MyD88 mRNA Research Articles

NALP3 inflammasome activation is MYD88-, but not TLR4-dependent and involves both parenchymal and bone marrow derived cells in non-alcoholic steatohepatitis (NASH)

Background and Aims: Endogenous danger signals from damaged hepatocytes in NASH might up-regulate and activate the inflammasome, that is a multiprotein complex that leads to IL-1ß secretion. We reported NALP3 inflammasome up-regulation activation in NASH. Here, we aimed to investigate the role of toll-like receptor 4 (TLR4) and the common TLR adaptor, MyD88 in the activation of NALP3 inflammasome. Furthermore, we aimed to investigate which cell types are involved in the inflammasome activation in the liver in NASH. Methods: Wild type (WT), TLR4- and MyD88-deficient (KO) mice, and WT mice transplanted with MyD88 KO bone marrow (WT/MyD88KO-BM) or MyD88KO mice with WT bone marrow (MyD88KO/WT-BM) received methionine-choline-deficient (MCD) or -supplemented (MCS) diet. Results: MCD diet-induced NASH resulted in up-regulation of TLR4, MyD88 and NALP3 inflammasome (NALP3, ASC, pro-caspase-1) mRNA, and mature IL-1β (17kDa) protein and caspase-1 activation in the liver compared to MCS diet feeding in WT mice. Total body deficiency in TLR4 although attenuated the MCD diet-induced liver injury and steatosis, but did not prevent the either the NALP3 up-regulation or the IL-1β production. MyD88 is a common adaptor molecule of most of the TLRs, including TLR4, but also TLR9. Total body deficiency in MyD88 prevented up-regulation of NALP3 mRNA and mature IL-1β production and significantly attenuated liver damage and steatosis in NASH. Selective MyD88-deficiency in bone marrow-derived or parenchymal cells prevented NALP3 mRNA up-regulation and attenuated IL-1β production in both chimeras (WT/MyD88KO-BM and MyD88KO/WT-BM)). Interestingly, WT/MyD88KO-BM mice were protected from MCD diet induced steatosis and liver damage, but liver injury was comparable between MCD diet-fed WT and MyD88KO/WT-BM mice suggesting that BM-derived cells contribute to NASH in a MyD88-dependent manner. Conclusions: We demonstrate for the first time that NALP3 inflammasome activation requires MyD88-dependent signaling in the MCD diet-induced NASH, but the deficiency of TLR4 is not enough to prevent the infammasome priming and activation. Finally, we show that both bone marrow-derived and parenchymal cells contribute to IL-1β production and the absence of MyD88 in bone marrow-derived cells attenuates steatohepatitis.

Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Abstract Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+/+ mice and ran Real Time-PCR or Western Blot for different Toll-like Receptor Pathway components. Results: qRT-PCR showed that Smad3−/-− mice have a significantly lower quantity of Bacteroides sp; the introduction of Helicobacter hepaticus results in a significant increase in B. distasonis and a significant decrease in B. thetaiotaomicron in Smad3−/− mice. These data suggest that loss of SMAD3 and the addition of Helicobacter hepaticus alters the colonic microflora. Previous studies illustrated the importance of epithelial signaling in microbial induced inflammation. To examine if Smad3 is altering the Toll-like receptor pathway we examined RT-PCR of the cecum and found a significant increase in Tlr4, Irak4, Cd14, Myd88, Nfkb, Cox2, and Nos2 mRNA in the Smad3−/−− mice compared to Smad3+/+ mice. Western blots confirmed these data by showing that cecal tissue from Smad3−/− mice has increased IRAK4 and pNF-κB. Conclusion: Smad3−/− mice show heightened Toll-like receptor pathway activity which leads to a hyper-inflammatory response of the mucosa to endogenous microbes. Further, loss of SMAD3 results in dysbiosis of the gut microbiota. These findings suggest that SMAD3 plays a role in microbial-host homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 407. doi:1538-7445.AM2012-407

Expression of intestinal myeloid differentiation primary response protein 88 (Myd88) following experimental traumatic brain injury in a mouse model

