Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

A Role for cis Interaction between the Inhibitory Ly49A receptor and MHC class I for natural killer cell education.

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

The role of oxidative stress during inflammatory processes.

Abstract The production of various reactive oxidant species in excess of endogenous antioxidant defense mechanisms promotes the development of a state of oxidative stress, with significant biological consequences. In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation, and thus contributes to the pathophysiology of a number of debilitating illnesses, such as cardiovascular diseases, diabetes, cancer, or neurodegenerative processes. Oxidants affect all stages of the inflammatory response, including the release by damaged tissues of molecules acting as endogenous danger signals, their sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families, and the activation of signaling pathways initiating the adaptive cellular response to such signals. In this article, after summarizing the basic aspects of redox biology and inflammation, we review in detail the current knowledge on the fundamental connections between oxidative stress and inflammatory processes, with a special emphasis on the danger molecule high-mobility group box-1, the TLRs, the NLRP-3 receptor, and the inflammasome, as well as the transcription factor nuclear factor-κB.

N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

An allele-specific, stochastic gene expression process controls the expression of multiple Ly49 family genes and generates a diverse, MHC-specific NK cell receptor repertoire.

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the Dd (or Dk)-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Ação da IL-18 sobre a expressão do receptor dectina-1 e produção de citocinas por monócitos humanos desafiados com diferentes cepas de Paracoccidioides brasiliensis

Estudos têm demonstrado que o reconhecimento inicial de microrganismos é mediado por receptores celulares expressos em células da imunidade inata denominados receptores de reconhecimento de padrões (PRRs). Assim, a interação entre moléculas de superfície dos patógenos e receptores homólogos presentes na membrana celular de monócitos, modula a fagocitose, a ativação da célula e conseqüentemente a produção de citocinas. Trabalhos têm demonstrado a importância da estimulação de receptores toll-like 2 e 4 (TLR2 e TLR4), receptores de manose (MR) e dectina-1 de monócitos, tanto em infecções bacterianas como fúngicas, culminando com indução de produção de várias citocinas. A IL-18 é uma citocina indutora de IFN-g e possui uma ação extremamente importante, por ser capaz de promover tanto uma resposta do tipo Th1 ou Th2, dependendo do contexto de estimulação e do microambiente de citocinas. Em trabalho recente demonstramos que a IL-18 possui uma ação importante sobre o aumento da expressão de MR em monócitos humanos, e o reconhecimento do Paracoccidioides brasiliensis via esse receptor causaria aumento do crescimento fúngico no interior da célula. Dessa forma, os objetivos do presente projeto foram: 1) Avaliar a ação da IL- 18 sobre a expressão do receptor dectina-1 por monócitos humanos desafiados in vitro com diferentes cepas do P. brasiliensis; b) Avaliar a participação da dectina-1 na indução da produção de IL-18, TNF-a e IL-10 por monócitos desafiados in vitro com diferentes cepas do P. brasiliensis. Assim, monócitos de indivíduos normais tratados in vitro com IL-18 foram desafiados com diferentes de cepas do P. brasiliensis, e a expressão do receptor dectina-1 foi avaliada pela técnica de citometria de fluxo. A dosagem de IL-18, TNF-a e IL-10 no sobrenadante de cultura de monócitos desafiados com P. brasiliensis foi realizada utilizando a técnica de ELISA . Os resultados mostraram que a ...

Influência do receptor TLR-2 no processo de fagocitose do fungo Sporothrix schenckii

A esporotricose é uma micose provocada pelo fungo Sporothrix schenckii, resultante da inoculação direta de conídios e hifas do fungo. A doença se desenvolve habitualmente na pele, no tecido subcutâneo, podendo através dos vasos linfáticos, comprometer órgãos internos. Nos últimos anos tem-se voltado à atenção à participação do receptor Toll Like-2 no reconhecimento e fagocitose de diversos antígenos fungicos. A partir disto, o objetivo deste estudo foi estudar a influência do receptor Toll Like-2 no processo de fagocitose do Sporothrix schenckii em camundongos C57BL/6. Este estudo avaliou o processo de internalização do Sporothrix schenckii por macrófagos murinos obtidos de camundongos C57BL/6 WT e C57BL/6 TLR-2 KO. Os experimentos foram realizados através da utilização de lâminas de cultura celular e microscopia ótica. Além de analisar a viabilidade de células fúngicas internalizadas através de ensaios de sobrevivência. Como resultados pudemos observar a significativa importância do TLR-2 no processo de internalização deste microrganismo, uma vez que o estudo evidenciou uma eficácia de internalização acima de 85% dos macrófagos obtidos de animas selvagens em relação aos obtidos de animais TLR-2 KO. Além disso, através de ensaios de sobrevivência foram obtidos resultados que sugerem a ausência de viabilidade das células fúngicas depois de fagocitadas

Avaliação do envolvimento do receptor dectina-1 na atividade fungicida e na produção de metabólitos do oxigênio por neutrófilos e monócitos humanos desafiados com Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin-18 increases TLR4 and mannose receptor expression and modulates cytokine production in human monocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)