Recognition of hepatitis C virus RNA by toll like receptors 7 and 8 : implications for the initiation of innate immune response

Le virus de l’hépatite C (VHC) est un virus à ARN simple brin positif (ssARN) qui se replique dans le foie. Deux cents millions de personnes sont infectées par le virus dans le monde et environ 80% d’entre elles progresseront vers un stade chronique de l’infection. Les thérapies anti-virales actuelles comme l’interféron (IFN) ou la ribavirin sont de plus en plus utilisées mais ne sont efficaces que dans la moitié des individus traités et sont souvent accompagnées d’une toxicité ou d’effets secondaires indésirables. Le système immunitaire inné est essentiel au contrôle des infections virales. Les réponses immunitaires innées sont activées suite à la reconnaissance par les Pathogen Recognition Receptors (PRRs), de motifs macromoléculaires dérivés du virus appelés Pathogen-Associated Molecular Patterns (PAMPs). Bien que l'activation du système immunitaire par l'ARN ou les protéines du VHC ait été largement étudiée, très peu de choses sont actuellement connues concernant la détection du virus par le système immunitaire inné. Et même si l’on peut très rapidement déceler des réponses immunes in vivo après infection par le VHC, l’augmentation progressive et continue de la charge virale met en évidence une incapacité du système immunitaire à contrôler l’infection virale. Une meilleure compréhension des mécanismes d’activation du système immunitaire par le VHC semble, par conséquent, essentielle au développement de stratégies antivirales plus efficaces. Dans le présent travail nous montrons, dans un modèle de cellule primaire, que le génome ARN du VHC contient des séquences riches en GU capables de stimuler spécifiquement les récepteurs de type Toll (TLR) 7 et 8. Cette stimulation a pour conséquence la maturation des cellules dendritiques plasmacytoïdes (pDCs), le production d’interféron de type I (IFN) ainsi que l’induction de chémokines et cytokines inflammatoires par les différentes types de cellules présentatrices d’antigènes (APCs). Les cytokines produites après stimulation de monocytes ou de pDCs par ces séquences ssARN virales, inhibent la production du virus de façon dépendante de l’IFN. En revanche, les cytokines produites après stimulation de cellules dendritiques myéloïdes (mDCs) ou de macrophages par ces mêmes séquences n’ont pas d’effet inhibiteur sur la production virale car les séquences ssARN virales n’induisent pas la production d’IFN par ces cellules. Les cytokines produites après stimulation des TLR 7/8 ont également pour effet de diminuer, de façon indépendante de l’IFN, l’expression du récepteur au VHC (CD81) sur la lignée cellulaire Huh7.5, ce qui pourrait avoir pour conséquence de restreindre l’infection par le VHC. Quoiqu’il en soit, même si les récepteurs au VHC comme le CD81 sont largement exprimés à la surface de différentes sous populations lymphocytaires, les DCs et les monocytes ne répondent pas aux VHC, Nos résultats indiquent que seuls les macrophages sont capables de reconnaître le VHC et de produire des cytokines inflammatoires en réponse à ce dernier. La reconnaissance du VHC par les macrophages est liée à l’expression membranaire de DC-SIGN et l’engagement des TLR 7/8 qui en résulte. Comme d’autres agonistes du TLR 7/8, le VHC stimule la production de cytokines inflammatoires (TNF-α, IL-8, IL-6 et IL-1b) mais n’induit pas la production d’interféron-beta par les macrophages. De manière attendue, la production de cytokines par des macrophages stimulés par les ligands du TLR 7/8 ou les séquences ssARN virales n’inhibent pas la réplication virale. Nos résultats mettent en évidence la capacité des séquences ssARN dérivées du VHC à stimuler les TLR 7/8 dans différentes populations de DC et à initier une réponse immunitaire innée qui aboutit à la suppression de la réplication virale de façon dépendante de l’IFN. Quoiqu’il en soit, le VHC est capable d’échapper à sa reconnaissance par les monocytes et les DCs qui ont le potentiel pour produire de l’IFN et inhiber la réplication virale après engagement des TLR 7/8. Les macrophages possèdent quant à eux la capacité de reconnaître le VHC grâce en partie à l’expression de DC-SIGN à leur surface, mais n’inhibent pas la réplication du virus car ils ne produisent pas d’IFN. L’échappement du VHC aux défenses antivirales pourrait ainsi expliquer l’échec du système immunitaire inné à contrôler l’infection par le VHC. De plus, la production de cytokines inflammatoires observée après stimulation in vitro des macrophages par le VHC suggère leur potentielle contribution dans l’inflammation que l’on retrouve chez les individus infectés par le VHC.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Stimulation of IL-6 Cytokines in Fibroblasts by Toll-like Receptors 2

