NOD2 in hepatocytes engages a liver-gut axis to protect against steatosis, fibrosis, and gut dysbiosis during fatty liver disease in mice.

Obesity promotes nonalcoholic fatty liver disease (NAFLD). The intestinal microbiota contributes to NAFLD progression through a gut-to-liver pathway that promotes inflammation and fibrosis. Gut microbiota-derived factors can travel to the liver and activate immune responses in liver resident cells to promote inflammation and NAFLD. Little is known about bacterial sensors or immune responses that can protect against NAFLD. We tested whether the bacterial cell wall sensor nucleotide-binding oligomerization domain-containing (NOD)2 protects against diet-induced NAFLD in mice. Whole body deletion of NOD2 exacerbated liver steatosis and fibrosis in mice fed a NAFLD-promoting diet. Mice with a hepatocyte-specific deletion of NOD2 (Nod2-/-HKO) also had higher liver steatosis and fibrosis compared with littermate wild-type mice (WT) fed a NAFLD-promoting diet. Hepatocyte-specific NOD2 deletion altered the composition of the gut microbiome. Nod2-/-HKO mice had increased relative abundance of Clostridiales and lower Erysipelotrichaceae among other changes in cecal bacteria compared with littermate WT mice. Hepatocyte-specific NOD2 deletion altered a transcriptional program of liver inflammation, metabolism, and fibrosis. Nod2-/-HKO mice had higher levels of transcripts involved in lipid and cholesterol metabolism. Nod2-/-HKO mice had higher transcript levels of transforming growth factor-ß and collagen isoforms, which coincided with higher levels of liver collagen compared with WT mice. These data show that bacterial cell wall sensing within hepatocytes can engage retrograde cross-talk from the liver to the gut, where liver immunity communicates with the gut to influence the intestinal host-microbe relationship during diet-induced NAFLD, and NOD2 within the hepatocyte confers protection from liver steatosis and fibrosis.

RIPK2 Dictates Insulin Responses to Tyrosine Kinase Inhibitors in Obese Male Mice.

Tyrosine kinase inhibitors (TKIs) used in cancer are also being investigated in diabetes. TKIs can improve blood glucose control in diabetic cancer patients, but the specific kinases that alter blood glucose or insulin are not clear. We sought to define the role of Receptor Interacting Serine/Threonine Kinase 2 (RIPK2) in mouse models of insulin resistance. We tested the TKI gefitinib, which inhibits RIPK2 activity, in wild-type (WT), Nod1-/-, Nod2-/-, and Ripk2-/- mice fed an obesogenic high-fat diet. Gefitinib lowered blood glucose during a glucose tolerance test (GTT) in a nucleotide-binding oligomerization domain (NOD)-RIPK2-independent manner in all obese mice. However, gefitinib lowered glucose-stimulated insulin secretion only in obese Ripk2-/- mice. Gefitinib had no effect on insulin secretion in obese WT, Nod1-/-, or Nod2-/- mice. Hence, genetic deletion of Ripk2 promoted the insulin-sensitizing potential of gefitinib, since this TKI lowered both blood glucose and insulin only in Ripk2-/- mice. Gefitinib did not alter the inflammatory profile of pancreas, adipose, liver, or muscle tissues in obese Ripk2-/- mice compared with obese WT mice. We also tested imatinib, a TKI that does not inhibit RIPK2 activity, in obese WT mice. Imatinib lowered blood glucose during a GTT, consistent with TKIs lowering blood glucose independently of RIPK2. However, imatinib increased glucose-stimulated insulin secretion during the glucose challenge. These data show that multiple TKIs lower blood glucose, where actions of TKIs on RIPK2 dictate divergent insulin responses, independent of tissue inflammation. Our data show that RIPK2 limits the insulin sensitizing effect of gefitinib, whereas imatinib increased insulin secretion.

The regenerating family member 3 ß instigates IL-17A-mediated neutrophil recruitment downstream of NOD1/2 signalling for controlling colonisation resistance independently of microbiota community structure.

OBJECTIVE: Loss of the Crohn's disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB. DESIGN: Colonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces. RESULTS: Primary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3ß by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3. CONCLUSIONS: These results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

The synergistic effect of NOD2 and TLR4 on the activation of autophagy in human submandibular gland inflammation.

BACKGROUND: Sialadenitis is a nonneoplastic disease that causes salivary dysfunction. Autophagy may be involved in helping protect salivary function when the salivary gland is impaired; this process is primarily activated by sensors of innate immunity, such as Toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors. The role of these pattern recognition receptors (PRRs) in the regulation of salivary gland tissue defense and homeostasis has been underappreciated. This study hypothesized that NOD2 and TLR4 have a synergistic effect on the activation of autophagy in human submandibular gland (HSG) inflammation. METHODS: Submandibular gland inflammation was modeled by treating HSG cell lines in vitro with muramyl dipeptide (MDP) and lipopolysaccharide (LPS) for 24 hours. The mRNA and protein expression of NOD2, TLR4 and autophagy-related proteins (ATG5, LC3, Beclin1) were evaluated by real-time PCR and Western blot. Immunohistochemistry and double immunofluorescence were used to analyze the presence, distribution and colocalization of the aforementioned indicators in HSG tissues. RESULT: The mRNA and protein expression of autophagy-related proteins were significantly increased in HSG cells costimulated with LPS and MDP for 24 hours. NOD2, TLR4 and the autophagy-related proteins were also highly expressed in residual acini and dilated ducts of chronic submandibular sialadenitis tissues. In addition, PRRs and autophagy markers were obviously colocalized in chronic submandibular sialadenitis tissues and HSG cells. CONCLUSION: TLR4 and NOD2 have unique expression sites in salivary glands, and they may synergistically activate autophagy in salivary glands under conditions of inflammation.

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity.

BACKGROUND: Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2 mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. METHODS: Depletion of CD4 T cells in Nod2 mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. RESULTS: In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-α and interferon-γ secretion by CD4 T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. CONCLUSIONS: Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

NOD2 promotes endothelial-to-mesenchymal transition of glomerular endothelial cells via MEK/ERK signaling pathway in diabetic nephropathy.

Endothelial-to-mesenchymal transition (EndMT) of glomerular vascular endothelial cells (GEnCs) is now considered to play a critical role in diabetic nephropathy (DN). NOD2 is newly discovered to be closely related to DN renal injury. However, the relationship between NOD2 and EndMT of GEnCs has never been reported. In the present study, we found that NOD2 over-expression was positively correlated with the severity of DN injury in human renal biopsy samples. Immunohistochemical staining of DN renal slices showed gradual absence of endothelial character and gain of mesenchymal character, both of which were associated with NOD2 over-expression. In high glucose stimulated GEnCs, NOD2 was increased. What's more, over-expression and activation of NOD2 could both promote EndMT of GEnCs. On the other hand, silencing of NOD2 markedly attenuated EndMT induced by high glucose. Mechanically, we further found that MEK/ERK signaling pathway was involved in NOD2-regulated EndMT. Collectively, our results indicate that NOD2 has a regulatory role in EndMT via activation of MEK/ERK in high glucose-treated GEnCs. Targeting this pathway is a promising strategy for intervention of DN endothelial dysfunction.

IKKα controls ATG16L1 degradation to prevent ER stress during inflammation.

Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.

Role of nucleotide-binding oligomerization domain 1 (NOD1) and its variants in human cytomegalovirus control in vitro and in vivo.

Induction of nucleotide-binding oligomerization domain 2 (NOD2) and downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2) by human cytomegalovirus (HCMV) is known to up-regulate antiviral responses and suppress virus replication. We investigated the role of nucleotide-binding oligomerization domain 1 (NOD1), which also signals through RIPK2, in HCMV control. NOD1 activation by Tri-DAP (NOD1 agonist) suppressed HCMV and induced IFN-ß. Mouse CMV was also inhibited through NOD1 activation. NOD1 knockdown (KD) or inhibition of its activity with small molecule ML130 enhanced HCMV replication in vitro. NOD1 mutations displayed differential effects on HCMV replication and antiviral responses. In cells overexpressing the E56K mutation in the caspase activation and recruitment domain, virus replication was enhanced, but in cells overexpressing the E266K mutation in the nucleotide-binding domain or the wild-type NOD1, HCMV was inhibited, changes that correlated with IFN-ß expression. The interaction of NOD1 and RIPK2 determined the outcome of virus replication, as evidenced by enhanced virus growth in NOD1 E56K mutant cells (which failed to interact with RIPK2). NOD1 activities were executed through IFN-ß, given that IFN-ß KD reduced the inhibitory effect of Tri-DAP on HCMV. Signaling through NOD1 resulting in HCMV suppression was IKKα-dependent and correlated with nuclear translocation and phosphorylation of IRF3. Finally, NOD1 polymorphisms were significantly associated with the risk of HCMV infection in women who were infected with HCMV during participation in a glycoprotein B vaccine trial. Collectively, our data indicate a role for NOD1 in HCMV control via RIPK2- IKKα-IRF3 and suggest that its polymorphisms predict the risk of infection.

