PI
W/O NC_1.011
100 101 102 103 104
FL1-H
W/O PC_1.014
100 101 102 103 104
FL1-H W/O HSP_1.017
100 101 102 103 104
FL1-H
W/O MTC_1.020
100 101 102 103 104
FL1-H
Figure5. HpHsp60 dose not induce PBMC apoptosis. (A) Quadrant plots show
the apoptosis status of HpHsp60-treated PBMCs. (W/O stands for “treat without IL-2”; NC: Cell only; PC: PBMCs activated with anti-CD3; HSP: PBMC treated with anti-CD3 and HpHsp60. MTC: mitomycin C (500µg/ml), as the positive control for the apoptosis.) The lower and upper right quarters are represent early and late phases of apoptosis respectively. (B) The percentages of apoptosis were calculated according to the flowcytomery analysis.Figure 6
Fresh PBMCs : Treated PBMCs
Anti-CD3-treated cell
Fresh PBMCs : Treated PBMCs
Anti-CD3-treated cell HpHsp60-treated cell
*
Figure6. The inhibition is through a cell-dependent manner. PBMCs were
seeded with or with IL2 and activated by OKT3 only or treated with HpHsp60 simultaneity. After four days, treated PBMCs were collected and co-culture with fresh PBMCs and OKT3 in different ratio for another four days incubation. Then MTT assay was taken to evaluate the proliferation. (A) Cells treated with HpHsp60 but without IL-2 (gray bar) significantly inhibited the fresh PBMCs proliferation compared to the cells treated with anti-CD3 only (white bar). * P< 0.05. (B) The inhibition was abolished when the treated cell were co-culture with IL-2 before mixed with allogenic fresh PBMCs.Figure 7
Figure7. The inhibition is not through a supernatant-dependent manner.
PBMCs were seeded with or with IL-2 and activated by OKT3 only or treated with HpHsp60 (200ng) simultaneity. After four days, supernatants were collected and co-culture with fresh PBMC and OKT3 in different ratio for another four days incubation. Then MTT assay was taken to evaluate the proliferation. PC:
supernatants were collected from anti-CD3 activated group. HpHsp60: supernatants were collected from HpHsp60-treated group. (A) Supernatants without IL-2. (B) Supernatants with IL-2.
Figure 8
Figure8. T cell percentage of HpHsp60-treated PBMCs. The treated PBMCs
were verified their T cell population. The chart showed that almost 93% of HpHsp60-treated cell were T cell. The data shown here was one representative of three independent experiments.Counts
CD3
+W/O NC_CD3_3.016
100 101 102 103 104 FL1-H
M1
W/O PC_CD3_4.023
100 101 102 103 104 FL1-H
M1
W/O HSP_CD3_3.011
100 101 102 103 104 FL1-H
M1
64% 83% 93%
Figure 9 A.
B.
W/O HSP_FoxP3.006 W/O HSP_FoxP3.006
100 101 102 103 104
FL1-H
100 101 102 103 104
FL1-H W/O PC_FoxP3.004
W/O PC_FoxP3.004
100 101 102 103 104
FL1-H
100 101 102 103 104
FL1-H W/O NC_FoxP3.002
W/O NC_FoxP3.002
100 101 102 103 104
FL1-H
100 101 102 103 104
FL1-H
Percentages of CD4+ FoxP3+ T cell (%)
*
C.
0 1 2 3 4 5 6 7
NC PC HpHsp60 FoxP3 mRNA Relative Expression
Figure 9. Treatment with HpHsp60 increases the percentage of regulatory T cell. (A) Percentage of regulatory T cell in CD4
+ cells were assay by the FoxP3 intracellular staining. One representative of three independent experiments was shown here. (B) The percentages of Treg cells of three independent experiments were calculated. Treatment with HpHsp60 significantly increased the CD4+FoxP3+ regulatory T cells about 16 % compared to the cells treated with OKT3 only. (From 56% for the PC group to 72% for the HpHsp60 group). * P <0.05. (C) FoxP3 expression was verified at mRNA level by real-time PCR. Relative expression was normalized with ß-actin. FoxP3 mRNA expression was
up-Figure 10
Figure 10. TGF-ß signaling pathway may be involved in the proliferative inhibition. PBMCs were seeded in 96-well pre-coated anti-CD3 antibodies
(2*105/well) and treated with HpHsp60 and with or without TGF-ß inhibitors. After four days incubation, MTT assay was used to detect the proliferation of PBMCs.The different symbols stand for different PMBCs samples obtained from different donors. A row line represented mean value of each condition. (N=4) (A) Mean value of proliferation index: PC=100%, HpHsp60=78%, HpHsp60+SB431542 (1µM)=86%, SB431542=100%. (B) Mean value of proliferation index: PC=100%, HpHsp60=78%, HpHsp60+SIS3 (0.1µM)=79%, SIS3 (0.1µM)=105%.
Figure 11
Percentage of CD4+ FoxP3+ cells (%)
NCPC
NC PC HpHsp60 HpHsp60 + SB431542 Percentage of CD4+ FoxP3+ cells (%)
Figure 11. TGF-ß signaling pathway is involved in the generation of HpHsp60-induced Treg cells. (A) Time course of Treg generation. Intracellular FoxP3
staining assay at different time points demonstrated percentages of CD4+ FoxP3+Treg cells. (N=2) (B) Treatment with 1µM SB431542 significantly reduced the generation of Treg cells. (N=3) *, P < 0.05.
*
*
Figure 12
Figure 12. TGF-ß signaling pathway may be involved in the proliferative inhibition caused by HpHsp60-induced Treg cells. PBMCs were seeded as the
same condition with the proliferation assay. 2 days after treatment with HpHsp60, SB431542 (1µM) or SIS3 (0.1µM) were added. Proliferation was evaluated by the MTT assay on the forth day. (A) SB431542. Proliferation index of HpHsp60 = 78%.Proliferation index of HpHsp60 + SB431542 = 91%. (N=3) * P < 0.05. (B) SIS3.
Proliferation index of HpHsp60 = 78%. Proliferation index of HpHsp60 + SIS3 = 76%. (N=3)
Reference
1. Warren, J. R. and Marshall, B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. (Letter). Lancet. 1983; 1:1273-5.
2. Pisani, P., Parkin, D. M., Munoz, N., et al. Cancer and infection: estimates of the attributable fraction in 1900. Cancer Epidemiology,Biomarkers and
Preventio. 1997;6:389–400.
3. Peek, Jr., Richard, M. and Martin J. B. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat. Rev. Cancer. 2002; 2:28–37.
4. Peterson, W. L. Helicobacter pylori and peptic ulcer disease. N. Engl. J. Med.
1991; 324:1043–1048.
5. Uemura, N., S. Okamoto, S. Yamamoto, N. Matsumura, S. Yamaguchi, M.
Yamakido, K. Taniyama, N. Sasaki, and R. J. Schlemper. Helicobacter
pylori infection and the development of gastric cancer. N. Engl. J. Med. 2001;
345:784–789.
6. Correa, P. Human gastric carcinogenesis: a multistep and multifactorial process. Cancer. Res. 1992; 52:6735–6740.
7. Dixon, M. F., et al. Classification and grading of gastritis. The updated Sydney system. Am. J. Surg. Pathol. 1996; 20:1161–1181.
8. Suzuki, T., et al. Localization of antigen- presenting cells in Helicobacter
pylori-infected gastric mucosa. Pathol. Int. 2002; 52:265–271.
9. Lundgren, A., et al. Helicobacter pylori-specific CD4+T cells home to and accumulate in the human Helicobacter pylori-infected gastric mucosa. Infect.
Immun. 2005; 73:5612–5619.
10. Quiding-Jarbrink, M., et al. CD4+and CD8+T cell responses in Helicobacter
pylori-infected individuals. Clin. Exp. Immunol. 2001; 123:81–87.
11. Bamford, K.B., et al. Lymphocytes in the human gastric mucosa during
Helicobacter pylori have a T helper cell 1 phenotype. Gastroenterology.
1998; 114:482-492.
12. Haeberle, H.A., et al. Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa. Infect. Immun. 1997; 65: 4229-4235.
13. Umehara, s., et al. Effects of Helicobacter pylori CagA protein on the growth and survival of B lymphocytes, the origin of MALT lymphama.
Oncogene. 2003; 22:8337-8342.
14. Beata Paziak-Doman´ ska., et al. Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori. Infections Cellular Immunology. 2000;
202:136–139.
15. Orsini, B., et al. Helicobacter pylori cag pathogenicity island is associated expression of interleukin-4 (IL-4) mRNA and modulation of the IL-4delta2
6667.
16. Gebert, B., et al. Helicobacter pylori vacuolating cytotoxin toxin inhibits T cell lymphocyte activation. Science. 2003; 301:1099-1102.
17. Montecucco, C. and M. de Bernard. Immunosuppressive and proinflammatory activities of the VacA toxin of Helicobacter pylori. J. Exp.
Med. 2003; 198:1767-1771.
18. Gobert, A. P., et al. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival. Proc. Natl.
Acad. Sci. 2001; 98:13844-13849.
19. Gewirtz, A. T., et al. Helicobacter pylori flagellin evades toll-like receptor 5-mediated innate immunity. J. Infect. Dis. 2004; 189:1914-1920.
20. Backhed, F., et al. Gastric mucosal recognition of Helicobacter pylori is independent of Toll-like receptor 4. J. Infect. Dis. 2003; 187:829-836.
21. Covacci, A., et al. Molecular charaterization of the 128- kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc. Natl. Acad. Sci. 1993; 90: 5791-5795.
22. Montecucco, C., et al. Helicobacter pylori vacuolating cytotoxin: cell intoxication and anion-specific channel activity. Curr.Top. Microbiol.
Immunol. 2001; 257:113-129.
23. Knipp, U., et al. Partial characterization of a cell proliferation-inhibiting
24. Chen, W., et al. Inhibition of mitogen-induced murine lymphocyte proliferation by Helicobacter pylori cell-free extract. J. Gastroenterol.
