Yes-associated protein induces age-dependent inflammatory signaling in the pulmonary endothelium

Abstract

Acute lung injury (ALI) causes the highly lethal acute respiratory distress syndrome (ARDS) in children and adults, for which therapy is lacking. Children with pediatric ARDS have a mortality rate that is about half of adults with ARDS. Improved ALI measures can be reproduced in rodent models with juvenile animals, suggesting that physiologic differences may underlie these outcomes. Here, we show that pneumonia-induced ALI caused inflammatory signaling in the endothelium of adult mice, which depended on Yes-associated protein (YAP). This signaling was not present in 21-day-old weanling mice. Transcriptomic analysis of lung endothelial responses revealed nuclear factor-kappa B (NF-κB) as significantly increased with ALI in adult versus weanling mice. Blockade of YAP signaling protected against inflammatory response, hypoxemia, and NF-κB nuclear translocation in response to Pseudomonas aeruginosa pneumonia in adult mice. Our results demonstrate an important signaling cascade in the lung endothelium of adult mice that is not present in weanlings. We suggest other pathways may also exhibit age-dependent signaling, which would have important implications for ARDS therapeutics in the adult and pediatric age groups.NEW & NOTEWORTHY Like human patients, adult mice get worse lung injury than juveniles. In pneumonia-induced lung injury, Yes-associated protein is more highly expressed in the endothelium of adult mice than juveniles, causing more NF-κB nuclear translocation and inflammation. This could partly explain better outcomes in kids with pediatric acute respiratory distress syndrome as compared with adults with ARDS.

Keywords: NF-κB; PARDS; Yes-associated protein; acute lung injury; lung endothelium.

Figure 1.. Responses to intranasal Pseudomonas aeruginosa in weanling and adult mice.

Analyses were performed 24h after intranasal instillation of 2.5 × 10⁵ CFU of P. aeruginosa or PBS. (A-C, E) BAL was obtained by instillation of ice cold PBS intratracheally. (A) BAL protein was determined by BCA assay. (B-C) BAL cell and neutrophil counts were determined with Vetscan hematology analyzer. (D) ELVW was determined in separate experiments. (E) BAL cytokines were measured by multiplex array (Eve Technologies). (F) SpO₂ was determined in anesthetized mice using a MouseOx^® Pulse Oximeter. Data are shown as mean±SE. Number of replicates are indicated by dots (≥4 for all groups). BAL, bronchoalveolar lavage; 3wo, 3 week-old mice; Pseudomonas, Pseudomonas aeruginosa strain K; PMNs, neutrophils; EVLW, extravascular lung water; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; SpO₂, non-invasive O₂ saturation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Multiple comparisons were analyzed by two-way ANOVA; paired differences were compared by unpaired 2-tailed t test.

Figure 2.. YAP expression responses to P. aeruginosa-induced lung injury in weanling and adult mice.

Representative gels and scatter plots show immunoblotting and densitometry of enriched cytoplasmic (A) and nuclear (B) fractions of whole lung, and enriched lung endothelial cells (C) and epithelial cells (D). All animals were treated with i.n. PBS or P. aeruginosa 24 hours before lung removal. Antibodies used were YAP rabbit monoclonal, GAPDH mouse monoclonal, HDAC mouse monoclonal, and actin rabbit polyclonal. Lanes were run on the same gel. Number of replicates are indicated by dots (≥4 for all groups). 3wo, 3 week-old mice; n.s., not significant. *p < 0.05, ****p<0.0001. Differences between groups were compared by t test.

Figure 3.. YAP mediates P. aeruginosa-induced lung injury in adult mice.

Mice were injected intravenously with YAP-targeted siRNA or non-targeting control siRNA (NTC) 24h before all mice were treated with P. aeruginosa intranasal instillation. (A) Representative gel and scatter plots show immunoblotting and densitometry of whole lung cytoplasmic fraction. Antibodies used were YAP rabbit monoclonal and GAPDH mouse monoclonal. There was high variability of lung YAP expression, but statistical significance remained when the highest outlier from the NTC-treated group was removed from the analysis. (B) BAL cell counts were determined with Vetscan hematology analyzer. (C) BAL IL-6 was measured by multiplex array. (D) SpO₂ was determined using a MouseOx^® Pulse Oximeter. Number of replicates are indicated by triangles (≥3 for all groups). BAL, bronchoalveolar lavage; 3wo, 3 week-old mice; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; SpO₂, non-invasive O₂ saturation. *p<0.05, ***p<0.001, ****p<0.0001. Paired differences were compared by t test.

Figure 4.. YAP expression drives endothelial NF-κB responses in P. aeruginosa-infected adult mice.

(A) Principal component analysis of four treatment groups. PC1, principal component 1; blue, saline-treated; orange, P. aeruginosa-treated. •, adult; x, weanling. (B,C) Top pathways in which the adult response to P. aeruginosa infection is higher (B) and lower (C) than in weanling mice. Select pathways are highlighted in red. (D,E) Representative gel and scatter plots show immunoblotting and densitometry of whole lung cytoplasmic and nuclear enriched fractions: (D) 24h after PBS or P. aeruginosa instillation and (E) following pretreatment with YAP siRNA by tail vein injection and P. aeruginosa instillation. Antibodies used were NF-κB p65 rabbit monoclonal, YAP rabbit monoclonal, HDAC mouse monoclonal, and actin rabbit polyclonal. Number of replicates are indicated by dots (≥3 for all groups). NF-κB, nuclear factor-kappa B; NTC, non-targeting control siRNA; siRNA, YAP-targeted siRNA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Paired differences were compared by t test.