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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: [email protected]; (R.M.) [email protected]
(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.
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
A C
FU/L
iver
L. pneumophila Coinfectedd2 d4 d2 d4
CFU
/Spl
een
B
L. pneumophila Coinfectedd2 d4 d2 d4
C D
PFU
s/K
idne
y
PFU
/Spl
een
Influenza Coinfectedd2 d4 d2 d4
Influenza Coinfectedd2 d4 d2 d4
ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
0 1 2 3 4101
103
105
107C
FU/g
days after L.p. infection
LegionellaCoinfected
LegionellaCoinfected
∆ flaA
∆ dotA
0
50
100%
sur
viva
l
days after L.p. infection
A
0 2010**
B
D
Ctrl.NAi
0 4 8102
104
106
days post influenza infection
PFU
/ml Ctrl.
NAi
* *
days after L.p. infection
100
90
80
70
% te
mp
0 2 4 6
C
days after L.p. infection
100
90
80
70
% w
eigh
t
0 2 4 6
Ctrl.NAi
**
*
Ctrl.NAi
CCL7
0
80
160
fold
incr
ease
CXCL13
050
100150
0
100
200CXCL10
1 3
days after L.p. infection
1 3 1 3
InfluenzaLegionellaCoinfected
0
100
200CXCL1
1 3
1 3
400
200
0
CCL3
0100200300
1 3
CCL4
CXCL2
CCL2
0
80
160
1 3
2000
600400
1 3
0204060
1 3
**
**
* *
* * *
* * **CCL8
1 3
Nos2*
0
40
80
1 3
G-CSF*
*0
60
120 40
20
0
*
1 3
TNFα
BA
InfluenzaLegionellaCoinfected
% s
urvi
val
0 10 20
100
50
days after L.p. infection
% s
urvi
val
0 10
100
50
days after L.p. infection
0
InfluenzaLegionellaCoinfected
20** **
C
InfluenzaLegionellaCoinfected
InfluenzaLegionellaCoinfected
+ DEX d+2
% s
urvi
val
0 10 20
100
50
days after L.p. infection
0**
D
0 10days after L.p. infection
20
100
50
0% s
urvi
val Ctrl.
NAC
Rag2-/-
IFNαR1-/-
Adamts2
0
0.5
1.0
Stra13
00.40.81.2
Aifm1
0
0.81.2
0.4
Hif3a
0
20
10
Timp4
0
2
4
Gcnt2
0
20
10
0
4
8 Mdk
fold
incr
ease
/uni
nfec
ted
influenzaLegionellacoinfected
0
1
Slpi2
Vegfc
0
2
4
Hmox1
0
20
10
0
200
400Itga1Itgb7
0
20
10
0
2
4Mmp2
0
4
8 Mmp9
* ** *
*
**
*** **
** * *
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