TLR10: An Intriguing Toll-Like Receptor with Many Unanswered Questions.

BACKGROUND: Toll-like receptors (TLRs) are one of the first pattern recognition receptors found in the innate immune system. The TLR family has 12 members (TLR1-TLR9, TLR11-TLR13) in mice and 10 members (TLR1-TLR10) in humans, with TLR10 being the latest identified. SUMMARY: Considerable research has been performed on TLRs; however, TLR10 is known as an orphan receptor for the lack of information on its signalling, role, and ligands. Even though there are recent studies pointing towards the potential TLR10 ligands, their function and signalling pathway are yet to be determined. KEY MESSAGES: This review gives an insight into recent findings on TLR10's pro- and anti-inflammatory properties, with the goal of outlining existing results and indicating future research topics on this receptor.

Toll-like receptor 10 has a role in human macrophage response against Streptococcus pneumoniae.

Toll-like receptors (TLRs) are evolutionarily conserved pathogen-associated molecular pattern recognition receptors, and play a critical role in early response against invading pathogens. Even though TLRs have been widely studied, very little is known about the expression and function of TLR10. Till date, neither any data are available on expression of TLR10 in human lungs nor there is any information on function of TLR10 in macrophages. Streptococcus pneumoniae are Gram-positive, alpha-hemolytic, and major causative agent of pneumonia, ear infections, sinus infections, and meningitis. We examined the role of TLR10 in innate immune response to S. pneumoniae infection in U937 cell line-derived human macrophages. We found a significant increase in TLR10 mRNA and protein expression in S. pneumoniae challenged macrophages. TLR10 knockdown resulted in significant reduction of IL-1ß, IL-8, IL-17, and TNF-α but not IL-10 expression in infected macrophages. TLR10 knockdown in macrophages reduced nuclear translocation of NF-κB during S. pneumoniae challenge but did not affect the phagocytosis of the bacteria. Taken together, we report the first data on TLR10's role in macrophage response against S. pneumoniae.

Toll-like receptor 10 expression in B cells is negatively correlated with the progression of primary Sjögren's disease.

Primary Sjögren's Disease (pSjD) is considered a B cell-mediated disease. Toll-like receptor 10 (TLR10) is highly expressed in human B cells, indicating that TLR10 probably plays a vital role in pSjD. We examined TLR10 expression in peripheral B subsets of pSjD patients and analyzed their association with disease activity. We observed that TLR10 expression in total, naïve, memory, and switched memory B cells was significantly increased in low-activity pSjD patients as compared with healthy controls and high-activity patients. TLR10 expression in the above mentioned B subsets (except naïve B) was negatively correlated with serum levels of anti-SSA antibody and BAFF, respectively. Moreover, a higher proportion of high-activity pSjD patients was observed in TLR10 low- than high-expressed patients. Our study concluded that TLR10 expression in CD19+ B and memory B was negatively correlated with pSjD disease activity, suggesting that TLR10 might take part in the progression of pSjD.

Distinctive probiotic features share common TLR2-dependent signalling in intestinal epithelial cells.

The underlying mechanisms of probiotics and postbiotics are not well understood, but it is known that both affect the adaptive and innate immune responses. In addition, there is a growing concept that some probiotic strains have common core mechanisms that provide certain health benefits. Here, we aimed to elucidate the signalization of the probiotic bacterial strains Lactobacillus paragasseri K7, Limosilactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and Lactiplantibacillus plantarum WCFS1. We showed in in vitro experiments that the tested probiotics exhibit common TLR2- and TLR10-dependent downstream signalling cascades involving inhibition of NF-κB signal transduction. Under inflammatory conditions, the probiotics activated phosphatidylinositol 3-kinase (PI3K)/Akt anti-apoptotic pathways and protein kinase C (PKC)-dependent pathways, which led to regulation of the actin cytoskeleton and tight junctions. These pathways contribute to the regeneration of the intestinal epithelium and modulation of the mucosal immune system, which, together with the inhibition of canonical TLR signalling, promote general immune tolerance. With this study we identified shared probiotic mechanisms and were the first to pinpoint the role of anti-inflammatory probiotic signalling through TLR10.

TLR2, TLR4 and TLR10 Shape the Cytokine and Chemokine Release of H. pylori-Infected Human DCs.

Helicobacter pylori (H. pylori) is a stomach pathogen that persistently colonizes the gastric mucosa, often leading to chronic inflammation and gastric pathologies. Although infection with H. pylori is the primary risk factor for gastric cancer, the underlying mechanisms of pathogen persistence and consequential chronic inflammation are still not well understood. Conventional dendritic cells (cDCs), which are among the first immune cells to encounter H. pylori in the gastric lining, and the cytokines and chemokines they secrete, contribute to both acute and chronic inflammation. Therefore, this study aimed to unravel the contributions of specific signaling pathways within human CD1c+ cDCs (cDC2s) to the composition of secreted cytokines and chemokines in H. pylori infection. Here, we show that the type IV secretion system (T4SS) plays only a minor role in H. pylori-induced activation of cDC2s. In contrast, Toll-like receptor 4 (TLR4) signaling drives the secretion of inflammatory mediators, including IL-12 and IL-18, while signaling via TLR10 attenuates the release of IL-1ß and other inflammatory cytokines upon H. pylori infection. The TLR2 pathway significantly blocks the release of CXCL1 and CXCL8, while it promotes the secretion of TNFα and GM-CSF. Taken together, these results highlight how specific TLR-signaling pathways in human cDC2s shape the H. pylori-induced cytokine and chemokine milieu, which plays a pivotal role in the onset of an effective immune response.

Expression and Function of Toll-Like Receptor 10 (TLR10) in Diffuse Large B Cell Lymphoma, Acute Myeloid Leukemia, and Glioma.

BACKGROUND Toll-like receptor (TLR) family members are part of the major pathogen-recognition system for innate immunity. TLR10, the only remaining orphan receptor with an unknown ligand, has been poorly studied in tumors, and its functional and clinical relevance are unclear. MATERIAL AND METHODS We analyzed TLR10 expression data in The Cancer Genome Atlas (TCGA) by established computational approaches (UALCAN, GEPIA, CGGA, and TIMER) and confirmed them by immunohistochemistry analysis. RESULTS Bioinformatics analysis showed that TLR10 was most highly expressed in diffuse large B cell lymphoma (DLBC), acute myeloid leukemia (LAML), and glioblastoma multiforme (GBM) patients. A data-mining study also revealed that TLR10 levels were positively correlated with WHO grade in glioma, and patients with high TLR10 levels showed shorter overall survival (OS) and disease-free survival (DFS) times than patients with low TLR10 levels. TISIDB and TIMER data showed that TLR10 expression was significantly positively correlated with immune infiltrates, especially infiltrating levels of B cells. Importantly, immunohistochemistry analysis revealed that TLR10 expression was a potential biomarker for distinguishing CNS-DLBC (also known as primary central nervous system lymphoma, PCNSL) from GBM. CONCLUSIONS Taken together, these results suggest that TLR10 could serve as a promising theranostic target for patients with glioma and is a potential biomarker for distinguishing PCNSL from GBM.

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.

Expression profile of Toll­like receptors in human breast cancer.

Toll­like receptors (TLRs) are the most widely studied pattern recognition receptors. Mounting evidence suggests an important association between TLRs and the occurrence and development of breast cancer. Thus, targeting these receptors may be a potential strategy for breast cancer treatment. The current study analyzed the data of 1,215 patients with breast cancer obtained from The Cancer Genome Atlas (TCGA) database. It was observed that, in addition to TLR6, TLR7 and TLR8, the expression of the remaining TLRs in breast cancer tissues was lower than that in normal tissues. In addition, TLR3 and TLR9 displayed significantly different expression levels in ER­/PR­negative breast cancer compared with the control tissues, while TLR5 expression was significantly reduced in HER2­enriched breast cancer. Furthermore, TLR10 exhibited lower expression levels in advanced stages of the disease as compared with that observed in earlier stages. Survival analysis revealed that the expression of TLR4 and TLR7 had a significant impact on survival, and higher expression levels suggested worse prognosis. Finally, the expression levels of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 were correlated with those of the inflammatory cytokines interleukin­1ß and tumor necrosis factor­α, while the expression levels of TLR3, TLR7, TLR8 and TLR9 were correlated with those of interferon­ß and C­X­C motif chemokine ligand 10. Taken together, the current study results suggest that TLR expression may serve as a biomarker of cancer pathogenesis and progression, and may provide new insights for the treatment of breast cancer through the regulation and targeting of TLRs.

