Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in VSports app下载. gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2008 Feb;118(2):545-59.
doi: 10.1172/JCI33145.

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis (VSports)

Tomohiro Watanabe  1 , Naoki Asano, Peter J Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J Fuss, Atsushi Kitani, Warren Strober
Affiliations

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis

Tomohiro Watanabe et al. J Clin Invest. 2008 Feb.

Abstract

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease. VSports手机版.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Systemic administration of MDP prevents the development of TNBS colitis.
C57BL/10 mice were administered MDP or PBS (i.p.; see Methods) on days –3, –2, and –1 and then challenged with intrarectal TNBS on day 0. (A) Changes in body weight in mice treated with PBS or MDP (n = 10) and challenged with intrarectal administration of TNBS. **P < 0.01 compared with mice treated with PBS. (B) H&E-stained colonic tissue of mice harvested on day 4. Histology of PBS-treated mice showed massive infiltration of mononuclear cells as well as destruction of crypt architecture (top row); histology of MDP-treated mice showed almost normal colonic tissue with minimal infiltration of mononuclear cells (bottom row). Original magnification, ×100. (C) Histology score of the colonic tissue of the mice harvested on day 4.
Figure 2
Figure 2. MDP administration reduces the TLR-induced cytokine responses of MLN and colonic LP cells from mice with TNBS colitis.
(A) Colon LP lymphocytes (1 × 106/ml) isolated from the mice on day 3 were stimulated with PGN, Pam3CSK4, LPS, and CpG in the presence of IFN-γ (20 ng/ml); cultured supernatants were collected at 48 hours and analyzed for cytokine production. *P < 0.05; **P < 0.01 when supernatants from MDP-treated mice were compared with supernatants from PBS-treated mice. (B) MLN cells (1 × 106/ml) isolated from mice with TNBS colitis on day 3 were stimulated with anti-CD3 (1 μg/ml) and a broad range of TLR ligands. Cultured supernatants were collected at 48 hours and analyzed for cytokine production. *P < 0.05; **P < 0.01 when supernatants of MDP-treated mice were compared with supernatants of PBS-treated mice. (C) MLN cells and colon LP lymphocytes isolated from the mice on day 3 were stimulated with anti-CD3 (1 μg/ml); cultured supernatants were collected at 48 hours and analyzed for IFN-γ production. **P < 0.01 when supernatants from MDP-treated mice were compared with supernatants from PBS-treated mice. (D) Evaluation of NF-κB activation in MLN cells isolated from the mice on day 3 and stimulated with LPS or PGN. Nuclear extracts were prepared from MLN cells isolated from PBS- or MDP-treated mice and stimulated with LPS or PGN for 2 hours and then subjected to EMSA. (E) Nuclear extracts obtained in D assayed for p65 and c-Rel activation using NF-κB transcription factor ELISA. *P < 0.05; **P < 0.01 compared with nuclear extracts from PBS-treated mice.
Figure 3
Figure 3. MDP administration prevents DSS colitis.
NOD2-intact (NOD2+/+) and NOD2-deficient (NOD2–/–) mice were treated with drinking water containing 4% DSS for 6 days (days 0–5). At the early phase of colitis induction (days 0, 1, 2), mice were administered MDP (i.p.) or PBS every day. (A) Changes of body weight in PBS- or MDP-injected mice. *P < 0.05; **P < 0.01, time point values of NOD2-intact mice administered MDP compared with NOD2-intact mice administered PBS. (B) Serum amyloid A (SAA) levels and colitis scores of mice (see Methods) on day 7. SAA levels were determined by ELISA. *P < 0.05, NOD2-intact mice administered MDP compared with mice administered PBS.
Figure 4
Figure 4. Cytokine production by MLN cells in mice treated with DSS.
(A) MLN cells (1 × 106/ml) isolated from NOD2+/+ and NOD2–/– mice on day 5 were stimulated with a broad range of TLR ligands. Cultured supernatants were collected at 48 hours and analyzed for cytokine production by ELISA. *P < 0.05 when supernatants were compared with supernatants from mice treated with PBS (white bars). (B) Activation of NF-κB in MLN cells isolated from NOD2+/+ and NOD2–/– mice on day 5 following stimulation with LPS or PGN. Nuclear extracts were prepared from MLN cells isolated from PBS- or MDP-treated mice and stimulated with LPS or PGN for 2 hours and then subjected to EMSA. (C) Nuclear extracts obtained in B assayed for p65 and c-Rel activation using NF-κB transcription factor ELISA. *P < 0.05 compared with nuclear extracts from PBS-treated mice.
