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Introduction: Toll-like- receptors (TLR) control important aspects of innate and adaptive immune responses. Renal cells are among the non-immune cells that express (TLR). Therefore, their activation might be implicated in renal tubulo-interstitial injury., Aim: The study aimed to compare TLR9 expression in patients with primary membranous nephropathy (MN) to patients with lupus membranous nephropathy., Methods: Kidney sections from 10 Lupus nephritis (LN) patients and ten patients with primary MN were analyzed by immunohistochemistry using anti-human TLR9 antibody., Results: Results showed that TLR9 expression was weak and exclusively tubular in primary MN patients' biopsies. There was a significant difference between LN patients' biopsies and primary MN patients' biopsies. TLR9 expression was more diffused in LN patients' specimen than in those with primary MN., Conclusion: This study focuses on molecular level pathogenesis of MN. The data suggest that the receptors TLR9 may play role in tubulointerstitial injury in the pathogenesis of LN but not primary membranous nephropathy.

Aim: Toll-like receptor 9 (TLR9) can recognize the DNA fragments released from chemotherapy-treated cancer cells in tumor tissues and induce an inflammatory response. The aim of the present study was to evaluate the survivability benefit of TLR9 expression levels as a potential prognostic marker in breast cancer patients treated with neoadjuvant chemotherapy (NACT). Methods: To study the expression of TLR9 in breast tumor, immunohistochemical (IHC) analyses were performed on two patient cohorts, with NACT ( n  = 19) and without NACT ( n  = 23). To corroborate the findings from the in-house cohort, we also used publicly available datasets including SurvExpress (GSE 20685) and the Kaplan-Meier plotter tool (GSE 16446) to analyze the relationship between the expression of TLR9 and overall survivability for NACT. Results: The IHC analyses of our inhouse cohort demonstrated that TLR9 was expressed in both malignant breast epithelial cancer cells as well as in the adjacent stromal cells. The IHC results also indicated that, the percentage of malignant epithelial cells (54.76%) expressing TLR9 was higher than in the adjacent stromal compartment (11.9%). We also observed an increase in the expression levels of TLR9 in the patients who were given NACT ( p  = 0.0379). Further, the analysis of publicly available datasets demonstrated that elevated TLR9 expression was related to increased overall survival in patients treated with NACT. Conclusions: In this study, we show for the first time that elevated TLR9 tissue expression levels in breast cancer may serve as a prognostic marker for patients treated with NACT and could potentially be used to select the neoadjuvant regime.

An active metabolite of vitamin A, all-trans retinoic acid (ATRA), is known to exert immunomodulatory functions. This study investigates the possible immune potentiating effect of ATRA on NF-κB activity in human monocytic THP-1 cells after exposure to unmethylated CpG DNA ODN2006. We observed that challenge with ODN2006 significantly enhanced the NF-κB activity of PMA-differentiated THP-1 cells. ATRA synergistically enhanced NF-κB activity of cells, in a concentration- and time-dependent manner. The enhanced NF-κB activity of PMA-differentiated THP-1 cells after ODN2006 challenge was dependent on the RAR/RXR pathway. To determine the mechanism involved in increasing in the NF-κB activity of stimulated THP-1 cells, we examined the effects of PMA and ATRA on the expression of TLR9 (a receptor of ODN2006) in THP-1 cells. PMA treatment significantly enhanced both the intracellular and cell surface expression of TLR9, while ATRA alone showed no effect. However, ATRA synergistically enhanced the cell surface TLR9 expression of PMA-differentiated cells. To determine whether the ATRA-enhanced NF-κB activity is due to the enhanced cell surface TLR9 expression, we examined NF-κB activity after treatment with anti-TLR9 blocking antibody. Results revealed that the anti-TLR9 antibody treatment almost completely reverses the ATRA-enhanced NF-κB activity, suggesting that ATRA enhances NF-κB activity through upregulation of the cell surface TLR9 expression in PMA-differentiated and unmethylated CpG challenged THP-1 cells.

Curcumin treatment was reported to delay the progression of OA, but its underlying mechanism remains unclear. In this study, we aimed to investigate the molecular mechanism underlying the role of curcumin in OA treatment. Accordingly, by conducting MTT and flow cytometry assays, we found that the exosomes derived from curcumin-treated MSCs helped to maintain the viability while inhibiting the apoptosis of model OA cells. Additionally, quantitative real-time PCR and Western blot assays showed that the exosomes derived from curcumin-treated MSCs significantly restored the down-regulated miR-143 and miR-124 expression as well as up-regulated NF-kB and ROCK1 expression in OA cells. Mechanistically, curcumin treatment decreased the DNA methylation of miR-143 and miR-124 promoters. In addition, the 3' UTRs of NF-kB and ROCK1 were proven to contain the binding sites for miR-143 and miR-124, respectively. Therefore, the up-regulation of miR-143 and miR-124 in cellular and mouse OA models treated with exosomes remarkably restored the normal expression of NF-kB and ROCK1. Consequently, the progression of OA was attenuated by the exosomes. Our results clarified the molecular mechanism underlying the therapeutic role of MSC-derived exosomes in OA treatment., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Toll-like receptor9 (TLR9), a member of pattern recognition receptors, play an important role in tumor immunologic surveillance. However, the clinical impact of TLR9 and programmed cell death-ligand 1 (PD-L1) in peripheral T-cell lymphomas (PTCL) remains unclear. In this study, we examined the expression of TLR9 and PD-L1 by immunohistochemical staining in patients with PTCL, and evaluated the clinical significance between expression and clinicopathological features. We found that the rates of high expression of TLR9 and PD-L1 on tumor cells were 65.3% and 45.8% in PTCL, respectively. TLR9 expression was associated with PD-L1 expression in PTCL. Moreover, TLR9 expression was associated with gender, ECOG score, Ki-67 expression, while PD-L1 expression was associated with the number of extranodal involvement and platelet count. High expression of either TLR9 or PD-L1 indicated a poor survival rate for patients with PTCL. Multivariate analysis confirmed that high expression of TLR9 and PD-L1 were unfavorable prognostic factors for patients with PTCL. Thus, TLR9 and PD-L1 expression might be important on the point of prognostic markers in PTCL., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

This study aims to investigate the associations of hypomethylation and over expression of the TLR9 gene with systemic lupus erythematosus (SLE). Fifteen SLE patients who were diagnosed and not treated, were selected as cases, and 32 healthy subjects were enrolled as controls. DNA and total RNA of peripheral blood mononuclear cells (PBMCs) were extracted. The methylation status of the promoter region CpG motifs of the TLR9 gene was quantitatively measured using bisulfite sequencing PCR, and the mRNA expression of the TLR9 gene was determined using real-time fluorescent quantitative PCR. The methylation level of the 10 TLR9 CpG motifs of gene did not show difference between cases and controls (P>0.05). By contrast, we observed an abnormal increase of TLT9 mRNA expression in patients (P=9.379×10(-8)), which was significantly correlated with SLEDAI (Systemic Lupus Erythematosus Disease Activity Index) (P=9.018×10(-7)). The change of TLR9 gene expression may play an important role in the pathogenesis of SLE.

