Toll-like receptors: cellular signal transducers for exogenous molecular patterns causing immune responses

Innate immunity initiates protection of the host organism against invasion and subsequent multiplication of microbes by specific recognition. Germ line-encoded receptors have been identified for microbial products such as mannan, lipopeptide, peptidoglycan (PGN), lipoteichoic acid (LTA), lipopolysaccharide (LPS), and CpG-DNA. The Drosophila Toll protein has been shown to be involved in innate immune response of the adult fruitfly. Members of the family of Toll-like receptors (TLRs) in vertebrates have been implicated as pattern recognition receptors (PRRs). Ten TLRs are known and six of these have been demonstrated to mediate cellular activation by distinct microbial products. TLR4 has been implicated as activator of adaptive immunity, and analysis of systemic LPS responses in mice led to the identification of LPS-resistant strains instrumental in its identification as a transmembrane LPS signal transducer. Structural similarities between TLRs and receptor molecules involved in immune responses such as CD14 and the IL-1 receptors (IL-1Rs), as well as functional analysis qualified TLR2 as candidate receptor for LPS and other microbial products. Targeted disruption of the TLR9 gene in mice led to identification of TLR9 as CpG-DNA signal transducer. Involvement of TLR5 in cell activation by bacterial flagellin has been demonstrated. Further understanding of recognition and cellular signaling activated through the ancient host defense system represented by Toll will eventually lead to means for its therapeutic modulation.     

TLR2 engagement on CD8 T cells lowers the threshold for optimal antigen-induced T cell activation

TLR have a crucial role in the detection of microbial infection in mammals. Until recently, most investigations on TLR have focused on cells of the innate immune system and on the role of TLR in the initiation of antigen-specific responses following recognition of microbial products by APC. Here, we report that murine T cells express TLR1, TLR2, TLR6, TLR7 and TLR9 mRNA. Using CD8 T cells from F5 TCR-transgenic mice, we demonstrate that the lipopeptide Pam(3)CysSK(4) (Pam), a synthetic analog of bacterial and mycoplasmal lipoproteins that recognizes TLR1/2 complex, costimulates antigen-activated T cells. Costimulation with Pam permits an increased cell proliferation and survival associated with a sustained CD25 expression and an enhanced expression of Bcl-xL anti-apoptotic protein. In addition, we show that costimulation with Pam up-regulates IFN-gamma production but also granzyme B secretion and cytotoxic activity of antigen-activated T cells, indicating that TLR2 engagement enhances the major effector functions of CD8 T cells. Finally, we demonstrate that TLR2 engagement on T cells lowers the activation threshold for costimulatory signals delivered by APC.     

TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.     

LPS-mediated production of pro/anti-inflammatory cytokines and eicosanoids in whole blood samples: biological effects of +896A/G TLR4 polymorphism in a Sicilian population of healthy subjects

Toll-like receptors (TLRs) are the principal mediators of rapid microbial recognition: the lipopolysaccharide (LPS) receptor TLR4 seems to have a paradigmatic role. Single nucleotide polymorphisms (SNPs) in the TLR4 gene, such as +896A/G, known to attenuate receptor signaling, have been described. The +896A/G SNP is significantly less frequent in patients with myocardial infarction, Alzheimer's disease or prostate cancer, whereas it is overrepresented in centenarians. To clarify and confirm the biological effects of +896A/G SNP and its role in the pathophysiology of age-related diseases and longevity, we assessed the levels of IL-6, TNF-α, IL-10 and eicosanoids (LTB4 and PGE2) in LPS-stimulated whole blood samples in vitro of 50 young healthy Sicilians, screened for the presence of this SNP. To evaluate the possible influence of SNPs in PTGS2 and 5-Lo genes on eicosanoid production, the enrolled individuals were also genotyped for −765G/C PTGS2 and −1708G/A 5-Lo SNPs. Both pro-inflammatory cytokines and eicosanoids were significantly lower in carriers bearing the TLR4 mutation, whereas the anti-inflammatory IL-10 values were higher. On the basis of data reported herein, some suggestions can be drawn. First, pathogen load, by interacting with the host genotype, determines the type and intensity of inflammatory responses, according to the pro-inflammatory status and tissue injury, implicated in the pathophysiology of major age-related diseases. Second, adequate control of inflammatory response might reduce the risk of these diseases, and, reciprocally, might increase the chance of extended survival in an environment with reduced antigen (that is, pathogen) load.     

Genetic variation within TLR10 is associated with Crohn's disease in a New Zealand population

Toll-like receptors (TLRs) play an important role in the induction and regulation of the innate immune system and have been implicated in both infectious and inflammatory diseases. Recently the first association of TLR10 with Crohn's disease (CD) was reported. Here, we attempted to validate this association, using a candidate gene single nucleotide polymorphism (SNP) study of TLR10 in CD. We identified tagging SNPs, and genotyped these SNPs in a Caucasian New Zealand dataset consisting of 406 CD patients and 638 controls. In this sample, we were able to demonstrate an association between CD and several different TLR10 SNPs and haplotypes. Phenotypic analysis showed an association with early age at first diagnosis, inflammatory and ileocolonic CD behavior, requirement of bowel resection, and extra intestinal manifestations. This study provides evidence to suggest that genetic variation in TLR10 plays a role in interindividual differences in CD susceptibility and clinical outcome.     