Figure 5
Figure 5. DSS colitis in MDP-treated NOD2-deficient mice reconstituted with intact or frameshift NOD2.
NOD2-deficient (NOD2–/–) mice were treated with drinking water containing 5.5% DSS for 6 days (days 0–5). At an early phase of colitis induction (days 0, 1, 2), mice were administered MDP and HVJ-encapsulated plasmid (see Methods). (A) Changes in body weight in MDP-administered NOD2-deficient mice reconstituted with intact NOD2, frameshift NOD2, or control empty vector. Weights of MDP-administered NOD2-deficient mice given DSS are shown as a control. **P < 0.01, time point values of intact-NOD2–reconstituted mice compared with control empty vector–reconstituted mice. (B) H&E-stained colonic tissue of the mice harvested on day 7. Original magnification, ×50.
Figure 6
Figure 6. Human and mouse DCs prestimulated with MDP exhibit reduced cytokine and chemokine production when stimulated with TLR ligands.
(A) Human monocyte–derived DCs (1 × 106/ml) from 6 healthy donors were preincubated with MDP or medium for 24 hours and then stimulated with a broad range of TLR ligands alone or in combination with MDP for an additional 24 hours. Cultured supernatants were collected at 24 hours and analyzed for cytokine and chemokine production by ELISA. *P < 0.05; **P < 0.01 compared with supernatants from DCs preincubated with medium and stimulated with TLR ligands alone (light blue bars). (B) CD11c+ DCs (1 × 106/ml) derived from bone marrow cells from NOD2-intact (NOD2+/+) and NOD2-deficient (NOD2–/–) mice were preincubated with MDP (50 μg/ml) or medium alone for 24 hours and stimulated with a broad range of TLR ligands. Cultured supernatants were collected at 24 hours and analyzed for cytokine production by ELISA. *P < 0.05; **P < 0.01 when supernatants were compared with NOD2-intact DCs preincubated with medium and stimulated with TLR ligands (light blue bars). (C) OVA323-339 peptide–specific CD4+ T cells (OT-II) were purified from the spleens of OT-II transgenic mice; OT-II cells (1 × 106/ml) were cocultured with NOD2-intact or NOD2-deficient BMDCs (2 × 106/ml) in the presence of a broad range of TLR ligands and OVA peptide (0.5 μM); cultured supernatants were collected at 72 hours and analyzed for IFN-γ production by ELISA. *P < 0.05; **P < 0.01 compared with supernatants from NOD2-intact DCs preincubated with medium and stimulated with TLR ligands (light blue bars).
Figure 7
Figure 7. NOD2 stimulation is associated with upregulation of IRF4 expression.
(A) NF-κB activation in human monocyte–derived DCs. DCs were preincubated with MDP or medium for 24 hours and then stimulated with LPS, PGN, or flagellin for 2 hours; nuclear extracts of the cells were then obtained and subjected to gel-shift assays; results shown are representative of those obtained with 2 healthy donors. (B) Upregulation of IRF4 in MDP-stimulated human monocyte–derived DCs. Whole-cell extracts prepared from DCs incubated with MDP, LPS, or medium for 24 hours were immunoblotted with Abs against the indicated components; results shown are representative of those obtained in 3 healthy donors. (C) IRF4 expression in monocyte-derived DCs transfected with IRF4 siRNA. DCs were transfected with 2 μg of IRF4 siRNA, IRAK-M siRNA, or control siRNA using the Amaxa nucleofection method; 16 hours after transfection, DCs were stimulated with MDP, LPS, or medium for 24 hours, at which point whole-cell extracts were prepared and subjected to immunoblotting with Abs against the indicated components. (D) Effects of IRF4 or IRAK-M siRNA transfection on cytokine production by human monocyte–derived DCs. DCs (2 × 107/ml) from 6 healthy donors were transfected with IRF4 siRNA, IRAK-M siRNA, or control siRNA, as described in C. After 24 hours of culture with MDP, LPS, or medium, DCs were stimulated with PGN, Pam3CSK4, or LPS for another 24 hours; cultured supernatants were assayed for IL-12p40 by ELISA. *P < 0.05; **P < 0.01 compared with DCs transfected with control siRNA and preincubated with medium (white bars).
Figure 8
Figure 8. Mechanism of NOD2-induced IRF4 inhibition of TLR signaling.
