2 µg/g of Poly I:C for further experiments. In addition, the frequencies of IL-15Rα expression on splenocytes and lymph node cells from 0.2 µg/g of Poly I:C treated mice (n=5) were also up-regulated than that found from PBS administered control mice (n=3)(3.6 ±1.92 vs. 5.9 ±1.90%, p=0.1; 5.2 ±1.01 vs. 6.7 ±1.45%, p=0.2) but data was not significant.

Fig. 2. The expression of IL-15Rα were up-regulated by Poly I:C injection in a dose dependent manner in normal mice. 5 weeks aged male normal mice were randomly placed into the following treatment groups: PBS, Poly I:C 0.05, 0.2, 1 µg/g body weight for each group. (A) The mice were injected i.p. with PBS or Poly I:C for 2 times at 3-days interval.

Two days after the last injection, the mice were sacrificed and PBMC were isolated, and the expression of IL-15Rα was analyzed by Flow cytometry. (B) The data show the frequency of IL-15Rα in cells isolated from PBMC, spleens, and lymph nodes after Poly I:C or PBS injection.

C. Poly I:C ameliorates HSV-induced BD symptoms

To determine whether Poly I:C can improve the BD symptoms, we conducted intra-peritoneal injection of Poly I:C or PBS into BD mice for four times with three days interval (Fig. 3A). Before and 2 weeks after the last injection, the changes of BD symptoms in BD mice were photographed. The cutaneous symptoms were improved in Poly I:C treated BD mice compared to PBS injected BD mice (Fig. 3B). In Poly I:C injected BD mice, BD symptoms were ameliorated in 15 of 24 BD mice (63%) or not changed in 5 of 24 BD mice (21%) whereas deteriorated in 2 of 24 BD mice (8%) or died in 2 of 24 BD mice (8%).

However, in PBS injected BD mice, the BD symptoms were ameliorated in 3 of 20 BD mice (15%) or not changed in 7 of 20 BD mice (35%) whereas deteriorated in 6 of 20 BD mice (30%) or died in 4 of 20 BD mice (20%) (Table 1). Poly I:C administered BD mice with cutaneous symptoms showed diminished lesion size by 20% to 100% of the total area. The change of BD symptoms in BD mice was observed and scored according to the severity score of BD patients, which is outlined in the BD Current Activity Form. The scoring was followed by the changes of superficial symptoms because it is impossible to communicate with mice. In Poly I:C injected group, the severity score was decreased from 2.57 ±0.66 to 1.98 ±1.19 at 2 weeks after the first injection (n=22). Whereas, in the PBS injected group, the severity score was increased from 2.94 ±0.87 to 3.07 ±0.98 at 2 weeks after the first injection (n=14). At 2 weeks after the first injection, the severity score significantly lower in Poly I:C injected BD mice (n=22) than PBS injected BD mice (n=14)(1.98 ± 1.19 vs. 3.07

±0.98, p=0.006) (Fig. 3C).

Fig. 3. The change of symptoms in BD mice after injection of Poly I:C. (A) The used experimental schedule throughout this study. Poly I:C or PBS was injected i.p. into BD mice for 4 times at 3-days interval, and the symptoms were photographed and the severity score was analyzed. (B) The symptoms of BD mice were compared before and after injected with Poly I:C or PBS. (C) The severity score of Poly I:C or PBS injected BD mice. The disease score was estimated according to the Patients Index Score, Behcet’s disease current activity form 2006, ICBD.

Table 1. The change of symptoms in BD mice after injection of Poly I:C.

D. Poly I:C up-regulates the frequencies of IL-15Rα but not IL-7Rα (CD127) in BD mice.

To confirm the frequencies of IL-15Rα in Poly I:C injected BD mice, the cells were isolated from PBMC, spleen, and lymph nodes at 2 and 17 days after injection. As has been reported Poly I:C elicited a brief phase of T cell proliferation peaking at day 2 post-injection and was followed for 7 to 32 days (Zhang, et al., 1998; West, et al., 2011). At 2 days after the last injection, the frequencies of IL-15Rα from Poly I:C injected group were significantly increased than PBS injected group in PBMC (16.8±2.6 vs. 9.2±3.5%, p<0.001), spleen (12.9±2.8 vs. 4.6±0.1%, p=0.001), and lymph nodes (21.8±2.3 vs. 9.6±6.0%, p<0.001). At 17 days after the last injection, the frequencies of IL-15Rα from Poly I:C injected group was significantly increased than PBS injected group in PBMC (27.4 ±9.8 vs. 9.2 ±3.5%, p<0.0001) but recovered in spleen (5.2 ±2.1 vs. 4.6 ±0.1%, p=0.7) and lymph node (11.0

±11.0 vs. 9.6 ±6.0%, p=0.7) (Fig. 4A). In PBMC of Poly I:C injected group, the up-regulated expression of IL-15Rα were sustained until 17 days (at 17 days vs. at 2 days, 27.4 ±9.8 vs.

