Nucleotide-Binding Oligomerization Domain 1 (NOD1) Agonists Prevent SARS-CoV-2 Infection in Human Lung Epithelial Cells through Harnessing the Innate Immune Response.

The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-κB and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells.

Alterations in immune and antioxidant gene networks by gamma-d-glutamyl-meso-diaminopimelic acid in bovine mammary epithelial cells are attenuated by in vitro supply of methionine and arginine.

Nucleotide-binding oligomerization domain (NOD)-like receptor 1 (NOD1) is a cytosolic pattern recognition receptor with a crucial role in the innate immune response of cells triggered by the presence of compounds such as gamma-d-glutamyl-meso-diaminopimelic acid (iE-DAP) present in the peptidoglycan of all gram-negative and certain gram-positive bacteria. Methionine (Met) and arginine (Arg) are functional AA with immunomodulatory properties. In the present study, we aimed to assess the effect of increased Met and Arg supply on mRNA abundance of genes associated with innate immune response, antioxidant function, and AA metabolism during iE-DAP challenge in bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 per treatment) were precultured in modified medium for 12 h with the following AA formulations: ideal profile of AA (control), increased Met supply (incMet), increased Arg supply (incArg), or increased supply of Met plus Arg (incMetArg). Subsequently, cells were challenged with or without iE-DAP (10 µg/mL) for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4. Greater mRNA abundance of NOD1, the antioxidant enzyme SOD1, and AA transporters (SLC7A1 and SLC3A2) was observed in the incMet cells after iE-DAP stimulation. Although increased Met alone had no effect, incMetArg led to greater abundance of the inflammatory cytokine IL-6, and the antioxidant enzyme GPX1 after iE-DAP stimulation. The increased Arg alone downregulated NOD1 after iE-DAP stimulation, coupled with a downregulation in the AA transporters mRNA abundance (SLC7A1, SLC7A5, SLC3A2, and SLC38A9), and upregulation in GSS and KEAP1 mRNA abundance. Overall, the data indicated that increased supply of both Met and Arg in the culture medium were more effective in modulating the innate immune response and antioxidant capacity of BMEC during in vitro iE-DAP stimulation.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

NOD1 modulates decidual stromal cell function to maintain pregnancy in the early trimester.

We sought to explore the functions and modulated factors of NOD1 in normal decidual stromal cells (DSCs) derived from the first trimester pregnancy and whether existed different expression of NOD1 between normal and unexplained recurrent pregnancy loss (URPL) in DSCs. Twenty-six patients with normal pregnancies that required abortion and 12 URPL patients at first trimester were enrolled for the study. As a result, we found lower levels of NOD1 in the DSCs derived from URPL compared with those from normal early trimester pregnancy. Furthermore, increased NOD1 expression in the normal DSCs induced apoptosis and increased monocyte chemotactic protein-1 (MCP-1) and IL-1ß (interleukin 1 beta) secretion but decreased their invasion capacity. In addition, several cytokines such as IL-1ß, tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-17 (IL-17) were present at the maternal-fetal interface in RPL and were found to regulate NOD1 expression in primary DSCs. Our study indicates that RPL may be associated with NOD1 aberrant expression in DSCs, which plays a significant role in maintaining pregnancy via infection control and regulation of immune responses that might affect the pregnancy outcome. We expect that our results will bring more comprehensively understanding about the connection between NOD1 and RPL for researchers.

Role of peptide transporter 2 and MAPK signaling pathways in the innate immune response induced by bacterial peptides in alveolar epithelial cells.

AIMS: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549. MAIN METHODS: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium. KEY FINDINGS: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response. SIGNIFICANCE: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.

The Role of the p38-MNK-eIF4E Signaling Axis in TNF Production Downstream of the NOD1 Receptor.

Activation of nucleotide-binding oligomerization domain (NOD) 1 and NOD2 by muropeptides triggers a complex transcriptional program in innate immune cells. However, little is known about posttranscriptional regulation of NOD1- and NOD2-dependent responses. When stimulated with a prototypic NOD1 agonist, N-acetylglucosaminyl-N-acetylmuramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid (GM-triDAP), human monocyte-derived macrophages (MDM) produced an order of magnitude more TNF, IL-6, and pro-IL-1ß than did monocyte-derived dendritic cells (MDDC), despite similar NOD1 expression, similar cytokine mRNA kinetics, and comparable responses to LPS. TNF production by GM-triDAP-activated MDM was independent of autocrine IL-1. However, GM-triDAP-activated MDM translated TNF mRNA more efficiently than did MDDC. As an underlying mechanism, NOD1 triggering in MDM caused a more potent and long-lasting activation of the signaling axis involving p38 MAPK, MAPK-interacting kinase (MNK), and eukaryotic translation initiation factor 4E, which is a critical regulator of translation. Furthermore, MNK controlled TNF mRNA abundance in MDDC and MDM upon NOD1 triggering. NOD1-dependent responses were more sensitive to MNK inhibition than were TLR4-dependent responses. These results demonstrate the importance of the p38-MNK-eukaryotic translation initiation factor 4E axis in TNF production downstream of NOD1.

The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue.

Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. RESULTS: The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. CONCLUSIONS: Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.

Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm.

Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.

Modulatory roles of interferon-γ through indoleamine 2, 3-dioxygenase induction in innate immune response of dental pulp cells.

INTRODUCTION: Marked infiltration of inflammatory cells such as activated T cells producing interferon-γ (IFN-γ) is observed in severe pulpitis. However, the roles of IFN-γ in the innate immune response of dental pulp have not been reported. Indoleamine 2, 3-dioxygenase (IDO) is a regulator of immune responses, and the IDO expression is induced by IFN-γ in many cells whose expression in dental pulp is unknown. The purpose of this study was to determine the role of IFN-γ in the immune response through microbial pattern recognition receptors (PRRs) such as Toll-like receptors or nucleotide-binding oligomerization domain-like receptors on the production of proinflammatory cytokines such as CXCL10 and interleukin (IL)-6 and the expression of IDO in cultured human dental pulp cells (HDPCs). METHODS: HDPCs were established from explant cultures of healthy pulp tissues. CXCL10 and IL-6 production was determined using enzyme-linked immunosorbent assay. Confirmation of IDO localization in dental pulp tissues was examined using immunohistochemistry. IDO expression in HDPCs was analyzed by immunoblot. RESULTS: IFN-γ significantly up-regulated CXCL10 and IL-6 production in the HDPCs stimulated with ligands for PRRs in a concentration-dependent manner. The expression of IDO was detected in inflamed pulp tissue. In addition, IFN-γ in combination with the PRR ligands enhanced IDO expression in HDPCs compared with IFN-γ alone. Moreover, CXCL10 production in IFN-γ-stimulated HDPCs was inhibited by an IDO inhibitor. CONCLUSIONS: This study showed the synergistic effects by IFN-γ on cytokine production and IDO expression in HDPCs, suggesting that IFN-γ may modulate the innate immune response of dental pulp.

Differential expression of AP-1 proteins in human myometrium after spontaneous term labour onset.

OBJECTIVES: The aims of this study were (i) to determine the localisation of activator protein (AP)-1 family members (cFos, FosB, cJun, JunB and JunD) in human myometrium; and (ii) to determine the effect of human term labour on the expression of AP-1 family of transcription factors in myometrium. STUDY DESIGN: This localised the AP-1 family members cFos, FosB, cJun, JunB and JunD in human myometrium was performed by immunohistochemistry. The effect of term labour on the expression of these family members at the mRNA and protein level was assessed by qRT-PCR and Western blotting, respectively. The effect of pro-inflammatory stimuli on AP-1 transcriptional activity was assessed using a luciferase assay in primary human myometrial cells. RESULTS: Immunohistochemical expression of cFos, FosB, cJun, JunB and JunD were all present in human myometrial tissue and displayed cytoplasmic staining. FosB and JunD also displayed nuclear staining. Term labour was associated with an increase in cFos and JunB mRNA and protein expression. On the other hand, JunD mRNA and protein expression was decreased with labour. FosB mRNA was increased with labour, but there was no change at the protein level. There was no change in cJun mRNA or protein expression. AP-1 transcriptional activity was increased in human myometrial cells by the pro-inflammatory cytokine TNF-α. There was, however, no effect of the bacterial products lipopolysaccharide (LPS; TLR4 ligand), iE-DAP (NOD1 ligand), MDP (NOD2 ligand), FSL-1 (TLR2 ligand) or flagellin (TLR5 ligand) on AP-1 transcriptional activity. CONCLUSION: This study shows that human labour is associated with changes in AP-1 family members. Further studies are required to determine the exact role of the AP-1 family members in myometrium.

Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils.

