Bacterial Toll-like Receptor Research Articles

Lactic acid bacteria secrete toll like receptor 2 stimulating and macrophage immunomodulating bioactive factors

Lactic acid bacteria (LAB) can support host health and crosstalks between ligands on bacterial cells and Toll-like receptors (TLR) are known as key inducers of these health effects. LAB-secreted bioactive factors also possess health-promoting properties but the underlying mechanisms remain to be identified. Here, we report that secreted factors of different LAB strains induce cytokine (IL-10 and TNF-α) production by THP1-differentiated human macrophages in a species- and strain-dependent manner. LAB-secreted products also drive immune-stimulating and anti-pathogenic M1-macrophage polarization. Moreover, bacterial supernatants induce species- and strain-dependent TLR2 activation. Several bacterial strains such as L. casei CCFM9 and L. brevis CCFM498 cannot activate TLR signaling, but their secreted products strongly activate TLR2. This indicates that LAB-secreted components may further boost the functional effects of bacterial strains. Our results confirm direct interactions of LAB-secreted components with TLRs, and provide novel insights in how LABs may provide immune-modulation.

Increased grp78 transcription is correlated to reduced tlr4 transcription in patients surviving sepsis.

Regulated transcriptional readthrough during stress maintains genome structure and ensures access to genes that are necessary for cellular recovery. A broad number of genes, including of the bacterial sensor Toll-like receptor 4 (TLR-4), are markedly transcribed on initiating the systemic inflammatory response. Here we study the transcriptional patterns of tlr4 and of its modulator grp78 during human sepsis, and establish their correlations with the outcome of patients. We measured the daily tlr4 and grp78 RNA expression levels in peripheral blood of septic patients, immediately after admission to intensive care, and modeled these RNA values with a sine damping function. We obtained negative correlations between the transcription of tlr4 and grp78 RNA in the survivor group. In contrast, such relation is lost in the deceased patients. Loss of transcriptional homeostasis predicted by our model within the initial 4days of hospitalization was confirmed by death of those patients up to 28days later.

Caspase-8 promotes c-Rel–dependent inflammatory cytokine expression and resistance against Toxoplasma gondii

Caspase-8 is a key integrator of cell survival and cell death decisions during infection and inflammation. Following engagement of tumor necrosis factor superfamily receptors or certain Toll-like receptors (TLRs), caspase-8 initiates cell-extrinsic apoptosis while inhibiting RIPK3-dependent programmed necrosis. In addition, caspase-8 has an important, albeit less well understood, role in cell-intrinsic inflammatory gene expression. Macrophages lacking caspase-8 or the adaptor FADD have defective inflammatory cytokine expression and inflammasome priming in response to bacterial infection or TLR stimulation. How caspase-8 regulates cytokine gene expression, and whether caspase-8-mediated gene regulation has a physiological role during infection, remain poorly defined. Here we demonstrate that both caspase-8 enzymatic activity and scaffolding functions contribute to inflammatory cytokine gene expression. Caspase-8 enzymatic activity was necessary for maximal expression of Il1b and Il12b, but caspase-8 deficient cells exhibited a further decrease in expression of these genes. Furthermore, the ability of TLR stimuli to induce optimal IκB kinase phosphorylation and nuclear translocation of the nuclear factor kappa light chain enhancer of activated B cells family member c-Rel required caspase activity. Interestingly, overexpression of c-Rel was sufficient to restore expression of IL-12 and IL-1β in caspase-8-deficient cells. Moreover, Ripk3-/-Casp8-/- mice were unable to control infection by the intracellular parasite Toxoplasma gondii, which corresponded to defects in monocyte recruitment to the peritoneal cavity, and exogenous IL-12 restored monocyte recruitment and protection of caspase-8-deficient mice during acute toxoplasmosis. These findings provide insight into how caspase-8 controls inflammatory gene expression and identify a critical role for caspase-8 in host defense against eukaryotic pathogens.

Improving our mechanistic understanding of the indirect effects of CMV infection in transplant recipients.

Cytomegalovirus (CMV) is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal, and viral infections. However, the pathogenesis of this phenomenon is poorly understood. We determined whether inflammatory responses to different Toll-like receptor (TLR) ligands are blunted during CMV infection in solid-organ transplant (SOT) patients. Peripheral blood mononuclear cells from 38 SOT patients with and without CMV were incubated in the presence of various viral, fungal, and bacterial TLR ligands. Cytokines were measured in the supernatant by multiplex enzyme-linked immunosorbent assay. Patients had blunted cytokine responses to bacterial, fungal, and viral ligands during CMV infection when compared to the absence of CMV infection. This was independent of viral load, clinical presentation of CMV infection or immunosuppression, supporting the clinical observation in SOT recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. Moreover, in the absence of CMV infection, patients with subsequent CMV infection had lower cytokines in response to TLR ligands compared to those without subsequent CMV infection, suggesting that inherent differences in patients not directly related to CMV also contribute to this increased susceptibility. In summary, these data provide novel ex vivo evidence to support indirect effects of CMV.

In Vitro Induction of T Helper 17 Cells by Synergistic Activation of Human Monocyte-Derived Langerhans Cell-Like Cells with Bacterial Agonists.

In the case of epidermal barrier disruption, pathogens encounter skin-resident Langerhans cells (LCs) and are recognized by pathogen recognition receptors such as Toll-like receptors (TLRs). As the majority of microorganisms exhibit more than one TLR ligand, the mechanisms of subsequent T cell differentiation are complex and far from clear. In this study, we investigated combinatory effects on Th cell polarization by bacterial cell wall compounds peptidoglycan (PGN) and lipopolysaccharide (LPS) and by bacterial nucleic acid (DNA). Expression of maturation markers CD40, CD80, HLA-DR and CCR7 and the release of IL-1β, IL-6 and IL-23 was strongly enhanced by simultaneous exposure to PGN, LPS and DNA in LCs. As all these factors were potential Th17 driving cytokines, we investigated the potency of combinatory TLR stimuli to induce Th17 cells via LC activation. High amounts of IL-17A and IL-22, key cytokines of Th17 cells, were detected. By intracellular costaining of IL-17+T cells, IL-22− (Th17) and IL-22+ (immature Th17) cells were identified. Interestingly, one population of LPS stimulated cells skewed into IL-9+Th cells, and LPS synergized with PGN while inducing high IL-22. In conclusion, our data indicates that when mediated by a fine-tuned signal integration via LCs, bacterial TLR agonists synergize and induce Th17 differentiation.

Association between small intestinal bacterial overgrowth and toll-like receptor 4 in patients with pancreatic carcinoma and cholangiocarcinoma

Multiple factors have been linked to pathogenesis of pancreatic cancer and cholangiocarcinoma. Until now, few studies have investigated the role of small intestinal bacterial overgrowth (SIBO) and toll-like receptor 4 (TLR-4) signaling in these diseases. This study aimed to examine the relationship between the prevalence of SIBO and the TLR-4 expression in patients with pancreatic carcinoma and cholangiocarcinoma. A total of 90 human subjects suffering from pancreatic carcinoma (n=30), cholangiocarcinoma (n=30), and healthy controls (n=30) were enrolled in the study. A glucose hydrogen breath test (GHBT) was used to evaluate SIBO. The TLR4 protein expression was measured by immunohistochemistry (IHC). The positive rate of SIBO was 63.3% in the pancreatic cancer group and 46.7% in patients with cholangiocarcinoma, which was significantly greater than 13.3% in the healthy control group (p<0.05). An IHC analysis revealed that the TLR-4 protein expression in the SIBO-positive pancreatic carcinoma patients was significantly higher than that in the SIBO-negative patients (p<0.05), and the same result was in the cholangiocarcinoma subjects. In addition, a correlation analysis identified the positive relationship between the prevalence of SIBO and the TLR-4 protein expression in pancreatic carcinoma (r=0.489), and the same result was in the cholangiocarcinoma subjects. Our findings indicate a high prevalence of SIBO in pancreatic carcinoma and cholangiocarcinoma, and SIBO displays a positive correlation with the TLR-4 expression, suggesting that SIBO could be a risk factor for the pathogenesis of pancreatic carcinoma and cholangiocarcinoma, in which the TLR4 signaling may be involved.

Loss of Forkhead Box O3 Facilitates Inflammatory Colon Cancer: Transcriptome Profiling of the Immune Landscape and Novel Targets.

Background & AimsDiminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth.MethodsFOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3).ResultsIn mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth.ConclusionsWe identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

Bacterial contamination hypothesis: a new concept in endometriosis.

BackgroundEndometriosis is a multifactorial disease that mainly affects women of reproductive age. The exact pathogenesis of this disease is still debatable. The role of bacterial endotoxin (lipopolysaccharide, LPS) and Toll‐like receptor 4 (TLR4) in endometriosis were investigated and the possible source of endotoxin in the pelvic environment was examined.MethodsThe limulus amoebocyte lysate test was used to measure the endotoxin levels in the menstrual fluid and peritoneal fluid and their potential role in the growth of endometriosis was investigated. Menstrual blood and endometrial samples were cultured for the presence of microbes. The effect of gonadotrophin‐releasing hormone agonist (GnRHa) treatment on intrauterine microbial colonization (IUMC) and the occurrence of endometritis was investigated.Main findings (Results)Lipopolysaccharide regulates the pro‐inflammatory response in the pelvis and growth of endometriosis via the LPS/TLR4 cascade. The menstrual blood was highly contaminated with Escherichea coli and the endometrial samples were colonized with other microbes. A cross‐talk between inflammation and ovarian steroids or the stress reaction also was observed in the pelvis. Treatment with GnRHa further worsens intrauterine microbial colonization, with the consequent occurrence of endometritis in women with endometriosis.ConclusionFor the first time, a new concept called the “bacterial contamination hypothesis” is proposed in endometriosis. This study's findings of IUMC in women with endometriosis could hold new therapeutic potential in addition to the conventional estrogen‐suppressing agent.

Lactobacillus plantarum induces genomic DNA-dependent and TLR9-mediated elafin secretion from Caco-2 cells

Lactobacilli show anti-inflammatory effects in the human intestine, and their genomic DNA was identified as one of the anti-inflammatory components. Increased levels of the natural protease inhibitor elafin in the intestine plays an important role in protection against intestinal inflammation. However, there have been no previous reports regarding whether lactobacilli increase elafin levels. This study was performed to investigate whether Lactobacillus plantarum induces elafin secretion from the human epithelial colorectal adenocarcinoma cell line, Caco-2. Moreover, we examined the roles of bacterial genomic DNA and toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, in this effect. Elafin secretion from Caco-2 cells by live and heat-killed L. plantarum was measured. The analysis was also performed using DNase-treated L. plantarum and genomic DNA extracted from L. plantarum. We examined the role of TLR9 in elafin secretion by L. plantarum and its genomic DNA by suppressing TLR9 expression using RNAi in Caco-2 cells. Heat-killed L. plantarum time- and dose-dependently increased elafin secretion, whereas live L. plantarum had no such effect. The elafin secretion by heat-killed L. plantarum was partially abolished by DNase treatment of the bacterium. In addition, L. plantarum genomic DNA also increased elafin secretion. Furthermore, suppression of TLR9 expression partially or completely abolished elafin secretion by heat-killed L. plantarum and its genomic DNA. Our results indicated that heat-killed L. plantarum induced genomic DNA-dependent and TLR9-mediated elafin secretion. The anti-inflammatory effects of lactobacilli may be mediated by increases in the levels of elafin in the intestine.

Coagulation factor 9-deficient mice are protected against dextran sulfate sodium-induced colitis.

ABSTRACTPatients with inflammatory bowel disease (IBD) are susceptible to thromboembolism. Interestingly, IBD occurs less frequently in patients with inherited bleeding disorders. Therefore, we analyzed whether F9-deficiency is protective against the onset of acute colitis in a genetic hemophilia B mouse model. In the 3.5% dextran sulfate sodium (DSS)-induced colitis model, F9-deficient mice were protected from body-weight loss and had a reduced disease activity score. We detected decreased colonic myeloperoxidase activity and decreased CXCL1 levels in DSS-treated F9-deficient mice compared with wild-type (WT) littermate controls, indicating decreased neutrophil infiltration. Remarkably, we identified expression of coagulation factor IX (FIX) protein in small intestinal epithelial cells (MODE-K). In epithelial cell cultures, cellular FIX protein expression was increased following stimulation with the bacterial Toll-like receptor agonists lipopolysaccharide, macrophage-activating lipopeptide-2 and Pam3CSK4. Thus, we revealed a protective role of F9-deficiency in DSS-induced colitis and identified the intestinal epithelium as a site of ectopic FIX.This article has an associated First Person interview with the first author of the paper.

Estradiol alters the immune-responsiveness of cervical epithelial cells stimulated with ligands of Toll-like receptors 2 and 4.

The mucosa of the female reproductive tract plays a pivotal role in host defence. Pregnancy must alter immunological mechanisms at this interface to protect the conceptus. We sought to determine how estradiol (E2) alters the immune-responsiveness of cervical epithelial cells to ligand stimulation of Toll-like receptor (TLR)-2 and -4. Human ectocervical epithelial cells (HECECs) were cultured and co-incubated with two concentrations of E2 and peptidoglycan (PGN) or lipopolysaccharide (LPS) over durations that ranged between 10 minutes and 18 hours. Cytometric Bead Array was performed to quantify eight cytokines in the supernatant fluid. In response to PGN, HECECs co-incubated with E2 released lesser quantities of IL-1ß and IFNγ, higher levels of RANTES, and variable levels of IL-6 and IL-8 than those not exposed to E2. In contrast, HECECs co-incubated with LPS and E2 secreted increased levels of IL-1ß, IL-6, IL-8, and IFNγ at 2 and 18 hours than HECECs not exposed to E2, and reduced levels of RANTES at same study time-points. Estradiol alters the immune-responsiveness of cultured HECECs to TLR2 and TLR4 ligands in a complex fashion that appears to vary with bacterial ligand, TLR subtype, and duration of exposure. Our observations are consistent with the functional complexity that this mucosal interface requires for its immunological roles.

Transcriptome Analysis Reveals Comprehensive Insights into the Early Immune Response of Large Yellow Croaker (Larimichthys crocea) Induced by Trivalent Bacterial Vaccine.

Vaccination is an effective and safe strategy for combating bacterial diseases in fish, but the mechanisms underlying the early immune response after vaccination remain to be elucidated. In the present study, we used RNA-seq technology to perform transcriptome analysis of spleens from large yellow croaker (Larimichthys crocea) induced by inactivated trivalent bacterial vaccine (Vibrio parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). A total of 2,789 or 1,511 differentially expressed genes (DEGs) were obtained at 24 or 72 h after vaccination, including 1,132 or 842 remarkably up-regulated genes and 1,657 or 669 remarkably down-regulated genes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichments revealed that numerous DEGs belong to immune-relevant genes, involved in many immune-relevant pathways. Most of the strongly up-regulated DEGs are innate defense molecules, such as antimicrobial peptides, complement components, lectins, and transferrins. Trivalent bacterial vaccine affected the expressions of many components associated with bacterial ligand–depending Toll-like receptor signaling pathways and inflammasome formation, indicating that multiple innate immune processes were activated at the early period of vaccination in large yellow croaker. Moreover, the expression levels of genes involved in antigen processing were also up-regulated by bacterial vaccine. However, the expression levels of several T cell receptors and related CD molecules and signal transducers were down-regulated, suggesting that the T cell receptor signaling pathway was rapidly suppressed after vaccination. These results provide the comprehensive insights into the early immune response of large yellow croaker to vaccination and valuable information for developing a highly immunogenic vaccine against bacterial infection in teleosts.

Identification of TLR2/TLR6 signalling lactic acid bacteria for supporting immune regulation.

Although many lactic acid bacteria (LAB) influence the consumer’s immune status it is not completely understood how this is established. Bacteria-host interactions between bacterial cell-wall components and toll-like receptors (TLRs) have been suggested to play an essential role. Here we investigated the interaction between LABs with reported health effects and TLRs. By using cell-lines expressing single or combination of TLRs, we show that LABs can signal via TLR-dependent and independent pathways. The strains only stimulated and did not inhibit TLRs. We found that several strains such as L. plantarum CCFM634, L. plantarum CCFM734, L. fermentum CCFM381, L. acidophilus CCFM137, and S. thermophilus CCFM218 stimulated TLR2/TLR6. TLR2/TLR6 is essential in immune regulatory processes and of interest for prevention of diseases. Specificity of the TLR2/TLR6 stimulation was confirmed with blocking antibodies. Immunomodulatory properties of LABs were also studied by assessing IL-10 and IL-6 secretion patterns in bacteria-stimulated THP1-derived macrophages, which confirmed species and strain specific effects of the LABs. With this study we provide novel insight in LAB specific host-microbe interactions. Our data demonstrates that interactions between pattern recognition receptors such as TLRs is species and strain specific and underpins the importance of selecting specific strains for promoting specific health effects.

