Toll-like receptor expression in C3H/HeN and C3H/HeJ mice during Salmonella enterica serovar Typhimurium infection

Here, we have investigated the mRNA expression of Toll-like receptor 2 (TLR-2), TLR-4, and MD-2 in spleens and livers of C3H/HeN mice (carrying wild-type TLR-4) and C3H/HeJ mice (carrying mutated TLR-4) in response to Salmonella infection. During Salmonella infections, TLR-4 is activated, leading to increased TLR-2 and decreased TLR-4 expression

PENGARUH SUPLEMENTASI ISOFLAVON KEDELAI TERHADAP WANITA PENDERITA AKNE VULGARIS : Kajian Jumlah Lesi, Dihydrotestosterone, Toll-Like Receptor-2, dan Interleukin-8

Latar belakang : Akne vulgaris (AV) merupakan penyakit inflamasi kulit yang paling sering dijumpai. Isoflavon kedelai telah terbukti sebagai antiandrogen dan antiinflamasi. Tujuan penelitian ini membuktikan pengaruh isoflavon kedelai terhadap lesi AV akibat penurunan kadar dihydrotestosterone (DHT), Toll-like receptor-2 (TLR-2), Interleukin-8 (IL-8) pada wanita penderita AV. Metoda : Randomized pre and post test control design. Penelitian pendahuluan dengan besar sampel 25 orang, dirandomisasi dalam kelompok plasebo, isoflavon 40 mg, 80 mg, 120 mg, 160 mg, lama penelitian 4 minggu. Penelitian lanjutan dengan 40 sampel, dirandomisasi dalam kelompok kontrol dan perlakuan, lama penelitian 12 minggu. Variabel bebas isoflavon kedelai, varibel terikat lesi AV dan variabel antara adalah DHT, TLR-2, dan IL-8. Hasil : Lesi AV pada awal penelitian pendahuluan 100,178,92 dan akhir penelitian 78,945,85 terdapat penurunan bermakna (t:2,525)(p:0,019). Delta kelompok isoflavon 160 mg lebih besar dari plasebo, isoflavon 40 mg, 80 mg, dan 120 mg (Z:-2,611)(p: 0,009). Lesi AV pada awal penelitian lanjutan 97,8547,614 dan akhir penelitian 59,3845,549, terdapat penurunan yang bermakna (Z:-4,300)(p:0,000). DHT awal penelitian 307,6150,38 pg/ml dan akhir penelitian 283,5253,13 pg/ml, terdapat penurunan yang bermakna (Z:-2,204)(p:0,027). TLR-2 awal penelitian 4539,82862 pg/ml dan akhir penelitian 3944,63592,42 pg/ml terdapat penurunan bermakna (Z:-3,624)(p:0,000). IL-8 awal penelitian 411,3167,137 pg/ml dan akhir penelitian 311,09103,917 pg/ml, terdapat penurunan bermakna (Z:-4,557) (p:0,000). Simpulan : Pemberian suplementasi isoflavon kedelai dengan variasi dosis selama 4 minggu, dapat menurunkan lesi AV dan diperoleh dosis yang paling baik yaitu dosis 160 mg serta pemberian selama 12 minggu menyebabkan penurunan bermakna terhadap lesi AV, DHT, TLR-2, dan IL-8 dibandingkan terapi standar. Kata kunci : Akne vulgaris, isoflavon kedelai, lesi AV, DHT, TLR-2, IL-8. Background. Acne vulgaris (AV) is the most common feature of skin inflammatory diseases. Soy isoflavone has proven as an anti-androgen and an anti-inflammation. The purpose of this research was to prove the effect of soy isoflavone in AV lesion that caused by the decreased level of dihydrotestoreone (DHT), Toll-like receptor-2 (TLR-2), Interleukin-8 (IL-8) in women with AV. Methods. This research used randomized pre and post test control design. The first research had 25 samples, randomized in 5 groups: placebo, isoflavone 40 mg, 80 mg, 120 mg, 160 mg group in 4 weeks length of research. The last research had 40 samples, randomized in controled and threated group in 12 weeks length of research. The independent variables was soy isoflavone, the dependent variables was AV, the confounding variables were DHT, TLR-2, and IL-8. Results. There was a significant degradation (t=2.525; p=0.019) of AV lesion in the early (100.1+78.92) and in the end of the first research (78.9+45.85). Delta of isoflavone 160 mg group more than other groups (Z=-2.61; p=0.009). There was a significant degradation (Z=-4.300; p=0.000) of AV lesion in the early (97.85+47.614) and in the end of the last research (59.38+45.5). DHT level had a signficant decrease (Z=-2.204; p=0.027) from the first research (307.6+150.38 pg/ml) to the last research (283.5+253.13 pg/ml). TLR-2 level had a signficant decrease (Z=-3.624; p=0.000) from the first research (4539.8+2862 pg/ml) to the last research (3944.6+3592.42 pg/ml). IL-8 level had a signficant decrease (Z=-4.557; p=0.000) from the first research (411.31+67.137 pg/ml) to the last research (311.09+103.917 pg/ml). Conclusion. The administration of soy isoflavone with variances of doses in 4 weeks can decrease AV lesion and the best dose was 160 mg, and the administration in 12 weeks can make significant degradation of AV lesion and significant decreased level of DHT, TLR-2, and IL-8 compared by standard therapy. Keywords: Acne vulgaris, soy isoflavone, AV lesion, DHT, TLR-2, IL-

