TLR-sepsis&MV-Villar et al-Supplementary Material
- Supplementary Material -
Mechanical Ventilation Modulates Toll-like Receptor Signaling Pathway in a Sepsis-Induced Lung Injury Model
Running Title: TLR, sepsis, and VILI
1,2,3 Jesús Villar, MD, PhD
1,2 Nuria Cabrera, PhD
1,2 Milena Casula, PhD
1,4 Carlos Flores, PhD
1,5Francisco Valladares, PhD
1,6Mercedes Muros, PhD
1,7 Lluis Blanch, MD, PhD
3,8,9 Arthur S. Slutsky,MD
10,11Robert M. Kacmarek, RRT, PhD
From
(1) CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain;
(2) Multidisciplinary Organ Dysfunction Evaluation Research Network, Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;
(3) Keenan Research Center at the Li Ka Shing Knowledge Institute of St. Michael´s Hospital, Toronto, Canada;
(4) Research Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain;
(5) Department of Anatomy, Pathology & Histology, University of La Laguna, Tenerife, Canary Islands, Spain;
(6) Department of Clinical Biochemistry, Hospital Universitario NS de Candelaria, Tenerife, Spain;
(7) Critical Care Center, Corporació Sanitaria Parc Taulí, Sabadell, Spain;
(8) Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Canada;
(9) Adjunct Professor, King Saud University, Riyadh, Saudi Arabia.
(10) Department of Respiratory Care, Massachusetts General Hospital, Boston, Massachusetts, USA;
(11) Department of Anesthesia, Harvard Medical School, Boston, MA, USA.
METHODS
Histological examination
At the end of the 4-hour ventilation period, a midline thoracotomy/laparotomy was performed in the first 6 surviving rats from each group and the abdominal vessels were transected. The heart and lungs were removed en bloc. The lungs were isolated from the heart, the trachea was cannulated and the right lung was fixed by intratracheal instillation of 3 ml of 10% neutral buffered formalin. After fixation, the lungs were floated in 10% formalin for a week. Lungs were then serially sliced from apex to base and specimens were embedded in paraffin, then cut (3 m thickness), stained with hematoxylin-eosin and examined under light microscopy. A pathologist (FV), blinded to the experimental history of the lungs, performed the histological evaluation. Three random sections of the right lung from each animal were examined with particular reference to alveolar and interstitial damage defined as cellular inflammatory infiltrates, pulmonary edema, disorganization of lung parenchyma, alveolar rupture, and/or hemorrhage. A semiquantitative morphometric analysis of lung injury was performed in 3 random sections from the right lung of each animal by scoring from 0 to 4 (none, light, moderate, severe, very severe) for each parameter (cellular inflammatory infiltrates, pulmonary edema, disorganization of lung parenchyma, alveolar rupture, and/or hemorrhage). A total histological lung injury score was obtained by adding the individual scores in every animal and averaging the total values in each group. To confirm the identification and localization of cell types, we performed additional lung tissue staining with specific antibodies for alveolar type II cells (Thyroid and Lung Epithelial Transcription Factor, TTF-1, DAKO, Glostrup, Denmark) and for lung macrophages (CD68 antibody, DAKO, Glostrup, Denmark). Slides were viewed using a Nikon Optiphot microscope (Tokyo, Japan) and werephotographed with a Nikon Digital Sight DS-5M camera (Tokyo, Japan) at x200 magnification.
RNA extraction and reverse transcription
Left lung were excised, washed with saline, frozen in liquid nitrogen, and stored at –80ºC for subsequent RNA extraction. Lungs were homogenized and total lung tissue RNA was extracted using TRIreagent (Sigma, Hamburg, Germany) and DNase I digestion (Amersham Biosciences, Essex, United Kingdom) [1]. Five g of RNA were subsequently used to synthesize cDNA using the First Strand cDNA synthesis kit (Roche, Basel, Switzerland). Expression levels of tumour necrosis factor-alpha (TNF-), interleukin-6 (IL6), and IRAK3 genes for all samples were determined by using SYBR green I (Molecular Probes, Leiden, The Netherlands) and the iCycler iQ Real-Time detection System (Bio-Rad Laboratories, CA). The -actin gene was amplified and used as a housekeeping gene. Real-Time amplification reactions were performed using previously published primer pairs [2,3], except for the IRAK3 gene whose primers were designed for rat-mouse-human cross-species gene specific amplification (5’-catctgtggtacatgccagaag-3’ and 5’-ccagagagaagagctttgcag-3’). Relative expression levels were obtained from three serial dilutions of cDNA (each by triplicate) using the CT method. All fragments were checked for specificity by direct sequencing of both strands with an ABI PRISM 310 Genetic Analyzer using Big Dye Terminator kit v 3.1 (Applied Biosystems, CA).
