Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion

J Immunol. 2023 Feb 1;210(3):322-334. doi: 10.4049/jimmunol.2200444.


Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caspases / metabolism
  • Escherichia coli / metabolism
  • Extracellular Vesicles* / metabolism
  • Humans
  • Inflammasomes / metabolism
  • Lipopolysaccharides* / pharmacology
  • Macrophages* / metabolism
  • NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
  • Nerve Growth Factors / metabolism
  • Toll-Like Receptor 4 / metabolism


  • Caspases
  • Inflammasomes
  • Lipopolysaccharides
  • Nerve Growth Factors
  • NINJ1 protein, human
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Toll-Like Receptor 4