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NOD2-mediated suppression of CD55 on neutrophils enhances C5a generation during polymicrobial sepsis.

Oh SJ, Kim JH, Chung DH - PLoS Pathog. (2013)

Bottom Line: Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans.Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis.SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2-/- mice. Nod2-/- mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2-/- mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.

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SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis.(A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2−/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2−/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2−/−) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80−Ly6-G+ cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c]). Results shown are representative of three independent experiments except for (E) (mean and SEM).
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ppat-1003351-g006: SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis.(A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2−/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2−/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2−/−) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80−Ly6-G+ cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c]). Results shown are representative of three independent experiments except for (E) (mean and SEM).

Mentions: Upon activation, NOD2 oligomerizes and recruits RIP2 via CARD-CARD interaction, triggering IκB phosphorylation and NF-κB activation [27], [28]. SB203580, an inhibitor of RIP2 and P38 [29], inhibited MDP-mediated IL-1β and IL-10 production by total peritoneal cells from WT mice (Fig. 6A). Furthermore, Nod2−/− mice showed lower levels of RIP2 expression and phosphorylation, and P38 phosphorylation in total peritoneal cells during sepsis than did WT mice (Fig. 6B). Upon SB203580 injection, WT mice exhibited reduced RIP2 expression and phosphorylation, and P38 phosphorylation in total peritoneal cells and serum and peritoneal IL-1β, IL-10, and C5a levels during sepsis, whereas these were unaffected in Nod2−/− mice (Fig. 6B and C). Moreover, SB203580 administration to WT mice increased CD55 expression levels in F4/80−Ly-6G+ neutrophils, and increased survival rates during sepsis (Fig. 6D and E). These findings suggest that NOD2 blockade inhibits C5a generation by enhancing CD55 expression on neutrophils, depending on IL-1β and IL-10 production by neutrophils and resulting in increased survival rates.


NOD2-mediated suppression of CD55 on neutrophils enhances C5a generation during polymicrobial sepsis.

Oh SJ, Kim JH, Chung DH - PLoS Pathog. (2013)

SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis.(A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2−/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2−/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2−/−) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80−Ly6-G+ cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c]). Results shown are representative of three independent experiments except for (E) (mean and SEM).
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getmorefigures.php?uid=PMC3649968&req=5

ppat-1003351-g006: SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis.(A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2−/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2−/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2−/−) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80−Ly6-G+ cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c]). Results shown are representative of three independent experiments except for (E) (mean and SEM).
Mentions: Upon activation, NOD2 oligomerizes and recruits RIP2 via CARD-CARD interaction, triggering IκB phosphorylation and NF-κB activation [27], [28]. SB203580, an inhibitor of RIP2 and P38 [29], inhibited MDP-mediated IL-1β and IL-10 production by total peritoneal cells from WT mice (Fig. 6A). Furthermore, Nod2−/− mice showed lower levels of RIP2 expression and phosphorylation, and P38 phosphorylation in total peritoneal cells during sepsis than did WT mice (Fig. 6B). Upon SB203580 injection, WT mice exhibited reduced RIP2 expression and phosphorylation, and P38 phosphorylation in total peritoneal cells and serum and peritoneal IL-1β, IL-10, and C5a levels during sepsis, whereas these were unaffected in Nod2−/− mice (Fig. 6B and C). Moreover, SB203580 administration to WT mice increased CD55 expression levels in F4/80−Ly-6G+ neutrophils, and increased survival rates during sepsis (Fig. 6D and E). These findings suggest that NOD2 blockade inhibits C5a generation by enhancing CD55 expression on neutrophils, depending on IL-1β and IL-10 production by neutrophils and resulting in increased survival rates.

Bottom Line: Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans.Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis.SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2-/- mice. Nod2-/- mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2-/- mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.

Show MeSH
Related in: MedlinePlus