NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability

Abstract

The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic ALI in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP-1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type-II cells (AT-II) and THP-1 co-cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase-1, IL-1β cleavage, and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short-hairpin RNA silencing of inflammasome components abrogated ceramide-induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide-induced alveolar epithelial permeability in AT-II. Collectively, our results demonstrate for the first time that ceramide-induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation.

Figure1. Hyperoxia induced lung injury causes increase of intracellular ceramide levels

Ceramide concentrations were analyzed in homogenized lung tissues at different stages of hyperoxia-induced acute lung injury. C57BL/6 mice were exposed to hyperoxia (100% O₂) for 0, 24, 48 and 72h time points and lung tissues were isolated and analyzed for ceramide levels by the DAG kinase assay. Asterisks indicate significant differences compared to the respective time points of normoxia mice at 0h (n=6) (mean s.d.; ^**P < 0.01, ^***P < 0.001). The noted experiments are representative of a minimum of three similar evaluations.

Fig 2. Effect of ceramide on mouse alveolar epithelial and macrophages

(A) Dose-response curve showing survival of alveolar epithelial type-II cells (AT-II) after exposure to varying concentrations of ceramide ranging from 10 μM to 50 μM for 12 h. Cell viability was analyzed by using trypan blue exclusion. Mouse alveolar macrophages were isolated and treated with 30 μM of ceramide for the indicated time points and levels of (B) IL-1β, (C) IL-6 and (D) TNF-α were analyzed by ELISA. Asterisks indicate significant differences compared to the respective time points of control and ceramide treatment (mean s.d.; ^**P < 0.01). The noted experiments are representative of a minimum of three similar evaluations.

Fig 3. Inflammasome silence blunted ceramide induced cytokine production in THP-1

(A)Inflammasome activation was assessed by analyzing caspase-1 cleavage using Western blot analysis. shRNA silencing of NALP-3 abrogated ceramide-induced caspase-1 cleavage (upper panel) as well as secretion of IL-1β in ceramide treated (30 μM) THP-1 cells (lower panel, graph). ATII cells were grown in 24-well transwell plates until the cellular monolayers reached confluence. THP-1 cells were added to the appropriate ATII monolayers, cocultured for 6 h, and exposed to ceramide for 12 h. In some experiments, THP-1 cells were transfected with control shRNA or NALP-3 shRNA and then added to a monolayer of ATII cells. The media contents were collected and (B) IL-1β, (C) TNF-α and (D) IL-6 proinflammatory cytokines were analyzed in extracellular media by ELISA. Asterisks indicate significant differences compared to the AT-II (^***P < 0.001) and compared to control shRNA (mean s.d.; ^**P < 0.01). The noted experiments are representative of a minimum of three similar evaluations.

Fig 4. Inflammasome silencing inhibited ceramide induced transepithelial albumin flux in alveolar epithelial type II cells

(A) ATII cells were grown in 24-well transwell plates until the cellular monolayers reached confluence. THP-1 cells were added to the appropriate ATII monolayers, cocultured for 6 h, and exposed to ceramide for 12 h. In some experiments, THP-1 cells were transfected with control shRNA or NALP-3 shRNA and then added to a monolayer of ATII cells. Transepithelial albumin flux across each monolayer was determined by the addition of [¹²⁵I]-labeled human serum albumin to each upper compartment. The contents from the lower compartment were collected and counted in a Wizard γ-counter. *p<0.01 relative to AT-II cells; ^#p < 0.05, relative to control shRNA. (B) Effect of inflammasome silencing on TER in AT-II and THP-1 cells. ATII cells were grown in 24-well transwell plates until the cellular monolayers reached confluence. THP-1 cells were added to the appropriate ATII monolayers, cocultured for 6 h, and exposed to ceramide for 12 h. In some experiments, THP-1 cells were transfected with control shRNA or NALP-3 shRNA and then added to a monolayer of ATII cells. TER was measure by an epithelial voltammeter. *p<0.01 relative to AT-II cells; **p < 0.05, relative to NALP-3 shRNA. The noted experiments are representative of a minimum of three similar evaluations.

Fig 5. Proposed scheme for the role of inflammasome in ceramide-induced alveolar permeability and pulmonary edema

In hyperoxia, ceramide accumulation causes inflammasome activation and IL-1β production in macrophages. IL-1β further causes epithelial damage and releases other cytokines such as IL-6 and TNF-α which causes epithelial permeability and pulmonary edema. Silencing of NALP-3 within the inflammasome caused blunting of the cytokine production which leads to suppression of permeability and pulmonary edema.