RESUMO
Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) are double-stranded RNA (dsRNA)-recognizing receptors that mediate innate immune responses to virus infection. However, the roles played by these receptors in the pathogenesis of avian viruses are poorly understood. In this study, we generated TLR3 and MDA5 single knockout (SKO) and TLR3-MDA5 double knockout (DKO) quail fibroblast cells and examined dsRNA receptor-mediated innate immune responses in vitro. The knockout cells were then stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or influenza A virus. Endosomal stimulation of TLR3 by adding poly(I:C) in cell culture media or cytoplasmic stimulation of MDA5 by transfecting poly(I:C) resulted in significant increases of TLR3, MDA5, interferon (IFN) ß, and interleukin 8 gene expression levels in wild type (WT) cells. Endosomal poly(I:C) treatment induced a higher level expression of most of the genes tested in MDA5 SKO cells compared with WT cells, but not in TLR3 SKO and DKO cells. Cytoplasmic transfection of poly(I:C) led to significant upregulation of all four genes in WT, TLR3 SKO, and MDA5 SKO cells at 8 hr posttransfection and negligible gene expression changes in TLR3-MDA5 DKO cells. Upon infection with a strain of influenza virus with compromised IFN antagonistic capability, WT cells produced the highest amount of biologically active type I IFN followed by TLR3 SKO and MDA5 SKO cells. DKO cells did not produce detectable amounts of type I IFN. However, the IFN did not induce an antiviral state fast enough to block virus replication, even in WT cells under the experimental conditions employed. In summary, our data demonstrate that TLR3 and MDA5 are the key functional dsRNA receptors in quail and imply their coordinated roles in the induction of innate immune responses upon virus infection.
Evaluación de las respuestas inmunitarias mediadas por TLR3 y MDA5 utilizando células de fibroblastos de codorniz con genes eliminados. El receptor tipo Toll 3 (TLR3) y el gene 5 asociado a la diferenciación de melanoma (MDA5) son receptores de reconocimiento de ARN de doble cadena (dsRNA) que median las respuestas inmunitarias innatas a la infección por virus. Sin embargo, no se conocen bien las funciones que desempeñan estos receptores en la patogenia de los virus aviares. En este estudio, se generaron células de fibroblastos de codorniz con eliminación simple de los genes TLR3 y MDA5 (SKO) y eliminación doble de los genes TLR3-MDA5 (DKO) y se examinaron las respuestas inmunitarias innatas mediadas por el receptor de dsRNA in vitro. Posteriormente, las células con genes eliminados se estimularon con un ligando sintético de ARN de doble cadena poliinosínico: ácido policitidílico [poli (I: C)] o con el virus de la influenza A. La estimulación endosómica de TLR3 mediante la adición de poli(I: C) en medios de cultivo celular, o la estimulación citoplásmica de MDA5 mediante la transfección de poli(I: C), dieron como resultado aumentos significativos de los niveles de expresión de los genes para TLR3, MDA5, interferón (IFN) ß e interleucina 8 en células de tipo silvestre (WT). El tratamiento con poli(I: C) endosómico indujo un nivel de expresión más alto de la mayoría de los genes analizados en las células con eliminación simple del gene MDA5 en comparación con las células silvestres, pero no en las células con eliminación simple del gene TLR3 y con eliminación doble de genes. La transfección citoplásmica de poli(I: C) condujo a una regulación positiva significativa de los cuatro genes en las células silvestres, en las células con eliminación simple del gene TLR3 y en las células con eliminación simple del gene MDA5 a las ocho horas posteriores a la transfección y cambios insignificantes en la expresión de genes en las células con eliminación doble de los genes TLR3 y MDA5. Durante la infección con una cepa del virus de la influenza con una capacidad antagonista para IFN comprometida, las células silvestres produjeron la mayor cantidad de IFN de tipo I biológicamente activo, seguidas de las células con eliminación simple del gene TLR3 y de las células con eliminación simple del gene MDA5. Las células con eliminación doble de genes no produjeron cantidades detectables de IFN de tipo I. Sin embargo, el IFN no indujo un estado antiviral lo suficientemente rápido como para bloquear la replicación del virus, incluso en células silvestres bajo las condiciones experimentales empleadas. En resumen, los datos de este estudio demuestran que TLR3 y MDA5 son los receptores de ARN de doble cadena funcionales clave en la codorniz e implican sus funciones coordinadas en la inducción de respuestas inmunitarias innatas durante la infección por virus.
