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Background

Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.

Methods

TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins.

Results

TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).

Conclusion

Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.

Pseudomonas aeruginosa, an opportunistic respiratory pathogen that infects the majority of patients with cystic fibrosis, initiates host inflammatory responses through interaction with airway epithelial cells. The Toll-like receptors (TLRs) are a family of pathogen pattern recognition receptors that play key roles in host innate immunity. In this study we aimed to determine whether TLRs mediate the interaction between P. aeruginosa and airway epithelial cells. Individual murine TLRs (TLR1 to TLR9) and dual combinations of these TLRs that activate an NF-κB-driven luciferase reporter in response to PAO1 were screened in HEK 293 cells. TLR5, TLR2, a combination of TLR1 and TLR2, or a combination of TLR2 and TLR6 responded to PAO1. Another P. aeruginosa strain, strain PAK, activated TLR5 similarly, while the isogenic flagellin-deficient strain PAK/fliC and the flagellum-free bacterium Haemophilus influenzae failed to activate TLR5. Reverse transcription-PCR was used to probe the presence of multiple TLRs (including TLR5) in primary human airway epithelial cells (HAECs). Immunostaining with TLR5 antibodies showed that TLR5 was expressed in HAECs and on the apical surface of the human trachea epithelium. In HAECs, PAO1, PAK, and Burkholderia cepacia, but not flagellin-deficient strain PAK/fliC or a B. cepacia fliC mutant, activated the NF-κB reporter. Dominant negative TLR5 specifically blocked the response to P. aeruginosa but not to the response to lipoteichoic acid, a specific ligand of TLR2. We also determined that MyD88, IRAK, TRAF6, and Toll-interacting protein (Tollip), but not TIRAP, were involved in the TLR-mediated response to P. aeruginosa in HAECs. These findings demonstrate that the airway epithelial receptor TLR5 senses P. aeruginosa through its flagellin protein, which may have an important role in the initiation of the host inflammatory reaction to clear the invading pathogen.

Background

Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.

Methods and Findings

Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.

Conclusions

Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model.

Editors' Summary

Background.

Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated.

Why Was This Study Done?

It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches.

What Did the Researchers Do and Find?

The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage.

What Do These Findings Mean?

These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040248.

Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish)

The UK Health Protection Agency provides information for the public and health professionals on melioidosis

Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)

Mycoplasma lipoproteins are recognized by Toll-like receptors (TLR), but TLRs' role in responses to infection are unknown. Mycoplasma pulmonis is a naturally occurring respiratory pathogen in mice. In the current study, we used TLR-transfected HEK cells and TLR2−/− bone marrow-derived dendritic cells to demonstrate TLR2-mediated events are important in the initial host-mycoplasma interactions promoting cytokine responses. As we found alveolar macrophages expressed TLR1, TLR2 and TLR6 mRNAs, a role for TLR2 in innate immune clearance in lungs was examined. Three days post-infection, TLR2−/− mice had higher M. pulmonis numbers in lungs, but not in nasal passages. However, TLR2−/− mice had higher lung cytokine levels, indicating TLR2-independent mechanisms are also involved in host responses. Thus, TLR2 plays a critical role in the ability of innate immunity to determine M. pulmonis numbers in the lung, and it is likely that early after respiratory infection that TLR2 recognition of M. pulmonis triggers initial cytokine responses of host cells.

SUMMARY

Toll-like receptors (TLR) have a critical role in innate immunity against pathogens. We investigated the cytokine response to TLR stimulation in peripheral blood cells of subjects infected with hepatitis C virus (HCV) and / or human immunodeficiency virus (HIV) in the Women Interagency HIV Study (WIHS) cohort. Interleukin (IL)-6 in response to TLR3 and TLR4 ligands such as polyinosinic-polycytidylic acid and lipopolysaccharide was significantly compromised in HCV-infected women. High spontaneous secretion of IL-6 suggested pre-existing cell activation as a factor mediating reduced responses to TLR3 and TLR4 stimulation. To a lesser extent, tumour necrosis factor-α and IL-1β responses to TLR stimulation were also compromised. Monocytes, but not B cells or NK cells, were identified as the cell population spontaneously secreting cytokines and also as the cells responding to TLR stimulation. These results highlight a functional defect in antigen-presenting cells of women with HCV infection or co-infection. In women with existing HIV co-infection, decreased cytokine function of antigen-presenting cells suggests another mechanism contributing to immune dysfunction in addition to the HIV-associated CD4 defect.

