NLRP1-Dependent Pyroptosis Leads to Acute Lung Injury and Morbidity in Mice

Martina Kovarova, Pamela R. Hesker, Leigh Jania, MyTrang Nguyen, John N. Snouwaert, Zhidan Xiang, Stephen E. Lommatzsch, Max T. Huang, Jenny P.-Y. Ting and Beverly H. Koller
J Immunol August 15, 2012, 189 (4) 2006-2016; DOI: https://doi.org/10.4049/jimmunol.1201065

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Generation of a Nlrp1-deficient mouse line. (A) A schematic of the endogenous Nlrp1b locus, the targeting vector designed to disrupt the gene, and the expected structure of the gene after homologous recombination. (B) Southern blot analysis of DNA from ES cells (upper panel) and from tail biopsies of pups generated from the intercross of mice carrying the modified Nlrp1b locus (lower panel) verified correct recombination of the plasmid with the endogenous locus and transmission of this allele through the germline. The expected 9.5-kb XbaI fragment is observed in wild-type ES cells and in mice carrying a wild-type Nlrplb allele. Homologous recombination introduces a novel XbaI site reducing the size of the DNA fragment that binds to the probe to 7.7 kb. (C) RNA was prepared from the jejunum of 129 and Nlrp1b−/− mice, and expression of all three Nlrp1 genes was examined by PCR using primers specific for each gene. As expected, 129S6 mice express only Nlrp1b, and this expression was not observed on analysis of RNA prepared from mice homozygous for the mutant allele.

  • FIGURE 2.
    FIGURE 2.

    Lethal toxin mediated cell death and IL-1β and IL-1α cytokine release from macrophages. (A) BMDMs from 129+/+ and Nlrp1b−/− mice were incubated with LT for 4 h and cell viability was determined with the WST-1 reagent. (B–D) Thioglycolate-elicited peritoneal macrophages were primed with 100 ng/ml ultrapure LPS overnight and subsequently incubated for 6 h in presence of 1 μg/ml LT. Cell death was determined by LDH activity in supernatant and expressed as the percentage of total cellular LDH activity (B). IL-1β (C) and IL-1α (D) release to supernatant was detected by ELISA. n = 4 for 129+/+, Nlrp1b−/−, Nlrp3−/−; n = 3 for Casp-1−/−. *p < 0.05, **p < 0.01, ***p < 0.001.

  • FIGURE 3.
    FIGURE 3.

    NLRP1b is not required for IL-1β release induced by MDP or PGN. (A) Thioglycolate-elicited peritoneal macrophages were primed with 100 ng/ml ultrapure LPS for 6 h and incubated in OPTIMEM with indicated concentration of TiO2, MDP, PGN, or combinations of those stimuli for 16 h. IL-1β from supernatants was detected by ELISA; n = 4. (B) Thioglycolate-elicited peritoneal macrophages were primed as in (A) follow by treatment with 1 mg/ml L18-MDP, 5 mg/ml TiO2, or 5 mg/ml TiO2 and 10 mg/ml MDP for 16 h. IL-1β was detected by ELISA in supernatants; n = 4 (C, D) 129+/+ (▪) and Nlrp1b−/− (□) mice were injected i.p. with 5 μg LPS. Three hours later, mice were i.p. injected with 50 μg L18–MDP. Serum and peritoneal lavage fluid (PLF) was collected 1.5 h postinjection with L18–MDP. The total number of inflammatory cells in peritoneal lavage fluid (60% neutrophils) (C) and IL-1β in the serum, and PLF measured by ELISA (D) were determined; n = 13 for 129+/+, 14 for Nlrp1b−/−, #p < 0.0001.

  • FIGURE 4.
    FIGURE 4.

    Nlrp1b is required for LT-mediated release of IL-1β and IL-1α in the lung. 129+/+ (▪) and Nlrp1b−/− (□) mice were treated with 0.3 mg/kg LPS or PBS i.t. at 0 h, and with PBS, 1 μg LT, or 50 μl 100 mM ATP i.t. at 2 h. BAL fluid was collected at 4 h after the initial treatment. IL-1β (A) and IL-1α (B) in the BAL determined by ELISA. (C) Processed IL-1β and caspase-1 proteins from concentrated BAL fluid analyzed by Western blot, 20 μg protein per line, two representative results from five independent experiments shown.***p < 0.001, #p < 0.0001, n = 1 for PBS, n = 10 for LPS/LT, n = 4 for LPS/ATP.

  • FIGURE 5.
    FIGURE 5.

    Nlrp1b−/− mice are protected from lethal toxin-induced lung injury. 129+/+ (▪) and Nlrp1b−/− (□) mice were treated with 20 μg LT i.t. BAL fluid was collected at 0, 2, 6, 16, and 24 h after treatment. MIP-2 (A), KC (B), IL-1β (C), and CXCL15/lungkine (D) in the BAL fluid were detected by ELISA. (E) Western blot analysis of HMGB1 protein in concentrated BAL fluid; GAPDH was used as a loading control, 20 μg protein per line. Results shown are representative of three independent experiments. (F) The total number of inflammatory cells in BAL fluid. (G) Cell death in BAL fluid assessed by LDH activity. (H) Vascular permeability evaluated as a concentration of albumin in the BAL fluid, measured by colorimetric assay. (I) MPO level in BAL cell pellet collected from 129+/+ and Nlrp1b−/− mice 48 and 72 h after LT treatment. (J–N) FACS analysis of whole lung cell suspension from 129+/+ (J, L) and Nlrp1b−/− mice 16-h post-LT treatment (K, M). CD45+ cells were gated and analyzed for Mac-1 and GR-1 epitope (J, K) or Mac-1 and CCR-2 epitope (L, M). (J)–(M) are representative of three independent experiments, quantitative analysis of CD45+Mac-1+CCR2+ cells (N). n = 2 per genotype at 0 h, n = 5/genotype at 2 h, n = 6/genotype at 6 h, n = 3/genotype at 16 h, n = 7/genotype at 24–72 h. *p < 0.05, **p < 0.01, ***p < 0.001.

  • FIGURE 6.
    FIGURE 6.

    Histological analysis of LT-induced lung injury. Micrographs of H&E-stained sections of lungs harvested from 129+/+ and Nlrp1b−/− mice after treatment with 20 μg LT i.t. Lungs were harvested at the indicated times (A–H), at 48 h (I, L), or at 72 h (J, K, M, N). Scale bars, 250 μm (A–H); 10 μm (I–N). Original magnification ×2 (A–H), ×40 (I–N). KO, Knockout; WT, wild-type.

  • FIGURE 7.
    FIGURE 7.

    LT-induced lung injury is dependent on caspase-1 but not IL-1β. (A–E) Casp-1+/+ (black) and Casp-1−/− (shade) mice were treated with PBS as a vehicle or 20 μg LT i.t. BAL fluid was collected at 72 h after treatment. (A) The total number of inflammatory cells collected in the BAL fluid. (B) Cell death in BAL fluid determined by LDH activity in supernatants and expressed as the percentage of total cellular LDH activity. (C) Vascular permeability evaluated as a concentration of albumin in the BAL fluid, measured by colorimetric assay. KC (D) and CXCL15/lungkine (E) in the BAL fluid were detected by ELISA. n = 2 mice/genotype for vehicle treatment and n = 10 mice/genotype for LT treatment. (F and G) Il1β+/+ (black) and Il1β−/− (striped) mice were treated as above. (F) The total number of inflammatory cells collected in BAL fluid. (G) The percentage of weight lost 72 h after treatment. n = 2/genotype for vehicle treatment and n = 9 mice/genotype for LT treatment. *p < 0.05, **p < 0.01, #p < 0.0001.

  • FIGURE 8.
    FIGURE 8.

    LT activates caspase-1 in the lung independently on P2X7 receptor. (A and B) 129+/+, Nlrp1b−/−, and Casp-1−/− mice were treated with 20 μg LT by i.t., BAL fluid was collected 2 h after treatment, and cells were stained with fluorescently labeled caspase inhibitors and with 7-AAD; n = 4/genotype. Cells were analyzed on FACS and compared with untreated controls. (A) Data are expressed as the difference in median fluorescent intensity (MFI) in the LT-treated mice and untreated mice. (B) A representative overlay of the untreated (gray filled) and LT-treated (black line) cell populations. (C–F) BALB/c or P2X7−/− mice were treated with 20 μg LT i.t. BAL fluid was collected at 72 h after treatment. (C) The total number of inflammatory cells collected in the BAL fluid. (D) Vascular permeability evaluated as a concentration of protein in the BAL fluid, measured by bicinchoninic acid assay. (E) Cell death in BAL fluid determined by LDH activity in supernatants. (F) KC in the BAL fluid was detected by ELISA, n = 5 mice/genotype. *p < 0.05, **p < 0.01, ***p < 0.001 compared with WT.

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