Identification of Oxidative Stress and
Toll-like Receptor 4 Signaling
as a Key Pathway of Acute Lung Injury
Yumiko Imai,1,16,* Keiji Kuba,1,2G. Greg Neely,1Rubina Yaghubian-Malhami,1Thomas Perkmann,3Geert van Loo,4
Maria Ermolaeva,4,5Ruud Veldhuizen,6Y.H. Connie Leung,8Hongliang Wang,9Haolin Liu,9Yang Sun,9
Manolis Pasparakis,4,5Manfred Kopf,10Christin Mech,11Sina Bavari,11J.S. Malik Peiris,8Arthur S. Slutsky,12,13
Shizuo Akira,14Malin Hultqvist,15Rikard Holmdahl,15John Nicholls,7Chengyu Jiang,9Christoph J. Binder,3
and Josef M. Penninger1,*
1IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohrgasse 3, A-1030 Vienna, Austria
2Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai 2-3-10, Chiyoda-ku, Tokyo 101-0062, Japan
3CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences and Institute of Medical and Chemical Laboratory
Diagnostics, Medical University of Vienna, Wa ¨hringer Gu ¨rtel 18-20, A-1090 Vienna, Austria
4European Molecular Biology Laboratory (EMBL), Mouse Biology Unit, Via Ramarini 32, Monterotondo-Scalo, Rome 00016, Italy
5Institute for Genetics, University of Cologne, Zu ¨lpicher Str. 47, D-50674 Cologne, Germany
6Lawson Health Research Institute, Departments of Medicine and Physiology and Pharmacology, The University of Western Ontario,
London Ontario, N6A 4V2, Canada
7Department of Pathology
8Department of Microbiology
University of Hong Kong, SAR
9National Key Laboratory of Medical Molecular Biology, School of Basic Medicine Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing 100005, China
10Molecular Biomedicine, Swiss Federal Institute of Technology, Wagistrasse 27, CH-8952 Zu ¨rich-Schlieren, Switzerland
11US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701, USA
12Interdepartmental Division of Critical Care Medicine
13Department of Critical Care, St. Michael’s Hospital
University of Toronto, Toronto M5B 1W8, Canada
14Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
15Medical Inflammation Research, MBB, Karolinska Institutet, Stockholm, Sweden and Medical Inflammation Research BMC I11 Lund,
16Present address: The Global COE program, Akita University School of Medicine, Akita 010-8543, Japan.
*Correspondence: firstname.lastname@example.org (J.M.P.), email@example.com (Y.I.)
Multiple lung pathogens such as chemical agents,
H5N1 avian flu, or SARS cause high lethality due to
acute respiratory distress syndrome. Here we report
that Toll-like receptor 4 (TLR4) mutant mice display
natural resistance to acid-induced acute lung injury
(ALI). We show that TLR4-TRIF-TRAF6 signaling is
a key disease pathway that controls the severity of
ALI. The oxidized phospholipid (OxPL) OxPAPC was
identified to induce lung injury and cytokine produc-
tion by lung macrophages via TLR4-TRIF. We ob-
served OxPL production in the lungs of humans and
animals infected with SARS, Anthrax, or H5N1. Pul-
monary challenge with an inactivated H5N1 avian in-
fluenza virus rapidly induces ALI and OxPL formation
in mice. Loss of TLR4 or TRIF expression protects
mice from H5N1-induced ALI. Moreover, deletion of
ncf1, which controls ROS production, improves the
severity of H5N1-mediated ALI. Our data identify
oxidative stress and innate immunity as key lung
injury pathways that control the severity of ALI.
In 2003, severe acute respiratory syndrome (SARS) spread rap-
idlyfromChinathroughout Asiato Canada(WorldHealthOrgani-
zation, 2008). Most patients who died of SARS developed acute
respiratory distress syndrome (ARDS) (Lew et al., 2003)—the
most severe form of acute lung injury (ALI) (Ware and Matthay,
2000). ARDS was also the cause of death in millions of people
the world primarily infecting poultry and migratory birds. The re-
ported death rates of H5N1 avian influenza infections in humans
pandemic (Beigel et al., 2005; World Health Organization, 2005).
