NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

Abstract

Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes.

Figure 1. NLRP3 protects the alveolar barrier during pneumococcal pneumonia and PLY treatment.

(a–e) 8–10 week old female mice were intranasally infected with 5 × 10⁶ S. pneumoniae PN36 for 24 h. (a) Lung permeability was quantified by measuring the HSA BALF/serum ratio after i.v. injection of HSA. Bacterial loads in BALF (b), neutrophil infiltration (c), as well as IL-1β (d) and IL-18 production (e) were measured 24 h post infection. (f,g) 8–10 week old female mice were intranasally infected with 5 × 10⁶ S. pneumoniae D39 for 24 h and lung permeability was quantified by measuring the HSA BALF/serum ratio in BALF after i.v. injection of HSA (f). Bacterial loads 24 h p.i were measured in BALF (g). (h) Isolated perfused and ventilated lungs from WT and Nlrp3−/− mice were intratracheally treated with PBS or PLY (0,2 μg/ml), and lung permeability was determined 30 min after stimulation by measuring leakage of HSA from the perfusion system to the broncho-alveolar space. Values are given as mean ± SEM; n = 7–11. *p < 0.05, **p < 0.01, n.s. = not significant.

Figure 2. NLRP3 enhances resistance of alveolar epithelial cell monolayers against PLY-dependent interruption.

MLECs and AECs from WT (black line) and Nlrp3−/− (red line) mice were isolated and their purity was determined by flow cytometry (a,b). MLECs (c) and AECs (d), as well as AECs co-cultured with alveolar macrophages (AMΦ) (e) were grown on ECIS electrodes. The cell monolayers were treated with 1 μg/mL PLY and resistance was measured continuously for 2 h by ECIS. Values represent a mean of at least 4 independent experiments performed in duplicates and are given as mean ± SEM. Statistical significance of the resistance drop was measured 30 min after PLY stimulation (indicated by grey dotted line). *p < 0.05, **p < 0.01, n.s. = not significant.

Figure 3. The protective effect of NLRP3 for the alveolar permeability is independent of inflammasomes, IL-1β and IL-18.

(a–c). Permeability of isolated perfused and ventilated lungs from WT, Asc−/−, Il1a/b−/− and Il18−/− was measured by HSA ELISA. (d–f) AECs isolated from WT, Casp1/11−/−, Il1a/b−/− and Il18−/− mice and grown on gold electrodes were treated with 1 μg/ml PLY and resistance was measured continuously for 2 hours. (g) Isolated perfused and ventilated lungs from WT and Nlrp3−/−J (Jax^® mice) mice were intratracheally treated with PBS or PLY (0.2 μg/lung), and lung permeability was determined 30 min after stimulation by measuring leakage of HSA from the perfusion system to the broncho-alveolar space. Values represent a mean of 4–12 independent experiments and are given as mean ± SEM. Statistical significance of the resistance drop was measured 30 min after PLY stimulation (indicated by grey dotted line). *p < 0.05, **p < 0.01, n.s. = not significant.

Figure 4. NLRP3 enhances attachment of alveolar epithelial cells.

Attachment (a) as well as junctional stability (b) upon PLY treatment of WT and Nlrp3−/− AECs was determined by measuring the alpha (α) and Rb parameters using ECIS. Values represent a mean of ten independent experiments performed in duplicates. (c,d) AECs were stained with the membrane dye DiO and subsequently subjected to TIRFM life imaging. Pictures were taken at 30 min after adding medium without (−) and 45 min after changing media containing PLY (1 μg/ml). (c) Representative DiO-TIRF and DIC images are shown. (d) Particle analysis was performed from four fields from two independent experiments and values were normalized to cell numbers and average control values. (e,f,g) Cell death of WT and Nlrp3−/− AEC treated for 30 min with PLY (1 μg/ml) was measured by LDH release (e), Sytox staining (f) and AnnexinV staining (g). (e,f,g) Data represent the mean of three independent experiments. All data are given as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant.