Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis
2005; Elsevier BV; Volume: 167; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61240-0
ISSN1525-2191
AutoresMario Perl, Chun‐Shiang Chung, Joanne Lomas‐Neira, Tina-Marie Rachel, Walter L. Biffl, William G. Cioffi, Alfred Ayala,
Tópico(s)Phagocytosis and Immune Regulation
ResumoApoptosis 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-α, tumor necrosis factor-α, 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-α, IL-6, IL-10, interferon-γ, 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. 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-α, tumor necrosis factor-α, 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-α, IL-6, IL-10, interferon-γ, 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. Acute lung injury (ALI) can be differentiated into direct ALI that evolves from direct injury to the lung, caused by pneumonia, aspiration, and lung trauma, or indirect ALI that evolves from extrapulmonary injury, caused by nonpulmonary sepsis and trauma (ie, hemorrhagic shock, fractures, and so forth).1Bersten AD Edibam C Hunt T Moran J Incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three Australian states.Am J Respir Crit Care Med. 2002; 165: 443-448Crossref PubMed Scopus (358) Google Scholar So far, a substantial role for primed neutrophils in inducing indirect ALI has been indicated. Activation of neutrophils by inflammatory stimuli delays apoptosis2Parsey MV Kaneko D Shenkar R Abraham E Neutrophil apoptosis in the lung after hemorrhage or endotoxemia: apoptosis and migration are independent of IL-1beta.Clin Immunol. 1999; 91: 219-225Crossref PubMed Scopus (42) Google Scholar, 3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar and enhances their respiratory burst capacity.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Adoptive transfer of primed neutrophils induces ALI in a septic environment3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar and prevention of neutrophil priming or their immigration into the lung attenuates ALI.4Lomas-Neira JL Chung CS Grutkoski PS Miller EJ Ayala A CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.J Leukoc Biol. 2004; 76: 58-64Crossref PubMed Scopus (66) Google Scholar, 5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar In contrast, the process of programmed cell death in the lung has been suggested to be a major pathogenetic factor in models of direct ALI6Kitamura Y Hashimoto S Mizuta N Kobayashi A Kooguchi K Fujiwara I Nakajima H Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice.Am J Respir Crit Care Med. 2001; 163: 762-769Crossref PubMed Scopus (255) Google Scholar, 7Vernooy JH Dentener MA van Suylen RJ Buurman WA Wouters EF Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: no role for tumor necrosis factor-alpha and infiltrating neutrophils.Am J Respir Cell Mol Biol. 2001; 24: 569-576Crossref PubMed Scopus (93) Google Scholar as well as in acute respiratory distress syndrome.8Bardales RH Xie SS Schaefer RF Hsu SM Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury.Am J Pathol. 1996; 149: 845-852PubMed Google Scholar, 9Guinee Jr, D Brambilla E Fleming M Hayashi T Rahn M Koss M Ferrans V Travis W The potential role of BAX and BCL-2 expression in diffuse alveolar damage.Am J Pathol. 1997; 151: 999-1007PubMed Google Scholar After lipopolysaccharide-induced ALI in mice, epithelial cells display an augmented expression of Fas along with an increased migration of Fas ligand (FasL)-expressing inflammatory cells into the alveolar space.6Kitamura Y Hashimoto S Mizuta N Kobayashi A Kooguchi K Fujiwara I Nakajima H Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice.Am J Respir Crit Care Med. 2001; 163: 762-769Crossref PubMed Scopus (255) Google Scholar In patients, acute respiratory distress syndrome is associated with increased levels of FasL in the bronchoalveolar lavage fluid inducing apoptosis in distal lung epithelial cells via the activation of the death receptor pathway.10Matute-Bello G Liles WC Steinberg KP Kiener PA Mongovin S Chi EY Jonas M Martin TR Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).J Immunol. 