Modulation of monocytes by bioactive lipid anandamide in multiple sclerosis involves distinct Toll-like receptors

Monocytes are believed to be involved in the immunopathogenesis of multiple sclerosis (MS). The aim of this study was to investigate their role in MS and their immunomodulation by the endocannabinoid system (ECS), a novel target for the treatment of this disease. We compared the level of cytokine production from monocytes in healthy subjects and MS patients upon stimulation with viral or bacterial Toll-like receptors (TLR) and we evaluated the ECS immunomodulatory role in these cells. Here we show that MS monocytes produced more TNF-α, IL-12 and IL-6 following activation of TLR2/4 with LPS or of TLR5 with flagellin, as opposed to TLR7/8 stimulation with R848. Furthermore AEA, the main endocannabinoid, suppressed cytokine production and release from healthy monocytes upon stimulation with both bacterial and viral TLR receptors but not in cells from MS patients, where its immunosuppressive activity was TLR7/8-dependent. Altered expression levels of key ECS members in MS monocytes paralleled these data. Our data disclose a distinct immunomodulatory effect of AEA and an alteration of AEA-related members of the ECS in monocytes from MS patients that involves viral but not bacterial TLR. These findings not only may help to better understand the role of monocytes in MS immunopathogenesis but also could be of help to exploit new endocannabinoid-based drugs that target innate immune cells.

Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.

Necrotizing enterocolitis (NEC) is the most frequent and lethal disease of the gastrointestinal tract of preterm infants. At present, NEC is thought to develop in the premature host in the setting of bacterial colonization, often after administration of non-breast milk feeds, and disease onset is thought to be due in part to a baseline increased reactivity of the premature intestinal mucosa to microbial ligands as compared with the full-term intestinal mucosa. The increased reactivity leads to mucosal destruction and impaired mesenteric perfusion and partly reflects an increased expression of the bacterial receptor Toll-like receptor 4 (TLR4) in the premature gut, as well as other factors that predispose the intestine to a hyper-reactive state in response to colonizing microorganisms. The increased expression of TLR4 in the premature gut reflects a surprising role for this molecule in the regulation of normal intestinal development through its effects on the Notch signalling pathway. This Review will examine the current approach to the diagnosis and treatment of NEC, provide an overview of our current knowledge regarding its molecular underpinnings and highlight advances made within the past decade towards the development of specific preventive and treatment strategies for this devastating disease.

Aberrant Expression of Bacterial Pattern Recognition Receptor NOD2 of Basophils and Microbicidal Peptides in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease, associated with basophil infiltration into skin lesions and Staphylococcus aureus (S. aureus)-induced inflammation. Pattern recognition receptors (PRRs), including microbicidal peptide human neutrophil α-defensins (HNP) and dermcidin, can exert immunomodulating activity in innate immunity and skin inflammation. We investigated the plasma concentration of HNP and dermcidin, the expression of bacterial toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors of basophils and plasma concentration and ex vivo induction of AD-related inflammatory cytokines and chemokines using ELISA and flow cytometry, in AD patients and control subjects. Plasma concentrations of HNP, dermcidin and AD-related Th2 chemokines CCL17, CCL22 and CCL27 were significantly elevated in AD patients compared with controls (all p < 0.05). Plasma concentrations of CCL27 and CCL22 were found to correlate positively with SCORing atopic dermatitis (SCORAD), objective SCORAD, % area affected, lichenification and disease intensity, and CCL27 also correlated positively with pruritus in AD patients (all p < 0.05). Protein expressions of NOD2 but not TLR2 of basophils were significantly down-regulated in AD patients compared with controls (p = 0.001). Correspondingly, there were lower ex vivo % inductions of allergic inflammatory tumor necrosis factor-α, IL-6 and CXCL8 from peripheral blood mononuclear cells upon NOD2 ligand S. aureus derived muramyl dipeptide stimulation in AD patients comparing with controls. The aberrant activation of bacterial PRRs of basophils and anti-bacterial innate immune response should be related with the allergic inflammation of AD.

