www.sciencemag.org/cgi/content/full/science.1233632/DC1

Supplementary Materials for

Role of Tissue Protection in Lethal Respiratory Viral-Bacterial

Coinfection

Amanda M. Jamieson,* Lesley Pasman, Shuang Yu, Pia Gamradt, Robert J. Homer,

Thomas Decker, Ruslan Medzhitov*

*Corresponding author. E-mail: ruslan.medzhitov@yale.edu; (R.M.) amanda.jamieson@univie.ac.at

(A.M.J.)

Published 25 April 2013 on Science Express

DOI: 10.1126/science.1233632

This PDF file includes:

Supplementary Text

Figs. S1 to S6

Full Reference List

Supplementary Materials

Mice

C57Bl/6 (B6) mice were obtained from National Cancer Institute and Jackson Laboratory and were

between 6 and 12 weeks of age. Rag2-/- Tlr2-/-/Tlr4-/-, Myd88-/-, Nos2-/-, and IFN r1-/- were bred in the

Yale School of Medicine animal facility or provided by biomodels Austria in specific pathogen free

conditions. All experiments were performed in accordance with the relevant institutional animal care

and use guidelines.

Bacterial and Viral Strains

The A/WSN/33 (H1N1) strain of influenza A virus was obtained from the laboratory of Dr. Akiko Iwasaki

and was propagated using MDCK cell as described in (32). Unless otherwise indicated mice were

infected with the JR32 flaA strain 3 days after infection with influenza virus. The LP01 flaA,

LP01 dotA, and thymidine auxotroph strain LP02 L. pneumophila strains were obtained from Dr. Craig

Roy and the LP02 flaA dotA L. pneumophila strain used were provided by Dr. Russell Vance, and were

grown as described in (33).

Infectious Models

For intranasal infections of influenza and Legionalla mice were anaesthetized with a ketamine/xylazine

mixture and the pathogen was administered dropwise. Mice were infected with a sublethal dose of 300

PFU of influenza in a volume of 30 l intranasally, and 1X106 L. pneumophila was administered in a

volume of 40 l. For AREG experiments mice were injected I.P. daily with 10 g of AREG (Peprotech).

Quantification of Viral and Bacterial Loads

Viral titers in the lungs were determined by titration of organ homogenate on MDCK cells and plaque

forming units (PFUs) were quantified as described in (32). L pneumophila levels were determined by

plating titrated amounts of organ homogenate on CYE plates as described in (21).Organs were

homogenized using the Polytron PT100 in 1ml of appropriate buffer for PFUs or 10ml of sterile water for

L. pneumophila for CFUs.

BAL Collection, Cytospin Analysis, and BALF Albumin Measurement

Bronchalveolar lavages were performed as described elsewhere (21). Briefly mice were euthanized and

the trachea was exposed. Using plastic tubing threaded over a 26 gauge needle attached to a 1ml

syringe .5ml of PBS was slowly injected intratracheally into the lung, and slowly removed into the

syringe. Tubes containing the fluid were centrifuged at 1300 rpm to separate the supernatant from the

cells. The supernatant was used to measure cytokine levels as described below and albumin levels.

Albumin levels were determined using the mouse albumin ELISA kit f(Immunology Consultants

Laboratory Inc.) according to manufacturers instructions. The cells were suspended at a concentration of

1X106 cells/ml, and 100 l were spun onto slides at 700 rpm for 7 minutes, followed by autofix at 700

rpm for 3 minutes. After desiccation for 30 minutes the cells were stained using the diff-quick staining

method according to manufacturer’s instructions, and differential cell counts were performed.

Measurement of Cytokines and Chemokines Expression Levels

Cytokine and chemokine levels were determined by quantitative PCR according to manufacturers’

instructions. Briefly, RNA was isolated from organs using the RNA-bee reagent and cDNA was produced

using the superscript III reagent after treatment with DNase. This cDNA was used with the Stratagene

qPCR machine. Serum and BALF cytokines and chemokine levels were determined using the Luminex

(Millipore) system according to manufacturer’s instructions.

Histology

Organs used for histology were fixed overnight in FormaldeFresh™ or paraformaldehye, and embedded

in paraffin for sectioning. Pathology scoring was done in a blind randomized manner. The scoring system

was as follows: 0= no airway necrosis, 1= focal necrosis, 2= some confluence of necrosis in larger

airways, 3= confluent airway necrosis in most airways.

Statistical Analysis

Statistical analysis was general done with either prism graphpad or Microsoft excel. The logrank test,

student t-test, or ANOVA was performed where appropriate.

Supplemental Figures

Fig. S1. Systemic bacterial and viral load. CFU levels in the (A) liver and (B) spleen are below the limit of

detection. PFU levels in the (C) kidney and (B) spleen are below the limit of detection.

Fig. S2. Growth of bacterial strains in vivo. CFUs in the lung of JR32∆flaA, and LP01∆dotA 1 hour, 3 days,

and 4 days after infection.

Fig. S3. (A) Survival of mice with NAi treatment. (B) Temperature loss after NAi treatment (C) Weight

loss of mice treated with NAi (D) Viral PFUs days post influenza infection following treatment with NAi .

Fig. S4. Expression of immune response genes in lungs from mice infected with influenza virus,

JR32∆flaA alone, or JR32∆flaA 3 days after influenza virus infection 1 day and 3 days after bacterial

infection.

Fig. S5. Survival of (A) Rag2-/-

mice and (B) Ifnar1-/-

mice singly infected or coinfected with JR32∆flaA 3

days after influenza virus infection. (C) Survival of singly and coinfected mice treated with

dexamethasone 2 days after bacterial infection. (D) Survival of coinfected mice treated with NAC.

Fig. S6. Expression of tissue repair genes in lungs from mice infected with influenza virus,

JR32∆flaAalone, or JR32∆flaA 3 days after influenza virus infection 1 day and 3 days after bacterial

infection.

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