Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.     

Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam₃CSK₄) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre-treatment of the cells with 10 μg/mL of a TLR2-specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam₃CSK₄ response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam₃CSK₄ response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ-induced tumor necrosis factor alpha production in a human monocytic cell line.     

TLR9-dependent and independent pathways drive activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1-2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders.     

Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (> or =50 microg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.     

VIP balances innate and adaptive immune responses induced by specific stimulation of TLR2 and TLR4

Crohn's disease (CD) is a chronic intestinal inflammatory pathology, which develops as a result of innate immune signals, such as the activation of Toll-like receptors (TLRs), and adaptive immune signals, including Th1 cytokine release. We have recently demonstrated in TNBS-induced colitis, a murine model of CD, that VIP plays a homeostatic role, by reducing TNBS-induced TLR2 and TLR4 expression to control levels. The purpose of this paper is to elucidate for the first time, the physiological relevance of VIP specific control of innate and adaptive immune responses through TLR2 and TLR4 ligands. In addition, we investigated the effect of VIP on regulatory activity of T regulatory (Treg) cells in the TNBS-colitis model. First, we found that VIP downregulated the inflammatory response elicited in mesenteric lymph node cell cultures by treatment with the TLR2 ligand Pam3Cys, or the TLR4 ligand lipopolysaccharide (LPS), reducing the production of the chemokine CXCL1. Also, treatment with VIP impaired the induction of Th1 responses by decreasing p70 interleukin (IL)-12 and interferon gamma (IFN-γ) levels after TLR2/TLR4 stimulation in culture. Besides, VIP treatment restored in vivo the numbers of TLR2 and TLR4 positive CD4⁺CD25⁺ T lymphocytes, augmented by TNBS administration, and increased the expression of molecules involved in regulatory T cell function, such as Foxp3 and TGF-β. In conclusion, the ability of VIP to down-regulate uncontrolled inflammation by targeting TLR-mediated responses and regulatory T cell activity could be used as a new alternative therapy for intestinal inflammatory/autoimmune disorders.     

Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells

Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-kappaB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands.     

Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach

Extensive evidence suggests that the self-assembly of amyloid-beta peptide (Aβ) is a nucleation-dependent process that involves the formation of several oligomeric intermediates. Despite neuronal toxicity being recently related to Aβ soluble oligomers, results from aggregation studies are often controversial, mainly because of the low reproducibility of several experimental protocols. Here a multimethodological study that included atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy (FLM), mass spectrometry techniques (matrix-assisted laser desorption/ionization time-of-flight [MALDI–TOF] and electrospray ionization quadrupole time-of-flight [ESI–QTOF]), and direct thioflavin T (ThT) fluorescence spectroscopy were enabled to set up a reliable and highly reproducible experimental protocol for the characterization of the morphology and dimension of Aβ 1–42 (Aβ42) aggregates along the self-assembly pathway. This multimethodological approach allowed elucidating the diverse assembly species formed during the Aβ aggregation process and was applied to the detailed investigation of the mechanism of Aβ42 inhibition by myricetin. In particular, a very striking result was the molecular weight determination of the initial oligomeric nuclei by MALDI–TOF, composed of up to 10 monomers, and their morphology by AFM.     

Tie2 signalling through Erk1/2 regulates TLR4 driven inflammation

The inflammatory response is essential for eradication of lipopolysaccharide (LPS) presenting microbial invaders but requires exquisite regulation to prevent detrimental vascular inflammation. Endothelial cells play active roles in both the initiation of inflammation, through the detection of LPS by Toll-like Receptor 4 (TLR4), and the resolution of inflammation, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 attenuates LPS-TLR4 driven inflammation is poorly understood. To investigate the effects of Tie2 on TLR4 signalling, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine (F) substitutions in the cytoplasmic domain. Tie2 attenuated LPS induced Nf-κB activation in a manner requiring Tie2 kinase activation, the carboxy-terminal tyrosine residue Y1100 and downstream Erk1/2 signalling. Tyrosine 1100 was also required for the Tie2 dependent decrease in expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and stabilization of the Nf-κB inhibitor, IκBα. In contrast, upregulation of known TLR4 antagonist miRNA-146b-5p required all three tyrosine phosphorylation sites in Tie2. Finally, we confirmed in an in vivo model that activation of Tie2 signalling reduces LPS mediated inflammation. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 mediated inflammation.     

