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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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Nucleotide-binding oligomerization domain (Nod2)−/− mice exhibit attenuated cecal ligation and puncture (CLP)-induced sepsis through suppression of C5a generation.To induce sepsis, the cecum of wild-type (WT) or Nod2−/− mice was ligated and punctured. (A) Serum and peritoneal C5a and C3a levels were evaluated in WT and Nod2−/− mice 24 h after CLP (n = 4). (B) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.951[not significant], bP = 0.0077, cP = 0.0141, log-rank test; WT [n = 12], WT mice injected with recombinant C5a [n = 8], Nod2−/− [n = 8], Nod2−/− mice injected with recombinant C5a [ n = 8]) (C) Peritoneal cells obtained from WT (n = 4), Nod2−/− (n = 4), or Nod2−/− mice injected with recombinant C5a (n = 6) 4 and 12 h after CLP were incubated with LPS or PBS for 6 h and cytokine levels were measured. The responsiveness of peritoneal cells to LPS was determined by estimating the ratios of individual cytokines produced in response to LPS versus PBS. (D) Serum D-dimer levels were measured in WT (n = 5), Nod2−/−(n = 5), Nod2−/− mice injected with recombinant C5a (n = 4) 24 h after CLP. (E) Phagocytosis activity of peritoneal cells obtained from mice 24 h after CLP was determined by measuring the percentage of cells with intracellular FITC-conjugated E. coli after 15 min incubation. (n = 3) (F) Bacterial CFUs were counted using blood and liver homogenates obtained from mice 24 h after CLP. (n = 3) *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c–f]) for WT B6 vs. Nod2−/− mice and Nod2−/− vs. Nod2−/− mice injected with recombinant C5a. Results shown are representative of three independent experiments except for (B) (mean and SEM).
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ppat-1003351-g001: Nucleotide-binding oligomerization domain (Nod2)−/− mice exhibit attenuated cecal ligation and puncture (CLP)-induced sepsis through suppression of C5a generation.To induce sepsis, the cecum of wild-type (WT) or Nod2−/− mice was ligated and punctured. (A) Serum and peritoneal C5a and C3a levels were evaluated in WT and Nod2−/− mice 24 h after CLP (n = 4). (B) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.951[not significant], bP = 0.0077, cP = 0.0141, log-rank test; WT [n = 12], WT mice injected with recombinant C5a [n = 8], Nod2−/− [n = 8], Nod2−/− mice injected with recombinant C5a [ n = 8]) (C) Peritoneal cells obtained from WT (n = 4), Nod2−/− (n = 4), or Nod2−/− mice injected with recombinant C5a (n = 6) 4 and 12 h after CLP were incubated with LPS or PBS for 6 h and cytokine levels were measured. The responsiveness of peritoneal cells to LPS was determined by estimating the ratios of individual cytokines produced in response to LPS versus PBS. (D) Serum D-dimer levels were measured in WT (n = 5), Nod2−/−(n = 5), Nod2−/− mice injected with recombinant C5a (n = 4) 24 h after CLP. (E) Phagocytosis activity of peritoneal cells obtained from mice 24 h after CLP was determined by measuring the percentage of cells with intracellular FITC-conjugated E. coli after 15 min incubation. (n = 3) (F) Bacterial CFUs were counted using blood and liver homogenates obtained from mice 24 h after CLP. (n = 3) *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c–f]) for WT B6 vs. Nod2−/− mice and Nod2−/− vs. Nod2−/− mice injected with recombinant C5a. Results shown are representative of three independent experiments except for (B) (mean and SEM).

Mentions: To investigate whether NOD2 regulates C5a generation during sepsis, we performed CLP in wild-type (WT) and Nod2−/− mice. Serum and peritoneal C5a levels were lower in Nod2−/− than in WT mice during sepsis, whereas C3a levels were similar (Fig. 1A). All Nod2−/− mice were alive up to 10 days after CLP, whereas all WT mice died within 2 days. However, ELISA system for C5a might detect C5 in some cases, although cross reactivity for C5a and C5 in detection system does not usually occur. Thus, to confirm harmful effects of NOD2-mediated C5a generation on sepsis, we injected WT and Nod2−/− mice with recombinant (r)C5a during CLP-induced sepsis. Injection of Nod2−/− mice with rC5a decreased survival rates during sepsis, whereas rC5a had no effect on survival of WT mice (Fig. 1B). These findings suggest that NOD2-mediated C5a generation contributes to sepsis development and severity. NOD2 was recently reported to affect composition of the host microbiota in mice and humans [21]. To rule out differences in the cecal bacterial composition of WT and Nod2−/− mice might affect sepsis, cecal contents obtained from WT or Nod2−/− mice were injected i.p. into WT and Nod2−/− mice after their ceca had been ligated but not punctured and WT and Nod2−/− mice were cohoused for 4 weeks. The survival rates after injection of Nod2−/− or WT cecal contents were lower in WT mice than Nod2−/− mice (Fig. S1). Moreover, survival rates of cohoused WT mice were lower than cohoused Nod2−/− mice during CLP-induced sepsis (Fig. S1), indicating that difference in intestinal microbiota between two mouse groups minimally contributes to septic responses because gut microbiota of cohoused mice are replaced by each other [22]. Collectively, these findings suggest that NOD2-mediated signals enhance generation of C5a, but not C3a, thereby enhancing the systemic inflammatory response.


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

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

Nucleotide-binding oligomerization domain (Nod2)−/− mice exhibit attenuated cecal ligation and puncture (CLP)-induced sepsis through suppression of C5a generation.To induce sepsis, the cecum of wild-type (WT) or Nod2−/− mice was ligated and punctured. (A) Serum and peritoneal C5a and C3a levels were evaluated in WT and Nod2−/− mice 24 h after CLP (n = 4). (B) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.951[not significant], bP = 0.0077, cP = 0.0141, log-rank test; WT [n = 12], WT mice injected with recombinant C5a [n = 8], Nod2−/− [n = 8], Nod2−/− mice injected with recombinant C5a [ n = 8]) (C) Peritoneal cells obtained from WT (n = 4), Nod2−/− (n = 4), or Nod2−/− mice injected with recombinant C5a (n = 6) 4 and 12 h after CLP were incubated with LPS or PBS for 6 h and cytokine levels were measured. The responsiveness of peritoneal cells to LPS was determined by estimating the ratios of individual cytokines produced in response to LPS versus PBS. (D) Serum D-dimer levels were measured in WT (n = 5), Nod2−/−(n = 5), Nod2−/− mice injected with recombinant C5a (n = 4) 24 h after CLP. (E) Phagocytosis activity of peritoneal cells obtained from mice 24 h after CLP was determined by measuring the percentage of cells with intracellular FITC-conjugated E. coli after 15 min incubation. (n = 3) (F) Bacterial CFUs were counted using blood and liver homogenates obtained from mice 24 h after CLP. (n = 3) *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c–f]) for WT B6 vs. Nod2−/− mice and Nod2−/− vs. Nod2−/− mice injected with recombinant C5a. Results shown are representative of three independent experiments except for (B) (mean and SEM).
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ppat-1003351-g001: Nucleotide-binding oligomerization domain (Nod2)−/− mice exhibit attenuated cecal ligation and puncture (CLP)-induced sepsis through suppression of C5a generation.To induce sepsis, the cecum of wild-type (WT) or Nod2−/− mice was ligated and punctured. (A) Serum and peritoneal C5a and C3a levels were evaluated in WT and Nod2−/− mice 24 h after CLP (n = 4). (B) The percentages of surviving mice were estimated during CLP-induced sepsis (aP = 0.951[not significant], bP = 0.0077, cP = 0.0141, log-rank test; WT [n = 12], WT mice injected with recombinant C5a [n = 8], Nod2−/− [n = 8], Nod2−/− mice injected with recombinant C5a [ n = 8]) (C) Peritoneal cells obtained from WT (n = 4), Nod2−/− (n = 4), or Nod2−/− mice injected with recombinant C5a (n = 6) 4 and 12 h after CLP were incubated with LPS or PBS for 6 h and cytokine levels were measured. The responsiveness of peritoneal cells to LPS was determined by estimating the ratios of individual cytokines produced in response to LPS versus PBS. (D) Serum D-dimer levels were measured in WT (n = 5), Nod2−/−(n = 5), Nod2−/− mice injected with recombinant C5a (n = 4) 24 h after CLP. (E) Phagocytosis activity of peritoneal cells obtained from mice 24 h after CLP was determined by measuring the percentage of cells with intracellular FITC-conjugated E. coli after 15 min incubation. (n = 3) (F) Bacterial CFUs were counted using blood and liver homogenates obtained from mice 24 h after CLP. (n = 3) *P<0.05, **P<0.01, ***P<0.001 (two-tailed unpaired t-test [a, c–f]) for WT B6 vs. Nod2−/− mice and Nod2−/− vs. Nod2−/− mice injected with recombinant C5a. Results shown are representative of three independent experiments except for (B) (mean and SEM).
Mentions: To investigate whether NOD2 regulates C5a generation during sepsis, we performed CLP in wild-type (WT) and Nod2−/− mice. Serum and peritoneal C5a levels were lower in Nod2−/− than in WT mice during sepsis, whereas C3a levels were similar (Fig. 1A). All Nod2−/− mice were alive up to 10 days after CLP, whereas all WT mice died within 2 days. However, ELISA system for C5a might detect C5 in some cases, although cross reactivity for C5a and C5 in detection system does not usually occur. Thus, to confirm harmful effects of NOD2-mediated C5a generation on sepsis, we injected WT and Nod2−/− mice with recombinant (r)C5a during CLP-induced sepsis. Injection of Nod2−/− mice with rC5a decreased survival rates during sepsis, whereas rC5a had no effect on survival of WT mice (Fig. 1B). These findings suggest that NOD2-mediated C5a generation contributes to sepsis development and severity. NOD2 was recently reported to affect composition of the host microbiota in mice and humans [21]. To rule out differences in the cecal bacterial composition of WT and Nod2−/− mice might affect sepsis, cecal contents obtained from WT or Nod2−/− mice were injected i.p. into WT and Nod2−/− mice after their ceca had been ligated but not punctured and WT and Nod2−/− mice were cohoused for 4 weeks. The survival rates after injection of Nod2−/− or WT cecal contents were lower in WT mice than Nod2−/− mice (Fig. S1). Moreover, survival rates of cohoused WT mice were lower than cohoused Nod2−/− mice during CLP-induced sepsis (Fig. S1), indicating that difference in intestinal microbiota between two mouse groups minimally contributes to septic responses because gut microbiota of cohoused mice are replaced by each other [22]. Collectively, these findings suggest that NOD2-mediated signals enhance generation of C5a, but not C3a, thereby enhancing the systemic inflammatory response.

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