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Interleukin-1 is the primary initiator of pulmonary inflammation following liver injury in mice

Departments of ¹Surgery, Section of Abdominal Transplantation, and ²Immunology, Washington University School of Medicine, St. Louis, Missouri; and ³Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 5 January 2007 ; accepted in final form 24 May 2007

	ABSTRACT

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Hepatic injury can lead to systemic and pulmonary inflammation through activation of NF-B-dependent pathways and production of various proinflammatory cytokines. The exact mechanism remains unknown, although prior research suggests interleukin-1 (IL-1) plays an integral role. Cultured murine alveolar macrophages were used to identify an optimized IL-1-specific short interfering RNA (siRNA) sequence, which then was encapsulated in liposomes and administered intraperitoneally to transgenic HLL mice (5'-HIV-LTR-Luciferase). A 35% hepatic mass cryoablation in HLL and IL-1 receptor 1 knockout mice (IL1R1KO) was performed as a model for liver-induced pulmonary inflammation. IL-1 siRNA pretreatment effectively and significantly reduced circulating IL-1 levels at 4 h post-hepatic injury. IL-6 also was suppressed in mice with impaired IL-1 signaling pathways. NF-B activation in the noninjured liver of HLL reporter mice pretreated with IL-1 siRNA was found to be reduced compared with controls. Pulmonary NF-B activity in this group also was diminished relative to controls. C-X-C chemokine levels in the lung remained significantly lower in IL-1 pathway-deficient mice. Similarly, lung myeloperoxidase content was unchanged from baseline at 24 h post-liver injury in IL-1 siRNA-treated animals, whereas all other control groups demonstrated marked pulmonary neutrophilic infiltration. In conclusion, liver injury-induced lung inflammation in this model is mediated predominantly by IL-1. Knockdown of IL-1 expression before hepatic injury led to significant reductions in both cytokine production and NF-B activation. This translated to reduced pulmonary neutrophil accumulation. Pretreatment with IL-1 siRNA may represent a novel intervention for preventing liver-mediated pulmonary inflammation.

systemic inflammatory response; cytokines; NF-B; cryoablation

The technique of hepatic cryoablation involves the use of a supercooled probe to produce local destruction of liver tumors through the disruption of cell membranes (7). The use of this method of ablation has been largely abandoned due to a morbidity rate approaching 60%, with the lungs being particularly susceptible to injury (19, 32, 33, 36). In light of the clinical similarities in the lung injury patterns that occur in patients following hepatic cryoablation and prolonged cold ischemia during orthotopic liver transplantation, cryoinjury may represent a form of hepatic I/R injury. As such, hepatic cryoablation is a simplified and reproducible model for investigating the pathophysiology of liver-mediated pulmonary inflammation, without such confounding factors as portal hypertension (as occurs during portal clamping techniques) or acute rejection (observed following transplantation). Previously, we demonstrated that members of the IL-1 cytokine family may mediate the ensuing lung inflammation following liver injury (17). The current study further explored this concept utilizing both short interfering RNA (siRNA) directed against IL-1 and homozygous IL-1 receptor 1 knockout (IL1R1KO) mice. We report the novel finding that IL-1 is an early initiator of liver injury-induced lung inflammation and that blocking IL-1 signaling using RNA interference can prevent the remote organ inflammatory response.

	MATERIALS AND METHODS

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In vitro-based siRNA sequence selection. An immortalized murine alveolar macrophage cell line (MH-S; American Type Culture Collection, Bethesda, MD) was grown under standard cell culture conditions using RPMI 1640 media supplemented with heat-inactivated 10% FBS and 0.05 nM 2-mercaptoethanol. MH-S cells constitutively express IL-1, with greater expression observed following LPS-based stimulation (12, 24). Cells underwent transfection with each IL-1 siRNA or a nonsense siRNA as described below, with nontransfected or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-only exposure used as positive controls. Cultures were maintained for 24–72 h, following which the cells were pelleted and the supernatant removed for further analysis. IL-1 was measured in the culture supernatants using ELISA (R&D Systems, Minneapolis, MN). Following the selection of the siRNA sequence exhibiting the greatest degree of IL-1 knockdown, additional testing was performed to select the appropriate siRNA:DOTAP ratio and siRNA working concentration.

siRNA selection and preparation. Three IL-1-specific siRNA sequences initially were selected from the Dharmacon (Lafayette, CO) siRNA library as potential candidates with high likelihoods of knockdown based on in silico screening techniques (28). The 21-nucleotide sequences each included 3'-dUdU overhangs and underwent counter ion sodium exchange, dialysis, and endotoxin screening before administration. The mRNA targets were murine IL-1 sequence A (5'-GAA AGA AUC UAU ACC UGU C-3'), murine IL-1 sequence B (5'-GCU CCG AGA UGA ACA ACA A-3'), and murine IL-1 sequence C (5'-GUG UGG AUC CCA AGC AAU A-3'). Transfection was accomplished using a cationic liposomal carrier molecule (DOTAP; Roche Applied Science, Mannheim, Germany) after incubation with 100 nM siRNA at 26°C for 15 min (35, 37). The siRNA:DOTAP mixture then was diluted in either OptiMEM media (for cell culture; Invitrogen, Carlsbad, CA) or phosphate-buffered physiological saline solution (for in vivo administration). Transfection vehicle without siRNA (DOTAP-only) was administered to control for potential toxicity, and nonsense siRNA (siCONTROL RISC-Free, Dharmacon) transfected under identical conditions to the IL-1-specific siRNA was utilized as a control for non-specificity.

Animal care and surgical techniques. Transgenic 5'-HIV-LTR-Luciferase (HLL) mice were utilized for all siRNA-based experiments. Mice were housed in pathogen-free conditions with a 12-h diurnal light cycle and access to water and food ad libitum. Breeding pairs on a C57Bl/6 background were inbred, with subsequent generations used for this study (ages 8–13 wk). Additionally, IL-1 receptor 1 homozygous knockout (IL1R1KO) mice with C57Bl/6 backgrounds were purchased from Jackson Laboratories (Bar Harbor, ME) and housed under similar conditions. All experiments were approved by the Washington University Animal Studies Committee and conducted in accordance with criteria outlined by the National Institutes of Health in "The Guide for the Care and Use of Laboratory Animals."

Experimental animals received an intraperitoneal dose of 7.5 nmol siRNA (either IL-1-specific or nonsense siRNA) with DOTAP transfection agent and phosphate-buffered saline in a total volume of 1.5 ml 24 h before liver injury. The vehicle-only group received an identical volume containing only DOTAP. Sham animals were given 1.5 ml of PBS only. A separate group of animals received the siRNA:DOTAP mixture but did not undergo liver injury; they were anesthetized only and then killed at the designated times as controls. All experimental groups contained four to six animals.

Our model of hepatic injury using cryoablation has been well characterized (7, 16, 17). In brief, mice are anesthetized with an intramuscular injection of a ketamine (87 mg/kg body wt) and lidocaine (13 mg/kg) cocktail. Active warming to maintain normothermia is accomplished using a heating pad. Following sterile preparation and using aseptic instruments, a midline laparotomy is performed, the liver is mobilized, and the bowel is protected from cryoinjury using surgical sponges. Cryoablation of the left lateral section of the liver is accomplished utilizing a 3-mm diameter probe containing recirculating liquid nitrogen delivered by a commercially available device (Candela, Wayland, MA). A single freeze-thaw cycle reliably produces ablation of 35% of the liver by mass. The abdomen is irrigated with 2 ml of warmed physiological saline and closed in two layers. Animals are subsequently killed at designated times using an overdose of anesthetic with rapid bilateral pneumothoraces. This hepatic injury reliably induces an acute, progressive, inflammatory lung injury with a mortality rate of 45% at 24 h (1, 7, 38). Mice with evidence of intra-abdominal injury other than hepatic injury (e.g., hemoperitoneum or inadvertent gastrointestinal cryoinjury) were excluded from analysis. Blood was drawn from the inferior vena cava and centrifuged for plasma extraction. Tissue samples of the nonablated hepatic remnant and lungs were snap-frozen in liquid nitrogen and stored at –80°C in DNase/RNase-free containers.