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Efeito da própolis sobre a produção de citocinas e expressão do receptor TLR-4 por camundongos submetidos a estresse

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Expressão e localização de receptores Toll-like -1, -2, -4 e -6 em membranas corioamnióticas de gestações complicadas por corioamnionite histológica

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Expressão dos receptores Toll-like em carcinoma espinocelular de orofaringe

Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe.

Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells.

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.

TLR 2 modulates inflammation in zymosan-induced arthritis in mice

Résumé L'objectif de cette étude est la compréhension des mécanismes sous-jacents à l'inflammation articulaire dans un modèle murin d'arthrite induite par le zymosan (ZIA). En particulier, la participation du récepteur Toll 2 (TLR2) et du complément C3 a été recherchée. L'inflammation articulaire a été quantifiée par l'accumulation de Technetium (Tc) in vivo, et par histologie des articulations arthritiques. Les réponses humorales et cellulaires induites par le zymosan ont été quantifiées par la prolifération lymphocytaire in vitro et par la mesure de la production d'anticorps dirigés contre le zymosan in vivo. L'inflammation associée à l'arthrite induite au zymosan est, d'après le Tc-uptake, d'aspect biphasique, avec un pic après 1 jour, puis une deuxième phase plus tardive. La deuxième phase persiste jusqu'au 24 ème jour et est associée au développement d'une immunité spécifique contre le zymosan. Les souris déficientes pour TLR-2 présentent une réduction significative de l'inflammation articulaire précoce (jour 1) et tardive (jour 24), ainsi qu'une nette diminution de l'infiltrat inflammatoire dans la membrane synoviale. De plus, la prolifération de cellules du ganglion lymphatique ainsi que le taux d'IgG dirigés contre le zymosan sont diminués de façon significative après 25 jour d'arthrite chez les souris déficientes en TLR2 par rapport aux souris sauvages contrôles. Par contraste, chez les souris déficientes pour C3 on n'observe pas de différence dans l'uptake de Tc ou le scoring histologique par rapport à la lignée sauvage. Ces résultats montrent que l'arthrite induite au zymosan n'est pas seulement un modèle d'inflammation aigue, mais que l'inflammation synoviale persiste même après 25 jours. Ce modèle implique à la fois des mécanismes d'immunité innée et acquise. Le signalling via TLR 2 semble jouer in rôle dans l'immunité au zymosan et pourrait être responsable de la nature biphasique de ce modèle d'arthrite. Abstract The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dentin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dentin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

Toll-Interacting Protein Modulates Gut Flora Composition, Epithelial Barrier Integrity and Susceptibility to Colitis

Toll-like receptor ( TLR) s ignals are key to maintaining hostmicrobial i nteractions. T he T oll-interacting-protein (Tollip) is a ubiquitously-expressed inhibitor of inflammasome a nd TLR signaling. W e hypothesized that T ollip might control g ut homeostasis. G enetic ablation of T ollip d id not lead to spontaneous colitis b ut h ad d ramatic c onsequences on t he intestinal expression of the α-defensin cryptidin 4 and the C-type lectin R EGIIIβ. These c hanges were associated with intestinal dysbiosis a nd e nhanced colonization b y segmented filamentous bacteria - a k ey p ro-inflammatory component of the microbiota. Tollip deficiency increased susceptibility to dextran sulfate sodium (DSS) colitis and aggravated chronic Th17-driven colitis in IL-10-/- mice. Flora d epletion w ith a ntibiotics in T ollip-/- mice w as not sufficient to restore DSS colitis susceptibility and deletion of Tollip in n on-hematopoietic c ells using bone-marrow chimeras w as sufficient to increase s usceptibility t o DSS colitis. After D SS administration, we o bserved several e pithelial defects i n Tollip-/- mice including early tight junctions disruption, increased epithelial apoptosis, and increased intestinal permeability. Overall, our data show that T ollip significantly impacts intestinal h omeostasis by controlling b acterial ecology and intestinal r esponse to chemical and immunological stresses.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9.Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS).Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Propolis Immunomodulatory Action In Vivo on Toll-Like Receptors 2 and 4 Expression and on Pro-Inflammatory Cytokines Production in Mice