BackgroundTraumatic brain injury (TBI) can cause gastrointestinal dysfunction and increase intestinal permeability. Nuclear factor kappa B (NF-κB) has been shown to be associated with these intestinal events, but it is not well known how NF-κB is activated in the intestine after TBI. Based on previous studies, we hypothesize that myeloid differentiation primary response protein 88 (Myd88) may have an important role in NF-κB activation in the intestine, which mediates the inflammation and ultimately results in acute intestinal mucosal injury. MethodsWe randomly divided adult male C57BL/6 mice into control groups and TBI groups at different time points. We induced a closed head injury model by weight drop (a 333-g metal rod dropping from a 2.5-cm height). We detected Myd88 protein level and NF-κB binding activity in ileum tissue by Western blot and electrophoretic mobility shift assay, respectively. Meanwhile, we detected the mRNA levels of Myd88, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and intercellular adhesion molecule-1 by real-time polymerase chain reaction. ResultsThe Myd88 protein and mRNA levels, as well as NF-κB binding activity in the ileum tissue, significantly increased at 6 h after TBI, peaked at 3 d, and remained elevated by 5 d post-injury. The levels of TNF-α, IL-1β, and intercellular adhesion molecule-1 also remarkably increased after TBI. There was a positive relationship between the expression of Myd88 and that of NF-κB, TNF-α, and IL-1β. ConclusionsTraumatic brain injury induced a rapid and persistent up-regulation of Myd88, NF-κB, and proinflammatory cytokines in the intestine. This up-regulation which might have an important role in the pathogenesis of acute intestinal mucosal injury.

Fragments of cell-free DNA increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway, and suppress apoptosis

Human mesenchymal stem cells (MSCs) are widely used in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism still remain unanswered. In lesion regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We have shown that in vitro contact of adipose-derived MSCs with cfDNA fragments the increase of transcription activity (evaluated by the increase of total cellular RNA and rRNA) is observed. GC-rich cfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway causing up-regulation of expression of TLR9 and MyD88, the TLR9-signaling pathway adapter. AT-rich DNA fragments did not increase the TLR9 expression, but did increase the level of MyD88 mRNA. So we suggest that AT-DNA acts via some other receptors, which, nevertheless, activate MyD88-dependent signalling in MSCs. We have also shown that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. Thus, cfDNA can significantly influence the functional activity of MSC by activating the TLR9- and MyD88-dependent signal pathways and by lowering the apoptosis level.

Silencing gene of TLR4 down-regulates the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus conidia stimulation

Objective To study the role of TLR2 and TLR4 signal transduction in RAW264.7 monocyte-macrophages stimulated by Aspergillus fumigatus conidia,and to investigate the expression of TLR2 signal transduction after silencing gene of TLR4.Methods Macrophages were randomly divided into normal group ( N group),normal+stimulated with Aspergillus fumigatus conidia ( N +Af group ),normal + transfected with TLR4-siRNA [ TLR4 (RNAi) group ],normal+transfected with TLR4-siRNA +stimulated with Aspergillus fumigatus conidia[ TLR4(RNAi) +Af group].RT-PCR and Western blot were used to assay expression levels of TLR2,TLR4,MyD88 mRNA and pro-inflammatory cytokines TNF-α protein when macrophages were stimulated 12 h by Aspergillus fumigatus conidia after tranfected 24 h with TLR4-siRNA by technology of RNAi.Results ( 1 ) Compared with N group,the expression of TLR2,TLR4,MyD88 mRNA and TNF-αprotein in N+Af group significantly increased before silencing gene of TLR4.(2) Silencing efficiency of macrophates was up to 83％ after transfected with TLR4-siRNA.(3)The expression of TLR2,MyD88 mRNA in TLR4 (RNAi) group significantly decreased contrast with normal group.Meanwhile the expression of TLR2,MyD88 mRNA and TNF-α protein also obviously reduced in TLR4(RNAi) +Af group when compared with N +Af group.Compared with TLR4 (RNAi) group,the expression of MyD88 mRNA in TLR4 (RNAi) +Af group significantly increased.However,the expression of TLR2 mRNA and TNF-α protein have no significant change after silencing gene of TLR4.Conclusion Signaling pathway of TLR2 and TLR4 in macrophages was activated by given stimulus of Aspergillusfumigatus conidia and exerted the effect of anti-Aspergillus fumigatus spores stimulation through the release of pro-inflammatory cytokines TNF-α.Meanwhile,silencing gene of TLR4 down-regulate the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus conidia stimulation,and it found that TLR4 played an more important role by contrast with TLR2. Key words: TLR2 ; TLR4 ; TLR4-siRNA ; Aspergillus fumigatus conidia; Regulation mechanism

Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1h after LPS stimulation and remained significantly higher up to 48h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.

MyD88 Could Promote Multiple Myeloma Cells Growth and Survival

Abstract 5086Toll like receptors (TLRs) are the major agents for innate immunity, which recognize invading microbial products. In previous study, we proved that activated TLR4 and TLR9 could promote MM cells growth and activated NF-κB signaling played a crucial role for ligands of TLR4 and TLR9 via increasing IL-6 autocrine. It was reported that MyD88 could play the key role in TLR signaling pathway and TLR4 and TLR9 shared the same MyD88-dependent pathway. In this study, we observed that expression of MyD88 mRNA in primary MM cells was higher than normal plasma cells and most of MM cell lines had a high MyD88 transcripts. Futher experiments demonstrated that transient over-expression of MyD88 could promote MM primary cells and MM cell lines growth and production of IL-6, IL-1, TNF-α, IFN-γ, VEGF and TGF-β. Moreover, down-regulation of MyD88 by sepecific virus based siRNA plamids in MM cell lines preliminarily resulted in reduced MM cells proliferation and increased MM cells apoptosis. These results highly suggested that MyD88 played a crucial role for TLR ligands to promote MM cells growth and survival, which make it possible to develop TLR system and MyD88 gene as the MM therapy target in the future. Disclosures:No relevant conflicts of interest to declare.

MyD88-Dependent Expansion of An Immature GR-1+CD11b+ Population From Donor Bone Marrow Reduces Early Mortality After Allogeneic Hematopoietic Stem Cell Transplantation,

Abstract 4011Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity. MyD88 is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the toll-like receptor (TLR) family. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs.We therefore investigated how the MyD88-dependent expansion of MDSCs from donor bone marrow (BM) contributes to protection of acute GVHD. To test this, we employed an intestinal GVHD murine model, C57BL/6 (H-2b) → B6D2F1 (H-2b/d), which differs at major and minor histocompatibility loci. Lethally irradiated recipient mice were transplanted with wild-type (WT) or MyD88 knock out (KO) mice T cell-depleted (TCD)-BM together with WT spleen T cells. Morbidity and mortality of GVHD was significantly worse in recipients of MyD88 KO TCD-BM with higher intestinal pathologic grading. Animals that underwent syngeneic HSCT did not show early mortality regardless of presence of MyD88 in BM, which ruled out myelosuppression-associated toxicity. The expression of Gr-1+CD11b+ in blood, mesenteric lymph nodes and liver on day 13 was significantly reduced in the recipients of MyD88 KO TCD-BM compared with those of WT TCD-BM while the percentage of donor T cells infiltrating colon and liver was significantly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of MyD88 KO TCD-BM. Injection of MDSCs from BM of non-tumor bearing donor markedly inhibited GVHD lethality in recipients of MyD88 KO TCD-BM. Moreover, in vivo administration of lipopolysaccharide (LPS), a TLR ligand, to donor mice expanded GR-1+CD11b+ in BM with enhanced expression of MyD88 mRNA. Recipients of TCD-BM from WT mice injected LPS showed attenuated GVHD severity as measured by weight loss and survival compared to those of TCD-BM from WT mice injected diluent.In summary, MyD88-dependent expansion of GR-1+CD11b+ population from donor TCD-BM appears to be critical for survival after allo-HSCT. Incomplete expansion of GR-1+CD11b+ population in target organs correlates with decreased apoptosis and increased infiltration of donor T cells into the target organs. Disclosures:No relevant conflicts of interest to declare.