Ex vivo determination of NF-B activity. The 5'-HIV-LTR is a known NF-B binding sequence, and its upstream location from the luciferase gene allows direct quantification of NF-B-DNA interaction in HLL mice using luciferase activity measurements (2). Luciferase activity was determined in organ homogenates with slight modification from previously described techniques (23, 29). Briefly, 30-mg portions of frozen lung or liver tissue were homogenized in reporter lysis buffer 1x (Promega, Madison, WI), subjected to three freeze-thaw cycles, and centrifuged at 10,000 g to extract cytoplasmic protein. Protein extract (50 µl) was combined with 100 µl of reconstituted luciferase assay substrate (Promega), followed by measurement of relative light units (RLUs) using a standard luminometer (Berkhold Detection Systems, Oak Ridge, TN). Luciferase activity was normalized to protein concentration, as determined by the Bradford method with spectrophotometric analysis (4).

Determination of pulmonary neutrophilic response. A myeloperoxidase (MPO) assay was used to quantify the degree of acute lung injury at 24 h after hepatic injury. The functional MPO assay is a reliable indicator of the degree of neutrophilic infiltration, particularly in the lungs (18, 30). Frozen pulmonary tissue (100 mg) was homogenized using a tissue rotastator in a 1 M potassium phosphate buffer containing 10% hexadecyltrimethylammonium bromide (Sigma, St. Louis, MO), sonically disrupted, and centrifuged at 1,700 g for 30 min. Supernatant (5 µl) was added to a 0.1 M potassium phosphate buffer solution supplemented with 10% bovine serum albumin, 7.5% sodium bicarbonate, 1 M HEPES, and 10x HBSS. A solution of 0.05% H₂O₂ and o-dianisidine initiated the functional assay and resulting color shift, and this reaction was terminated by the addition of 1% sodium azide after 15 min. Spectrophotometric shift at 460 nm was determined at 5 and 20 min in duplicate. Results were calculated as the change in optical density per minute per milligram of protein to yield a relative quantification of neutrophil content and subsequently normalized to noninjured control values.

ELISA for cytokines. Commercial ELISA kits for murine IL-1, IL-6, macrophage inflammatory protein-2 (MIP-2), and keratinocyte-derived chemokine (KC) were used per the manufacturer's instructions to quantify these proinflammatory cytokines/chemokines in both serum and tissue (R&D Systems). For tissue measurements, total protein was extracted from snap-frozen mouse lung tissue by homogenization in a 150 mM sodium chloride buffer containing HEPES (1 M), EDTA (0.2 M), PMSF in ethanol, and Nonidet P-40. Protein concentrations were measured using the Bradford assay. All samples were measured in duplicate.

Statistical analysis. Comparisons between groups were made using analysis of variance with the Newman-Keuls post-hoc analysis. Data are expressed as means ± SD. A P value less than 0.05 is considered significant.

	RESULTS

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Effective knockdown of IL-1 in murine alveolar macrophages using RNA interference. Variable IL-1 knockdown in the MH-S cell line was achieved using siRNA relative to vehicle-only control. Varying the concentration of the DOTAP transfection vehicle between 3.7 and 7.4 µl/µg siRNA did not affect the degree of IL-1 knockdown in the transfected MH-S cells in the absence of LPS stimulation (data not shown); subsequently, the lower concentration of DOTAP was used for the remaining in vitro and all in vivo experimentation to diminish possible off-target effects from the vehicle itself. Longer exposures to IL-1 siRNA resulted in greater knockdown as measured by ELISA (Fig. 1). Murine IL-1 siRNA sequence C exhibited the greatest cytokine knockdown, with almost undetectable IL-1 levels at 72 h posttransfection. Dose-response testing using siRNA sequence C following Escherichia coli LPS stimulation demonstrated a direct relationship between siRNA concentration and the degree of IL-1 suppression (at 72 h of continuous exposure: control, 118.1 ± 6.7; 1 nM, 111.8 ± 5.8; 10 nM, 96.8 ± 11.7; 100 nM, 92.2 ± 4.9 pg/ml). On the basis of its superior knockdown of IL-1, siRNA sequence C was utilized for all subsequent in vivo experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Varying degrees of IL-1 knockdown following transfection of IL-1-specific short interfering RNA (siRNA) in cultured murine alveolar macrophages (MH-S). Results are depicted for 3 different siRNA sequences at 100 nM, relative to a control consisting only of transfection agent following either 24 or 72 h of continuous exposure.