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, infects epithelial surfaces and establishes latency in the central nervous system, where astrocytes are a major immune cell type. Here, we report changes that occur in the expression of pathogen recognition receptors, such as Toll-like receptors, DNA and RNA sensors, interferons, and interferon-stimulated genes, when astrocytes are infected with HSV-1 strain F. We observed upregulation of Toll-like receptors 2, 6 and 9, MDA5, and DAI along with an increase in the expression of type I interferons and interferon-stimulated genes such as IFIT1, IFIT3 and RNase L. These genes encode proteins that mediate the antiviral immune response.

The Toll-like receptor family belongs to the group of pathogen recognition receptors which is responsible for the discrimination of self and non-self pathogen-associated molecular patterns (PAMP's). Toll-like receptors play an important role in the innate immunity and defects in protein expression or polymorphism is linked to various diseases such as Systemic Lupus Erythematosus (SLE). The elucidation of the underlying mechanism is crucial for future treatment and therapeutics of toll-like receptor linked diseases. Herein, we report the cell-free synthesis of human Toll-like receptor 9 (hTLR9) using CHO lysate and the continuous exchange cell-free (CECF) synthesis platform. The functionality of this protein was demonstrated by an ELISA binding assay using the ectodomain of TLR9 (TLR9-ECD)., Competing Interests: The authors have declared that no competing interests exist.

Objective: The aim of this study was to investigate the expression levels of toll-like receptor 9 (TLR9) and interleukin-23 (IL-23) in renal tissue and serum of patients with lupus nephritis (LN), and to explore their clinical correlation., Patients and Methods: LN patients and healthy controls were enrolled in the experimental group and the control group, respectively. Blood samples, serum, and peripheral blood mononuclear cells (PBMCs) were collected. Renal lesion tissues and adjacent normal tissues of LN patients were harvested from a renal tissue biopsy. Serum level of IL-23 was detected by enzyme-linked immunosorbent assay (ELISA). Expression of IL-23 in PBMCs was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Meanwhile, TLR9 expression in renal tissues was accessed by immunohistochemistry staining. Subsequently, 24-h protein urine, renal tubular pathological activity index, erythrocyte sedimentation rate (ESR), serum complement C3 level, and blood albumin level of LN patients were recorded. Also, the correlation between TLR9 expression and these pathological indexes was measured by correlation analysis., Results: Serum level of IL-23 in LN patients was significantly higher than that of healthy controls. Similarly, the mRNA and protein expressions of IL-23 in PBMCs of LN patients were markedly higher than those of healthy controls. IL-23 expression was positively correlated with renal tubular pathological activity index of LN patients. Meanwhile, TLR9 was highly expressed in renal tissues of LN patients. Furthermore, TLR9 expression was positively correlated with 24-h protein urine, renal tubular pathological activity index and ESR, whereas negatively correlated with serum complement C3 level and blood albumin level of LN patients., Conclusions: IL-23 is highly expressed in the serum of LN patients, and its expression is closely related to the occurrence of LN. Also, the expression of TLR9 is up-regulated in the tubulointerstitium of LN patients, which is correlated with relevant clinical indexes.

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Plasmacytoid dendritic cells (pDCs) were described to accumulate in the skin of patients with psoriasis and to be recruited into the dermis upon allergen challenge in atopic dermatitis. Activation of pDCs in the skin has been identified as an important initiator of psoriasis development. Ribonuclease (RNase) 7 is one of the major antimicrobial peptides secreted by keratinocytes and is expressed in significantly higher amounts in lesional skin of patients with atopic dermatitis or psoriasis than in healthy individuals. The skin-derived antimicrobial peptides human ß-defensin 2 and LL-37 indirectly stimulate the activity of skin pDCs, but to our knowledge, an immunomodulatory potential of RNase 7 has not yet been reported. We show here that RNase 7 enables human pDCs to recognize self-DNA and promotes their rapid sensing of bacterial DNA. This very fast innate immune response was sufficient to up-regulate the expression of several antiviral IFN-stimulated genes in human peripheral blood mononuclear cells and to inhibit an infection of primary human keratinocytes with herpes simplex virus 1. RNase 7 was a markedly stronger trigger for IFN-α expression in human pDCs than the other antimicrobial peptides. Our data indicate that RNase 7 exhibits potent immunomodulatory functions and supports the efficient recognition of microbial infections by human skin-infiltrating pDCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL.

Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells. [BMB Reports 2018; 51(1): 27-32].

Purpose: The aim of this work was to evaluate the association of single nucleotide polymorphisms in TLR9 (-1486 T/C [rs187084], -1237T/C [rs5743836] and G2848A [rs352140]) with HPV infection, squamous intraepithelial lesions, and uterine cervical neoplasm in a Mexican population. Additionally, the peripheral expression of TLR9 was evaluated to evaluate the differences in the TLR9 expression associated with every genotype in the locus -1486 of the TLR9 gene. The serum concentration of TLR9 was evaluated in a randomly selected subsample., Methods: Genotyping was performed using predesigned 5' endonuc lease assays and the association of the polymorphisms with the diagnosis groups were assessed by performing multinomial regression models. The relative expression of TLR9 in peripheral blood mononuclear cells was evaluated by real-time polymerase chain reaction and the association of the level of TLR9 expression with the diagnosis was evaluated by performing multinomial regression models. The serum concentration of TLR9 was evaluated in a subsample of patients diagnosed with uterine cervical neoplasm by ELISA., Results: The results showed that genotype TT in the -1486 locus of TLR9 was significantly associated with HPV infection (OR = 3.25, 95% CI 1.12-9.46), squamous intraepithelial cervical lesion (OR = 3.76, 95% CI 1.36-10.41), and uterine cervical neoplasm (OR = 5.30, 95% CI 1.81-15.55). Moreover, the highest level of TLR9 expression was significantly associated with a greater risk for developing squamous intraepithelial cervical lesion and uterine cervical neoplasm. The serum TLR9 concentration was higher in patients with uterine cervical cancer than in controls., Conclusion: Our findings indicate that genotype TT in the -1486 locus of the TLR9 gene could comprise a risk genotype for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Further studies with larger samples are needed to evaluate if the peripheral expression of TLR9 could be used as a biomarker of uterine cervical neoplasm progression.