The high lethality of H5N1 or SARS infections as well as their
economic and social impact makes it paramount to explore
common disease mechanisms and novel therapeutic targets of
ARDS. Moreover, many viruses including H5N1 mutate readily
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 235
and produce resistant strains that are no longer controlled by
vaccines or antiviral drugs. The pathology of ARDS in SARS-
coronavirus (Lew et al., 2003) and H5N1 avian influenza virus in-
accumulation of inflammatory cells, edema formation, and
a marked increase in cytokines. The same clinical syndrome of
acute lung failure isobserved in multiple other pathogenic condi-
tions including sepsis, gastric acid aspiration, or pulmonary in-
fections with Anthrax spores (Hudson et al., 1995; Guarner
et al., 2003). Similar ARDS symptoms can be observed in multi-
ple species ranging from birds, rodents, tigers, and primates to
humans. Given the common pathology, we wondered whether
there might be a conserved injury pathway for ARDS.
TLR4 Is a Susceptibility Gene for ALI
To identify endogenous genesthat control the severity of ALI, we
tested inbred mouse strains for their propensity to develop ALI in
response to acid aspiration, a model that recapitulates the acute
phase of human ARDS (Imai et al., 2005; Nagase et al., 2000).
Acid aspiration in mice results in rapid impairment of lung func-
tion as determined by increased lung elastance, a measure of
the change in pressure achieved per unit change in volume, rep-
resenting the stiffness of the lungs, decreased blood oxygena-
tion, and alveolar wall thickening, bleeding, inflammatory cell in-
available online). The severity of acid-induced ALI was compara-
ble among various inbred mouse strains with one exception:
C3H/HeJ mice exhibited reduced lung elastance. Moreover,
C3H/HeJ mice exhibited better blood oxygenation and reduced
inflammation (Figures S1A–S1C).
challenge dueto amutationinthe intracellulardomainof Toll-like
receptor 4 (TLR4) (Tlr4Lps-d) that interferes with TLR4 signaling
(Poltorak et al., 1998). To test whether this TLR4 mutation might
be involved in ALI severity, we assessed acid-induced ALI in
C3H/HeOuJ mice, a C3H-congenic mouse strain that expresses
lost in C3H/HeOuJ mice (Figures S1A–S1D). To confirm that
TLR4 determines disease severity, we triggered acid-mediated
ALI in tlr4?/?mice (Hoshino et al., 1999). Genetic inactivation of
tlr4 markedly attenuated ALI as determined by improved lung
elastance (Figure 1A), reduced edema formation (Figure 1B),
and ameliorated histological changes (Figure 1C). Bone marrow
chimeras showed that the severity of ALI is controlled by TLR4
expression on hematopoietic cells (Figures 1D–1F). Lung pathol-
ogy were not affected in tlr3 (Yamamoto et al., 2003) or tlr9
(Hemmi et al., 2000) mutant mice (Figures S2A and S2B). These
data identify TLR4 as a susceptibility gene for ALI.
Acute Lung Injury Is Mediated
via TLR4-TRIF-TRAF6-NF-kB Signaling
TLR4 stimulation is relayed to cellular responses via different
adaptors such as MyD88 and TRIF (Akira et al., 2006). Surpris-
ingly, genetic deletion of myd88 (Adachi et al., 1998) had no ap-
parent effect on the severity of ALI (Figures S2C and S2D). In
contrast, trif?/?mice (Yamamotoetal.,2003)exhibited markedly
improved lung function (Figure 2A), lung pathology (Figure 2B),
and lung edema (Figure S3A). TRIF signals either through IKK-3
and activation of IRF3 or via TRAF6-mediated NF-kB activation
(Sato et al., 2003). To dissect the TLR4-TRIF activation pathway,
we conducted experiments using irf3 and traf6 knockout mice.
Deletion of irf3 (Sato et al., 2000) did not show a significant
change in acid-mediated ALI (Figures S2E and S2F). We next
generated myeloid cell-specific traf6 mutant mice using Lys-
Cre-mediated deletion of a TRAF6flox/floxallele (TRAF6MC-KO).
