Lipopolysaccharide inhibits Th2 lung inflammation induced by house dust mite allergens in

Transcription

1 Online data supplement Lipopolysaccharide inhibits Th2 lung inflammation induced by house dust mite allergens in mice J. Daan de Boer, Joris J.T.H. Roelofs, Alex F. de Vos, Regina de Beer, Marcel Schouten, Tijmen J. Hommes, Arie. J. Hoogendijk, Onno J. de Boer, Ingrid Stroo, Jaring S. van der Zee, Cornelis van t Veer and Tom van der Poll. Material and methods Bronchoalveolar lavage and tissue handling BAL was performed by exposing the trachea through a midline incision and BALF was harvested by instilling and retrieving 1 ml (in aliquots of 500 μl) of sterile saline 0.9%. Cell counts were determined for each BALF sample in a hemocytometer (Beckman Coulter, Fullerton, CA) and differential cell counts were performed on cytospin preparations stained with Giemsa stain (Diff Quick; Dade Behring AG, Düdingen, Switzerland). In the second experiment non lavaged lungs were homogenized in 5 volumes of sterile 0.9% saline using a tissue homogenizer (Biospec Products, Bartlesville, OK) or fixed in 10% formalin. Immunohistochemistry Lungs were fixed in 10% formalin and embedded in paraffin; 4 μm thick sections were stained with hematoxylin eosin. The following parameters were scored: peribronchial inflammation, interstitial inflammation, endothelialitis, edema and pleuritis. Each parameter was graded on a scale of 0 to 4 (0: absent, 1: mild, 2: moderate, 3: severe, 4: very severe).

2 The total pathology score was expressed as the sum of the score for all parameters. In a similar way Periodic Acid Schiff (PAS) D staining for carbohydrates in mucus was performed to quantify the amount of mucus. The amount of mucus per lung section was scored by an experienced histopathologist in a semiquantitative fashion on a scale form 0 3. In addition, eosinophil staining was performed using a monoclonal antibody against major basic protein (MPB, kindly provided by Dr. Nancy Lee and Prof. James Lee, Mayo Clinic Arizona, Scottsdale, USA). Entire sections were digitized with a slide scanner using the 10x objective (Olympus dotslide, Tokyo, Japan) and images were exported in the TIFF format for quantification. Eosinophil influx was determined by measuring the MPB immunopositive area by digital image analysis (ImageJ 1.46, National Institute of Health, Bethesda, Maryland), and was subsequently expressed as a percentage of the total lung area. The average of ten pictures was used for analysis of eosinophilic pulmonary influx. Assays Lung cytokines interleukin (IL) 4, IL 5, IL 6, IL 10, IL 13, IL 17, IL 33, interferon (IFN) γ, KC and E selectin were determined by ELISA (R&D Systems, Abingdon, United Kingdom). TN3 capture antibody for the mouse tumor necrosis factor (TNF) α ELISA was kindly provided by Dr. Steven OldeDaminck, Department of Surgery, Maastricht (the Netherlands), (TNF) α standard and detection antibodies were from R&D Systems (Abingdon, United Kingdom). Plasma total IgE was measured using rat anti mouse IgE capture antibody and biotin rat antimouse IgE as detection antibody, purified mouse IgE was used for standard (BD Biosciences Pharmingen, Breda, the Netherlands). Thrombin antithrombin complexes (TATc) were determined as marker of coagulation activation by ELISA (Affinity Biologicals, Ancaster, Ontario, Canada). Complement activation in BALF was determined by C3a and C5a ELISAs. In

3 short, purified rat anti mouse C3a (clone I ) or purified rat anti mouse C5a (clone I ) were used as capture antibody, and biotin rat anti mouse C3a (clone I87 419) or biotin rat anti mouse C5a (clone I52 278) as detection antibody for C3a and C5a, respectively (all from BD Biosciences). A standard curve for C3a and C5a was generated by using serial dilutions of an in house standard of maximally activated mouse serum. This standard was prepared by incubating normal mouse serum at 37 o C for 1 week in the presence of sodium azide. Total protein in BALF was measured using a colorimetric assay (Biorad Laboratories B.V., Veenendaal, the Netherlands), using Bovine Serum Albumin as standard. All assays were performed according to the manufacturer s recommendations.

4 Figure E1: Study design and treatment groups. Mice were treated with saline, HDM (25 µg) and/or LPS ( µg) intranasally: on day 0, 1 and 2 mice received 100% of the HDM and/or LPS dose indicated; on day 14, 15, 18 and 19 mice received 25% of the HDM and/or LPS dose indicated. On day 21 mice were euthanized and samples harvested for analysis Figure E2: Complement activation. Mean (± SE) concentrations of C3a and C5a in broncholalveolar lavage fluid. * P < 0.05 for the difference with saline. P value in the figure relates to LPS effect on the HDM response. Figure E3: Total protein in BALF. Mean (± SE) concentrations of total protein in broncholalveolar lavage fluid. * P < 0.05 for the difference with saline, ND for not detectable

5

6 C3a C5a * P = * P = 0.07 AU 0.05 AU HDM(µg) LPS(µg) HDM(µg) LPS(µg)

7 1.5 Total protein in BALF P = 0.09 mg/ml n=2 ND HDM(µg) LPS(µg)