A large subset of oropharyngeal squamous cell carcinomas (OPSCCs) is associated with HPV infection and has better outcome than non-viral-related tumors. Various malignancies also carry a role for TLRs, key activators of inflammation and innate immunity. We examined the expression of TLRs in OPSCC, and their association with HPV status and treatment outcome. TLR 5, 7, 9, and p16 were studied by immunohistochemistry and HPV status was detected with in situ hybridization in 202 tumors of consecutively treated OPSCC patients using tissue microarray method. The relations between TLR expression and HPV status, p16 expression, clinicopathological factors, and survival were analyzed. TLR 5, 7, and 9 expression patterns differed between HPV-positive and -negative tumors, and they were statistically significantly associated with history of smoking, heavy drinking, tumor site, grade, size (T), metastasis (N), and stage. Moreover, in HPV-positive tumors the expression of TLR 5 and 7 correlated with tumor recurrence. After adjustment, among HPV-positive OPSCC patients, high TLR 5 and low TLR 7 expression were associated with poor disease-specific survival. Our results indicate that TLR 5 and 7 may have a role in the prognostication of HPV-positive OPSCC, however, further studies are needed to clarify the comprehensive role of these TLRs in OPSCC.

Clinically, systemic inflammatory response syndrome (SIRS) occurs after serious trauma or sepsis. In sepsis, neutrophils are the major effector cells responsible for eliminating pathogens. However, the role of neutrophils in development of SIRS, especially in local inflammatory area, is controversial. In this study, we established a SIRS mouse model characterized with cytokine-mediated lethal shock by intraperitoneal injection of oligodexynucleotides containing CpG motifs (CpG ODN) in D-galactosamine (D-GalN) sensitized mice based on our previous work and found that abundant neutrophils were rapidly recruited into the peritoneal cavity, where some neutrophils expressed surface TLR9 (sTLR9), defined as sTLR9 + neutrophils. Along with the progression of SIRS, the expression of sTLR9 in sTLR9 + neutrophils isolated from peritoneal lavage cells (PLCs) was declined in accompany with an increase in the level of the inflammatory cytokine TNFα and a decrease in the level of the anti-inflammatory cytokine IL-10 in Ly6G + PLCs. When using CCT ODN, an oligodeoxynucleotide with CCT repeats, which we have previously shown to be capable of rescuing mice from lethal shock, the expression of sTLR9 on neutrophils was significantly elevated. Adoptive therapy using early recruited neutrophil-rich PLCs containing sTLR9 + neutrophils that express high levels of sTLR9, could rescue mice from SIRS. Overall, the data reveal that the early recruited sTLR9 + neutrophils may, at least in the area of local inflammation, play a protective role during SIRS development and provide a method to rescue the patients with severe SIRS via the up-regulation of sTLR9 levels on the surface of neutrophils or via adoptive therapy with protective sub-populations of neutrophils., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus 5 (BHV5) infection of young cattle is frequently associated with fatal neurological disease and, as such, represents an attractive model for studying the pathogenesis of viral-induced meningoencephalitis. Following replication in the nasal mucosa, BHV5 invades the central nervous system (CNS) mainly through the olfactory pathway. The innate immune response triggered by the host face to virus replication through the olfactory route is poorly understood. Recently, an upregulation of conserved pathogen-associated molecular pattern, as Toll-like receptors (TLRs), has been demonstrated in the CNS of BHV5 experimentally infected cows. A new perspective to understand host-pathogen interactions has emerged elucidating microRNAs (miRNAs) network that interact with innate immune response during neurotropic viral infections. In this study, we demonstrated a link between the expression of TLRs 3, 7, and 9 and miR-155 transcription in the olfactory bulbs (OB) of 16 cows suffering from acute BHV5-induced neurological disease. The OBs were analyzed for viral antigens and genome, miR-155 and TLR 3, 7, and 9 expression considering three major regions: olfactory receptor neurons (ORNs), glomerular layer (GL), and mitral cell layer (ML). BHV5 antigens and viral genomes, corresponding to glycol-C gene, were detected in all OBs regions by fluorescent antibody assay (FA) and PCR, respectively. TLR 3, 7, and 9 transcripts were upregulated in ORNs and ML, yet only ORN layers revealed a positive correlation between TLR3 and miR-155 transcription. In ML, miR-155 correlated positively with all TLRs studied. Herein, our results evidence miR-155 transcription in BHV5 infected OB tissue associated to TLRs expression specifically ORNs which may be a new window for further studies.

Background and aims : TLR9 deletion protects against steatohepatitis due to choline-amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods : We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type ( Wt ), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9 -/- , and Tlr9 -/- foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results : Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9 -/- mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9 -/- foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9 +/+ .foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9 -/- mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt Livers from Ath-fed Tlr9 -/- mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt , and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology., Conclusion: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Pancreatic cancer remains one of the deadliest malignancies in the world. Inflammatory response and tumor environment are thought to play a major role in its pathogenesis. Knowledge on TLR expression and impact on patient survival in pancreatic cancer is limited. The study's aim was to clarify the role of different TLRs in pancreatic cancer. TLR2, TLR4, and TLR9 expression was investigated in 65 surgically resected pancreatic ductal adenocarcinoma specimens by immunohistochemistry. The association between TLR expression, clinical parameters, and local inflammatory response to the tumor was assessed using chi-square test. Relation between patient survival and TLR expression was calculated with multivariable Cox regression, adjusted for age, sex, and tumor stage. We found TLR2, TLR4, and TLR9 to be expressed in pancreatic cancer. There was no association between TLR expression and tumor stage, tumor size, lymph node metastasis, or tumor necrosis. Contrary to our initial hypothesis, high cytoplasmic TLR9 expression was associated with longer patient survival, and multivariate analysis identified low TLR9 expression as an independent risk factor for cancer-specific death (HR 3.090, 95% CI 1.673-5.706). The results suggest that high TLR9 expression in pancreatic ductal adenocarcinoma indicates improved prognosis. The prognostic effect of TLR9 might be associated with bacterial exposure, but this needs further evidence.