1999; 163: 2217-2225PubMed Google Scholar Similarly, high doses of Fas-activating antibody have been found to induce lung epithelial apoptosis in mice.11Matute-Bello G Winn RK Jonas M Chi EY Martin TR Liles WC Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation.Am J Pathol. 2001; 158: 153-161Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Interestingly, modulation of the Fas/FasL systems not only affects lung apoptosis but also results in alteration of local pulmonary inflammation.6Kitamura Y Hashimoto S Mizuta N Kobayashi A Kooguchi K Fujiwara I Nakajima H Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice.Am J Respir Crit Care Med. 2001; 163: 762-769Crossref PubMed Scopus (255) Google Scholar, 11Matute-Bello G Winn RK Jonas M Chi EY Martin TR Liles WC Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation.Am J Pathol. 2001; 158: 153-161Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12Matute-Bello G Frevert CW Liles WC Nakamura M Ruzinski JT Ballman K Wong VA Vathanaprida C Martin TR Fas/Fas ligand system mediates epithelial injury, but not pulmonary host defenses, in response to inhaled bacteria.Infect Immun. 2001; 69: 5768-5776Crossref PubMed Scopus (63) Google Scholar However, so far the role of the extrinsic death (Fas) receptor pathway in mediating apoptosis and inflammation in ALI caused by extrapulmonary injury remains unclear. To address this question we attempted to shed light on the contribution of gene products of the extrinsic death (Fas) receptor pathway to the pathology of shock-induced septic ALI by using organ-specific gene knockdown. In this regard, RNA interference has become a powerful tool in selectively silencing mammalian genes. The relatively small amounts required for their gene-specific silencing abilities, as well as the longevity of silencing, are promising characteristics associated with potential in vivo siRNA administration. Nonetheless, there have been only a limited number of reports on siRNA administration in vivo. In these studies, intravenous administration of caspase-8-siRNA prevented acute liver failure in mice13Zender L Hutker S Liedtke C Tillmann HL Zender S Mundt B Waltemathe M Gosling T Flemming P Malek NP Trautwein C Manns MP Kuhnel F Kubicka S Caspase 8 small interfering RNA prevents acute liver failure in mice.Proc Natl Acad Sci USA. 2003; 100: 7797-7802Crossref PubMed Scopus (357) Google Scholar and RNA interference targeting Fas was protective in a murine model of fulminant hepatitis.14Song E Lee SK Wang J Ince N Ouyang N Min J Chen J Shankar P Lieberman J RNA interference targeting Fas protects mice from fulminant hepatitis.Nat Med. 2003; 9: 347-351Crossref PubMed Scopus (1014) Google Scholar In addition, in vivo delivery of Fas- or caspase-8-siRNA improves survival in septic mice.15Wesche-Soldato DE Chung CS Lomas-Neira J Doughty LA Gregory SH Ayala A In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice.Blood. 2005; 106: 2295-2301Crossref PubMed Scopus (164) Google Scholar However, these approaches are not organ-specific and either require viral preparations13Zender L Hutker S Liedtke C Tillmann HL Zender S Mundt B Waltemathe M Gosling T Flemming P Malek NP Trautwein C Manns MP Kuhnel F Kubicka S Caspase 8 small interfering RNA prevents acute liver failure in mice.Proc Natl Acad Sci USA. 2003; 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418: 38-39Crossref PubMed Scopus (956) Google Scholar Thus, more localized and thereby in part organ-specific approaches have evolved, including intraperitoneal19Sorensen DR Leirdal M Sioud M Gene silencing by systemic delivery of synthetic siRNAs in adult mice.J Mol Biol. 2003; 327: 761-766Crossref PubMed Scopus (427) Google Scholar and intra-alveolar delivery.5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar, 20Massaro D Massaro GD Clerch LB Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice.Am J Physiol. 2004; 287: L1066-L1070Google Scholar, 21Zhang X Shan P Jiang D Noble PW Abraham NG Kappas A Lee PJ Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis.J Biol Chem. 2004; 279: 10677-10684Crossref PubMed Scopus (239) Google Scholar However, these studies 1) did not evaluate siRNA targeting of a potentially therapeutic protein,20Massaro D Massaro GD Clerch LB Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice.Am J Physiol. 