TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.     

Central role for MyD88 in the responses of microglia to pathogen-associated molecular patterns

Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-alpha, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll-like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation ( tviA ), biosynthesis ( tviBCDE ) and export ( vexABCDE ) of the Vi capsule. Expression of the Vi capsule in S.  Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor-alpha (TNF-α) and IL-6 production by murine bone-marrow derived macrophages. Production of TNF-α and IL-6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4^−/− mice with S.  Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S.  Typhimurium induced expression of TNF-α and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S.  Typhimurium reduced TNF-α and iNOS expression to levels observed after infection with a S.  Typhimurium msbB mutant. In contrast, no differences in TNF-α expression between the S.  Typhimurium wild type and strains expressing the Vi-capsule or carrying a mutation in msbB were observed after infection of TLR4^−/− mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S.  Typhimurium lipopolysaccharide by TLR4.     

Toll-like receptor 4 is involved in neuroprotection afforded by ischemic preconditioning

It has been demonstrated that a short ischemic event (ischemic preconditioning, IPC) results in a subsequent resistance to severe ischemia (ischemic tolerance, IT). We have recently demonstrated the role of innate immunity and in particular of toll-like receptor (TLR) 4 in brain ischemia. Several evidences suggest that TLR4 might also be involved in IT. Therefore, we have now used an in vivo model of IPC to investigate whether TLR4 is involved in IT. A 6-min temporary bilateral common carotid arteries occlusion was used for focal IPC and it was performed on TLR4-deficient mice (C57BL/10ScNJ) and animals that express TLR4 normally (C57BL/10ScSn). To assess the ability of IPC to induce IT, permanent middle cerebral artery occlusion was performed 48 h after IPC. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological scores. IPC caused neuroprotection as shown by a reduction in infarct volume and better outcome in mice expressing TLR4 normally. TLR4-deficient mice showed less IPC-induced neuroprotection than wild-type animals. Western blot analysis of tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) showed an up-regulation in the expression of these proteins in both substrains of mice measured 18, 24 and 48 h after IPC, being higher in mice with TLR4. Similarly, nuclear factor-kappa B (NF-κB) activation was observed 18, 24 and 48 h after IPC, being more intense in TLR4-expressing mice. These data demonstrate that TLR4 signalling is involved in brain tolerance as shown by the difference in the percentage of neuroprotection produced by IPC between ScSn and ScNJ (60% vs. 18%). The higher expression of TNF-α, iNOS and cyclooxygenase-2 and NF-κB activation in mice expressing TLR4 is likely to participate in this endogenous neuroprotective effect.     

Interferon-γ, interleukin-1 and tumour necrosis factor-α synthesis during experimental murine staphylococcal infection

Abstract Several exotoxins of Staphylococcus aureus were shown to modulate the host immune system by stimulation of monokine release. BALB/c mice infected intravenously (i.v.) with live cells if S. aureus , strain Cowan 1, had a detectable serum level of TNF-α at 3, 4 and 5 h after injection. When S. epidermidis (strain F3380, clinical isolate) was used to infect mice, the level of TNF-α was lower (the detection limit of the cytotoxicity assay with WEHI cells was 40 pg ml⁻). Kinetics of TNF synthesis was different from that observed in experimental infections caused by Gram-negative bacteria. Similarly to TNF-α, IL-1α appears in a measureable level at 3 h after i.v. injection of bacteria. The highest serum level of IFN-γ was observed 12 h after infection with both S. aureus and S. epidermidis . A quantity ten times more of S. epedermidis than of S. aureus cells was required to induce similar levels of TNF-α and IFN-γ administered in vivo in four daily doses followed by infection of S. aureus resulted in increased elimination of bacteria from the spleen, liver and peritoneal cavity of mice.     

Immunomodulatory effects of etanercept in a model of brain injury act through attenuation of the acute-phase response

TNF-α has proved to be a successful target in the treatment of many peripheral inflammatory diseases, but the same interventions worsen immune-mediated CNS disease. However, anti-TNF-α strategies may offer promise as therapy for non-immune CNS injury. In this study, we have microinjected IL-1β or lipopolysaccharide (LPS) into the rat brain as a simple model of brain injury and have systemically administered the TNF-α antagonist etanercept to discover whether hepatic TNF-α, produced as part of the acute-phase response to CNS injury, modulates the inflammatory response in the brain. We report a significant reduction in neutrophil numbers recruited to the IL-1β- or LPS-challenged brain as a result of TNF-α inhibition. We also show an attenuation in the levels of hepatic mRNA including TNF-α mRNA and of TNF-α-induced genes, such as the chemokines CCL-2, CXCL-5, and CXCL-10, although other chemokines elevated by the injury were not significantly changed. The reduction in hepatic chemokine synthesis results in reduced numbers of circulating neutrophils, and also a reduction in the numbers recruited to the liver as a consequence of brain injury. These findings suggest that TNF-α inhibitors may reduce CNS inflammatory responses by targeting the hepatic acute-phase response, and thus therapies for brain injury need not cross the blood–brain barrier to be effective.     

A toll-like receptor 2 pathway regulates the Ppargc1a/b metabolic co-activators in mice with Staphylococcal aureus sepsis

Activation of the host antibacterial defenses by the toll-like receptors (TLR) also selectively activates energy-sensing and metabolic pathways, but the mechanisms are poorly understood. This includes the metabolic and mitochondrial biogenesis master co-activators, Ppargc1a (PGC-1α) and Ppargc1b (PGC-1β) in Staphylococcus aureus (S. aureus) sepsis. The expression of these genes in the liver is markedly attenuated inTLR2(-/-) mice and markedly accentuated in TLR4(-/-) mice compared with wild type (WT) mice. We sought to explain this difference by using specific TLR-pathway knockout mice to test the hypothesis that these co-activator genes are directly regulated through TLR2 signaling. By comparing their responses to S. aureus with WT mice, we found that MyD88-deficient and MAL-deficient mice expressed hepatic Ppargc1a and Ppargc1b normally, but that neither gene was activated in TRAM-deficient mice. Ppargc1a/b activation did not require NF-kβ, but did require an interferon response factor (IRF), because neither gene was activated in IRF-3/7 double-knockout mice in sepsis, but both were activated normally in Unc93b1-deficient (3d) mice. Nuclear IRF-7 levels in TLR2(-/-) and TLR4(-/-) mice decreased and increased respectively post-inoculation and IRF-7 DNA-binding at the Ppargc1a promoter was demonstrated by chromatin immunoprecipitation. Also, a TLR2-TLR4-TRAM native hepatic protein complex was detected by immunoprecipitation within 6 h of S. aureus inoculation that could support MyD88-independent signaling to Ppargc1a/b. Overall, these findings disclose a novel MyD88-independent pathway in S. aureus sepsis that links TLR2 and TLR4 signaling in innate immunity to Ppargc1a/b gene regulation in a critical metabolic organ, the liver, by means of TRAM, TRIF, and IRF-7.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

Interleukin-12 P40 induces the expression of TNF-α in microglia and macrophages

Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α in BV-2 microglial cells. This induction of TNF-α production was accompanied by an induction of TNF-α mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-κB and C/EBPb is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPb. Activation of NF-κB as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-α by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.
Acknowledgements:   This study was supported by NIH grants (NS39940 and AG19487).     

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

S. aureus-dependent microglial activation is selectively attenuated by the cyclopentenone prostaglandin 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2)

Microglial activation is a hallmark of brain abscess. The continual release of proinflammatory mediators by microglia following bacterial challenge may contribute, in part, to the destruction of surrounding normal tissue characteristic of brain abscess. Therefore, attenuating chronic microglial activation during the course of CNS bacterial infections may have therapeutic benefits. The purpose of this study was to evaluate the ability of the natural peroxisome proliferator-activated receptor (PPAR)-gamma agonist 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2) to modulate microglial activation in response to Staphylococcus aureus, one of the main etiologic agents of brain abscess in humans. 15d-PGJ2 was a potent inhibitor of proinflammatory cytokine (IL-1beta, TNF-alpha, IL-12 p40) and CC chemokine (MIP-1beta, MCP-1) production in primary microglia, but had no effect upon the expression of select CXC chemokines (MIP-2, KC). 15d-PGJ2 also selectively inhibited the S. aureus-dependent increase in microglial TLR2, CD14, MHC class II, and CD40 expression, whereas it had no effect on the co-stimulatory molecules CD80 and CD86. Microarray analysis revealed additional inflammatory mediators modulated by 15d-PGJ2 in primary microglia following S. aureus exposure, the majority of which were chemokines. These results suggest that suppressing microglial activation through the use of 15d-PGJ2 may lead to the sparing of damage to normal brain parenchyma that often results from brain abscess.     

CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.     

