TLR3 and TLR7 are involved in expression of IL-23 subunits while TLR3 but not TLR7 is involved in expression of IFN-beta by Theiler's virus-infected RAW264.7 cells

Theiler's murine encephalomyelitis virus (TMEV) infects macrophages and causes demyelinating disease (DD) in certain mouse strains. IL-23 p19/p40 and IFN-beta, which are both expressed by macrophages in response to TMEV, could contribute to or prevent DD. Because TMEV may induce macrophages' cytokines through TLR3 and TLR7 (toll-like receptors), their role in TMEV-induced IL-23 and IFN-beta expression by the RAW264.7 macrophage cell line was determined following infection with TMEV or stimulation with the poly (I:C) or loxoribine. TMEV infection or stimulation with poly (I:C), a TLR3 agonist, or loxoribine, a TLR7 agonist, induced expression of IL-23 and IFN-beta in RAW264.7 cells. In addition, TMEV infection increased expression of TLR3 and TLR7 in RAW264.7 cells. Transfection of RAW264.7 cells with shRNA plasmid vectors expressing siRNA specific for TLR3 or TLR7 concomitantly decreased expression of TLR3 or TLR7, respectively, and TMEV-induced p19 mRNA, p19 protein, and IL-23 p19/p40. Transfection with TLR7-shRNA plasmids reduced expression of TMEV-induced p40 mRNA and p40 protein. However, transfection with TLR3-shRNA plasmids increased expression of TMEV-induced p40 mRNA but decreased p40 protein. In addition, transfection with TLR3-shRNA plasmids but not TLR7-shRNA plasmids decreased expression of TMEV-induced IFN-beta mRNA. Thus TLR3 and TLR7 contribute to TMEV-induced IL-23 p19 and p40, while TLR3 contributes to TMEV-induced IFN-beta.     

CylA is a sequence-specific protease involved in toxin biosynthesis

Journal of Industrial Microbiology & Biotechnology - CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As...     

RP105 involved in activation of mouse macrophages via TLR2 and TLR4 signaling

RP105 is a member of the toll-like receptor family of proteins that transmits an activation signal in B cells, playing a role in regulation of B cell growth and death; in macrophages and dendritic cells, RP105 is a specific inhibitor of TLR4 signaling. RP105 is uniquely important for regulating TLR4-dependent signaling. It also proved that RP105 is closely related to TLR2 in macrophage activation by Mycobacterium tuberculosis lipoproteins. The aim of our study is to investigate the role of RP105 in mouse macrophages activation of TLR4 and TLR2 signaling by lipopolysaccharides (LPS) and Pam3CysSerLys4 (Pam3CSK4) alone or in combination, and the interaction between TLR2 and TLR4 signaling through RP105. Our results indicate that besides exhibiting negative regulation of TNF-α and IL12-p40 secretion in macrophage activated by LPS, RP105 is also involved in macrophages activation by Pam3CSK4 through TLR2 signaling and exhibited regulation to IL-10 and RANTES production by mouse peritoneal macrophage activated by Pam3CSK4. In macrophages activation by LPS and Pam3CSK4 in combination, TLR2 signaling can overcome RP105-mediated regulation of TLR4 signaling. Thus, our data demonstrate that not only TLR4 signaling, but also RP105 appears to be an essential accessory for immune responses through TLR2 signaling. The function of TLR2 and TLR4 in response to TLR ligands could be associated with each other by RP105. These results can help us understanding the unique role of RP105 in macrophages response to TLR ligands.     

Dual or triple activation of TLR7, TLR8, and/or TLR9 by single-stranded oligoribonucleotides

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.     

Minisatellite binding protein Msbp-1 is a sequence-specific single-stranded DNA-binding protein.

Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes.     

Extracellularly delivered single-stranded viral RNA causes neurodegeneration dependent on TLR7

Innate immune receptors represent an evolutionarily ancient system that allows organisms to detect and rapidly respond to pathogen- and host-derived factors. TLRs are predominantly expressed in immune cells and mediate such a response. Although this class of pattern recognition receptors is involved in CNS disorders, the knowledge of ligands leading to activation of TLRs and to subsequent CNS damage is limited. We report in this study that ssRNA causes neurodegeneration and neuroinflammation dependent on TLR7 in the CNS. TLR7 is not only expressed in microglia, the major immune cells of the brain, but also in neurons of the CNS. Extracellularly delivered ssRNA40, an oligoribonucleotide derived from HIV and an established ligand of TLR7, induces neuronal cell death dependent on TLR7 and the central adapter molecule MyD88 in vitro. Activation of caspase-3 is involved in neuronal damage mediated by TLR7. This cell-autonomous neuronal cell death induced by ssRNA40 is amplified in the presence of microglia that mount an inflammatory response to ssRNA40 through TLR7. Intrathecal administration of ssRNA40 causes widespread neurodegeneration in wild-type but not in TLR7(-/-) mice, confirming that neuronal cell death induced by ssRNA40 through TLR7 occurs in vivo. Our results point to a possible mechanism through which extracellularly delivered ssRNA contributes to CNS damage and determine an obligatory role for TLR7 in this pathway.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8

Although TLR7 and TLR8 are phylogenetically and structurally related, their relative functions are largely unknown. The role of TLR7 has been established using TLR7-deficient mice and small molecule TLR7 agonists. The absence of TLR8-selective agonists has hampered our understanding of the role of TLR8. In this study TLR agonists selective for TLR7 or TLR8 were used to determine the repertoire of human innate immune cells that are activated through these TLRs. We found that TLR7 agonists directly activated purified plasmacytoid dendritic cells and, to a lesser extent, monocytes. Conversely, TLR8 agonists directly activated purified myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells (GM-CSF/IL-4/TGF-beta). Accordingly, TLR7-selective agonists were more effective than TLR8-selective agonists at inducing IFN-alpha- and IFN-regulated chemokines such as IFN-inducible protein and IFN-inducible T cell alpha chemoattractant from human PBMC. In contrast, TLR8 agonists were more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as TNF-alpha, IL-12, and MIP-1alpha. Thus, this study demonstrated that TLR7 and TLR8 agonists differ in their target cell selectivity and cytokine induction profile.     

Kir3.1 channel is functionally involved in TLR4-mediated signaling

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1β, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1^KD THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.     

Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages

The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH₂-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1 and IL-6. PI3K and JNK are also important for the production of IL-1. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1 via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages. cytokine; toll-like receptor-4; c-Jun NH₂-terminal kinase     

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.     

A human DNA-binding protein is methylation-specific and sequence-specific.

A nuclear protein isolated from human placenta, methylated DNA-binding protein (MDBP), binds selectively to DNA enriched in 5-methylcytosine. We now demonstrate that MDBP is a sequence-specific, as well as methylation-specific, DNA-binding protein. From ten restriction fragments of pBR322 DNA methylated with human DNA methyltransferase, one was bound to MDBP very much more strongly than any of the others. For this preferential binding to MDBP, the DNA had to be methylated. By a DNase I protection experiment (DNase I footprinting), a 22-base sequence within this methylated restriction fragment was shown to be specifically protected by MDBP. The sequence-specificity of MDBP coupled with its dependence on DNA methylation suggests that this is one of the proteins which modulates important functions of human DNA methylation in vivo.     

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.