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 intra-tracheal (I.T.) delivery in mice primarily effects lung EpiCs, but not lung ECs; while intravenous (I.V.) 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 hemorrhage followed by septic challenge [Hem-CLP]), PD-L1 expression on pulmonary ECs or EpiCs was significantly up-regulated in the iALI mice at 24h post-septic insult. After documenting the selective ability of I.T. vs. I.V. delivery of PD-L1siRNA to inhibit PD-L1 expression on EpiCs vs. ECs, respectively, we observed that the iALI-induced elevation of cytokine/chemokine levels (in the broncho-alveolar lavage fluid, lung lysates or plasma), lung MPO and caspase-3 activities could largely only be inhibited by I.V., but not I.T. delivery of PD-L1 siRNA. Moreover, I.V., but not I.T., delivery led to a preservation of normal tissue architecture, lessened pulmonary edema and reduced neutrophils influx induced by iALI. In addition, 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-g 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.