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.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

High-glucose conditions attenuate the response of macrophages to Legionella pneumophila infection by inhibiting NOD1 and MAPK signaling.

BACKGROUND: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo. METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR. RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05). CONCLUSION: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.

Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Tuberculosis is the leading cause of death from an infectious disease worldwide, partially due to a lack of access to drug treatments in certain countries where the disease is common. The only available tuberculosis vaccine ­ known as the BCG vaccine ­ is useful for preventing cases in young children, but is ineffective in teenagers and adults. So, there is a need to develop new vaccines that offer better, and longer lasting, durable protection in people of all ages. During an infection, our immune system recognizes markers known as PAMPs on the surface of bacteria, viruses or other disease-causing pathogens. The recognition of PAMPs by the immune system enables the body to distinguish foreign invading organisms from its own cells and tissues, thus triggering a response that fights the infection. If the body encounters the infectious agent again in the future, the immune system is able to quickly recognize and eliminate it before it can cause disease. Vaccines protect us by mimicking the appearance of the pathogen to trigger the first immune response without causing the illness. The BCG vaccine contains live bacteria that are closely related to the bacterium responsible for tuberculosis called Mycobacterium tuberculosis. Both M. tuberculosis and the live bacteria used in the BCG vaccine are able to hide an important PAMP, known as the NOD-1 ligand, from the immune system, making it harder for the body to detect them. The NOD-1 ligand forms part of the bacterial cell wall and modifying the BCG bacterium so it cannot disguise this PAMP may lead to a new, more effective vaccine. To investigate this possibility, Shaku et al. used a gene editing approach to develop a modified version of the BCG bacterium which is unable to hide its NOD-1 ligand when treated with a specific drug. Immune cells trained with the modified BCG vaccine were more effective at controlling the growth of M. tuberculosis than macrophages trained using the original vaccine. Furthermore, mice vaccinated with the modified BCG vaccine were better able to limit M. tuberculosis growth in their lungs than mice that had received the original vaccine. These findings offer a new candidate vaccine in the fight against tuberculosis. Further studies will be needed to modify the vaccine for use in humans. More broadly, this work demonstrates that gene editing can be used to expose a specific PAMP present in a live vaccine. This may help develop more effective vaccines for other diseases in the future.

Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1.

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.

DEFB114 protein enhances host resistance to fungal infection through the NOD1/2-ATG16L1-NF-κB signaling pathway.

The overuse of antibiotics has led to the enhanced resistance of many pathogenic bacteria, posing a threat to human health. Therefore, there is a need to develop green and safe alternatives to antibiotics. Beta-defensins play a crucial role in host defense against pathogens and have multifunctional properties, exerting key roles in innate and adaptive immunity, as well as non-immune processes. In this study, a 210 bp long cDNA sequence of yak DEFB114 gene was amplified and successfully expressed in a prokaryotic system. The DEFB114 protein exhibited significant inhibitory effects on the growth of Aspergillus fumigatus in vitro. When co-cultured with yak macrophages, DEFB114 protein enhanced macrophage phagocytic activity and increased nucleic acid fluorescence intensity (P < 0.05). DEFB114 protein also enhanced the activity of yak macrophages stimulated by inactivated Aspergillus fumigatus spores, increased the release of nitric oxide (NO), and promoted the expression of genes such as γ-actin, Lgals, Man2b, and Capg (P < 0.05). In mice experiments, DEFB114 protein promoted resistance against Aspergillus fumigatus infection, by regulating the NOD1/2-ATG16L1-NF-κB pathway to modulate the host immune response and exert its anti-infective effects. In summary, the yak DEFB114 protein could inhibit the growth of Aspergillus fumigatus and enhance the animal's resistance to pathogenic microorganisms, thereby having significant implications in the treatment and prevention of fungal infections.

NOD1 deficiency ameliorates the progression of diabetic retinopathy by modulating bone marrow-retina crosstalk.

