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A novel crosstalk between TLR4- and NOD2-mediated signaling in the regulation of intestinal inflammation.

Kim H, Zhao Q, Zheng H, Li X, Zhang T, Ma X - Sci Rep (2015)

Bottom Line: Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies.Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense.To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, Sheng Yushou Center of Cell Biology and Immunology and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China 200240.

ABSTRACT
Although Toll-like receptor 4 (TLR4)- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated signaling mechanisms have been extensively studied individually, the crosstalk between them in the regulation of intestinal mucosal defense and tissue homeostasis has been underappreciated. Here, we uncover some novel activities of NOD2 by gene expression profiling revealing the global nature of the cross-regulation between TLR4- and NOD2-mediated signaling. Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies. This balancing act is mediated through receptor-interacting serine/threonine kinase 2, and the transcriptional regulator CCAAT/enhancer-binding protein α (C/EBPα) via its serine 248 phosphorylation by Protein Kinase C. Mice deficient in C/EBPα in the hematopoietic compartment are highly susceptible to chemically induced experimental colitis in an IL-12-dependent manner. Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense. To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases.

No MeSH data available.


Related in: MedlinePlus

CD-associated NOD2 mutants selectively inhibit IL-12p35 transcription.(a) Western blot analysis of human NOD2 and its variants. The expression vectors contain a FLAG tag. Following transient transfection in duplicates, whole cell lysates were analyzed with a FLAG-specific mAb. Although cropping was used in the final image presentation for the purpose of saving space (the red-box defines the cropped boundary), all lanes were taken from the same gel in the original ordering (see Supplemental Fig. 1s for the original gel). (b) Human macrophage cell line THP-1 was transiently transfected with expression vectors for human NOD2 or its CD-associated variants as indicated. Cells were then stimulated with LPS (500 ng/ml) in the presence or absence of MDP (5 μg/ml) for 6 h, harvested and mRNA expression of IL-12p35, p40 and IL-10 analyzed by real time RT-PCR. pcDNA3 was used as an empty control vector. Data represent four independent experiments. (c) Luciferase activity in lysates of cells transfected with the human IL12p35 promoter-luciferase reporter, together with various molar ratios of vectors encoding NOD2 and 1007fs, then stimulated for 7 h with LPS before harvesting. Data are summary of 3 independent experiments. (d) IFN-γ production by human PBMCs from five healthy controls and three CD patients homozygous for 1007fs.
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f8: CD-associated NOD2 mutants selectively inhibit IL-12p35 transcription.(a) Western blot analysis of human NOD2 and its variants. The expression vectors contain a FLAG tag. Following transient transfection in duplicates, whole cell lysates were analyzed with a FLAG-specific mAb. Although cropping was used in the final image presentation for the purpose of saving space (the red-box defines the cropped boundary), all lanes were taken from the same gel in the original ordering (see Supplemental Fig. 1s for the original gel). (b) Human macrophage cell line THP-1 was transiently transfected with expression vectors for human NOD2 or its CD-associated variants as indicated. Cells were then stimulated with LPS (500 ng/ml) in the presence or absence of MDP (5 μg/ml) for 6 h, harvested and mRNA expression of IL-12p35, p40 and IL-10 analyzed by real time RT-PCR. pcDNA3 was used as an empty control vector. Data represent four independent experiments. (c) Luciferase activity in lysates of cells transfected with the human IL12p35 promoter-luciferase reporter, together with various molar ratios of vectors encoding NOD2 and 1007fs, then stimulated for 7 h with LPS before harvesting. Data are summary of 3 independent experiments. (d) IFN-γ production by human PBMCs from five healthy controls and three CD patients homozygous for 1007fs.

Mentions: To further investigate the molecular mechanisms whereby the NOD2 mutations may impact on IL-12 gene expression we expressed recombinant NOD2 and the three major CD-associated mutants: 1007fs, R702W, and G908R. These mutants were expressed at mRNA and protein levels almost as efficiently as the WT NOD2 (Fig. 8a). In contrast to the enhancing activity of the low-dose NOD2, the three mutants inhibited p35 transcription in the absence of MDP (Fig. 8b, upper), but not p40 (middle), nor IL-10 (lower). Thus, it appears that the NOD2 mutants have an “acquired activity” selectively inhibiting p35 gene transcription in a MDP-independent manner.