Assuntos
Codorniz , Receptor 3 Toll-Like , Animais , Fibroblastos , Imunidade Inata , Poli I-C/farmacologia , Receptor 3 Toll-Like/genéticaRESUMO
Maternal immune priming is the transfer of immunity from mother to offspring, which may reduce the offspring's risk of disease from a pathogen that previously infected its mother. Maternal immune priming has been described in at least 25 invertebrate taxa, including Crassostrea gigas. Larvae of C. gigas have improved survival to Ostreid herpesvirus (OsHV-1) if their mothers are either infected with OsHV-1 or were injected with a virus mimic called poly(I:C). However, fitness costs associated with maternal immune priming in C. gigas are unknown. Here, we show C. gigas larvae produced from poly(I:C)-treated mothers are smaller, and have higher total bacteria and Vibrio loads compared to control larvae. These results suggest that the improved offspring survival of C. gigas to OsHV-1 due to maternal immune priming with poly(I:C) is potentially traded off with other important life history traits, such as larval growth rate and destabilisation of the microbiome.
Assuntos
Crassostrea/imunologia , Vírus de DNA/fisiologia , Aptidão Genética/genética , Tolerância Imunológica , Imunidade Inata/genética , Animais , Crassostrea/genética , Herança Materna , Poli I-C/farmacologiaRESUMO
A skin fibroblast cell line WE-skin11f from walleye (Sander vitreus) was used to study the impact of temperature (26 °C, 20 °C, 14 °C, or 4 °C) on the transcript levels of genes involved in the endogenous antigen processing and presentation pathway (EAPP), which is an important antiviral pathway of vertebrates. Partial coding sequences were found for 4 previously unidentified walleye EAPP members, calreticulin, calnexin, erp57, and tapasin, and the constitutive transcript levels of these genes in WE-skin11f was unchanged by culture incubation temperature. The viral mimic poly (I:C) and viral haemorrhagic septicaemia virus (VHSV) IVb were used to study possible induction of EAPP transcripts (b2m, mhIa, and tapasin). The walleye cells were exquisitely sensitive to poly (I:C), losing adherence and viability at concentrations greater than 100 ng/mL, particularly at suboptimal temperatures. VHSV IVb viral particles were produced from infected WE-skin11f cells at 20 °C, 14 °C, and 4 °C but with much lower production at 4 °C. Under conditions where their impact on the viability of WE-skin11f cultures was slight, poly (I:C) and VHSV IVb were shown to induce b2m, mhIa, and tapasin transcript°s at 26 °C and 20 °C respectively. However, at 4 °C, the up-regulation of EAPP transcript levels was either delayed or completely impaired when compared to the 26 °C and 20 °C control temperatures of the respective experiments. These in vitro results suggest that suboptimal temperatures may be capable of modulating the regulation of the EAPP in walleye cells during viral infection.
Assuntos
Apresentação de Antígeno/genética , Proteínas de Peixes/imunologia , Percas/imunologia , Animais , Linhagem Celular , Fibroblastos , Novirhabdovirus/fisiologia , Percas/genética , Poli I-C/farmacologia , Temperatura , Transcrição GênicaRESUMO
Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4⯰C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.