IL-1 siRNA suppresses systemic cytokine response after liver injury.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Sterile hepatic cryoinjury results in significant IL-1 induction in both 5'-HIV-LTR-Luciferase (HLL) and IL-1 receptor 1 knockout (IL1R1KO) mice. Serum levels of IL-1 were undetectable before liver injury (time 0). *P < 0.05 vs. baseline (time 0), P < 0.001 vs. baseline (time 0).

View larger version (12K): [in this window] [in a new window]   Fig. 3. IL-1-specific siRNA significantly impairs production of serum IL-1 at 4 h post-liver injury. No differences in IL-1 were observed between control groups or the IL1R1KO mice following liver injury. Serum levels of IL-1 were undetectable in the uninjured groups. *P < 0.001 vs. uninjured controls, P < 0.05 vs. IL-1 siRNA group. NS, nonsense.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Lack of IL-1 receptor activation, due to either siRNA-medicated knockdown of IL-1 or lack of IL-1 receptors (IL1R1KO mice), results in reduced serum IL-6. Production of IL-6 is mediated largely via IL-1 signaling pathways. *P < 0.05 vs. uninjured controls.

Suppression of IL-1 production reduces hepatic and pulmonary NF-B activation.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Pretreatment with IL-1-specific siRNA prevented the significant increases in pulmonary NF-B activation observed in control groups after hepatic injury. Baseline values were determined from uninjured, untreated controls. *P < 0.05 vs. baseline, P < 0.01 vs. baseline, and P < 0.01 vs. IL-1 siRNA group. RLU, relative light units.

Reduction in pulmonary NF-B activation ameliorates the subsequent neutrophil influx.

View larger version (13K): [in this window] [in a new window]   Fig. 6. The increase in pulmonary KC chemokine generation observed after liver injury was blunted in the IL-1-specific siRNA-pretreated mice and not observed at all in IL1R1KO mice. *P < 0.01 vs. baseline, P < 0.01 vs. IL1R1KO mice.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Pulmonary myeloperoxidase (MPO) content, as a marker for neutrophil infiltration, was unchanged from baseline in mice with impaired IL-1 signaling pathways at 24 h post-liver injury. Results normalized to uninjured control groups. *P < 0.05 vs. IL-1-specific siRNA group.

	DISCUSSION

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Moderate to large volume hepatic cryoablation induces a systemic inflammatory state affecting multiple organ systems, but the majority of research has focused on the heterogeneous acute lung injury that is characterized by a patchy, mixed neutrophil and macrophage infiltrate (1, 6, 7, 17). Since similar lung injury does not occur following resection or radiofrequency ablation of an equivalent liver volume, cryo-induced injury may represent a variant of I/R injury initiated by the nonablated liver remnant (7). Prior research has shown that IL-1 reaches pathological levels within hours of hepatic injury and that members of the IL-1 signaling pathway are upregulated within the lung in response to hepatic cryoablation (17). These findings were confirmed in the current study, where IL-1 was detectable in serum in both the HLL and IL1R1KO mice following liver injury. This cytokine acts through two cell membrane surface receptors; IL-1 receptor 1 transduces a signal, whereas IL-1 receptor 2 binds the cytokine but is nonfunctioning, presumably acting as a "decoy" receptor (3, 10, 14). Mice deficient in receptor 1 produce normal levels of IL-1 but lack the subsequent downstream effects (15, 20). Accordingly, we observed equivalent IL-1 production following liver injury in the IL1R1KO mice as in the untreated controls. In comparison, pretreatment using IL-1-specific siRNA resulted in a blunted (but non-zero) IL-1 response.