A novel crosstalk between TLR4- and NOD2-mediated signaling in the regulation of intestinal inflammation.

Kim H, Zhao Q, Zheng H, Li X, Zhang T, Ma X - Sci Rep (2015)

CD-associated NOD2 mutants selectively inhibit IL-12p35 transcription.(a) Western blot analysis of human NOD2 and its variants. The expression vectors contain a FLAG tag. Following transient transfection in duplicates, whole cell lysates were analyzed with a FLAG-specific mAb. Although cropping was used in the final image presentation for the purpose of saving space (the red-box defines the cropped boundary), all lanes were taken from the same gel in the original ordering (see Supplemental Fig. 1s for the original gel). (b) Human macrophage cell line THP-1 was transiently transfected with expression vectors for human NOD2 or its CD-associated variants as indicated. Cells were then stimulated with LPS (500 ng/ml) in the presence or absence of MDP (5 μg/ml) for 6 h, harvested and mRNA expression of IL-12p35, p40 and IL-10 analyzed by real time RT-PCR. pcDNA3 was used as an empty control vector. Data represent four independent experiments. (c) Luciferase activity in lysates of cells transfected with the human IL12p35 promoter-luciferase reporter, together with various molar ratios of vectors encoding NOD2 and 1007fs, then stimulated for 7 h with LPS before harvesting. Data are summary of 3 independent experiments. (d) IFN-γ production by human PBMCs from five healthy controls and three CD patients homozygous for 1007fs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495563&req=5

f8: CD-associated NOD2 mutants selectively inhibit IL-12p35 transcription.(a) Western blot analysis of human NOD2 and its variants. The expression vectors contain a FLAG tag. Following transient transfection in duplicates, whole cell lysates were analyzed with a FLAG-specific mAb. Although cropping was used in the final image presentation for the purpose of saving space (the red-box defines the cropped boundary), all lanes were taken from the same gel in the original ordering (see Supplemental Fig. 1s for the original gel). (b) Human macrophage cell line THP-1 was transiently transfected with expression vectors for human NOD2 or its CD-associated variants as indicated. Cells were then stimulated with LPS (500 ng/ml) in the presence or absence of MDP (5 μg/ml) for 6 h, harvested and mRNA expression of IL-12p35, p40 and IL-10 analyzed by real time RT-PCR. pcDNA3 was used as an empty control vector. Data represent four independent experiments. (c) Luciferase activity in lysates of cells transfected with the human IL12p35 promoter-luciferase reporter, together with various molar ratios of vectors encoding NOD2 and 1007fs, then stimulated for 7 h with LPS before harvesting. Data are summary of 3 independent experiments. (d) IFN-γ production by human PBMCs from five healthy controls and three CD patients homozygous for 1007fs.
Mentions: To further investigate the molecular mechanisms whereby the NOD2 mutations may impact on IL-12 gene expression we expressed recombinant NOD2 and the three major CD-associated mutants: 1007fs, R702W, and G908R. These mutants were expressed at mRNA and protein levels almost as efficiently as the WT NOD2 (Fig. 8a). In contrast to the enhancing activity of the low-dose NOD2, the three mutants inhibited p35 transcription in the absence of MDP (Fig. 8b, upper), but not p40 (middle), nor IL-10 (lower). Thus, it appears that the NOD2 mutants have an “acquired activity” selectively inhibiting p35 gene transcription in a MDP-independent manner.

Bottom Line: Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies.Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense.To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, Sheng Yushou Center of Cell Biology and Immunology and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China 200240.

ABSTRACT
Although Toll-like receptor 4 (TLR4)- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated signaling mechanisms have been extensively studied individually, the crosstalk between them in the regulation of intestinal mucosal defense and tissue homeostasis has been underappreciated. Here, we uncover some novel activities of NOD2 by gene expression profiling revealing the global nature of the cross-regulation between TLR4- and NOD2-mediated signaling. Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies. This balancing act is mediated through receptor-interacting serine/threonine kinase 2, and the transcriptional regulator CCAAT/enhancer-binding protein α (C/EBPα) via its serine 248 phosphorylation by Protein Kinase C. Mice deficient in C/EBPα in the hematopoietic compartment are highly susceptible to chemically induced experimental colitis in an IL-12-dependent manner. Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense. To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases.

No MeSH data available.


Related in: MedlinePlus