Serotonin Receptor Type 3 Antagonists Improve Obesity-Associated Fatty Liver Disease in Mice

Obesity is a major cause for nonalcoholic fatty liver disease (NAFLD). Previous studies suggested that alterations in intestinal motility and permeability contribute to the development of NAFLD. Serotonin and serotonin receptor type 3 (5-HT(3)R) are key factors in the regulation of intestinal motility and permeability. Therefore, we studied the effect of the 5-HT(3)R antagonists tropisetron and palonosetron on the development of NAFLD in leptin-deficient obese mice. Four-week-old ob/ob mice and lean controls were treated for 6 weeks orally with tropisetron or palonosetron at 0.2 mg/kg per day. We determined markers of liver damage and inflammation, portal endotoxin levels, and duodenal concentrations of serotonin, serotonin-reuptake transporter (SERT), occludin, and claudin-1. Tropisetron treatment significantly reduced liver fat content (-29%), liver inflammation (-56%), and liver cell necrosis (-59%) in ob/ob mice. The beneficial effects of tropisetron were accompanied by a decrease in plasma alanine aminotransferase and portal vein plasma endotoxin levels, an attenuation of enhanced MyD88 and tumor necrosis factor-α mRNA expression in the liver, and an increase of tight junction proteins in the duodenum. Tropisetron treatment also caused a reduction of elevated serotonin levels and an increase of SERT in the duodenum of ob/ob mice. Palonosetron had similar effects as tropisetron with regard to the reduction of liver fat and other parameters. Tropisetron and palonosetron are effective in attenuating NAFLD in a genetic mouse model of obesity. The effect involves the intestinal nervous system, resulting in a reduction of endotoxin influx into the liver and subsequently of liver inflammation and fat accumulation.

MyD88 Deficiency Ameliorates β-Amyloidosis in an Animal Model of Alzheimer's Disease

The accumulation of β-amyloid protein (Aβ) in the brain is thought to be a primary etiologic event in Alzheimer's disease (AD). Fibrillar Aβ plaques, a hallmark of AD abnormality, are closely associated with activated microglia. Activated microglia have contradictory roles in the pathogenesis of AD, being either neuroprotective (by clearing harmful Aβ and repairing damaged tissues) or neurotoxic (by producing proinflammatory cytokines and reactive oxygen species). Aβ aggregates can activate microglia by interacting with multiple toll-like receptors (TLRs), the pattern-recognition receptors of the innate immune system. Because the adapter protein MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88(-/-)) on Aβ accumulation and microglial activation in an AD mouse model. MyD88 deficiency decreased Aβ load and microglial activation in the brain. The decrease in Aβ load in an MyD88(-/-) AD mouse model was associated with increased and decreased protein expression of apolipoprotein E (apoE) and CX3CR1, respectively, compared with that in an MyD88 wild-type AD mouse model. These results suggest that MyD88 deficiency may reduce Aβ load by enhancing the phagocytic capability of microglia through fractalkine (the ligand of CX3CR1) signaling and by promoting apoE-mediated clearance of Aβ from the brain. These findings also suggest that chronic inflammatory responses induced by Aβ accumulation via the MyD88-dependent signaling pathway exacerbate β-amyloidosis in AD.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freund's adjuvant. Mice were treated with HA intraperitoneally daily for 30 days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

Enhanced Expression of Toll-Like Receptor 4 and MyD88 is Associated With Disease Progression in Human Nonalcoholic Fatty Liver Disease

Background/Aims: Activation of Toll-like receptors (TLRs) and their downstream genes, such as tumor necrosis factor-alpha (TNFα) and interleukin-1beta (IL1β), are known to be implicated in various liver diseases such as alcoholic liver disease and viral hepatitis. Recent studies using knockout mice reveal a major contribution of TLRs to the pathogenesis of nonalcoholic steatohepatitis (NASH). However, their roles in human NASH development have not been clarified. As such, we aimed to investigate the expression of TLRs in nonalcoholic fatty liver disease (NAFLD) using human liver samples. Methods: Ninety-eight NAFLD patients who underwent percutaneous liver biopsy in Shinshu University Hospital from 2006 to 2009 were enrolled. They were diagnosed as having simple steatosis (SS, n = 15), mild NASH (fibrosis stage 1-2, n = 59), or advanced NASH (fibrosis stage 3-4, n = 24). Liver biopsy specimens from healthy liver transplantation donors (n = 6) were also used as a control. Total RNA was extracted from biopsied liver tissues, and the mRNA levels of TLR1-9 and the related genes were assessed by quantitative polymerase chain reaction. The relationship between the mRNA levels and fibrosis stage were also examined. Results: Among TLRs, the mRNA levels of TLR4 only were significantly higher in the advanced NASH group than in the normal group and mild NASH group (P = 0.034 and 0.041, respectively). Additionally, MyD88 mRNA levels were significantly increased in the mild NASH group compared with the normal group (P = 0.032). Hepatic TLR4 levels were positively correlated with those of TNFα (r = 0.531, P < 0.001) and IL1β (r = 0.383, P < 0.019), and the MyD88 levels were also correlated with TNFα (r = 0.438, P = 0.001). Finally, the mRNA levels of TLR4 and MyD88 demonstrated significant positive correlations with fibrosis stage (r = 0.329 and 0.470, respectively; both P < 0.001). Conclusion: Increased expression of TLR4/ MyD88 may be associated with the progression of human NAFLD.

Expression of myeloid differentiation primary response protein 88 (Myd88) in the cerebral cortex after experimental traumatic brain injury in rats

A growing body of evidence indicates that Toll-like receptors (TLRs) and Interleukin-1 (IL-1) family have been shown to be involved in the damaging inflammatory processes associated with stroke, infection, neoplasia, and other diseases in the central nervous system. Myeloid differentiation primary response protein 88 (Myd88) is a critical adaptor protein that transmits signals for TLRs and IL-1 family. Therefore, this study aimed to detect the expression of Myd88 protein and mRNA in a rat weight-dropping trauma model and to clarify the role of Myd88 after traumatic brain injury (TBI). A total of fifty-four Sprague Dawley (SD) rats were randomly divided into control group and TBI groups at hours 6, 12 and on day 1, day 2, day 3, and day 7. The TBI groups suffered experimental TBI by improved Feeney model. Myd88 expression is measured by Reverse Transcription PCR (RT-PCR), Western blot analysis and immunohistochemistry; and nuclear factor-kappaB (NF-κB) binding activity by electrophoretic mobility shift assay (EMSA); The levels of tumor necrosis factor-α (TNF-α) and Interleukin 1β (IL-1β) were measured by enzyme linked immunosorbent assay (ELISA) and the intercellular adhesion molecule-1 (ICAM-1) expression by immunohistochemistry. The expression of Myd88 in the injured brain was dramatically increased through 6 h and 7 days postinjury, and peaked on 3 days. NF-κB, TNF-α, IL-1β and ICAM-1 also ascended significantly after TBI. Our data demonstrated that Myd88 was increasingly expressed in a parallel time course to the up-regulation of NF-κB, proinflammatory cytokines and ICAM-1 and there was a highly positive relationship among them. These findings might have important implications during the administration of specific Myd88 antagonists in order to prevent or reduce inflammatory response after TBI.

