RESUMEN
Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of beclin-1, atg16l1 and atg12, indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of il-6, il-10, il-12, tnf-α, ifn-γ and tgf-ß genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.
Asunto(s)
Autofagia , Células Dendríticas , Equinococosis , Echinococcus granulosus , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Animales , Echinococcus granulosus/inmunología , Autofagia/inmunología , Equinococosis/inmunología , Equinococosis/parasitología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones , Lectinas Tipo C/metabolismo , Citocinas/metabolismo , Femenino , Autofagosomas/inmunología , Autofagosomas/metabolismoRESUMEN
In an attempt to find new anti-echinococcal drugs, resveratrol (Rsv) effectiveness against the larval stages of Echinococcus granulosus and E. multilocularis was evaluated. The in vitro effect of Rsv on parasites was assessed via optical and electron microscopy, RT-qPCR and immunohistochemistry. In vivo efficacy was evaluated in murine models of cystic (CE) and alveolar echinococcosis (AE). The impact of infection and drug treatment on the mouse bone marrow hematopoietic stem cell (HSC) population and its differentiation into dendritic cells (BMDCs) was investigated via flow cytometry and RT-qPCR. In vitro treatment with Rsv reduced E. granulosus metacestode and protoscolex viability in a concentration-dependent manner, caused ultrastructural damage, increased autophagy gene transcription, and raised Eg-Atg8 expression while suppressing Eg-TOR. However, the intraperitoneal administration of Rsv was not only ineffective, but also promoted parasite development in mice with CE and AE. In the early infection model of AE treated with Rsv, an expansion of HSCs was observed followed by their differentiation towards BMCDs. The latter showed an anti-inflammatory phenotype and reduced LPS-stimulated activation compared to control BMDCs. We suggest that Rsv ineffectiveness could have been caused by the low intracystic concentration achieved in vivo and the drug's hormetic effect, with opposite anti-parasitic and immunomodulatory responses in different doses.
RESUMEN
Immune evasion is a hallmark of persistent echinococcal infection, comprising modulation of innate immune cells and antigen-specific T cell responses. However, recognition of Echinococcus granulosus by dendritic cells (DCs) is a key determinant of the host's response to this parasite. Given that mTOR signaling pathway has been described as a regulator linking metabolism and immune function in DCs, we reported for the first time in these cells, global translation levels, antigen uptake, phenotype, cytokine transcriptional levels, and splenocyte priming activity upon recognition of the hydatid fluid (HF) and the highly glycosylated laminar layer (LL). We found that LL induced a slight up-regulation of CD86 and MHC II in DCs and also stimulated the production of IL-6 and TNF-α. By contrast, HF did not increase the expression of any co-stimulatory molecules, but also down-modulated CD40 and stimulated the expression of the anti-inflammatory cytokine IL-10. Both parasitic antigens promoted protein synthesis through mTOR activation. The use of rapamycin decreased the expression of the cytokines tested, empowered the down-modulation of CD40 and also reduced splenocyte proliferation. Finally, we showed that E. granulosus antigens increase the amounts of LC3-positive structures in DCs which play critical roles in the presentation of these antigens to T cells.
Asunto(s)
Antígenos Helmínticos/inmunología , Células Dendríticas/inmunología , Equinococosis/inmunología , Echinococcus granulosus/inmunología , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Animales , Autofagosomas/inmunología , Autofagosomas/metabolismo , Proliferación Celular , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/parasitología , Equinococosis/parasitología , Echinococcus granulosus/fisiología , Femenino , Citometría de Flujo , Ratones , Microscopía Confocal , Linfocitos T/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The secretion of extracellular vesicles (EVs) in helminth parasites is a constitutive mechanism that promotes survival by improving their colonization and adaptation in the host tissue. In the present study, we analyzed the production of EVs from supernatants of cultures of Echinococcus granulosus protoscoleces and metacestodes and their interaction with dendritic cells, which have the ability to efficiently uptake and process microbial antigens, activating T lymphocytes. To experimentally increase the release of EVs, we used loperamide, a calcium channel blocker that increases the cytosolic calcium level in protoscoleces and EV secretion. An exosome-like enriched EV fraction isolated from the parasite culture medium was characterized by dynamic light scattering, transmission electron microscopy, proteomic analysis and immunoblot. This allowed identifying many proteins including: small EV markers such as TSG101, SDCBP, ALIX, tetraspanins and 14-3-3 proteins; proteins involved in vesicle-related transport; orthologs of mammalian proteins involved in the immune response, such as basigin, Bp29 and maspardin; and parasite antigens such as antigen 5, P29 and endophilin-1, which are of special interest due to their role in the parasite-host relationship. Finally, studies on the EVs-host cell interaction demonstrated that E. granulosus exosome-like vesicles were internalized by murine dendritic cells, inducing their maturation with increase of CD86 and with a slight down-regulation in the expression of MHCII molecules. These data suggest that E. granulosus EVs could interfere with the antigen presentation pathway of murine dendritic cells inducing immunoregulation in the host. Further studies are needed to better understand the role of these vesicles in parasite survival and as diagnostic markers and new vaccines.