16.8 ±2.6%, p=0.045). In spleen and lymph nodes of Poly I:C injected group, up-regulated IL-15Rα was decreased to control level at 17 days.

The frequencies of IL-7Rα expression in spleen cells were isolated from BD mice (n=5) were significantly lower than BDN mice (n=5)(28.5±3.8 vs. 20.0±1.4%, p=0.002) but not different in PBMC (42.6±12.5 vs. 37.8±7.8%, p=0.5) and lymph nodes cells (33.4±11.2 vs. 39.0±5.7%, p=0.3) between BD mice and BDN mice. Actually, IL-15 and IL-7 cytokines were involved with the generation and maintenance of memory CD8+ T cells (Kimberly, et al., 2003). These cytokines interact with IL-15Rα and IL-7Rα respectively, as has been

reported, the down-regulation of IL-15Rα and IL-7Rα expression is a contributing factor for the poor survival of memory CD8+ T cells in the airways (Shen, et al., 2008). To determine whether the injection of Poly I:C into BD mice can induce not only IL-15Rα but also IL-7Rα, we analyzed the frequencies of IL-7Rα in the same method. At 2 days after the last injection, the frequencies of IL-7Rα from Poly I:C injected group were not different compared with PBS injected group in PBMC (Poly I:C vs. PBS, 41.9 ±18.1 vs. 45.7 ±20.0%, p=0.7), spleen (26.1 ±6.4 vs. 28.0 ±12.9%, p=0.8), and lymph nodes (45.6 ±12.6 vs. 41.8 ±14.8%, p=0.6).

At 17 days after the last injection, the frequencies of IL-7Rα from Poly I:C injected group were also not different compared with PBS injected group in PBMC (Poly I:C vs. PBS, 51.0

±20.4 vs. 45.7 ±20.0%, p=0.6), spleen (23.4 ±14.0 vs. 28.0 ±12.9%, p=0.5), and lymph nodes (47.5 ±12.3 vs. 41.8 ±14.8%, p=0.4) (Fig. 4B). Poly I:C elevates the level of IL-15Rα but not IL-7Rα in BD mice. We next investigated the serum level of IL-7 and IL-15 by ELISA to determine whether the serum level of IL-7 and IL-15 was changed in BD mice after Poly I:C injection. Serum levels of IL-7 were 46.08±38.67 pg/ml in BDN (n=12) and 32.84±10.41 pg/ml in BD (n=13) by ELISA. The level of IL-7 was not different between BD mice and BDN mice (p=0.2). Serum IL-7 levels were elevated after injection of Poly I:C in BD mice at 2 days after the last injection (Poly I:C (n=6) vs. PBS (n=8))(48.8 ±18.22 vs.

34.33 ±13.12 pg/ml, p=0.1) and at 17 days after the last injection (Poly I:C (n=6) vs. PBS (n=8))(54.75 ±15.89 vs. 34.33 ±13.12 pg/ml, p=0.02) (Fig. 4C). Although the frequency of IL-7Rα were not affected, the serum level of IL-7 significantly up-regulated by Poly I:C injection in BD mice. The serum level of 15 was not analyzed because reliable mouse

IL-Fig. 4. The expression of IL-15Rα, IL-7Rα in PBMC, splenocytes and lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C in BD mice. The cells were isolated from PBMC, spleen and lymph nodes at 2 and 17 days after the last injection of Poly I:C or PBS. The expression of IL-15Rα and IL-7Rα were measured by Flow cytometry.

The data show the frequencies of IL-15Rα (A) and IL-7Rα (B) in PBMC, splenocytes, and lymph node cells from PBS or Poly I:C injected BD mice. (C) The serum levels of IL-7 were measured by ELISA in BDN, Poly I:C or PBS injected BD mice.