Cardiopulmonary Bypass Down-Regulates NOD Signaling and Inflammatory Response in Children with Congenital Heart Disease.

In the present study, we aimed to examine the impact of cardiopulmonary bypass (CPB) on expression and function of NOD1 and NOD2 in children with congenital heart disease (CHD), in an attempt to clarify whether NOD1 and NOD2 signaling is involved in the modulation of host innate immunity against postoperative infection in pediatric CHD patients. Peripheral blood samples were collected from pediatric CHD patients at five different time points: before CPB, immediately after CPB, and 1, 3, and 7 days after CPB. Real-time PCR, Western blot, and ELISA were performed to measure the expression of NOD1 and NOD2, their downstream signaling pathways, and inflammatory cytokines at various time points. Proinflammatory cytokine IL-6 and TNF-α levels in response to stimulation with either the NOD1 agonist Tri-DAP or the NOD2 agonist MDP were significantly reduced after CPB compared with those before CPB, which is consistent with a suppressed inflammatory response postoperatively. The expression of phosphorylated RIP2 and activation of the downstream signaling pathways NF-κB p65 and MAPK p38 upon Tri-DAP or MDP stimulation in PBMCs were substantially inhibited after CPB. The mRNA level of NOD1 and protein levels of NOD1 and NOD2 were also markedly decreased after CPB. Our results demonstrated that NOD-mediated signaling pathways were substantially inhibited after CPB, which correlates with the suppressed inflammatory response and may account, at least in part, for the increased risk of postoperative infection in pediatric CHD patients.

Nod2 Deficiency Leads to a Specific and Transmissible Mucosa-associated Microbial Dysbiosis Which Is Independent of the Mucosal Barrier Defect.

BACKGROUND AND AIMS: Crohn's disease [CD] is a complex disorder characterised by an inappropriate immune response, impaired barrier function and microbial dysbiosis. Mutations in nucleotide oligomeriation domain 2 [NOD2] are CD risk factors. Increase of intestinal permeability, CD4+ T cell infiltration, and bacterial dysbiosis are also seen in Nod2-knockout [Nod2 KO] mice. However, the specificity and relationship between these Nod2-associated abnormalities remain largely unexplored. METHODS: Wild-type [WT], Nod1-knockout [Nod1 KO] and Nod2 KO mice were analysed in parallel. Microbial composition was defined by 454-pyrosequencing of bacterial 16S rRNA genes. Mucin and antimicrobial peptide expression was assessed by RT-PCR. Cell populations from Peyer's patches were determined by flow cytometry. Ussing chambers were used to measure intestinal permeability and bacterial translocation. Finally, to explore the impact of colonisation with mother's microbiota at birth, analyses were also performed in Nod2 KO and WT mice born from WT surrogate mothers after embryo transfer. RESULTS: Nod2 KO mice exhibited colonic bacterial dysbiosis different from WT and Nod1 KO mice. Altered expression of antimicrobial peptides and mucins in ileum and colon was associated with the microbial composition. Bacterial composition of Nod2 KO and WT mice obtained by embryo transfer was similar to that observed in Nod2 KO mice, arguing for a dominant effect of Nod2 KO-associated dysbiosis. In contrast, increased levels of CD4+ T cells and gut barrier defects across Peyer's patches were specific to Nod2 deficiency and independent of Microbial dysbiosis. CONCLUSIONS: Nod2 deficiency is associated with a specific dominant dysbiosis which does not drive mucosal tissue and immune alterations.

Respective Roles of Hematopoietic and Nonhematopoietic Nod2 on the Gut Microbiota and Mucosal Homeostasis.

BACKGROUND: NOD2 mutations are associated with Crohn's disease (CD). Both CD (in human) and Nod2 deficiency (in mice) are characterized by increased mucosal CD4 T-cells, an altered permeability and a microbial dysbiosis. However, the respective roles of the gut epithelial and immune compartments on the phenotype are not known. METHODS: Microbial composition, epithelial peptide secretion, intestinal permeability, and immune cell composition of Peyer patches were studied in Nod2 knock-out mice transplanted with wild-type bone marrow cells and vice versa. RESULTS: The nonhematopoietic cells control the microbiota composition and epithelial secretion of mucins and antimicrobial peptides. These parameters are correlated with recurrent associations between bacterial species and luminal products. In contrast, Nod2 in the hematopoietic compartment regulates the epithelial permeability and the gut-associated lymphoid tissue independently of the bacterial composition. CONCLUSIONS: The immune system and the gut permeability in one hand and the microbial and epithelial peptide compositions in the other hand are separate couples of interdependent parameters, both controlled by Nod2 in either the hematopoietic or nonhematopoietic lineages.

