Research ArticleInflammasomes

The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1

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Science Signaling  14 May 2019:
Vol. 12, Issue 581, eaau0615
DOI: 10.1126/scisignal.aau0615
  • Fig. 1 BCAP interacts with Flightless-1 and LRRFIP2 in macrophages.

    (A and B) MS analysis of proteins associated with BCAP in lysates from WT or BCAP−/− bone marrow–derived macrophages (BMDMs) immunoprecipitated (IP) for BCAP. Tabulated data (A) are the average numbers of unique and total peptides from each protein. Mean peak area quantification (B) for each BCAP, Flightless-1, and LRRFIP2 peptide isolated is representative of three independent experiments. The intensity of each peptide was summed to generate an overall protein quantification score (peak area). (C) Western blot analysis of the indicated proteins in WT or BCAP−/− macrophages lysates immunoprecipitated for BCAP. Blots (top) are representative of three independent experiments. Quantified band intensity values (bottom) of Flightless-1 and LRRFIP2 are means ± SEM pooled from all experiments. ***P < 0.001 by two-sided unpaired t test.

  • Fig. 2 BCAP interacts with NLRP3 inflammasome components through its N-terminal domain.

    (A and B) Western blot analysis of the indicated proteins in lysates from HEK293T cells cotransfected with plasmids encoding BCAP-Flag, Flightless-1, LRRFIP2–hemagglutinin (HA), NLRP3, ASC, and pro–caspase-1–red fluorescent protein (RFP) as indicated and immunoprecipitated for Flag. Black arrowhead in (B) indicates LRRFIP2 band. (C) Flag-tagged BCAP deletion mutants used in (D). (D) Western blot analysis of HEK293T cells cotransfected with plasmids encoding BCAP-Flag deletion mutants and Flightless-1. Lysates were immunoprecipitated for Flag and immunoblotted with anti-Flag and anti–Flightless-1 antibodies. Data are representative of three independent experiments. NTD, N-terminal domain; AR, ankyrin repeats; IVD, intervening domain; CTD, C-terminal domain.

  • Fig. 3 Increased NLRP3 inflammasome activation in the absence of BCAP.

    (A) Immunofluorescence staining for active caspase-1 (left), enzyme-linked immunosorbent assay (ELISA) analysis of IL-1β secretion (middle), and lactate dehydrogenase (LDH) assay of cell death (right) in BMDMs primed with LPS for 4 hours and then treated with nigericin for 30 min. Data are means + SEM pooled from three experiments. (B and C) Western blot analysis for pro–caspase-1 (B) and gasdermin D (C) cleavage in cell lysates of LPS-primed nigericin-treated BMDMs. Blots (top) are representative of three independent experiments, and band intensity values (bottom) are means + SEM pooled from experiments. (D) Live-cell imaging of cell death in LPS-primed nigericin-treated BMDMs. Data are means ± SEM of three independent experiments. (E) Western blot analysis of indicated proteins in lysates from cells treated for 16 hours with LPS. Blots (left) are representative of three experiments, and quantified band intensity values are means + SEM pooled from all experiments. (F) LDH assay of cell death in BMDMs primed with TNF for 6 hours and treated with nigericin for 30 min. Data are means ± SEM of three independent experiments. (G) Western blot analysis of indicated proteins from lysates of BMDMs primed with TNF for 6 hours. Blots (left) are representative of three independent experiments, and quantified band intensity values (right) are means + SEM pooled from all experiments. nd, not detected; ns, P > 0.05. *P < 0.05, **P < 0.01, by two-sided unpaired t test.

  • Fig. 4 Increased Yersinia-mediated inflammasome activation in the absence of BCAP.

    (A) Immunofluorescence staining for active caspase-1 (casp-1) (left), ELISA analysis of IL-1β secretion (middle), and LDH assay of cell death (right) in BMDMs primed with LPS overnight and then infected with Ypstb∆ for 90 min. Data are means + SEM pooled from four independent experiments. (B) Western blot analysis of pro–caspase-1 cleavage in lysates of BMDMs primed with LPS overnight and then infected with Ypstb∆ for 90 min. Blots (top) are representative of three independent experiments, and quantified band intensity values (bottom) are means + SEM pooled from all experiments. (C and D) Bacterial burden in the spleens of WT and BCAP−/− mice on day 4 after infection with YpstbΔYopM (C) or Ypstb (D). Data and median values (line) of at least 16 mice are from two independent experiments. *P < 0.05, **P < 0.01, by two-sided unpaired t test (A and B) and Mann-Whitney test (C and D).

  • Fig. 5 BCAP inhibits NLRC4 inflammasomes.