Cytokine serum levels
At the end of the 4 h experimental period, 2 ml of blood was collected from the same 6 surviving rats in each group by cardiac puncture and centrifuged for 15 min at 3,000 rpm. Sera were divided into aliquot portions and frozen at –80ºC. TNF-and IL-6 protein concentrations in serum were measured by enzyme-linked immunosorbent assay (ELISA) in dilutions that allowed interpolation from a simultaneously run standard curve. Levels of TNF-and IL-6 were measured with a commercially available ELISA, specific for rats (Cytoscreen, Biosource International, Camarillo, CA). Results were analyzed using an ELx800 NB Universal Microplate Reader (Bio-Tek Instruments, Winooski, Vermont, USA). TNF-and IL-6 concentrations are expressed as pg/ml. The threshold sensitivity was 8 pg/ml for IL-6 and 4 pg/ml for TNF-.
Total protein extraction and Western inmunoblotting
Lung tissue from 6 surviving rats in each group were processed for total protein using ice-cold Nonidet P-40 lysis buffer containing 1% Nonidet P-40, 25 mM Tris-HCl (pH=7.5), 150 mM sodium chloride, 1 mM EDTA, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenyl-methylsulfonyl fluoride plus Protease Inhibitor Cocktail (Roche MolecularBiochemicals, Basel, Switzerland). After centrifuging at 12,000 rpm, the supernatant was subjected to electrophoresis on 10% SDS-PAGE gel, transferred to PVDF membranes, and blocked with 10% skim milk in Tris-buffered saline plus 0.1% Tween 20 (TBS-T). After incubation with TLR2, TLR4, IĸBα, IRAK-3 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; Abcam, Cambridge, UK) blots were incubated with secondary antibody linked to HRP (Goat anti-rabbit IgG-HRP; Santa Cruz Biotechnology, Santa Cruz, CA). Specific antibodies were detected with a chemiluminescence kit (Amersham ECL Western Blotting Detection Reagents, GE Healthcare, Chalfont St Giles, UK). Chemiluminescence was visualized by exposure to X-ray films. Then, membranes were stripped using Restore Western Blot Stripping Buffer and re-probed with β-actin primary antibody (Cell Signaling Technology) and the same secondary antibody to verify protein loading. Densitometric quantification of data was performed using the Scion Image software package.
Immunohistochemistry for IRAK-3
Immunohistochemical stains were performed by applying a standard avidin–biotin complex (ABC) technique. Paraffin-embedded rat lung sections (5 μm thick) were deparaffinized and rehydrated with alcohol series to distilled water. For antigen retrieval, slides were placed in citrate buffer solution (0.01 M, pH 6.0) and microwaved at 800 W for 4-7 min. Fresh frozen sections (5 μm) of rat lung were mounted onto glass slides, fixed in acetone, air dried, and rehydrated in PBS. After blocking endogenous peroxidase activity (10 min in 0,3% hydrogen peroxide), slides were incubated for 1 hour at room temperature with the rabbit polyclonal anti-IRAK-3 antibody (Abcam, Cambridge, UK), then washed in PBS and incubated for 10 min with biotinylated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Following another washing cycle, slides were incubated for 13 min at room temperature with horseradish peroxidase (HRP)-conjugated streptavidin (Zymed, San Francisco, CA), and for 20 minutes at room temperature with AEC+/substrate Chromogen (Dako, Hamburg, Germany). Finally, sections were rinsed in distilled water, counterstained with hematoxylin (Dako, Hamburg, Germany), washed in running tap water, and mounted with mounting media. Slides were viewed using an Olympus (BX50) microscope and werephotographed with an Olympus Camedia digital camera at x400 magnification.
RESULTS
Staining with specific antibodies for type II epithelial cells and lung macrophages
Positive staining of rat lung tissue with TTF-1 and CD68 confirmed that the shape of the cells and localization of alveolar type II cels and lung macrophages were in agreement with current knowledge (Figure S1).
E.S.M. REFERENCES
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- Herrera MT, Toledo C, Valladares F, Muros M, Díaz-Flores L, Flores C, Villar J (2003) Positive end-expiratory pressure modulates local and systemic inflammatory responses in a sepsis-induced lung injury model. Intensive Care Med 29:1345-1353
- Patak E, Candenas ML, Pennefather JN, Ziccone S, Lilley A, Martín JD, Flores C, Mantecón AG, Story ME, Pinto FM (2003) Tachykinins and tachykinin receptors in human uterus. Br J Pharmacol 139:523-532
LEGENDS FOR THE SUPPLEMENTARY FIGURES
Figure S1. Positive cytoplasmic staining for TTF-1 in type II alveolar epithelial cells (A), and for CD68 in alveolar and interstitial macrophages (B), confirming the identification and localization of cell types. Hematoxylin was used as counterstaining (blue/violet color). Original magnification x400.
FIGURE S1