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-α production was abolished in Irak-1–deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-κB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1–deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-α induction in pDCs.

Toll-like receptors play an important role in innate and adaptive immunity and in balancing immune responses with tolerance. TLR2 is related to protection against allergies and allergic asthma by sensing pathogen associated patterns as lipoproteins and lipopeptides. A constant Th1 triggering is thought to prevent Th2 related disorders.

TLR2 is expressed on a variety of cells, both structural as well as immune cells. Importantly, TLR2 is also expressed on dendritic cells, which are thought to be one of the key players of initiating and maintaining immune responses. Therefore, TLR2 on dendritic cells is a good target for modulating immunity either to Th1 or Th2 responses, or induction of tolerance.

TLR2 agonists show high immunomodulatory and adjuvantic capacity. This makes TLR2 agonisation a promising approach for pharmaceutical intervention of allergic disorders.

Dendritic cells (DCs) play a crucial role in the immune responses against infections by sensing microbial invasion through toll-like receptors (TLRs). In humans, two distinct DC subsets, CD11c− plasmacytoid DCs (PDCs) and CD11c+ myeloid DCs (MDCs), have been identified and can respond to different TLR ligands, depending on the differential expression of cognate TLRs. In this study, we have examined the effect of TLR-7 ligands on human DC subsets. Both subsets expressed TLR-7 and could respond to TLR-7 ligands, which enhanced the survival of the subsets and upregulated the surface expression of costimulatory molecules such as CD40, CD80, and CD86. However, the cytokine induction pattern was distinct in that PDCs and MDCs produced interferon (IFN)-α and interleukin (IL)-12, respectively. In response to TLR-7 ligands, the Th1 cell supporting ability of both DC subsets was enhanced, depending on the cytokines the respective subsets produced. This study demonstrates that TLR-7 exerts its biological effect in a DC subset-specific manner.

Summary

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their co-receptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression extended also to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen- mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.

Author Summary

Influenza A virus, a negative stranded RNA, can cause severe illness in humans and animals and stimulates many receptors including Toll like receptors 7 (TLR7). TLR signaling induces maturation of dendritic cells and the production of a variety of inflammatory cytokines that are crucial for both innate and adaptive immunity. TLR7 is an intracellular receptor, which resides in endosomes and senses viruses to trigger host defence. Previous data have shown that TLR9 requires proteolysis to be functional but it is unclear whether other intracellular TLRs (TLR3 and TLR7) are also subject to degradation. Here, we used a protease deficient mouse model to show the in vivo importance of TLR7 processing in influenza infection. Inflammation monitored by cytokine release and adaptive immunity measured by cross priming of CD8+ T cells was significantly reduced in infected protease-deficient animals in comparison to control animals. We showed that TLR7 requires a proteolytic cleavage by a cysteine endopeptidase in order to be functional. Our findings indicate that TLR7 processing mediated by a protease, asparagynil endopeptidase, is critical for inducing robust anti-influenza immune responses. Given our results, targeting TLR7 response in the lungs through proteases may offer new therapeutic potential in pulmonary infection.

Although oral coinfections (e.g., periodontal disease) are highly prevalent in human immunodeficiency virus type 1-positive (HIV-1+) patients and appear to positively correlate with viral load levels, the potential for oral bacteria to induce HIV-1 reactivation in latently infected cells has received little attention. We showed that HIV-1 long terminal repeat (LTR) promoter activation can be induced by periodontopathogens in monocytes/macrophages; nevertheless, the mechanisms involved in this response remain undetermined. Since Toll-like receptor 2 (TLR2), TLR4, and TLR9 activation have been involved in HIV-1 recrudescence, we sought to determine the role of these TLRs in HIV-1 reactivation induced by the periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis using BF24 monocytes/macrophages stably transfected with the HIV-1 promoter driving chloramphenicol acetyltransferase (CAT) expression and THP89GFP cells, a model of HIV-1 latency. We demonstrated that TLR9 activation by F. nucleatum and TLR2 activation by both bacteria appear to be involved in HIV-1 reactivation; however, TLR4 activation had no effect. Moreover, the autocrine activity of tumor necrosis factor alpha (TNF-α) but not interleukin-1β (IL-1β) produced in response to bacteria could impact viral reactivation. The transcription factors NF-κB and Sp1 appear to be positively regulating HIV-1 reactivation induced by these oral pathogens. These results suggest that oral Gram-negative bacteria (F. nucleatum and P. gingivalis) associated with oral and systemic chronic inflammatory disorders enhance HIV-1 reactivation in monocytes/macrophages through TLR2 and TLR9 activation in a mechanism that appears to be transcriptionally regulated. Increased bacterial growth and emergence of these bacteria or their products accompanying chronic oral inflammatory diseases could be risk modifiers for viral replication, systemic immune activation, and AIDS progression in HIV-1+ patients.

