The role of Toll-like receptor 10 in modulation of trained immunity.

Toll-like receptor 10 (TLR10) is the only member of the human Toll-like receptor family with an inhibitory function on the induction of innate immune responses and inflammation. However, its role in the modulation of trained immunity (innate immune memory) is unknown. In the present study, we assessed whether TLR10 modulates the induction of trained immunity induced by ß-glucan or bacillus Calmette-Guérin (BCG). Interleukin 10 receptor antagonist production was increased upon activation of TLR10 ex vivo after BCG vaccination, and TLR10 protein expression on monocytes was increased after BCG vaccination, whereas anti-TLR10 antibodies did not significantly modulate ß-glucan or BCG-induced trained immunity in vitro. A known immunomodulatory TLR10 missense single-nucleotide polymorphism (rs11096957) influenced trained immunity responses by ß-glucan or BCG in vitro. However, the in vivo induction of trained immunity by BCG vaccination was not influenced by TLR10 polymorphisms. In conclusion, TLR10 has a limited, non-essential impact on the induction of trained immunity in humans.

TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection.

Toll-like receptors (TLRs) play a crucial role in innate immunity and provide a first line of host defense against invading pathogens. Of the identified human TLRs, TLR10 remains an orphan receptor whose ligands and functions are poorly understood. In the present study, we sought to evaluate the level of TLR10 expression in breast milk (BM) and explore its potential function in the context of HIV-1 infection. We evaluated HIV-1-infected (Nigerian: n = 40) and uninfected (Nigerian: n = 27; Canadian: n = 15) BM samples for TLR expression (i.e., TLR10, TLR2, and TLR1) and report here that HIV-1-infected BM from Nigerian women showed significantly higher levels of TLR10, TLR1, and TLR2 expression. Moreover, the level of TLR10 expression in HIV-1-infected BM was upregulated by over 100-fold compared to that from uninfected control women. In vitro studies using TZMbl cells demonstrated that TLR10 overexpression contributes to higher HIV-1 infection and proviral DNA integration. Conversely, TLR10 inhibition significantly decreased HIV-1 infection. Notably, HIV-1 gp41 was recognized as a TLR10 ligand, leading to the induction of IL-8 and NF-κBα activation. The identification of a TLR10 ligand and its involvement in HIV-1 infection enhances our current understanding of HIV-1 replication and may assist in the development of improved future therapeutic strategies.

Beyond disease susceptibility-Leveraging genome-wide association studies for new insights into complex disease biology.

Genetic studies in complex diseases have been highly successful, but have also been largely one-dimensional: predominantly focusing on the genetic contribution to disease susceptibility. While this is undoubtedly important-indeed it is a pre-requisite for understanding the mechanisms underlying disease development-there are many other important aspects of disease biology that have received comparatively little attention. In this review, I will discuss how existing genetic data can be leveraged to provide new insights into other aspects of disease biology, why such insights could change the way we think about complex disease, and how this could provide opportunities for better therapies and/or facilitate personalised medicine. To do this, I will use the example of Crohn's disease-a chronic form of inflammatory bowel disease that has been one of the main success stories in complex disease genetics. Indeed, thanks to genetic studies, we now have a much more detailed understanding of the processes involved in Crohn's disease development, but still know relatively little about what determines the subsequent disease course (prognosis) and why this differs so considerably between individuals. I will discuss how we came to realise that genetic variation plays an important role in determining disease prognosis and how this has changed the way we think about Crohn's disease genetics. This will illustrate how phenotypic data can be used to leverage new insights from genetic data and will provide a broadly applicable framework that could yield new insights into the biology of multiple diseases.

TLR10 suppresses the activation and differentiation of monocytes with effects on DC-mediated adaptive immune responses.

