Paracoccidioides brasiliensis lipids modulate macrophage activity via Toll-dependent or independent mechanisms

The macrophages are the first host cells that interact with the fungus Paracoccidioides brasiliensis, but the main mechanisms that regulate this interaction are not well understood. Because the role played by P. brasiliensis lipids in macrophage activation was not previously investigated, we aimed to assess the influence of diverse lipid fractions from P. brasiliensis yeasts in this process. The possible participation of TLR2 and TLR4 signaling was also evaluated using TLR2- and TLR4-defective macrophages. Four lipid-rich fractions were studied as follows: F1, composed by membrane phospholipids and neutral lipids, F2 by glycolipids of short chain, F3a by membrane glycoproteins anchored by glycosylphosphatidylinositol (GPI) groups, and F3b by glycolipids of long chain. All assayed lipid fractions were able to activate peritoneal macrophages and induce nitric oxide (NO) production. Importantly, the F1 and F3a fractions exerted opposite effects in the control of P. brasiliensis uptake and killing, but both fractions inhibited cytokines production. Furthermore, the increased NO production and expression of costimulatory molecules induced by F3a was shown to be TLR2 dependent although F1 used Toll-independent mechanisms. In conclusion, our work suggests that lipid components may play a role in the innate immunity against P. brasiliensis infection using Toll-dependent and independent mechanisms to control macrophage activation.

Differential expression of toll-like receptor mRNAs in recurrent aphthous ulceration

BACKGROUND: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. METHODS: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. RESULTS: The analysis of mRNA expression levels in oral lesion showed significant (P < 0.01) overexpression of the TLR2(similar to 6-fold) gene and decreased expression of the TLR3 (similar to 5-fold) and TLR5 (similar to 6-fold) genes in comparison with healthy oral mucosa. The analysis of mRNA expression in PBMC indicated a down-regulation of TLR5 gene expression in the cells from RAU patients (P < 0.05; similar to 2-fold). CONCLUSION: Our results support the hypothesis that a subset of RAU patients has fewer TLR expression that have been tentatively implicated in antiinflammatory effects. This derangement of TLR gene expression may cause an overlay exuberant inflammation reaction in situations where normal individuals are resistant. J Oral Pathol Med (2012) 41: 8085

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Toll-like receptor 9 activation: a novel mechanism linking placenta-derived mitochondrial DNA and vascular dysfunction in pre-eclampsia

Emerging evidence suggests that in addition to being the 'power houses' of our cells, mitochondria facilitate effector responses of the immune system. Cell death and injury result in the release of mtDNA (mitochondrial DNA) that acts via TLR9 (Toll-like receptor 9), a pattern recognition receptor of the immune system which detects bacterial and viral DNA but not vertebrate DNA. The ability of mtDNA to activate TLR9 in a similar fashion to bacterial DNA stems from evolutionarily conserved similarities between bacteria and mitochondria. mtDNA may be the trigger of systemic inflammation in pathologies associated with abnormal cell death. PE (pre-eclampsia) is a hypertensive disorder of pregnancy with devastating maternal and fetal consequences. The aetiology of PE is unknown and removal of the placenta is the only effective cure. Placentas from women with PE show exaggerated necrosis of trophoblast cells, and circulating levels of mtDNA are higher in pregnancies with PE. Accordingly, we propose the hypothesis that exaggerated necrosis of trophoblast cells results in the release of mtDNA, which stimulates TLR9 to mount an immune response and to produce systemic maternal inflammation and vascular dysfunction that lead to hypertension and IUGR (intra-uterine growth restriction). The proposed hypothesis implicates mtDNA in the development of PE via activation of the immune system and may have important preventative and therapeutic implications, because circulating mtDNA may be potential markers of early detection of PE, and anti-TLR9 treatments may be promising in the management of the disease.

Toll-like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rats

Activation of TLRs (Toll-like receptors) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of CVDs (cardiovascular diseases). Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study, we hypothesize that inhibition of TLR4 decreases BP (blood pressure) and improves vascular contractility in resistance arteries from SHR (spontaneously hypertensive rats). TLR4 protein expression in mesenteric resistance arteries was higher in 15-week-old SHR than in age-matched Wistar controls or in 5-week-old SHR. To decrease the activation of TLR4, 15-week-old SHR and Wistar rats were treated with anti-TLR4 (anti-TLR4 antibody) or non-specific IgG control antibody for 15 days (1 mu g per day, intraperitoneal). Treatment with anti-TLR4 decreased MAP (mean arterial pressure) as well as TLR4 protein expression in mesenteric resistance arteries and IL-6 (interleukin 6) serum levels from SHR when compared with SHR treated with IgG. No changes in these parameters were found in treated Wistar control rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to NA (noradrenaline) compared with IgG-treated SHR. Inhibition of COX (cyclo-oxygenase)-1 and COX-2, enzymes related to inflammatory pathways, decreased NA responses only in mesenteric resistance arteries of SHR treated with IgG. COX-2 expression and TXA(2) (thromboxane A(2)) release were decreased in SHR treated with anti-TLR4 compared with IgG-treated SHR. Our results suggest that TLR4 activation contributes to increased BP, low-grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

Toll-like receptor 9 activation in neutrophils impairs chemotaxis and reduces sepsis outcome