Nod-like receptors are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two Nod-like receptor family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected intraperitoneally with thioglycollate, and then peritoneal neutrophils were isolated 4 hr after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonists, respectively. The level of cytokines [interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)] and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in wild-type (WT) neutrophils. Increased production of cytokines and chemokines with MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-κB and MAPK in WT neutrophils, but not in Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and Toll-like receptor (TLR) agonists (Pam3 CSK4 and lipopolysaccharide) exerted synergistic effects on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-α, CXCL1 and CCL2 in neutrophils in response to Gram-negative bacteria. Our findings suggest that the Nod2-Rip2 axis may contribute to the innate immune response of neutrophils against bacterial infection.

A TNF-α-CCL20-CCR6 axis regulates Nod1-induced B cell responses.

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate within the spleen, and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

Beyond toll-like receptors: Porphyromonas gingivalis induces IL-6, IL-8, and VCAM-1 expression through NOD-mediated NF-κB and ERK signaling pathways in periodontal fibroblasts.

To investigate whether oligomerization domains (NODs) are involved in Porphyromonas gingivalis-induced interleukin (IL)-6, IL-8, and vascular cell adhesion molecule (VCAM)-1 expression beyond Toll-like receptors (TLRs), we investigated the role of NOD1/2 in P. gingivalis-induced IL-6, IL-8, and VCAM-1 expression in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs). The mechanism was explored by activation and silence of NODs, electrophoretic mobility shift assay (EMSA), and pathway blockade assays. Results showed that P. gingivalis could induce NOD1, NOD2, IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLs at mRNA and protein levels. Activation of NOD1/2 by agonists could clearly upregulate the expression of these genes, while silence of NOD1/2 could remarkably attenuate them. EMSA and blockade of NF-κB and extracellular-signal-regulated kinase (ERK)1/2 pathway assays also verified that the two pathways were involved in NOD1/2-mediated IL-6, IL-8, and VCAM-1 expression. In conclusion, our findings demonstrated that P. gingivalis induced IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLCs through NOD1/2-mediated NF-κB and ERK1/2 signaling pathways beyond TLRs.

Activation of nucleotide oligomerization domain containing protein 1 induces lipolysis through NF-κB and the lipolytic PKA activation in 3T3-L1 adipocytes.

Obesity is associated with chronic inflammation. Toll-like receptors (TLR) and NOD-like receptors (NLR) are two families of pattern recognition receptors that play important roles in the immune response and inflammation in adipocytes. Activation of TLR4 has been shown to stimulate lipolysis from adipose tissue or adipocytes. However, effects of activation of nucleotide-oligomerization domain containing protein 1 (NOD1), one of the prominent members of NLRs, on adipocyte lipolysis have not been studied. Here we report that NOD1 activation by the synthetic ligands (Tri-DAP and C12-iEDAP) stimulated lipolysis in 3T3-L1 adipocytes in a time- and dose-dependent manner. C12-iEDAP-induced lipolysis was attenuated with NOD1 siRNA knockdown, demonstrating the specificity of the effects. Moreover, inhibition of the protein kinase A (PKA)/hormone sensitive lipase (HSL) and NF-κB pathways by the pharmacological inhibitors attenuated the lipolytic effects of C12-iEDAP. Furthermore, we show NOD1 activation induced PKA activation independent of cAMP production and inhibition of NF-κB pathways attenuated phosphorylation of selected PKA lipolytic targets (phosphorylation of Perilipin Ser 517 and HSL Ser 563). Taken together, our results demonstrate a novel role of NOD1 activation, via NF-κB/PKA lipolytic activation, in inducing lipolysis in adipocytes and suggest that NOD1 activation may contribute to dyslipidemia in obesity.

Synthesis of conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid derivatives as ligands of nucleotide-binding oligomerization domain protein 1 (Nod1).

Nod1, an important member of the pattern recognition receptor family, remains a virtually unexploited target. Harnessing its innate immune stimulatory properties still remains an unfulfilled goal of medicinal chemistry. Nucleotide-binding oligomerization domain protein 1 (Nod1) agonists have been shown to boost the inflammatory responses against pathogenic microbes and could thus constitute a new class of broad spectrum antimicrobial agents. To gain additional insight into the structure/activity relationships of Nod1 agonistic compounds, a series of novel, conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) analogs have been designed and synthesized. Ramos-Blue cells expressing Nod1 were used to screen and validate our compounds for their Nod1-agonist activity. Their immunomodulatory properties were subsequently determined in vitro, by evaluating their capacity to induce pro-inflammatory cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC), by themselves and in synergy with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand. The synthesized iE-DAP analogs were shown to possess immuno-enhancing properties as a result of their potent and specific Nod1-agonistic effect. The activity of the compound exhibiting the greatest capacity to induce pro-inflammatory cytokine release from PBMC surpassed that of lauroyl-γ-D-glutamyl-meso-diaminopimelic acid (C12-iE-DAP).

