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A4160 - Murine Model of Prenatal Cigarette Smoke Exposure Leads to Increased Cytotoxicity of Lung Natural Killer Cells in Offspring
Author Block: J. Arroyo Morales1, H. Alikaj2, V. R. Stolberg2, M. R. Kady2, J. L. Curtis3, C. M. Freeman4; 1University of Michigan, Ann Arbor, MI, United States, 2VA Healthcare System, Ann Arbor, MI, United States, 3Internal Medicine, Univ of Michigan Hlth System, Ann Arbor, MI, United States, 4Univ of Michigan, Ann Arbor, MI, United States.
BACKGROUND: Prenatal cigarette smoke (CS) exposure impairs post-natal lung development and predisposes to obstructive lung diseases via incompletely defined mechanisms. We have demonstrated increased in vitro killing of autologous lung epithelial cells by adult lung natural killer (NK) cells from both humans with COPD (relative to smokers without obstruction) and from CS-exposed mice (relative to air-exposure). We hypothesized that prenatal CS exposure programs offspring lung NK cells for enhanced post-natal cytotoxicity. METHODS: We exposed female C57BL/6 mice to either CS or air (one hour/day, five days/week) for four weeks prior to breeding. Exposures continued throughout gestation but were halted when pups were born. Without further exposure, pups were euthanized at four weeks to collect lung tissue. We reserved a portion of whole lung to characterize lung immune cell populations using flow cytometry; from the remaining lung, we isolated DX5+ NK cells, pan-dendritic cells (DCs), and CD326+ epithelial cells via immunomagnetic bead separation. We co-cultured NK cells and epithelial cells (5:1 ratio for four hours), then assayed epithelial cell apoptosis using Annexin-V plus 7-AAD staining and flow cytometry. In some experiments, lung NK cells were co-cultured overnight with lung DC before adding epithelial cells. In all experiments, epithelial cells were cultured alone as a reference. RESULTS: Pups exposed to prenatal CS had increased proportions of both lung NK cells and lung CD103+ DCs (as a percent of all lung leukocytes) compared to mice exposed only to air during gestation. In two independent experiments, lung NK cells from pups undergoing prenatal CS exposure were also more cytotoxic towards autologous lung epithelial cells than control pups (15.0 ± 3.3% versus 2.6 ± 0.9% specific cytotoxicity). There were no differences between male and female pups in either parameter. We next analyzed whether DCs from prenatal CS-exposed mice could prime naïve NK cells (from syngeneic non-exposed mice), analogous to the ability we have seen for lung DCs in human COPD to prime autolgous peripheral blood NK cells. Co-culture with DCs from air-exposed pups did not increase NK cytotoxicity. However, co-culture with lung DCs from pups that had undergone prenatal CS exposure increased cytotoxicity over NK cells alone, from 2.6 ± 0.9% to 15.4 ± 5.5%. CONCLUSIONS: These data imply that CS exposure in utero increases lung NK cytotoxicity even without further CS exposure; this effect appears in part to be mediated by DC priming. NK cell dysfunction could impact immediate and long-term respiratory health.