E. Poly I:C induces CD8+CD44+ memory T cells in BD mice

There has been reported, IL-15Rα on bone marrow derived cells trans-present IL-15 and it mediates the basal proliferation of memory CD8+ T cells (Schluns, et al., 2004). We previously observed the frequencies of CD8+CD44+ and CD8+CD62L- memory T cells frequencies of memory cell types were measured by flow cytometry. At 2 days after the last injection, the frequency of CD4+CD44+ memory T cells in Poly I:C injected BD mice significantly increased than PBS injected BD mice in spleen (Poly I:C vs. PBS, 24.1 ±5.9 vs.

15.9 ±8.4%, p=0.03) and lymph node (44.7 ±6.8 vs. 33.7 ±10.9%, p=0.02) but was not significant at 17 days after the last injection in PBMC (Poly I:C vs. PBS, 34.2 ±16.3 vs. 22.8

±16.6%, p=0.1), spleen (26.6 ±14.1 vs. 15.9 ±8.4%, p=0.07), and lymph nodes (41.5 ±12.6 vs. 33.7 ±10.9%, p=0.5) (Fig. 5A). Whereas, the frequencies of CD8+CD44+ memory T cells were not different between Poly I:C and PBS injected group at 2 days after the last injection in PBMC (Poly I:C vs. PBS, 9.4 ±2.4 vs. 7.2 ±4.0%), spleen (3.8 ±1.2 vs. 3.4

±2.0%) and lymph node (7.8 ±2.6 vs. 10.5 ±5.5%). At 17 days after the last injection, the frequencies of CD8+CD44+ memory T cells from Poly I:C injected group were significantly increased than PBS injected group in PBMC (Poly I:C vs. PBS, 18.6 ±9.6 vs. 7.2 ±4.0%, p=0.003) and spleen (7.2 ±1.5 vs. 3.4 ±2.0%, p=0.0008) though there was no difference in

lymph node (14.0 ±9.2 vs. 10.5 ±5.5%, p=0.3) (Fig. 5B). The injection of Poly I:C significantly elevated CD8+CD44+ memory T cells in BD mice. We conducted to determine whether injection of Poly I:C in BD mice can induce not only CD8+CD44+ memory T cells but also CD4+CD62L- or CD8+CD62L- memory T cells. CD4+CD62L- and CD8+CD62L- memory T cells were measured in the same method. The frequencies of CD4+CD62L- memory T cells were not different between Poly I:C and PBS injected group in PBMC, spleen and lymph nodes (Fig. 5C). Whereas, the frequencies of CD8+CD62L- memory T cells significantly increased in Poly I:C injected group than PBS injected group at 2 days (Poly I:C vs. PBS, 8.8 ±2.4 vs. 2.4 ±1.6%, p=0.0004) and 17 days (10.9 ±3.9 vs. 2.4 ±1.6%, p=0.0006) after the last injection in PBMC but there was no difference in spleen and lymph nodes (Fig. 5D).

Fig. 5. The frequencies of memory T cells in PBMC, splenocytes and lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C in BD mice. The cells were isolated from PBMC, spleen and lymph nodes at 2 and 17 days after the last injection of Poly I:C. The cells were stained for memory T cell markers and analyzed by Flow cytometry.

The data show the frequencies of CD4+CD44+ (A), CD8+CD44+ (B), CD4+CD62L- (C), CD8+CD62L- (D) in PBMC, splenocytes, and lymph node cells after Poly I:C injection.

F. Poly I:C up-regulate CD122(IL-15/2Rβ) and CD8+CD122+ T cells in BD mice

IL-15 binding to IL-15Rα likely leads to recruitment of CD122(IL-15/2Rβ) and common γ chain, and initiation of signaling cascades from these shared chains (Bamford, et al., 1994). We next investigated the frequency of CD122-expressing cells in Poly I:C injected BD mice to determine whether the expression of CD122 that demanded to signaling cascades of IL-15-15Rα complexes could be induced by Poly I:C.

The frequencies of CD122-expressing cells in PBMC (8.4±7.6 vs. 15.3±9.7%, p=0.2) and lymph nodes cells (4.6±2.0 vs. 5.7±1.1%, p=0.3) did little different between BD mice and BDN mice. At 2 days after the last injection, the frequencies of CD122-expressing cells were not different between Poly I:C and PBS injected BD mice in PBMC (Poly I:C vs. PBS, 12.1±4.8 vs. 8.4±7.6%) and lymph nodes (7.3±4.4 vs. 4.6±2.0%). At 17 days after the last injection, the frequencies of CD122-expressing cells from Poly I:C injected group were significantly elevated than PBS injected group in PBMC (Poly I:C vs. PBS, 25.9±12.0 vs.