2004; 287: L1066-L1070Google Scholar 2) were performed as a pretreatment scenario,19Sorensen DR Leirdal M Sioud M Gene silencing by systemic delivery of synthetic siRNAs in adult mice.J Mol Biol. 2003; 327: 761-766Crossref PubMed Scopus (427) Google Scholar, 21Zhang X Shan P Jiang D Noble PW Abraham NG Kappas A Lee PJ Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis.J Biol Chem. 2004; 279: 10677-10684Crossref PubMed Scopus (239) Google Scholar or 3) did not sufficiently address the quest-ion of which cells were the target of the administered siRNA.5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar, 20Massaro D Massaro GD Clerch LB Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice.Am J Physiol. 2004; 287: L1066-L1070Google Scholar, 21Zhang X Shan P Jiang D Noble PW Abraham NG Kappas A Lee PJ Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis.J Biol Chem. 2004; 279: 10677-10684Crossref PubMed Scopus (239) Google Scholar Thus, the feasibility of local gene silencing in the lung in a critical condition still remains to be demonstrated. In this context, we tested the hypothesis that postshock pulmonary administration of Fas- and caspase-8-siRNA attenuates apoptosis and inflammation in indirect ALI after hemorrhagic shock and sepsis. Our data will show that organ-specific delivery of siRNA into the lung is a feasible approach. It can be performed as a posttreatment after the onset of hemorrhagic shock and is not associated with the induction of toll-like receptor or protein kinase PKR-mediated inflammation. We will demonstrate that pulmonary epithelial cells are a main target for siRNA delivered into the lung. In addition, down-regulation of Fas but not caspase-8 in these cells reduces pulmonary apoptosis and lung inflammation, decreases pulmonary neutrophil influx, and attenuates ALI, indicating the pathophysiological relevance of the Fas receptor pathway in indirect ALI. Male transgenic C57BL/6-TgN(ACTbEGFP)1Osb mice (Jackson Laboratories, Bar Harbor, ME) expressing green fluorescent protein (GFP) and inbred male C3H/HeN mice (Charles River, Wilmington, MA), 8 to 9 weeks old, were used. C3H/HeN mice were used in all experiments except for the initial proof of principle experiments due to our previous experience with this strain in the pathogenesis of ALI after hemorrhagic shock and sepsis.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Lomas-Neira JL Chung CS Grutkoski PS Miller EJ Ayala A CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.J Leukoc Biol. 2004; 76: 58-64Crossref PubMed Scopus (66) Google Scholar, 5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar C57BL/6-TgN(ACTbEGFP)1Osb mice were used for proof of principle experiments with GFP-siRNA because mice ubiquitously expressing GFP-protein were not available on the C3H/HeN background. Animals were allowed free access to water and food before and after procedures. A day-night cycle of 12 hours was applied. Experiments described in this article were performed in accordance with National Institutes of Health guidelines and with approval from the Animal Use Committee of Rhode Island Hospital. C57BL/6-TgN(ACTbEGFP)1Osb mice were subjected to intratracheal delivery of siRNA targeting GFP mRNA (Dharmacon, Lafayette, CO), C3H/HeN mice were challenged with hemorrhagic shock, intratracheal delivery of siRNA (see below), and cecal ligation and puncture. The hemorrhage model we have used for these experiments has been previously described.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar In brief, C3H/HeN mice were anesthetized with isoflurane, restrained in supine position, and catheters were inserted into both femoral arteries. Anesthesia was discontinued and blood pressure was continuously monitored through one catheter attached to a blood pressure analyzer (MicroMed, Louisville, KY). When fully awake, as determined by a mean blood pressure of ∼110 mmHg, the mice were bled throughout a 5- to 10-minute period to a mean blood pressure of 30 mmHg (±5 mmHg) and were kept stable for 90 minutes. Immediately after hemorrhage, mice were resuscitated (intravenously) with Ringer's lactate at four times the drawn blood volume. The catheters were then removed, the vessels were ligated, and the incisions were closed. Sham-operated controls (sham) had their femoral arteries ligated and were restrained for periods of time equal to their hemorrhaged counterparts, but no catheters were inserted and no blood was shed. Intratracheal delivery of agents was performed as described previously.5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar, 22McKinley L Kim J Bolgos GL Siddiqui J Remick DG Reproducibility of a novel model of murine asthma-like pulmonary inflammation.Clin Exp Immunol. 