M. Kojima1, T. Chan1, J. Gimenes1, B. Eliceiri1, A. Baird1, T. Costantini1, R. Coimbra1 1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns And Acute Care Surgery/Department Of Surgery,San Diego, CA, USA
Introduction: Acute lung injury (ALI) is a common cause of morbidity in patients following severe injury. Studies have shown that mesenteric lymph (ML) carries gut-derived inflammatory mediators to the lung and serves as the inciting event in ALI. Alveolar macrophages (AM), a lung resident macrophage, play a critical role in the development of ALI. We have recently demonstrated that exosomes, nano-sized extracellular vesicles, are present in ML and have the ability to trigger NF-κB activation in human monocytic cells in vitro. We hypothesized that exosomes in post-injury ML induce pro-inflammatory cytokine production in AM and contributes to post-injury ALI.
Methods: Male rats underwent cannulation of the femoral artery, jugular vein and ML duct prior to trauma/hemorrhagic shock (T/HS; mean arterial pressure 35 mmHg for 60 min), followed by resuscitation with shed blood and two times normal saline. The ML was collected before hemorrhagic shock (pre-shock) and after T/HS (post-T/HS) for isolation of exosomes by differential centrifugation. In vitro AM were stimulated with exosomes harvested from pre-shock or post-T/HS ML for 6 or 12 hours for measurement of cytokine production by quantitative reverse transcription PCR (qRT-PCR). ML Exosomes from each experimental group (2 x 109 exosomes/g) were also intravenously injected into male naïve C57BL/6 mice to assess in vivo biologic activity. Lung injury was evaluated by measuring histologic lung injury, vascular permeability and immune cell recruitment in bronchoalveolar lavage (BAL) and lung tissue.
Results: Exosomes released into post-T/HS ML increased the gene expression of pro-inflammatory cytokines (TNF-a and CINC-1; see figure), NF-kB (5-fold increase at 6h vs. pre-shock; p<0.001) and iNOS (4-fold increase at 12h vs. pre-shock; p<0.001) in AM in vitro. Compared to pre-shock ML exosomes, the in vivo injection of post-T/HS ML exosomes resulted in increased histological lung injury score, a 2-fold increase in Evan’s blue dye lung permeability (0.058 ± 0.003 to 0.117 ± 0.011 mg/g tissue; p<0.05) as well as an increase of wet-to-dry ratio (4.608 ± 0.071 to 5.202 ± 0.149; p<0.05). Exosomes in post-T/HS ML also induced increased recruitment of neutrophils (CD45+Ly-6G+) and macrophages (CD45+CD11c+) in BALF and lung parenchyma determined by flow cytometry (p<0.05 vs. pre-shock).
Conclusion: Exosomes released into post-T/HS ML caused an inflammatory response from AM in vitro and ALI in vivo. Our findings define the critical role of ML exosomes as a biologically active mediator of ALI after severe injury.
