Toll-like Receptor Signaling Cascade Research Articles

Microbial Toll/interleukin 1 receptor proteins: A new class of virulence factors

Quite a number of microbes possess genes which encode for proteins containing a Toll/interleukin 1 receptor domain. This domain is key for the physical interaction of eukaryotic Toll-like receptors with their adaptor molecules like MyD88 enabling innate immune cells to recognize invading pathogens and to initiate appropriate defense responses. Recent findings imply that microbial Toll/interleukin 1 receptor proteins impair Toll-like receptor signaling. As a consequence, secretion of pro-inflammatory cytokines is dampened, and microbial replication is enhanced. This group of proteins can thus be classified as a new family of virulence factors able to modulate the Toll-like receptor signaling cascade. This review summarizes current knowledge of the biology of this fascinating group of molecules.

Negative Regulation of Toll-like Receptors Signaling Pathways*

Toll-like receptors (TLRs) are key mediators of both innate and adaptive immunity by recognizing and eliciting responses to invading pathogens. The activation of TLRs must be stringently controlled in order to avoid exaggerated expression of signaling components as well as pro-inflammatory cytokines that can devastate the host,resulting in chronic inflammatory diseases,autoimmune disorders and aid in the pathogenesis of TLR-associated diseases. Therefore,it is essential that negative regulators act at multiple levels within TLR signaling cascades in order to synchronize the activation and negative regulation of signal transduction to limit potentially harmful immunological consequences. A summary of the various mechanisms employed by negative regulators of TLRs signaling to ensure the appropriate modulation of both immune and inflammatory responses was provided.

Diversification of TOLLIP isoforms in mouse and man

The Toll-interacting protein TOLLIP is an ubiquitin-binding protein that interacts with several components of the Toll-like receptor signaling cascade. The canonical protein consists of three annotated domains: an N-terminal TBD-loop-coil domain that mediates protein-protein interactions, a C2 domain that targets TOLLIP to the endosome, and a CUE domain at the C-terminus that binds monoubiquitin. TOLLIP has been described primarily in trafficking of the interleukin-1 receptor (IL1R) and turnover of the interleukin-1 receptor-associated kinase (IRAK), so it is an essential regulator of inflammatory signaling. Here we describe the expression of numerous alternate transcripts from mouse and human TOLLIP, which are predicted to generate at least five variant proteins between the two species. Most of the variant proteins are predicted to have altered N-terminal domains, altered TBD-loop-coil domains, or a truncated C2 domain. A mouse-specific variant arises from an alternate termination exon, and the resulting protein lacks the CUE domain. Two transcripts arising from alternate initiating exons are highly conserved between mouse and human but exhibit different patterns of expression. The consequent protein isoforms retain (TOLLIP.A) or lack (TOLLIP.D) the protein-binding TBD, so are predicted to traffic monoubiquitinated proteins to alternate protein complexes within the endosomal compartment. In summary, the widespread and inducible expression of Tollip isoforms predicts diversification of its function in rodent and human immune systems. Alternate splicing of critical signaling molecules such as Tollip may provide one mechanism behind the broad repertoire of responses generated by cells of the innate immune system in response to infection.

Characterization of two key molecules of teleost innate immunity from rainbow trout ( Oncorhynchus mykiss): MyD88 and SAA

Toll-like receptors (TLR) are relevant for piscine innate immunity. TLR activation recruits several downstream factors regulating the expression of immunorelevant genes. We have characterized two key factors of innate immunity from rainbow trout: MyD88 as an adaptor protein interacting directly with TLRs, and serum amyloid A as an effector molecule induced by the activated Toll-like receptor signaling cascade. Both factors share a remarkable high degree of structural conservation with their mammalian orthologs suggesting that innate immune defense mechanisms may also functionally be conserved between fish and mammals.

