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.     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

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.     

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.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

MyD88 functions as a negative regulator of TLR3/TRIF-induced corneal inflammation by inhibiting activation of c-Jun N-terminal kinase

The adaptor molecule MyD88 is necessary for responses to all Toll-like receptors except TLR3 and a subset of TLR4 signaling events, which are mediated by the adaptor molecule TRIF. To determine the role of TRIF in host inflammatory responses, corneal epithelium of C57BL/6, TLR3(-/-), TRIF(-/-), and MyD88(-/-) mice was abraded and stimulated with the synthetic TLR3 ligand poly(I:C). We found that poly(I:C) induced a pronounced cellular infiltration into the corneal stroma, which was TLR3- and TRIF-dependent. Unexpectedly, the inflammatory response was exacerbated in MyD88(-/-) mice, with enhanced neutrophil and F4/80(+) cell infiltration into the corneal stroma and elevated corneal haze, which is an indicator of loss of corneal transparency. To determine whether MyD88-dependent inhibition of TLR3/TRIF responses is a general phenomenon, we examined cytokine production by MyD88(-/-) bone marrow-derived macrophages; however, no significant difference was observed between MyD88(+/+) or MyD88(-/-) macrophages. In contrast, human corneal epithelial cells (HCECs) transfected with MyD88 small interfering RNA had significantly increased (2.5-fold) CCL5/RANTES production compared with control HCECs, demonstrating a negative regulatory role for MyD88 in TLR3/TRIF responses in these cells. Finally, knockdown of MyD88 in HCECs resulted in increased phosphorylation of c-Jun N-terminal kinase (JNK), but not p38, IRF-3, or NF-kappaB. Consistent with this finding, the JNK inhibitor SP600125, but not p38 inhibitor SB203580, ablated this response. Taken together, these findings demonstrate a novel JNK-dependent inhibitory role for MyD88 in the TLR3/TRIF activation pathway.     

Phosphatidylinositol 3-kinase activation attenuates the TLR2-mediated macrophage proinflammatory cytokine response to Francisella tularensis live vaccine strain

An inadequate innate immune response appears to contribute to the virulence of Francisella tularensis following pulmonary infection. Studies in mice suggest that this poor response results from suppression of proinflammatory cytokine production early during infection, but the mechanisms involved are not understood. PI3K is known to regulate proinflammatory cytokine expression, but its exact role (positive versus negative) is controversial. We sought to clarify the role of PI3K in regulating proinflammatory signaling and cytokine production during infection with F. tularensis live vaccine strain (LVS). In this study, we demonstrate that the induction of TNF and IL-6 expression by LVS in mouse bone marrow-derived macrophages was markedly enhanced when PI3K activity was inhibited by either of the well-known chemical inhibitors, wortmannin or LY294002. The enhanced cytokine expression was accompanied by enhanced activation of p38 MAPK and ERK1/2, both of which were critical for LVS-induced expression of TNF and IL-6. LVS-induced MAPK activation and cytokine production were TLR2- and MyD88- dependent. PI3K/Akt activation was MyD88-dependent, but was surprisingly TLR2-independent. LVS infection also rapidly induced MAPK phosphatase-1 (MKP-1) expression; PI3K and TLR2 signaling were required. Peak levels of MKP-1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. These data suggest that infection by LVS restrains the TLR2-triggered proinflammatory response via parallel activation of PI3K, leading to enhanced MKP-1 expression, accelerated deactivation of MAPKs, and suppression of proinflammatory cytokine production. This TLR2-independent inhibitory pathway may be an important mechanism by which Francisella suppresses the host's innate immune response.     

Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

Cutting edge: TLR2 is a functional receptor for acute-phase serum amyloid A

Induced secretion of acute-phase serum amyloid A (SAA) is a host response to danger signals and a clinical indication of inflammation. The biological functions of SAA in inflammation have not been fully defined, although recent reports indicate that SAA induces proinflammatory cytokine expression. We now show that TLR2 is a functional receptor for SAA. HeLa cells expressing TLR2 responded to SAA with potent activation of NF-kappaB, which was enhanced by TLR1 expression and blocked by the Toll/IL-1 receptor/resistance (TIR) deletion mutants of TLR1, TLR2, and TLR6. SAA stimulation led to increased phosphorylation of MAPKs and accelerated IkappaBalpha degradation in TLR2-HeLa cells, and results from a solid-phase binding assay showed SAA interaction with the ectodomain of TLR2. Selective reduction of SAA-induced gene expression was observed in tlr2-/- mouse macrophages compared with wild-type cells. These results suggest a potential role for SAA in inflammatory diseases through activation of TLR2.     

Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.     

Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins

The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1(-/-)) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1(-/-) cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-kappaB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.     

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.     

Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2

The pattern recognition receptor Toll-like receptor 2 (TLR2) has been implicated in the response to several human viruses, including herpes simplex viruses (types 1 and 2) and cytomegalovirus. We demonstrated that varicella-zoster virus (VZV) activates inflammatory cytokine responses via TLR2. VZV specifically induced interleukin-6 (IL-6) in human monocytes via TLR2-dependent activation of NF-kappaB, and small interfering RNA designed to suppress TLR2 mRNA reduced the IL-6 response to VZV in human monocyte-derived macrophages. Unlike other herpesviruses, the cytokine response to VZV was species specific. VZV did not induce cytokines in murine embryonic fibroblasts or in a mouse cell line, although VZV did activate NF-kappaB in a human cell line expressing a murine TLR2 construct. Together, these results suggest that TLR2 may play a role in the inflammatory response to VZV infection.