Hepatol. 2000;15:1000-1006.
25. Arthur, L. H., et al. Two families of chaperonin: physiology and Mmchanism.
Annu. Rev. Cell Dev. Biol. 2007; 23:115-145.
26. Wieten, L., et al. Cell stress induced HSP are targets of regulatory T cells: a role for HSP inducing compounds as anti-inflammatory immuno-modulators?
FEBS Lett. 2007; 581:3716-3722.
27. A. Kinnunen., et al.Heat shock protein 60 specific T-Cell response in Chlamydial Infections. Scand. J. Immunol. 2001; 54:76-81.
28. Haque, M. A., et al. Suppression of adjuvant arthritis in rat by induction of oral tolerance to mycobacterial 65-kDa heat hock protein. Eur. I. Immunol.
1996; 26:2022-2032.
29. Phadnis, S. H., et al. Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis. Infect. Immun. 1996;
64:905–912.
30. Zhao, Y., et al. Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR) 2 and mitogen-activated protein (MAP) kinase pathway in human monocytes. J. Med. Microbiol. 2007;
56:154-164.
interleukin-6 production by macrophages via a Toll-like receptor (TLR)-2,TLR-4, and myeloid differentiation factor 88-independent mechanism. J.
Biol. Chem. 2004; 279:245-250.
32. Lin, C. Y., et al. Characterizing the polymeric status of Helicobacter pylori heat shock protein 60. Biochem. Biophys. Res. Commun. 2009; 388:283-289.
33. Sakaguchi, S., et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of asingle mechanism of self-tolerance causes various autoimmunediseases. J. Immunol.
1995; 155:1151–1164.
34. Hori, S., et al. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003; 299:1057–1061.
35. Fontenot, J. D., et al. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 2003; 4:330–336.
36. Khattri, R., et al. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 2003; 4:337–342.
37. Rad, R., et al. CD25+/Foxp3+ T cells regulate gastric inflammation and
Helicobacter pylori colonization in vivo. Gastroenterology. 2006;
131:525-37.
38. Jang, T. J., et al. The number of Foxp3-positive regulatory T cells is increased in Helicobacter pylori gastritis and gastric cancer. Pathol. Res.
39. Anna, L., et al. Mucosal FoxP3-expressing CD4+CD25high regulatory T cells in Helicobacter pylori-infected patients. Infection and Immunity. 2005;
73:523–531.
40. Arne, K., et al. Naturally occurring regulatory T cells (CD4+, CD25high, FoxP3+) in the antrum and cardia are associated with higher H. pylori colonization and increased gene expression of TGF-β
1. Helicobacter . 2008;
13: 295–303.
41. Peti, M. D., et al. Depressed responsiveness of peripheral blood mononuclear cells to heat-shock proteins in periodontitis patients. J. Dent. Res. 1999;
78:1393-1400.
42. Zanin-Zhorov A., et al. Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling. J. Clin. Invest. 2006;
116:2022-32.
43. Chen, Z. M., et al. IL-10 and TGF-beta induce alloreactive CD4+ CD25-T cells to acquire regulatory cell function. Blood. 2003; 101:5076–5083.
44. N. J. Laping, et al. Inhibition of Transforming Growth Factor (TGF)-ß1-induced extracellular matrix with a novel inhibitor of the TGF-ßType I receptor kinase activity: SB-431542. Mol. Pharmacol. 2002; 62:58–64.
45. Gareth, J., et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-ß superfamily type I activin receptor-like kinase (ALK)
46. Shigeo, M., et al. SB-431542 and gleevec inhibit transforming growth factor-ß-induced proliferation of human osteosarcoma cells. Cancer Research. 2003;
63:7791–7798.
47. Jong, S. K., et al. New regulatory mechanisms of TGF-ß receptor function.
Trends in Cell Biology. 2009;19: 385-394.
48. Sean, L., et al. The Type II transforming growth factor-ß receptor autophosphorylates not only on serine and threonine but also on tyrosine residues. The Journal of Biological Chemistry. 1997; 272: 14850-14859.
49. Matt, K. L., et al. TGF-ß activates Erk MAP kinase signalling through direct phosphorylation of ShcA. The EMBO Journal. 2007; 26: 3957-3967.
50. Hanks, S. K. and Hunter, T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J.
1995; 9: 576–596.
51. Galliher, A. J. and Schiemann, W. P. Src phosphorylates Tyr284 in TGF-b type II receptor and regulates TGF-ß stimulation of p38 MAPK during breast cancer cell proliferation and invasion. Cancer Res. 2007; 67: 3752–3758.
52. Samuel, H., et al. P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg. Plos One. 2008; 3: e3302.
53. Henric, S. A. and Kerstin, S. MAP kinase p38 and its relation to T cell anergy and suppressor function of regulatory T cells. Cell Cycle. 2008;
54. Henric, S. A., et al. Activation of MAPkinase p38 is critical for the cell-cycle–controlled suppressor function of regulatory T cells. Blood. 2007; 109:
4351-4359.