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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 (NOD)2-mediated signals induce IL-1β and IL-10 production by peritoneal neutrophils during cecal ligation and puncture (CLP)-induced sepsis.(A) Serum and peritoneal IL-6, IFN-γ, TNF-α, IL-1β, and IL-10 levels were measured in WT (n = 4) and Nod2−/− (n = 4) mice 24 h after CLP. (B) Peritoneal cells were obtained from WT or Nod2−/− mice 4–6 h after injection with thioglycollate and incubated with or without MDP for 24 h. The IL-1β and IL-10 concentrations in culture supernatants were measured. (C) Serum and peritoneal IL-1β and IL-10 levels were estimated in WT (n = 4) and Nod2−/− (n = 4) mice 4, 12, and 24 h after CLP using ELISA. (D) The NOD2 expression pattern was estimated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells of WT mice 4, 12, and 24 h after CLP by real-time PCR. (E) IL-1β and IL-10 transcript levels were evaluated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells from WT and Nod2−/− mice 4, 12, and 24 h after CLP. (F) Intracellular IL-1β and IL-10 expression in gated F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells was analyzed by flow cytometry 2, and 12 h after CLP, respectively. (n = 3) *P<0.05, **P<0.01, ***P<0.001 for WT vs. Nod2−/− mice (two-tailed unpaired t-test [a, b], two-way ANOVA [c, e]) Results shown are representative of three independent experiments (mean and SEM).
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ppat-1003351-g002: Nucleotide-binding oligomerization domain (NOD)2-mediated signals induce IL-1β and IL-10 production by peritoneal neutrophils during cecal ligation and puncture (CLP)-induced sepsis.(A) Serum and peritoneal IL-6, IFN-γ, TNF-α, IL-1β, and IL-10 levels were measured in WT (n = 4) and Nod2−/− (n = 4) mice 24 h after CLP. (B) Peritoneal cells were obtained from WT or Nod2−/− mice 4–6 h after injection with thioglycollate and incubated with or without MDP for 24 h. The IL-1β and IL-10 concentrations in culture supernatants were measured. (C) Serum and peritoneal IL-1β and IL-10 levels were estimated in WT (n = 4) and Nod2−/− (n = 4) mice 4, 12, and 24 h after CLP using ELISA. (D) The NOD2 expression pattern was estimated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells of WT mice 4, 12, and 24 h after CLP by real-time PCR. (E) IL-1β and IL-10 transcript levels were evaluated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells from WT and Nod2−/− mice 4, 12, and 24 h after CLP. (F) Intracellular IL-1β and IL-10 expression in gated F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells was analyzed by flow cytometry 2, and 12 h after CLP, respectively. (n = 3) *P<0.05, **P<0.01, ***P<0.001 for WT vs. Nod2−/− mice (two-tailed unpaired t-test [a, b], two-way ANOVA [c, e]) Results shown are representative of three independent experiments (mean and SEM).

Mentions: To explore the mechanism by which NOD2 enhances C5 generation during sepsis, the serum and peritoneal levels of various cytokines in WT and Nod2−/− mice were estimated after CLP. The serum and peritoneal IL-1β and IL-10 levels of WT mice were significantly higher than those of Nod2−/− mice, whereas IL-6 and IFN-γ levels in WT mice were similar to those in Nod2−/− mice (Fig. 2A). Serum TNF-α levels were higher in WT than Nod2−/− mice, whereas its peritoneal levels were similar in two mouse groups. To estimate IL-1β and IL-10 production by peritoneal cells, we obtained total peritoneal cells from WT and Nod2−/− mice 4–6 h after injection with thioglycollate. Upon MDP, an NOD2 agonist treatment, WT peritoneal cells produced IL-1β and IL-10, whereas NOD2-deficient cells produced minimal IL-1β and IL-10 (Fig. 2B). A kinetic analysis revealed that IL-1β and IL-10 levels in peritoneal fluid peaked 4 and 12 h after CLP, respectively, and then decreased gradually (Fig. 2C). Serum IL-1β levels peaked 12 h after CLP and were significantly higher in WT than Nod2−/− mice at 24 h, whereas serum IL-10 levels in WT mice increased continuously from 4 to 24 h after CLP, and were significantly higher than those in Nod2−/− mice. Subset analysis revealed that F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages were major cells infiltrated into peritoneum during sepsis (Fig. S2) and the numbers of these cells was similar in Nod2−/− and WT mice (data not shown). The percentages of neutrophils peaked and macrophages showed the lowest percentages in Nod2−/− and WT mice 4 h after CLP, and the percentages of these cells were similar in WT and Nod2−/− mice before, 4, 12, and 24 h after CLP (Fig. S2). Based on these findings, NOD2 expression was investigated in sorted F4/80+Ly-6G− macrophages and F4/80−Ly-6G+ neutrophils from peritoneum of WT mice with CLP (Fig. 2D). Real-time PCR analysis revealed that NOD2 expression in F4/80−Ly-6G+ neutrophils was constitutive and sustained during sepsis, whereas F4/80+Ly-6G− macrophages showed low expression levels of NOD2 before, and 4 and 12 h after CLP, but highly expressed NOD2 24 h after CLP, which was consistent with expression pattern of NOD2 in blotting assay (S3A and B). These findings indicate that F4/80−Ly-6G+ neutrophils rather than F4/80+Ly-6G− macrophages in the peritoneum predominantly express NOD2 during early and intermediate stages of sepsis. Consistent with the kinetics of the IL-1β and IL-10 levels, peritoneal F4/80−Ly-6G+ neutrophils from WT mice showed high IL-1β mRNA levels at 4 and 24 h, but low transcriptional levels at 12 h (Fig. 2E). In contrast, peritoneal F4/80+Ly-6G− macrophages produced high levels of IL-1β at 24 h. Unlike IL-1β, peritoneal F4/80−Ly-6G+ neutrophils from WT mice predominantly produced IL-10 at 12 h. Although the kinetics of IL-1β and IL-10 production were similar in Nod2−/− and WT mice, individual cytokine levels were much lower in the former, which were consistent with results in intracellular staining (Fig. 2F). To investigate whether peritoneal neutrophils produce IL-1β and IL-10 during sepsis, peritoneal F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages from Nod2−/− and WT mice with CLP were obtained, sorted, and cultured for 24 h without stimulation. F4/80−Ly-6G+ neutrophils from WT mice produced larger amount of IL-1β and IL-10 than WT F4/80+Ly-6G− macrophages did (Fig. S4A). In contrast, F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages from Nod2−/− mice minimally produced IL-1β and IL-10. Furthermore, neutrophil depletion using anti-Ly-6G mAb in WT mice reduced the levels of IL-1β and IL-10 in serum and peritoneum, which was dependent on depletion time points (Fig. S4B and C). Combined in vitro and depletion experiments, it is suggested that peritoneal neutrophils rather than macrophages directly produce IL-1β and IL-10 at different time points during sepsis, although neutrophils might interact with macrophages or monocytes to produce various cytokines during CLP-induced sepsis.


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 (NOD)2-mediated signals induce IL-1β and IL-10 production by peritoneal neutrophils during cecal ligation and puncture (CLP)-induced sepsis.(A) Serum and peritoneal IL-6, IFN-γ, TNF-α, IL-1β, and IL-10 levels were measured in WT (n = 4) and Nod2−/− (n = 4) mice 24 h after CLP. (B) Peritoneal cells were obtained from WT or Nod2−/− mice 4–6 h after injection with thioglycollate and incubated with or without MDP for 24 h. The IL-1β and IL-10 concentrations in culture supernatants were measured. (C) Serum and peritoneal IL-1β and IL-10 levels were estimated in WT (n = 4) and Nod2−/− (n = 4) mice 4, 12, and 24 h after CLP using ELISA. (D) The NOD2 expression pattern was estimated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells of WT mice 4, 12, and 24 h after CLP by real-time PCR. (E) IL-1β and IL-10 transcript levels were evaluated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells from WT and Nod2−/− mice 4, 12, and 24 h after CLP. (F) Intracellular IL-1β and IL-10 expression in gated F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells was analyzed by flow cytometry 2, and 12 h after CLP, respectively. (n = 3) *P<0.05, **P<0.01, ***P<0.001 for WT vs. Nod2−/− mice (two-tailed unpaired t-test [a, b], two-way ANOVA [c, e]) Results shown are representative of three independent experiments (mean and SEM).
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ppat-1003351-g002: Nucleotide-binding oligomerization domain (NOD)2-mediated signals induce IL-1β and IL-10 production by peritoneal neutrophils during cecal ligation and puncture (CLP)-induced sepsis.(A) Serum and peritoneal IL-6, IFN-γ, TNF-α, IL-1β, and IL-10 levels were measured in WT (n = 4) and Nod2−/− (n = 4) mice 24 h after CLP. (B) Peritoneal cells were obtained from WT or Nod2−/− mice 4–6 h after injection with thioglycollate and incubated with or without MDP for 24 h. The IL-1β and IL-10 concentrations in culture supernatants were measured. (C) Serum and peritoneal IL-1β and IL-10 levels were estimated in WT (n = 4) and Nod2−/− (n = 4) mice 4, 12, and 24 h after CLP using ELISA. (D) The NOD2 expression pattern was estimated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells of WT mice 4, 12, and 24 h after CLP by real-time PCR. (E) IL-1β and IL-10 transcript levels were evaluated in sorted F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells from WT and Nod2−/− mice 4, 12, and 24 h after CLP. (F) Intracellular IL-1β and IL-10 expression in gated F4/80−Ly-6G+ and F4/80+Ly-6G− peritoneal cells was analyzed by flow cytometry 2, and 12 h after CLP, respectively. (n = 3) *P<0.05, **P<0.01, ***P<0.001 for WT vs. Nod2−/− mice (two-tailed unpaired t-test [a, b], two-way ANOVA [c, e]) Results shown are representative of three independent experiments (mean and SEM).