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Profiles of gene polymorphisms in cytokines and toll-like receptors with higher risk for gastric cancer

Background: Chronic inflammation and gastric carcinogenesis show a close association, so gene polymorphisms that modify the intensity of the inflammatory response may contribute to variations in gastric cancer risk. Aims: The purpose of this study was to investigate the combined effect of the pro- and anti-inflammatory cytokines and toll-like receptors polymorphisms on the chronic gastritis and gastric cancer risk in a Brazilian population sample. Methods: We evaluated 669 DNA samples (200 of gastric cancer [GC], 229 of chronic gastritis [CG], and 240 of healthy individuals [C]). Ten polymorphisms were genotyped: IL-1RN and TLR2 -196 to -174 del using the allele-specific PCR method and TNF-A (rs1800629; rs1799724), TNF-B (rs909253), IL-8 (rs4073; rs2227532), IL-10 (rs1800872) and TLR4 (rs4986790; rs4986791) using PCR-RFLP. Results: Polymorphisms TNF-A-308G/A, IL-8-251A/T, TNF-B + 252A/G and TLR4 + 1196C/T were not associated with risk of any gastric lesion. However, an association with increased risk for GC was observed for polymorphisms IL-1RNL/2 (p < 0.001), TNF-A-857C/T (p = 0.022), IL-8-845T/C (p < 0.001), IL-10-592C/A (p < 0.001), TLR2ins/del (p < 0.001), and TLR4 + 896A/G (p = 0.033). In CG, an association was observed only with polymorphisms IL-1RNL/2 and IL-10-592A/C (p < 0.001 for both). A combined analysis of these six polymorphisms associated with GC revealed a profile with two to four combined genotypes which confer a higher risk of gastric carcinogenesis, with an OR increased 2.95-fold to 50.4-fold, highlighting the combinations IL-1RN2/TNF-A-857T/IL-8-845C, IL-1RN2/IL-8-845C/TLR2del, IL-1RN2/IL-10-592A/TLR4 + 896G, IL-10-592A/TLR2del/ TLR4 + 896G, and IL-1RN2/TNFA-857T/IL8-845C/TLR2del. Conclusions: Our findings evidenced that the combined effect of polymorphisms in genes involved in the inflammatory process may potentiate the risk of gastric cancer, thus emphasizing the importance of evaluating multiple polymorphisms together. © 2012 Springer Science+Business Media New York.

Relevance of the Myeloid Differentiation Factor 88 (MyD88) on RANKL, OPG, and Nod Expressions Induced by TLR and IL-1R Signaling in Bone Marrow Stromal Cells

The myeloid differentiation factor 88 (MyD88) plays a pivotal role in Toll-like receptor (TLR)- and interleukin-1 receptor (IL-1R)-induced osteoclastogenesis. We examined the role of MyD88 on p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and nucleotide-binding oligomerization domain (Nod) induction by lipopolysaccharide (LPS) and IL-1 beta, and their effect on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production in bone marrow stromal cell (BMSC). RANKL, Nod1, Nod2, NF-κB, and p38 protein levels were determined by Western blot. Nod2 was stimulated with muramyl dipeptide (MDP) prior to TLR4 stimulation with LPS. MyD88 deficiency markedly inhibited RANKL expression after LPS stimulation and increased OPG messenger RNA (mRNA) production. Also, MyD88 was necessary for NF-κB and p38 MAPK activation. MDP alone did not induce RANKL and OPG expressions; however, when combined with LPS, their expressions were significantly increased (p < 0.05). Our results support that MyD88 signaling has a pivotal role in osteoclastogenesis thought NF-κB and p38 activation. Nod2 and especially Nod1 levels were influenced by MyD88.

Relevance of the Myeloid Differentiation Factor 88 (MyD88) on RANKL, OPG, and Nod Expressions Induced by TLR and IL-1R Signaling in Bone Marrow Stromal Cells

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