8.4±7.6%, p=0.01) and lymph nodes (7.8±3.3 vs. 4.6±2.0%, p=0.05) (Fig. 6A). In result, the frequencies of CD122-expressing cells were significantly up-regulated in lymph nodes cells isolated form Poly I:C injected BD mice at 17 days after the last injection.

CD8+CD122+ T cells are newly identified regulatory T cells (Saitoh, et al., 2007) and reported the effect which involved anti-inflammatory responses (Rifa'i, et al., 2008) in the recovery phase of EAE mouse model (Lee, et al., 2008). The frequencies of CD8+CD122+ T cells in lymph nodes cells of BD mice (n=6) were significantly lower than BDN mice (n=5)(1.5±0.7 vs. 3.8±1.6%, p=0.009) but not different in PBMC between BD mice and

BDN mice (4.0±2.4 vs. 5.7±2.6%, p=0.4). At 2 days after the last injection, the frequencies of CD8+CD122+ cells were not different between Poly I:C and PBS injected BD mice in PBMC (Poly I:C vs. PBS, 3.9±3.1 vs. 6.1±1.3%) and lymph nodes (4.0±2.9 vs. 3.3±0.1%, p=0.07). At 17 days after the last injection, the frequencies of CD8+CD122+ cells from Poly I:C injected group (n=4) were significantly increased than PBS injected group (n=6) in lymph node (3.6±1.2 vs. 1.5±0.7%, p=0.008) though there was no difference in PBMC (5.8±1.0 vs. 4.0±2.4%, p=0.2) (Fig. 6B). In the result, the frequencies of CD8+CD122+

regulatory T cells were significantly higher in lymph nodes cells isolated from BDN mice compared with BD mice. Administration of Poly I:C into BD mice up-regulated the frequencies of CD8+CD122+ regulatory T cells in lymph nodes cells.

Fig. 6. The frequencies of CD122(IL-15/2Rβ) and CD8+CD122+ T cells in lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C into BD mice. The cells were isolated from lymph nodes at 2 or 17 days after the last injection of Poly I:C. The frequencies of CD122- and CD8+CD122+-expressing cells were analyzed by Flow cytometry. The data shows the frequencies of CD122+ (A), CD8+122+ cells in PBMC and lymph node cells (B) and the dot plot of CD8+CD122+ T cells in lymph node cells from BDN, PBS or Poly I:C injected BD mice group (C).

G. Poly I:C down-regulates IL-23 receptor and IL-17A in BD mice

Recent studies have showed that the IL-23R gene is strongly associated with Behcet’s disease (Jiang, et al., 2010; Mizuki, et al., 2010; Remmers, et al., 2010) as well as several autoimmune diseases, such as Crohn’s disease (Duerr, et al., 2006), Rheumatoid arthritis (Hollis-Moffatt, et al., 2009), and Ankylosing spondylitis (Rueda, et al., 2008; Sung, et al., 2009). The IL-23R is essential for the terminal differentiation of IL-17-producing effector T helper cells in vivo (McGeachy, et al., 2009). Th17 cells are also present in human patients with various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis (Matusevicius, et al., 1999), systemic lupus erythematous (Wong, et al., 2000), and asthma (Hashimoto, et al., 2005). Therefore, we investigated the mRNA level of IL-23R and IL-17A cytokine serum level from BD, BDN and Poly I:C injected BD mice. IL-23R mRNA levels in lymph nodes of BD mice were up-regulated when compared to BDN by RT-PCR. At 2 and 17 days after the last injection, IL-23R mRNA levels in lymph nodes down-regulated in Poly I:C injected BD mice compared to BDN mice (Fig. 7A). Th17 cells mainly produce IL-17A, therefore the serum level of IL-17A was measured by ELISA. Serum levels of IL-17A were 9.87±4.46 pg/ml in BDN (n=9) and 21.30±11.79 pg/ml in BD (n=12) by ELISA. As a result, the serum IL-17A levels were higher in BD mice than BDN mice (p=0.006). Serum IL-17A levels were down-regulated by injection of Poly I:C in BD mice at 2 days after the last injection though statistically not significant (Fig. 7B) (Poly I:C (n=5) vs. PBS (n=6))(11.70

±1.60 vs. 18.32 ±9.95 pg/ml, p=0.18). At 17 days after the last injection, serum IL-17A levels were returned to PBS injected control levels.

Fig. 7. mRNA expression of IL

I:C injected BD mice. Lymph nodes cells and serum were collected from Poly I:C injected BD mice isolated at 2

expression was analyzed by RT ELISA .

