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MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.

ABSTRACT: Human neutrophil peptide 1 (HNP-1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP-1 expression in epithelial cell lines.Gentamicin protection assay using Salmonella typhimurium was performed in Caco-2 cells. The mRNA level was determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and defensin expression was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappaB activation was assessed using pIV luciferase and Renilla plasmids. A NOD2 mutant was generated by site-directed mutagenesis.Among the defensins tested, only HNP-1 expression is induced in colonic epithelial model HCT116 cells after MDP-LD stimulation. HNP-1 secretion is significantly increased after MDP-LD stimulation in the cell supernatant of intestinal epithelial cells expressing endogenous NOD2, but not in cells that lack endogenous NOD2 expression. HNP-1 is required for NOD2-dependent NF-kappaB activation after MDP-LD stimulation since hnp-1 siRNA transfection abrogated the response to MDP-LD stimulation. The antibacterial function of NOD2 against S. typhimurium was impaired when expression of HNP-1 was blocked by siRNA.HNP-1 secretion depends on NOD2 stimulation by MDP-LD and contributes to antibacterial activity in intestinal epithelial cells expressing endogenous NOD2, but not NOD2 3020insC mutant associated with increased susceptibility to Crohn's disease.

SUBMITTER: Yamamoto-Furusho JK

PROVIDER: S-EPMC2895931 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.

Yamamoto-Furusho Jesus K JK Barnich Nicolas N Hisamatsu Tadakazu T Podolsky Daniel K DK

Inflammatory bowel diseases 20100501 5

<h4>Background</h4>Human neutrophil peptide 1 (HNP-1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP-1 expression in epithelial cell lines.<h4>Methods</h4>Gentamicin protection assay using Salmo ...[more]

PMID: 19856414

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Project description:The NOD-like receptors are cytoplasmic proteins that sense microbial by-products released by invasive bacteria. Although NOD1 and NOD2 are functionally expressed in cells from oral tissues and play a role triggering immune responses, the role of NOD2 receptor in the bone resorption and in the modulation of osteoclastogenesis is still unclear. We show that in an experimental model of periodontitis with Porphyromonas gingivalis W83, NOD2(-/-) mice showed lower bone resorption when compared to wild type. Quantitative polymerase chain reaction analysis revealed that wild-type infected mice showed an elevated RANKL/OPG ratio when compared to NOD2(-/-) infected mice. Moreover, the expression of 2 osteoclast activity markers-cathepsin K and matrix metalloproteinase 9-was significantly lower in gingival tissue from NOD2(-/-) infected mice compared to WT infected ones. The in vitro study reported an increase in the expression of the NOD2 receptor 24 hr after stimulation of hematopoietic bone marrow cells with M-CSF and RANKL. We also evaluated the effect of direct activation of NOD2 receptor on osteoclastogenesis, by the activation of this receptor in preosteoclasts culture, with different concentrations of muramyl dipeptide. The results show no difference in the number of TRAP-positive cells. Although it did not alter the osteoclasts differentiation, the activation of NOD2 receptor led to a significant increase of cathepsin K expression. We confirm that this enzyme was active, since the osteoclasts resorption capacity was enhanced by muramyl dipeptide stimulation, evaluated in osteoassay plate. These results show that the lack of NOD2 receptor impairs the bone resorption, suggesting that NOD2 receptor could contribute to the progression of bone resorption in experimental model of periodontitis. The stimulation of NOD2 by its agonist, muramyl dipeptide, did not affect osteoclastogenesis, but it does favor the bone resorption capacity identified by increased osteoclast activity.

Project description:Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-β-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating proinflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment.

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MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.

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MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.

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