Growth and constitutive expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88 function (165.6)

Abstract Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors in vivo, but not in vitro. Here we explored whether the effects of LPS were dependent upon TLR4 and Myd88 using RNA interference. Two clones with reduced TLR4 mRNA and protein expression and two clones with reduced Myd88 mRNA and protein expression were generated, as well as a clone expressing shRNA specific for lacZ as a control. Significantly, tumors with reduced TLR4 or Myd88 expression no longer exhibited decreased growth rates in vivo following LPS treatment indicating that the decreased growth rate of the 4T1 tumor following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more significantly, tumor cells with reduced levels of TLR4 or Myd88 exhibited slower growth rates in vitro and in vivo relative to the parental 4T1 and lacZ controls, and reducing Myd88 expression was sufficient to decrease CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function was enough to alter tumor growth and CCL2 and CCL5 expression we used a Myd88 homodimerization inhibitory peptide. The data revealed that inhibiting Myd88 function was sufficient enough to impair growth of the tumor cells, and decrease constitutive expression of CCL2 and CCL5. These data imply that Myd88 is necessary for growth of murine mammary carcinomas, and expression of at least two proinflammatory chemokines.

The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response.

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

Human Neonatal Peripheral Blood Leukocytes Demonstrate Pathogen-Specific Coordinate Expression of TLR2, TLR4/MD2, and MyD88 During Bacterial Infection In Vivo

Toll-like receptors (TLRs) play important roles in infection. We have previously reported TLR2 is up-regulated in neonatal Gram-positive (G+) bacteremia, whereas TLR4 is up-regulated in neonatal Gram-negative (G-) bacteremia. For functional signaling, TLR4 requires myeloid differentiation (MD)-2, and both TLR2 and TLR4 signal need myeloid differentiation factor (MyD88). However, it is unknown whether newborns can enhance expression of MD-2 and MyD88 with bacterial infection in coordination with TLR expression. We characterized neonatal peripheral blood leukocyte expression of MD-2 and MyD88 in relation to TLR2/4 in newborns. TLR2 mRNA expression by PBMCs and TLR2 protein expression by monocytes and granulocytes were significantly increased in the G+ bacteremia group. TLR4 mRNA on PMBCs and protein expression on monocytes and granulocytes were significantly increased in the G- bacterial group. Remarkably, although, MyD88 mRNA was increased in all patients with documented bacterial infection and correlated with both TLR2 and TLR4, MD-2 mRNA was selectively increased in G- bacterial group, wherein it correlated with TLR4 but not with TLR2 mRNA. Our findings demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2-MyD88 pathway in G+ bacterial infection and the TLR4/MD2/MyD88 pathway in G- bacterial infection, suggesting key roles for innate immune pathway in neonatal responses to bacterial infection.

Avian-specific TLRs and downstream effector responses to CpG-induction in chicken macrophages

Chickens possess toll-like receptor (TLR15), a pattern recognition receptor (PRR) absent in mammals. We characterized the regulation and mechanism of CpG responsiveness via TLRs in chicken macrophage HD11 cells. TLR15 was significantly upregulated after induction with B- and C-type CpG oligonucleotides (ODN), tripalmitoylated lipopeptide (PAM3CSK4), Escherichia coli- and Salmonella enteritidis-derived lipopolysaccharide (LPS). In response to CpG-ODN inhibitor, TLR15 and IL1B were downregulated, but TLR21 was upregulated. IL1B was upregulated with CpG-ODN and downregulated after inhibitor treatment. The results suggest that responsiveness to different types of CpG-ODN in chicken macrophages requires multiple receptors, each with unique variation in expression. We utilized RNA interference (RNAi) technology to examine myeloid differentiation primary response gene (MyD88) dependency of TLR15 and TLR21. HD11 macrophages transfected with multiple MyD88-target siRNAs exhibited 70% decrease in MyD88 mRNA expression. IL1B was upregulated with CpG induction in cells with no reduction of MyD88 mRNA levels, but not in cells with 70% MyD88 reduction. Therefore, induction through TLR15 in response to CpG-ODN operates via the MyD88-dependent pathway in chicken macrophages.

