RESUMEN
Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1ß secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1ß in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1ß was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1ß and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1ß by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1ß in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1ß through at least two separate mechanisms: by targeting pro-IL-1ß for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Autofagia/fisiología , Interleucina-1beta/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Antibacterianos/farmacología , Células Presentadoras de Antígenos/citología , Autofagia/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Femenino , Interleucina-1beta/genética , Ligandos , Lipopolisacáridos/farmacología , Lisosomas/genética , Macrófagos/citología , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos BALB C , NADPH Oxidasa 2 , NADPH Oxidasas , Proteína con Dominio Pirina 3 de la Familia NLR , Sirolimus/farmacología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Activation of CD1d-restricted invariant NKT (iNKT) cells with the glycolipid α-galactosylceramide (α-GalCer) confers protection against disease in murine models, however, clinical trials in humans have had limited impact. We synthesized a novel thioglycoside analogue of α-GalCer, denoted α-S-GalCer, and tested its efficacy for stimulating human iNKT cells in vitro. α-S-GalCer stimulated cytokine release by iNKT cells in a CD1d-dependent manner and primed CD1d(+) target cells for lysis. α-S-GalCer-stimulated iNKT cells induced maturation of monocyte-derived dendritic cells into antigen-presenting cells that released IL-12 and small amounts of IL-10. The nature and potency of α-S-GalCer and α-GalCer in human iNKT cell activation were similar. However, in contrast to α-GalCer, α-S-GalCer did not activate murine iNKT cells in vivo. Because of its enhanced stability in biological systems, α-S-GalCer may be superior to α-GalCer as a parent compound for developing adjuvant therapies for humans.
Asunto(s)
Galactosilceramidas/inmunología , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/inmunología , Tiogalactósidos/inmunología , Tioglicósidos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD1d/inmunología , Línea Celular , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Galactosilceramidas/química , Galactosilceramidas/farmacología , Células HeLa , Humanos , Interferón gamma/sangre , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-4/sangre , Interleucina-4/inmunología , Interleucina-4/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismo , Tiogalactósidos/química , Tiogalactósidos/farmacología , Tioglicósidos/química , Tioglicósidos/farmacologíaRESUMEN
CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.