Abstract
Objective CD14+ monocytes, play pivotal role, in SLE pathogenesis through enhanced production of B-cell activating factor (BAFF), a major cytokine driving B-cell maturation into autoantibodies-secreting plasma cells. Lupus monocytes demonstrate deregulated autophagy and inflammatory phenotype upon IFNα stimulation.1 3 Notably, autophagy has been implicated in the secretion of inflammatory cytokines (so-called secretory autophagy),2 however, its possible involvement in BAFF production remains unexplored. We herein sought to delineate the role of secretory autophagy in BAFF secretion by SLE and IFNα-stimulated CD14+ monocytes.
Methods To evaluate unconventional BAFF secretion from IFNα- stimulated CD14+ monocytes we administered Brefeldin A (ER-Golgi transport inhibitor) in vitro and BAFF, IL1β and TNFα secretion were measured by ELISA. BAFF expression was also assessed by flow cytometry. The contribution of secretory autophagy was examined, in IFNα/SLE sera – exposed CD14+ monocytes, by gene silencing studies (for Atg5/Rab8a/Rab11) and by ex vivo treatment with Bafilomycin A1, 3MA and GW4869 (autophagy and exosome release inhibitors). In a translational approach, we isolated SLE patient monocytes and measured BAFF expression both at mRNA (RT- PCR) and protein levels (ELISA). The potential BAFF-LC3B-CD63 co localization was evaluated by immunofluorescence.
Results IFNα-stimulated CD14+ monocyte cell cultures in the presence of brefeldin A, (conventional secretory pathway inhibitor), failed to inhibit BAFF secretion; instead, interference with the autophagy secretory machinery through silencing of Atg5, Rab8a/Rab11, or the pharmacological inhibitors Bafilomycin, 3MA and GW4869, resulted in significant reduction of BAFF secretion. By contrast, induction of autophagy by starvation or mTOR inhibition (rapamycin) correlated with increased BAFF production. Importantly, IFNα stimulated and active SLE CD14+ monocytes produced copious amounts of BAFF and exhibited significant colocalization of BAFF with the autophagy marker LC3B and the exosome marker CD63 (figure 1).
Conclusions Our data provide strong evidence that, under the effect of IFNα, BAFF secretion by monocytes engages in an autophagy/exosome- based secretory pathway. Pending experimental procedures corroborating the presence of BAFF within the exosomes will strengthen our working hypothesis. Delineation of the BAFF secretory pathway may pave the way for novel targeted treatments in SLE.
Autophagy -dependent secretion of BAFF in SLE patient-derived or IFNα stimulated CD14+ monocytes (a) Representative confocal image of immunofluorescence microscopy of anti-LC3A/B, anti- CD63 and anti- BAFF (green: BAFF, red: LC3A/B, magenta: CD63) blue: 4′,6-diamidino-2-phenylindole (DAPI)/DNA) are shown. Scale bars: 10μm. Pearson’s co-localization coefficient for BAFF CD63 and LC3A/B. Each dot represents an independent SLE donor from 100 cells each. (n=7) and bar plots show the mean ± SEM expression. ****P < 0.0001 (two-tailed, Mann-Whitney test). (b) BAFF secreted levels by CD14+ monocytes cultured with 20% SEL active sera +/- Bafilomycin or stimulated with IFNα (c).
Acknowledgement The research work is supported by the Hellenic Foundation for Research and Innovation (HFRI) under the 3rd Call for HFRI PhD Fellowships (Fellowship Number: 6729).
References
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