Contribution of Nitric Oxide to CpG-Mediated Protection against Listeria monocytogenes
http://www.100md.com
感染与免疫杂志 2005年第6期
CBER/FDA, Bethesda, Maryland 20892
ABSTRACT
Immunostimulatory CpG oligodeoxynucleotides (ODN) improve host resistance to listeriae. CpG ODN trigger immune cells to produce gamma interferon and "prime" host cells to secrete nitric oxide in response to bacterial exposure. CpG treatment does not protect inducible nitric oxide synthase 2 knockout mice, indicating that NO is critical to CpG-mediated protection against listeriae.
TEXT
CpG motifs present in bacterial DNA trigger lymphocytes, dendritic cells, and macrophages to produce a variety of immunoprotective cytokines, chemokines, and antibodies (Abs) (1, 9, 14, 16, 21). Synthetic oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of bacterial DNA to protect the host from infection (5, 8, 11, 12, 15, 18, 23). For example, CpG ODN significantly reduce host susceptibility to Listeria monocytogenes. This immunoprotective effect peaks several days after CpG ODN administration and persists for several weeks (5, 10, 12, 15).
CpG-activated macrophages produce various immunoprotective factors, including large amounts of nitric oxide (NO) (6, 7). Macrophage-derived NO contributes to the clearance of listeria infection (2, 3). Mice lacking inducible nitric oxide synthase 2 (NOS2) are highly susceptible to listeriae (17), consistent with NO playing an important role in host control of this pathogen.
To examine whether NO contributes to CpG-mediated protection against listeriae, 6- to 8-week-old C57BL/6 mice (NCI, Frederick, MD) were treated intraperitoneally (i.p.) with 50 μg of CpG (GCTAGACGTTAGCGT) or control (GCTAGAGCTTAGGCT) ODN. Peritoneal cells were isolated 3 days later by lavage and cultured in vitro with 2 x 105 CFU/ml of heat-killed listeriae (HKL). Nitrite levels in culture supernatants were quantified using the Griess reagent (Sigma, St. Louis, MO) and comparison to a standard curve generated using NaNO2.
As seen in Fig. 1A, CpG ODN had no effect on basal levels of NO production. However, cells from CpG-treated mice produced significantly more NO when cultured with HKL than cells from mice treated with control ODN (P < 0.001). This increased response developed within 1 day of ODN administration, peaked on day 3, and persisted through day 7 (Fig. 1B) (P < 0.01), mirroring the kinetics of CpG-induced protection against listeriae.
Mice treated with CpG (but not control) ODN survived challenged with 10 50% lethal doses (LD50s) of listeriae (Fig. 2) (P < 0.001) (5, 12, 15). The role of NO was further defined by comparing the rates of survival of NOS2 knockout (KO) and wild-type (WT) mice (Jackson Laboratory, Bar Harbor, ME). NOS2 KO mice were more susceptible to listeria infection than WT mice (1 LD50 = 103 CFU in NOS2 mice but 104 CFU in WT mice). Moreover, CpG ODN treatment failed to protect NOS2 KO mice from listeria challenge (Fig. 2) (P < 0.001).
CpG treatment also activates host cells to secrete gamma interferon (IFN-) and increases serum IFN- levels, as determined by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay (P < 0.001) (Fig. 3; methods used to obtain the results in this figure are described in reference 13). The importance of IFN- to NO production was examined by culturing peritoneal cells from CpG-treated mice with HKL plus 0.5 ng/ml of neutralizing Abs against IFN-, tumor necrosis factor alpha, interleukin-12, or interleukin-18 (R & D Systems, Minneapolis, MN). As seen in Fig. 4A, only anti-IFN- Abs significantly reduced NO production (P < 0.001).
To confirm the importance of IFN- to NO production, CpG ODN were administered to IFN- and NOS2 KO mice (Jackson Laboratory). Whereas CpG treatment "primed" immune cells from normal mice to produce NO when exposed to HKL, no such effect was observed on cells from either KO strain (Fig. 4B) (P < 0.001). Moreover, CpG ODN treatment failed to protect IFN- KO mice from listeria infection (Fig. 5).
