Intramembrane attenuation of the TLR4-TLR6 dimer impairs receptor assembly and reduces microglia-mediated neurodegeneration.

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aß peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aß-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aß. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.

Cell type-specific regulatory effects of glucocorticoids on cutaneous TLR2 expression and signalling.

Glucocorticoids (GCs) induce Toll-like receptor (TLR) 2 expression and synergistically upregulate TLR2 with pro-inflammatory cytokines or bacteria. These paradoxical effects have drawn attention to the inflammatory initiating or promoting effects of GCs, as GC treatment can provoke inflammatory skin diseases. Here, we aimed to investigate the regulatory effects of GCs in human skin cells of different epidermal and dermal layers. We found that Dex induced TLR2 expression mainly in undifferentiated and less in calcium-induced differentiated keratinocytes but not in HaCaT cells or fibroblasts, however, Dex reduced TLR1/6 expression. Stimulation with Dex under inflammatory conditions further increased TLR2 but not TLR1 or TLR6 levels in keratinocytes. Increased ligand-induced interaction of TLR2 with MyD88 and expression of the adaptor protein TRAF6 indicated enhanced TLR2 signalling, whereas TLR2/1 or TLR2/6 signalling was not increased in Dex-pretreated keratinocytes. GC-increased TLR2 expression was negatively regulated by JNK MAPK signalling when stimulated with Propionibacterium acnes. Our results provide novel insights into the molecular mechanisms of glucocorticoid-mediated expression and function of TLR2 in human skin cells and the understanding of the mechanisms of corticosteroid side effects.

Vibrio cholerae porin OmpU induces LPS tolerance by attenuating TLR-mediated signaling.

Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio choleraeporin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. choleraeOmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway.

Lipopeptides are signaled by Toll-like receptor 1, 2 and 6 in endolysosomes.

Toll-like receptors (TLRs) recognize a variety of microbial products and induce defense responses. Pathogen sensing by TLRs occurs either on the cell surface or in endolysosomes. TLR-dependent responses are greatly influenced by the site of pathogen sensing. TLR heterodimers TLR1/TLR2 and TLR2/TLR6 recognize tri- or diacylated microbial lipopeptides, respectively. Although TLR1, 2 and 6 are believed to localize on the cell surface of immune cells, little is known about where lipopeptides are signaled. In this study, we established mAbs to TLR1, 2 and 6. TLR1, 2 and 6 were expressed on the surface of B cells, monocytes and dendritic cells in a manner dependent on a TLR-specific chaperone PRAT4A (protein associated with TLR4 A). Cell surface localization of TLR1 or TLR6 was not necessarily required for TLR2 response. Furthermore, a dynamin inhibitor 'Dynasore' abolished the lipopeptide responses by preventing lipopeptide internalization into LAMP-1 and LAMP-2 positive compartments. Our findings suggest that lipopeptides elicit TLR1/2 and TLR2/6 signaling in the endolysosomes, but not on the cell surface.

Toll-like receptor 2/6-dependent stimulation of mesenchymal stem cells promotes angiogenesis by paracrine factors.

Reconstruction of critical size bone defects represents a major challenge in orthopaedic surgery. Insufficient angiogenesis is a limiting factor for engraftment of large-scale tissue transplants. Transplantation or stimulation of local mesenchymal stem cells (MSCs) represents a potential solution to enhance angiogenesis. We recently identified angiogenic properties for the Toll-like receptor (TLR) 2/6 agonist MALP-2 and now investigated if MALP-2 could be used to stimulate MSCs in order to promote angiogenesis in vitro and in vivo. Human MSCs from the bone marrow of healthy subjects were isolated, cultured and expanded in vitro and were shown to be positive for mesenchymal stem cells markers as well as for the MALP-2 receptors TLR2 and TLR6. MALP-2 directly enhanced migration but not proliferation of human MSCs. Conditioned medium from MALP-2 stimulated MSCs significantly increased proliferation, migration and tube formation of endothelial cells. Analysis of the conditioned medium from MSCs revealed that MALP-2 stimulation enhanced the secretion of several chemokines and growth factors including vascular endothelial growth factors (VEGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Finally, we studied MALP-2 effects on MSCs in a sheep model of tissue engineering in vivo. Therefore, MSCs were isolated from the iliac crest of black head sheep and co-cultivated with MALP-2 ex vivo. Implantation of autologous MSCs within a scaffold cylinder into the M. latissimus dorsi significantly enhanced vessel density of these constructs after 6 months. We here present the first evidence that TLR2/6-dependent stimulation of MSCs promotes angiogenesis in vitro and in vivo offering a novel strategy for therapeutic angiogenesis, e.g., for tissue engineering of bone.

Gram-positive and Gram-negative bacteria synergize with oxidants to release CXCL8 from innate immune cells.

We have recently demonstrated that oxidants can activate monocytes via an action on Toll-like receptor (TLR) 2; however, it is unclear what functional consequence this has on immune surveillance for Gram-negative and -positive bacteria. Gram-negative and -positive bacteria and their related pathogen-associated molecular patterns (PAMPs) are sensed by TLR4 and TLR2, respectively. In the current study, we used a human monocyte cell line to show that oxidants prime cells to subsequent challenge with Gram-negative or -positive bacteria as well as PAMPs specific for TLR4 (LPS), TLR2/1 (Pam(3)CSK4), TLR2/6 (FSL-1), Nod1 (FK565), and Nod2 (MDP Lys 18). Similarly, activation of TLR4 with LPS primed for subsequent activation of cells by agonists of the TLR2/6 or TLR2/1 complex. However, no synergy was noted when cells were costimulated with Pam(3)CSK4 and FSL-1. We then tested blood (and isolated monocytes) derived from healthy smokers, which is oxidant primed, making it more sensitive to bacterial or PAMP stimulation when compared with blood of nonsmokers. Thus an oxidant stimulation, possibly via an action on TLR2 or associated transduction pathways, provides a signal that initiates inflammatory responses and sensitizes cells to pathogenic insults.

The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein, the macrophage infectivity potentiator, through TLR2/TLR1/TLR6 and CD14.

Chlamydiae components and signaling pathway(s) responsible for the production of proinflammatory cytokines by human monocytes/macrophages are not clearly identified. To this aim, Chlamydia trachomatis-inactivated elementary bodies (EB) as well as the following seven individual Ags were tested for their ability to induce the production of proinflammatory cytokines by human monocytes/macrophages and THP-1 cells: purified LPS, recombinant heat shock protein (rhsp)70, rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2). Aside from EB, rMip displayed the highest ability to induce release of IL-1beta, TNF-alpha, IL-6, and IL-8. rMip proinflammatory activity could not be attributed to Escherichia coli LPS contamination as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B (50 microg/ml), and different serum requirement. We have recently demonstrated that Mip is a "classical" bacterial lipoprotein, exposed at the surface of EB. The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Native Mip was able to induce TNF-alpha and IL-8 secretion, whereas a nonlipidated C20A rMip variant was not. Proinflammatory activity of rMip was unaffected by heat or proteinase K treatments but was greatly reduced by treatment with lipases, supporting a role of lipid modification in this process. Stimulating pathways appeared to involve TLR2/TLR1/TLR6 with the help of CD14 but not TLR4. These data support a role of Mip lipoprotein in pathogenesis of C. trachomatis-induced inflammatory responses.

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins.

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host's immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-alpha and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.