Abstract Rationale Gram-negative bacteremia following systemic Pseudomonas aeruginosa (PA) infection causes high mortality due to acute lung injury (ALI). However, bacteria-endothelial interactions underlying ALI-causing lung endothelial barrier failure remain unclear. Here, we tested the hypothesis that endothelial internalization of PA forms membrane pores, hence barrier failure. Methods We induced systemic infection in mice by intraperitoneal (i.p.) inoculation of fluorescent, GFP-labelled, PA (GFP-PA, 4x106 cfu). After 48 hours we carried out real-time confocal microscopy (RCM) of the lung to determine GFP-PA interactions with calcein red (CR)-loaded lung endothelium. To determine barrier properties, we assessed vascular leak of intravenously injected 70kDa FITC-dextran (FD70). We assessed survival of mice given intravenous injection of the enhancer of cell cortical f-actin, TAT-V12Rac1 (PMID: 33749665) 7 hours after PA inoculation (n = 10). Control mice did not receive the enhancer. Results RCM imaging of live lung microvessels revealed GFP-PA as fluorescent particles attached to the endothelial surface, indicating that peritoneally injected bacteria reached the lungs. Endothelium-internalized bacteria were evident in confocal optical sections obtained at planes that included the cell interior. Membrane pore formation, marked by the loss of cytosolic CR fluorescence, was evident at sites of GFP-PA internalization in the lung endothelium. Intravenous administration of FD70 led to rapid fluorescence extravasation, indicating loss of endothelial barrier properties. In survival assays, 80% of mice succumbed within three days of PA inoculation (p 0.01 vs. PBS control). In contrast, treatment with TAT-V12Rac1 markedly improved survival, reducing mortality to 30% (p 0.01). Conclusions We demonstrate, for the first time, that within 48 hours of i.p. inoculation, GFP-PA is internalized in the lung endothelium, inducing membrane pore formation, loss of endothelial barrier properties and high mortality. Notably, enhancing the endothelial actin fence by intravenous V12Rac1 injection markedly abrogated the mortality. We propose, strengthening the endothelial actin fence may protect against bacteremia-induced endothelial barrier failure in sepsis-associated ALI. (Support: HL36024). This abstract is funded by: NHLBI
Islam et al. (Fri,) studied this question.