The Zinc Finger Protein Gfi1 Controls TLR4-Mediated Inflammatory Response by Directly Antagonizing NF-κB Transcription Factor

Inflammatory responses are complex and comprise multiple mediators including cytokines such as TNF-alpha (TNF-α) and IL-1beta. These cytokines are synthesized and secreted in response to signaling by plasma membrane receptors of the Toll-like receptor (TLR) family. A central downstream element of TLR-dependent signaling is the transcription factor NF-kappaB (NF-κB), which plays a pivotal role in controlling the proper sequence of events during an inflammatory response. In unstimulated cells, NF-κB is bound to inhibitory IkappaB (IκB) proteins and remains sequestered in the cytoplasm. Stimulation of TLRs triggers a signaling cascade that leads to phosphorylation and proteasomal degradation of IκB, resulting in the translocation of NF-κB to the nucleus, where it acts as a transcriptional activator of target genes. To keep the innate immune system under control, the TLR signaling cascade is under a tight control of many positive and negative regulators. We have previously shown that the transcription factor Growth Factor Independence 1 (Gfi1) represents a novel factor limiting the inflammatory immune response including TNF-α. Gfi1-deficient (Gfi1−/−) mice show a very strong systemic response to the TLR4 ligand and endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here, we investigated the molecular mechanism of this exaggerated TNF-α production in the absence of Gfi1. It is known that endotoxin stimulation results in the activation of the transcription factor NF-κB through TLR4, leading to TNF-α production. This activation also resulted in rapid and de novo expression of Gfi1 in the nucleus in a time- and dose-dependent manner. The expression of Gfi1 was not due to feedback regulation from secreted TNF, since TNF-deficient macrophages were also able to upregulate Gfi1 mRNA following LPS stimulation. As expected, LPS stimulation of Gfi1−/− macrophages resulted in significantly higher levels of TNF-α mRNA, and secreted TNF-α cytokine. Strikingly and in contrast to most known negative regulators of TLRs, Gfi1 did not affect the activity or the expression levels of the cytoplasmic components of TLR signaling pathway. Additionally, NF-κB phosphorylation and nuclear translocation post- LPS treatment were intact in both Gfi1−/− and Gfi1+/+ macrophages. Immunoprecipitation analysis from cells endogenously expressing Gfi1 and NF-κB or over-expressing these two proteins post transfection, clearly revealed a direct interaction between Gfi1 and the p65 subunit of NF-κB. Immunofluorescence staining of macrophages post-LPS treatment confirmed direct interaction of these two proteins in the nucleus at the endogenous level. Gfi1 represses transcription by binding to DNA recognition sequences in target gene promoters. Thus, aiming to investigate the effect of Gfi1 expression on NF-κB nuclear signaling, we found that LPS treatment enhances NF-κB DNA binding activity in Gfi1−/− macrophages as compared to Gfi1+/+ cells. Furthermore, over expression of Gfi1 protein resulted in negative regulation of NF-κB mediated gene activation in a dose-dependent manner. Chromatin immune precipitation with anti-p65 antibodies from LPS stimulated Gfi1+/+ and Gfi1−/− macrophages revealed enhanced NF-κB promoter occupancy at the TNF gene in Gfi1−/− macrophages as compared to Gfi1+/+ cells. In conclusion, our findings reveal a novel function for Gfi1 in the innate immune response by directly antagonizing NF-κB function. This molecular perceptive of TNF-α regulation during inflammation may provide an attractive strategy for therapeutic intervention in chronic inflammatory diseases and certain cancers.

Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli1

This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01-5 mug/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.

Human Immunodeficiency Virus Infection Alters Tumor Necrosis Factor Alpha Production via Toll-Like Receptor-Dependent Pathways in Alveolar Macrophages and U1 Cells

Human immunodeficiency virus (HIV)-positive persons are predisposed to pulmonary infections, even after receiving effective highly active antiretroviral therapy. The reasons for this are unclear but may involve changes in innate immune function. HIV type 1 infection of macrophages impairs effector functions, including cytokine production. We observed decreased constitutive tumor necrosis factor alpha (TNF-alpha) concentrations and increased soluble tumor necrosis factor receptor type II (sTNFRII) in bronchoalveolar lavage fluid samples from HIV-positive subjects compared to healthy controls. Moreover, net proinflammatory TNF-alpha activity, as measured by the TNF-alpha/sTNFRII ratio, decreased as HIV-related disease progressed, as manifested by decreasing CD4 cell count and increasing HIV RNA (viral load). Since TNF-alpha is an important component of the innate immune system and is produced upon activation of Toll-like receptor (TLR) pathways, we hypothesized that the mechanism associated with deficient TNF-alpha production in the lung involved altered TLR expression or a deficit in the TLR signaling cascade. We found decreased Toll-like receptor 1 (TLR1) and TLR4 surface expression in HIV-infected U1 monocytic cells compared to the uninfected parental U937 cell line and decreased TLR message in alveolar macrophages (AMs) from HIV-positive subjects. In addition, stimulation with TLR1/2 ligand (Pam(3)Cys) or TLR4 ligand (lipopolysaccharide) resulted in decreased intracellular phosphorylated extracellular signal-regulated kinase and subsequent decreased transcription and expression of TNF-alpha in U1 cells compared to U937 cells. AMs from HIV-positive subjects also showed decreased TNF-alpha production in response to these TLR2 and TLR4 ligands. We postulate that HIV infection alters expression of TLRs with subsequent changes in mitogen-activated protein kinase signaling and cytokine production that ultimately leads to deficiencies of innate immune responses that predispose HIV-positive subjects to infection.

