{
  "abstract": "Objectives In SLE, type I interferon (IFNa) stimulates monocytes to produce high levels of B-cell activating factor (BAFF), a cytokine that drives B-cell maturation. IFNa also induces deregulation of autophagy in SLE monocytes, contributing to aberrant inflammatory responses . Notably, autophagy has been implicated in the secretion of cytokines such as IL-1b .Since BAFF lacks a signal peptide directing it to the canonical ER/Golgi secretory pathway, we investigated whether IFNa promotes its release through an unconventional, autophagy-dependent mechanism involving extracellular vesicles (EVs).Methods BAFF, IL1b, and TNFa secretion by monocytes was quantified by ELISA, and membrane bound BAFF expression was assessed by flow cytometry. The contribution of secretory autophagy and EVs was examined in IFNa- or SLE serum–exposed monocytes through gene silencing (Atg5, Rab8a, Rab11; involved in autophagosome formation, secretory autophagy and exosome release), and ex vivo treatment with Bafilomycin A1, 3-MA, or GW4869 (autophagy and EV release inhibitors). SLE patient monocytes were analyzed for BAFF expression (RT-PCR, ELISA). BAFF–LC3B–CD63 colocalization was evaluated by confocal microscopy.Results Administration of the ER/Golgi transport inhibitor brefeldin A failed to inhibit BAFF secretion. Instead, interference with the secretory autophagy machinery, through silencing of Atg5, Rab8a, or Rab11, or treatment with the pharmacological inhibitors Bafilomycin A1, chloroquine (CQ), or 3-MA, significantly reduced release of BAFF and IL1b, without affecting TNFa production. Moreover, inhibition of multivesicular body formation by GW4869 also decreased BAFF secretion. SLE monocytes produced approximately double the levels of BAFF compared to healthy individuals. Importantly, SLE monocytes exhibited significantly higher colocalization of BAFF with autophagy marker LC3B and EV marker CD63 compared to healthy controls (mean Pearson’s coefficient 0.65 vs. 0.28, ****P < 0.0001), which was reduced following 3 months of Anifrolumab treatment ( figure 1).Abstract PT1:01 Figure 1Autophagy –dependent secretion of BAFF in SLE patient-derived or IFNa 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. (a,b) 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). (c) BAFF secreted levels by CD14+ monocytes cultured with 20% SEL active sera +/- Bafilomycin or stimulated with IFNa (c).Conclusions Our study unveils a mechanism by which IFNa trigger SLE monocytes to release BAFF through the autophagy/EV-based secretory pathway. Elucidating the role of exophagic steps in BAFF-mediated B cell autoantibody production will provide further evidence to strengthen our hypothesis. This novel route of IFNa – induced exophagy of BAFF may pave the way for the development of targeted therapies aimed to improving outcomes in SLE.",
  "authors": [
    {
      "affiliations": [
        "Laboratory of Rheumatology, Autoimmunity and Inflammation, University of Crete, Medical School, Iraklio, Greece"
      ],
      "name": "Dimitra Nikoleri"
    },
    {
      "affiliations": [
        "Uppsala University, Department of Medical Sciences, Rheumatology, Uppsala, Sweden"
      ],
      "name": "Maija-Leena Eloranta"
    },
    {
      "affiliations": [
        "Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital and Lausanne Unive, Lausanne, Switzerland"
      ],
      "name": "Spiros Georgakis"
    },
    {
      "affiliations": [
        "Infections and Immunity, Institute of Molecular Biology and Biotechnology, Foundation for Research and TechnologyHellas, Iraklio, Greece"
      ],
      "name": "Aikaterini Gkirtzimanaki"
    },
    {
      "affiliations": [
        "Uppsala University, Department of Medical Sciences, Rheumatology, Uppsala, Sweden"
      ],
      "name": "Dag Leonard"
    },
    {
      "affiliations": [
        "Laboratory of Rheumatology, Autoimmunity and Inflammation, University of Crete, Medical School, Iraklio, Greece",
        "Infections and Immunity, Institute of Molecular Biology and Biotechnology, Foundation for Research and TechnologyHellas, Iraklio, Greece",
        "Rheumatology Department, University General Hospital, Iraklio, Greece"
      ],
      "name": "Prodromos Sidiropoulos"
    },
    {
      "affiliations": [
        "Uppsala University, Department of Medical Sciences, Rheumatology, Uppsala, Sweden"
      ],
      "name": "Lars Rönnblom"
    },
    {
      "affiliations": [
        "Department of Medical Sciences, Uppsala University,Hospital, Uppsala, Sweden"
      ],
      "name": "Fariborz Mobarrez"
    },
    {
      "affiliations": [
        "Laboratory of Rheumatology, Autoimmunity and Inflammation, University of Crete, Medical School, Iraklio, Greece",
        "Infections and Immunity, Institute of Molecular Biology and Biotechnology, Foundation for Research and TechnologyHellas, Iraklio, Greece",
        "Rheumatology Department, University General Hospital, Iraklio, Greece"
      ],
      "name": "George Bertsias"
    }
  ],
  "title": "PT1:01 IFN-alpha-stimulated monocyte secretion of B cell activating factor (BAFF) is mediated through autophagy and extracellular vesicles in systemic lupus erythematosus (SLE)",
  "uid": "fc1f5703-667a-57e3-8b02-eb9f0e4d1f98"
}