The Kampo medicine “Daikenchuto (TU-100)” prevents bacterial translocation and hepatic fibrosis in a rat model of biliary atresia

Biliary atresia is the most common cause of end-stage liver disease in children. It is known that bile duct ligation contributes to liver fibrosis via bacterial translocation (BT) and toll-like receptor 4 (TLR4) signaling of hepatic stellate cells (HSCs). We have reported previously that the traditional Japanese medicine, "Dai-kenchu-to (TU-100)," a form of "Kampo medicine" prevents BT in rats exposed to the stress of fasting. The aim of this study was to clarify the effect of TU-100 on a rat model of biliary atresia using bile duct ligation. Bile duct ligation and subsequent daily oral administration of TU-100 was performed in 6-week-old rats. The rats were killed at 3, 7, or 14days after bile duct ligation to evaluate the liver injury, occurrence of BT, and hepatic fibrosis. As an invitro experiment, we isolated fresh HSCs from the rats undergoing bile duct ligation. After cell attachment, TU-100 and its 3 component herbs (eg, processed ginger, ginseng radix, and Japanese pepper) were added, and the expressions of Alpha actin2 (acta2), Alpha-1 type I collagen (colIa1), and tissue inhibitor of metalloproteinase 1 (timp1) were analyzed. Invivo experiments demonstrated that oral administration of TU-100 decreased liver injury and atrophy of intestinal mucosa BT, hepatic fibrosis, and hepatic expression of alpha smooth muscle actin (αSMA) and TLR4, compared with rats that underwent bile duct ligation only. Invitro experiments showed that administration of TU-100 or the component herbs inhibited the expressions of acta2, colIa1, and timp1 in the HSCs. TU-100 prevented BT, activation of HSCs, and subsequent hepatic fibrosis. TU-100 may prevent progression of hepatic fibrosis in children with biliary atresia and improve prognosis.

Small intestinal bacterial overgrowth and toll-like receptor signaling in patients with non-alcoholic fatty liver disease.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll-like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD. A total of 142 subjects composed of NAFLD (n = 60, mean age 38.7 ± 10.4 years), chronic viral hepatitis (CVH) (n = 32, mean age 39.5 ± 10.6 years), and healthy volunteers (n = 50, mean age 36.56 ± 4.2 years) were enrolled in the study. Duodenal fluid was taken endoscopically in 32 prospective patients with NAFLD for evaluation of SIBO. Hepatic mRNA expression of TLR4, CD14, TLR2, NF-κβ, and MD2 and protein expression of TLR4 and TLR2 were studied in 64 patients (NAFLD = 32, CVH = 32) by RT-PCR and immunohistochemistry, respectively. Serum levels of TNF-α, adiponectin, insulin, and endotoxins were also evaluated. Small intestinal bacterial overgrowth was present in 12 (37.5%) out of 32 patients with NAFLD with Escherichia coli as the predominant bacterium. In comparison with those without SIBO, patients with SIBO had significantly higher endotoxin levels and higher CD14 mRNA, nuclear factor kappa beta mRNA, and TLR4 protein expression. Patients with NASH had significantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison with patients without NASH. Serum levels of TNF-α, endotoxins, and insulin were significantly higher and of adiponectin lower in NAFLD in comparison with CVH and healthy volunteers. Our study provides the first direct evidence of role of SIBO and endotoxemia and its relation with TLR signaling genes and liver histology in patients with NAFLD.

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation.

ABSTRACTLinear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation.

Small Intestinal Bacterial Overgrowth and Toll-Like Receptor Signaling in Patients With Nonalcoholic Fatty Liver Disease: Presidential Poster

Small Intestinal Bacterial Overgrowth and Toll-Like Receptor Signaling in Patients With Nonalcoholic Fatty Liver Disease: Presidential Poster