The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype.

Cellular senescence is a stress response program characterized by a robust cell cycle arrest and the induction of a proinflammatory senescence-associated secretory phenotype (SASP) that is triggered through an unknown mechanism. Here, we show that, during oncogene-induced senescence (OIS), the Toll-like receptor 2 (TLR2) and its partner TLR10 are key mediators of senescence in vitro and in murine models. TLR2 promotes cell cycle arrest by regulating the tumor suppressors p53-p21CIP1, p16INK4a, and p15INK4b and regulates the SASP through the induction of the acute-phase serum amyloids A1 and A2 (A-SAAs) that, in turn, function as the damage-associated molecular patterns (DAMPs) signaling through TLR2 in OIS. Last, we found evidence that the cGAS-STING cytosolic DNA sensing pathway primes TLR2 and A-SAAs expression in OIS. In summary, we report that innate immune sensing of senescence-associated DAMPs by TLR2 controls the SASP and reinforces the cell cycle arrest program in OIS.

IRAK2 and TLR10 confer risk of Hashimoto's disease: a genetic association study based on the Han Chinese population.

Hashimoto's disease (HD) is one of the major clinical subtypes of autoimmune thyroid disease. Both environmental and genetic factors contribute to the pathogenesis of HD. Previous evidence has shown that both IRAK2 and TLR10 are potential candidate susceptibility genes for HD. In this study, a total of 3654 Chinese women, including 973 HD cases and 2681 healthy controls, were recruited. Thirty-three tag single nucleotide polymorphisms (SNPs) in IRAK2 and TLR10 were genotyped. Genetic association analyses at both the single-marker and haplotype levels were performed. Gene-by-gene interaction analyses were also conducted in case-only samples, as well as eQTL analyses for significant SNPs based on data extracted from the GTEx database. We identified that two SNPs, rs165501 (OR = 1.20, P = 0.0008, IRAK2) and rs10004195 (OR = 1.23, P = 0.0001, TLR10), were identified to be significantly associated with HD. Rs10004195 was significantly associated with the gene expression of TLR10 in human pituitary tissues (P = 2.00 × 10-4), while rs165501 was significantly associated with the expression of IRAK2 in human thyroid tissues (P = 3.10 × 10-6). No significant results were obtained in the gene-by-gene interaction analyses. Our findings suggest that both IRAK2 and TLR10 play important roles in the onset and development of HD.

Toll-like receptor 10 (TLR10) exhibits suppressive effects on inflammation of prostate epithelial cells.

Prostate inflammation (PI) is closely related to the development and progression of chronic prostatic diseases: benign prostatic hyperplasia and prostate cancer. Toll-like receptor (TLR) 2 has been reported to be associated with inflammatory diseases, such as infections, autoimmune diseases, and cancers. Meanwhile, TLR10, which can form heterodimers with TLR2, has been considered an orphan receptor without an exact function. The present study therefore aims to examine the effects of TLR2 and TLR10 on PI. Prostate samples and clinical data were obtained from the patients diagnosed with benign prostatic hyperplasia. The inflammatory cell model was established by adding lipopolysaccharide to RWPE-1 cells. Prostate tissues/cells were examined by histological, molecular, and biochemical approaches. Both TLR2 and TLR10 were found to be expressed in prostate tissues and RWPE-1 cells. mRNA/protein expression levels of TLR2 and TLR10 were both positively correlated with prostate tissue inflammatory grades. Lipopolysaccharide-stimulated RWPE-1 cells expressed higher levels of TLR2, TLR10, high mobility group box 1 (HMGB1), phospho-nuclear factor kappa-light-chain-enhancer of activated B-cells P65 (phospho-NF-κB P65), interleukin (IL)-6, and IL-8 than control cells. Moreover, HMGB1, phospho-NF-κB P65, IL-6, and IL-8 were downregulated after TLR2 knockdown and upregulated after TLR10 knockdown in RWPE-1 cells. TLR2 stimulation can activate the inflammatory signaling cascade in prostate epithelial cells. Conversely, TLR10 exhibited suppressive effects on inflammation. With antagonistic functions, both TLR2 and TLR10 were involved in PI. TLR10 could be a novel target in modulating inflammatory signal transduction of prostate epithelial cells.