(A) Whole-cell extracts were prepared from THP1 cells stimulated with MDP or LPS for 24 hours and then immunoblotted with Abs against IRF4, IRAK-M, and actin. (B) THP1 cells (5 × 105/ml) were prestimulated with MDP, LPS, or medium for 24 hours and stimulated with TLR ligands; cultured supernatants were collected at 24 hours and analyzed for cytokine production by ELISA. *P < 0.05 compared with the concentrations of cytokines by cells preincubated with medium and stimulated with TLR ligands (white bars). (C) Physical interactions between IRF4 and RICK, MyD88, and TRAF6. Whole-cell extracts of HEK293 cells transfected with vectors (2 μg) expressing FLAG-tagged human IRF4 and HA-tagged human MyD88 or with untagged RICK, TRAF6, or TRAF2 were immunoprecipitated with anti-FLAG–conjugated beads and then immunoblotted with anti-HA Abs or with anti-RICK, -TRAF6, or -TRAF2. (D) Negative regulation of NF-κB by IRF4. HT-29 cells (1 × 105/96-well plate) transfected with pNF-κB–Luc (50 ng) and pSV–β-galactosidase (10 ng) were cotransfected with vectors expressing human RICK (200 ng), human MyD88 (200 ng), or human TRAF6 (200 ng) with or without an IRF4-expressing vector (50 ng, 200 ng, 1000 ng). *P < 0.05; **P < 0.01 compared with cells without IRF4 transfection (white bars). (E) Physical interaction of IRF4 and RICK in MDP-prestimulated human DCs. DCs were cultured with MDP or medium for 24 hours and then stimulated with Pam3CSK4 for an additional hour; whole-cell extracts were prepared and then immunoprecipitated with anti-IRF4 Abs and immunoblotted with anti-RICK Abs.
Figure 9
Figure 9. Systemic administration of MDP prevents the development of TNBS colitis by upregulating IRF4 expression.
Mice administered intrarectal TNBS on day 0 were injected with MDP or PBS i.p. on days –3, –2, and –1 and also administered 100 μg of HVJ-encapsulated control siRNA or IRF4 siRNA by intrarectal instillation on days –2, –1, 0, and 1. (A) IRF4 expression in CD11b+ myeloid cells from MLNs and spleens from mice on day 0 (top); IRF4 expression in whole-cell extracts of CD11b+ myeloid cells from MLNs isolated from mice treated with IRF4 siRNA on day 0 (bottom). (B) Changes of body weight in mice treated with MDP and siRNAs. **P < 0.01 compared with body weight of mice treated with PBS. (C) H&E-stained colonic tissue of mice harvested on day 4. Histology of PBS-treated mice and IRF4 siRNA–treated mice showed massive infiltration of mononuclear cells as well as destruction of crypt architecture; histology of control siRNA-treated mice showed almost normal colon tissue with minimal infiltration of mononuclear cells. Original magnification, ×100. (D) MLN cells (1 × 106/ml) isolated from mice on day 4 were stimulated with a broad range of TLR ligands; cultured supernatants were collected at 48 hours and analyzed for cytokine production by ELISA. *P < 0.05; **P < 0.01 compared with the concentrations of cytokines from PBS-treated mice (white bars).
Figure 10
Figure 10. IRF4 signaling is necessary for the suppression of DSS colitis.
IRF4-intact (IRF4+/+) and IRF4-deficient (IRF4–/–) mice were treated with 5% DSS in drinking water for 6 days (days 0–5). At an early phase of colitis induction (days 0, 1, 2), mice were administered MDP or PBS (i.p.). (A) Weight curves of IRF4–/– or IRF+/+ mice administered MDP or PBS. **P < 0.01 compared with PBS-injected IRF4+/+ mice. (B) Histology score of IRF4–/– mice treated with PBS or MDP on day 7. (C) MLN cells (1 × 106/ml) isolated from IRF4+/+ and IRF4–/– mice on day 7 were stimulated with PGN, LPS, or CpG; cultured supernatants were collected at 48 hours and analyzed for cytokine production by ELISA.
See this image and copyright information in PMC

References (VSports最新版本)

    1. Strober W., Murray P.J., Kitani A., Watanabe T. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat. Rev. Immunol. 2006;6:9–20. - PubMed
    1. Watanabe T., Kitani A., Strober W. NOD2 regulation of Toll-like receptor responses and the pathogenesis of Crohn’s disease. Gut. 2005;54:1515–1518. - PMC - PubMed
    1. Inohara Chamaillard, onald C., Nunez G. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev. Biochem. 2005;74:355–383. - PubMed
    1. Hugot J.P., et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature. 2001;411:599–603. - PubMed
    1. Ogura Y., et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature. 2001;411:603–606. - PubMed

V体育官网入口 - Publication types

MeSH terms