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. 2022 Oct 6;23(10):e54277.
doi: 10.15252/embr.202154277. Epub 2022 Jul 28.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis

Dhruv Chauhan #  1 , Dieter Demon #  2   3 , Lieselotte Vande Walle  2   3 , Oonagh Paerewijck  2 , Annalisa Zecchin  1 , Leslie Bosseler  4 , Karin Santoni  5 , Rémi Planès  5 , Silvia Ribo  2   3 , Amelie Fossoul  2   3 , Amanda Gonçalves  3   6 , Hanne Van Gorp  2   3 , Nina Van Opdenbosch  1   2   3 , Filip Van Hauwermeiren  1   2   3 , Etienne Meunier  5 , Andy Wullaert  2   3   7 , Mohamed Lamkanfi  2
Affiliations

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis

Dhruv Chauhan et al. EMBO Rep. .

Abstract

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1β secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1β in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1β are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Keywords: NETosis; gasdermin D; inflammasome; neutrophil; pyroptosis.

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Figures

Figure 1
Figure 1. Neutrophil‐intrinsic NLRP3A350V activation promotes auto‐inflammatory pathology in mice
  1. A, B

    Neutrophils isolated from tamoxifen‐treated Nlrp3 A350V/wt CreT+ and Nlrp3 A350V/wt CreT− mice were incubated with LPS (3 h) before culture media were analysed for secreted IL‐1β (A), and cell lysates and culture media were immunoblotted for β‐actin, caspase‐1 and IL‐1β (B). Cytokine values represent mean ± SEM of n = 4 biological repeats. Statistical significance was analysed by two‐way ANOVA and P‐values were corrected for multiple comparisons (Bonferroni). **P ≤ 0.01.

  2. C

    Representative pictures of Nlrp3 A350V/wt MRP8‐CreTg and Nlrp3 A350V/wt MRP8‐Cre− mice on days 4 and 8 after birth. Left side in each picture: Nlrp3 A350V/wt MRP8‐Cre− mice; right side in each picture: Nlrp3 A350V/wt MRP8‐CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3 A350V/wt MRP8‐CreTg mice.

  3. D

    Survival curves of Nlrp3 A350V/wt MRP8‐CreTg (n = 22) and littermate Nlrp3 A350V/wt MRP8‐Cre− (n = 21) mice. Statistical significance was analysed by the log‐rank Mantel–Cox test. ****P ≤ 0.0001.

  4. E

    Representative H&E‐stained sections of the spleen (top, scale bar: 100 μM) and liver (bottom, scale bar: 200 μM) of Nlrp3 A350V/wt MRP8‐CreTg and littermate Nlrp3 A350V/wt MRP8‐Cre− mice. Spleens of Nlrp3 A350V/wt MRP8‐Cre− control mice (top left) display characteristic white pulp (WP) regions (example shown surrounded by dashed line) composed of lymphoid follicles and periarteriolar lymphoid sheaths (PALS), which at this magnification are recognized by dark‐purple staining, large aggregates of lymphocytes surrounding the central artery (CA) and their distinctive separation from the red‐staining red pulp (RP). WP was largely absent in spleens of Nlrp3 A350V/wt MRP8‐CreTg (top middle). Megakaryocytes (MK) are indicated for easier recognition of areas of extramedullary haematopoiesis (EMH). Most of the spleen parenchyma of Nlrp3 A350V/wt MRP8‐CreTg animals was replaced by EMH of mostly myeloid lineage, which can be recognized by the medium‐sized cells with more cytoplasm, giving a lighter appearance (inset). In both the overview picture (top middle picture) and inset (top right) of Nlrp3 A350V/wt MRP8‐CreTg spleen, a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker‐stained, smaller cells (asterisk, *). Liver sections of both Nlrp3 A350V/wtMRP8‐Cre− control mice and diseased Nlrp3 A350V/wt MRP8‐CreTg pups displayed comparable EMH of the erythroid lineage (bottom left picture, indicated with yellow arrows). Liver sections of Nlrp3 A350V/wt MRP8‐CreTg pups also showed a modest increase in EMH of the myeloid lineage (bottom middle picture and inset (scale bar: 100 μM) in the bottom right, indicated with red arrowheads), as well as hepatocyte enlargement.

  5. F–I

    H&E‐stained histological sections of the spleen (F, G) or liver (H, I) of Nlrp3 A350V/wt MRP8‐Cre− (Cre−, n = 8) and Nlrp3 A350V/wt MRP8‐CreTg (CreTg, n = 6) mice were assigned a score of 0–5 for lymphoid depletion (F) or EMH (G, H) or hepatocyte enlargement (I) by a board‐certified pathologist. Values represent mean ± SEM of n = 6–8 biological repeats. Statistical significance was analysed by the Mann–Whitney U‐test. ***P ≤ 0.001.

