Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria.

Abstract

Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.

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INFECTION AND IMMUNITY, Jan. 2005, p. 532–545
0019-9567/05/$08.00?0
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 1
doi:10.1128/IAI.73.1.532–545.2005
Central Role of Toll-Like Receptor 4 Signaling and Host Defense in
Experimental Pneumonia Caused by Gram-Negative Bacteria
Jill R. Schurr,1Erana Young,1Pat Byrne,1Chad Steele,2Judd E. Shellito,1and Jay K. Kolls2*
Section of Pulmonary/Critical Care Medicine and the Gene Therapy Program, Louisiana State University Health
Sciences Center, New Orleans, Louisiana,1and Department of Pediatrics, Children’s Hospital
of Pittsburgh, Pittsburgh, Pennsylvania2
Received 29 July 2004/Returned for modification 7 September 2004/Accepted 2 October 2004
Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise
role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has
not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene
expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of
Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dis-
semination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4
signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection,
C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which
were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and
chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical
adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the
gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast
majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that
this subsequent gene expression determines survival of the host.
It is now known that recognition of lipopolysaccharide (LPS)
by the host is mediated by Toll-like receptor 4 (TLR4) and is
responsible for the early innate immune response to this agent
(3, 23). However, the precise role that TLR4 plays in coordi-
nating gene expression in response to an intact gram-negative
infection where LPS is only one of many virulence factors
remains unclear. It has been previously reported in experimen-
tal animal models of TLR deficiency that TLR4-deficient mice
are more susceptible to Salmonella sp. infection (10) and that
TLR2-deficient mice are more susceptible to Staphylococcus
aureus infection (29). In both models, reduced survival was
associated with uncontrolled bacterial growth.
The genomic response of purified cell populations such as
dendritic cells (15) and macrophages (21) in response to puri-
fied bacterial ligands like LPS or whole organisms have re-
vealed distinct genetic programs. Huang and colleagues
showed that dendritic cells respond to Escherichia coli, Can-
dida albicans, and influenza virus infection with both distinct
and shared gene expression profiles. E. coli infection resulted
in the greatest number of gene expression changes, with 118 of
685 genes being unique to E. coli and 166 being shared be-
tween influenza, C. albicans, and E. coli. Nau and colleagues
showed similar findings of shared transcriptional programs in
human macrophages but also demonstrated that LPS resulted
in a gene expression profile similar to that of live E. coli,
suggesting that LPS controls the dominant response in macro-
phages to this organism (21, 22). Furthermore, that group
showed that infection of human peripheral blood mononuclear
cells with Mycobacterium tuberculosis poorly induced interleu-
kin-12 (IL-12) p40 and IL-15 compared to E. coli or S. aureus,
which may in part explain immune evasion by this organism
(21).
Despite these data, it remains unclear on a genomic scale
what the contribution of TLR4 signaling is in the lung in
response to infection by gram-negative bacteria. To address
this question, we performed gene expression profiling using
whole lungs of TLR4-deficient C3H/HeJ mice and C3H/HeN
mice with intact TLR4 as well as resistant (C57BL/6) and
susceptible (129/SvJ) strains of mice in an experimental model
of Klebsiella pneumoniae infection. We chose this organism
because it is capable of eliciting pneumonia with very small
inocula (35, 36), and the growth curves of this bacteria are
similar in these mouse strains from time 0 to 16 h, such that
changes in gene expression would not be due to changes in
organism burden over this time course (36). We chose C3H
mice since the C3H/HeJ mutation was initially characterized in
these mice by using C3H/HeN as a control which is nearly
isogenic, with the exception of the TLR4 mutation (28).
TLR4?/?mice have been made on the 129/SvJ background
with subsequent backcrossing to C57BL/6; however, significant
129/SvJ alleles still exist in this strain (14). Moreover, since
129/SvJ mice show susceptibility to infections caused by gram-
negative bacteria independent of TLR4, as outlined below,
data from TLR4?/?mice may be confounded by these 129
alleles.
By using susceptible TLR4 mutant C3H/HeJ mice (23), it
was shown that only 42 genes out of 14,700 genes were signif-
icantly induced by twofold or more at 4 h compared to a total
of 184 genes in resistant C57BL/6 mice or 130 genes in resis-
tant C3H/HeN mice which were induced. These data demon-
* Corresponding author. Mailing address: Children’s Hospital of
Pittsburgh, 3705 Fifth Ave., Suite 3765, Pittsburgh, PA 15213. Phone:
(412) 692-5184. Fax: (412) 692-6645. E-mail: jay.kolls@chp.edu.
532

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strate that TLR4 is critical for gene induction and accounts for
over 81% of acute gene expression changes in C57BL/6 mice
and over 74% of acute gene expression changes in C3H/HeN
mice. Moreover, we identified 67 genes that were shared be-
tween resistant C57BL/6 and C3H/HeN mice which were
clearly TLR dependent. Although TLR4 signaling was critical
in early gene expression, hierarchal clustering showed that
TLR4 mutant mice “catch up” by 16 h, as evidenced by the fact
that gene expression profiles in C3H/HeJ mice at 16 h cluster
with C57BL/6 and C3H/HeN mice with intact TLR at 4 h. This
result may be due to other TLR pathways such as TLR2 or
TLR9 which recognize lipopeptides (25) and CpG DNA (12),
respectively.
MATERIALS AND METHODS
Mice. C3H/HeJ and 129/SvJ male mice (6 to 8 weeks old) were obtained from
Jackson Laboratory (Bar Harbor, Maine), while C3H/HeN and C57BL/6 male
mice (6 to 8 weeks old) were obtained from the National Cancer Institute
(Frederick, Md.). All mice were maintained according to protocol approved by
the Institutional Animal Care and Use Committee. Mice were provided with
sterile food, water, and filtered air and were housed with 12-h light-dark cycles
in the Louisiana State University Health Sciences Center Animal Care Facility.
Bacteria. K. pneumoniae strain ATCC 43816 serotype 2 (American Type
Culture Collection, Rockville, Md.) bacteria were grown in 100 ml of tryptic soy
broth (Difco, Sparks, Md.) for 18 h at 37°C. After 18 h, 1 ml of the culture was
added to a fresh 100 ml of tryptic soy broth and grown for 2 h at 37°C. The
culture was centrifuged at 2,700 ? g for 15 min, and the supernatant was
discarded. The bacterial pellet was washed twice with phosphate-buffered saline
(PBS) and serially diluted to the desired concentration. The concentration of
bacteria was measured by calculating the number of CFU on tryptic soy agar
plates (Remel, Lenexa, Kans.).
Experimental animal procedures. All mice were anesthetized with 50 ?l of
PBS-diluted ketamine-xylazine (50 to 150 mg/kg). The mice were intratracheally
inoculated with 104CFU of K. pneumoniae/ml in a 50-?l volume. At 0, 4, and
16 h postinoculation, the mice were euthanized, the heart and both lungs were
excised, and the right ventricle was flushed with PBS. The right lung was isolated,
homogenized in 1 ml of Trizol (Invitrogen, Carlsbad, Calif.), and placed at
?80°C.
Preparation of labeled cRNA. RNA was extracted from tissues in Trizol (In-
vitrogen) according to manufacturer’s protocol. The SuperScript Choice system
(GIBCO/BRL) in combination with a T7-(T)24DNA primer (5?-GGCCAGTG
AATTGTAATACGACTCACTATAGGGAGGCGG-d(T)24-3?; Integrated DNA
Technologies) was used to synthesize cDNA from total RNA. The first-strand DNA
FIG. 1. Panel A: reduced survival of mice deficient in TLR4 signaling. 129/SvJ, C3H/HeJ, C3H/HeN, and C57BL/6N (n ? 10 mice per group)
were challenged with intratracheal inoculation of 104CFU of K. pneumoniae, and the survival rate was recorded every 24 h. An ? denotes significant
difference (P ? 0.05; log-rank test) compared to C57BL/6 mice. Panel B: lung bacterial burden in each mouse strain over time (n ? 4 to 6 mice
per time point). An * indicates a P value of ?0.05 compared to C57BL/6 mice.
