The Nalp3 inflammasome is essential for the development of silicosis

Abstract

Inhalation of crystalline silica and asbestos is known to cause the progressive pulmonary fibrotic disorders silicosis and asbestosis, respectively. Although alveolar macrophages are believed to initiate these inflammatory responses, the mechanism by which this occurs has been unclear. Here we show that the inflammatory response and subsequent development of pulmonary fibrosis after inhalation of silica is dependent on the Nalp3 inflammasome. Stimulation of macrophages with silica results in the activation of caspase-1 in a Nalp3-dependent manner. Macrophages deficient in components of the Nalp3 inflammasome were incapable of secreting the proinflammatory cytokines interleukin (IL)-1beta and IL-18 in response to silica. Similarly, asbestos was capable of activating caspase-1 in a Nalp3-dependent manner. Activation of the Nalp3 inflammasome by silica required both an efflux of intracellular potassium and the generation of reactive oxygen species. This study demonstrates a key role for the Nalp3 inflammasome in the pathogenesis of pneumoconiosis.

Fig. 1.

Silica induces IL-1β secretion from LPS-primed macrophages. (A–C) Macrophages from WT mice were stimulated with either 50 μg/cm² silica or 50 ng/ml LPS. Culture supernatants were collected at the indicated time after stimulation; TNFα (A) and IL-6 (B) release into culture supernatants was measured by ELISA. (C) IL-1β and IL-12p40 expression in silica- or LPS-stimulated macrophages was determined by RT-PCR. (D–F) Macrophages from WT mice were either primed with 50 ng/ml LPS or left untreated. Macrophages were stimulated with the indicated dose of silica (D), asbestos (E), or titanium dioxide (F), and culture supernatants were collected 4 h later. IL-1β release into culture supernatants was measured by ELISA. (G) Human alveolar macrophages were either primed with 50 ng/ml LPS or left untreated. Macrophages were stimulated with 50 μg/cm² silica or 40 μg/cm² asbestos for 4 h, and IL-1β release into culture supernatants was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two separate experiments.

Fig. 2.

Silica- and asbestos-induced IL-1β secretion is dependent on Nalp3. LPS-primed macrophages from WT, caspase-1-, ASC-, Nalp3-, or IPAF-deficient mice were stimulated with 50 μg/cm² silica. Culture supernatants were collected at 4 h or at the indicated time after stimulation (A–C) and IL-1β and IL-18 release into culture supernatants was measured by ELISA (A and B). Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of three (A) and two (B) separate experiments. (C) Lysates from LPS-primed WT, ASC-, NALP3-, or IPAF-deficient macrophages stimulated with 50 μg/cm² silica for the indicated times were immunoblotted with antibodies against the p10 subunit of caspase-1. (D) LPS-primed macrophages from WT and NALP3-deficient mice were stimulated with either 40 μg/cm² asbestos or 50 μg/cm² titanium dioxide. Culture supernatants were collected 6 h later and IL-1β release into culture supernatants was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of three separate experiments. *, Nondetectable.

Fig. 3.

Silica-induced IL-1β secretion requires internalization of silica, a potassium efflux, and generation of reactive oxygen species. (A) LPS-primed WT macrophages were stimulated with the indicated amount of silica. Culture supernatants were collected 4 h later and cytotoxicity was measured by lactate dehydrogenase (LDH) release and expressed as a percentage of LDH release by Triton X-100 detergent. (B) LPS-primed WT macrophages were incubated with 10 μM cytochalasin B or 20 μM cytochalasin D for 10 min before the addition of 50 μg/cm² silica or 5 mM ATP. (C) LPS-primed macrophages were incubated in either high Na⁺- or high K⁺-containing medium and then stimulated with either 50 μg/cm² silica or 5 mM ATP. ATP-treated cells had their medium replaced with fresh medium after 20 min. Four hours after initial stimulation, culture supernatants were collected and IL-1β release was measured by ELISA. (A–C) Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two separate experiments. (D and E) WT and Nalp3−/− macrophages were stimulated with the indicated amount of silica, and ROS production was measured by luminol-amplified chemiluminescence. RLU, relative light units. (F and G) LPS-primed WT macrophages were incubated with 10 μM or the indicated amount of diphenyleneiodonium chloride (DPI) for 10 min before the addition of 50 μg/cm² silica. Four hours after initial stimulation, culture supernatants were collected and IL-1β release was measured by ELISA (F). Determinations were performed in triplicate and expressed as the mean ± SEM and are representative of three separate experiments. At the indicated time after stimulation lysates were collected and immunoblotted with antibodies against the p10 subunit of caspase-1, caspase-3, and actin (G). *, Nondetectable.

Fig. 4.

Decreased inflammation and collagen deposition in ASC- and Nalp3-deficient mice after intranasal instillation of silica. WT, ASC−/−, and Nalp3−/− mice received either intranasal PBS or silica. Three months after the initial challenge, lungs were stained with H&E to examine inflammation (A) or trichrome stain to examine collagen deposition (B). H&E-stained sections were scored in a blinded fashion for cellular tissue infiltration (*, P = 0.039; **, P = 0.0062 compared with WT by one-tailed unpaired Student's t test). Images were obtained at ×10 (A) and ×20 (B) magnification and are representative of two mice per group for PBS and five mice per group for silica.