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TLR4 activation primes human macrophages for non-canonical caspase-4 inflammasome-induced extracellular vesicle secretion

ABSTRACT: Human macrophages secrete extracellular vesicles loaded with numerous immunoregulatory proteins. Here we employed high throughput quantitative proteomics to characterize the modulation of vesicle-mediated protein secretion during non-canonical caspase-4/5-dependent inflammasome activation. We show that human macrophages activate robust caspase-4- dependent extracellular vesicle secretion upon transfection of LPS, and this process is also partially dependent on NLRP-3 and caspase-5. Similar effect occurs with delivery of the LPS with E. coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from ATP-induced vesiculation, and is dependent on autocrine interferon signal associated with TLR4 activation. TLR4 activation preceding the non-canonical 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 bioinformatic methods, we identify 15-deoxy-delta-12,14-prostaglandin J2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating potential new therapeutic potential of these anti-inflammatory drugs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Macrophage

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD033002 | Pride | 2022-12-01

REPOSITORIES: Pride

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Proteomic and bioinformatic characterization of proteins secreted conventionally and unconventionally from human primary macrophages upon LPS transfection

Project description:The non-canonical caspase-4/5 inflammasome has been only very recently characterized. It is expressed by macrophages, the principal effector cells of the innate immunity system, and is activated by gram-negative bacteria, ER stress, and UV radiation. Activation of this inflammasome results in pyroptosis, inflammatory cell death, and secretion of pro-inflammatory cytokines of the interleukin-1 family. In our model we transfected human primary macrophages with Lipopolysaccharide, a cell wall component of gram-negative bacteria, to simulate bacteria entering the cell and thus activating the caspase-4/5 inflammasome. We then performed high-throughput proteomics to identify proteins secreted during the stimulation. The secretome was separated into two fractions using size-exclusion centrifugation, with a 100 kDa cut-off we enriched extracellular vesicles (EVs) and the flow through was concentrated over 10 kDa to yield the rest-secretome (RS) samples. The EV and RS samples of LPS transfected cells were then compared to untreated cells.

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