Expression of TLR-2 in hepatocellular carcinoma is associated with tumour proliferation, angiogenesis and Caspase-3 expression

Aims: Unlike other Toll-like receptors (TLRs), the role of toll like receptor 2 (TLR-2) in the pathogenesis of chronic liver disease and hepatocellular carcinoma (HCC) is not well studied. We, therefore, set out to investigate the expression of TLR-2 in different chronic liver disease states along with other markers of cell death, cellular proliferation and tissue vascularisation Methods and results: Immunohistochemistry was performed on liver tissue microarrays comprising hepatitis, cirrhosis and HCC patient samples using antibodies against TLR-2, Ki-67, Caspase-3 and VEGF. This was done in order to characterise receptor expression and translocation, apoptosis, cell proliferation and vascularisation. Cytoplasmic TLR-2 expression was found to be weak in 5/8 normal liver cases, 10/19 hepatitis cases and 8/21 cirrhosis patients. Moderate to strong TLR-2 expression was observed in some cases of hepatitis and cirrhosis. Both, nuclear and cytoplasmic TLR-2 expression was present in HCC with weak intensity in 11/41 cases, and moderate to strong staining in 19/41 cases. Eleven HCC cases were TLR-2 negative. Surprisingly, both cytoplasmic and nuclear TLR-2 expression in HCC were found to significantly correlate with proliferative index (r = 0.24 and 0.37), Caspase-3 expression (r = 0.27 and 0.38) and vascularisation (r = 0.56 and 0.23). Further, nuclear TLR-2 localisation was predominant in HCC, whereas cytoplasmic expression was more prevalent in hepatitis and cirrhosis. Functionally, treatment of HUH7 HCC cells with a TLR-2 agonist induced the expression of cellular proliferation and vascularisation markers CD34 and VEGF. Conclusions: Our results demonstrate a positive correlation between the expression of TLR-2 and other markers of proliferation and vascularisation in HCC which suggests a possible role for TLR-2 in HCC pathogenesi

Toll like receptor 2 and 4 expression in peripheral blood mononuclear cells of multiple sclerosis patients

Background: Multiple sclerosis (MS) is a T cell mediated autoimmune disease with unknown etiology. Appropriate MS therapeutic strategies need thorough understanding of both disease etiology and pathogenesis mechanisms. Ligation of TLR-2 and TLR-4 stimulates the production of several cytokines leading to CNS autoimmunity and neurodegenerative diseases. Objective: To find a relationship between MS disability and TLR-2 and TLR-4 expression on mononuclear cells in the blood of MS patients. Methods: Forty-five new case (NC) MS patients (33 females and 12 males) and 45 age and gender-matched healthy controls (HC) were recruited to the study. PBMCs were prepared and the expressions of TLR-2 and TLR-4 were assessed by flowcytometry technique using appropriate monoclonal antibodies. Results: Our results showed that the expression of TLR-2 and TLR-4 proteins in the patients group was significantly higher than that of healthy controls. TLR-2 but not TLR-4 was correlated with expanded disability status scale (EDSS) scores. Conclusion: High expressions of TLR-2 and TLR-4 may represent a state of innate immune activation in patients with MS. © 2014, Shiraz University of Medical Sciences