Differential expression of TLRs 2, 4, 9, iNOS and TNF-α and arginase activity in peripheral blood monocytes from glucantime unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis caused by Leishmania tropica.

BACKGROUND: Detection of the mechanism of host/parasite interactions in unresponsive forms of anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica is helpful for immunotherapy and vaccine development. In the present study, the gene expression of toll-like receptors (TLRs), TNF-α, iNOS and also arginase (ARG) activity in monocytes from Glucantime unresponsive in comparison to responsive patients infected with L. tropica was investigated. METHODS: In this case-control study, patients with unresponsive (n = 10) and responsive (n = 10) ACL were recruited. Gene expression of TLR2, TLR4, TLR9, TNF-α and iNOS was analyzed in L. tropica-exposed monocytes. The level of ARG activity in both isolated promastigotes and the lysates of monocytes was also determined. RESULTS: L. tropica-exposed monocytes represented higher expression of all three TLRs and TNF-α and lower expression of iNOS compared to unexposed ones in both groups of patients. Results revealed a significant down-regulation of TLR2 and TNF-α and up-regulation of TLR9 expression in unresponsive isolates in comparison to responsive ones. Besides, ARG level showed a significant increase in L. tropica-stimulated monocytes and cultured promastigotes from unresponsive isolates versus responsive ones. CONCLUSIONS: The decreased TLR2, TLR4, TNF-α and iNOS and the increased level of TLR9 expression in L. tropica-exposed monocytes from unresponsive isolates and also the increment in ARG activity in their promastigotes and monocytes, might possibly be involved in the severity of the disease and leading to Glucantime unresponsiveness.

Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10.

Toll-like receptor (TLR)-10 remains an orphan receptor without well-characterized ligands or functions. Here, we reveal that TLR10 is predominantly localized to endosomes and binds dsRNA in vitro at endosomal pH, suggesting that dsRNA is a ligand of TLR10. Recognition of dsRNA by TLR10 activates recruitment of myeloid differentiation primary response gene 88 for signal transduction and suppression of interferon regulatory factor-7 dependent type I IFN production. We also demonstrate crosstalk between TLR10 and TLR3, as they compete with each other for dsRNA binding. Our results suggest for the first time that dsRNA is a ligand for TLR10 and propose novel dual functions of TLR10 in regulating IFN signaling: first, recognition of dsRNA as a nucleotide-sensing receptor and second, sequestration of dsRNA from TLR3 to inhibit TLR3 signaling in response to dsRNA stimulation.

A role for TLR10 in obesity and adipose tissue morphology.

Toll like receptors (TLRs) are expressed in adipose tissue and promote adipose tissue inflammation during obesity. Recently, anti-inflammatory properties have been attributed to TLR10 in myeloid cells, the only member of the TLR family with inhibitory activity. In order to assess whether TLR10-induced inhibition of inflammation may be protective during the development of obesity and metabolic abnormalities we used transgenic human TLR10 mice (hTLR10tg) and wild type (WT) controls on a C57B6J background. HFD-feeding enhanced TLR10 expression in the adipose tissue, and HFD-fed hTLR10tg mice displayed reduced adipocyte size, adipose tissue weight, and a trend toward lower plasma insulin levels compared to WT mice. In humans, obese individuals with polymorphisms in the TLR10 gene displayed reduced macrophage infiltration in the adipose tissue accompanied by a trend to lower leptin levels and higher adiponectin levels in plasma. In healthy individuals with the same polymorphisms in the TLR10 gene we did not observe any difference in plasma concentrations of leptin and adiponectin. We conclude that TLR10 impacts adipose tissue morphology in obesity. Larger studies in humans are warranted to assess its potential value as therapeutic target in metabolic syndrome and type 2 diabetes.

