The NLRP3 inflammasome is critically involved in the development of bronchopulmonary dysplasia

Abstract

The pathogenesis of bronchopulmonary dysplasia (BPD), a devastating lung disease in preterm infants, includes inflammation, the mechanisms of which are not fully characterized. Here we report that the activation of the NLRP3 inflammasome is associated with the development of BPD. Hyperoxia-exposed neonatal mice have increased caspase-1 activation, IL1β and inflammation, and decreased alveolarization. Nlrp3(-/-) mice have no caspase-1 activity, no IL1β, no inflammatory response and undergo normal alveolarization. Treatment of hyperoxia-exposed mice with either IL1 receptor antagonist to block IL1β or glyburide to block the Nlrp3 inflammasome results in decreased inflammation and increased alveolarization. Ventilated preterm baboons show activation of the NLRP3 inflammasome with increased IL1β:IL1ra ratio. The IL1β:IL1ra ratio in tracheal aspirates from preterm infants with respiratory failure is predictive of the development of BPD. We conclude that early activation of the NLRP3 inflammasome is a key mechanism in the development of BPD, and represents a novel therapeutic target for BPD.

Figure 1. Nlrp3^−/− mice are protected from neonatal hyperoxia-induced inflammation and decreased alveolarization

All data are expressed as mean ± s.e.m. with n = 6–12 animals per group for each experiment, (a) Mice exposed to 85% O₂ had a 45-fold increase in IL1β mRNA. (b) In BAL, WT mice exposed to hyperoxia had increased mature IL1β protein, whereas Nlp3^−/− mice had no detectable mature IL1β. (c) Lung caspase-1 activity was increased threefold in WT mice exposed to 85% O₂, but was undetectable in Nlrp3^−/− mice, (d) Hyperoxia-exposed WT mice had a 35-fold increase in p20-cleaved caspase-1. Animals in 21% O₂ and Nlrp3^−/− mice exposed to 85% O₂ had no detectable p20. (e) On PN15, WT lungs obtained had decreased secondary septation, whereas Nlrp3^−/− mice had normal alveolarization (Scale bar, 400 µm). (f) WT mice exposed to 85% O₂ had decreased radial alveolar count (RAC), whereas Nlrp3^−/− mice had normal RAC. (g) MPO activity of lavage cell pellets was increased maximally on day 10 in WT animals exposed to 85% O₂, but was unchanged in WT mice kept in 21% O₂ and in Nlrp3^−/− mice in either condition, (h) NAG activity of lavage cell pellets was increased on day 15 in WT animals exposed to 85% O₂, but was unchanged in WT mice kept in 21% O₂ and in Nlrp3^−/− mice under either condition. (i) Single-cell preparations of lungs from WT or Nlrp3^−/− mice were subjected to flow cytometry to identify total white cells (CD45), neutrophils (Ly6G), lymphocytes (CD4), macrophages (F4/80) and M1 macrophages (CD86). WT mice, but not Nlrp3^−/− mice, exposed to hyperoxia had increased absolute numbers and per cent total CD45+ cells and M1 macrophages. (j) By immunofluorescence, WT mice had high expression of YM1 that decreased with exposure to 85% O₂. This decrease was significantly less in Nlrp3^−/− mice in 85% O₂. (k) By immunofluorescence, expression of CD11c was low to undetectable in WT mice and increased with 85% O₂. This increase was blunted in Nlrp3^−/− mice in 85% O₂. (I) Steady-state mRNA content of iNOS confirmed the CD11c findings. (m) WT mice exposed to 85% O₂ had increased TUNEL-positive cells, whereas Nlrp3^−/− had no evidence of apoptosis in any condition.

Figure 2. WT mice exposed to 85% O₂ are protected when treated with either recombinant IL1ra to block IL1β receptor or glyburide to block formation of the Nlrp3 inflammasome

All data are presented as mean ± s.e.m. with n = 6–12 animals per group. (a) The lungs of WT mice exposed to 85% O₂ had equally increased caspase-1 activity in both vehicle and IL1ra-treated mice. (b) All mice in 85% O₂, both vehicle- and IL1ra-treated, showed an eightfold increase in p20-cleaved caspase-1. None of the 21% O₂ animals had the p20-cleaved protein. (c) Vehicle-treated mice exposed to 85% O₂ had decreased secondary septation. Mice exposed to 85% O₂ and treated with IL1ra had improved alveolarization as compared with vehicle-treated controls. (d) Exposure of WT mice to 85% O₂ resulted in decreased RAC. Treatment of hyperoxia-exposed mice with IL1ra significantly improved RAC. (e) MPO activity of lavage cell pellets was increased in vehicle-treated, 85% O₂-exposed mice and this was abrogated with treatment with IL1ra. (f) The NAG activity of lavage cell pellets was increased in vehicle-treated, 85% O₂-exposed mice, and treatment with IL1ra prevented this increase. (g) WT mice in 85% O₂ had increased caspase-1 activity either with vehicle or glipizide treatment. Mice in 85% O₂ treated with glyburide caspase-1 activity similar to 21% O₂ animals. (h) Only 85% O₂-exposed animals given glipizide, and not those treated with glyburide, had increased p20-cleaved caspase-1. (i) On PN15, WT lungs exposed to 85% O₂ with either vehicle or glipizide had decreased secondary septation, whereas 85% O₂-exposed mice treated with glyburide had normal alveolarization (Scale bar, 400 µm). (j) Exposure to 85% O₂ and treatment with either vehicle or glipizide results in decreased RAC. Treatment with glyburide, normalized RAC. (k) MPO activity was increased in BAL cell pellets from 85% O₂-exposed mice treated with glipizide, whereas mice treated with glyburide had no increase in MPO. (I) The NAG activity of BAL cell pellets was increased in glipizide-treated, 85% O₂-exposed mice. However, treatment of hyperoxia-exposed mice with glyburide prevented this increase in NAG activity.

Figure 3. Increased IL1β, IL1ra and IL1β:IL1ra ratio in a non-human primate model of BPD

All data are expressed as mean ± s.e.m. with n = 6–8 animals per group for each experiment. Preterm baboon tracheal aspirates or lung tissue obtained from 125-day gestation animals ventilated with appropriate ventilation and oxygenation for 14 days (125d + 14d PRN), and either at 125 or 140 days gestation animals as controls (125d GC; 140d GC) were examined as described in Methods section. (a) IL1β mRNA was elevated 60-fold in 125-day preterm baboons ventilated for 14 days as compared with either 125- or 140-day gestational age controls. (b). Caspase-1 activity was elevated twofold in the lungs of ventilated preterm baboons as compared with the gestational age controls. (c) Cleaved p20 caspase-1 was increased twofold in ventilated preterm baboons as compared with gestational age controls. (d-f) Tracheal aspirate concentrations of IL1β and IL1ra normalized to the Secretory Component of IgA (SCIgA), and the IL1β:IL1ra ratio in the preterm baboon model, (d) BAL IL1β/SCIgA was increased threefold in ventilated preterm baboons as compared with 125- and 140-day gestational controls. (e) BAL IL1ra/SCIgA decreased from 125 to 140 days in gestational age controls. Ventilated preterm baboons had IL1ra/SCIgA levels intermediate between the two gestational ages. (f) The ratio of IL1β to IL1ra in the BAL was increased eightfold in ventilated preterm baboons as compared with 125- and 140-day gestational age controls. (g-i) Changes in IL1β/SCIgA, IL1ra/SCIgA and IL1β:IL1ra ratio were determined in tracheal aspirate samples obtained on days 2, 6 and 14 after birth. (g) Although there was a trend to increase from day 2 to day 6, there were no significant differences in IL1β/SCIgA after birth. (h) IL1ra/SCIgA decreased significantly between day 6 and day 14 after birth. (i) The ratio of IL1β to IL1ra steadily increased after birth such that it was significantly elevated by day 14 after birth.

Figure 4. Tracheal aspirate IL1β, IL1ra and N-acetyl glucosaminidase activity are associated with adverse outcome in preterm infants intubated for respiratory failure

(a–c) Box plots of log-transformed IL1β, IL1ra and IL1β:IL1ra ratio in tracheal aspirates obtained between days 1 and 3 segregated by adverse outcome (death or BPD at 36 weeks PMA). Each biomarker was significantly elevated between days 1 and 3 in infants that subsequently developed adverse outcome at 36 weeks PMA. (d) Box plot of log-transformed NAG activity between days 7 and 10 segregated by adverse outcome. NAG activity at 710 days was increased in those infants that developed death or BPD at 36 weeks PMA. (e–g) Comparisons of the contribution of gestational age and each of the tracheal aspirate biomarkers to the probability of adverse outcome at each gestational age. Using the 50th percentile value for each biomarker at 1–3 days, the probability of adverse outcome at each gestational age was determined and compared with the contribution of gestational age alone. Gestational age alone contributed 40% to the probability of adverse outcome at each gestational age. For infants <27 weeks gestation, each biomarker (IL1β, IL1ra and IL1β:IL1ra ratio) doubled the probability of adverse outcome above that of gestational age alone.