Cathepsin L3 from Fasciola hepatica induces IL-1β and IL-18 secretion in a NLRP3 dependent manner on dendritic cells

CELIAS, DAIANA PAMELA; SILVANE LEONARDO; CORVO I; FRESNO MANUEL; ARRANZ ALICIA; PRUZZO CESAR; SANABRIA RODRIGO; . CERVI L.

Mexico

Congreso; XII Congress of the Latin American Association of Immunology ALAI.XXIII Congress of the Mexican Society of Immunology ? SMI.; 2018

ALAI.? SMI.

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<0.05). While, DC cultured with rvCL3, did not produce IL-1β or IL-18, indicating that the enzymatic activity of CL3 could be involved in this effect. Other cytokines measured were not found.In order to know whether CL3 is taken by DC and located in some intracellular compartment or simply remained and fulfilled its role from the extracellular medium, experiments using confocal microscopy were performed. We determine that labeled CL3 is internalized by DC, with a weak colocalization to EEA and LAMP positive compartment being mostly targeted to the cytosol.On the other hand, we investigated whether IL-1β and IL-18 secretion induced by CL3 in DC cultures, could be enhanced by the addition of a first or second signal as LPS or ATP, respectively. We observed that CL3 in combination with LPS or ATP, did not promote a higher IL-1β and IL-18 secretion. Therefore, CL3 alone would be triggering the necessary mechanisms for IL-1β and IL-18 production. Then we studied whether CL3 favored NF-κB activation by western blot. We did not observe IκBα degradation, which indicate NF-κB activation, when DC were cultured with CL3, unlike the control, LPS-treated DC. Therefore, CL3 could be stimulating a non-canonical activation of the inflammasome. Moreover, we wonder whether two key components in inflammasome assembly, NLRP3 and caspase, are required for CL3-induced inflammasome activation in DC. For this, DC from NLRP3 or caspase 1/11 deficient mice (and WT DC as control) were cultured with CL3 or LPS. We observed that IL-1β and IL-18 secretion by DC from NLRP3 deficient mice, was partially diminished, whereas, in the absence of caspase 1/11, the production of these cytokines was not modified. These results suggest that IL-1β and IL-18 production, promoted by CL3, is partially dependent on NLRP3 and independent on caspase 1/11 activity. Besides, we evaluated whether CL3 is able to directly cleave pro-IL-1β. In an enzymatic digestion assay, measurable amounts of mature IL-1β were detected when pro-IL-1β was incubated with CL3, and IL-1β levels produced were dependent on CL3 concentration. In contrast, mature IL-1β was not detected when pro-IL-1β was exposed to rvCL3. Furthermore, we observed that CL3-treated DC were capable to induce IL-13 and IFN-γ secretion in splenocytes in allogeneic cultures (p<0,05). This effect was not observed when rvCL3 treated-DC where cultured with allogeneic splenocytes. Interestingly, CL3-treated DC from NLRP3KO mice showed a diminished capacity to stimulateIFN-γ production of allogeneic splenocytes in comparison with CL3-treated DC from Wild type mice. Our data show that CL3 is endocytosed into DC and is able to cleave pro-IL-1β and pro-IL-18 to their mature and biologically active forms, promoting their release to the extracellular environment, being CL3 cysteine protease activity involved this process. These events are partially dependent on NLRP3 and independent of caspase activity, suggesting that CL3 could exert on DC a caspase like activity undescribed so far. In turn, CL3 treated-DC induce a particular expression of cytokine profile (IFN-γ and IL-13) in allogeneic cultures.