  6. J–L

    Cytokine secretion analysis (Luminex) of serum samples obtained on day 7 from Nlrp3 A350V/wt MRP8‐Cre− (Cre−, n = 8) and Nlrp3 A350V/wt MRP8‐CreTg (CreTg, n = 6) pups. Values represent mean ± SEM of n = 6–8 biological repeats. Statistical analysis of serum cytokine levels was performed by the Welch's t‐test. *P ≤ 0.05; **P ≤ 0.01.

Source data are available online for this figure.
Figure EV1
Figure EV1. Neutrophil NLRP3A350V activation promotes auto‐inflammation in mice
  1. A

    Representative H&E‐stained sections of the heart (scale bar: 200 μM), small intestine (scale bar: 200 μM) and colon (scale bar: 100 μM) of Nlrp3 A350V/wt MRP8‐CreTg and littermate Nlrp3 A350V/wt MRP8‐Cre‐ mice. A mild EMH (arrows) is present in the heart sections of Nlrp3 A350V/wt MRP8‐CreTg mice.

  2. B

    An expert pathologist graded the presence of EMH in H&E‐stained histological sections of the heart of Nlrp3 A350V/wt MRP8‐Cre‐ (Cre‐, n = 8) and Nlrp3 A350V/wt MRP8‐CreTg (CreTg, n = 6) pups using a score of 0–5. Values represent mean ± SEM of n = 6–8 biological repeats. Statistical analysis of histological damage score was determined by Mann–Whitney U‐test. ***P < 0.001.

  3. C–Q

    Cytokine and chemokine concentrations in serum samples obtained on day 7 from Nlrp3 A350V/wt MRP8‐Cre‐ (Cre‐, n = 8) and Nlrp3 A350V/wt MRP8‐CreTg (CreTg, n = 6) pups. Values represent mean ± SEM of biological repeats. Statistical analysis of serum cytokine levels was performed using Welch's t‐test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure EV2
Figure EV2. Analysis of neutrophil enrichment by negative selection
  1. A–C

    Representative flow cytometry plots of neutrophils isolated from the bone marrow of C57Bl6/N (A), B6Nlrp1b+ (B) and AIM2 KO (C) mice. Left plots: identification of CD11b+ enriched population; right panels: Ly6G+/Ly6C+ neutrophils on gated CD11b+ cells. Data are representative of n = 3 biological repeats.

Figure 2
Figure 2. Canonical inflammasomes driving IL‐1β maturation and secretion in neutrophils
  1. A–H

    LPS‐primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 h) (A, B), TcdA (3 h) (C, D), infected with F. tularensis (18 h) (E, F) or stimulated with FlaTox (3 h) (G, H). IL‐1β secretion levels were determined in culture media (A, C, E, G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro‐IL‐1β (~39 kDa) into mature IL‐1β (p17) (B, D, F, H). Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats. Statistical significance was analysed by two‐way ANOVA: *P ≤ 0.05; **P ≤ 0.01. P‐values were corrected for multiple comparisons (Bonferroni).

Source data are available online for this figure.
Figure EV3
Figure EV3. Analysis of canonical inflammasome‐induced IL‐1β secretion
  1. A, B

    LPS‐primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with ATP (1 h). IL‐1β secretion levels were determined in culture media (A); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro‐IL‐1β (~39 kDa) into mature IL‐1β (p17) (B). Cytokine values represent mean ± SEM of n = 3 biological repeats. Statistical significance was determined by two‐way ANOVA: **P ≤ 0.01. P‐values were corrected for multiple comparisons (Bonferroni).

  2. C–E

    Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium (3 h). IL‐1β secretion levels were determined in culture media (C, E), and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro‐IL‐1β (~39 kDa) into mature IL‐1β (p17) (D). Cytokine values represent mean ± SEM of n = 3 biological repeats. Statistical significance was determined by two‐way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ns = non‐significant. P‐values were corrected for multiple comparisons (Bonferroni).

  3. F

    LPS‐primed human blood neutrophils were left untreated (Mock) or infected with S. Typhimurium (MOI 0.5) for 3 h in the presence or absence of VX‐765 before IL‐1β secretion levels in culture media were determined. Cytokine data represent mean ± SEM of n = 4 donors. Statistical significance was determined by one‐way ANOVA: **P ≤ 0.01. P‐values were corrected for multiple comparisons (Bonferroni).

Source data are available online for this figure.
Figure EV4
Figure EV4. LPS protects neutrophils from spontaneous PMP
  1. A–F

    Neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LPS. PMP was assessed by measurement of SYTOX Green incorporation over time (A–F). Values represent mean ± SEM of three independent biological repeats (A–C, E, F). Statistical significance was determined by non‐parametric wilcoxon two‐tailed t‐test: *P ≤ 0.05; **P ≤ 0.01. (D) Data are depicted as individual data points with a mean of n = 2 biological repeats.

Figure 3
Figure 3. Canonical inflammasomes promote plasma membrane permeabilization (PMP) in neutrophils
  1. A–D

    LPS‐primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), TcdA (B), ATP (C) or FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n = 3 biological repeats. Statistical significance was analysed by two‐way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. P‐values were corrected for multiple comparisons (Bonferroni).

  2. E

    LPS‐primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 h. Data are representative of n = 3 biological repeats.

  3. F

    B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole‐cell lysates (Lys) were immunoblotted for the release of cytosolic GAPDH into culture media. Data are representative of n = 3 biological repeats.

Source data are available online for this figure.
Figure 4
Figure 4. GSDMD drives canonical inflammasome‐induced PMP and IL‐1β secretion in neutrophils
  1. A–I

    LPS‐primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 h) (A, D, G), FlaTox (3 h) (B, E, H) or nigericin (100 min) (C, F, I).

  2. A–C

    Whole‐cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro‐IL‐1β (~39 kDa) into mature IL‐1β (p17) and cleaved GSDMD (p30). * With Western blots indicates non‐specific cross‐reactivity of the antibody. Data are representative of n = 3 (A, B) or n = 2 (C) biological repeats.

  3. D–F

    PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n = 3 (D, E) or n = 4 (F) biological repeats. Statistical significance was analysed by two‐way ANOVA: **P ≤ 0.01; ***P ≤ 0.001. P‐values were corrected for multiple comparisons (Bonferroni).

  4. G–I

    IL‐1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n = 3 (G, I) or n = 4 (H) biological repeats. Statistical significance was analysed by two‐way ANOVA: *P ≤ 0.05; **P ≤ 0.01.

Source data are available online for this figure.
Figure 5
Figure 5. PMA‐induced NETosis requires NADPH oxidase activity, but not GSDMD
  1. A–H

    LPS‐primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 h) (A–D), nigericin (100 min) (E–G) or TcDA (3 h) (H). Whole‐cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro‐IL‐1β (~39 kDa) into mature IL‐1β (p17) and cleaved GSDMD (p30) (A), and extracellular release of β‐actin and GAPDH (C–F, H). * With Western blots indicates non‐specific bands. (B, G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n = 3 biological repeats. Statistical significance was analysed by two‐way ANOVA: ***P ≤ 0.001; **P ≤ 0.01; ns = non‐significant. P‐values were corrected for multiple comparisons (Bonferroni).

Source data are available online for this figure.
Figure EV5
Figure EV5. Caspase‐1 is dispensable for PMA‐induced NETosis
  1. A–C

    LPS‐primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with PMA (3 h) before IL‐1β was measured in culture media (A). (B) Representative confocal micrographs of Mock‐ and PMA‐stimulated neutrophils of the indicated genotypes stained with DAPI (dark‐blue nuclei) and SYTOX Green (light blue) taken at the indicated time points (scale bar: 20 μM). (C) PMP was assessed by measurement of SYTOX Green incorporation over time.

  2. D, E

    LPS‐primed human blood neutrophils were left untreated (Mock) or stimulated with PMA in the presence or absence of VX‐765. PMP was determined by SYTOX green uptake over time (D) and cell death was measured by LDH release in culture media 3 h post‐stimulation (E).

Data information: Error bars represent mean ± SEM of n = 3 (A) or n = 4 (C–E) biological repeats. (C–E) Statistical significance was determined by two‐way ANOVA (C, D) or one‐way ANOVA (E): *P ≤ 0.05; **P ≤ 0.01; ns = non‐significant. P‐values were corrected for multiple comparisons (Bonferroni).
Figure 6
Figure 6. Caspase‐1 and GSDMD drive S. Typhimurium‐induced neutrophil pyroptosis
  1. A–H

    Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM. (B) Representative confocal micrographs of untreated (Mock), S. Typhimurium‐infected or PMA‐stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM). (C, D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1 and 3 h post‐infection. (E) Cell death was measured by LDH assay 3 h post‐infection. (F) IL‐1β secretion levels were determined by Luminex assay 1 h post‐infection. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark‐blue nuclei) and SYTOX Green (light blue) taken at different time points post‐infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time.

Data information: In E, F and H, values represent mean ± SEM of n = 3 biological repeats. Statistical significance was analysed by two‐way ANOVA: **P ≤ 0.01; ***P ≤ 0.001. P‐values were corrected for multiple comparisons (Bonferroni). Other data are representative of n = 3 (A, B, G) or n = 2 (C, D) biological repeats. S. Typh.: S. Typhimurium. Source data are available online for this figure.
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