Background

Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/Principal Findings

We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/Significance

These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.

Author Summary

Dengue virus (DENV) infections cause a broad spectrum of clinical manifestations, ranging from self-limited fever to severe disease, such as dengue hemorrhagic fever (DHF) that can be fatal. The pathogenesis of severe dengue is associated with an inadequate immune response characterized by the over-production of cytokines and other inflammatory components. However, little is known about the role of the innate immune response in the progression to hemorrhagic manifestations. TLRs are among the most important components of innate immunity and are responsible for initiating a response against a variety of pathogens, including viruses. Recent studies suggest that TLRs may contribute to disease pathogenesis. Here we aimed to explore the role of these receptors in dengue disease progression. To this end, we examined the expression of several TLRs and of co-stimulatory molecules in monocytes and DCs from dengue patients. A link between TLRs expression and the severity of dengue was observed: patients with dengue fever express higher levels of TLR3 and TLR9 than patients with DHF. This could be crucial for the host defense against dengue virus or disease progression. In addition, expression of CD80 and CD86 was altered in DCs of severe dengue cases. We show that interferon type I production is also altered in vitro through TLR9. This suggests that dengue virus affects the interferon response through this signaling pathway.

Background

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.

Methodology/Principal Findings

TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.

Conclusions/Significance

These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.

OBJECTIVES

Toll-like receptors (TLR) are innate immune sensors that are integral to resisting chronic and opportunistic infections. Mounting evidence implicates TLR polymorphisms in susceptibilities to various infectious diseases, including HIV-1. We investigated the impact of TLR single-nucleotide polymorphisms (SNPs) on clinical outcome in a sero-incident cohort of HIV-1-infected volunteers.

DESIGN

We analyzed TLR SNPs in 201 antiretroviral treatment-naïve HIV-1 infected subjects from a longitudinal sero-incident cohort with regular follow-up intervals (median follow-up 4.2 years, interquartile range 4.4). Subjects were stratified into two groups according to either disease progression, defined as peripheral blood CD4+ T-cell decline over time, or peak and setpoint viral load.

METHODS

Haplotype-tagging SNPs from TLR2, TLR3, TLR4, and TLR9 were detected by mass-array genotyping, and CD4+ T-cell counts and viral load measurements were determined prior to antiretroviral therapy initiation. The association of TLR haplotypes with viral load and rapid progression was assessed by multivariate regression models using age and sex as covariates.

RESULTS

Two TLR4 SNPs in strong linkage disequilibrium (1063A/G [D299G] and 1363C/T [T399I]) were more frequent among subjects with high peak viral load compared to low/moderate peak viral load (OR=6.65, 95% CI 2.19–20.46, P<0.001; adjusted P=0.002 for 1063A/G). In addition, a TLR9 SNP previously associated with slow progression was found less frequently among subjects with high viral setpoint compared to low/moderate setpoint (OR=0.29, 95% CI 0.13–0.65, P=0.003, adjusted P=0.04).

CONCLUSION

This study suggests a potentially new role for TLR4 polymorphisms in HIV-1 peak viral load and confirms a role for TLR9 polymorphisms in disease progression.

Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we showed that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The C-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG, and, when expressed in Tlr9−/− dendritic cells, restored CpG-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5′ 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002) and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC)were measured by evaluating CD86, CD40 and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV− individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-α and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-α. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV− individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I∶C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I∶C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I∶C)-inhibited replication of DENV2. The poly(I∶C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I∶C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I∶C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.