Background: Toll-like receptor 9 (TLR9) is a key immunotherapy target for glioblastoma (GBM). This study explored the correlation of TLR9 expression with tumor location and survival outcomes in patients with supratentorial GBM., Methods: We retrospectively identified 46 patients with supratentorial GBMs and divided them into those with high TLR9 (TRL9 High ) and low TLR9 (TRL9 Low ) levels. The 2 groups were compared by patients' ages, sex, preoperative Karnofsky Performance Scale (KPS) score, resection extent, tumor location, progression-free survival (PFS), and overall survival (OS)., Results: The TLR9 expression percentages for the GBM specimens were TRL9 High : 72% (33/46) and TRL9 Low : 28% (13/46). The 2 groups showed no differences in patient age (P = 0.147) and sex (χ 2  = 0.002, P = 0.966), preoperative KPS score (χ 2  = 0.033, P = 0.855), or resection extent (χ 2  = 2.405, P = 0.121). Location differed significantly, with 85% (11/13) of TRL9 Low tumors in the left hemisphere and 45% (15/33) of TRL9 High tumors on the same side (χ 2  = 5.82, P = 0.016). The TRL9 Low group had a median PFS of 612 days (range, 77-926 days), significantly longer than the TRL9 High group (355 days; range, 105-861 days; P = 0.042). Median OS of the TRL9 Low group (733 days; range, 163-969 days) was also significantly longer than in the TRL9 High group (396 days; range, 135-1024 days; P = 0.020)., Conclusions: Patients with TRL9 Low supratentorial GBM tend to have longer survival than those with higher TLR9 expression. Such tumors show a location preference for the left hemisphere., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background/aims: Mitochondrial DNA (mtDNA), acting as a newly found 'danger-associated molecular patterns' (DAMPs), is released into circulation upon tissue injury and performs as a considerable activator of inflammation and immune response. However, the role of circulating mtDNA in experimental autoimmune myocarditis (EAM) as well as Toll like receptor4 (TLR4) mediated cardiac inflammation and injury remains unknown., Methods: A model of EAM was established in BALB/c mice by immunization with porcine cardiac myosin. Lipopolysaccharide (LPS) was used to stimulate TLR4 activation in EAM mice and H9C2 cells., Results: LPS stimulation significantly aggravated cardiac inflammation and tissue injury in EAM, as demonstrated by increased myocardium inflammatory cell infiltration, and up-regulated inflammatory cytokines and troponin I(TnI) level in serum. Circulating mtDNA level was increased in EAM and TLR4 activation led to a greater elevation, which may be related to Reactive oxygen species (ROS) stress involved mtDNA damage characterized by reduced mtDNA copy number in myocardium tissue. In addition, the expression of Toll like receptor9 (TLR9), a ligand of mtDNA, was significantly up-regulated in the myocardium of EAM and EAM LPS group; meanwhile, TLR9 inhibition by ODN 2088 caused an inhibited apoptosis in LPS treated H9C2 cells. Moreover, in EAM and EAM LPS group, simultaneously giving ODN 2088 treatment significantly ameliorated cardiac inflammation and tissue injury compared with untreated group., Conclusion: Increased circulating mtDNA combined with upregulated TLR9 expression may corporately play a role in EAM as well as TLR4 activation mediated cardiac inflammation and injury., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

CpG-oligodeoxynucleotide (CpG-ODN) is not only reported to protect against airway hyper responsiveness but is also known as a potent vaccine adjuvant for anti-tumor therapy. Little is known about the effect of CpG-ODN in mice with radiation-induced lung fibrosis (RILF), a common late stage form of tissue damage that occurs after thorax radiotherapy (RT). Here, we evaluated the immunomodulatory effects of CpG-ODN on the development of RILF. Mice were divided into four groups: (1) RT, single dose of 12Gy to the whole thorax; (2) CpG, only intraperitoneal injection of CpG-ODN for total 5 weeks; (3) RT+CpG, irradiation plus CpG-ODN treatment before and after irradiation for total 5 weeks; and (4) control (CTL): No RT or CpG-ODN treatment. In this study, we found that CpG-ODN treatment attenuated lung fibrosis and collagen deposition by increasing the number of M1 macrophagocytes, levels of Type-2 cytokines and TGF-β. CpG-ODN administration up-regulated the expression of TLR9 and STAT1 phosphorylation and reversed the expression of Type-2 immune response key transcription factor GATA-3. Activation of the JAK-STAT1 signaling pathway further enhanced M1 macrophage differentiation and Type-1 cytokine production. This study reveals the mitigating effect of early exposure to CpG-ODN on lung injury caused by irradiation in mice. The potential mechanism of action may be related to enhancement of Type-1 immunity. In conclusion, CpG-ODN may be a potential therapeutic target to treat RILF., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Staphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA., Results: Osteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress., Conclusions: In this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.

Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.

The expression of Toll-like receptor (TLR)-9, a pathogen recognition receptor that recognizes unmethylated CpG sequences in microbial DNA molecules, is linked to the pathogenesis of several lung diseases. TLR9 expression and signaling was investigated in animal and cell models of chronic obstructive pulmonary disease (COPD). We observed enhanced TLR9 expression in mouse lungs following exposure to cigarette smoke. Tlr9(-/-) mice were resistant to cigarette smoke-induced loss of lung function as determined by mean linear intercept, total lung capacity, lung compliance, and tissue elastance analysis. Tlr9 expression also regulated smoke-mediated immune cell recruitment to the lung; apoptosis; expression of granulocyte-colony stimulating factor (G-CSF), the CXCL5 protein, and matrix metalloproteinase-2 (MMP-2); and protein tyrosine phosphatase 1B (PTP1B) activity in the lung. PTP1B, a phosphatase with anti-inflammatory abilities, was identified as binding to TLR9. In vivo delivery of a TLR9 agonist enhanced TLR9 binding to PTP1B, which inactivated PTP1B. Ptp1b(-/-) mice had elevated lung concentrations of G-CSF, CXCL5, and MMP-2, and tissue expression of type-1 interferon following TLR9 agonist administration, compared with wild-type mice. TLR9 responses were further determined in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoker, smoker, and COPD donors, and then cultured at air liquid interface. NHBE cells from smokers and patients with COPD expressed more TLR9 and secreted greater levels of G-CSF, IL-6, CXCL5, IL-1β, and MMP-2 upon TLR9 ligand stimulation compared with cells from nonsmoker donors. Although TLR9 combats infection, our results indicate that TLR9 induction can affect lung function by inactivating PTP1B and upregulating expression of proinflammatory cytokines., (Copyright © 2016 the American Physiological Society.)

Background: Toll-like receptors (TLRs) are pattern-recognizing proteins involved in innate immunity and they seem to regulate both cancer progression and inhibition. In oral cancer, TLR activation has been linked to invasion. To define the role of TLR-2, TLR-4, and TLR-9 in oral tongue squamous cell carcinoma (OTSCC), we studied their expression in vivo in OTSCC tumor samples, as well as in vitro in cell invasion model., Methods: We used immunohistochemistry to compare the expression of TLR-2, TLR-4, and TLR-9 in 21 primary Stage I-II OTSCCs, neck metastases, and recurrent tumors. In addition, we used myoma organotypic invasion assay to evaluate the effect of GIT27 (4,5-dihydro-3-phenyl-5-isoxasoleaceticacid) on the invasion of the HSC-3 OTSCC cell line., Results: TLR-2, TLR-4, and TLR-9 were expressed in most tumors. Nuclear TLR-2 expression occurred more often in primary tumors than in neck metastases or recurrent tumors of the neck, whereas nuclear TLR-4 expression and cytoplasmic TLR-9 expression were higher in primary tumors than in local recurrent tumors. GIT27 did not affect the invasion of HSC-3 OTSCC cells, but a myoma organotypic invasion assay revealed that the expression of TLR-2 and TLR-4 was stronger in deeper-invading cells., Conclusions: TLR-2, TLR-4, and TLR-9 were expressed in primary tumors, neck metastases as well as in recurrent tumors of OTSCC. Thus, these receptors seem to play a role in both the development and progression of tongue carcinoma. These TLRs may also contribute to the invasive potential of OTSCC., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs) and play an important role in the antiviral response. To determine the effect of common carp TLR9 (CcTLR9) overexpression on the expression of down-stream interferon associated immune factors in epithelioma papulosum cyprini (EPC) cells, may provide useful information for the further investigation on the anti-virus effect mediated by TLR9 in fish. In this study, we constructed an overexpression vector, pEGFP-N1-CcTLR9, by cloning the CcTLR9 gene and inserting it into an expression vector pEGFP-N1. Both plasmids DNA of pEGFP-N1 and pEGFP-N1-CcTLR9 were transfected into EPC cells, and the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin mRNA at 0, 6, 12, 24, 48 and 72h post-transfection were determined by real-time quantitative PCR (Q-PCR). Overexpression of the CcTLR9 gene in EPC cells upregulates the expression of IRF3, IRF7, ISG15, Mx1, PKR, and Viperin mRNA, and this was more significant for Viperin, ISG15, and IRF7, and least significant for PKR. Thus, fish TLR9 activates IRF7 signaling to induce I-IFN secretion and the subsequent upregulation of IFN-stimulated genes., (Copyright © 2016. Published by Elsevier B.V.)