VOL. 73, 2005 ROLE OF TLR4 SIGNALING AND HOST DEFENSE IN PNEUMONIA533

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synthesis reaction mixture contained 5 ?g of total RNA, 100 pmol of T7-(T)24
primer, 500 ?M each deoxynucleoside triphosphate, and 200 U of reverse transcrip-
tase (Superscript II Reverse; Gibco/BRL). The reaction mixture was incubated for
1 h at 42°C. Second-strand cDNA synthesis was carried out at 16°C for 2 h in a total
volume of 170 ?l using 10 U of E. coli DNA ligase, 40 U of E. coli DNA polymerase
I, and 2 U of E. coli RNase H in the presence of 200 ?M each deoxynucleoside
triphosphate. Following the second-strand cDNA synthesis, 10 U of T4 DNA poly-
merase was added, and the samples were incubated for 5 min at 16°C. The reaction
was stopped by the addition of 0.5 M EDTA, and samples were phenol-chloroform
extracted by using Phase-Lock gels (Eppendorf 5 Prime, Boulder, Colo.). Samples
were then precipitated overnight at ?20°C with 0.5 volumes of 7.5 M ammonium
acetate and 2.5 volumes of 100% ethanol. With this double-stranded DNA serving
as a template, a biotinylated antisense cRNA was synthesized by using the Enzo
Bioarray High-Yield RNA transcript labeling kit (Affymetrix). Reactions were run
according to the manufacturer’s instructions. The reaction mixture was incubated at
37°C for approximately 5 h. Samples were then precipitated overnight at ?20°C and
subsequently resuspended in 20 ?l of diethyl pyrocarbonate-treated water. Forty
micrograms of biotinylated antisense cRNA was fragmented by heating the sample
to 94°C for 35 min in a volume of 40 ?l of fragmentation buffer containing 40 mM
Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate.
Microarray analysis of lung RNA. To determine which gene expression pro-
files correlated with host resistance to K. pneumoniae and which of those genes
were regulated by TLR4 in the lung, microarray analysis of lung RNA at time
points early in the infection (4 and 16 h) was performed. Mice were euthanized
at 0, 4, and 16 h (n ? 4 to 9 mice per time point) postinoculation, the right lung
was harvested and homogenized, and the total RNA was isolated. Replicate
samples were individually prepared and hybridized onto separate microarrays as
outlined below. Subsequent enzymatic reactions were carried out with 5 ?g of
total RNA to generate labeled and fragmented cRNA which were then hybrid-
ized to Affymetrix MGU74AV2 microarrays.
Microarray processing. U74Av2 chips (Affymetrix) were prehybridized with
200 ?l of 1? hybridization buffer (100 mM MES, 1 M Na?, 20 mM EDTA,
0.01% Tween 20) for 10 min at 45°C in an Affymetrix Genechip Hybridization
Oven 640 at 60 rpm. Hybridization was done in a final volume of 300 ?l con-
taining 15 ?g of fragmented biotinylated cRNA, 50 pmol of control oligonucle-
otide B2 (Affymetrix), eukaryotic hybridization controls (Affymetrix), 0.1 mg of
herring sperm DNA/ml, and 0.5 mg of acetylated bovine serum albumin/ml in 1?
hybridization buffer. The samples were heated to 95°C for 5 min and 45°C for an
additional 5 min and then briefly spun down. Two hundred microliters of the
hybridization cocktail was added to the standard arrays, and hybridizations were
carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After
hybridization, the solutions were removed, and the arrays were washed by using
a fluidics station (Affymetrix). Hybridized arrays were stained for 10 min at 25°C
with streptavidin-R phycoerythrin (10 ?g/ml; Molecular Probes), followed by
staining with biotinylated goat anti-streptavidin antibody (3 ?g/ml; Sigma Chem-
ical) for 10 min at 25°C. Genechips were then stained once again with strepta-
vidin-R phycoerythrin for 10 min at 25°C. Probe arrays were scanned with a
confocal laser scanner (Agilent) at a wavelength of 570 nm. Pixel intensities were
then measured, and expression signals were analyzed by using a commercial
software package (Microarray Suite [MAS], version 5.0; Affymetrix). LIMS ver-
sion 3.0 (Affymetrix), Data Mining Tools (DMT) version 3.0 (Affymetrix), and
Genespring version 6.0 (Silicon Genetics) were used to perform data analysis.
