RESUMEN
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
Asunto(s)
Antígeno B7-H1/inmunología , Hígado/inmunología , Sepsis/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo/inmunología , Hepatopatías/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regulación hacia Arriba/inmunologíaRESUMEN
Sepsis mouse models revealed thymus atrophy, characterised by decreased thymus weight and loss of thymocytes due to apoptosis. Mice suffered from lymphopenia, a lack of T cells in the periphery, which attenuates their ability to fight against recurring and secondary infections during sepsis progression. Key players in thymus atrophy are IL-6, which is directly involved in thymus involution, and the sphingosine-1-phosphate - sphingosine-1-phosphate receptor 1 signaling, influencing thymocytes emigration. In healthy individuals a sphingosine-1-phosphate (S1P) gradient from lymphoid organs to the circulatory system serves as signal for mature T cell egress. In the present study we investigated, whether inhibition of S1P generation improves thymus involution. In sepsis, induced by cecal ligation and puncture (CLP), S1P in the thymus increased, while it decreased in serum, thus disrupting the naturally occurring S1P gradient. As a potential source of S1P we identified increased numbers of apoptotic cells in the thymic cortex of septic mice. Pharmacological inhibition of the S1P generating sphingosine kinases, by 4- [[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol (SK I-II), administered directly following CLP, prevented thymus atrophy. This was reflected by lymphocytosis, diminished apoptosis, decreased IL-6 expression, and an unaltered thymus weight. In addition SK I-II-treatment preserved the S1P balance and prevented S1P-dependent internalization of the sphingosine-1-phosphate receptor 1. Our data suggest that inhibition of sphingosine kinase and thus, S1P generation during sepsis restores thymic T cell egress, which might improve septic outcome.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Lisofosfolípidos/sangre , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Sepsis/patología , Esfingosina/análogos & derivados , Timocitos/metabolismo , Timo/patología , Aminofenoles/farmacología , Animales , Atrofia/patología , Atrofia/prevención & control , Ciego/cirugía , Modelos Animales de Enfermedad , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Linfocitosis , Linfopenia , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/sangre , Esfingosina/metabolismo , Tiazoles/farmacología , Timocitos/citologíaRESUMEN
Translating murine data to the human situation, we proposed that the level of peroxisome proliferator-activated receptor γ (PPARγ) expression in T cells from septic patients correlates with clinical outcome. In this preliminary report, we analyzed PPARγ mRNA expression in CD3 T cells derived from blood of a very small number of septic patients (nâ=â18) on various days up to 2 weeks after the initial diagnosis. CD3 T cell count was determined by flow cytometry. T cells from nâ=â11 healthy donors were included as controls. Maximal PPARγ mRNA expression was observed on the day of sepsis diagnosis (day 0; 5,896â±â1,523 copies PPARγ mRNA/25âng mRNA, Pâ<â0.05 vs. controls). In contrast, the number of CD3 T cells was significantly decreased in septic patients compared with healthy controls (296â±â31 vs. 1,803â±â134 T cells/µL blood, Pâ<â0.001). Setting two arbitrary limits: patients with a PPARγ expression in T cells higher than 7,000 copies/25âng mRNA, of whom five of six patients died during the ICU stay, and patients with a T cell count below 100 T cells/µL blood, of whom five of eight patients died, we identified a correlation between sepsis survival and low T cell number, paired with high T cell-specific PPARγ expression. Among all 18 sepsis patients, four fulfilled the criteria for both arbitrary settings and all four of these patients subsequently died. We suggest that both high PPARγ expression in T cells and low absolute T cell number in blood of septic patients may have the potential as a new prognostic marker for a poor sepsis outcome.
Asunto(s)
PPAR gamma/sangre , Sepsis/diagnóstico , Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Complejo CD3/metabolismo , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Hospitales Universitarios , Humanos , Unidades de Cuidados Intensivos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Sensibilidad y Especificidad , Sepsis/sangre , Sepsis/mortalidad , Análisis de SupervivenciaRESUMEN
Understanding of the physiological role of peroxisome proliferator-activated receptor gamma (PPARγ) offers new opportunities for the treatment of cancers, immune disorders and inflammatory diseases. In contrast to PPARγ agonists, few PPARγ antagonists have been studied, though they do exert immunomodulatory effects. Currently, no therapeutically useful PPARγ antagonist is commercially available. The aim of this study was to identify and kinetically characterise a new competitive PPARγ antagonist for therapeutic use. A PPARγ-dependent transactivation assay was used to kinetically characterise (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB) in kidney, T and monocytic cell lines. Cytotoxic effects were analysed and intracellular accumulation of MTTB was assessed by tandem mass spectrometry (LC-MS/MS). Potential interactions of MTTB with the PPARγ protein were suggested by molecular docking analysis. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), MTTB exhibited competitive antagonism against rosiglitazone in HEK293T and Jurkat T cells, with IC50 values in HEK293T cells of 4.3µM and 1.6µM, using the PPARγ ligand binding domain (PPARγ-LBD) and the full PPARγ protein, respectively. In all cell lines used, however, MTTB showed much higher intracellular accumulation than GW9662. MTTB alone exhibited weak partial agonistic effects and low cytotoxicity. Molecular docking of MTTB with the PPARγ-LBD supported direct interaction with the nuclear receptor. MTTB is a promising prototype for a new class of competitive PPARγ antagonists. It has weak partial agonistic and clear competitive antagonistic characteristics associated with rapid cellular uptake. Compared to commercially available PPARγ modulators, this offers the possibility of dose regulation of PPARγ and immune responses.
Asunto(s)
Cinamatos/farmacología , PPAR gamma/antagonistas & inhibidores , Quinolinas/farmacología , Anilidas/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Luciferasas/metabolismo , Simulación del Acoplamiento Molecular , PPAR gamma/agonistas , PPAR gamma/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacología , Activación TranscripcionalRESUMEN
NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8(+) T cell activation by using an antigen-driven coculture model consisting of Nrf2(-/-) and Nrf2(+/+) bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8(+) T cells. OT-1 CD8(+) T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257-264)-pulsed Nrf2(-/-) BMDMΦ with transgenic OT-1 CD8(+) T cells attenuated CD8(+) T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8(+) T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2(-/-) cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2(-/-) cocultures via addition of glutathione ester, N-acetylcysteine, ß-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2(+/+) BMDMΦ mimicked the effect of Nrf2(-/-) BMDMΦ on CD8(+) T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2(-/-) BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8(+) T cell function.
Asunto(s)
Médula Ósea/inmunología , Linfocitos T CD8-positivos/inmunología , Cistina/metabolismo , Glutatión/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Macrófagos/inmunología , Factor 2 Relacionado con NF-E2/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis , Western Blotting , Médula Ósea/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/metabolismo , Técnicas para Inmunoenzimas , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Estrés Oxidativo , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages.