Critical role for the NLRP3 inflammasome during acute lung injury

Abstract

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger-associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1β and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1β, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Ab-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K(+), increased intracellular Ca(2+) concentration, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1β levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models, suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug.

Figure 1

The NLRP3 inflammasome and caspase-1 mediate LPS-induced ALI. Wild type or the indicated knockout mice received 60 μg LPS (i.t.) under anesthesia. Sham mice received sterile saline. BALFs were harvested 8 hours later and levels of A) albumin, B) IL-1β, and C) TNF were determined by ELISA. D) Neutrophil numbers in BALFs were counted (n ≥ 5 mice per group). E) Lung epithelial barrier integrity in ALI. Tight junctional (Tj) proteins claudin-3 and occludin immunostaining in lung of mice subjected to LPS-induced ALI (Wt ALI) showed defragmented and low intensity staining in alveolar septa (arrowhead) indicating the loss of Tj between epithelial cells, compared with control (Wt sham) where both claudin-3 and occludin had a linear staining pattern (arrows). ALI in caspase-1^−/− and NLRP3^−/− mice induced only partial changes in linear staining of claudin-3 and occludin, indicating only limited alteration of Tj and lung epithelial barrier. Scale bar is 50 μm. Left hand side, H & E features of wild type sham lung, wild type ALI, and ALI in caspase-1^−/− or NLRP3^−/− mice. See text for details.

Figure 2

IL-1β production is dependent on the presence of macrophages and neutrophils during ALI. A) IL-1β levels measured in BALF at time points during LPS-induced ALI (n ≥ 5 mice per group). B) IL-1β presence in BALF during ALI (8 hours) in control (PBS Lipo.) or macrophage depleted (Clodr Lipo.) mice (n ≥ 5 mice per group). C) IL-1β presence in BALF during ALI (8 hours) in control or neutrophil depleted mice (n ≥ 6 mice per group). D) IL-1β levels in culture supernatants from mouse peritoneal macrophages (left side) or neutrophils (right side) treated in the presence or absence of LPS (100 ng/ml) for 4 hrs followed by ATP (1 mM) for 45 min. Results are triplicate samples representative of 3 independent experiments.

Figure 3

Neutrophils are the source of extracellular histones during LPS-induced ALI. A) Histone detection (BWA3 antibody) in tissue sections from sham or ALI lung. The scale bar in A is for all images. B) Histones measured in BALF at time points during LPS-induced ALI (n ≥ 5 mice per group). C) Histone presence in BALF during ALI (8 hours) in control or neutrophil depleted mice (n ≥ 6 mice per group). D) ALI (6 hours) lung tissue sections were labeled for histones (BWA3 antibody) as well as neutrophils (Ly6G). Most histone-associated cells were neutrophils (arrows), however a small number of histone-associated cells were not neutrophils (arrowheads). The scale bar in D is for all images. E) Quantitative analysis of histone-associated cell subytpes. Tissues sections were labeled for histones as well as neutrophils (Ly6G), alveolar macrophages/dendritic cells (CD11c), or type II epithelial cells (surfactant A). The percentage of histone-associated cells that co-labeled with the cell type specific marker is shown (n=3 mice).

Figure 4

Release of histones by neutrophils in vitro. A) Purified bone marrow neutrophils were incubated in the presence of PMA (50 ng/ml), C5a (1 μg/ml), or vehicle (untreated) for 90 min. Cells were fixed and histones were labeled with BWA3 antibody (H2A/H4). Images are representative of 2 independent experiments. The scale bar in A is for all images. B) Higher magnification images of C5a-treated neutrophils as described in A. The scale bar in B is for both images. C) Quantitation of histones present in culture supernatants of neutrophils treated as described in A (n=4 samples).

Figure 5

Extracellular histones activate the inflammasome. A–B) Albumin and IL-1β levels in BALF 6 hours following i.t. administration of histones or BSA (250 μg). Sham animals received sterile PBS (n ≥ 5 mice per group). C) LPS-induced ALI in the presence of histone H2A/H4 neutralizing antibody (Anti-H2A/H4) or isotype control antibody. IL-1β levels found in BALF after 8 hours is shown (n=6–7 per group). D) RT-PCR analysis of IL-1β mRNA from mouse peritoneal macrophages treated in vitro for 3 hours with LPS or histones. E–F) Macrophages or neutrophils (5 × 10⁵ each) were primed with LPS (100 ng/ml) for 4 hours, then treated with ATP or various concentrations of histones (Hist) for 45 min. The level of IL-1β in culture supernatants was determined by ELISA. G) Macrophages were treated as described in E. Cell lysates or culture supernatants were analyzed by Western blot for the presence of IL-1β. H) Macrophages were treated as in E, but histone preparations were pretreated with DNAse (10 U/ml), RNAse (10 U/ml) or proteinase K (20 U/ml) prior to cell treatment. In vitro experiments are triplicate samples representative of ≥ 3 independent experiments.

Figure 6

Mechanism of inflammasome activation by extracellular histones. A) Macrophages were primed with LPS (100 ng/ml), then treated with ATP (1 mM) or histones (50 μg/ml). IL-1β levels in supernatants were determined by ELISA. B) Macrophages were treated with histones (50 μg/ml) for 1 hour and forward scatter (cell size) was measured by flow cytometry. C) Macrophages were analyzed as in B, at time points following histone addition. Mean forward scatter (arbitrary units, AU), representing cell swelling, was determined from > 2 × 10⁴ cells per sample. Results are triplicate samples representative of 2 independent experiments. D–F and H–K) Macrophages were treated as described in A. As indicated, cells were pretreated with inhibitors 15–30 min prior to ATP or histones. G) Macrophage ROS production in response to histones (n=4 mice) determined by flow cytometry (CellRox Red). Unless otherwise indicated, in vitro experiments are triplicate samples representative of ≥ 3 independent experiments.

Figure 7

Properties of individual histones. Inflammasome activation and cell swelling assays were performed as described for Fig. 6. The ability of individual histones to A) activate the inflammasome or B) induce swelling of macrophages is shown. Results are triplicate samples representative of 3 independent experiments.

Figure 8

The NLRP3 inflammasome is required for full extracellular histone presence during LPS-induced ALI (8 hours). A) Histone levels found in BALF from wild type or NLRP3^−/− mice (n ≥ 5 mice per group). B) Histone levels found in BALF from wild type or caspase-1^−/− mice (n ≥ 5 mice per group).

Figure 9

Evidence that histone/inflammasome-mediated inflammation is conserved among ALI models. A) Albumin, histone, and IL-1β levels observed in BALF 6 hours after the i.t. instillation of C5a (500 ng). Sham animals received sterile PBS (n=4 mice per group). B) Albumin, histone, and IL-1β levels found in BALF 6 hours after the distal airway deposition of IgG immune complexes. Sham animals received only anti-BSA (n=5 mice per group).

Figure 10

Model of the interactions between extracellular histones and the NLRP3 inflammasome during ALI. 1) Neutrophils are the source of extracellular histones during ALI (Fig. 3 and 4). 2) Extracellular histones activate the NLRP3 inflammasome (Fig. 5). 3) The NLRP3 inflammasome is required for optimal neutrophil influx (Fig. 1D) and histone presence (Fig. 8).