Microarray data analysis. Microarray data were generated by using Affymetrix
(http://www.affymetrix.com) protocols. Absolute expression transcript levels
were normalized for each chip by globally scaling all probe sets to a target signal
intensity of 500. Three statistical algorithms (detection, change call, and signal
log ratio) were then used to identify differential gene expression in experimental
and control samples. The detection metric (presence, absence, or marginal) for
a particular gene was determined by using default parameters of the MAS
software. Transcripts that were absent under both control and experimental
conditions were eliminated from further consideration. Statistical significance of
signals between the control and experimental conditions (P ? 0.05) for individual
transcripts was determined by using the t test and Mann-Whitney test. Batch
analyses in which pairwise comparisons between individual experimental and
control chips were made in order to generate a change call and a signal log ratio
value for each transcript were performed with MAS. We defined a positive
change call as one in which greater than 50% of the change calls for any one
transcript were increased or marginally increased for upregulated genes and
decreased or marginally decreased for downregulated genes. Finally, the median
value of the signal log ratios from each comparison file was calculated. Signal log
ratio values were converted from log2and expressed as fold changes. In addition,
only those genes that met the above-mentioned criteria and that had a median
signal log ratio of greater than or equal to 1 for upregulated transcripts and less
than or equal to 1 for downregulated transcripts were kept in the final list of
genes.
For hierarchical clustering, genes were included in the final lists if they passed
the following filter requirements when the 4- or 16-h time point was compared to
the 0-h controls within the same strain: elimination of Absent to Absent genes
FIG. 2. Cluster analysis showing relative RNA message levels of
genes found differentially expressed in C57BL/6N mice in response to
K. pneumoniae intratracheal challenge at 0, 4, and 16 h postchallenge.
Gene lists were generated by using algorithms from Affymetrix MAS
version 5.0 and mined by using Affymetrix DMT version 3.0, and lists
were imported into Genespring version 6.0 in order to generate a
cluster. Only those genes that were significantly upregulated and that
had a greater than threefold change in gene expression in all strains
were used to generate a hierarchical cluster using a standard correla-
tion. A total of 90 genes met these requirements (see Table S1 in the
supplementalmaterial[http://www.medschool.lsuhsc.edu/genetics
/genechip_record.asp]). All strains at the zero-hour time point clus-
tered together. Overall gene expression is similar for 129/SvJ and
C3H/HeJ mice at 4 h in that they share a common node. Interestingly,
the 16-h time point for C3H/HeJ shows a gene expression pattern
similar to that of C57BL/6N at just 4 h, indicating that key TLR4-
dependent immunomodulators produced within 4 h of K. pneumoniae
challenge contribute to the overall survival phenotype.
FIG. 3. Venn diagram displaying genes upregulated at 4 h post-
challenge that are both common and unique to three strains of mice:
C57BL/6N, C3H/HeN, and C3H/HeJ (LPS hyporesponsive). Data
were analyzed by using Affymetrix software MAS version 5.0 and DMT
version 3.0. Genes that were included had to be significantly upregu-
lated at 4 h versus the zero-hour time point and had to have a fold
change between these two conditions of two or higher. Lists were
imported into Genespring software version 6.0 (Silicon Genetics) for
graphic illustration. The list of 29 genes unique to C3H/HeJ mice are
TLR4 independent in nature, whereas the list of 67 genes shared
between C57BL/6N and C3H/HeN mice are dependent and should
correlate with overall resistance patterns in mice.
534 SCHURR ET AL.INFECT. IMMUN.

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TABLE 1. Mean TLR4-dependent gene fold changes categorized by function
Affymetrix probe ID Gene symbol
Genbank
accession no.
