Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

Abstract

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.

Figure 1

Frozen lung (A, B) and liver (C) tissue sections from transgenic mice overexpressing GFP 24 hours after intratracheal administration of PBS (A1, B1, C1) or GFP-siRNA (A2, B2, C2). Mice having received GFP-siRNA display reduced pulmonary but not hepatic green fluorescence. Nuclear counterstaining in B and C with DAPI. Original magnifications: ×400 (A, B); ×200 (C).

Figure 2

Uptake of siRNA into pulmonary epithelial cells, but not macrophages 24 hours after intratracheal instillation. Cy5-labeled Fas-siRNA (red) (A1), the corresponded nuclear counterstaining (blue) (A2), and both channels merged with differential interference contrast (A3) show cellular uptake of siRNA. Cy5-labeled Fas-siRNA (red) (B1), cytokeratin-18 staining (green) (B2), and both channels merged (B3) identify up-taking cells as pulmonary epithelium. Cy5-labeled Fas-siRNA (red) (C1), CD115 staining for macrophages (green) (C2), and both channels merged (C3) show that siRNA was not taken up into lung macrophages. Original magnifications, ×400.

Figure 3

Lung tissue IFN-α (A), IL-6 (B), and TNF-α (C) concentrations at 18 hours after intratracheal instillation of polyinosinic-polycytidylic acid [poly(I:C)], GFP-siRNA, PBS, or caspase-8-siRNA (C-8) show that administration of siRNA did not induce IRF/NF-κB-mediated inflammation in the lung. Mediator levels were measured by ELISA (IFN-α) or cytometric bead array (TNF-α and IL-6). Values are means ± SEM of three to four animals in each group; one-way analysis of variance followed by Student-Newman-Keuls test, *P < 0.05 versus poly(I:C).

Figure 4

Lung tissue Fas mRNA expression and GAPDH-related integrated density values (IDTs) of this expression (A) and lung tissue caspase-8 mRNA expression and GAPDH-related IDT of this expression (B) 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock (Hem+Sepsis), sepsis alone, or sham procedures (individual lanes represent individual animals). Animals received either Fas-siRNA (Fas) or GFP-siRNA (GFP) (here serving as a control) intratracheally. Only the combined insult of hemorrhagic shock and sepsis increased Fas and caspase-8 mRNA expression identifying them as potential therapeutic targets. Administration of Fas- or caspase-8-siRNA significantly reduced mRNA expression of Fas or caspase-8, respectively. Values for IDT are means ± SEM of three animals in each group; *P < 0.05 versus GFP-siRNA administration after sham hemorrhage and sham sepsis (Sham GFP), ^#P < 0.05 versus GFP-siRNA administration after sham hemorrhage and sepsis (Sepsis GFP), ⁺P < 0.05 versus Fas siRNA (Fas) or caspase-8 (C-8) treatment after the double insult of hemorrhage and sepsis (Hem+Sepsis Fas, Hem+Sepsis C-8).

Figure 5

Lung tissue IL-6 (A), TNF-α (B), MCP-1 (C) concentrations per mg of total protein 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock or sham procedures display a significant increase associated with the injury. Instillation of Fas- but not caspase-8-siRNA 4 hours after hemorrhage ameliorated this inflammation when compared to control siRNA. Mediator levels were measured by cytometric bead array. One-way analysis of variance followed by Student-Newman-Keuls test; *P < 0.05 versus sham, #, P < 0.05 versus Fas-siRNA treatment.

Figure 6

Lung tissue IL-10 (A), IFN-γ (B), and IL-12 (C) concentrations per mg of total protein 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock or sham procedures display a significant increase associated with the injury. Instillation of Fas- but not caspase-8-siRNA 4 hours after hemorrhage ameliorated this inflammation when compared to control siRNA. Mediator levels were measured by cytometric bead array. Values are means ± SEM of eight animals in each group; one-way analysis of variance followed by Student-Newman-Keuls test; *P < 0.05 versus sham, #, P < 0.05 versus Fas-siRNA treatment.

Figure 7

Caspase-3 activity in lung tissue (A) was significantly increased 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock when compared to sham animals. A: Fas-siRNA but not caspase-8-siRNA administration 4 hours after hemorrhage significantly reduced caspase-3 activity in lung samples; *, < 0.05 versus sham, ^#, < 0.05 versus Fas-siRNA treatment. Lung immunostaining displayed a considerable number of cells positive for active caspase-3 (B1) as well as positive cells for M-30 (C1) in response to hemorrhagic shock and sepsis. However, in response to Fas-siRNA treatment only a few cells were positive for caspase-3 (B2) and virtually no cells for M-30 (C2). Original magnifications, ×400.

Figure 8

Lung tissue MPO staining 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and administration of GFP-siRNA (A) or hemorrhage and administration of GFP-siRNA and sepsis (B) or hemorrhage and administration of Fas-siRNA and sepsis (C). Fas-siRNA treatment markedly reduced the number of MPO-stained cells (neutrophils) present in the lung in response to hemorrhagic shock and sepsis. Alexa Fluor 594-labeled MPO-stained cells (red) (A1–C1), the corresponding nuclear counterstaining (blue) (A2–C2), and both channels merged (A3–C3) display the relative change in the number of MPO-positive cells observed in a typical field. Original magnifications, ×400.

Figure 9

Representative H&E preparation of lung tissue slides from animals 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and GFP-siRNA administration (A) or hemorrhage and GFP-siRNA administration and sepsis (B) or hemorrhage and administration Fas-siRNA and sepsis (C). Fas-siRNA administration typically reduced lung congestion and inflammatory infiltrates and improved alveolar architecture after hemorrhagic shock and sepsis (C). Original magnifications: ×200 (A1, B1, C1); ×400 (A2, B2, C2).