Notch signaling is activated by TLR stimulation and regulates macrophage functions

Notch signaling is a well-conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll-like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up-regulation of Notch1 in primary and macrophage-like cell lines. Notch1 up-regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by gamma-secretase using a gamma-secretase inhibitor (GSI), the expression of Notch1 was down-regulated to basal levels. This treatment significantly modulated expression of TNF-alpha, IL-6, and IL-10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up-regulated in GSI-treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF-kappaBp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro-inflammatory responses, through activation of NF-kappaB.

Toll-like receptor 4 deficiency: Smaller infarcts, but nogain in function

BackgoundIt has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT).ResultsInfarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R.ConclusionThese results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.

ACTIVATION OF SRC FAMILY TYROSINE KINASES AND PHOSPHORYLATION OF VAV1 DOWNSTREAM FROM MYD88 AND TRAF6 IN MURINE MACROPHAGES.

Background We and others have reported that exposure of macrophages to LPS, CpG DNA, and other Toll-like receptor (TLR) ligands triggers the activation of multiple src family tyrosine kinases (SFKs) and the subsequent phosphorylation of vav1. However, the mechanism(s) leading to the activation of SFKs downstream from TLRs are unknown. Purpose We sought to determine whether SFK activation and vav1 phosphorylation occur downstream from two key signaling intermediates in the TLR cascade, MyD88 and TRAF6. Methods We used two subclones of RAW 264.7 murine macrophages that have been engineered to express forms of MyD88 and TRAF6, respectively, that can be activated upon addition of the antibiotic coumermycin, leading to the production of inflammatory mediators, including tumor necrosis factor (TNF). Selective inhibitors of SFKs (PP1, PP2, SU6656) were used to test the involvement of these kinases in stimulation of TNF secretion downstream from MyD88 and TRAF6. Finally, we used immunoblotting to detect phosphorylation of vav1 on tyrosine 174 (an event known to be mediated by SFKs). Results TLR-ligand-independent activation of either MyD88 or TRAF6 led to robust TNF secretion in these RAW subclones. Preincubation with any of the three SFK inhibitors resulted in dramatic reductions in the magnitude of TNF secretion by these cells (75-85% less in response to MyD88 activation; 50-60% less in response to TRAF6 activation). Finally, the tyrosine phosphorylation of vav1 on tyrosine 174 was detected within 10 to 20 minutes of coumermycin-induced activation of either MyD88 or TRAF6. Conclusions Our data indicate that one or more SFKs are activated downstream from the TLR signaling cascade components MyD88 and TRAF6 in macrophages, leading to the phosphorylation of vav1 and playing an important role in the up-regulation of TNF production.

Alternate transcription of the Toll-like receptor signaling cascade

A systematic analysis of the FANTOM3 mouse cDNA dataset provides transcriptional evidence of widespread alternate splicing in the Toll-like receptor signaling pathway.

The Effect of Adenosine on TLR Signaling Pathways in Human Monocytic THP-1 Cells.

Adenosine is an important metabolite that serves as a potent regulator of inflammation and mediates various biological functions in different cell types. Adenosine inhibits the proinflammatory actions of inflammatory and immune cells via interaction with its receptors, particularly A2A receptors. Adenosine receptors belong to the seven-transmembrane G-protein coupled receptors and via the different G proteins transfer signals through different effectors including adenyl cyclase, PKA, PKC, PI3K, and MAP kinases. The mechanisms by which the adenosine regulates immune responses and how adenosine receptor pathways interact with other signaling pathways are currently unknown. Toll-like receptors (TLRs) of the innate immune cells recognize conserved microbial structures, such as bacterial lipopolysaccharide and viral double-stranded RNA, and activate signaling pathways that result in innate immune responses against microbial infections. Fcγ receptors of the innate cells play a critical role in the clearance of pathogens, regulation of inflammation and co-ordination of the immune response. We seek to understand the interaction between adenosine (via adenosine receptors) and TLR- and Fcγ R- mediated signaling pathways. We have initiated study on the effect of adenosine and lipopolysaccharide (LPS) on expression of TLR2, TLR4, FcγRI and Fcγ RII receptors in the human monocytic cell line THP-1. We incubated cells with 100μM adenosine for three hours at 37°C and assayed the expression of receptors using flow cytometry. Our results suggest that adenosine increases the expression of TLR2 and TLR4 in both undifferentiated cells and the cells induced to become macrophages by phorbol ester. Incubation with adenosine for 24 hours further increases the expression of TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. Similarly, incubation with LPS for three hours increases the expression TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. In contrast, the expression level of FcγRI and FcγRII receptors do not change in the presence of either adenosine or LPS. These observations suggest that adenosine specifically enhances expression of TLRs but not Fcγ receptors. To further understand the interaction between adenosine and TLR pathways, we are continuously investigating the effect of adenosine on expression and the protein modification (e.g. phosphorylation) of TLR2, TLR4, and the molecules in the TLR signaling cascades including MyD88, IRAK and MAP kinases using real-time RT-PCR and western blotting.

Functional role for toll-like receptors in atherosclerosis and arterial remodeling.

Activation of inflammatory cascades is causally related to the development of atherosclerotic disease. Toll-like receptors are innate immune receptors that recognize pathogen-associated molecular patterns. In this review the pathways by which toll-like receptors might play a role in the development and progression of atherosclerosis will be discussed according to recent literature. Toll-like receptors are expressed in atherosclerotic tissue. Next to pathogens, endogenous toll-like receptor ligands have been linked with the development of arterial occlusive disease. In mouse models of hyperlipidemia, a potential role for the toll-like receptor pathway has been suggested in hypercholesterolemia-induced atherosclerosis. Recent in-vitro studies revealed a mechanism by which toll-like receptor ligation results in a strong inhibition of cholesterol efflux from macrophages. In addition, oxidized lipoproteins interact with toll-like receptors. Furthermore, activation of the apoptotic cascade, which is important during atherogenesis, enhances the toll-like receptor pathway resulting in upregulation of proinflammatory cytokines. Human epidemiologic studies have linked TLR4 polymorphism with atherosclerosis. However, data on the association between atherosclerosis progression and TLR4 polymorphisms are conflicting. Next to plaque growth, arterial remodeling is an important determinant of luminal narrowing in atherosclerosis. Recently, a possible role for TLR4 signaling in arterial remodeling has been revealed in mouse models. A clarification of the molecule [corrected] mechanisms by which the toll-like receptor signaling cascade influences atherosclerosis might [corrected] lead to novel strategies to intervene in the development of this life-threatening disease.

Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus

We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus. ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells. Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages. The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated. The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice. In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP. The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-κB. Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages. Taken together, our results demonstrate that ASP, isolated from a cell culture of A. senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-κB.

Osteoclast differentiation by human osteoblastic cell line SaOS-2 primed with bacterial lipid A

We examined the responses of human osteoblastic cell line SaOS-2 to bacterial lipid A, a bioactive center of lipopolysaccharide, during osteoclast differentiation of human peripheral blood mononuclear cells (PBMC). SaOS-2 cells expressed mRNA for Toll-like receptor (TLR) 4, MD-2, CD14, and myeloid differentiation factor 88, whereas they failed to express mRNA for TLR2. Escherichia coli-type synthetic lipid A (compound 506) induced cytokine mRNA expression and nuclear factor (NF)-κB activation in SaOS-2 cells. Compound 506 also increased the expression of receptor activator of NF-κB ligand. Further, cells primed with compound 506 augmented the differentiation of PBMC into osteoclastic cells, and the effect was inhibited by anti-TLR4 monoclonal antibody. These findings suggest that the TLR signaling cascade in osteoblastic cells is involved in regulating the function of osteoclastogenesis.

IL-1 Receptor-Associated Kinase 4 Is Essential for IL-18-Mediated NK and Th1 Cell Responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.