RESUMEN
Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1ß). STEC stimulated neutrophils to release elevated levels of IL-1ß through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1ß secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1ß secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1ß secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli O157 , Síndrome Hemolítico-Urémico , Interleucina-1beta , Escherichia coli Shiga-Toxigénica , Humanos , Caspasas , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Neutrófilos , Interleucina-1beta/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.
Asunto(s)
Glioblastoma , Linfopoyetina del Estroma Tímico , Adulto , Humanos , Células Cultivadas , Citocinas/metabolismo , Neutrófilos/metabolismo , Microambiente Tumoral , Factor A de Crecimiento Endotelial VascularRESUMEN
PURPOSE: γδ T lymphocytes are non-conventional T cells that participate in protective immunity and tumor surveillance. In healthy humans, the main subset of circulating γδ T cells express the TCRVγ9Vδ2. This subset responds to non-peptide prenyl-pyrophosphate antigens such as (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP). This unique feature of Vγ9Vδ2 T cells makes them a candidate for anti-tumor immunotherapy. In this study, we investigated the response of HMBPP-activated Vγ9Vδ2 T lymphocytes to glioblastoma multiforme (GBM) cells. METHODS: Human purified γδ T cells were stimulated with HMBPP (1 µM) and incubated with GBM cells (U251, U373 and primary GBM cultures) or their conditioned medium. After overnight incubation, expression of CD69 and perforin was evaluated by flow cytometry and cytokines production by ELISA. As well, we performed a meta-analysis of transcriptomic data obtained from The Cancer Genome Atlas. RESULTS: HMBPP-stimulated γδ T cells cultured with GBM or its conditioned medium increased CD69, intracellular perforin, IFN-γ, and TNF-α production. A meta-analysis of transcriptomic data showed that GBM patients display better overall survival when mRNA TRGV9, the Vγ9 chain-encoding gene, was expressed in high levels. Moreover, its expression was higher in low-grade GBM compared to GBM. Interestingly, there was an association between γδ T cell infiltrates and TNF-α expression in the tumor microenvironment. CONCLUSION: GBM cells enhanced Th1-like profile differentiation in phosphoantigen-stimulated γδ T cells. Our results reinforce data that have demonstrated the implication of Vγ9Vδ2 T cells in the control of GBM, and this knowledge is fundamental to the development of immunotherapeutic protocols to treat GBM based on γδ T cells.
Asunto(s)
Glioblastoma , Medios de Cultivo Condicionados , Difosfatos , Humanos , Activación de Linfocitos , Perforina , Receptores de Antígenos de Linfocitos T gamma-delta , Células TH1 , Microambiente Tumoral , Factor de Necrosis Tumoral alfaRESUMEN
γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation.
Asunto(s)
Elastasa de Leucocito/inmunología , Activación de Linfocitos , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3/inmunología , Humanos , Interferón gamma/inmunología , Lectinas Tipo C/inmunología , Neutrófilos/citología , Receptor PAR-1/inmunología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN-γ, and the proliferation of γδ T cells induced by (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil-derived ROS. We also show that the ROS-generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi-directional cross-talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.
Asunto(s)
Neutrófilos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Cultivadas , Humanos , Activación de Linfocitos/inmunología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1ß) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1ß processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1ß processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1ß; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1ß out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1ß secretion might constitute a physiological mechanism to control IL-1ß-dependent inflammatory processes where neutrophils play a crucial role.
Asunto(s)
Inflamasomas/inmunología , Interleucina-1beta/inmunología , NADPH Oxidasas/inmunología , Especies Reactivas de Oxígeno/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Línea Celular , Femenino , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Mieloblastina/genética , Mieloblastina/inmunología , Mieloblastina/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno/metabolismoRESUMEN
To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation.
Asunto(s)
Células Dendríticas/metabolismo , NADPH Oxidasas/metabolismo , Fagosomas/metabolismo , Proteínas de Unión al GTP rab/fisiología , Animales , Células Dendríticas/citología , Células Dendríticas/ultraestructura , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Microscopía Inmunoelectrónica , Ovalbúmina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
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
Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4(+) T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4(+) T cells from CLL patients, similarly in T cells from ZAP-70(+) to ZAP-70(-) patients. Autologous nurse-like cells establish a close contact with CD4(+) T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Quimiocina CXCL12/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos/inmunología , Antígenos CD/biosíntesis , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Separación Celular , Quimiocina CXCL12/metabolismo , Citometría de Flujo , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/metabolismo , Microscopía ConfocalRESUMEN
Shiga toxin producing Escherichia coli (STEC) are foodborne pathogens that release Shiga toxin (Stx), virulence factor responsible for the development of Hemolytic Uremic Syndrome (HUS). Stx causes endothelial cell damage, which leads to platelets deposition and thrombi formation within the microvasculature. It has been described that Stx activates blood cells and induces the shedding of proinflammatory and prothrombotic microvesicles (MVs) containing the toxin. In this sense, it has been postulated that MVs containing Stx2 (MVs-Stx2+) can contribute to the physiopathology of HUS, allowing Stx2 to reach the target organs while evading the immune system. In this work, we propose that circulating MVs-Stx2+ can be a potential biomarker for the diagnosis and prognosis of STEC infections and HUS progression. We developed a rat HUS model by the intraperitoneal injection of a sublethal dose of Stx2 and observed: decrease in body weight, increase of creatinine and urea levels, decrease of creatinine clearance and histological renal damages. After characterization of renal damages, we investigated circulating total MVs and MVs-Stx2+ by flow cytometry at different times after Stx2 injection. Additionally, we evaluated the correlation of biochemical parameters such as creatinine and urea in plasma with MVs-Stx2+. As a result, we found a significant circulation of MVs-Stx2+ at 72 and 96 h after Stx2 injection, nevertheless no correlation with creatinine and urea plasma levels were detected. Our results suggest that MVs-Stx2+ may be an additional biomarker for the characterization and diagnosis of HUS progression. A further analysis is required in order to validate MVs-Stx2+ as biomarker of the disease.