CpG ODN stimulate a complex immunomodulatory cascade involving multiple cell types, cytokines, and chemokines (1, 9, 14, 16, 21). This complexity impeded efforts to identify the contribution of specific factors to CpG-mediated host protection. Current findings establish that IFN--dependent NO production is a critical component of host resistance to listeriae elicited by CpG ODN.
Intracellular bacteria take up residence in host macrophages to avoid immune surveillance/destruction (19, 20, 22). These pathogens are cleared when the macrophages are activated to produce bactericidal factors, such as NO. Evidence that IFN--dependent NO contributes to protection against listeriae is provided by Fig. 2, showing that CpG treatment protects WT but not NOS2 KO mice from infection (Fig. 2) (P < 0.001). Similarly, Datta et al. recently showed that NO contributes to CpG-mediated resistance against leishmania (4). The possibility of a role for IFN- in the regulation of NO production is supported by the finding that anti-IFN- Abs selectively blocked NO secretion by CpG-stimulated cells (Fig. 4A). In addition, cells from CpG-treated IFN- KO mice failed to produce NO in response to HKL (Fig. 4B). These findings confirm and extend previous results indicating that CpG ODN up-regulate the production of IFN-, which in turn modulates the host's ability to produce NO in response to infection (4).
Present address: RIMO, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
REFERENCES
1. Ballas, Z. K., W. L. Rasmussen, and A. M. Krieg. 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157:1840-1845.
2. Beckerman, K. P., H. W. Rogers, J. A. Corbett, R. D. Schreiber, M. L. McDaniel, and E. R. Unanue. 1993. Release of nitric oxide during the T cell-independent pathway of macrophage activation. Its role in resistance to Listeria monocytogenes. J. Immunol. 150:888-895.
3. Boockvar, K. S., D. L. Granger, R. M. Poston, M. Maybodi, M. K. Washington, J. B. Hibbs, Jr., and R. L. Kurlander. 1994. Nitric oxide produced during murine listeriosis is protective. Infect. Immun. 62:1089-1100.
4. Datta, N., S. Mukherjee, L. Das, and P. K. Das. 2003. Targeting of immunostimulatory DNA cures experimental visceral leishmaniasis through nitric oxide up-regulation and T cell activation. Eur. J. Immunol. 33:1508-1518.
5. Elkins, K. L., T. R. Rhinehart-Jones, S. Stibitz, J. S. Conover, and D. M. Klinman. 1999. Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria. J. Immunol. 162:2291-2298.
6. Gao, J. J., E. G. Zuvanich, Q. Xue, D. L. Horn, R. Silverstein, and D. C. Morrison. 1999. Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages. J. Immunol. 163:4095-4099.
7. Ghosh, D. K., M. A. Misukonis, C. Reich, D. S. Pisetsky, and J. B. Weinberg. 2001. Host response to infection: the role of CpG DNA in induction of cyclooxygenase 2 and nitric oxide synthase 2 in murine macrophages. Infect. Immun. 69:7703-7710.
8. Gramzinski, R. A., D. L. Doolan, M. Sedegah, H. L. Davis, A. M. Krieg, and S. L. Hoffman. 2001. Interleukin-12- and gamma interferon-dependent protection against malaria conferred by CpG oligodeoxynucleotide in mice. Infect. Immun. 69:1643-1649.
9. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.
10. Ito, S., K. J. Ishii, H. Shirot, and D. M. Klinman. 2004. CpG oligodeoxynucleotides improve the survival of pregnant and fetal mice following Listeria monocytogenes infection. Infect. Immun. 72:3543-3548.
11. Juffermans, N. P., J. C. Leemans, S. Florquin, A. Verbon, A. H. Kolk, P. Speelman, S. J. H. van Deventer, and T. van der Poll. 2002. CpG oligodeoxynucleotides enhance host defense during murine tuberculosis. Infect. Immun. 70:147-152.