2004; 136: 224-231Crossref PubMed Scopus (24) Google Scholar Four hours after resuscitation mice were anesthetized lightly with isoflurane and restrained in supine position with their head reclined. The tongue of the animals was pulled out gently to prevent swallow reflex and siRNA or placebo was administered in the oral cavity. Mice were maintained in the described position until siRNA or placebo was aspirated (usually between 5 and 10 seconds). Polymicrobial sepsis was induced in mice by the method described previously.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Mice were anesthetized with isoflurane, shaved at the abdomen, and scrubbed with betadine. A midline incision (∼1 cm) was made below the diaphragm to expose the cecum. The cecum was ligated and punctured twice with a 22-gauge needle and gently compressed to extrude a small amount of cecal contents through the punctured holes. The cecum was returned to the abdomen, and the incision was closed in layers with 6-0 Ethilon suture (Ethicon, Somerville, NJ). The animals were then resuscitated with 0.8 ml of lactated Ringer's solution by subcutaneous injection. This procedure usually results in ∼65% mortality of the mice throughout the period of 10 days, however does not induce significant lung injury as described previously, if not preceded by hemorrhagic shock.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Lomas-Neira JL Chung CS Grutkoski PS Miller EJ Ayala A CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.J Leukoc Biol. 2004; 76: 58-64Crossref PubMed Scopus (66) Google Scholar, 5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar First we evaluated, as a proof of principle, whether intratracheal delivery of siRNA was a feasible therapeutic approach and which cell type was the in vivo target of this delivery method. One hundred μg of GFP-siRNA in 100 μl of phosphate-buffered saline (PBS) or PBS only were administered intratracheally into male transgenic C57BL/6-TgN(ACTbEGFP)1Osb mice (n = 3/group). Lungs, livers, and spleens were harvested 24 hours thereafter for morphological assessment and quantification of fluorescence intensity on frozen sections. In addition, male C3H/HeN mice (n = 3/group) received 100 μg of Fas-siRNA labeled with a Cy5-fluorochrome in 100 μl of PBS or PBS only. The lungs of these animals were harvested 24 hours thereafter for immunohistochemistry on frozen lung sections. To shed light on possible proinflammatory properties of in vivo siRNA delivery a separate set of male C3H/HeN mice were randomized into four groups (n = 3 to 4 each). One hundred μg of polyinosinic-polycytidylic acid sodium salt [poly(I:C)] (Sigma, St. Louis, MO) in 100 μl of PBS as a positive control, 100 μg of GFP-siRNA in 100 μl of PBS, 100 μg of caspase-8-siRNA in 100 μl of PBS, or 100 μl of PBS were administered intratracheally. For these experiments, caspase-8-siRNA was chosen over Fas-siRNA because the latter had revealed anti-inflammatory properties in preliminary experiments that could have acted as a potential confounder in the experiments. Lung, liver, and plasma were harvested 18 hours after instillation and interferon (IFN)-α, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were quantified in tissue homogenates and plasma. So far it remains unclear through which pathways short-interfering RNA signal. Thus, IFN-α was chosen as an inflammatory readout, because it is produced on activation of TLR-323Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4796) Google Scholar [recognizing double-stranded RNA and poly(I:C)] and TLR-724Hornung V Guenthner-Biller M Bourquin C Ablasser A