Salivary immunity in elderly individuals presented with Candida-related denture stomatitis

Objectives: Elderly individuals with Candida-related denture stomatitis (DS) present with a reduced defence against Candida albicans. This study evaluated levels of antimicrobial mediators in the elderly DS saliva and salivary neutrophils' activation characteristics compared with elderly and young without DS. Methods: Salivary peroxidases (SPO) and elastase activities (ELA), nitric oxide (NO), transforming growth factor beta (TGF-beta), IL-6 and CCL3 production were determined in saliva from elderly with or without DS, and young control individuals. TLR4, CXCR1, CD11b, CD16 and CD32 expression on salivary neutrophils were evaluated. Correlations between number and apoptosis rate of salivary neutrophils, enzymatic activities and cytokine levels were determined. Results: Elderly DS individuals exhibited the lowest SPO and ELA activities. Also, the activity of both enzymes was low in elderly without DS. Although both elderly groups showed higher salivary NO and TGF-beta levels compared to young control groups, elderly DS presented the highest salivary NO, TGF-beta, IL-6 and CCL3 levels. Decreased percentages of salivary TLR4(+) and CD16(+) neutrophils were detected in both elderly groups. Although these damages could influence the establishment and persistence of DS, the highest levels of salivary IL-6 and CCL3 in elderly DS could be preventing more serious complications.

MyD88 Signaling Pathway Is Involved in Renal Fibrosis by Favoring a T(H)2 Immune Response and Activating Alternative M2 Macrophages

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4+ T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (TH2)-prone inflammatory milieu in a MyD88- dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished TH2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10+ and CD206+ CD11bhigh cells, at 7 d after surgery. We evaluated the role of a TH2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF- β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and TH1:TH2 balance, as TH2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Effects of Continuous Erythropoietin Receptor Activator in Sepsis-Induced Acute Kidney Injury and Multi-Organ Dysfunction

Background: Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI. Methods: Rats were randomized into three groups: control; CLP; and CLP+CERA (5 mu g/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting-to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-kappa B)-and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1 beta, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection. Results: Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-kappa B was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats. Conclusion: CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

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.

Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57BL/6 mouse

Background: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. Methods: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce experimental cerebral malaria (ECM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. Results: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from ECM. Nevertheless, infection with parasites of the ANKA Delta pm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. Conclusions: Infection of pregnant C57BL/6 females with K173, NK65 and ANKA Delta pm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.

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 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.

TLR2- and 4-independent immunomodulatory effect of high molecular weight components from Ascaris suum

Components of high molecular-weight (PI) obtained from Ascaris suum extract down-regulate the Th1/Th2-related immune responses induced by ovalbumin (OVA)-immunization in mice. Furthermore, the PI down-modulates the ability of dendritic cells (DCs) to activate T lymphocytes by an IL-10-mediated mechanism. Here, we evaluated the role of toll like receptors 2 and 4 (TLR2 and 4) in the modulatory effect of PI on OVA-specific immune response and the PI interference on DC full activation. An inhibition of OVA-specific cellular and humoral responses were observed in wild type (WT) or in deficient in TLR2 (TLR2(-/-)) or 4 (TLR4(-/-)) mice immunized with OVA plus PI when compared with OVA-immunized mice. Low expression of class II MHC, CD40, CD80 and CD86 molecules was observed in lymph node (LN) cells from WT, TLR2(-/-) or TLR4(-/-) mice immunized with OVA plus PI compared with OVA-primed cells. We also verified that PI was able to modulate the activation of DCs derived from bone marrow of WT, TLR2(-/-) or TLR4(-/-) mice induced in vitro by agonists of TLRs, as observed by a decreased expression of class II MHC and costimulatory molecules and by low secretion of pro-inflammatory cytokines. Its effect was accompanied by IL-10 synthesis. In this sense, the modulatory effect of PI on specific-immune response and DC activation is independent of TLR2 or TLR4.