Objectives: To investigate the role of toll-like receptor 9 on sepsis-induced failure of neutrophil recruitment to the site of infection. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Model of polymicrobial sepsis induced by cecal ligation and puncture in wild-type and toll-like receptor 9-deficient mice. Measurements and Main Results: Toll-like receptor 9-deficient mice with cecal ligation and puncture-induced severe sepsis did not demonstrate failure of neutrophil migration and consequently had a low systemic inflammatory response and a high survival rate. Upon investigating the mechanism by which toll-like receptor 9-deficiency prevents the failure of neutrophil migration, it was found that neutrophils derived from toll-like receptor 9-deficient mice with cecal ligation and puncture induced severe sepsis expressed high levels of chemokine C-X-C motif receptor 2 (CXCR2) and had reduced induction of G-protein-coupled receptor kinase 2. Conclusions: These findings suggest that the poor outcome of severe sepsis is associated with toll-like receptor 9 activation in neutrophils, which triggers G-protein-coupled receptor kinase 2 expression and CXCR2 downregulation. These events account for the reduction of neutrophil migration to the site of infection, with consequent spreading of the infection, onset of the systemic inflammatory response, and a decrease in survival. (Crit Care Med 2012; 40:2631-2637)

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma

Background Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

Toll-like Receptor 2 Knockout Mice Showed Increased Periapical Lesion Size and Osteoclast Number

Introduction: The aim of this study was to characterize the formation and progression of experimentally induced periapical lesions in TLR2 knockout (TLR2 KO) mice. Methods: Periapical lesions were induced in molars of 28 wild type (WT) and 27 TLR2 KO mice. After 7, 21, and 42 days, the animals were euthanized, and the mandibles were subjected to histotechnical processing. Hematoxylin-eosin-stained sections were examined under conventional light microscopy for the description of pulpal, apical, and periapical features and under fluorescence microscopy for the determination of the periapical lesion size. The subsequent sections were evaluated by tartrate resistant acid phosphatase histoenzymology (osteoclasts), Brown and Brenn staining (bacteria), and immunohistochemistry (RANK, RANKL, and OPG). Data were analyzed by the Mann-Whitney U and Kruskal-Wallis tests (alpha = 0.05), Results: The WT group showed significant differences (P < .05) in the periapical lesion size and the osteoclast number between 7 and 42 days and between 21 and 42 days. In the TLR2 KO group, significant differences (P < .05) in the periapical lesion size and the osteoclast number were found between 7 days and the other periods. There was a significant difference (P < .05) between the 2 types of animal regarding the periapical lesion size, which was larger in the TLR2 KO animals. No significant differences (P > .05) were found between WT and TLR2 KO mice related to the pulpal, apical, and periapical features; bacteria localization; and immunohistochemical results (except for RANK expression). Conclusions: TLR2 KO animals developed larger periapical lesions with a greater number of osteoclasts, indicating the important role of this receptor in the host's immune and inflammatory response to root canal and periradicular infection. (J Endod 2012;38:803-813)

Toll-like receptor 3: implications for proinflammatory microenvironment in human breast cancer

Under many circumstances, the host constituents that are found in the tumor microenvironment support a malignancy network and provide the cancer cells with advantages in proliferation, invasiveness and metastasis establishment at remote organs. It is known that Toll like receptors (TLRs) are expressed not only on immune cells but also on cancer cells and it has suggested a deleterious role for TLR3 in inflammatory disease. Hypothesizing that altered IFN gamma signaling may be a key mechanism of immune dysfunction common to cancer as well CXCR4 is overexpressed among breast cancer patients, the mRNA expression of TLR3, CXCR4 and IFN gamma in breast cancer tumor tissues was investigated. No statistically significant differences in the expression of CXCR4 mRNA, IFN gamma and TLR3 between healthy and tumor tissues was observed, however, it was verified a positive correlation between mRNA relative expression of TLR3 and CXCR4 (p < 0.001), and mRNA relative expression of TLR3 was significantly increased in breast cancer tumor tissue when compared to healthy mammary gland tissue among patients expressing high IFN gamma (p = 0.001). Since the tumor microenvironment plays important roles in cancer initiation, growth, progression, invasion and metastasis, it is possible to propose that an overexpression of IFN gamma mRNA due to the pro-inflammatory microenvironment can lead to an up-regulation of CXCR4 mRNA and consequently to an increased TLR3 mRNA expression even among nodal negative patients. In the future, a comprehensive study of TLR3, CXCR4 and IFN gamma axis in primary breast tumors and corresponding healthy tissues will be crucial to further understanding of the cancer network.

Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: Participation of TNF-alpha, IL-1 beta and CXCL1/KC

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/Hej mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-alpha, IL-1 beta and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-alpha, IL-1 beta and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis. (C) 2011 Elsevier B.V. All rights reserved.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Electron Interactions with Disulfide Bridges

Because of its electronic properties, sulfur plays a major role in a variety of metabolic processes and, more in general, in the chemistry of life. In particular, S-S bridges between cysteines are present in the amino acid backbone of proteins. Protein disulfur radical anions may decay following different paths through competing intra and intermolecular routes, including bond cleavage, disproportionation, protein-protein cross linking, and electron transfer. Indeed, mass spectrometry ECD (electron capture dissociation massspectroscopy) studies have shown that capture of low-energy (<0.2 eV) electrons by multiply protonated proteins is followed by dissociation of S-S bonds holding two peptide chains together. In view of the importance of organic sulfur chemistry, we report on electron interactions with disulphide bridges. To study these interactions we used as prototypes the molecules dimethyl sulfide [(CH3)2S] and dimethyl disulfide [(H3C)S2(CH3)]. We seek to better understand the electron-induced cleavage of the disulfide bond. To explore dissociative processes we performed electron scattering calculations with the Schwinger Multichannel Method with pseudopotentials (SMCPP), recently parallelized with OpenMP directives and optimized with subroutines for linear algebra (BLAS) and LAPACK routines. Elastic cross sections obtained for different S-S bond lengths indicate stabilization of the anion formed by electron attachment to a σ*SS antibonding orbital, such that dissociation would be expected.