Assuntos
Temperatura Baixa/efeitos adversos , Fibroblastos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Oncorhynchus mykiss/imunologia , Aclimatação/imunologia , Animais , Apresentação de Antígeno , Linhagem Celular , Fibroblastos/metabolismo , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Indutores de Interferon/farmacologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Novirhabdovirus/imunologia , Oncorhynchus mykiss/virologia , Poli I-C/farmacologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The cytokine thymic stromal lymphopoietin (TSLP) is involved in the development and the progression of allergic diseases. It is mainly released by epithelial cells at barriers such as skin and gut in response to danger signals. Overexpression of TSLP in keratinocytes (KC) can provoke the development of a type 2 inflammatory response. Additionally, TSLP directly acts on sensory neurons and thereby triggers itch. Since histamine is also increased in lesions of inflammatory skin diseases, the aim of this study was to investigate possible effects of histamine as well as different histamine receptor subtype agonists and antagonists on TSLP production in KC. We therefore stimulated human KC with histamine in the presence or absence of the known TSLP-inductor poly I:C and measured TSLP production at protein as well as mRNA level. Histamine alone did not induce TSLP production in human KC, but pre-incubation with histamine prior to challenge with poly I:C resulted in a significant increase of TSLP production compared to stimulation with poly I:C alone. Experiments with different histamine receptor agonists (H1R: 2-pyridylethylamine; H2R: amthamine; H2R/H4R: 4-methylhistamine (4MH)) revealed a dominant role for the H4R receptor, as 4-MH in combination with poly I:C displayed a significant increase of TSLP secretion, while the other agonists did not show any effect. The increase in TSLP production by 4MH was blocked with the H4R antagonist JNJ7777120. This effect was reproducible also in the murine KC cell line MSC. Taken together, our study indicates a new role for the H4 receptor in the regulation of TSLP in keratinocytes. Therefore, blocking of the H4R receptor in allergic diseases might be promising to alleviate inflammation and pruritus via TSLP.
Assuntos
Citocinas/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Receptores Histamínicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Histamina/metabolismo , Humanos , Queratinócitos/metabolismo , Metilistaminas/farmacologia , Camundongos , Poli I-C/farmacologia , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Dendritic cell-based vaccines offer promise for therapy of ovarian cancer. Previous studies have demonstrated that oxidation of several antigens, including ovarian cancer cells, using hypochlorous acid strongly enhances their immunogenicity and their uptake and presentation by dendritic cells. The response of T cells and dendritic cells to autologous tumour from patients with active disease has not previously been investigated. Monocyte-derived dendritic cells were generated from patients with active disease and activated by co-culture with oxidised tumour cells and the TLR agonist poly I:C. The dendritic cells showed an activated phenotype, but secreted high levels of TGFß. Co-culture of the antigen-loaded dendritic cells with autologous T cells generated a population of effector T cells that showed a low level of specific lytic activity against autologous tumour, as compared to autologous mesothelium. The addition of neutralising antibody to TGFß in DC/T cell co-cultures increased the levels of subsequent tumour killing in three samples tested. Co-culture of monocytes from healthy volunteers with the ovarian cell line SKOV-3 prior to differentiation into dendritic cells reduced the ability of dendritic cells to stimulate cytotoxic effector cells. The study suggests that co-culture of dendritic cells with oxidised tumour cells can generate effector cells able to kill autologous tumour, but that the high tumour burden in patients with active disease may compromise dendritic cell and/or T cell function.
Assuntos
Células Dendríticas/transplante , Neoplasias Ovarianas/terapia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Monócitos/imunologia , Neoplasias Ovarianas/imunologia , Poli I-C/imunologia , Poli I-C/farmacologia , Linfócitos T/imunologia , Transplante Autólogo/imunologiaRESUMO
Western blotting with polyclonal antisera to polypeptides of the rainbow trout major histocompatibility (MH) genes and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare expression of MH genes in rainbow trout cell lines. One line was the spleen monocyte/macrophage-like RTS11, which grew loosely on plastic surfaces. Adherent cell lines were fibroblast-like RTG-2 from gonads and four epithelial-like cell lines from gill, intestine, liver and hepatoma: RTgill-W1, RTgutGC, RTL-W1, and RTH-149 respectively. All cell lines expressed a 45 kDa MHC class I alpha chain. All cell lines expressed beta-2-microglobulin (beta2m), which was at 11 kDa, but detection was abrogated following trypsinization prior to cell collection. All cell lines expressed transcripts for MH class II alpha and MH class II beta genes; however, MH class II polypeptides were expressed only in RTS11, the only cell line from a lineage of antigen-presenting cells. We report here that double stranded RNA up regulates beta2m and that these cell lines and antisera can be employed for studying MH regulation.