Toll-like Receptors 2 and 4 and Their Mutations in Patients with Otitis Media and Middle Ear Effusion

ObjectivesToll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion.MethodsMiddle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis.ResultsAmong the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20±60.94 ng/mL, 21.59±7.96 ng/mL and 11.55±16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile).ConclusionTLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME

Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis

<p>Abstract</p> <p>Background</p> <p>Previously we reported that mice deficient in toll-like receptor 4 (TLR-4) signalling were protected from diet-induced non-alcoholic steatohepatitis (NASH). Another member of the toll-like receptor family, TLR-2, has been shown to play a role in lipid trafficking via uptake of diacylated lipoproteins. However, a role for TLR-2 in NASH has not been elucidated. The objectives of the current study were to examine the influence of dietary fat quality and TLR-2 on NASH pathogenesis.</p> <p>Methods</p> <p>Steatohepatitis was induced in male Db, C57BL/6 and TLR-2^-/-mice by feeding an L-amino acid-defined diet that was deficient in methionine and choline (MCDD). Mice fed the base diet supplemented with methionine and choline (control diet; CD) were used as controls. To determine the role of fat quality, MCDD was enriched with polyunsaturated corn oil (PUFA) or coconut oil that is comprised mostly of saturated fat (SAFA); the total amount of each fat was 112.9 g/kg of diet. After 8 weeks of feeding CD or MCDD, hepatic steatosis, inflammation and necrosis were evaluated in histological sections. Total RNA was extracted from frozen liver samples and mRNA expression of TNFα, collagen α1, IL-10, peroxisome proliferator-activated receptor-γ (PPAR-γ), TLR-4, and CD14, was analyzed via real-time PCR. Protein levels of TLR-2 were analyzed by western blot.</p> <p>Results</p> <p>Panlobular macrovessicular steatosis and diffuse leukocyte infiltration were noted in PUFA-fed Db mice. Histological scores demonstrated significantly less steatosis, inflammation and necrosis in SAFA-fed mice of all mouse strains. However, compared to wild type mice, hepatocellular damage was notably more severe in TLR-2^-/-mice. Consistent with histological findings, mRNA expression of TNFα was elevated by approximately 3-fold in TLR-2^-/-mice; PPAR-γ expression was blunted in this strain compared to wild type. Expression of the matrix protein collagen αI was also significantly higher in TLR-2^-/-mice, indicating a pro-fibrogenic state. Sensitivity to steatohepatitis due to dietary fat or TLR-2 deficiency correlated significantly with alterations in the expression of TLR-4 as well as the co-receptor CD-14.</p> <p>Conclusions</p> <p>Our findings suggest that dietary saturated fat plays a protective role against MCDD-induced steatohepatitis, whereas TLR-2 deficiency exacerbated NASH. The mechanism underlying the response to dietary fat and TLR-2 likely involves altered signalling via the TLR-4 pathway.</p

Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses

Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life

Toll-like receptor (TLR) 2 and TLR4 gene expression in canine heart

Toll-like receptors (TLRs) are archetypal pattern recognition receptors of immediate importance for an efficacious innate immune response. TLRs exhibit marked differential tissue activity and their levels within a discrete cell type can be highly dynamic. Of 13 known mammalian paralogues, three TLRs have been identified in the dog. Although cardiac TLR expression has been reported in other species, this study is the first to present evidence that these innate immune receptors are expressed in the canine heart. Heart tissue samples from all four chambers were collected from healthy dogs immediately after euthanasia and stored at -80°C until analysis. Total RNA was extracted with TRI Regent. Specific primers were designed for amplification of canine TLR2 and TLR4 based on previously reported sequences for these genes. Reverse transcription was performed with M-MLV reverse transcriptase. PCR amplification was performed and PCR products analyzed by agarose gel electrophoresis. Bands were excised from the gel and the DNA isolated and cloned using the TA Cloning® Kit. The correct sequence for each product was verified by nucleotide sequencing. TLR4 expression was detected in the left ventricle and right atrium; TLR2 was detectable at low levels in the right atrium only. Identity of the RT-PCR products was confirmed by sequencing. Our findings show that at least two TLR paralogues- namely TLR2 and TLR4 - are expressed in the canine heart. Additional studies are warranted to determine these immune receptors' potential implication in the development of naturally occurring heart disease in the dog

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-γ secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-γ secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2