12. Klinman, D. M., J. Conover, and C. Coban. 1999. Repeated administration of synthetic oligodeoxynucleotides expressing CpG motifs provides long-term protection against bacterial infection. Infect. Immun. 67:5658-5663.
13. Klinman, D. M., and T. B. Nutman. 1994. ELIspot assay to detect cytokine-secreting murine and human cells sect. 6.19, p. 1-8. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology, 7th ed. Greene Publishing Associates, Brooklyn, N.Y.
14. Klinman, D. M., A.-K. Yi, S. L. Beaucage, J. Conover, and A. M. Krieg. 1996. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12 and interferon . Proc. Natl. Acad. Sci. USA 93:2879-2883.
15. Krieg, A. M., L. Love-Homan, A.-K. Yi, and J. T. Harty. 1998. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428-2434.
16. Krieg, A. M., A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546-549.
17. MacMicking, J. D., C. Nathan, G. Hom, N. Chartrain, D. S. Fletcher, M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N. Hutchinson, et al. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641-650.
18. Pyles, R. B., D. Higgins, C. Chalk, A. Zalar, J. Eiden, C. Brown, G. Van Nest, and L. R. Stanberry. 2002. Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection. J. Virol. 76:11387-11396.
19. Raupach, B., and S. H. Kaufmann. 2001. Immune responses to intracellular bacteria. Curr. Opin. Immunol. 13:417-428.
20. Shiloh, M. U., and C. F. Nathan. 2000. Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria. Curr. Opin. Microbiol. 3:35-42.
21. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and D. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167:3555-3558.
22. Vázquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Domínguez-Bernal, W. Goebel, B. González-Zorn, J. Wehland, and J. Kreft. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584-640.
23. Zimmermann, S., O. Egeter, S. Hausmann, G. B. Lipford, M. Rocken, H. Wagner, and K. Heeg. 1998. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160:3627-3630.(Shuichi Ito, Ken J. Ishii)
ABSTRACT
Immunostimulatory CpG oligodeoxynucleotides (ODN) improve host resistance to listeriae. CpG ODN trigger immune cells to produce gamma interferon and "prime" host cells to secrete nitric oxide in response to bacterial exposure. CpG treatment does not protect inducible nitric oxide synthase 2 knockout mice, indicating that NO is critical to CpG-mediated protection against listeriae.
TEXT
CpG motifs present in bacterial DNA trigger lymphocytes, dendritic cells, and macrophages to produce a variety of immunoprotective cytokines, chemokines, and antibodies (Abs) (1, 9, 14, 16, 21). Synthetic oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of bacterial DNA to protect the host from infection (5, 8, 11, 12, 15, 18, 23). For example, CpG ODN significantly reduce host susceptibility to Listeria monocytogenes. This immunoprotective effect peaks several days after CpG ODN administration and persists for several weeks (5, 10, 12, 15).
CpG-activated macrophages produce various immunoprotective factors, including large amounts of nitric oxide (NO) (6, 7). Macrophage-derived NO contributes to the clearance of listeria infection (2, 3). Mice lacking inducible nitric oxide synthase 2 (NOS2) are highly susceptible to listeriae (17), consistent with NO playing an important role in host control of this pathogen.
To examine whether NO contributes to CpG-mediated protection against listeriae, 6- to 8-week-old C57BL/6 mice (NCI, Frederick, MD) were treated intraperitoneally (i.p.) with 50 μg of CpG (GCTAGACGTTAGCGT) or control (GCTAGAGCTTAGGCT) ODN. Peritoneal cells were isolated 3 days later by lavage and cultured in vitro with 2 x 105 CFU/ml of heat-killed listeriae (HKL). Nitrite levels in culture supernatants were quantified using the Griess reagent (Sigma, St. Louis, MO) and comparison to a standard curve generated using NaNO2.
