Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

Abstract

Unconventional protein secretion represents an important process of the inflammatory response. The release of the pro-inflammatory cytokine interleukin (IL)-1β which burst during pyroptosis as a consequence of gasdermin D plasma membrane pore formation, can also occur through other unconventional secretion pathways dependent on caspase-1 activation. However, how caspase-1 mediates cytokine release independently of gasdermin D remains poorly understood. Here we show that following caspase-1 activation by different inflammasomes, caspase-1 cleaves early endosome autoantigen 1 (EEA1) protein at Asp^127/132. Caspase-1 activation also results in the release of the endosomal EEA1 protein in a gasdermin D-independent manner. EEA1 knock-down results in adecreased release of caspase-1 and IL-1β, but the pyroptotic release of other inflammasome components and lactate dehydrogenase was not affected. This study shows how caspase-1 control the release of EEA1 and IL-1β in a pyroptotic-independent manner.

Figure 1

Caspase-1 induces the release of EEA1. (A) Canonical NLRP3 activation by hypotonic solution (90 mOsm, 1 h) in LPS-primed THP-1. Cell lysate and concentrated cell-free supernatant were analyzed by immunoblot for EEA1, Rab5, LAMP1 and IL-1β. Arrows denotes a smaller product of EEA1 upon hypotonic stimulation. IL-1β released to the extracellular medium was also detected by ELISA (bottom). (B) Immunoblot analysis of EEA1 and caspase-1 on cell lysate and cell-free supernatant from LPS-primed THP-1 (wild-type, CASP1^−/− or CASP4^−/−) after hypotonic stimulation. Where indicated, Ac-YVAD was added to inhibit caspase-1. (C) In vitro processing assay for recombinant human EEA1 expressed in HEK293 cells and incubated with either recombinant caspase-1, or with concentrated supernatant or cell lysates from wild type (WT) or Casp1/11^−/− LPS-primed BMDM activated with nigericin. Where indicated, Ac-YVAD was added to inhibit caspase-1. Dashed line separates two different blots. (D) In vitro processing assay for recombinant human EEA1 WT, D132A, D127A, or D127A/D132A mutants expressed in HEK293 cells and incubated with recombinant caspase-1. (E) Representative high resolution deconvolved images of LPS-primed BMDMs after incubation with hypotonicity and stained with fluorescent probe for active caspase-1 (FLICA, green) and the endosome marker EEA1 (red) and nuclei (DAPI, blue); bar, 5 µm. (F) Co-immunoprecipitation of EEA1 and the p10 subunit of active caspase-1 in cell supernatants of LPS-primed THP-1 macrophages after NLRP3 stimulation with hypotonicity. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete membrane fragments are showed in all cases. Blots and pictures are representative of three to four independent experiments. ELISA data are presented as average ± sem from n = 3 independent experiments.

Figure 2

Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. (A) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11^−/− after stimulation with nigericin, ATP, E. coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). (B) LDH release from macrophages treated as in A. (C) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd^−/− after nigericin stimulation. (D) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd^−/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. (E,F) ELISA for released IL-1β (E) or LDH (F) from macrophages treated as in D. (G) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. (H) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

Figure 3

EEA1 is released during the alternative NLRP3 activation pathway in the absence of pyroptosis. (A) Immunoblot analysis of EEA1, caspase-1 and IL-1β on cell-free supernatant from human monocytes incubated during 4 h with 1 µg/ml of LPS. Blots are representative of three independent experiments. (B,C) ELISA for released IL-1β (B) or LDH (C) from isolated monocytes treated as in A. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments. *p ≤ 0.05.

Figure 4

EEA1 knock down results in a decrease release of IL-1β from macrophages without affecting pyroptosis. (A) Immunoblot analysis of EEA1, NLRP3, ASC, caspase-1 and IL-1β on cell lysate and cell-free supernatant from BMDMs transduced with non-target control (Non-target Lv) or EEA1 specific shRNA lentiviral particles (shRNA EEA1 Lv), primed with LPS and activated with ATP as indicated. This figure is representative of three independent experiments. (B) Densitometry quantification of EEA1 immunoblots shown in A. Relative EEA1 expression is normalized to β-actin expression. (C,D) ELISA for released IL-1β (C) or LDH (D) from BMDMs treated as in A. Nitrocellulose membranes were cropped after protein blotting and then inmunostained separately for each protein. Complete blots for IL-1β ans Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments. ns, not significant (p > 0.05); ***p < 0.0001; *p ≤ 0.05.