Asunto(s)
Células Dendríticas/metabolismo , Echinococcus granulosus/metabolismo , Endocitosis , Vesículas Extracelulares/metabolismo , Animales , Células Cultivadas , Dispersión Dinámica de Luz , Vesículas Extracelulares/química , Femenino , Immunoblotting , Ratones , Microscopía Electrónica de Transmisión , ProteómicaRESUMEN
Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 µM and 0.5 µM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases.
Asunto(s)
Autofagia/efectos de los fármacos , Bortezomib/farmacología , Equinococosis/parasitología , Echinococcus granulosus/efectos de los fármacos , Echinococcus granulosus/fisiología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Animales , Autofagosomas/metabolismo , Autofagia/genética , Biomarcadores , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Femenino , Expresión Génica , Larva , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transporte de Proteínas , Sirolimus/farmacología , Respuesta de Proteína Desplegada/genéticaRESUMEN
Macrophages are one of the most important HIV-1 target cells. Unlike CD4(+) T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1ß, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy.
Asunto(s)
Candida albicans/fisiología , VIH-1/fisiología , Macrófagos/microbiología , Macrófagos/virología , Replicación Viral , Antígenos CD4/metabolismo , Quimiocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Células Dendríticas/virología , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Leucocitos Mononucleares/virología , Ligandos , Macrófagos/metabolismo , Receptores CCR5/metabolismo , Latencia del VirusRESUMEN
Seminal plasma is not just a carrier for spermatozoa. It contains high concentrations of cytokines, chemokines, and other biological compounds that are able to exert potent effects on the immune system of the receptive partner. Previous studies have shown that semen induces an acute inflammatory response at the female genital mucosa after coitus. Moreover, it induces regulatory mechanisms that allow the fetus (a semiallograft) to grow and develop in the uterus. The mechanisms underlying these regulatory mechanisms, however, are poorly understood. In this study, we show that seminal plasma redirects the differentiation of human dendritic cells (DCs) toward a regulatory profile. DCs differentiated from human monocytes in the presence of high dilutions of seminal plasma did not express CD1a but showed high levels of CD14. They were unable to develop a fully mature phenotype in response to LPS, TNF-α, CD40L, Pam2CSK4 (TLR2/6 agonist), or Pam3CSK4 (TLR1/2 agonist). Upon activation, they produced low amounts of the inflammatory cytokines IL-12p70, IL-1ß, TNF-α, and IL-6, but expressed a high ability to produce IL-10 and TGF-ß. Inhibition of the PG receptors E-prostanoid receptors 2 and 4 prevented the tolerogenic effect induced by seminal plasma on the phenotype and function of DCs, suggesting that E-series PGs play a major role. By promoting a tolerogenic profile in DCs, seminal plasma might favor fertility, but might also compromise the capacity of the receptive partner to mount an effective immune response against sexually transmitted pathogens.