Background

Toll-like receptors have a key role in innate immune response to microbial infection. The toll-like receptor (TLR) family consists of ten identified human TLRs, of which TLR2 and TLR9 have been shown to initiate innate responses to herpes simplex virus type 1 (HSV-1) and TLR3 has been shown to be involved in defence against severe HSV-1 infections of the central nervous system. However, no significant activation of the TLR3 pathways has been observed in wild type HSV-1 infections. In this work, we have studied the TLR responses and effects on TLR gene expression by HSV-1 with Us3 and ICP4 gene deletions, which also subject infected cells to apoptosis in human monocytic (U937) cell cultures.

Results

U937 human monocytic cells were infected with the Us3 and ICP4 deletion herpes simplex virus (d120), its parental virus HSV-1 (KOS), the Us3 deletion virus (R7041), its rescue virus (R7306) or wild type HSV-1 (F). The mRNA expression of TLR2, TLR3, TLR4, TLR9 and type I interferons (IFN) were analyzed by quantitative real-time PCR. The intracellular expression of TLR3 and type I IFN inducible myxovirus resistance protein A (MxA) protein as well as the level of apoptosis were analyzed by flow cytometry. We observed that the mRNA expression of TLR3 and type I IFNs were significantly increased in d120, R7041 and HSV-1 (F)-infected U937 cells. Moreover, the intracellular expression of TLR3 and MxA were significantly increased in d120 and R7041-infected cells. We observed activation of IRF-3 in infections with d120 and R7041. The TLR4 mRNA expression level was significantly decreased in d120 and R7041-infected cells but increased in HSV-1 (KOS)-infected cells in comparison with uninfected cells. No significant difference in TLR2 or TLR9 mRNA expression levels was seen. Both the R7041 and d120 viruses were able to induce apoptosis in U937 cell cultures.

Conclusion

The levels of TLR3 and type I IFN mRNA were increased in d120, R7041 and HSV-1 (F)-infected cells when compared with uninfected cells. Also IRF-3 was activated in cells infected with the Us3 gene deletion viruses d120 and R7041. This is consistent with activation of TLR3 signaling in the cells. The intracellular TLR3 and type I IFN inducible MxA protein levels were increased in d120 and R7041-infected cells but not in cells infected with the corresponding parental or rescue viruses, suggesting that the HSV-1 Us3 gene is involved in control of TLR3 responses in U937 cells.

Background

HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host's response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.

Methods

Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.

Results

MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.

Conclusion

These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.

New and exciting insights into the importance of the innate immune system are revolutionizing our understanding of immune defense against infections, pathogenesis, and the treatment and prevention of infectious diseases. The innate immune system uses multiple families of germline-encoded pattern recognition receptors (PRRs) to detect infection and trigger a variety of antimicrobial defense mechanisms. PRRs are evolutionarily highly conserved and serve to detect infection by recognizing pathogen-associated molecular patterns that are unique to microorganisms and essential for their survival. Toll-like receptors (TLRs) are transmembrane signalling receptors that activate gene expression programs that result in the production of proinflammatory cytokines and chemokines, type I interferons and antimicrobial factors. Furthermore, TLR activation facilitates and guides activation of adaptive immune responses through the activation of dendritic cells. TLRs are localized on the cell surface and in endosomal/lysosomal compartments, where they detect bacterial and viral infections. In contrast, nucleotide-binding oligomerization domain proteins and RNA helicases are located in the cell cytoplasm, where they serve as intracellular PRRs to detect cytoplasmic infections, particularly viruses. Due to their ability to enhance innate immune responses, novel strategies to use ligands, synthetic agonists or antagonists of PRRs (also known as ‘innate immunologicals’) can be used as stand-alone agents to provide immediate protection or treatment against bacterial, viral or parasitic infections. Furthermore, the newly appreciated importance of innate immunity in initiating and shaping adaptive immune responses is contributing to our understanding of vaccine adjuvants and promises to lead to improved next-generation vaccines.

