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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 more potent than N-acetyl MDP at inducing proinflammatory cytokine production in macrophages. (A) Naive peritoneal macrophages from Nod2+/+ mice were stimulated for 6 h with LPS (top) or for 12 h with TDM (bottom) in combination with various concentrations of N-glycolyl or N-acetyl MDP. (B) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left unstimulated or were stimulated for 12 h with 10 µg/ml N-acetyl MDP alone, 10 µg/ml N-glycolyl MDP alone, TDM alone and a combination of N-acetyl MDP and TDM, or N-glycolyl MDP and TDM. The amount of TNF-α and IL-6 released in the supernatant was quantified by ELISA. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with no MDP added; **, P < 0.05 between N-glycolyl versus N-acetyl MDP.
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fig3: N-glycolyl MDP is more potent than N-acetyl MDP at inducing proinflammatory cytokine production in macrophages. (A) Naive peritoneal macrophages from Nod2+/+ mice were stimulated for 6 h with LPS (top) or for 12 h with TDM (bottom) in combination with various concentrations of N-glycolyl or N-acetyl MDP. (B) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left unstimulated or were stimulated for 12 h with 10 µg/ml N-acetyl MDP alone, 10 µg/ml N-glycolyl MDP alone, TDM alone and a combination of N-acetyl MDP and TDM, or N-glycolyl MDP and TDM. The amount of TNF-α and IL-6 released in the supernatant was quantified by ELISA. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with no MDP added; **, P < 0.05 between N-glycolyl versus N-acetyl MDP.

Mentions: It is known that MDP alone is a potent inducer of TNF-α mRNA, which remains untranslated, and that presence of LPS abrogates this translation block (Wolfert et al., 2002). To compare N-glycolyl and N-acetyl MDP, we stimulated mouse peritoneal macrophages with various concentrations of MDP along with a fixed concentration of LPS, and measured TNF-α and IL-6 production (Fig. 3 A, top). At all concentrations ranging from 0.5 to 10 µg/ml, N-glycolyl MDP was more stimulatory than N-acetyl MDP. In terms of potency, N-glycolyl MDP was active at a 10-fold lower concentration than N-acetyl MDP (0.5 vs. 5 µg/ml). Because Actinomycetes do not produce LPS, we repeated this experiment using M. tuberculosis–derived trehalose 6,6′-dimycolate (TDM), a molecule that has been shown to have synergistic immunological activity in combination with MDP (Masihi et al., 1985). Again, N-glycolyl MDP was more stimulatory than N-acetyl MDP and was active at a 10- to 20-fold lower concentration. (Fig. 3 A, bottom) The synergistic response to MDP plus TDM in Nod2+/+ cells was abrogated in Nod2−/− cells (Fig. 3 B). Thus, N-glycolyl MDP is more stimulatory and more potent than N-acetyl MDP at inducing proinflammatory cytokine production upon co-stimulation with nonmycobacteria- and mycobaceria-derived molecules.


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 more potent than N-acetyl MDP at inducing proinflammatory cytokine production in macrophages. (A) Naive peritoneal macrophages from Nod2+/+ mice were stimulated for 6 h with LPS (top) or for 12 h with TDM (bottom) in combination with various concentrations of N-glycolyl or N-acetyl MDP. (B) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left unstimulated or were stimulated for 12 h with 10 µg/ml N-acetyl MDP alone, 10 µg/ml N-glycolyl MDP alone, TDM alone and a combination of N-acetyl MDP and TDM, or N-glycolyl MDP and TDM. The amount of TNF-α and IL-6 released in the supernatant was quantified by ELISA. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with no MDP added; **, P < 0.05 between N-glycolyl versus N-acetyl MDP.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: N-glycolyl MDP is more potent than N-acetyl MDP at inducing proinflammatory cytokine production in macrophages. (A) Naive peritoneal macrophages from Nod2+/+ mice were stimulated for 6 h with LPS (top) or for 12 h with TDM (bottom) in combination with various concentrations of N-glycolyl or N-acetyl MDP. (B) Naive peritoneal macrophages from Nod2+/+ and Nod2−/− mice were either left unstimulated or were stimulated for 12 h with 10 µg/ml N-acetyl MDP alone, 10 µg/ml N-glycolyl MDP alone, TDM alone and a combination of N-acetyl MDP and TDM, or N-glycolyl MDP and TDM. The amount of TNF-α and IL-6 released in the supernatant was quantified by ELISA. Representative data from three independent replicates are shown (means ± SEM). *, P < 0.05 compared with no MDP added; **, P < 0.05 between N-glycolyl versus N-acetyl MDP.
Mentions: It is known that MDP alone is a potent inducer of TNF-α mRNA, which remains untranslated, and that presence of LPS abrogates this translation block (Wolfert et al., 2002). To compare N-glycolyl and N-acetyl MDP, we stimulated mouse peritoneal macrophages with various concentrations of MDP along with a fixed concentration of LPS, and measured TNF-α and IL-6 production (Fig. 3 A, top). At all concentrations ranging from 0.5 to 10 µg/ml, N-glycolyl MDP was more stimulatory than N-acetyl MDP. In terms of potency, N-glycolyl MDP was active at a 10-fold lower concentration than N-acetyl MDP (0.5 vs. 5 µg/ml). Because Actinomycetes do not produce LPS, we repeated this experiment using M. tuberculosis–derived trehalose 6,6′-dimycolate (TDM), a molecule that has been shown to have synergistic immunological activity in combination with MDP (Masihi et al., 1985). Again, N-glycolyl MDP was more stimulatory than N-acetyl MDP and was active at a 10- to 20-fold lower concentration. (Fig. 3 A, bottom) The synergistic response to MDP plus TDM in Nod2+/+ cells was abrogated in Nod2−/− cells (Fig. 3 B). Thus, N-glycolyl MDP is more stimulatory and more potent than N-acetyl MDP at inducing proinflammatory cytokine production upon co-stimulation with nonmycobacteria- and mycobaceria-derived molecules.

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