Change (fold) at 4 vs 0 h
Results in
C3H/HeJ
mice at 4
vs 0 ha
Description
C57BL/6 129/SvJ C3H/HeN
Cytokine/chemokine
101160_at
102218_at
102424_at
102629_at
102736_at
103486_at
104388_at
94755_at
95349_g_at
98772_at
94142_at
102914_s_at
Cxcl2
IL-6
Ccl3
Tnf
Ccl2
IL1b
Ccl9
IL1a
Cxc11
Cxc15
G-CSF
A1b
X53798
X54542
J04491
D84196
M19681
M15131
U49513
M14639
J04596
U27267
M13926
U23778
51.27
26.91
8.51
9.45
32.90
21.56
4.99
9.45
61.39
28.05
2.83
3.76
41.9
4.89
3.05
5.54
6.73
6.68
1.42
5.10
25.99
3.46
2.13
1.93
13.18
25.11
3.05
3.34
6.23
4.82
2.25
4.82
25.63
12.64
3.97
2.60
A
A
A
A
A
A
A
A
A
A
A
A
Chemokine (C-X-C motif) ligand 2/MIP-2
IL-6
Chemokine (C-C motif) ligand 3/MIP-1?
TNF
Chemokine (C-C motif) ligand 2/MCP-1
Interleukin 1?
Chemokine (C-C motif) ligand 9
IL-1?
Chemokine (C-X-C motif)/KC-GRO
Chemokine (C-X-C motif) ligand 5
Granulocyte colony-stimulating factor
Hematopoietic-specific early response A1-b
Receptors
102658_at
931198_at
93430_at
93871_at
102255_at
102663_at
101410_at
98088_at
99413_at
IL-12
Csf3r
Cmkor1
IL-1m
Osmr
Plaur
mCPE-R
CD14
CCR1
X59769
M58288
AF000236
L32838
AB015978
X62700
AB000713
X13333
U29678
12.38
4.41
4.06
8.06
3.03
4.03
4.06
7.84
6.96
3.39
1.58
1.67
3.18
1.33
2.19
1.88
2.82
3.05
8.63
3.20
3.78
2.89
2.08
2.77
5.35
4.17
2.63
A
A
A
A
Chg
NS
A
Fld?2 LPS receptor
?AChemokine (C-C) receptor 1
IL-1 receptor, type II
Colony-stimulating factor 3 receptor (granulocyte)
Chemokine orphan receptor 1
IL-1 receptor antagonist
Oncostatin receptor
Urokinase plasminogen activator receptor
CPE receptor
Apoptosis
101979_atGadd45g AF055638 3.342.172.20AGrowth arrest and DNA damage-inducible 45
gamma
TNF-?-induced protein 399392_at Tnfaip3 U19463 17.759.453.76A
Signal/transduction
102430_at
96747_at
98423_at
92232_at
92534_at
Myd88
Arhu
Gjb2
SOCS-3
GEM
X51397
AW121294
M81445
U88328
U10551
2.10
2.48
2.14
8.28
2.23
1.16
1.56
2.41
3.94
1.56
2.11
3.12
4.89
4.50
4.41
A
A
A
A
A
Myeloid differentiation primary response gene 88
ras homolog gene family, member U
Gap junction membrane channel protein beta 2
Suppressor of cytokine signaling 3
GTP binding protein
Adhesion
102805_at
103005_s_at
96752_at
Ceacam1
CD44
Icam1
M77196
X66084
M90551
2.89
3.07
2.28
1.11
1.48
1.47
3.53
2.08
2.51
A
A
Fld?2 Intercellular adhesion molecule
CEA-related cell adhesion molecule 1
CD44 antigen
Enzymes
102905_at
103341_at
104509_at
104671_at
99985_at
98473_at
94297_at
Casp4
Ctps
Ch25h
Ampd3
TxNR
Arg2
FK506 binding
protein 5
Adam8
Y13089
U49350
AF059213
D88994
AB027565
AF032466
U16959
2.57
2.89
9.25
3.05
3.70
4.20
6.02
1.05
1.40
2.19
1.67
1.92
2.39
2.57
2.25
2.01
2.75
2.22
3.56
3.89
2.81
A
A
A
A
Chg
A
A
Caspase 4, apoptosis-related cysteine protease
Cytidine 5?-triphosphate synthase
Cholesterol 25-hydroxylase
AMP deaminase 3
Thioredoxin reductase 1
Arginase type II
FK506 binding protein 5
103024_at X133353.184.418.75A A disintegrin and metalloprotease domain 8
Transcriptional factors
102955_at
104712_at
NFIL3/E4BP4
C-MYC
U83148
L00039
3.03
6.32
1.89
2.58
2.55
5.58
A
A
Nuclear factor, IL-3 regulated
Myelocytomatosis oncogene
Other
102780_at
103887_at
92315_at
93861_f_at
Tx01
MRP14
Slfn4
Endogenous murine
leukemia virus
modified polytropic
provirus
Z31362
M83219
AF099977
M17327
3.81
5.86
4.20
3.05
3.03
6.36
1.13
1.16
3.11
5.03
2.17
2.33
A
NS
A
A
Migration inhibitory factor-related protein 14
Schlafen4
Endogenous murine leukemia virus modified
polytropic provirus
Continued on following page
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(genes denoted to be absent in control and experimental conditions); statistical
significance by Student’s t test and Mann-Whitney test; change calls of “in-
creased” for upregulated genes and “decreased” for downregulated genes; and a
fold change of ?3 or ??3. Parametric (t test) and nonparametric (Mann-
Whitney test) statistical tests were performed on these data to obtain those genes
that were potentially normally distributed across groups and those that were not.
Self-organizing maps were generated by using DMT with a subset of genes that
were found to be up- and downregulated by the above-described methods in
C57BL/6 mice infected with K. pneumoniae at 4 h compared to the 0-h time
point. The same subset of genes was used for hierarchical clustering using
Genespring version 6.0 on all strains of mice at 0, 4, and 16 h following inocu-
lation.
Alveolar macrophage isolation. Male C57BL/6, C3H/HeN, C3H/HeJ, or 129/
SvJ mice were anesthetized with intraperitoneal pentobarbital and sacrificed by
exsanguination. Thereafter, the lungs were lavaged through an intratracheal
catheter with prewarmed (37°C) calcium and magnesium-free PBS supple-
mented with 0.6 mM EDTA. A total of 10 ml was used in each mouse in 0.5-ml
increments with a 30-s dwell time. The lavage fluids were pooled and centrifuged
at 300 ? g for 10 min, and the cells were collected. To ensure that each cell
preparation was enriched for macrophages, 10,000 cells were cytospun onto
slides and stained with hematoxylin and eosin. Cell preparations were generally
?98% enriched for alveolar macrophages.
Cytokine assays. Alveolar macrophages were isolated from C57BL/6, C3H/
HeN, C3H/HeJ, and 129/SvJ mice as described above. Macrophages were ad-
justed to 5 ? 104in RPMI 1640 medium with 10% fetal bovine serum and 1%
PenStrep (Gibco) and added to a volume of 100 ?l in individual wells of a 96-well
plate (in duplicate). The macrophages were allowed to attach for 1 h at 37°C, and
nonadherent cells were washed away. Thereafter, heat-killed K. pneumoniae
(70°C for 30 min) was added to experimental wells at 104CFU/well in a volume
of 100 ?l of RPMI 1640 medium with 10% fetal bovine serum and 1% PenStrep.
Controls included macrophages cultured in the presence of LPS (10 ?g/ml; List
Biological Laboratories, Campbell, Calif.) or medium alone (control for spon-
taneous production). The macrophage cultures were allowed to incubate for 6 h
at 37°C, 5% CO2, and thereafter, supernatants were harvested for quantification
of macrophage inflammatory protein 2 (MIP-2), IL-6, tumor necrosis factor
alpha (TNF-?), KC-GRO, and granulocyte colony-stimulating factor (G-CSF) by
using the Bio-Plex protein array system (Bio-Rad, Hercules, Calif.) according to
the manufacturer’s instructions. The concentrations of each cytokine and che-
mokine were determined by using Bio-Plex Manager version 3.0 software (Bio-
Rad). Data are expressed as picograms per milliliter.
Statistical analysis of lung CFU and cytokine protein and fold change data.
Data were analyzed by using StatView statistical software (Brainpower Inc.,
Calabasas, Calif.). Comparisons between groups were analyzed by analysis of
variance with a Scheffe follow-up test. Survival was analyzed by log-rank testing.
Significance was accepted at a P value of ?0.05.
RESULTS
Reduced survival of mice deficient in TLR4 signaling. Four
strains of mice were chosen to investigate the role of TLR4
signaling in host lung defense in a K. pneumoniae model:
C57BL/6, 129/SvJ, C3H/HeN, and C3H/HeJ. The C3H/HeJ
strain has a mutant TLR4 allele which results in defective
TLR4 signaling, rendering it hyporesponsive to LPS (23), while
the C3H/HeN mice have a normal TLR4 allele. Lung inocu-
lums were based on previous pilot studies, whereby the 50%
lethal dose at 12 days was determined to be 3 ? 104CFU in
C57BL/6 mice (data not shown). Four strains of mice were
inoculated intratracheally with 104CFU of K. pneumoniae
(ATCC 43816, serotype 2), and their survival was recorded in
24-h intervals for 96 h. Wild-type control mice, C57BL/6 and
C3H/HeN mice, had a 100 and 60% survival rate, respectively,
after 96 h, while the remaining susceptible strains had a 100%
mortality rate within the same time period. Of these strains,
the C3H/HeJ and 129/SvJ mice shared a phenotype of drasti-
cally reduced survival, with death resulting 48 to 72 h following
inoculation (Fig. 1A). Bacterial lung burdens of K. pneumoniae
were determined postinoculation in a separate group of ani-
mals at 4, 12, 16, and 24 h (n ? 4 to 6 mice per time point) by
quantitative culture of lung homogenates (Fig. 1B). As previ-
ously described for C57BL/6 and IL-17 receptor knockout mice
(36), all mice had similar lung bacterial burdens within the first
16 h, with C3H/HeJ and 129/SvJ mice developing statistically
significantly higher lung bacterial burdens at 24 h (Fig. 1B).
Moreover, bacterial dissemination was assessed by quantitative
cultures of spleen homogenates at the same time points. How-
ever, by 24 h, five out of six 129/SvJ, four out of six C3H/HeJ,
and two out of six C3H/HeN mice had spleen cultures that
were positive for K. pneumoniae, whereas zero out of six
C57BL/6 mice were bacteremic at this time point. By 48 h,
three out of six C3H/HeN and only one out of six C57BL/6
mice had positive spleen cultures. 129/SvJ and C3H/HeJ mice
were moribund at this time point and 100% bacteremic. Thus,
as previously observed in this model, mortality was associated
with bacterial dissemination from the lungs (36).
Microarray analysis of lung homogenates. Hierarchal clus-
ter analysis revealed distinct patterns among the four murine
strains at 4 h that correlate closely with the survival outcome as
seen in Fig. 1. Specifically, the mouse strains with the greatest
susceptibility to bacterial dissemination, 129/SvJ and C3H/HeJ,
clustered near each other at 4 h. Moreover, as visually seen in
the cluster (Fig. 2) the C3H/HeJ mice failed to upregulate a
number of genes by 4 h that were observed in C3H/HeN or
TABLE 1—Continued
Affymetrix probe ID Gene symbol
Genbank
accession no.
Change (fold) at 4 vs 0 h
Results in
C3H/HeJ
mice at 4
vs 0 ha
Description
C57BL/6 129/SvJ C3H/HeN
92471_i_at
103448_at
93573_at
99915_at
103328_at
93104_at
98067_at
97689_at
Slfn2
MRP8
MT1
SDGF
I-TRAF
BTG1
P21
Coagulation factor
III
AF099973
M83218
V00835
L41352
U59864
Z16410
U09507
M26071
2.36
5.86
7.89
2.46
2.39
2.07
2.19
3.36
1.95
6.50
5.70
2.33
1.62
1.48
2.10
3.39
2.30
4.56
6.23
2.53
2.33
2.38
2.66
2.35
A
NS
Fld?2 Metallothionein I
A Amphiregulin
A TRAF-interacting activator
Fld?2 B-cell-transclocation gene 1 protein
ACyclin-dependent kinase inhibitor 1A
Chg Coagulation factor III
Schlafen2
Intracellular Ca2?binding protein
aFold changes were not shown for those genes that had detection calls of absent (A) in the 4-h treated group; that had an inappropriate change call (Chg), such
that there was not a call of increased or marginally increased in greater than half of the comparisons made; that had a fold change less than 1 (Fld?2); or that were
not determined to be significant (NS) by parametric and nonparametric tests with a P cutoff value of ?0.05.
536 SCHURR ET AL.INFECT. IMMUN.