Background: Although a number of in vitro studies have shown that methamphetamine (METH) can increase HIV-1 replication in human immune cells, a direct link between METH use and HIV-1 pathogenesis remains to be determined among HIV-1 patients., Methods: According to the status of METH use and HIV-1 infection, we enrolled participants and divided them into four groups: METH+HIV+, METH-HIV+, METH+HIV-, and METH-HIV-. HIV viral loads and HIV-1-related cellular factors were measured and compared among different groups., Results: A total of 60 participants were enrolled into this study, 15 within each group. HIV viral loads in METH+HIV+ group were significantly higher than those in METH-HIV+ group, while CD4+ T cell counts had an inverse trend between the two groups (p<0.05). METH users or HIV-1 infected patients had lower CCR5+, CXCR4+ percentages in CD4+ T cells than METH-HIV- subjects (p<0.01). However, METH use had little effect on CD3 expression in PBMCs and the levels of MIP-1α, MIP-1β and IL-6 in PBMCs or plasma, which were increased by HIV-1 infection with or without METH. TLR-9 and IFN-α levels in PBMCs of METH users with or without HIV infection were higher than non-METH users (p<0.05)., Conclusions: METH use is associated with higher viral loads and lower CD4+ T cell counts in HIV-infected individuals. This finding may be mediated by activation of innate immunity (TLR-9, IFN-α) by METH use., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Synovial infiltration of monocytes is commonly associated with inflammation in rheumatoid arthritis (RA). Toll-like receptors (TLRs) are innate sensors that recognize cell debris and microbial components in host, a process contributing to maintain chronic inflammation in RA. We assessed the expression levels of TLR2 and TLR9 in monocyte subsets of active RA patients and characterized their cytokine profiles in response to synthetic and viral TLR2 and TLR9 agonists, including Epstein-Barr virus (EBV) which is suspected to contribute to RA symptoms., Methods: Prevalence of monocyte subsets CD14(++) CD16(-), CD14(+) CD16(+) and CD14(low) CD16(++) was evaluated in blood and synovial fluids of active RA patients and levels of TLR2 and TLR9 in monocyte subsets were measured by flow cytometry. Enriched monocytes derived from RA patients and healthy donors were stimulated in vitro with synthetic TLR2 and TLR9 agonists and with EBV particles or viral DNA. Intracellular cytokine profiles were determined in respective monocyte subsets. Finally, the presence of EBV genome was evaluated by real-time PCR in blood and synovial monocytes of RA patients., Results: Numbers of CD14(+) CD16(+) and CD14(low) CD16(++) were found to increase in blood of RA patients compared to healthy controls, while all three subsets were detected in synovial fluids. TLR2 is abundantly expressed on blood and synovial CD14(++) CD16(-) and CD14(+) CD16(+) monocytes from RA patients. Levels of TLR9 were increased on all three subsets of blood monocytes but markedly enhanced in monocytes isolated from synovial fluids. Compared to healthy controls, CD14(++) CD16(-) monocytes of RA patients displayed an enlarged capacity to produce proinflammatory cytokines after stimulation with synthetic TLR2 and TLR9 agonists while both CD14(++) CD16(-) and CD14(+) CD16(+) monocytes showed increased response to EBV stimulation. The presence of EBV genome was also detected in monocytes and neutrophils of a significant proportion of patients., Conclusion: Patients with active RA show an increased expression of TLR2 and TLR9 on monocyte subsets and display higher production of inflammatory cytokines in response to TLR agonists. The presence of EBV genome in monocytes and neutrophils reinforces the suspected role of the virus in the exacerbation of RA symptoms.

Objectives: There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown., Design, Setting, and Patients: We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls., Interventions: Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I., Measurements and Main Results: Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p < 0.0001). Toll-like receptor 2 expression was unchanged. Neutrophil phagocytic activity was decreased, and spontaneous oxidative burst increased in all patients with acetaminophen-induced acute liver failure compared with healthy controls (p < 0.0001). Neutrophil Toll-like receptor 9 expression correlated with plasma interleukin-8 and peak ammonia concentration (r = 0.6; p < 0.05) and increased with severity of hepatic encephalopathy (grade 0-2 vs 3/4) and systemic inflammatory response syndrome score (0-1 vs 2-4) (p < 0.05). Those patients with advanced hepatic encephalopathy (grade 3/4) or high systemic inflammatory response syndrome score (2-4) on day 1 had higher neutrophil Toll-like receptor 9 expression, arterial ammonia concentration, and plasma interleukin-8 associated with neutrophil exhaustion. Healthy neutrophil Toll-like receptor 9 expression increased upon stimulation with acetaminophen-induced acute liver failure plasma, which was abrogated by preincubation with deoxyribonuclease-I. Intracellular Toll-like receptor 9 was induced by costimulation with interleukin-8 and ammonia., Conclusion: These data point to neutrophil Toll-like receptor 9 expression in acetaminophen-induced acute liver failure being mediated both by circulating endogenous DNA as well as ammonia and interleukin-8 in a synergistic manner inducing systemic inflammation, neutrophil exhaustion, and exacerbating hepatic encephalopathy.

