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Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide.

Coulombe F, Divangahi M, Veyrier F, de Léséleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA - J. Exp. Med. (2009)

Bottom Line: In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion.Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy.Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada.

ABSTRACT
Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) alpha secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

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N-glycolyl MDP is a critical active constituent of the adjuvant-active mycobacterial cell wall. (A) Naive Nod2+/+ and Nod2−/− mice (n = 4 per genotype per immunization) were immunized i.p. with either M. smegmatis or M. smegmatisΔnamH and rechallenged 14 d later with the same organism. Saline-injected mice were used as a control. The frequency of IFN-γ–producing splenocytes (top) as well as total IFN-γ (middle) and IL-12p40 (bottom) production by these cells was quantified using ELISPOT and ELISA, respectively. (B) C57BL/6 mice (n = 3–4 per immunization) were immunized s.c. with the indicated emulsified preparations for 7 d. The frequency of OVA-specific IFN-γ–producing T cells in the draining lymph nodes was analyzed by ELISPOT of unstimulated and OVA-stimulated cells. Representative data from two independent replicates are shown (means ± SEM). *, P < 0.05.
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fig5: N-glycolyl MDP is a critical active constituent of the adjuvant-active mycobacterial cell wall. (A) Naive Nod2+/+ and Nod2−/− mice (n = 4 per genotype per immunization) were immunized i.p. with either M. smegmatis or M. smegmatisΔnamH and rechallenged 14 d later with the same organism. Saline-injected mice were used as a control. The frequency of IFN-γ–producing splenocytes (top) as well as total IFN-γ (middle) and IL-12p40 (bottom) production by these cells was quantified using ELISPOT and ELISA, respectively. (B) C57BL/6 mice (n = 3–4 per immunization) were immunized s.c. with the indicated emulsified preparations for 7 d. The frequency of OVA-specific IFN-γ–producing T cells in the draining lymph nodes was analyzed by ELISPOT of unstimulated and OVA-stimulated cells. Representative data from two independent replicates are shown (means ± SEM). *, P < 0.05.

Mentions: Our group recently showed that activation of the IFN-γ–IL-12 axis during mycobacterial infection is impaired in Nod2-deficient mice (Divangahi et al., 2008). To investigate the NOD2-dependent contribution of mycobacterial NamH to T cell activation, we performed a short-term immunization experiment using live WT M. smegmatis and namH-deficient M. smegmatis. 14 d after immunization, no bacteria could be detected in the mouse spleens (unpublished data). As shown in Fig. 5 A, the number of IFN-γ–producing splenocytes (top) and the level of IFN-γ production by these cells (middle) were significantly increased in Nod2+/+ compared with Nod2−/− cells after immunization with WT M. smegmatis. This NOD2 dependence was lost after immunization with namH-deficient M. smegmatis. Similar results were obtained when measuring IL-12p40 production by splenic antigen-presenting cells (Fig. 5, bottom).


Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide.

Coulombe F, Divangahi M, Veyrier F, de Léséleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA - J. Exp. Med. (2009)

N-glycolyl MDP is a critical active constituent of the adjuvant-active mycobacterial cell wall. (A) Naive Nod2+/+ and Nod2−/− mice (n = 4 per genotype per immunization) were immunized i.p. with either M. smegmatis or M. smegmatisΔnamH and rechallenged 14 d later with the same organism. Saline-injected mice were used as a control. The frequency of IFN-γ–producing splenocytes (top) as well as total IFN-γ (middle) and IL-12p40 (bottom) production by these cells was quantified using ELISPOT and ELISA, respectively. (B) C57BL/6 mice (n = 3–4 per immunization) were immunized s.c. with the indicated emulsified preparations for 7 d. The frequency of OVA-specific IFN-γ–producing T cells in the draining lymph nodes was analyzed by ELISPOT of unstimulated and OVA-stimulated cells. Representative data from two independent replicates are shown (means ± SEM). *, P < 0.05.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722178&req=5

fig5: N-glycolyl MDP is a critical active constituent of the adjuvant-active mycobacterial cell wall. (A) Naive Nod2+/+ and Nod2−/− mice (n = 4 per genotype per immunization) were immunized i.p. with either M. smegmatis or M. smegmatisΔnamH and rechallenged 14 d later with the same organism. Saline-injected mice were used as a control. The frequency of IFN-γ–producing splenocytes (top) as well as total IFN-γ (middle) and IL-12p40 (bottom) production by these cells was quantified using ELISPOT and ELISA, respectively. (B) C57BL/6 mice (n = 3–4 per immunization) were immunized s.c. with the indicated emulsified preparations for 7 d. The frequency of OVA-specific IFN-γ–producing T cells in the draining lymph nodes was analyzed by ELISPOT of unstimulated and OVA-stimulated cells. Representative data from two independent replicates are shown (means ± SEM). *, P < 0.05.
Mentions: Our group recently showed that activation of the IFN-γ–IL-12 axis during mycobacterial infection is impaired in Nod2-deficient mice (Divangahi et al., 2008). To investigate the NOD2-dependent contribution of mycobacterial NamH to T cell activation, we performed a short-term immunization experiment using live WT M. smegmatis and namH-deficient M. smegmatis. 14 d after immunization, no bacteria could be detected in the mouse spleens (unpublished data). As shown in Fig. 5 A, the number of IFN-γ–producing splenocytes (top) and the level of IFN-γ production by these cells (middle) were significantly increased in Nod2+/+ compared with Nod2−/− cells after immunization with WT M. smegmatis. This NOD2 dependence was lost after immunization with namH-deficient M. smegmatis. Similar results were obtained when measuring IL-12p40 production by splenic antigen-presenting cells (Fig. 5, bottom).

Bottom Line: In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion.Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy.Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada.

ABSTRACT
Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) alpha secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

Show MeSH
Related in: MedlinePlus