In vivo ethanol exposure down-regulates TLR2-, TLR4-, and TLR9-mediated macrophage inflammatory response by limiting p38 and ERK1/2 activation

Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-alpha and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-alpha synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-alpha levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-alpha production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-alpha production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-alpha production via signaling pathways, which showed unique characteristics.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

Differential effect of TLR2 and TLR4 on the immune response after immunization with a vaccine against Neisseria meningitidis or Bordetella pertussis

Neisseria meningitidis and Bordetella pertussis are Gram-negative bacterial pathogens that can cause serious diseases in humans. N. meningitidis outer membrane vesicle (OMV) vaccines and whole cell pertussis vaccines have been successfully used in humans to control infections with these pathogens. The mechanisms behind their effectiveness are poorly defined. Here we investigated the role of Toll-like receptor (TLR) 2 and TLR4 in the induction of immune responses in mice after immunization with these vaccines. Innate and adaptive immune responses were compared between wild type mice and mice deficient in TLR2, TLR4, or TRIF. TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. In contrast, immune responses were not lower in TLR2-/- mice but tended even to be higher after immunization. Together our data demonstrate that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required.     

Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1

The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.     

Unique properties of the chicken TLR4/MD-2 complex: selective lipopolysaccharide activation of the MyD88-dependent pathway

During evolution, mammals have evolved a powerful innate immune response to LPS. Chickens are much more resistant to LPS-induced septic shock. Herein we report that chickens sense LPS via orthologs of mammalian TLR4 and myeloid differentiation protein-2 (MD-2) rather than the previously implicated chicken TLR2 isoform type 2 (chTLR2t2) receptor. Cloning and expression of recombinant chTLR4 and chMD-2 in HeLa 57A cells activated NF-kappaB at concentrations of LPS as low as 100 pg/ml. Differential pairing of chicken and mammalian TLR4 and MD-2 indicated that the protein interaction was species-specific in contrast to the formation of functional human and murine chimeric complexes. The chicken LPS receptor responded to a wide variety of LPS derivatives and to the synthetic lipid A compounds 406 and 506. The LPS specificity resembled the functionality of the murine rather than the human TLR4/MD-2 complex. Polymorphism in chTLR4 (Tyr(383)His and Gln(611)Arg) did not influence the LPS response. Interestingly, LPS consistently failed to activate the MyD88-independent induction of IFN-beta in chicken cells, in contrast to the TLR3 agonist poly(I:C) that yielded a potent IFN-beta response. These results suggest that chicken lack a functional LPS-specific TRAM-TRIF (TRIF-related adapter molecule/TIR-domain-containing adapter-inducing IFN-beta) signaling pathway, which may explain their aberrant response to LPS compared with the mammalian species.     

TLR2/TLR4-independent neutrophil activation and recruitment upon endocytosis of nucleosomes reveals a new pathway of innate immunity in systemic lupus erythematosus

The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.     

Glycoprotein-dependent and TLR2-independent innate immune recognition of herpes simplex virus-1 by dendritic cells

Innate immune recognition is an important early event in the host response to herpes simplex virus-1 (HSV-1) infection. Dendritic cells (DC) play an important sentinel role in this recognition. Previous studies have shown that monocyte-derived DC (MDDC) respond to HSV-1 by up-regulation of costimulatory molecules and type I IFN release, but the molecular targets on the virus recognized by the DC have not been defined. In this study we show that MDDC recognize and respond to the four essential viral glycoproteins, gB, gD, and gHgL, independent of other viral proteins or nucleic acids. DC recognition of these four glycoproteins leads to the up-regulation of CD40, CD83, CD86, and HLA-DR and to the production of IFN-alpha and IL-10, but not IL-12p70. Glutaraldehyde-fixation and nonfunctional gH mutants were used to show that recognition of glycoproteins does not require membrane fusion. The nature of the recognition event was probed further by transfecting glycoproteins individually or in combination, by blocking individual proteins with Abs, or by using mutant gD constructs unable to bind to their known cognate receptors. Unexpectedly, MDDC responses were found to require expression of all four glycoproteins. Furthermore, gD mutants that cannot bind nectin-1 and/or herpesvirus entry mediator can still induce DC maturation. Finally, although HSV-1 can signal via the TLR2 receptor, this receptor does not mediate recognition of glycoproteins. Thus, the complex of the four essential HSV-1 entry glycoproteins on the cell surface can provide a target for innate immune recognition of this virus.