    (A) Immunofluorescence staining for active caspase-1 (left) and LDH assay of cell death (right) in unprimed BMDMs infected with S. Typhimurium for 30 min. Data are means + SEM pooled from three independent experiments. (B) Western blot analysis of pro–caspase-1 cleavage in lysates of BMDMs infected with S. Typhimurium for 30 min. Blots (left) are representative of three independent experiments, and quantified band intensity values are means + SEM pooled from all experiments. (C) ELISA analysis of IL-1β release from LPS-primed BMDMs infected with S. Typhimurium for 30 min. Data are means + SEM pooled from three independent experiments. (D) LDH assay of cell death in unprimed BMDMs treated with FlaTox for 20 min. Data are individual means from five independent experiments (connected with lines). (E) Live-cell imaging to measure cell death in BMDMs treated with RodTox. Data are means ± SEM of three independent experiments. (F) LDH assay of cell death in unprimed BMDMs transfected with calf thymus DNA for 3 hours. Data are means + SEM pooled from three independent experiments. (G) LDH assay of cell death in unprimed BMDMs treated with LPS and cholera toxin B for 3.5 hours. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by two-sided unpaired (A to C and E to G) and paired t test (D).

  • Fig. 6 BCAP delays caspase-1 recruitment into NLRP3 inflammasome foci.

    (A to F) Immunofluorescence analysis of NLRP3, ASC, or caspase-1 foci formation in BMDMs primed with LPS overnight and then infected with YpstbΔ. Yellow arrowheads indicate foci. Images (A, C, and E) are representative of three independent experiments, and quantification of the percentage of cells with foci of the indicated protein (B, D, and F) is means ± SEM from all experiments. (G and H) Immunofluorescence analysis of active caspase-1 and ASC in BMDMs primed with LPS overnight and then infected with YpstbΔ for 90 min. White arrowhead indicates preinflammasome focus containing ASC but not active caspase-1. Images (G) are representative of three independent experiments, and quantification of the percentage of cells with ASC foci with active caspase-1 (H) is means ± SEM from all experiments. *P < 0.05, ***P < 0.001, by two-sided unpaired t test.

  • Fig. 7 BCAP inhibition of NLRP3 inflammasome activation requires Flightless-1.

    (A) Western blot analysis of Flightless-1 in BMDMs transduced with lentiviruses encoding shRNA#1 (Flii) or NT shRNA. Blots (left) are representative of three independent experiments, and quantified KD efficiency values (right) are means + SEM from three independent experiments. (B) Immunofluorescence analysis of NLRP3, ASC, and active caspase-1 in WT BMDM transduced with NT or Flii-specific shRNA#1, primed with LPS overnight, and then infected with YpstbΔ. Data are means + SEM from three independent experiments. (C and D) Immunofluorescence analysis of active caspase-1 in WT and BCAP−/− BMDM transduced with NT shRNA or Flii-specific shRNA#1 and were either LPS-primed overnight and infected with YpstbΔ for 60 min (C) or LPS-primed for 4 hours and treated with nigericin for 30 min (D). Data are means + SEM of three independent experiments. (E and F) Western blot analysis of proteins immunoprecipitated with antibodies to Flightless-1 (E) or BCAP (F) in lysates of WT or BCAP−/− BMDM primed with LPS for 4 hours and then treated with nigericin for 15 min. Blots are representative of three independent experiments. ns, not significant. **P < 0.01, ***P < 0.001, by two-sided unpaired t test (A and B) or one-way analysis of variance (ANOVA) with Tukey’s post hoc test (C and D).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/581/eaau0615/DC1

    Fig. S1. BCAP YxxM tyrosines are not required for association with Flightless-1.

    Fig. S2. No difference in the expression of NLRP3 inflammasome components or soluble factors induced by LPS between WT and BCAP−/− macrophages.

    Fig. S3. BCAP−/− macrophages have increased caspase-1 activation after YpstbΔ infection.

    Fig. S4. No difference in NLRC4 or AIM2 expression between WT and BCAP−/− macrophages.

    Fig. S5. BCAP inhibition of inflammasome activation requires Flightless-1.

    Fig. S6. Model of NLRP3 inflammasome inhibition by BCAP and Flightless-1.

  • This PDF file includes:

    • Fig. S1. BCAP YxxM tyrosines are not required for association with Flightless-1.
    • Fig. S2. No difference in the expression of NLRP3 inflammasome components or soluble factors induced by LPS between WT and BCAP−/− macrophages.
    • Fig. S3. BCAP−/− macrophages have increased caspase-1 activation after YpstbΔ infection.
    • Fig. S4. No difference in NLRC4 or AIM2 expression between WT and BCAP−/− macrophages.
    • Fig. S5. BCAP inhibition of inflammasome activation requires Flightless-1.
    • Fig. S6. Model of NLRP3 inflammasome inhibition by BCAP and Flightless-1.

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