S80 The role of MyD88 in rhinovirus 1B infection

Introduction Rhinovirus (RV) are major triggers of acute asthma exacerbations and result in innate immune cell infiltration into the airways. Viral recognition by TLRs results in activation of pathways mediated by the adaptors MyD88 and TRIF, which predominantly control the production of proinflammatory cytokines and interferons respectively. We have previously shown that addition of the cytokine IL-1 (which also signals via MyD88) potentiates responses to the viral mimic Poly (I:C),[1][1] which acts in a MyD88-independent manner. This demonstrates the potential for IL-1signaling to impact on viral infection. In this study, we explored the ability of MyD88 to regulate responses to the natural viral pathogen, RV serotype 1B (RV-1B). Methods MyD88 was stably knocked down in the immortalised bronchial epithelial cell line, BEAS-2B, using a lentiviral transduction system containing shRNA targeted to MyD88. Wildtype or MyD88KD cells were stimulated, in the presence or absence of human monocytes, with TNFα and IL-1β, poly(I:C), LPS and gardiquimod (TLR3, TLR4 and TLR7/8 agonists, respectively), or infected with RV-1B. Selected experiments were carried out in the presence of IL-1ra. Changes in cytokine release were measured by ELISA. Rates of viral replication were measured using quantitative PCR. Results Costimulation of BEAS-2B cells with IL-1 and RV-1B caused a dramatic increase in proinflammatory (CXCL8), but not CCL5 production. MyD88KD cells with ∼70% reduction in MyD88 mRNA levels showed no impairment to TNFα or poly(I:C) stimulation, but significantly reduced responses to IL-1β. MyD88KD cells also had significantly impaired responses to RV-1B as assessed by production of CXCL8 and CCL5. Inhibition of RV-induced CXCL8 production could also be achieved by pre-treatment with the IL-1 antagonist, IL-1ra. IL-1β was not produced from RV-infected cells, implicating other members of the IL-1 family in the response of epithelial cells to viral infection. Viral replication was more marked in MyD88KD cells. Conclusion A reduction in MyD88 signalling modulates specific epithelial cell responses to rhinovirus, and thus may be an important target to control acute inflammation induced by human viral pathogens. ![Abstract S80 Figure 1][2]</img> Abstract S80 Figure 1 [1]: #ref-1 [2]: pending:yes

Dual targets guided screening and isolation of Kukoamine B as a novel natural anti-sepsis agent from traditional Chinese herb Cortex lycii

Treating sepsis remains challenging at present. Bacterial lipopolysaccharide (LPS) and bacterial DNA/CpG DNA are important pathogenic molecules and drug targets for sepsis. It is thus a promising strategy to treat sepsis by discovering agents that neutralize LPS and CpG DNA simultaneously. In this study, we present evidences of the biosensor based screening and isolation of active anti-sepsis fractions and monomers from traditional Chinese herbs using dual targets (LPS and CpG DNA) guided drug discovery strategy. Firstly, LPS or CpG DNA was immobilized on surfaces of cuvettes in the biosensor to establish a screening platform. Then, Cortex lycii with both highest affinities was selected out from one hundred and fourteen traditional Chinese herbs. In subsequent experiments, chromatography was utilized and coupled with the biosensor to purify fractions with a higher affinity for LPS and CpG DNA. In line with affinity assay, these fractions were shown to neutralize LPS and CpG DNA and inhibit their activity in vitro and in vivo. Lastly, the contributing monomer Kukoamine B (KB) was purified. KB neutralized LPS and CpG DNA in vitro. It inhibited TLR4, TLR9 and MyD88 mRNA expressions up-regulated by LPS and CpG DNA, and also attenuated the LPS and CpG DNA elicited nuclear translocation of NF-κB p65 protein in RAW264.7 cells. It also protected mice from lethal challenge of heat-killed E. coli, a mixture of LPS and CpG DNA. In conclusion, we presented a dual target guided discovery of a novel anti-sepsis agent KB from traditional Chinese herbs via combination of biosensor technology and chromatography methods.

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p < 0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p < 0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p < 0.05) and 2.1-fold (p < 0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A–I transfer. In vitro, supplementation of HDL or apo A–I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-010-0690-6) contains supplementary material, which is available to authorized users.