IL-23 receptor and serum protein levels of IL-17A

Lymph nodes cells and serum were collected from BDN, BD and ected BD mice isolated at 2 or 17 days after the last injection. (A)

expression was analyzed by RT-PCR. (B) The serum levels of IL-17A were measured by 17A in Poly BDN, BD and (A) IL-23R 17A were measured by

Ⅳ. DISCUSSION

In BD patients, the frequencies of CD45RO+ cells were lower than disease control group has been reported (Takase, et al., 2006) and also, in our laboratory we observed that the frequencies of CD8+CD44+ and CD8+CD62L- memory T cells in BD mice were significantly lower than BDN mice. Therefore, we investigated the expression of IL-15Rα and IL-7Rα which as known as association with generation of memory T cells (Kimberly, et al., 2003) in the HSV-induced BD mouse model. The expression of IL-15Rα was significantly lower in BD mice than BDN mice. Although serum level of IL-15 was not analyzed because mouse IL-15 ELISA kit was not available, IL-7 was not different between BD mice and BDN mice. Therefore, we focused in induction of IL-15Rα to up-regulate memory T cells in BD mice. There has been reported, the IL-15Rα mRNA induced during Poly I:C bystander responses in vivo (Lodolce, et al., 2001). It was also reported that administrated Poly I:C enhanced in type I IFN and TGFβ-mediated inflammation in systemic sclerosis (Rothstein, et al., 2010), airway inflammation in a rat model of asthma (Takayama, et al., 2011), and EAE (Experimental Autoimmune Uveitis) retinal autoimmunity model (Ren, et al., 2011). In contrast, many observations supports a protective role of Poly I:C supplementation in the diabetes prone BB rat (Sobel, et al., 1998), a murine Experimental autoimmune encephalomyelitis model (Touil, et al., 2006), and inflammatory arthritis model by IFN-α involvement (Yarilina, et al., 2007). In this experiment, we found low dose of Poly I:C effectively increased the level of IL-15Ra by dose dependent manner in vivo. Then, 0.2

µg/g of Poly I:C was injected intra-peritoneally into BD mice to induce IL-15Rα level. At 2 days after the last injection, we observed that cutaneous symptoms were particularly improved and the severity score of BD mice was decreased compared to PBS injected control group. In the same time, we observed the increased frequencies of IL-15Rα in PBMC and splenocytes significantly but there was no difference in the frequencies of IL-7Rα between Poly I:C and PBS injected BD mice group. Furthermore, the frequencies of CD8+CD44+ memory T cells were significantly increased in PBMC and splenocytes but the frequencies of CD4+CD44+ memory T cells were not different between Poly I:C and PBS injected BD mice group at 17 days after the last injection. In this result, CD8+CD44+

memory T cells were selectively up-regulated in BD mice by Poly I:C injection. Therefore, the increased CD8+CD44+ memory T cells could be correlated to IL-15Rα that was up-regulated by Poly I:C. Interestingly, although the frequency of IL-7Rα were not affected, the serum level of IL-7 significantly up-regulated by Poly I:C injection in BD mice. It is possible that the increased IL-7 cytokine also could be involved in up-regulation of CD8+CD44+ memory T cells. There are many papers associated with potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15 (Zhang, et al., 1998;

Becker, et al., 2002; Becker, et al., 2003;Oh, et al., 2004; Mortier, et al., 2009) but are not required for memory-phenotype CD4+ T cells (Tan, et al., 2002). In addition, the expression of IL-15Rα in PBMC was steadily elevated by 17 days in contrast with return to PBS control level in splenocytes and lymph node cells. As has been reported that IL-15 and IL-15Rα, that form stable complexes on the cell surface of activated monocytes, recycled after endosomal

Sato, et al., 2007). It has been thought that highly IL-15Rα-expressing cells such as monocytes and macrophages were abundant in blood compared to spleen and lymph nodes.

The expression of IL-15Rα on cell surfaces enable sustained IL-15 activity by recycling. In our result, also has shown steady expression of IL-15Rα in PBMC. As a result, IL-15Rα induced CD8+ memory T cells were up-regulated at 17 days and the improvement of cutaneous symptoms of BD mice was also observed after Poly I:C injection. Then, what kind of factors affect improvement in HSV-induced BD mice model in early phase after poly I:C injection? Recent data has been revealed that Poly I:C stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFN-β-dependent way

문서에서 Anti-inflammatory Role of IL-15Rα Up-regulated by Poly I:C in Herpes Simplex Virus-induced Behcet's Disease-like Mouse Model (페이지 19-0)