Poly (ADP-ribose) polymerases (PARPs) 1-3 regulate astrocyte activation

Besides their traditional role in maintaining CNS homeostasis, astrocytes also participate in innate immune responses. Indeed, we have previously demonstrated that astrocytes are capable of recognizing bacterial pathogens such as Staphylococcus aureus , a common etiologic agent of CNS infections, and respond with the robust production of numerous proinflammatory mediators. Suppression of Poly (ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme, has been shown to attenuate inflammatory responses in several cell types including mixed glial cultures. However, a role for PARP-1 in regulating innate immune responses in purified astrocytes and the potential for multiple PARP family members to cooperatively regulate astrocyte activation has not yet been examined. The synthetic PARP-1 inhibitor PJ-34 attenuated the production of several proinflammatory mediators by astrocytes in response to S. aureus stimulation including nitric oxide, interleukin-1 beta, tumor necrosis factor-alpha, and CCL2. The release of all four mediators was partially reduced in PARP-1 knockout (KO) astrocytes compared to wild-type cells. The residual inflammatory mediator expression detected in PARP-1 KO astrocytes was further blocked with PJ-34, suggesting either non-specific effects of the drug or actions on alternative PARP isoforms. Reduction in PARP-2 or PARP-3 expression by siRNA knock down revealed that these isoforms also contributed to inflammatory mediator regulation in response to S. aureus . Interestingly, the combined targeting of either PARP-1/PARP-2 or PARP-2/PARP-3 attenuated astrocyte inflammatory responses more effectively compared to knock down of either PARP alone, suggesting cooperativity between PARP isoforms. Collectively, these findings suggest that PARPs influence the extent of S. aureus -induced astrocyte activation.     

TNF-α reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor α (TNF-α). Therefore, this work was designed to explore the effect of TNF-α on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-α caused a marked reduction in the number of intracellular S. epidermidis , suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-κB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-κB inhibitors, SN50 or BAY 11–7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis . These results suggest that activation of the signaling pathway initiated by TNF-α could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-κB was shown not to be important for the internalization process of S. epidermidis .     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Probiotic genomic DNA reduces the production of pro-inflammatory cytokine tumor necrosis factor-alpha

The effect of Lactobacillus plantarum genomic DNA on lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, nuclear factor-kappa B activation, and the expressions of tumor necrosis factor-alpha, interleukin-1 receptor-associated kinase M, and the pattern recognition receptor were examined. Pretreatment of p-gDNA inhibited the phosphorylation of MAPKs and nuclear factor-kappa B, and also inhibited LPS-induced TNF-α production in response to subsequent LPS stimulation. L.?plantarum genomic DNA-mediated inhibition of signaling pathway and tumor necrosis factor-alpha was accompanied by the suppression of toll-like receptor (TLR) 2, TLR4, and TLR9 and the induction of interleukin-1 receptor-associated kinase M, a negative regulator of TLR. This study can extend our understanding of the biological function of probiotic genomic DNA as an anti-inflammatory agent.     

Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

Toll-like receptor (TLR) 2 induced through TLR4 signaling initiated by Helicobacter pylori cooperatively amplifies iNOS induction in gastric epithelial cells

Cell-surface Toll-like receptors (TLRs) initiate innate immune responses, such as inducible nitric oxide synthase (iNOS) induction, to microorganisms' surface pathogens. TLR2 and TLR4 play important roles in gastric mucosa infected with Helicobacter pylori (H. pylori), which contains lipopolysaccharide (LPS) as a pathogen. The present study investigates their physiological roles in the innate immune response of gastric epithelial cells to H. pylori-LPS. Changes in the expression of iNOS, TLR2, and TLR4, as well as downstream activation of mitogen-activated protein kinases and nuclear factor-kappaB (NF-kappaB), were analyzed in normal mouse gastric mucosal GSM06 cells following stimulation with H. pylori-LPS and interferon-gamma. Specific inhibitors for mitogen-activated protein kinases, NF-kappaB, and small interfering RNA for TLR2 or TLR4 were employed. The immunohistochemistry of TLR2 was examined in human gastric mucosa. H. pylori-LPS stimulation induced TLR2 in GSM06 cells, but TLR4 was unchanged. TLR2 induction resulted from TLR4 signaling that propagated through extracellular signal-related kinase and NF-kappaB activation, as corroborated by the decline in TLR4 expression on small interfering RNA treatment and pretreatment with inhibitors. The induction of iNOS and the associated nitric oxide production in response to H. pylori-LPS stimulation were inhibited by declines in not only TLR4 but also TLR2. Increased expression of TLR2 was identified in H. pylori-infected human gastric mucosa. TLR4 signaling initiated by H. pylori-LPS and propagated via extracellular signal-regulated kinase and NF-kappaB activation induced TLR2 expression in gastric epithelial cells. Induced TLR2 cooperated with TLR4 to amplify iNOS induction. This positive correlation may constitute a mechanism for stimulating the innate immune response against various bacterial pathogens, including H. pylori-LPS.