Severe trauma such as burn injury is often associated with a systemic inflammatory syndrome characterized by a hyperactive innate immune response and suppressed adaptive immune function. Dendritic cells (DCs), which sense pathogens via their Toll-like receptors (TLRs), play a pivotal role in protecting the host against infections. The effect of burn injury on TLR-mediated DC function is a debated topic and the mechanism controlling the purported immunosuppressive response remains to be elucidated. Here we examined the effects of burn injury on splenic conventional DC (cDC) and plasmacytoid DC (pDC) responses to TLR9 activation. We demonstrate that, following burn trauma, splenic cDCs’ cytokine production profile in response to TLR9 activation became anti-inflammatory dominant, with high production of IL-10 (>50% increase) and low production of IL-6, TNF-α and IL-12p70 (∼25–60% reduction). CD4+ T cells activated by these cDCs were defective in producing Th1 and Th17 cytokines. Furthermore, burn injury had a more accentuated effect on pDCs than on cDCs. Following TLR9 activation, pDCs displayed an immature phenotype with an impaired ability to secrete pro-inflammatory cytokines (IFN-α, IL-6 and TNF-α) and to activate T cell proliferation. Moreover, cDCs and pDCs from burn-injured mice had low transcript levels of TLR9 and several key molecules of the TLR signaling pathway. Although hyperactive innate immune response has been associated with severe injury, our data show to the contrary that DCs, as a key player in the innate immune system, had impaired TLR9 reactivity, an anti-inflammatory phenotype, and a dysfunctional T cell-priming ability. We conclude that burn injury induced impairments in DC immunobiology resulting in suppression of adaptive immune response. Targeted DC immunotherapies to promote their ability in triggering T cell immunity may represent a strategy to improve immune defenses against infection following burn injury.

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens. Macrophages sample the phagosomal content and orchestrate the innate immune response. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA and is activated by fungal DNA. Here we demonstrate that specific triggering of TLR9 recruitment to the macrophage phagosomal membrane is a conserved feature of fungi of distinct phylogenetic origins, including Candida albicans, Saccharomyces cerevisiae, Malassezia furfur, and Cryptococcus neoformans. The capacity to trigger phagosomal TLR9 recruitment was not affected by a loss of fungal viability or cell wall integrity. TLR9 deficiency has been linked to increased resistance to murine candidiasis and to restriction of fungal growth in vivo. Macrophages lacking TLR9 demonstrate a comparable capacity for phagocytosis and normal phagosomal maturation compared to wild-type macrophages. We now show that TLR9 deficiency increases macrophage tumor necrosis factor alpha (TNF-α) production in response to C. albicans and S. cerevisiae, independent of yeast viability. The increase in TNF-α production was reversible by functional complementation of the TLR9 gene, confirming that TLR9 was responsible for negative modulation of the cytokine response. Consistently, TLR9 deficiency enhanced the macrophage effector response by increasing macrophage nitric oxide production. Moreover, microbicidal activity against C. albicans and S. cerevisiae was more efficient in TLR9 knockout (TLR9KO) macrophages than in wild-type macrophages. In conclusion, our data demonstrate that TLR9 is compartmentalized selectively to fungal phagosomes and negatively modulates macrophage antifungal effector functions. Our data support a model in which orchestration of antifungal innate immunity involves a complex interplay of fungal ligand combinations, host cell machinery rearrangements, and TLR cooperation and antagonism.

Toll-like receptors (TLRs) are major components of the innate immune system that recognize the conserved molecular structures of pathogens (pathogen-associated molecular patterns; PAMPs). TLRs are found in many different cell types, ranging from epithelial to immunocompetent cells. TLR binding triggers the expression of several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators. This results in the activation of both the innate immune response (elevated expression of antiapoptotic proteins, proinflammatory cytokines, and antibacterial proteins), as well as the adaptive immune response (maturation of the dendritic cells, antigen presentation, etc.). In consequence of their ability to enhance the specific and nonspecific immune reactions of an organism, TLR agonists are widely used in the therapy of infectious diseases and, as adjuvants, in the therapy of malignant neoplasia. However, to date, TLRs have had the opposite effects on tumor progression. On the one hand, TLR ligands can suppress tumor growth. On the other hand, TLR agonists can promote the survival of malignant cells and increase their resistance to chemotherapy. The purpose of this review is to summarize the available data on the effects of TLRs and their agonists on tumor progression, as well as the mechanisms underlying the differences in the effects of TLRs on tumor growth.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.