BACKGROUND: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally, the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells, a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction. METHODS: We generated NOD1-/--Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow-retina axis. RESULTS: Elevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1-/--Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration, as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations, including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation, and the results further indicated that NOD1 had a protective effect against DR. Mechanistically, loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina, subsequently leading to diminished neutrophil chemoattraction and NETosis. CONCLUSIONS: The results of our study unveil, for the first time, the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation, culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

Fusobacterium nucleatum-triggered neutrophil extracellular traps facilitate colorectal carcinoma progression.

BACKGROUND: Fusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance. METHODS: Fn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments. RESULTS: Tissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively. CONCLUSIONS: Our findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.

An innate immune sensor wandering around - NOD1 promotes cell migration via non-canonical signaling.

NOD1 is a cytosolic immune receptor well known for recognizing intracellular bacteria and inducing innate immune responses. Upon ligand binding, it usually forms a complex with the serine/threonine kinase RIPK2 to activate the transcription factor NF-κB. Next to its role in pathogen recognition, NOD1 has been associated with cancer progression. In this regard, Hezinger et al. investigated a non-canonical role of NOD1 in cell migration. They discovered that NOD1 is crucial for the migration and chemotaxis of HeLa cells and identified HAX-1 as a novel interaction partner.

NOD1 cooperates with HAX-1 to promote cell migration in a RIPK2- and NF-ĸB-independent manner.

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.

NOD1 mediates interleukin-18 processing in epithelial cells responding to Helicobacter pylori infection in mice.

The interleukin-1 family members, IL-1ß and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1ß processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

NOD1 mediated D. pteronyssinus-induced allergic airway inflammation through RIP2/NF-κB.

BACKGROUND: Dermatophagoides pteronyssinus (D. pteronyssinus) is the main cause of allergic airway inflammation. As the earliest intracytoplasmic pathogen recognition receptors (PRR), NOD1 has been identified as key inflammatory mediator in NOD-like receptor (NLR) family. OBJECTIVE: Our primary aim is to elucidate whether NOD1 and its downstream regulatory proteins mediate D. pteronyssinus-induced allergic airway inflammation. METHODS: Mouse and cell models of D. pteronyssinus-induced allergic airway inflammation were established. NOD1 was inhibited in bronchial epithelium cells (BEAS-2B cells) and mice by cell transfection or application of inhibitor. The change of downstream regulatory proteins was detected by quantitative real-time PCR (qRT-PCR) and Western blot. The relative expression of inflammatory cytokines was evaluated by ELISA. RESULTS: The expression level of NOD1 and its downstream regulatory proteins increased in BEAS-2B cells and mice after treating with D. pteronyssinus extract, followed by the aggravation of inflammatory response. Moreover, inhibition of NOD1 decreased the inflammatory response, which also downregulated the expression of downstream regulatory proteins and inflammatory cytokines. CONCLUSIONS: NOD1 involves in the development of D. pteronyssinus-induced allergic airway inflammation. Inhibition of NOD1 reduces D. pteronyssinus-induced airway inflammation.

When SLC46A2 delivers cargo into keratinocytes.

How pattern recognition receptors NOD1 and NOD2 sense bacterial muropeptides from extracellular bacteria to drive keratinocyte inflammation remains unclear. In this issue of Immunity, Bharadwaj et al. show that the solute carrier 46A2 (SLC46A2) delivers DAP-muropeptides into the cytosol to drive NOD1 activation in keratinocytes and elicit skin inflammation during psoriasis.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Eugenol modulates the NOD1-NF-κB signaling pathway via targeting NF-κB protein in triple-negative breast cancer cells.

Background: The most aggressive subtype of breast cancer, triple-negative breast cancer (TNBC), has a worse prognosis and a higher probability of relapse since there is a narrow range of treatment options. Identifying and testing potential therapeutic targets for the treatment of TNBC is of high priority. Methods: Using a transcriptional signature of triple-negative breast cancer collected from Gene Expression Omnibus (GEO), CMap was utilized to reposition compounds for the treatment of TNBC. CCK8 and colony formation experiments were performed to detect the effect of the candidate drug on the proliferation of TNBC cells. Meanwhile, transwell and wound healing assay were implemented to detect cell metastasis change caused by the candidate drug. Moreover, the proteomic approach was presently ongoing to evaluate the underlying mechanism of the candidate drug in TNBC. Furthermore, drug affinity responsive target stability (DARTS) coupled with LC-MS/MS was carried out to explore the potential drug target candidate in TNBC cells. Results: We found that the most widely used medication, eugenol, reduced the growth and metastasis of TNBC cells. According to the underlying mechanism revealed by proteomics, eugenol could inhibit TNBC cell proliferation and metastasis via the NOD1-NF-κB signaling pathway. DARTS experiment further revealed that eugenol may bind to NF-κB in TNBC cells. Concludes: Our findings pointed out that eugenol was a potential candidate drug for the treatment of TNBC.

NOD1 activation in 3T3-L1 adipocytes confers lipid accumulation in HepG2 cells.

AIMS: Activation of specific innate immune receptors has been characterized to modulate nutrient metabolism in individual metabolic tissue directly or indirectly via secretory molecules. Activation of the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in adipocytes has been reported to induce lipolysis linked with insulin resistance and inflammatory response. These cues are positioned to modulate metabolic action in distal organs through paracrine/endocrine signaling. Here, we assessed the role of NOD1-mediated lipolysis and inflammatory response in adipocytes to affect lipid metabolism in hepatocytes. MAIN METHODS: Human hepatoma cells (HepG2) were exposed to conditioned medium obtained from 3 T3-L1 adipocytes pretreated with NOD1 ligand (iE-DAP) and the effects on lipid accumulation, inflammation and insulin response were assessed. Activation of mechanisms leading to hepatic lipid accumulation was investigated by gene expression analysis. KEY FINDINGS: The conditioned medium from NOD1-activated 3 T3-L1 adipocytes (CM-DAP) induced lipid accumulation in HepG2 cells, driven by both lipolysis and inflammatory responses. The CM-DAP-induced lipid accumulation was independent to de novo lipogenesis and resulted from the enhanced transport of fatty acids inside and consequent increase in rate of triglycerides synthesis in hepatocytes. Moreover, CM-DAP-induced lipid accumulation instigated the expression of the markers of fatty acid oxidation and VLDL assembly for the export of triglycerides from hepatocyte. Furthermore, CM-DAP-induced lipid accumulation was associated with induction of inflammatory response and impairment of insulin signaling in HepG2 cells. SIGNIFICANCE: Beyond showing liver-specific mechanisms to adipocytes-derived factors, our findings support the involvement of adipose tissue as a mediator in NOD1-mediated biological responses to modulate hepatic metabolism.

NOD1 and NOD2 Are Potential Therapeutic Targets for Cancer Immunotherapy.

The nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a group of intracellular proteins that are essential for controlling the host's innate immune response. The cytosolic nucleotide binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are the most widely investigated NLRs. As pattern recognition receptors (PRRs), NOD1 and NOD2 may recognize and bind endogenous damage associated molecular patterns (DAMPs) and external pathogenic associated molecular patterns (PAMPs), directing the activation of inflammatory caspases through engaging the adaptor protein RIP2, which further activates the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, thereby mediating host innate immunity and regulating the adaptive immunity. Previous research has identified NOD1 and NOD2 as key players in inflammatory disease and host-microbial defense. Despite numerous studies claiming that NOD1 and NOD2 are linked to tumorigenesis and tumor development, it is still unclear whether NOD1 and NOD2 act as cancer's friends or foes. In this review, we focus on concluding the current research progress on the role of NOD1 and NOD2 in a variety of cancers and discussing the potential reasons for the contradicting role of NOD1 and NOD2 in cancers. This review may help better understand the role of NOD1 and NOD2 in cancer and shed light on NOD1 and NOD2 as potential therapeutic targets for tumor immunotherapy.