Schlee M Uematsu S Noronha A Manoharan M Akira S de Fougerolles A Endres S Hartmann G Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat Med. 2005; 11: 263-270Crossref PubMed Scopus (1021) Google Scholar [single-stranded (ss) RNA and siRNA] via IRF translocation. Alternatively, TNF-α and IL-6 were quantified because activation of TLR-3, TLR-8 (ssRNA), and TLR-9 [cytidine-phosphate-guanoside(cpg)DNA] or induction of protein kinase PKR pathways promotes genes controlled by the nuclear factor (NF)-κB transcriptional regulator.25Robbins MA Rossi JJ Sensing the danger in RNA.Nat Med. 2005; 11: 250-251Crossref PubMed Scopus (37) Google Scholar, 26Heidel JD Hu S Liu XF Triche TJ Davis ME Lack of interferon response in animals to naked siRNAs.Nature Biotechnol. 2004; 22: 1579-1582Crossref Scopus (166) Google Scholar Finally, we examined whether intratracheal delivery of Fas- or caspase-8-siRNA would result in an attenuation of shock/sepsis-induced pulmonary inflammation, a decrease in lung apoptosis and neutrophil influx, and/or the protection of lung histology. To do this, male C3H/HeN mice were randomized into four groups (n = 18 each), subjected to hemorrhagic shock, treated with 75 μg of either Fas-, caspase-8-, or GFP(control)-siRNA in 100 μl of PBS or 100 μl of PBS, intratracheally, 4 hours thereafter and then challenged with polymicrobial sepsis 24 hours thereafter. Lung, liver, and plasma were harvested from mice 24 hours after induction of sepsis for quantification of cytokine levels, polymerase chain reaction (n = 8/group), determination of apoptosis (n = 6/group), or histological assessment of pulmonary architecture and neutrophil immigration (n = 4/group). The following inflammatory mediators were chosen based on our previous studies5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar to provide some index of the nature of the inflammatory and chemotactic milieu in the lung:3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Lomas-Neira JL Chung CS Grutkoski PS Miller EJ Ayala A CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.J Leukoc Biol. 2004; 76: 58-64Crossref PubMed Scopus (66) Google Scholar TNF-α, IL-6, IL-12, and IFN-γ were monitored as indicators of the early proinflammatory reaction, MCP-1, KC, and MIP-2 as representative chemotactic agents, and IL-10 to assess a co-existent anti-inflammatory response. Hemorrhagic shock followed by sepsis was chosen as a model of indirect/nonpulmonary-induced ALI because previous results have identified that it is potent in inducing ALI, which is at least in part a result of primed neutrophils.3Ayala A Chung CS Lomas JL Song GY Doughty LA Gregory SH Cioffi WG LeBlanc BW Reichner J Simms HH Grutkoski PS Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.Am J Pathol. 2002; 161: 2283-2294Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Lomas-Neira JL Chung CS Grutkoski PS Miller EJ Ayala A CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.J Leukoc Biol. 2004; 76: 58-64Crossref PubMed Scopus (66) Google Scholar, 5Lomas-Neira JL Chung CS Wesche DE Perl M Ayala A 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.J Leukoc Biol. 2005; 77: 846-853Crossref PubMed Scopus (116) Google Scholar As opposed to cecal ligation and puncture alone, which by itself induces pulmonary inflammation only the combination of hemorrhagic shock and polymicrobial sepsis leads to pulmonary inflammation plus increased lung apoptosis, significant neutrophil apoptosis, and gross pathohistological lung alterations under the described protocol. This does not exclude that if the severity of the polymicrobial sepsis was increased or a different mouse strain was used, cecal ligation and puncture alone could not lead to such alterations per se. C57BL/6-TgN(ACTbEGFP)1Osb mice, overexpressing GFP, were harvested 24 hours after intratracheal administration of GFP-siRNA. Frozen lung, liver, and spleen samples were cut into 6-μm sections at −20°C and fixed in acetone (Sigma). Tissue sections were initially incubated with 0.01% Sudan Black B (MT Biomedicals, Aurora, CO) in 70% ethanol (Sigma) for 5 minutes to reduce tissue autofluorescence. Lung, liver, and s
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