TLRs are important pattern-recognition receptors involved in the activation of innate immune responses against foreign pathogens. TLR10 is the only TLR family member without a known ligand, signaling pathway, or clear cellular function. Previous work has shown that TLR10 suppresses proinflammatory cytokine production in response to TLR agonists in a mixed human mononuclear cell population. We report that TLR10 is preferentially expressed on monocytes and suppresses proinflammatory cytokine production resulting from either TLR or CD40 stimulation. TLR10 engagement affects both the MAPK and Akt signaling pathways, leading to changes in the transcriptome of isolated human monocytes. Differentiation of monocytes into dendritic cells in the presence of an αTLR10 mAb reduced the expression of maturation markers and the induction of proinflammatory cytokines, again in response to either TLR or CD40 stimulation. Finally, in coculture experiments, TLR10 differentiated dendritic cells exhibited a decreased capacity to activate T cells as measured by IL-2 and IFN-γ production. These data demonstrate that TLR10 is a novel regulator of innate immune responses and of the differentiation of primary human monocytes into effective dendritic cells.

Epistatic effect of TLR-1, -6 and -10 polymorphisms on organic dust-mediated cytokine response.

Exposure to organic dust from agricultural environments is associated with inflammatory respiratory conditions. The putative causal agents in organic dust include viral, microbial and fungal components, which are recognized by the family of Toll-like receptors (TLRs) and drive host innate and adaptive responses. Our aim in this study was to determine whether responsiveness to organic dust among agricultural workers was dependent on polymorphisms in the TLR10-TLR1-TLR6 gene cluster. We stimulated whole blood from 509 agricultural workers with organic dust, triacyl lipopeptide N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4 (Pam3CSK4) and the diacyl-lipopeptide peptidoglycan. Several of the tagging polymorphisms and haplotypes conferred hyper-responsiveness to organic dust with an increase in interleukin-6 (IL-6; P<0.005), but not tumor necrosis factor-α (TNF-α), secretion. We conclude that genetic variation in the TLR10-TLR1-TLR6 gene cluster mediates responsiveness to organic dust, but indicates different signaling pathways for IL-6 and TNF-α. These studies provide new insight into the role of the TLR10-TLR1-TLR6 gene cluster and the innate immune response to organic dust.

TLR10 Is a B Cell Intrinsic Suppressor of Adaptive Immune Responses.

Toll-like receptors play a central role in the initiation of adaptive immune responses with several TLR agonists acting as known B cell mitogens. Despite thousands of publications on TLRs, the function of TLR10 remains unknown. We have found that Ab-mediated engagement of TLR10 on primary human B cells suppresses B cell proliferation, cytokine production, and signal transduction. When challenged with either a T independent or T dependent Ag, TLR10 transgenic mice exhibit diminished Ab responses. Adoptive transfer of splenic B cells into B cell-deficient mice revealed that the suppressive effects on Ag-specific humoral immune responses are entirely B cell intrinsic. Our results demonstrate that TLR10 has a functional role within the B cell lineage that is distinct from that of other TLR family members and may provide a potential therapeutic target for diseases characterized by dysregulated B cell activity.

A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis.

BACKGROUND: Toll-like receptor (TLR) family members are key players in inflammation. TLR10 has been poorly studied in chronic inflammatory disorders, and its clinical relevance in rheumatoid arthritis (RA) is as yet unknown. We aimed at identifying TLR10 variants within all coding regions of the gene in patients with RA as well as studying their functional and clinical significance. METHODS: TLR10 gene variants were studied by performing sequencing of 66 patients with RA and 30 control subjects. A selected variant, I473T, was then analyzed in 1654 patients and 1702 healthy control subjects. The capacity of this TLR10 variant to modify the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) was determined by using a luciferase reporter assay and analyzing the expression of NFkB target genes by quantitative polymerase chain reaction. Differences between groups were analyzed by using the Mann-Whitney U test and the unpaired two-tailed Student's t test. RESULTS: We detected ten missense variants in the TLR10 gene and focused on the I473T substitution based on allele frequencies and the predicted functional impact. I473T variant is not associated with susceptibility to RA, but it significantly correlates with erosive disease in patients seropositive for antibodies to citrullinated protein antigens (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to infliximab treatment as measured by the change in Disease Activity Score in 28 joints (p = 0.012) and by the European League Against Rheumatism criteria (p = 0.049). Functional studies showed that TLR10 reduced activation of the NFkB inflammatory pathway in hematopoietic cells, whereas the I473T variant lacked this inhibitory capacity. Consistently, after exposure to infliximab, cells expressing the I437T variant showed higher NFkB activity than cells carrying wild-type TLR10. CONCLUSIONS: A TLR10 allelic variant, I473T, has impaired NFkB inhibitory activity and is highly associated with disease severity and low response to infliximab in patients with RA.

RNA expression of TLR10 in normal equine tissues.

BACKGROUND: Toll like receptors are one of the major innate immune system pathogen recognition systems. There is little data on the expression of the TLR10 member of this family in the horse. RESULTS: This paper describes the genetic structure of the Equine TLR10 gene and its RNA expression in a range of horse tissues. It describes the phylogenetic analysis of the Equine TLR1,6,10,2 annotations in the horse genome, firmly identifying them in their corresponding gene clades compared to other species and firmly placing the horse gene with other TLR10 genes from odd-toed ungulates. Additional 3' transcript extensions to that annotated for TLR10 in the horse genome have been identified by analysis of RNAseq data. RNA expression of the equine TLR10 gene was highest in peripheral blood mononucleocytes and lymphoid tissue (lymph nodes and spleen), however some expression was detected in all tissues tested (jejunum, caudal mesenteric lymph nodes, bronchial lymph node, spleen, lung, colon, kidney and liver). Additional data on RNAseq expression of all equine TLR genes (1-4 and 6-10) demonstrate higher expression of TLR4 than other equine TLRs in all tissues. CONCLUSION: The equine TLR10 gene displays significant homology to other mammalian TLR10 genes and could be reasonably assumed to have similar fuctions. Its RNA level expression is higher in resting state PBMCs in horses than in other tissues.

[The Study of Altered Immune Status of UCMSC upon the Activation of TLR1/2 Pathway].

OBJECTIVE: To study whether the activation of Toll like receptor 1/2 (TLR1/2) has influence on the immune status of umbilical cord mesenchymal stem cell (UCMSC). METHODS: TLR1/2 agonist Pam3CSK4 was conducted in UCMSC-peripheral blood mononuclear cell (PBMC) co-culture system. Flow cytometry and lactate dehydrogenase (LDH) detection were used to measure the proliferation of PBMC and immune attack of PBMC to UCMSC, respectively. The detection of expressions of co-stimulator and stem cell markers of UCMSC upon TLR1/2 activation was completed by flow cytometry. Real-time PCR was introduced to assay the expression of many immune-related molecules in UCMSC. Cell differentiation staining was conducted to study the change of differentiation ability of UCMSC. RESULTS: Activation of TLR1/2 pathway in UCMSC increased the proliferation of PBMC and attack of PBMC to UCMSC, but without influenced the expressions of co-stimulator and stem cell markers. The results of real-time PCR showed that many immune-related molecules were dramatically induced in UCMSC upon TLR1/2 activation, such as Cinterleukin (IL)-2, IL-6, IL-10, IL-12, interferon-γ (IFN-γ), nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α)]. However, the activation of TLR1/2 had no influence on the differentiation ability of UCMSC. CONCLUSION: Activation of TLR1/2 altered the immune status of UCMSC, including increase the immune attack to UCMSC.

Toll-like receptor 10 expression in chicken, cattle, pig, dog, and rat lungs.

Toll-like receptors (TLRs) are conserved immune receptors that play critical roles in innate immunity and are known as "gate keepers" of immune system. TLR10 is identified as type 1 plasma membrane protein but the identity of its ligand remains unclear. Till date, no data are available on tissue and cell specific expression of TLR10 in normal and inflamed lungs of domestic animal species, and rat, which is commonly used as a model to study human diseases. We characterized a commercially available TLR10 antibody for use in cattle, pig, dog, chicken, and rat. Western blotting of total lung protein extracts from cattle, dog, pig and chicken showed a band of 85-100kDa, which is similar to the molecular weight of TLR10. The immuno-histochemical and immuno-electron microscopic data show TLR10 expression in vascular endothelium and smooth muscles in lungs of control and inflamed animals. Further, we found that expression of TLR10 in bovine neutrophils is altered upon treatment with Escherichia coli lipopolysaccharide. These data show TLR10 expression in the lungs of these mammalian species and that activation of bovine neutrophils alters the expression of TLR10.

Association of 4p14 TLR locus with antibodies to Helicobacter pylori.

A genome-wide association study among Europeans related polymorphisms of the Toll-like receptor (TLR) locus at 4p14 and the Fcγ receptor 2a locus at 1q23.3 to Helicobacter pylori serologic status. We replicated associations of 4p14 but not 1q23.3 with anti-Helicobacter pylori antibodies in 1402 Finnish males. Importantly, our analysis clarified that the phenotype affected by 4p14 is quantitative level of these antibodies rather than association with seropositivity per se. In addition, we annotated variants at 4p14 as expression quantitative trait loci (eQTL) associated with TLR6/10 and FAM114A1. Our findings suggest that 4p14 polymorphisms are linked to host immune response to H. pylori infection but not to its acquisition.

Polymorphisms in TLR1, TLR6 and TLR10 genes and the risk of Graves' disease.

Graves' disease (GD) is postulated to be caused by the combined effects of susceptibility genes and environmental triggers. Toll-like receptors (TLRs) play a role in the activation of innate and adaptive immune responses in mammalians. The aim of this study was to evaluate the potential association of polymorphisms in TLR1, TLR6 and TLR10 genes with GD in Chinese Cantonese population. Seven single nucleotide polymorphisms (i.e. rs4833095 and rs5743565 in TLR1; rs5743808 in TLR6; and rs4504265, rs11466655, rs11096957 and rs10856839 in TLR10) were evaluated in 332 GD patients and 351 unrelated controls from Chinese Cantonese population. SNP rs5743565 in TLR1 conferred a protective effect against GD. The minor allele G of rs5743565 decreased the risk of GD in all cases (odds ratio; ORG vs. A=0.72 (0.58-0.91); p=0.005; ptrend=0.004) and early onset patients (ORG vs. A=0.72 (0.56-0.91); p=0.007; ptrend=0.006). This study provided evidence that genetic variation rs5743565 in TLR1 might be associated with the decreased susceptibility of GD.

Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection.

Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNA-protein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.

Identification of TLR10 as a key mediator of the inflammatory response to Listeria monocytogenes in intestinal epithelial cells and macrophages.

Listeria monocytogenes is a Gram-positive bacterium that can cause septicemia and meningitis. TLRs are central receptors of the innate immune system that drive inflammatory responses to invading microbes such as L. monocytogenes. Although intestinal epithelial cells (IECs) represent the initial point of entry used by L. monocytogenes for infection, the innate immune response to L. monocytogenes in these cells has been poorly characterized to date. The aim of this study was to determine which TLRs are involved in mediating the immune response to L. monocytogenes in IECs. We performed an RNA interference screen of TLRs 1-10 in the HT-29 IEC cell line and observed the most significant reduction in chemokine output following silencing of TLR10. This effect was also observed in the macrophage cell line THP-1. The chemokines CCL20, CCL1, and IL-8 were reduced following knockdown of TLR10. Silencing of TLR10 resulted in increased viability of L. monocytogenes in both HT-29 and THP-1 cells. TLR10 was found to be predominantly expressed intracellularly in epithelia, and activation required viable L. monocytogenes. NF-κB activation was seen to require TLR2 in addition to TLR10. Taken together, these data indicate novel roles for TLR10 in sensing pathogenic infection in both the epithelium and macrophages and have identified L. monocytogenes as a source of ligand for the orphan receptor TLR10.

Association between C13ORF31, NOD2, RIPK2 and TLR10 polymorphisms and urothelial bladder cancer.

Several evidences have been published linking polymorphism in genes involved in chronic or recurrent inflammation with increased tumor risk and progression. Nevertheless the influence of innate immune receptors in urothelial cancer risk and characteristics has not been sufficient explored. We studied the possible association of polymorphisms in genes encoding NOD2, RIPK2, TLR10 and C13ORF31 with the risk, clinical/pathological characteristics and outcomes of urothelial cancer. We have found association between RIPK2 (rs42490) and cancer risk (AA vs AT&TT, p=0042). In addition, we found statistical differences in TLR10 (rs4129009) gen between low and high tumor infiltration stage (p=0.033). NOD2 (rs9302752) and RIPK2 (rs42490) were found to be associated with development of lymph node metastasis (p=0.011 and p=0.015). Importantly we detect association of TLR10 (Log Rank=0.035) and RIPK2 (Log Rank=0040) with overall survival. Multivariate Cox analysis revealed that both SNPs were survival prognosis factor independent of tumor stage and grade. Our results indicate that innate immunity receptors play a role in modulating urothelial cancer risk and progression.

Evolution of the bovine TLR gene family and member associations with Mycobacterium avium subspecies paratuberculosis infection.

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination.

Polymorphisms in the toll-like receptor 2 subfamily and risk of asthma: a case-control analysis in a Chinese population.

BACKGROUND AND OBJECTIVE: Cell activation through toll-like receptors (TLRs) has robust bipolar effects on host immunity and the pathogenesis of asthma. The TLR2 subfamily is a pivotal member of the TLR family. We sought to determine whether mutations in TLR2 subfamily genes affect the risk of asthma. METHODS: A total of 318 asthmatic patients and 352 nonasthmatic controls were recruited. Eight single-nucleotide polymorphisms in TLR2 subfamily genes were detected using GenomeLab SNPstream (Beckman Coulter, Fullerton, California, USA). RESULTS: We found that patients with the TLR2/rs7656411 TT variant homozygote had a significantly reduced risk of asthma when compared with those with the GG wild-type homozygote (adjusted odds ratio [OR], 0.63; 95% confidence interval (CI], 0.41-0.98; P = .036). Furthermore, a positive association was observed between the T allele of rs2381289 in TLR6 and allergic rhinitis in asthma (OR, 1.79; 95% CI, 1.10-2.91; P = .025), while the A allele of rs11466651 in TLRIO was negatively associated with allergic rhinitis (OR, 0.49; 95% CI, 0.26-0.95; P = .046). CONCLUSION: Our results indicate that a genetic variant in the TLR2 subfamily may play a role in susceptibility to asthma.

Molecular regulation of hepatic dendritic cell function and its relation to liver transplant outcome.

Studies on liver interstitial dendritic cells (DC) indicate that the maturation and function of these important antigen-presenting cells may be suppressed by continual exposure to microbial products from the gut, in particular, bacterial lipopolysaccharide. New evidence is emerging for a role of specific intracellular regulators of signal transduction and of cytokines in the hepatic microenvironment, which may contribute to a hyporesponsive state in liver DC. Analysis of signaling molecule expression within DC in liver transplant tissue is likely to uncover its relation to allograft outcome.

Identification of Toll-like receptors in the rat (Rattus norvegicus): messenger RNA expression in the male reproductive tract under conditions of androgen variation.

PROBLEM: Although the majority of Toll-like receptors (TLRs) are reported in many species, some of them are not yet described in the rat. Further, factors that govern Tlr expression in the male reproductive tract have received little attention. We attempt to identify and characterize Tlrs in the rat and determine the expression profile under conditions that affect male reproductive tract gene expression. METHOD OF STUDY: Rat Tlr5, Tlr10, and Tlr11 transcript sequences were submitted to GenBank and in silico characterization carried out using bioinformatics tools. RT-PCR analyses using gene specific primers for rat Tlr1-13 were carried out with RNA isolated from reproductive tract tissues of various experimental groups. RESULTS: Tlr5, Tlr10, and Tlr11 identified in this study share features that are characteristic of the known TLRs. Abundant Tlr expression was observed in the male reproductive tract of adult and developing rats. Further, Tlr expression was also observed in the epididymides of androgen ablated rats. CONCLUSION: Tlr5, Tlr10, and Tlr11 are ubiquitously expressed in the rat. Tlrs seem to be expressed during male reproductive tract development and under conditions of androgen ablation, suggesting the preparedness of the male reproductive tract to detect an infection under all conditions of androgen status.

Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells.

BACKGROUND: The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs) might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs), chemokine receptors (CCRs) and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB) DCs to find a possible explanation for the age-dependent severity of SARS. RESULTS: Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL), but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. CONCLUSION: The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted.