In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury

Abstract

Lung injury in trauma patients exposed to a secondary infectious/septic challenge contributes to the high morbidity/mortality observed in this population. Associated pathology involves a dys‐regulation of immune function, specifically, sequestration of activated polymorphonuclear neutrophils (PMN) in the lungs. The targeting of PMN is thought to involve the release of chemokines from cells within the local environment, creating a concentration gradient along which PMN migrate to the focus of inflammation. Keratinocyte‐derived chemokine (KC) and macrophage‐inflammatory protein‐2 (MIP‐2) are murine neutrophil chemokines identified as playing significant but potentially divergent roles in the pathogenesis of acute lung injury (ALI). In the current study, we examined the contribution of local pulmonary cells to the production of KC and MIP‐2 and the pathogenesis of ALI. We hypothesized that local silencing of KC or MIP‐2, via the local administration of small interference RNA (siRNA) against KC or MIP‐2, following traumatic shock/hemorrhage (Hem), would suppress signaling for PMN influx to the lung, thereby reducing ALI associated with a secondary septic challenge (cecal ligation and puncture). Assessment of siRNA local gene silencing was done in green fluorescent protein (GFP)‐transgenic, overexpressing mice. A marked suppression of GFP expression was observed in the lung 24 h following intratracheal (i.t.) instillation of GFP siRNA, which was not observed in the liver. To test our hypothesis, siRNA against KC or MIP‐2 (75 ug/C3H/Hen mouse) was instilled (i.t.) 2 h post‐Hem (35 mm Hg for 90 min, 4× LRS Rx.). Twenty‐four hours after, mice were subjected to septic challenge and then killed 24 h later. i.t. MIP‐2 siRNA significantly (P<0.05, ANOVA‐Tukey’s test, n=5–6/group) reduced tissue and plasma interleukin (IL)‐6, tissue MIP‐2 (enzyme‐linked immunosorbent assay), as well as neutrophil influx [myeloperoxidase (MPO) activity]. In contrast, KC siRNA treatment reduced plasma KC, tissue KC, and IL‐6 but produced no significant reduction in plasma IL‐6 or MPO. Neither treatment reduced tissue or plasma levels of tumor necrosis factor α compared with vehicle. These data support not only our hypothesis that local pulmonary chemokine production of MIP‐2, to a greater extent than KC, contributes to the pathogenesis of PMN‐associated ALI following Hem but also the use of siRNA as a potential therapeutic.