Upon stimulation with IL-1, intracellular inhibitory-B (IB) is phosphorylated and rapidly degraded, leading to translocation of NF-B to the nucleus and binding to the promoter regions of various proinflammatory genes. Increased hepatic NF-B activation occurs in a variety of liver injury models, including following cryoablation, where the remnant liver exhibits markedly elevated NF-B-mediated transcriptional activity (1, 38). In the HLL mice, IL-1 siRNA pretreatment resulted in NF-B activity within the nonablated liver remnant approximating only 30% of that seen in the untreated control groups. Likewise, in the lungs, much lower NF-B activity was observed in the siRNA group, with an even more pronounced difference observed at 24 h post-liver injury. It is unknown whether the large decrement of NF-B activity in the lungs is due solely to the initial blunted hepatic response or whether intraperitoneal siRNA delivery produced systemic (and pulmonary) knockdown of IL-1. Nonetheless, it is clear that treatment with IL-1 siRNA resulted in less NF-B activation in both the liver and the lungs following hepatic injury.

IL-6 induction by IL-1 accounts for the hepatic acute phase protein response and further propagates the proinflammatory cytokine cascade (13). We observed marked increases in serum IL-6 following hepatic injury in control animals, whereas both the IL-1 siRNA-treated and the IL1R1KO mice demonstrated statistically insignificant increases in this cytokine, presumably secondary to disruption of the IL-1 signaling pathway. Likewise, although IL-1 is not a direct chemoattractant for neutrophils, it mediates neutrophil recruitment through IL-1-induced synthesis of C-X-C chemokines such as KC and MIP-2 (5, 14, 22). The lack of IL-1 signaling, either through the use of siRNA or receptor knockout, resulted in diminished KC accumulation in the lungs at both 4 and 24 h after liver injury. Interestingly, animals receiving IL-1 siRNA exhibited an intermediate KC response, suggesting that knockdown (but not complete elimination) via RNA interference still permitted some degree of chemokine generation. The reductions in NF-B activity and KC generation within the lungs of non-control mice resulted in a less vigorous neutrophilic infiltration at 24 h post-liver injury, as evidenced by MPO quantification.

The potential utility of siRNA sequences as therapeutic agents has been previously described in the literature (11, 21, 25). The liver, in particular, seems an ideal target organ for RNA interference, due in part to its reticuloendothelial function and relative ease of transfection. To our knowledge, the current study is the first to demonstrate that RNA interference within the liver can prevent the ensuing pulmonary and systemic inflammatory response. Knockdown of IL-1 before hepatic injury resulted in reduced NF-B activation within the liver and the lungs, reduced levels of systemic IL-1 and IL-6, diminished pulmonary chemokine generation, and a reduction in neutrophil accumulation in the lungs. Although our model of hepatic cryoablation shares many similarities to graft I/R injury during liver transplantation, it is possible the systemic inflammatory responses are mediated through different pathways. Nonetheless, IL-1 appears to play a central role in liver injury-mediated inflammation, and siRNA represents an attractive therapeutic option for the prevention of this response.

	GRANTS

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This project was partially supported by National Institutes of Health Grants P01-HL-66196 and Digestive Diseases Research Core Center (DDRCC) Grant P30-DK-52574.

	ACKNOWLEDGMENTS

 
We appreciate the assistance of Dr. Sekhar Dharmarajan (Washington Univ., St. Louis, MO) with the development of the myeloperoxidase assay.

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. C. Chapman, Washington Univ. School of Medicine, 660 S. Euclid Ave., Campus Box 8109, St. Louis, MO 63110 (e-mail: chapmanw{at}wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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