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis.

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.

An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp.

Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation.

NLRs Challenge Impacts Tight Junction Claudins In Sertoli Cells.

AIM: The present study aims to investigate the NALP3 system and its effect on claudins in Sertoli cells using a mouse adult Sertoli cell line as a model. We focus on the Sertoli cell biology looking for the possible implications for male reproductive functions. MATERIALS AND METHODS: Adult Sertoli cells were transfected with NAPL3 siRNA and treated with NOD1 (ie-DAP) and NOD2 (MDP) receptor ligands. Two dimensional gel electrophoresis was performed on lysates of non-challenged and MDP-treated Sertoli cells. RESULTS: There were positive claudin-5 and claudin-11 expression levels on transcript (RT-qPCR) levels. Specific protein spots in 2D gels were detected after bioinformatics analysis. This study demonstrates direct induction of tight-junction proteins probably favouring junction stability. CONCLUSIONS: The innate immunity and tight-junction pathway integration probably have a protective role for both blood-testis immune barrier and spermatogenesis compartmentalisation maintained by the very same barrier. This integration also points the way for mechanistic research of the disturbances inflicted during an inflammatory response in the testis niche.

Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells.

Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1ß had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1ß and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1ß secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes.

NOD2 pathway via RIPK2 and TBK1 is involved in the aberrant catabolism induced by T-2 toxin in chondrocytes.

OBJECTIVE: This study aimed to identify the key intracellular pattern recognition receptor (PRR) and its role in the unbalanced extracellular matrix gene expressions of chondrocytes treated by T-2 toxin, a potential etiological factor for cartilage damages. DESIGN: Differential expressions of intracellular PRRs after T-2 toxin treatment were screened by RT-qPCR in chondrocytes. RNAi was used to knockdown the expression of NOD2 and its two downstream signal molecules, RIPK2, and TBK1, for observing the effects of NOD2 pathway on regulation of metabolism gene expressions by RT-qPCR. The matrix metalloproteinases (MMP) activity was determined by gelatin zymography. The inhibitor of NF-κB and ROS scavenger were exploited to analyze the mechanism of NOD2 up-regulation in chondrocytes treated with T-2 toxin. RESULTS: In chondrocytes treated with T-2 toxin, anabolism genes were down-regulated whereas catabolism genes were up-regulated, and NOD2 was identified as a significantly up-regulated gene. Intervening NOD2 expression via RNAi could ameliorate the down-regulation of anabolism genes, while inhibit the up-regulation of catablolism genes induced by T-2 toxin in chondrocytes. RNAi of RIPK2 and TBK1 in chondrocytes could obtain the similar outcome. Furthermore, up-regulation of NOD2 expression induced by T-2 toxin could be abrogated by pretreating the cells with inhibitors of NF-κB and scavenger of ROS. CONCLUSION: T-2 toxin could up-regulate NOD2 expression via ROS/NF-κB pathway and activate NOD2 signaling pathway. The up-regulated NOD2 would affect the metabolism gene expressions and MMP activity in chondrocytes via RIPK2 and TBK1. The findings add new insights into understanding NOD2 effects on chondrocytes treated with T-2 toxin.

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model.

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells.

Immunotherapy is a promising treatment for liver cancer. Here, we tested the ability of the attenuated hepatocellular carcinoma-specific Listeria vaccine (Lmdd-MPFG) to treat hepatocellular carcinoma (HCC) in a mouse model. Immunization with the vaccine caused a strong anti-tumor response, especially in mice reinfused with dendritic cells (DCs). In mice that were also administered DCs, tumor suppression was accompanied by the strongest cytotoxic T lymphocyte response of all treatment groups and by induced differentiation of CD4+ T cells, especially Th17 cells. Additionally, the Lmdd-MPFG vaccine caused maturation of DCs in vitro. We demonstrated the synergistic effect of TLR4 and NLRP3 or NOD1 signaling pathways in LM-induced DC activation. These results suggest that the Lmdd-MPFG vaccine is a feasible strategy for preventing HCC.