Phänotypische und funktionelle Eigenschaften humaner neonataler dentritischer Zellen

Humane Neugeborene leiden an einer erhöhten Anfälligkeit für Infektionskrankheiten. Die dendritischen Zellen (DC) werden für die beeinträchtigten neonatalen T-Helfer 1 (Th1) Immunantworten verantwortlich gemacht. Der Hauptaktivator der Differenzierung von Th1 Zellen ist Interleukin-12 (IL-12), ein Zytokin, welches in adulten, nicht aber in neonatalen DC vornehmlich nach Bindung der Toll-like Rezeptoren produziert wird. In der vorliegenden Arbeit wurde der potentielle Einfluss verschiedener TLR-Liganden auf die Initiierung neonataler Th1-Immunantworten untersucht. Einzelne TLR-Liganden induzierten die Reifung adulter DC, während die neonatalen DC erst nach simultaner Stimulierung von TLR3/TLR8 bzw. TLR4/TLR8 Reifungsmarker exprimierten. Der synergistische Effekt kombinierter TLR-Liganden zeigte sich sowohl in der adulten als auch der neonatalen Zytokinproduktion, wobei unterschiedliche Expressionsmuster für IL-1beta, IL-6, IL-8, IL-10, TNFalpha und vor allem IL-27 beobachtet wurden. Besonders IL-12p70 wurde von neonatalen DC ausschließlich nach kombinierter TLR-Stimulation produziert. Darüber hinaus wurde die Fähigkeit aktivierter DC analysiert, autologe T-Zellen zu polarisieren. Interessanterweise konnte beobachtet werden, dass die Überstände kombiniert stimulierter neonataler DC die Interferon-gamma Produktion in neonatalen naiven T-Zellen auslösten. Und somit konnte gezeigt werden, dass neonatale DC naive T-Zellen hinsichtlich einer Th1-Immunantwort polarisieren können. Letztendlich zeigen die Ergebnisse neue Möglichkeiten auf, potente Impfstrategien für Neugeborene zu entwickeln.

Die Synthese von Mucin-Glycopeptid-Vakzinkonjugaten und Adjuvanzien für eine potentielle Krebsimmuntherapie

Die vorliegende Arbeit beschäftigt sich mit der Synthese von unterschiedlichen tumorassoziierten Antigenen, die in Form von glycosylierten Aminosäurebausteinen durch Festphasensynthese in den tandem repeat des epithelialen Mucin MUC1 eingebaut wurden. Zur Erzeugung von selektiven Immunantworten wurden synthetische MUC1-Glycopeptide über einen nicht immunogenen Spacer mit einer Reihe von Immunstimulanzien umgesetzt. Es wurden hierbei zwei Ziele verfolgt. Zum einen sollten mit Hilfe von synthetischen MUC1-Glycopeptiden und Immunstimulanzien cytotoxische T-Zellen ausgebildet werden, die Tumorzellen mit überexprimiertem MUC1 am Wachstum hindern und sie in den programmierten Zelltod treiben. Zum anderen sollte mit Hilfe von TH2-Epitopen eine adaptive humorale B-Zell-vermittelte Immunantwort in Form von Antikörpern ausgelöst werden. Zur Ausbildung von cytotoxischen T-Zellen kamen Vakzinkonjugate zum Einsatz, die aus dem IL-1β(163 – 169), dem TLR2-Agonisten (Pam3Cys-Ser-Lys4) oder dem TLR4-Agonisten (spacergebundenes MPL A) bestehen und jeweils an MUC1-Glycopeptide konjugiert wurden. Darüber hinaus wurde eine neue moderne Syntheseroute für das MPL-A entwickelt, welches als Adjuvans in den verschiedenen Vakzinen, wie Cervarix®, Fendrix® und Stimuvax®, Verwendung findet und die Immunantwort der Vakzinkonstrukte nochmals verstärken sollte. Zur Induktion von Antikörpern kamen die OVA(323 – 339)-, die Tetanus-Toxoid- und die BSA-Konjugate zur Anwendung, an denen MUC1-Glycopeptide angeknüpft wurden.

Effects of TLR agonists on the hypoxia-regulated transcription factor HIF-1alpha and dendritic cell maturation under normoxic conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1alpha in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1alpha using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1alpha stabilization or activity through different mechanisms. Stabilization of HIF-1alpha protein by hypoxia or CoCl(2) did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1alpha controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1alpha in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1alpha under normoxic conditions with physiological roles that differ from those induced by hypoxia.

Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-(beta) and RANTES production

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN- . Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN- induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF- induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN- and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKK -, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN- induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN- . Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKK -mediated induction of IRF3 and concomitant IFN- and RANTES production.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.