CIBICI   14215
CENTRO DE INVESTIGACION EN BIOQUIMICA CLINICA E INMUNOLOGIA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Cathepsin L3 from Fasciola hepatica induces IL-1β and IL-18 secretion in a NLRP3 dependent manner on dendritic cells
Autor/es:
SANABRIA RODRIGO; FRESNO MANUEL; CELIAS, DAIANA PAMELA; PRUZZO CESAR; CORVO I; . CERVI L.; ARRANZ ALICIA; SILVANE LEONARDO
Lugar:
Mexico
Reunión:
Congreso; XII Congress of the Latin American Association of Immunology ALAI.XXIII Congress of the Mexican Society of Immunology ? SMI.; 2018
Institución organizadora:
ALAI.? SMI.
Resumen:
Cathepsin L3 (CL3) is a cystein protease with collagenolytic activity, highly expressed in the juvenile larval stage of the helminth parasite Fasciola hepatica. Its ability to degrade collagen might facilitate the migration of parasite through the host tissues . However, there is no information about its interaction with the immune system. On the other hand, numerous helminth-derived molecules have been described that are able to modulate dendritic cells (DC) activity, which in turn are capable to polarize the adaptive immune response2. Furthermore, several studies have suggested that inflammasome, a multiprotein oligomer that is one of the components of the innate immune system, could be involved in these effects3,4,5,6. The NLRP3 inflammasome is formed by a set of cytosolic proteins: a receptor (Nod)-like receptor family pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis-associated speck-like protein (ASC), and the pro-caspase-1. Upon stimulations, NLRP3 inflammasome components are assembled leading to the activation of caspase-1, which is responsible for the maturation of pro-inflammatory cytokines, such as bioactive interleukin-1β (IL-1β) and interleukin-18 (IL-18) 7. Taking into consideration all above, the aim of this work was to study the ability of CL3 to modulate the inflammasome activation in bone marrow-derived DC. DC were prepared by culturing bone marrow cells isolated from femurs of C57BL/6, Caspase-1/11KO or NLRP3KO mice in complete RPMI 1640 with GM-CSF from J558 cell line supernatant. To activate the DC, cells were cultured with a recombinant CL3 (20 µg/ml) or rvCL3 (variant inactive of CL3), (20 µg/m), both produced in Hansenula polymorpha expression system and subjected to an endotoxin removal with polymyxin columns; or with LPS (1 ug/ml) and ATP (5 mM). Cell viability was assessed by adding a MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), incubating the cells for 4h at 37°C and absorbance was read at 570 nm. Optical density of DC treated with medium alone was considered as 100% cell viability. IL-1β and IL-18 secretion were quantified in culture supernatants by ELISA. DC from C57BL/6, Caspase-1/11KO or NLRP3KO mice were then cultured with allogeneic splenocytes from BALB/c mice during 5 days and IL-4, IL-13 and IFN-γ were measurement in supernatants. Besides, CL3 (previously labeled with Alexa Fluor ® 488 Microscale Protein Labeling Kit) was added to DC preparations, and incubated for 30 minutes or 4 hours. Next, the cells were stained with EEA-1 (early endosomes) and LAMP (lysosomes), examined with an Olympus FV1200 confocal microscope and images were analyzed using ImageJ software. Further cell lysates (treated or not with CL3 or LPS as control) were analyzed by western blot to evaluated IκBα. On the other hand, a pro-IL-1β mouse recombinant, was incubated with CL3 or rvCL3 at different concentrations for 4 h and IL-1β levels were quantified by ELISA.Cell viability of DC treated with CL3, was not affected and significant amounts of IL-1β and IL-18 were detected in culture supernatants (p