Meat and fiber intake and interaction with pattern recognition receptors (TLR1, TLR2, TLR4, and TLR10) in relation to colorectal cancer in a Danish prospective, case-cohort study.

Background: Meat and dietary fiber are associated with increased and decreased risk of colorectal cancer (CRC), respectively. Toll-like receptors (TLRs) regulate the intestinal immune response in a complex interplay between the mucosal epithelium and the microbiota and may therefore be important modulators of diet-induced CRC together with other inflammatory mediators. Objective: Our aim was to investigate the association between functional TLR polymorphisms and risk of CRC and the interaction with dietary factors. Additionally, interactions with previously studied polymorphisms in IL10, IL1B, PTGS2, and NFKB1 were assessed in order to examine possible biological pathways in meat-induced CRC. Design: A nested case-cohort study of 897 CRC cases and 1689 randomly selected participants from the Danish prospective "Diet, Cancer and Health" study encompassing 57,053 persons was performed using Cox proportional hazard models and the likelihood ratio test. Results: We found associations between polymorphisms in TLR2 (P = 0.018) and TLR4 (P = 0.044) and risk of CRC per se, interactions between intake of red and processed meat (10 g/d) and polymorphisms in TLR1 (P-interaction = 0.032) and TLR10 (P-interaction = 0.026 and 0.036), and intake of cereals (50 g/d) and TLR4 (P-interaction = 0.044) in relation to risk of CRC. Intake of red and processed meat also interacted with combinations of polymorphisms in TLR1 and TLR10 and polymorphisms in NFKB1, IL10, IL1B, and PTGS2 (P-interaction; TLR1/rs4833095 × PTGS2/rs20417 = 0.021, TLR10/rs11096955 × IL10/rs3024505 = 0.047, TLR10/rs11096955 × PTGS2/rs20417 = 0.017, TLR10/rs4129009 × NFKB1/rs28362491 = 0.027, TLR10/rs4129009 × IL1B/rs4848306 = 0.020, TLR10/rs4129009 × IL1B/rs1143623 = 0.021, TLR10/rs4129009 × PTGS2/rs20417 = 0.027), whereas intake of dietary fiber (10 g/d) interacted with combinations of polymorphisms in TLR4, IL10, and PTGS2 (P-interaction; TLR4/rs1554973 × IL10/rs3024505 = 0.0012, TLR4/rs1554973 × PTGS2/rs20417 = 0.0041, TLR4/rs1554973 × PTGS2/rs5275 = 0.0064). Conclusions: Our study suggests that meat intake may activate TLRs at the epithelial surface, leading to CRC via inflammation by nuclear transcription factor-κB-initiated transcription of inflammatory genes, whereas intake of fiber may protect against CRC via TLR4-mediated secretion of interleukin-10 and cyclooxygenase-2. Our results should be replicated in other prospective cohorts with well-characterized participants. The trial was registered at www.clinicaltrials.gov as NCT03250637.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress.

BACKGROUND/AIMS: Metabolic diseases such as obesity and type-2 diabetes (T2D) are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR) such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. METHODS: TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC), or primary monocytes were treated with hydrogen peroxide (H2O2) for induction of reactive oxygen species (ROS)-mediated oxidative stress. Superoxide dismutase (SOD) activity was measured using a commercial kit. Data (mean±SEM) were compared using unpaired student's t-test and P<0.05 was considered significant. RESULTS: The adipose tissue TLR10 gene/protein expression was found to be significantly upregulated in obesity as well as T2D which correlated with body mass index (BMI). ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB)/mitogen activated protein kinase (MAPK) signaling as well as endoplasmic reticulum (ER) stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. CONCLUSION: Oxidative stress induces the expression of TLR10 which may represent an immune marker for metabolic inflammation.

Increased Expression of TLR10 in B Cell Subsets Correlates with Disease Activity in Rheumatoid Arthritis.

Toll-like receptor (TLR) 10, mainly expressed on B cells, has emerged as a modulatory receptor in inflammation. Nonetheless, the clinical significance of TLR10 in rheumatoid arthritis (RA) remains unclear. In this study, we explored the expression of TLR10 in B cells and B cell subsets in RA subjects and healthy controls (HCs) and determined its relevance to disease activity and inflammatory biomarkers. TLR10 levels in B cells and B cell subsets (CD19+CD27+, CD19+CD27-, CD27+IgD-, CD27+IgD+, CD27-IgD+, D27-IgD-, CD19+CD5+, and CD19+CD5-) and inflammatory biomarker concentrations in peripheral blood (PB) obtained from RA subjects and HCs were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The correlations of TLR10 expression with disease activity and inflammatory biomarkers were then analysed. Similar levels of TLR10 in all CD19+ B cells were observed in the RA subjects and HCs. Compared to that in the HCs, TLR10 was elevated significantly in the CD19+CD27-IgD- and CD19+CD5+ subsets in the RA subjects. In addition, almost all subsets expressing TLR10 were increased with disease activity. The present study reveals that enhanced TLR10 in B cell subsets is positively correlated with disease activity in RA subjects.

Differential Expression of Toll-Like Receptor Signaling Pathway Is Associated with Microscopic Polyangiitis in Peripheral Blood Neutrophils.

Constitutive or excessive activation of Toll-like receptor (TLR) signaling pathway can disrupt the body's immune tolerance to autoantigen, thus promoting the development of autoimmune disease. However, the expression profile of TLR signaling pathway in peripheral blood neutrophils in the pathogenesis of microscopic polyangiitis (MPA) remains unclear. Thus, improved understanding of the pathobiology of this disease may aid in the development of therapeutic targets for patients with MPA. In the present study, we assessed the expression of TLR signaling pathway-related genes in peripheral blood neutrophils in patients with MPA. PCR array analysis was performed on 20 patients with MPA and 12 healthy controls. Gene expression profile was performed using the human TLR for autoimmunity and inflammation PCR array of Genecopoeia, containing 84 genes related to TLR signaling pathway and six house-keeping genes. We then used quantitative real-time PCR to validate the array test. The array results identified 13 upregulated genes and 5 genes which were downregulated. The resulting qRT-PCR was consistent with the findings by PCR array. Our results suggest that peripheral blood neutrophils display changes in the expression of TLR signaling pathway-related genes associated with the pathogenesis of microscopic polyangiitis.

Listeria monocytogenes Inhibits Serotonin Transporter in Human Intestinal Caco-2 Cells.

Listeria monocytogenes is a Gram-positive bacterium that can cause a serious infection. Intestinal microorganisms have been demonstrated to contribute to intestinal physiology not only through immunological responses but also by modulating the intestinal serotonergic system. Serotonin (5-HT) is a neuromodulator that is synthesized in the intestinal epithelium and regulates the whole intestinal physiology. The serotonin transporter (SERT), located in enterocytes, controls intestinal 5-HT availability and therefore serotonin's effects. Infections caused by L. monocytogenes are well described as being due to the invasion of intestinal epithelial cells; however, the effect of L. monocytogenes on the intestinal epithelium remains unknown. The main aim of this work, therefore, was to study the effect of L. monocytogenes on SERT. Caco2/TC7 cell line was used as an enterocyte-like in vitro model, and SERT functional and molecular expression assays were performed. Our results demonstrate that living L. monocytogenes inhibits serotonin uptake by reducing SERT expression at the brush border membrane. However, neither inactivated L. monocytogenes nor soluble metabolites were able to affect SERT. The results also demonstrate that L. monocytogenes yields TLR2 and TLR10 transcriptional changes in intestinal epithelial cells and suggest that TLR10 is potentially involved in the inhibitory effect observed on SERT. Therefore, L. monocytogenes, through TLR10-mediated SERT inhibition, may induce increased intestinal serotonin availability and potentially contributing to intestinal physiological changes and the initiation of the inflammatory response.

Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression.

Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1ß), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C-C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1ß, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.