Assuntos
Complexo Principal de Histocompatibilidade/imunologia , Oncorhynchus mykiss/imunologia , RNA de Cadeia Dupla/farmacologia , Microglobulina beta-2/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Poli I-C/farmacologiaRESUMO
Dendritic cells (DCs) residing in skin are important sentinels for foreign antigens. Methods to facilitate studies of subsets of skin DCs are important to increase the understanding of various pathogens, allergens, topical treatments or vaccine components targeting the skin. In this study, we developed a new DC purification method using a skin graft mesher, clinically used for expansion of skin grafts, to accelerate processing of skin into nets that allowed efficient enzymatic disruption and single cell isolation. The reduction in processing time using the skin graft mesher enabled processing of larger skin samples and also limited the ex vivo handling of the specimens which is associated with maturation of DCs. In addition, a skin explant model to functionally monitor early events of antigen uptake by DC subsets in situ was developed. DCs isolated from epidermis represented a uniform CD1a(+) HLA-DR(+) CD11c(+) Langerin(+) DC-SIGN(-) DC-LAMP(int) DEC-205(int) Langerhans cell (LC) population whereas three subtypes of HLA-DR(+) CD11c(+) DCs were isolated from dermis based on their varying expression of CD1a. Epidermal LCs showed a significantly higher antigen uptake capacity of fluorescently-labelled ovalbumin (OVA) and dextran as compared to any of the dermal DC (dDC) subsets. In contrast, injection of antigen directly into skin explants followed by in situ imaging revealed that the majority of DCs with internalized antigen were localized in the dermis, likely as a consequence of the anatomical site for antigen delivery. These methods offer potency for various applications addressing antigen uptake, microbial DC interactions or other antigenic stimulation targeting the skin and can enhance our knowledge of basic DC biology in human skin.
Assuntos
Antígenos/imunologia , Separação Celular/métodos , Células de Langerhans/imunologia , Derme/citologia , Derme/imunologia , Dextranos/farmacologia , Endocitose , Células Epidérmicas , Epiderme/imunologia , Feminino , Humanos , Hidrazinas/farmacologia , Indutores de Interferon/farmacologia , Células de Langerhans/citologia , Ovalbumina/imunologia , Poli I-C/farmacologiaRESUMO
The aims of this study were (i) to identify alternative Mx stimulatory compounds in Atlantic salmon (Salmo salar L.) and to characterise the kinetics and intensity of the stimulated responses and (ii) to investigate the effect of temperature on such responses by semi-quantitative RT-PCR. Mx transcripts were measured in Atlantic salmon parr kept at 14 degrees C and injected with either LPS, the synthetic double-stranded polyribonucleotide poly I:C, Vibrio anguillarum serotypes I and II-ordalii bacterin, beta-glucan, whole yeast cells or yeast RNA. Sampling periods lasted until transcripts were undetectable or up to three weeks after immunisation. The effect of temperature on poly I:C-induced Mx response was studied by injecting parr kept at 6 degrees C. Newly hatched salmon fry were immersed once, twice or three times in the Vibrio bacterin diluted five or 10 times and sampled for three weeks. None of the yeast compounds induced Mx expression in Atlantic salmon parr. LPS induced a very low Mx response 2 and 3 days after injection. The Vibrio bacterin administered by injection in parr (but not by immersion in fry) resulted in strong Mx induction on days 2 and 3, disappearing by day 6. Poly I: C-induced Mx responses that were more intense and longer lasting than those induced by the bacterin, peaking on day 3 and lasting over 6 days, disappearing by day 9 at 14 degrees C. Lower temperature caused a longer lasting Mx response to poly I:C (at least 21 days), which peaked on days 7-14, with a similar intensity and no delayed onset as compared with the response at 14 degrees C. However, some toxicity of the poly I:C was indicated in treatments at 6 degrees C.