Yukari Fujimoto

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF‐κB through RICK, a caspase‐recruitment domain‐containing kinase. However, the mechanism by which RICK activates NF‐κB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine‐63‐linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK‐mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IκB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1‐induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2‐mediated NF‐κB activation.

Autophagic control of listeria through intracellular innate immune recognition in drosophila

Autophagy, an evolutionally conserved homeostatic process for catabolizing cytoplasmic components, has been linked to the elimination of intracellular pathogens during mammalian innate immune responses. However, the mechanisms underlying cytoplasmic infection-induced autophagy and the function of autophagy in host survival after infection with intracellular pathogens remain unknown. Here we report that in drosophila, recognition of diaminopimelic acid–type peptidoglycan by the pattern-recognition receptor PGRP-LE was crucial for the induction of autophagy and that autophagy prevented the intracellular growth of Listeria monocytogenes and promoted host survival after this infection. Autophagy induction occurred independently of the Toll and IMD innate signaling pathways. Our findings define a pathway leading from the intracellular pattern-recognition receptors to the induction of autophagy to host defense.

Muramyldipeptide and diaminopimelic acid‐containing desmuramylpeptides in combination with chemically synthesized Toll‐like receptor agonists synergistically induced production of interleukin‐8 in a NOD2‐ and NOD1‐dependent manner, respectively, in human monocytic cells in culture

Two types of synthetic peptidoglycan fragments, diaminopimelic acid (DAP)‐containing desmuramylpeptides (DMP) and muramyldipeptide (MDP), induced secretion of interleukin (IL)‐8 in a dose‐dependent manner in human monocytic THP‐1 cells, although high concentrations of compounds are required as compared with chemically synthesized Toll‐like receptor (TLR) agonists mimicking bacterial components: TLR2 agonistic lipopeptide (Pam3CSSNA), TLR4 agonistic lipid A (LA‐15‐PP) and TLR9 agonistic bacterial CpG DNA. We found marked synergistic IL‐8 secretion induced by MDP or DAP‐containing DMP in combination with synthetic TLR agonists in THP‐1 cells. Suppression of the mRNA expression of nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the synergistic IL‐8 secretion induced by DMP and MDP with these TLR agonists respectively. In accordance with the above results, enhanced IL‐8 mRNA expression and the activation of nuclear factor (NF)‐κB induced by MDP or DMP in combination with synthetic TLR agonists were markedly suppressed in NOD2‐ and NOD1‐silenced cells respectively. These findings indicated that NOD2 and NOD1 are specifically responsible for the synergistic effects of MDP and DMP with TLR agonists, and suggested that in host innate immune responses to invading bacteria, combinatory dual signalling through extracellular TLRs and intracellular NODs might lead to the synergistic activation of host cells.

Nod1/RICK and TLR Signaling Regulate Chemokine and Antimicrobial Innate Immune Responses in Mesothelial Cells

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-γ and cooperated with IFN-γ in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

Differential release and distribution of Nod1 and Nod2 immunostimulatory molecules among bacterial species and environments

Nod1 and Nod2 are intracellular proteins that are involved in recognition of bacterial molecules and their genetic variations have been linked to several inflammatory diseases that are strongly affected by environmental factors. However, the distribution of Nod1- and Nod2-stimulatory molecules in different bacterial species and environments is unknown. Here we established a quantitative bioassay to screen and characterize Nod1- and Nod2-stimulatory activities in different environmental sites and bacterial species. Using this system, we found that common environments including foods and soils contain high levels of Nod1- and Nod2-stimulatory activities. Several Bacillus species were identified to possess the highest Nod1-stimulatory activity among soil bacteria. Unlike other immunostimulatory molecules, the higher level of Nod1-stimulatory activity was found in the culture supernatant and not in extracts from whole cell bacteria. Nod1-stimulatory molecules were highly stable at extreme pH and boiling conditions and were synthesized in an amidase- and sltY-independent manner. These results suggest a novel mechanism by which bacteria present in the environment stimulate the host immune system through Nod1.

Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of β-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Chemically synthesized pathogen‐associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll‐like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti‐bacterial factors. In humans, four kinds of PGRPs (PGRP‐L, ‐Iα, ‐Iβ and ‐S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized  pathogen‐associated  molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli‐type tryacyl lipopeptide (Pam3CSSNA), E. coli‐type lipid A (LA‐15‐PP), diaminopimelic acid containing desmuramyl peptide (γ‐ d‐glutamyl‐meso‐DAP; iE‐DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP‐Iα and ‐Iβ, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll‐like receptor (TLR)2, TLR4, nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA‐15‐PP, iE‐DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)‐κB in the epithelial cells, and suppression of NF‐κB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1.

Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as muramyldipeptide; N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) and γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram-positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide-binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and β-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, ll-DAP, and dd-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity.

Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: Combined effects of lipid a acylation state and TLR4 polymorphisms on signaling

The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymorphic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-κB-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-κB activity in wild-type transfectants under the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-α, IL-6, IL-1β, and IL-10 production. Cytokine levels were significantly lower (∼20–90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant.