Blockade of endothelial, but not epithelial, cell expression of PD-L1 following severe shock attenuates the development of indirect acute lung injury in mice

This study sets out to establish the comparative contribution of PD-L1 expression by pulmonary endothelial cells (ECs) and/or epithelial cells (EpiCs) to the development of indirect acute lung injury (iALI) by taking advantage of the observation that treatment with naked siRNA by intratracheal delivery in mice primarily affects lung EpiCs, but not lung ECs, while intravenous delivery of liposomal-encapsulated siRNA largely targets vascular ECs including the lung, but not pulmonary EpiCs. We showed that using a mouse model of iALI [induced by hemorrhagic shock followed by septic challenge (Hem-CLP)], PD-L1 expression on pulmonary ECs or EpiCs was significantly upregulated in the iALI mice at 24 h post–septic insult. After documenting the selective ability of intratracheal versus intravenous delivery of PD-L1 siRNA to inhibit PD-L1 expression on EpiCs versus ECs, respectively, we observed that the iALI-induced elevation of cytokine/chemokine levels (in the bronchoalveolar lavage fluid, lung lysates, or plasma), lung myeloperoxidase and caspase-3 activities could largely only be inhibited by intravenous, but not intratracheal, delivery of PD-L1 siRNA. Moreover, intravenous, but not intratracheal, delivery led to a preservation of normal tissue architecture, lessened pulmonary edema, and reduced neutrophils influx induced by iALI. In addition, in vitro mouse endothelial cell line studies showed that PD-L1 gene knockdown by siRNA or knockout by CRISPR/Cas9-mediated gene manipulation, reduced monolayer permeability, and maintained tight junction protein levels upon recombinant IFN-γ stimulation. Together, these data imply a critical role for pulmonary vascular ECs in mediating PD-1:PD-L1–driven pathological changes resulting from systemic stimuli such as Hem-CLP.

Targeted Ablation of AIMP1 in Lipopolysaccharide-induced Acute Lung Injury

Background and Hypothesis: Infants with Bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, are more susceptible to acute lung injury (ALI). Endothelial-Monocyte Activating Polypeptide II (EMAP II, encoded by Aimp1) is a proinflammatory cytokine that originates from bronchiolar club cells and plays a role in the inflammatory response during BPD development. Prolonged EMAP II exposure has been shown to worsen BPD pathogenesis by activating alveolar macrophages. The targeted ablation of EMAP II in the bronchial club cells of a mouse model (Scgb1a1-ERTCre;Aimp1/fl/fl) is hypothesized to decrease the inflammatory effects of ALI. Experimental Design: Aged-matched littermate mice (ages ranging from 20-25 weeks) with Tamoxifen inducible, Cremediated, bronchial club cell specific ablation of Aimp1 (Scgb1a1-ERTCre;Aimp1/fl/fl, denoted cKO) or with only partial ablation (Scgb1a1-ERT2Cre;Aimp1/fl/wt denoted Ctrl) were given three doses of 120 micro-liters of 20mg/ml tamoxifen over a seven day period. 24 hours after the final dose they were administered a single intratracheal delivery of lipopolysaccharide (LPS) (5mg/kg) 24 hours later immunohistochemistry (IHC) for EMAP II and inflammation was assessed by immunoblotting (IB) for IL-6 in bronchoalveolar lavage (BAL) and cytospin of BAL. Results: IHC showed a decrease of EMAP II expression in the bronchioles of the cKO as compared to ctrl. IL-6 was increased 1.95 fold in the BAL fluid of cKO by IB. Cytospin analysis showed: (Cell Type: Ctrl%, cKo%), Macrophages: 4.66%, 4.70%, Mature neutrophils: 44.44%, 60.25%, Banded Neutrophils: 41.93%, 26.20%, Lymphocytes: 4.30%, 5.10%, Eosinophils: 2.51%, 2.10%, Monocytes: 2.15%, 1.64%. Conclusions: Ablation of bronchial club cell EMAP II, in LPS-induced ALI increased the amount of IL-6 and percentage of mature neutrophils in bronchoalveolar lavage fluid. No significant difference in macrophage were noted in either group. These findings suggest that EMAP II influences immune response time; however, more experiments would be required to establish a link.

Pharmacokinetics/Pharmacodynamics of Pulmonary Delivery of Colistin against Pseudomonas aeruginosa in a Mouse Lung Infection Model

ABSTRACT Colistin is often administered by inhalation and/or the parenteral route for the treatment of respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. However, limited pharmacokinetic (PK) and pharmacodynamic (PD) data are available to guide the optimization of dosage regimens of inhaled colistin. In the present study, PK of colistin in epithelial lining fluid (ELF) and plasma was determined following intratracheal delivery of a single dose of colistin solution in neutropenic lung-infected mice. The antimicrobial efficacy of intratracheal delivery of colistin against three P. aeruginosa strains (ATCC 27853, PAO1, and FADDI-PA022; MIC of 1 mg/liter for all strains) was examined in a neutropenic mouse lung infection model. Dose fractionation studies were conducted over 2.64 to 23.8 mg/kg of body weight/day. The inhibitory sigmoid model was employed to determine the PK/PD index that best described the antimicrobial efficacy of pulmonary delivery of colistin. In both ELF and plasma, the ratio of the area under the unbound concentration-time profile to MIC (fAUC/MIC) was the PK/PD index that best described the antimicrobial effect in mouse lung infection (R 2 = 0.60 to 0.84 for ELF and 0.64 to 0.83 for plasma). The fAUC/MIC targets required to achieve stasis against the three strains were 684 to 1,050 in ELF and 2.15 to 3.29 in plasma. The histopathological data showed that pulmonary delivery of colistin reduced infection-caused pulmonary inflammation and preserved the integrity of the lung epithelium, although colistin introduced mild pulmonary inflammation in healthy mice. This study showed pulmonary delivery of colistin provides antimicrobial effects against MDR P. aeruginosa lung infections superior to those of parenteral administrations. For the first time, our results provide important preclinical PK/PD information for optimization of inhaled colistin therapy.

Efficient intratracheal delivery of airway epithelial cells in mice and pigs

Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.