Toll-like receptor 9 in murine lupus: more friend than foe!

The immune response induced by the pathogen-associated-pattern recognition receptor toll-like receptor 9 (TLR9) upon binding of CpG motif-containing DNA has been widely accepted as an important pathway in the immune defense against microbial pathogens. In contrast, the role of TLR9 in anti-DNA antibody generation and the pathogenesis of systemic lupus erythematosus (SLE) remains controversial. Indeed, the in vivo situation might consist of a delicate balance between B-cell receptor and DNA receptor signaling. Most surprisingly, TLR9 deletion does not ameliorate but rather exacerbates pathology in murine models. Such observations warrant caution with therapeutic efforts to treat autoimmune diseases, especially SLE, via TLR modulation.     

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.     

Single Nucleotide Polymorphisms of Toll-Like Receptor 7 and Toll-Like Receptor 9 in Hepatitis C Virus Infection Patients from Central China

Purpose

To analyze the correlation of polymorphisms of toll-like receptor 7 (TLR7) (rs179009) and toll-like receptor 9 (TLR9) (rs187084) in hepatitis C virus (HCV) infections in the Han population.

Materials and Methods

The genotypes of TLR7IVS2-151 in HCV infection were detected by Sanger sequencing using polymerase chain reaction-restriction fragment length polymorphism to determine the TLR9 T-1486C single nucleotide polymorphisms (SNP) for all enrolled patients.

Results

We found no significant difference between males with spontaneous clearance of HCV versus those chronically infected [χ²=2.71, p=0.10, odd ratios (OR)=0.58, 95% confidence interval (CI) 0.31-1.11]. However, significant differences were found for the distribution of TLR7 (rs179009) in females (χ²=9.46, p=0.01). In females, a significant difference was also found between chronic hepatitis C and those with spontaneous clearance of HCV in terms of TLR7 IVS2-151G/A allele frequencies (χ²=9.50, p=0.00, OR=0.46, 95% CI 0.28-0.75). In HCV-infected patients, no significant association was found between the frequency of TLR9 genotypes and alleles.

Conclusion

The site of TLR7 IVS2-151 (rs179009) G/A may be a factor for susceptibility of chronic HCV in the female Han population. TLR9T-1486C (rs18084) SNP may not play a major role in HCV infection. However, individual risk profiles for HCV infection did vary by sex and this relationship should be further investigated.     

Polymorphisms of toll like receptors in the genetics of severe RSV associated diseases

Toll like receptors (TLRs) are an essential part of the innate immune response. So far, ten different TLRs were identified in humans. They recognize a wide range of microbial and viral pathogens. Infection by respiratory syncytial virus (RSV) is still a major health problem, about 2% of all children are hospitalised due to RSV bronchiolitis during their first 2 years of live. TLR4 has already been described in association with RSV associated diseases by us and others. Thus we were interested whether other TLRs are also involved in the genetics of severe RSV infection. We genotyped 19 polymorphisms in the autosomal TLRs, these are TLR1, 2, 3, 5, 6, 9 and 10. Association analyses by the Armitage's Trend test revealed weak association of one TLR9 promoter polymorphism with RSV infection (p = 0.013). In addition, association was found with TLR10 haplotypes (p = 0.024). We conclude from our data--that--although we can not rule out a minor involvement of TLR9 polymorphism and TLR10 haplotypes--TLRs other than TLR4 do not play a major role in the genetics of severe RSV associated diseases. Future studies should focus on additional genes of the innate immune response.     

Association of the polymorphism of the Toll‐like receptor (TLR)‐3 and TLR‐9 genes with hepatitis C virus‐specific cell‐mediated immunity outcomes among Egyptian health‐care workers

Variations in the immune response could explain resistance to hepatitis C virus (HCV) infection. Toll‐like receptor gene (TLR)‐3 is an innate detector of dsRNA viruses, and the TLR‐9 gene recognizes bacterial and viral unmethylated cytosine–phosphate–guanosine (CpG) motifs. We previously reported that the TLR‐3.rs3775290 CC genotype was associated with HCV chronicity and that the TLR‐9 gene played no major role in this infection. This study identified the role of TLR‐3.rs3775290 (c.1377C/T), TLR‐9.rs5743836 (?1237T→C) and TLR‐9.rs352140 (G2848A) gene polymorphisms in predicting the outcome of HCV‐specific cell‐mediated immunity (CMI) among Egyptian health‐care workers (HCWs). We enrolled 265 HCWs in this study and divided them into four groups. Group 1: 140 seronegative‐aviraemic HCWs; group 2: 20 seronegative‐viraemic HCWs; group 3: 35 subjects with spontaneously resolved HCV infection; and group 4: 70 chronic HCV HCWs (patients). All subjects were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis for the TLR‐3.rs3775290, TLR‐9.rs5743836 and TLR‐9.rs352140 single nucleotide polymorphisms (SNPs). We also quantified HCV‐specific CMI in the four groups using an interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay in response to nine HCV genotype 4a, overlapping 15mer peptide pools covering the whole viral genome. No statistically significant difference was found between CMI‐responding subjects with different HCV states and TLR‐3.rs3775290 or TLR‐9.rs352140 genotypes. However, there was a significant relationship between the outcome of the HCV‐specific CMI and the TLR‐9.rs5743836 genotype among the responding subjects (P = 0·005) and the chronic HCV patients (P = 0·044). In conclusion, TLR‐9.rs5743836 SNP, but not TLR‐3.rs3775290 or TLR‐9.rs352140 genotypes, could predict the outcome of HCV‐specific CMI responses among Egyptians infected with genotype‐4.     

Expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on canine neutrophils

The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.     

The innate immune response to DNA

As a component of all living cells and microbes, it is not surprising that organisms have evolved mechanisms to detect foreign or aberrant DNA and trigger an innate immune response. TLR9 is an endosomal membrane bound receptor that is widely studied and the best understood DNA sensor. However, the existence of TLR9-independent DNA sensing pathways have been recognized for many years. Recently, novel cytosolic DNA sensors have been uncovered that include ZBP1 or DNA-dependent activator of interferon-regulatory factors (DAI) and a DNA sensing inflammasome consisting of the HIN200 protein, absent in melanoma 2 (AIM2). In combination with TLR9, these receptors provide a diverse repertoire of mechanisms to alert the cell to microbial DNA and possibly aberrant host DNA leading to the activation of the innate immune system.     

TLR2-dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness

Streptococcus suis capsular type 2 is an important swine pathogen and an agent of zoonosis. Although meningitis is the most common form of disease, septicemia and septic shock are also frequently reported. Despite reports that CD14 is involved in the recognition of encapsulated S. suis by host cells, the mechanisms underlying exacerbated release of pro-inflammatory cytokines, which may have a negative impact on disease outcome, are unclear. Here, we demonstrated that stimulation of human monocytes by whole encapsulated S. suis or its purified cell wall components influences the relative expression of Toll-like receptor (TLR)-2 and CD14 mRNA. Moreover, this stimulation triggered the release of cytokines (tumor necrosis factor-alpha, IL-1beta and IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1), which was significantly reduced by antibody-mediated blocking of TLR2 but not TLR4. Mouse macrophages deficient in TLR2 also showed impaired cytokine responses to encapsulated bacteria. Given that this response was completely abrogated in myeloid differentiation factor 88 (MyD88)-deficient macrophages, other TLRs might also be involved. Furthermore, we demonstrated that the presence of capsular polysaccharide (CPS)-modulated S. suis interactions with TLRs. In the absence of CPS, uncovered cell wall components induced cytokine and chemokine production via TLR2-dependent as well as -independent pathways, whereas CPS contributes to MCP-1 production in a MyD88-independent manner. Overall, this study contributes to a better understanding of the inflammatory processes induced by an encapsulated pathogen and suggests that the relative expression of CPS, known to be modulated during bacterial invasion and dissemination in the host, might alter interactions with host cells and, consequently, the outcome of the inflammatory response.     

Leishmania expressed lipophosphoglycan interacts with Toll‐like receptor (TLR)‐2 to decrease TLR‐9 expression and reduce anti‐leishmanial responses

Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.     

Genetic modulation of TLR8 response following bacterial phagocytosis

Human Toll‐like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G; p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16⁺CD14⁺ differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8‐dependent microbicidal response of infected macrophages. Hum Mutat 31:1069–1079, 2010. © 2010 Wiley‐Liss, Inc.     

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.     

Murine TLR2 expression analysis and systemic antagonism by usage of specific monoclonal antibodies

Cellular recognition of immuno-stimulatory microbial products alarming the host immune system upon infection, as well as endogenous molecular patterns representing perturbation of regular homeostasis such as through necrosis of host cells is mediated by innate pattern recognition receptors to which toll-like receptors (TLRs) belong. A variety of agonists has been attributed to TLR2. We raised monoclonal antibodies (mAbs) toward the murine TLR2 extracellular domain (mT2ECD) in order to analyze murine TLR2 expression. Murine macrophages were stained TLR2-specifically with distinct mAbs as shown by flow cytometry, immuno precipitation, and immuno-cytochemical analysis. TLR2-specific murine macrophage activation was inhibited through pre-incubation with a mAb mT2.4 while another mTLR2-specific mAb mT2.7 did not affect cell activation through TLR2. Plasmon resonance based analysis showed inhibition of lipopeptide binding to mT2ECD if complex formation with mT2.4 preceded binding analysis. Systemic induction of IL-6, IL-12p40, and GROalpha/KC release to the serum upon lipopeptide challenge of mice was inhibited by systemic administration of mT2.4. Furthermore, 120 mg/kg of mT2.4 protected mice from lethal shock-like syndrome in an experimental low-dose model of septic shock. This result validates blockage of cell surface TLR2 for inhibition of immune cell over-activation upon microbial challenge.