Asunto(s)
Diferenciación Celular/fisiología , Células Dendríticas/inmunología , Tolerancia Inmunológica/fisiología , Monocitos/inmunología , Semen/inmunología , Adulto , Antígenos CD1/inmunología , Diferenciación Celular/efectos de los fármacos , Citocinas/inmunología , Células Dendríticas/citología , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Masculino , Monocitos/citología , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/inmunología , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/inmunologíaRESUMEN
The development of acidic environments is a hallmark of inflammatory processes of different etiology. We have previously shown that transient exposure to acidic conditions, similar to those encountered in vivo, induces the activation of neutrophils and the phenotypic maturation of dendritic cells. We here report that extracellular acidosis (pH 6.5) selectively stimulates the production and the secretion of IL-1ß by human monocytes without affecting the production of TNF-α, IL-6 and the expression of CD40, CD80, CD86, and HLA-DR. Stimulation of IL-1ß production by pH 6.5-treated monocytes was shown to be dependent on caspase-1 activity, and it was also observed using peripheral blood mononuclear cells instead of isolated monocytes. Contrasting with the results in monocytes, we found that pH 6.5 did not stimulate any production of IL-1ß by macrophages. Changes in intracellular pH seem to be involved in the stimulation of IL-1ß production. In fact, monocytes cultured at pH 6.5 undergo a fall in the values of intracellular pH while the inhibitor of the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride induced both, a decrease in the values of intracellular pH and the stimulation of IL-1ß production. Real time quantitative PCR assays indicated that monocytes cultured either at pH 6.5 or in the presence of 5-(N-ethyl-N-isopropyl)amiloride expressed higher levels of pro-IL-1ß mRNA suggesting that low values of intracellular pH enhance the production of IL-1ß, at least in part, by stimulating the synthesis of its precursor.
Asunto(s)
Espacio Extracelular/metabolismo , Interleucina-1beta/biosíntesis , Monocitos/metabolismo , Calcio/metabolismo , Caspasa 1/metabolismo , Supervivencia Celular , Citosol/metabolismo , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Espacio Intracelular/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/enzimología , FenotipoRESUMEN
Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.
Asunto(s)
Células Dendríticas/citología , Células Epiteliales/citología , Infecciones por VIH/terapia , VIH/metabolismo , Células CACO-2 , Línea Celular Tumoral , Quimiocinas/metabolismo , Medios de Cultivo/farmacología , Citocinas/metabolismo , Endosomas/metabolismo , Humanos , Inflamación , Interferones/metabolismo , Macrólidos/farmacología , FenotipoRESUMEN
Unprotected sexual intercourse between discordant couples is by far the most frequent mode of HIV-1 (human immunodeficiency virus type 1) transmission being semen the main vector for HIV-1 dissemination worldwide. Semen is usually considered merely as a vehicle for HIV-1 transmission. In this review we discuss recent observations suggesting that beyond being a carrier for virus particles semen markedly influences the early events involved in sexual transmission of HIV through the mucosal barriers.
Asunto(s)
Células Dendríticas/virología , Infecciones por VIH/transmisión , VIH-1/fisiología , Semen/fisiología , Semen/virología , Espermatozoides/virología , Femenino , Infecciones por VIH/virología , Humanos , Concentración de Iones de Hidrógeno , Masculino , Vagina/química , Virión , Acoplamiento ViralRESUMEN
Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (approximately 6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , VIH-1/inmunología , Heparitina Sulfato/inmunología , Espermatozoides/inmunología , Espermatozoides/virología , Adulto , Humanos , Concentración de Iones de Hidrógeno , Interleucina-10/inmunología , Interleucina-12/inmunología , Masculino , Persona de Mediana Edad , Semen/inmunología , Semen/virología , Acoplamiento ViralRESUMEN
HIV-1 intersubtype recombination is a very common phenomenon that has been shown to frequently affect different viral genomic regions. Vpr and Tat are viral proteins known to interact with viral promoter (LTR) during the replication cycle. This interaction is mainly involved in the regulation of viral gene expression, so, any structural changes in the LTR and/or these regulatory proteins may have an important impact on viral replication and spread. It has been reported that these genetic structures underwent recombination in BF variants widely spread in South America. To gain more insight of the consequences of the BF intersubtype recombination phenomenon on these different but functionally related genomic regions we designed and performed and in vitro study that allowed the detection and recovery of intersubtype recombinants sequences and its subsequent analysis. Our results indicate that recombination affects differentially these regions, showing evidence of a time-space relationship between the changes observed in the viral promoter and the ones observed in the Vpr/Tat coding region. This supports the idea of intersubtype recombination as a mechanism that promotes biological adaptation and compensates fitness variations.