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

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR–to–NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR–expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.IOS-1354935 - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); 1262934 - National Science Foundation (NSF); 2014-BSP - Arnold and Mabel Beckman Foundatio

TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure

Peculiarities of Tlr-2 (Arg753gln) and Tlr-4 (Asp299gly) Polymorphism Prevalence in Patients with Acute Brucelleis and Cardiovascular System Diseases

Currently, there are single data on the relationship between TLR-2 polymorphisms (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms and susceptibility to brucellosis. Therefore, the aim of the study was to determine the frequency of TLR-2 (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms in patients with acute brucellosis with cardiovascular lesions in the Republic of Azerbaijan. Materials and methods: 178 patients with a brucellosis clinic were examined. According to the criteria for inclusion in the study, only 120 people fully met all the criteria, which made up the main group. The control group consisted of 30 healthy individuals. TLR-2 (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms were also determined for all patients in both groups. In order to assess the state of the cardiovascular system, an electrocardiogram was recorded, blood pressure was measured, and an ultrasound scan of the heart was performed for all patients.Results: it was found that 93 patients (77.50±3.13 %) with acute brucellosis had some or other signs of impairment in the work of the cardiovascular system, identified clinically or as a result of functional examination. Among carriers of the Asp / Gly genotype of the TLR-4 gene, an increased risk of brucellosis with CVS diseases was determined (χ2=30.19; p <0.0001; OR=24.29; 95 % CI [5.45 – 108.37]), while the carriage of the homozygous genotype Asp / Asp, by contrast, had a protective effect on the development of brucellosis (OR=0.06, 95 % CI [0.02 – 0.20]). Among the carriers of the Arg / Gln genotype and the Gln / Gln genotype of the TLR-2 gene, a significantly increased risk of brucellosis with CVS diseases was determined (χ2=5.68; p=0.02; OR=3.10; 95 % CI [0.99 – 9.67]) and (OR=2.48; 95 % CI [0.53 – 11.61]), respectively. While the carriage of the homozygous Arg / Arg genotype, by contrast, was rarely seen in patients with brucellosis (OR=0.28, 95 % CI [0.10 – 0.74]).Conclusions: The Asp / Gly genotype of the TLR-4 gene was 12.7 times more frequently detected in patients with acute brucellosis with CVS diseases than in healthy individuals and 9.5 times more often than in patients without CVS diseases (p<0.05). The Arg / Gln genotype of the TLR-2 gene was 2.4 times more frequently detected in patients with acute brucellosis with CVS diseases than in healthy individuals (p<0.05)

Caspase-8 controls the gut response to microbial challenges by Tnf-alpha-dependent and independent pathways

Objectives: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear. Design: We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo. Results: TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α. Conclusions: Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies

Cooperative Stimulation of Dendritic Cells by Cryptococcus neoformans Mannoproteins and CpG Oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.National Institutes of Health (RO1 AI25780, RO1 AI37532, K08 AI 53542

Novel mutations in the toll like receptor genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

Toll like receptors play a central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in TLR1, TLR2 and TLR4 genes may change the PAMP reorganization ability which causes altered responsiveness to the bacterial pathogens. A case control study, performed to assess the association between TLR gene mutations and susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP), revealed novel mutations (TLR1 - Ser150Gly and Val220Met; TLR2 - Phe670Leu) that hindered either PAMP recognition or further downstream TLR pathway activation. A cytokine expression experiments (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the challenged mutant and wild type moDCs (mocyte derived dendritic cells) confirmed the negative impact of these mutations and altered TLR downstream activation. Further In silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. The most critical positions that may alter the pathogen recognition ability of TLR were: the 9th amino acid position in LRR motif (TLR1, LRR10) and 4th residue downstream to LRR domain (exta LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection

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-