Mentions: To explore the mechanism by which NOD2 enhances C5 generation during sepsis, the serum and peritoneal levels of various cytokines in WT and Nod2−/− mice were estimated after CLP. The serum and peritoneal IL-1β and IL-10 levels of WT mice were significantly higher than those of Nod2−/− mice, whereas IL-6 and IFN-γ levels in WT mice were similar to those in Nod2−/− mice (Fig. 2A). Serum TNF-α levels were higher in WT than Nod2−/− mice, whereas its peritoneal levels were similar in two mouse groups. To estimate IL-1β and IL-10 production by peritoneal cells, we obtained total peritoneal cells from WT and Nod2−/− mice 4–6 h after injection with thioglycollate. Upon MDP, an NOD2 agonist treatment, WT peritoneal cells produced IL-1β and IL-10, whereas NOD2-deficient cells produced minimal IL-1β and IL-10 (Fig. 2B). A kinetic analysis revealed that IL-1β and IL-10 levels in peritoneal fluid peaked 4 and 12 h after CLP, respectively, and then decreased gradually (Fig. 2C). Serum IL-1β levels peaked 12 h after CLP and were significantly higher in WT than Nod2−/− mice at 24 h, whereas serum IL-10 levels in WT mice increased continuously from 4 to 24 h after CLP, and were significantly higher than those in Nod2−/− mice. Subset analysis revealed that F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages were major cells infiltrated into peritoneum during sepsis (Fig. S2) and the numbers of these cells was similar in Nod2−/− and WT mice (data not shown). The percentages of neutrophils peaked and macrophages showed the lowest percentages in Nod2−/− and WT mice 4 h after CLP, and the percentages of these cells were similar in WT and Nod2−/− mice before, 4, 12, and 24 h after CLP (Fig. S2). Based on these findings, NOD2 expression was investigated in sorted F4/80+Ly-6G− macrophages and F4/80−Ly-6G+ neutrophils from peritoneum of WT mice with CLP (Fig. 2D). Real-time PCR analysis revealed that NOD2 expression in F4/80−Ly-6G+ neutrophils was constitutive and sustained during sepsis, whereas F4/80+Ly-6G− macrophages showed low expression levels of NOD2 before, and 4 and 12 h after CLP, but highly expressed NOD2 24 h after CLP, which was consistent with expression pattern of NOD2 in blotting assay (S3A and B). These findings indicate that F4/80−Ly-6G+ neutrophils rather than F4/80+Ly-6G− macrophages in the peritoneum predominantly express NOD2 during early and intermediate stages of sepsis. Consistent with the kinetics of the IL-1β and IL-10 levels, peritoneal F4/80−Ly-6G+ neutrophils from WT mice showed high IL-1β mRNA levels at 4 and 24 h, but low transcriptional levels at 12 h (Fig. 2E). In contrast, peritoneal F4/80+Ly-6G− macrophages produced high levels of IL-1β at 24 h. Unlike IL-1β, peritoneal F4/80−Ly-6G+ neutrophils from WT mice predominantly produced IL-10 at 12 h. Although the kinetics of IL-1β and IL-10 production were similar in Nod2−/− and WT mice, individual cytokine levels were much lower in the former, which were consistent with results in intracellular staining (Fig. 2F). To investigate whether peritoneal neutrophils produce IL-1β and IL-10 during sepsis, peritoneal F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages from Nod2−/− and WT mice with CLP were obtained, sorted, and cultured for 24 h without stimulation. F4/80−Ly-6G+ neutrophils from WT mice produced larger amount of IL-1β and IL-10 than WT F4/80+Ly-6G− macrophages did (Fig. S4A). In contrast, F4/80−Ly-6G+ neutrophils and F4/80+Ly-6G− macrophages from Nod2−/− mice minimally produced IL-1β and IL-10. Furthermore, neutrophil depletion using anti-Ly-6G mAb in WT mice reduced the levels of IL-1β and IL-10 in serum and peritoneum, which was dependent on depletion time points (Fig. S4B and C). Combined in vitro and depletion experiments, it is suggested that peritoneal neutrophils rather than macrophages directly produce IL-1β and IL-10 at different time points during sepsis, although neutrophils might interact with macrophages or monocytes to produce various cytokines during CLP-induced sepsis.

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