Objectives: Despite underlying pathologies leading to ICU admittance are heterogeneous, many patients develop a systemic inflammatory response syndrome often in the absence of microbial pathogens. Mitochondrial DNA that shows similarities to bacterial DNA may be released after tissue damage and activates the innate immune system by binding to toll-like receptor-9 on immune cells. The aim of this study was to analyze whether levels of mitochondrial DNA are associated with 30-day survival and whether this predictive value is modified by the expression of its receptor toll-like receptor-9., Design: Single-center, prospective, observational study., Setting: A tertiary ICU in a university hospital., Patients: Two hundred twenty-eight consecutive patients admitted to a medical ICU between August 2012 and August 2013., Interventions: None., Measurements and Main Results: Blood was taken within 24 hours after ICU admission, and the levels of circulating mitochondrial DNA were quantified by real-time polymerase chain reaction. Toll-like receptor-9 expression in monocytes was measured by flow cytometry. Median acute physiology and chronic health evaluation II score was 20, and 30-day mortality was 25%. Median mitochondrial DNA levels at admission were significantly higher in nonsurvivors when compared with survivors (26.9, interquartile range = 11.2-60.6 ng/mL vs 19.7, interquartile range = 9.5-34.8 ng/mL; p < 0.05). Patients with plasma levels of mitochondrial DNA in the highest quartile (mitochondrial DNA > 38.2 ng/mL) had a 2.6-fold higher risk (p < 0.001) of dying, independently of age, gender, diagnosis, and acute physiology and chronic health evaluation II score. Mitochondrial DNA improved the c-statistic of acute physiology and chronic health evaluation II score (p < 0.05) and showed enhancement in individual risk prediction indicated by a net reclassification improvement of 32.3% (p < 0.05). Stratification of patients according to toll-like receptor-9 expression above/below median demonstrated that only patients with high expression of toll-like receptor-9 showed an increased risk associated with increased mitochondrial DNA levels (odds ratio, 2.7; p < 0.01), whereas circulating mitochondrial DNA was not associated with mortality in patients with low toll-like receptor-9 expression (odds ratio, 1.1; p = 0.98)., Conclusions: Circulating levels of mitochondrial DNA at ICU admission predict mortality in critically ill patients. This association was in particular present in patients with elevated toll-like receptor-9 expression.

Aim: To investigate the therapeutic and immunoregulatory effects of 1,25-dihydroxyvitamin D (1,25(OH)D3) on 2,4,6-trinitrobenzenesulfonic acid (TNBS) -induced colitis in rats., Methods: Experimental colitis induced by enema administration of TNBS plus ethanol was treated with 5-aminosalicylic acid (5-ASA) and/or 1,25(OH)D3. Disease activity was measured using the disease activation index (DAI), colon macroscopic damage index (CMDI), histological colonic damage score, and myeloperoxidase (MPO) activity. The expression of toll-like receptor 9 (TLR9) in the colon was determined by reverse transcription-polymerase chain reaction and immunohistochemistry., Results: Rats with TNBS-induced colitis had significantly elevated DAI, CMDI, histological colonic damage score, and MPO activity (all P<0.001) compared to rats without colitis. Treatment with 5-ASA or 1,25(OH)D3 ameliorated colitis by lowering CMDI (P=0.049, P=0.040, respectively), histological colonic damage score (P=0.010, P=0.005, respectively), and MPO activity (P=0.0003, P=0.0013, respectively) compared with the TNBS group. Combined treatment with 5-ASA and 1,25(OH)D3 significantly decreased MPO activity (P=0.003). 1,25(OH)D3 attenuated colitis without causing hypercalcemia or renal insufficiency. TNBS significantly increased the number of TLR9 positive cells compared to control (P<0.010), while 5-ASA, 1,25(OH)D3, and combined treatment with 5-ASA and 1,25(OH)D3 significantly decreased it compared to TNBS group (all P<0.010). In TNBS group a moderate correlation was observed between MPO activity and the number of TLR9-positive cells (r=0.654, P<0.001)., Conclusion: TLR9 expression correlates with the extent of inflammation in TNBS-induced colitis. 1,25(OH)D3 relieves this inflammation possibly by decreasing TLR9 expression.

Background: The pregnant uterine microenvironment is repleted with Toll-like receptors (TLRs), however, their roles of these receptors in establishing tolerance to growing fetus are largely unknown., Result: Decidual TLR1, TLR3, TLR4, TLR8 and TLR9 gene expressions were significantly over-expressed in patients of spontaneous abortion compared with elective abortion with normal pregnancy. In particular, the expression of TLR4 and TLR9 mRNA was considerably higher than that of remaining TLRs. We mimic TLR9 signal with combination of its pathogenic ligand CpG ODN and antagonists ODN in a well-established abortion-prone CBA/J×DBA/2 model. CpG ODN dramatically boosted fetal loss and lowered the proportion of Regulatory cells (Treg cells) in vivo. CpG ODN directly triggered the impaired survival and increased activity of Caspase 8/3 of Treg cells in vitro. These effects were blocked by antagonist ODN., Conclusion: Excessive TLR9 signaling contributed to maternal-fetal tolerance disruption via an effect on Treg cell survival by activation of Caspase 8/3., (Copyright © 2015 Elsevier B.V. All rights reserved.)

TLRs are important molecules of innate immune response, those play central role in host pathogen interaction and recognition through pathogen associated molecular patterns (PAMPs). Previous studies have indicated the role of TLRs in many human malignancy and cervical cancer in terms of viral recognition and inflammatory changes in-vivo. The objective of this study was to evaluate the expression and localization of toll-like receptor (TLR) 2 and TLR9 in preinvasive and invasive cervical cancer patients and to investigate its use as a probable diagnostic tool for better management cervical cancer. This single institution study includes individuals with normal, precancerous lesions, cervical intraepithelial neoplastic (CIN) and invasive squamous cell carcinoma (SCC) of the cervix. Upon confirmation by histopathology, fluorescence based immunohistochemistry was performed in all patients for TLR2 and TLR9, followed by semi-quantitative estimation of the staining intensity and grade of expression. The expression pattern of TLR2 and TLR9 does not vary greatly from normal to precancerous lesions, but a significant variation was observed in advance stages, i.e. squamous cell carcinoma of the uterine cervix. Additionally the expression increased marginally in higher grades. In spite of their low difference in expression along different stages of cervical cancer, both TLR2 and TLR9 could detect the disease at an advance stages as depicted by the receiver operator characteristics curve analysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1), decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation., Importance: The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Toll-like receptor 9 (TLR9) is a cellular receptor, which recognizes bacterial and host-derived DNA. Stimulation of TLR9 induces cellular invasion via matrix metalloproteinase 13 (MMP-13). The aim of this study was to evaluate the role of TLR9 in invasion of oral tongue squamous cell carcinoma (OTSCC)., Methods: The effects of TLR9 ligands on oral squamous cell carcinoma cell lines were studied with invasion and migration assays, as well as in a myoma organotypic model., Results: The TLR9 ligand, CpG-ODN, increased invasion and migration in OTSCC lines. These effects were reduced by TLR9 siRNA or inhibition with TLR9 antibodies. Immunohistochemical analysis of tissues from 195 patients with OTSCC revealed that TLR9 was expressed in 181/195 carcinomas. The expression of TLR9 was higher in the malignant cells than in the normal epithelium. High TLR9 expression was associated with high MMP-13 expression and poor differentiation. High TLR9 expression was also identified as an independent predictor of poor prognosis (HR 1.810, 95% CI [1.053-3.112])., Conclusion: Toll-like receptor 9 mediates OTSCC invasion and migration in vitro and is an independent prognostic factor of OTSCC. Inhibition of TLR9 may be a novel therapeutic opportunity in oral cancer., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors, such as prostate cancers. However, targeting MDSCs proved challenging due to their phenotypic heterogeneity., Experimental Design: Myeloid cell populations were evaluated using flow cytometry on blood samples, functional assays, and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects, localized and metastatic castration-resistant prostate cancer patients., Results: Here, we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3, which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA, thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1, a downstream STAT3 target gene and a potent T-cell inhibitor., Conclusions: Overall, we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression., (©2015 American Association for Cancer Research.)

Acute lung injury is the most serious and fatal complication of the elderly patients with hip fracture, but the mechanisms are unknown. Recent studies demonstrated the mitochondrial DNA (mtDNA) release was associated with lung injury after trauma. This study aimed to examine the differential release of mtDNA between younger and elderly rats suffering from hip fracture and to investigate the possible mechanism of mtDNA in the lung injury induced by hip fracture. In the first part of the study, we investigated the effects of hip fracture on the rats. The elderly and younger rats, respectively, received hip fracture operations. The degree of lung injury was evaluated, toll-like receptor 9 (TLR9) and nuclear factor kappa B (NF-κB) were determined using Western blot, and mtDNA were analyzed by fluorescent quantitative polymerase chain reaction. In the second part of the study, we investigated the effects of mtDNA on the rats. The elderly and younger rats directly received intravenous injections with mtDNA. After 24 h, the specimens were collected and detected as the first part. Hip fracture resulted in significant mtDNA release, TLR9 and NF-κB p65 expression, and lung injury in the rats. Meanwhile, the mtDNA injection could indirectly induce lung injury. Compared to the younger ones, the elderly rats suffered more serious lung injury after hip fracture and mtDNA injection. These results suggest that the lung injury induced by hip fracture may be involved with the mtDNA release and its TLR9/NF-κB pathway.

Guillain-Barré syndrome (GBS) is a post-infectious autoimmune disease. Dendritic cells (DCs) can recognize the pathogen and modulate the host immune response. Exploring the role of DCs in GBS will help our understanding of the disease development. In this study, we aimed to analyze plasmacytoid and conventional DCs in peripheral blood of patients with GBS at different stages of the disease: acute phase as well as early and late recovery phases. There was a significant increase of plasmacytoid DCs in the acute phase (p=0.03 vs healthy donors). There was a positive correlation between percentage of plasmacytoid DCs and the clinical severity of patients with GBS (r=0.61, p<0.001). Quantitative polymerase chain reaction and flow cytometry confirmed the aberrant plasmacytoid DCs in GBS. Thus, plasmacytoid DCs may participate in the development of GBS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Studies indicate that the natural immune-related protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) plays an antitumor role in nasopharyngeal epithelial tissue. However, the detailed mechanism of the tumor-suppressor effect of SPLUNC1 in the inflammatory microenvironment of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) remains elusive. The aim of the present study was to explore how SPLUNC1 reduces the inflammatory response of NPC cells infected with EBV by regulating the Toll-like receptor (TLR)9/NF-κB signaling pathway. As detected by immunohistochemistry and western blotting, SPLUNC1 protein expression exhibited low or negative expression in the NPC epithelial samples/cells, while it demonstrated positive expression in normal nasopharyngeal epithelial tissues/cells; this pattern of expression was the contrary to that of TLR9. The poorly differentiated HNE2 cell line had the highest efficiency of transfer of infection with EBV by 'cell-to-cell' contact method. The group of EBV-infected HNE2 cells showed significantly higher activation of the expression of TLR9/NF-κB signaling pathway-associated factors (TLR9, CD14, MyD88, IKK, P-IKβα, P-NF-κB and NF-κB). The levels of inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α in the HNE2 cell group after EBV infection were higher than these levels in the uninfected cell group (P<0.05); Meanwhile, after EBV infection, the expression levels of TLR9/NF-κB pathway associated-protein and inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α in the HNE2/SPLUNC1 cell group were lower than these levels in the HNE2/Vector cell group (P<0.05). After EBV-DNA direct transfection, cytokine mRNA expression levels of TLR9, IL-6, IL-8, IL-1β and TNF-α in the HNE2 cell group were significantly higher than these levels in the NP69 cell group (P<0.05). The expression levels of these cytokines in the HNE2/SPLUNC1 cell group were obviously lower than these levels in the HNE2/Vector cell group (P<0.05). These results suggest that EBV infection of NPC cells can activate the TLR9/NF-κB signaling pathway, promote the release of inflammatory cytokines and consequently enhance the inflammatory response, while SPLUNC1 can weaken the inflammatory response induced by EBV infection in NPC cells through the regulation of the TLR9/NF-κB signaling pathway and control of the tumor inflammatory microenvironment.

B cells are efficiently activated by CpG oligodeoxynucleotides (ODNs) to produce pro-inflammatory cytokines and antibody (Ab). Here, we describe a so far unidentified, spleen tyrosine kinase (Syk)-dependent pathway, which is indispensable for CpG-induced human B cell activation. We show that triggering of B cells by CpG results in Syk and src kinase phosphorylation, proliferation, as well as cytokine and Ab production independent of the BCR. Notably, all these functions are abrogated when Syk is inhibited. We demonstrate that CpG-induced Syk activation originates from the cell surface in a TLR9-dependent manner. While inhibition of Syk does not influence the uptake of CpG ODNs, activation of the kinase is a prerequisite for the delivery of CpG into TLR9-containing endolysosomes and for the CpG-induced up-regulation of TLR9 expression. Our results reveal an alternative, Syk-dependent pathway of CpG-induced B cell stimulation, which is initiated at the plasma membrane and seems to be an upstream requirement for endosomal TLR9-driven B cell proliferation and differentiation.

Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age ("inflammaging") and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA. Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b(++) neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR(+) neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors. In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: There is no effective medication to date for herpes simplex virus encephalitis (HSE). In this study, we investigated the anti-inflammatory effect of chlorogenic acid (CGA) on herpes simplex virus (HSV)-1-induced responses in BV2 microglia., Main Methods: The cellular model was established with BV2 cells stimulated by HSV-1 and then treated with CGA at different concentrations. Cell viability was assayed by the MTT assay. The mRNA expression of Toll-like receptor (TLR)-2, TLR9 and myeloid differentiation factor88 (Myd88) was assayed by real-time quantitative PCR, and the protein expression was assayed by flow cytometry or Western blotting. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were measured by ELISA as well as real-time quantitative PCR. Nuclear NF-κB p65 protein was assayed by Western blotting., Key Findings: The cell survival rate was significantly improved after CGA treatment, and CGA prevented increases in TLR2, TLR9 and Myd88 following HSV-1 challenge in BV2 cells both at the mRNA and protein levels. Moreover, CGA could attenuate HSV-induced TNF-α and IL-6 release into the supernatant. The mRNA levels of TNF-α and IL-6 were also significantly inhibited by CGA. The expression of NF-κB p65 increased significantly in the nucleus in HSV-1-stimulated microglia but could be reduced by CGA., Significance: CGA inhibits the inflammatory reaction in HSE via the suppression of TLR2/TLR9-Myd88 signaling pathways. CGA may serve as an anti-inflammatory agent and provide a new strategy for treating HSE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aim: We sought to clarify whether the expression of toll-like receptors (TLR) in Merkel cell carcinoma (MCC) is linked to tumor and patient characteristics, especially the presence of Merkel cell polyoma virus (MCV)., Materials and Methods: The study comprised of 128 patients with data on Merkel cell polyomavirus (MCV) status and clinical features were included in the study. Immunohistochemistry for TLR expression was performed on tissue microarray (TMA) slides., Results: TLR 2, 4, 5, 7 and 9 expression was noted in most of the tumor specimens. Decreased expression of TLR 9 correlated strongly with MCV positivity. Cytoplasmic TLR 2 expression correlated with small tumor size, while nuclear TLR 2 and TLR 5 expressions with larger tumors. Increased nuclear TLR 4 expression and decreased TLR 7 expression were associated with older age., Conclusion: TLR 2, 4, 5, 7 and 9 appear to reflect certain clinicopathological variables and prognostic markers of MCC tumors., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Kawasaki disease (KD) is an acute febrile systemic vasculitis of childhood characterized by elevated levels of inflammatory mediators at the acute stage. High-dose intravenous immunoglobulin (IVIG) is well accepted as a conventional therapy for KD. The aim of the present study was to determine the expression level of Toll like receptors (TLRs) and their corresponding signaling mediators in PBMCs of IVIG-treated KD patients. TLR2, 3, 9 and signaling mediators, MyD88 and TRIF transcript levels were determined in PBMCs from 31 KD patients, before (acute phase), 2 weeks later (sub-acute phase) and 6 weeks later (convalescent phase) of IVIG therapy using real time PCR. The mean age of the patients was 3.6 years and 65% of subjects were male and 35% were female. 20 age-matched irrelevant febrile patients and 20 healthy subjects were included as control groups. Elevated levels of TLR2, MyD88, and TRIF gene transcripts were observed in the PBMCs at acute phase of untreated KD patients in compression with normal subjects. IVIG therapy resulted in significant decrease in TLR2, 3 and 9 (60-90%) as well as MyD88 and TRIF (60-70%) transcripts following 2 and 6 weeks. With Regard to significant up-regulation of MyD88 and TRIF at the acute phase of KD, our findings suggest TLR signaling pathway potential in KD pathogenesis and may also support the assumption of an infectious background in KD. Down-regulation of TLR members and corresponding mediators in IVIG treated patient suggest general TLR pathway suppression as a novel anti-inflammatory mechanism of IVIG.

Control of human papillomavirus (HPV) infection involves the activation of Toll-like receptors (TLRs), key components of the mucosal antiviral response. Available studies on TLR expression in HPV-positive cervical cells are limited and reported conflicting results. This study quantified TLR 2, 3, 4, 7 and 9 transcripts in low-risk (LR) and high-risk (HR) HPV-positive and HPV-negative cervical samples from 154 women attending a gynaecological clinic. Expression levels of TLR 2, 3, 4 and 7 did not differ among samples, whereas TLR9 levels were quite significantly higher in LR and marginally significant in HR HPV-positive samples, with respect to the HPV-negative samples. Interestingly, in a subgroup of women with documented previous HPV-infection, TLR9 levels were extremely higher in patients persistently positive to the same HPV genotype for more than 1 year, with respect to women who cleared HPV infection and to those re-infected with a different genotype. These findings implicate TLR9 in the response to LR and HR HPVs, including HPV 16 known to interfere with TLR9 transcription in cell lines. Elevated TLR9 levels without HPV clearance in persistently infected women could drive inflammation thereby contributing to cervical cancer risk., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Inflammatory bowel disease (IBD) is a complex immunological disorder characterized by chronic inflammation caused mainly by unknown factors. The interleukin-10 knockout (IL-10 KO) mouse is a well-established murine model of IBD which develops spontaneous intestinal inflammation that resembles Crohn's disease. In the present study, human adipose-derived mesenchymal stem cells (hAMSCs) were administrated to IL-10 KO mice to evaluate the anti-inflammatory effects of hAMSCs that may attenuate the progress of or treat IBD. After IBD was induced by feeding the IL-10 KO mouse a 125-250 ppm piroxicam mixed diet for 1 week, 2×10(6) hAMSCs were injected into the peritoneum and the mice were switched to a normal diet. After 1 week, the mice were sacrificed and tissue samples were harvested. Tissue scores for inflammation and inflammation-related genes expression were determined. The hAMSC-treated group showed significantly reduced inflammatory changes in histological analysis. Reverse transcription-PCR analysis showed that RANTES, Toll-like receptor 9, and IL-4 expression levels were not significantly different between the groups while IL-12, INF-γ, and TNF-α levels were significantly decreased in the hAMSC treated group. hAMSC attenuated IBD in the IL-10 KO mice by suppressing inflammatory cytokine expression, was mediated by the type 1 helper T cell pathway. Even though only a single injection of hAMSCs was delivered, the effect influenced chronic events associated with inflammatory changes and demonstrated that hAMSCs are a powerful candidate for IBD therapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) family may play important roles in inflammatory bowel disease. This study examined the expression of TLR2, TLR4 and TLR9 in the colonic tissues of patients with ulcerative colitis (UC) and explored their roles in the pathogenesis of UC. Colonic biopsies were taken from the colon of 30 patients with mild or moderate UC (at active phase) and 10 healthy controls during colonoscopy. TLR2, TLR4 and TLR9 protein expression levels were immunohistochemically detected. The mRNA expression levels of TLR2, TLR4 and TLR9 were assessed by reverse transcription polymerase chain reaction (RT-PCR). The disease activity index (DAI), colonoscopic and histologic grades and fecal microbial flora were determined. Histological examination showed that the intestinal mucous membrane of UC patients underwent acute inflammation changes. Immunohistochemistry exhibited that the expression levels of TLR2, TLR4 and TLR9 in colon epithelia and inflammatory cells were higher in UC patients than in control group (P<0.01). The mRNA expression levels of TLR2, TLR4 and TLR9 were increased in UC patients but were not detected in the normal controls. Expression levels of TLR2, TLR4 and TLR9 were positively correlated, and bore close correlation with DAI, colonoscopic and histologic grades and fecal microbial flora. An important mechanism of UC might be that abnormal activation of mucosal immunity by intestinal dysbacteriosis caused dysregulation of TLRS that mediates innate immunity.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.