Tubular expression of Toll-like receptor 9 in lupus and primary membranous nephropathy.

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.

Toll-Like Receptor 9 Expression Levels in Breast Carcinoma Correlate with Improved Overall Survival in Patients Treated with Neoadjuvant Chemotherapy and Could Serve as a Prognostic Marker.

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.

Curcumin reinforces MSC-derived exosomes in attenuating osteoarthritis via modulating the miR-124/NF-kB and miR-143/ROCK1/TLR9 signalling pathways.

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.

All-trans retinoic acid increases NF-κB activity in PMA-stimulated THP-1 cells upon unmethylated CpG challenge by enhancing cell surface TLR9 expression.

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.

TLR9 expression is associated with PD-L1 expression and indicates a poor prognosis in patients with peripheral T-cell lymphomas.

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.

Expression Level of TLR9, but not Hypomethylation, Is Correlated With SLE Disease Activity.

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.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study.

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.

Cell-free synthesis of human toll-like receptor 9 (TLR9): Optimization of synthesis conditions and functional analysis.

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).

Correlation analysis of TLR9 and IL-23 expression in patients with lupus nephritis.

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.

Innate immune response in astrocytes infected with herpes simplex virus 1.

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.

Toll-like receptors 2, 4, and 9 expressions over the entire clinical and immunopathological spectrum of American cutaneous leishmaniasis due to Leishmania(V.) braziliensis and Leishmania (L.) amazonensis.

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-ß 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-ß+ 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.

Dual effects of a CpG-DNAzyme targeting mutant EGFR transcripts in lung cancer cells: TLR9 activation and EGFR downregulation.

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].

RNase 7 Strongly Promotes TLR9-Mediated DNA Sensing by Human Plasmacytoid Dendritic Cells.

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.

Toll-like receptor 5 and 7 expression may impact prognosis of HPV-positive oropharyngeal squamous cell carcinoma patients.

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.

TLR9 gene polymorphism -1486T/C (rs187084) is associated with uterine cervical neoplasm in Mexican female population.

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.

Expression of miR-155 associated with Toll-like receptors 3, 7, and 9 transcription in the olfactory bulbs of cattle naturally infected with BHV5.

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.

Protective role of surface Toll-like receptor 9 expressing neutrophils in local inflammation during systemic inflammatory response syndrome in mice.

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.

TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.

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.

The Impact of Circulating Mitochondrial DNA on Cardiomyocyte Apoptosis and Myocardial Injury After TLR4 Activation in Experimental Autoimmune Myocarditis.

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.

High toll-like receptor (TLR) 9 expression is associated with better prognosis in surgically treated pancreatic cancer patients.

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.