The synthesis of active and stable diaminopimelate analogues of the lantibiotic peptide lactocin S.

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.

NOD-like receptors and RIG-I-like receptors in human eosinophils: activation by NOD1 and NOD2 agonists.

NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) are newly discovered pattern-recognition receptors. They detect substructures of bacterial peptidoglycan and viral RNA, respectively, thereby initiating an immune response. However, their role in eosinophil activation remains to be explored. The aim of this study was to characterize the expression of a range of NLRs and RLRs in purified human eosinophils and assess their functional importance. Expression of NOD1, NOD2, NLRP3, RIG-I and MDA-5 was investigated using real-time reverse transcription PCR, flow cytometry and immunohistochemistry. The effects of the corresponding agonists iE-DAP (NOD1), MDP (NOD2), alum (NLRP3) and poly(I:C)/LyoVec (RIG-I/MDA-5) were studied in terms of cytokine secretion, degranulation, survival, expression of adhesion molecules and activation markers, and chemotactic migration. Eosinophils expressed NOD1 and NOD2 mRNA and protein. Low levels of RIG-I and MDA-5 were found, whereas expression of NLRP3 was completely absent. In accordance, stimulation with iE-DAP and MDP was found to induce secretion of interleukin-8, up-regulate expression of CD11b, conversely down-regulate CD62 ligand, increase expression of CD69 and induce migration. The MDP also promoted release of eosinophil-derived neurotoxin, whereas iE-DAP failed to do so. No effects were seen upon stimulation with alum or poly(I:C)/LyoVec. Moreover, the NOD1-induced and NOD2-induced activation was mediated via the nuclear factor-κB signalling pathway and augmented by interleukin-5 and granulocyte-macrophage colony-stimulating factor, but not interferon-γ. Taken together, the NLR system represents a novel pathway for eosinophil activation. The responses are enhanced in the presence of cytokines that regulate T helper type 2 immunity, suggesting that the NLRs constitute a link between respiratory infections and exacerbations of allergic disease.

Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts.

OBJECTIVE: Human periodontal ligament fibroblasts (HPDLFs) play a crucial role in protecting against oral bacteria in periapical tissue. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signalling. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein for TLR-mediated nuclear factor-κB (NF-κB) signalling pathway activation that induces the production of pro-inflammatory cytokines. The aim of this study was to investigate the expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs. METHODS: The expression of TLR2, TLR4, NOD1, and NOD2 was measured by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and immunostaining. HPDLFs were stimulated with TLR and NOD agonists. Then, the expression of TRAF6 was measured by real-time PCR and western blot. Concentrations of IL-1ß, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Finally, by using small interfering RNA (siRNA) for TRAF6, we analysed the production of IL-1ß, IL-6, and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists. RESULTS: We found clear mRNA and protein expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) were markedly up-regulated upon the activation of TLRs and NODs. Furthermore, the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines. We also found TRAF6 suppression resulted in reduced IL-1ß, IL-6, and IL-8 expression upon TLR and NOD agonists challenge. CONCLUSION: These findings indicated that TLR2, TLR4, NOD1, and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria, and a combination of signalling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs. In addition, TLR and NOD signalling involving TRAF6 contribute to inflammatory responses in HPDLFs.

Divergent responses to peptidoglycans derived from different E. coli serotypes influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages.

BACKGROUND: Pathogen-associated molecular patterns (PAMPs) are structural components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) from bacterial cell walls. PAMP-recognition by the host results in an induction of defence-related genes and often the generation of an inflammatory response. We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. RESULTS: Transcript profiling was assessed using function-targeted cDNA microarray hybridisation (n = 36) and results show differential responses to both PGNs that are both time and treatment dependent. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this, Gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high similarity to a generalised inflammatory priming response where multiple functional classes are related to ribosome biogenesis or cellular metabolism. Prostaglandin release was induced by both PGNs and macrophages were significantly more sensitive to PGN-O111:B4 as suggested from microarray data. CONCLUSION: Responses at the level of the transcriptome and the inflammatory outcome (prostaglandin synthesis) highlight the different sensitivity of the macrophage to slight differences (serotype) in peptidoglycan structure. Such divergent responses are likely to involve differential receptor sensitivity to ligands or indeed different receptor types. Such changes in biological response will likely reflect upon pathogenicity of certain serotypes and the development of disease.