As seen in Fig. 1A, CpG ODN had no effect on basal levels of NO production. However, cells from CpG-treated mice produced significantly more NO when cultured with HKL than cells from mice treated with control ODN (P < 0.001). This increased response developed within 1 day of ODN administration, peaked on day 3, and persisted through day 7 (Fig. 1B) (P < 0.01), mirroring the kinetics of CpG-induced protection against listeriae.
Mice treated with CpG (but not control) ODN survived challenged with 10 50% lethal doses (LD50s) of listeriae (Fig. 2) (P < 0.001) (5, 12, 15). The role of NO was further defined by comparing the rates of survival of NOS2 knockout (KO) and wild-type (WT) mice (Jackson Laboratory, Bar Harbor, ME). NOS2 KO mice were more susceptible to listeria infection than WT mice (1 LD50 = 103 CFU in NOS2 mice but 104 CFU in WT mice). Moreover, CpG ODN treatment failed to protect NOS2 KO mice from listeria challenge (Fig. 2) (P < 0.001).
CpG treatment also activates host cells to secrete gamma interferon (IFN-) and increases serum IFN- levels, as determined by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay (P < 0.001) (Fig. 3; methods used to obtain the results in this figure are described in reference 13). The importance of IFN- to NO production was examined by culturing peritoneal cells from CpG-treated mice with HKL plus 0.5 ng/ml of neutralizing Abs against IFN-, tumor necrosis factor alpha, interleukin-12, or interleukin-18 (R & D Systems, Minneapolis, MN). As seen in Fig. 4A, only anti-IFN- Abs significantly reduced NO production (P < 0.001).
To confirm the importance of IFN- to NO production, CpG ODN were administered to IFN- and NOS2 KO mice (Jackson Laboratory). Whereas CpG treatment "primed" immune cells from normal mice to produce NO when exposed to HKL, no such effect was observed on cells from either KO strain (Fig. 4B) (P < 0.001). Moreover, CpG ODN treatment failed to protect IFN- KO mice from listeria infection (Fig. 5).
CpG ODN stimulate a complex immunomodulatory cascade involving multiple cell types, cytokines, and chemokines (1, 9, 14, 16, 21). This complexity impeded efforts to identify the contribution of specific factors to CpG-mediated host protection. Current findings establish that IFN--dependent NO production is a critical component of host resistance to listeriae elicited by CpG ODN.
Intracellular bacteria take up residence in host macrophages to avoid immune surveillance/destruction (19, 20, 22). These pathogens are cleared when the macrophages are activated to produce bactericidal factors, such as NO. Evidence that IFN--dependent NO contributes to protection against listeriae is provided by Fig. 2, showing that CpG treatment protects WT but not NOS2 KO mice from infection (Fig. 2) (P < 0.001). Similarly, Datta et al. recently showed that NO contributes to CpG-mediated resistance against leishmania (4). The possibility of a role for IFN- in the regulation of NO production is supported by the finding that anti-IFN- Abs selectively blocked NO secretion by CpG-stimulated cells (Fig. 4A). In addition, cells from CpG-treated IFN- KO mice failed to produce NO in response to HKL (Fig. 4B). These findings confirm and extend previous results indicating that CpG ODN up-regulate the production of IFN-, which in turn modulates the host's ability to produce NO in response to infection (4).
Present address: RIMO, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
REFERENCES
1. Ballas, Z. K., W. L. Rasmussen, and A. M. Krieg. 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157:1840-1845.
2. Beckerman, K. P., H. W. Rogers, J. A. Corbett, R. D. Schreiber, M. L. McDaniel, and E. R. Unanue. 1993. Release of nitric oxide during the T cell-independent pathway of macrophage activation. Its role in resistance to Listeria monocytogenes. J. Immunol. 150:888-895.
3. Boockvar, K. S., D. L. Granger, R. M. Poston, M. Maybodi, M. K. Washington, J. B. Hibbs, Jr., and R. L. Kurlander. 1994. Nitric oxide produced during murine listeriosis is protective. Infect. Immun. 62:1089-1100.
4. Datta, N., S. Mukherjee, L. Das, and P. K. Das. 2003. Targeting of immunostimulatory DNA cures experimental visceral leishmaniasis through nitric oxide up-regulation and T cell activation. Eur. J. Immunol. 33:1508-1518.
5. Elkins, K. L., T. R. Rhinehart-Jones, S. Stibitz, J. S. Conover, and D. M. Klinman. 1999. Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria. J. Immunol. 162:2291-2298.
6. Gao, J. J., E. G. Zuvanich, Q. Xue, D. L. Horn, R. Silverstein, and D. C. Morrison. 1999. Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages. J. Immunol. 163:4095-4099.
7. Ghosh, D. K., M. A. Misukonis, C. Reich, D. S. Pisetsky, and J. B. Weinberg. 2001. Host response to infection: the role of CpG DNA in induction of cyclooxygenase 2 and nitric oxide synthase 2 in murine macrophages. Infect. Immun. 69:7703-7710.
8. Gramzinski, R. A., D. L. Doolan, M. Sedegah, H. L. Davis, A. M. Krieg, and S. L. Hoffman. 2001. Interleukin-12- and gamma interferon-dependent protection against malaria conferred by CpG oligodeoxynucleotide in mice. Infect. Immun. 69:1643-1649.
9. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.
10. Ito, S., K. J. Ishii, H. Shirot, and D. M. Klinman. 2004. CpG oligodeoxynucleotides improve the survival of pregnant and fetal mice following Listeria monocytogenes infection. Infect. Immun. 72:3543-3548.
11. Juffermans, N. P., J. C. Leemans, S. Florquin, A. Verbon, A. H. Kolk, P. Speelman, S. J. H. van Deventer, and T. van der Poll. 2002. CpG oligodeoxynucleotides enhance host defense during murine tuberculosis. Infect. Immun. 70:147-152.
12. Klinman, D. M., J. Conover, and C. Coban. 1999. Repeated administration of synthetic oligodeoxynucleotides expressing CpG motifs provides long-term protection against bacterial infection. Infect. Immun. 67:5658-5663.
13. Klinman, D. M., and T. B. Nutman. 1994. ELIspot assay to detect cytokine-secreting murine and human cells sect. 6.19, p. 1-8. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology, 7th ed. Greene Publishing Associates, Brooklyn, N.Y.
14. Klinman, D. M., A.-K. Yi, S. L. Beaucage, J. Conover, and A. M. Krieg. 1996. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12 and interferon . Proc. Natl. Acad. Sci. USA 93:2879-2883.
15. Krieg, A. M., L. Love-Homan, A.-K. Yi, and J. T. Harty. 1998. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428-2434.
16. Krieg, A. M., A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546-549.
17. MacMicking, J. D., C. Nathan, G. Hom, N. Chartrain, D. S. Fletcher, M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N. Hutchinson, et al. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641-650.
18. Pyles, R. B., D. Higgins, C. Chalk, A. Zalar, J. Eiden, C. Brown, G. Van Nest, and L. R. Stanberry. 2002. Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection. J. Virol. 76:11387-11396.
19. Raupach, B., and S. H. Kaufmann. 2001. Immune responses to intracellular bacteria. Curr. Opin. Immunol. 13:417-428.
20. Shiloh, M. U., and C. F. Nathan. 2000. Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria. Curr. Opin. Microbiol. 3:35-42.
21. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and D. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167:3555-3558.
22. Vázquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Domínguez-Bernal, W. Goebel, B. González-Zorn, J. Wehland, and J. Kreft. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584-640.
23. Zimmermann, S., O. Egeter, S. Hausmann, G. B. Lipford, M. Rocken, H. Wagner, and K. Heeg. 1998. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160:3627-3630.(Shuichi Ito, Ken J. Ishii)