Asunto(s)
Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Ratas , Animales , Toxina Shiga II/toxicidad , Creatinina , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/patología , Urea , BiomarcadoresRESUMEN
Aplidin is a novel cyclic depsipeptide, currently in Phase II/III clinical trials for solid and hematologic malignancies. The aim of this study was to evaluate the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common leukemia in the adult. Although there have been considerable advances in the treatment of CLL over the last decade, drug resistance and immunosuppression limit the use of current therapy and warrant the development of novel agents. Here we report that Aplidin induced a dose- and time-dependent cytotoxicity on peripheral blood mononuclear cells (PBMC) from CLL patients. Interestingly, Aplidin effect was markedly higher on monocytes compared to T lymphocytes, NK cells or the malignant B-cell clone. Hence, we next evaluated Aplidin activity on nurse-like cells (NLC) which represent a cell subset differentiated from monocytes that favors leukemic cell progression through pro-survival signals. NLC were highly sensitive to Aplidin and, more importantly, their death indirectly decreased neoplasic clone viability. The mechanisms of Aplidin-induced cell death in monocytic cells involved activation of caspase-3 and subsequent PARP fragmentation, indicative of death via apoptosis. Aplidin also showed synergistic activity when combined with fludarabine or cyclophosphamide. Taken together, our results show that Aplidin affects the viability of leukemic cells in two different ways: inducing a direct effect on the malignant B-CLL clone; and indirectly, by modifying the microenvironment that allows tumor growth.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Depsipéptidos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucocitos Mononucleares/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/patología , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclofosfamida/administración & dosificación , Depsipéptidos/administración & dosificación , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Péptidos Cíclicos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
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
Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.
Asunto(s)
Autofagia , Caspasa 1 , Interleucina-1beta , Neutrófilos , Serina Proteasas , Autofagia/genética , Autofagia/inmunología , Caspasa 1/genética , Caspasa 1/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Serina Proteasas/genética , Serina Proteasas/inmunologíaRESUMEN
The hemolytic uremic syndrome associated with diarrhea, a consequence of Shiga toxin (Stx)-producing Escherichia coli infection, is a common cause of pediatric acute renal failure in Argentina. Stx type 2a (Stx2a) causes direct damage to renal cells and induces local inflammatory responses that involve secretion of inflammatory mediators and the recruitment of innate immune cells. γδ T cells constitute a subset of T lymphocytes, which act as early sensors of cellular stress and infection. They can exert cytotoxicity against infected and transformed cells, and produce cytokines and chemokines. In this study, we investigated the activation of human peripheral γδ T cells in response to the incubation with Stx2a-stimulated human glomerular endothelial cells (HGEC) or their conditioned medium, by analyzing in γδ T lymphocytes, the expression of CD69, CD107a, and perforin, and the production of TNF-α and IFN-γ. In addition, we evaluated by confocal microscopy the contact between γδ T cells and HGEC. This analysis showed an augmentation in cellular interactions in the presence of Stx2a-stimulated HGEC compared to untreated HGEC. Furthermore, we observed an increase in cytokine production and CD107a expression, together with a decrease in intracellular perforin when γδ T cells were incubated with Stx2a-treated HGEC or their conditioned medium. Interestingly, the blocking of TNF-α by Etanercept reversed the changes in the parameters measured in γδ T cells incubated with Stx2a-treated HGEC supernatants. Altogether, our results suggest that soluble factors released by Stx2a-stimulated HGEC modulate the activation of γδ T cells, being TNF-α a key player during this process.
Asunto(s)
Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Niño , Células Endoteliales , Humanos , Toxina Shiga II , Linfocitos TRESUMEN
Ibrutinib is a BTK/ITK inhibitor with efficacy for the treatment of various lymphoid cancers, including CLL. Considering that innate and adaptative immune defects are a dominant feature of CLL patients, we evaluated whether in vitro ibrutinib affects the survival and function of neutrophils and γδ T cells, key players of the early immune response against microbes. Neutrophils and γδ T cells were obtained from peripheral blood of healthy donors and CLL patients. We found that ibrutinib reduces the production of reactive oxygen species (ROS) and bacteria killing capacity, and slightly impairs neutrophil extracellular traps (NETs) production without affecting bacteria-uptake and CD62L-downregulation induced by fMLP or aggregated IgG. In addition, ibrutinib reduces γδ T cell activation and CD107a degranulation induced by phosphoantigens or anti-CD3. These findings are in agreement with previous data suggesting that ibrutinib interferes with the protective immune response to pathogens, particularly Mycobacteria and Aspergillus.
Asunto(s)
Neutrófilos , Linfocitos T , Adenina/análogos & derivados , Humanos , Activación de Linfocitos , Piperidinas , Especies Reactivas de OxígenoRESUMEN
In recent years a non-neuronal cholinergic system has been described in immune cells, which is often usually activated during the course of inflammatory processes. To date, it is known that Acetylcholine (ACh), a neurotransmitter extensively expressed in the airways, not only induces bronchoconstriction, but also promotes a set of changes usually associated with the induction of allergic/Th2 responses. We have previously demonstrated that ACh polarizes human dendritic cells (DC) toward a Th2-promoting profile through the activation of muscarinic acetylcholine receptors (mAChR). Here, we showed that ACh promotes the acquisition of an inflammatory profile by murine DC, with the increased MHC II IAd expression and production of two cytokines strongly associated with inflammatory infiltrate and tissue damage, namely TNF-α and MCP-1, which was prevented by blocking mAChR. Moreover, we showed that ACh induces the up-regulation of M3 mAChR expression and the blocking of this receptor with tiotropium bromide prevents the increase of MHC II IAd expression and TNF-α production induced by ACh on DC, suggesting that M3 is the main receptor involved in ACh-induced activation of DC. Then, using a short-term experimental murine model of ovalbumin-induced lung inflammation, we revealed that the intranasal administration of ACh-treated DC, at early stages of the inflammatory response, might be able to exacerbate the recruitment of inflammatory mononuclear cells, promoting profound structural changes in the lung parenchyma characteristic of chronic inflammation and evidenced by elevated systemic levels of inflammatory marker, TNF-α. These results suggest a potential role for ACh in the modulation of immune mechanisms underlying pulmonary inflammatory processes.
Asunto(s)
Acetilcolina/metabolismo , Células Dendríticas/inmunología , Lesión Pulmonar/inmunología , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Pulmón/citología , Pulmón/inmunología , Pulmón/patología , Lesión Pulmonar/sangre , Lesión Pulmonar/diagnóstico , Ratones , Ovalbúmina/inmunología , Cultivo Primario de Células , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.
Asunto(s)
Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Proteínas de Escherichia coli/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Microvasos/efectos de los fármacos , Toxina Shiga II/toxicidad , Subtilisinas/toxicidad , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Sinergismo Farmacológico , Células Endoteliales/inmunología , Células Epiteliales/inmunología , Síndrome Hemolítico-Urémico , Humanos , Riñón/irrigación sanguíneaRESUMEN
Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1-4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1ß in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.
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Trampas Extracelulares/inmunología , Fiebre/inmunología , Interleucina-1beta/inmunología , Interleucina-8/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Trampas Extracelulares/microbiología , Femenino , Fiebre/microbiología , Fiebre/patología , Humanos , Masculino , Neutrófilos/microbiología , Neutrófilos/patología , Infecciones por Pseudomonas/patologíaRESUMEN
γδ T cells are non-conventional lymphocytes which show several properties of innate immune cells. They present a limited TCR repertoire and circulate as cells with a pre-activated phenotype thus being able to generate rapid immune responses. γδ T cells do not recognize classical peptide antigens, their TCRs are non-MHC restricted and they can respond to pathogen-associated molecular patterns and to cytokines in absence of TCR ligands. They also recognize self-molecules induced by stress, which indicate infection and cellular transformation. All these features let γδ T cells act as a first line of defense in sterile and non-sterile inflammation. γδ T cells represent 1-10% of circulating lymphocytes in the adult human peripheral blood, they are widely localized in non-lymphoid tissues and constitute the majority of immune cells in some epithelial surfaces, where they participate in the maintenance of the epithelial barriers. γδ T cells produce a wide range of cytokines that orchestrate the course of immune responses and also exert high cytotoxic activity against infected and transformed cells. In contrast to their beneficial role during infection, γδ T cells are also implicated in the development and progression of autoimmune diseases. Interestingly, several functions of γδ T cells are susceptible to modulation by interaction with other cells. In this review, we give an overview of the γδ T cell participation in infection and autoimmunity. We also revise the underlying mechanisms that modulate γδ T cell function that might provide tools to control pathological immune responses.