Deletion of TRAF6 was confirmed in cultured bone marrow-
derived macrophages (Figure 2C). Inactivation of TRAF6 in
TRAF6MC-KOmice resulted in alleviation of ALI (Figures 2D, 2E,
and S3B). To determine whether mutations of TLR4, TRIF, or
TRAF6 affect NF-kB activation, we performed in situ signaling
experiments to detect nuclear accumulation of Ser276-phos-
phorylated NF-kBp65 indicative of NF-kB activation (Vermeulen
et al., 2002). Whereas acid aspiration triggered NF-kBp65
activation in WT and myd88?/?mice, much less nuclear
NF-kBp65 translocation occurred in lungs from tlr4?/?, trif?/?,
or TRAF6MC-KOmice (Figure 2F). NF-kB activation was largely
restricted to macrophages. Taken together, our genetic results
identify TLR4-TRIF-TRAF6-NF-kB as a key signaling pathway
that links acid injury to the severity of ALI.
IL-6 Mediates Acute Lung Injury
Local cytokine production at the site of injury is one of the hall-
marks of ALI (Martin, 1999). The molecular basis of how such
cytokine storms are mediated by local injury and whether such
injury-released cytokines actually contribute to disease severity
of ARDS are still unknown. To examine whether TLR4 signaling
may be involved in local cytokine, we performed multiple cyto-
kine arrays from normal lungs and lung tissue following acid
aspiration. The cytokines IL-6, IL-1b, and KC (IL-8) were upregu-
lated by acid treatment in WT mice, but we failed to observe up-
regulation of TNFa, IL-2, IFN-g, IL-1a, IL-12p40, IL-12p70, IL-17,
RANTES, IL-9, Eotaxin, or MIP-1b (Figure S4 and data not
shown). IL-3, IL-4, IL-5, IL-13, G-CSF, GM-CSF, MCP-1, and
MIP-1a were below the detection limit of our assay. IL-10 nega-
tively correlated with the severity of ALI (Figure S4E). In particu-
lar, IL-6 levels closely reflected the severity of ALI in resistant
tlr4?/?, trif?/?, and TRAF6MC-KOmice and injury-prone mice
myd88?/?and irf3?/?mice (Figure 2G). To test if IL-6 is indeed
involved in the pathogenesis of ALI, we studied il-6?/?mice
hibited significant improvement of lung function (Figure 2H), lung
edema formation (Figure S3C), and decreased lung pathologies
(Figure2I). Althoughthesedata donotexclude the importanceof
other cytokines, our genetic results show that loss of IL-6 allevi-
ates the severity of ALI (Figure 2J).
Oxidative Stress and Formation of Oxidized
Phospholipids in ALI
Since we used acid aspiration to trigger ALI, a key question
emerged: what triggers TLR4 under such injury conditions? To
rule out the possibility of LPS contamination, we first treated
mice with polymyxin B to neutralize LPS (Jacobs and Morrison,
1977). Polymyxin B did not alter disease severity in acid-induced
ALI (Figure S5A). Moreover, in line with previous data from our
236 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
group (Imai et al., 2005) and others (Nagase et al., 2000), instilla-
tion of LPS alone into lungs does not worsen lung function in our
acute lung failure model (Figure S5B). We next stimulated alveo-
lar macrophages from WT, tlr4?/?, myd88?/?, or trif?/?mice with
bronchoalveolar lavage (BAL) fluid from normal mice (BAL con-
trol), BAL fluid from lungs of acid-treated WT mice (BAL acid),
or LPS. In line with our genetic data, BAL fluid-induced IL-6
production was mediated via TLR4 and TRIF, but not MyD88
(Figure 3A). By contrast, LPS-induced IL-6 production in alveolar
macrophages was dependent on TLR4, MyD88, and TRIF
(Figure 3A). Thus, other TLR4-activating agents rather than
LPS are generated during the course of ALI.
Since the lung air-liquid interfaceisexposed to theaerobic en-
vironment and therefore susceptible to oxidation, we speculated
that ALI could result from the local generation of reactive oxygen
species (ROS) and the subsequent formation of oxidized phos-
olar macrophages following in vivo acid challenge (Figure 3B).
Acid treatment also triggered enhanced TLR4 surface expres-
sion (Figure 3C). To demonstrate the role of OxPLs in vivo we
used the well-characterized mAb EO6 (Friedman et al., 2002).
This mAb specifically detects the phosphocholine (PC) head-
group of phospholipids in which the sn2 side chain has been
oxidized but does not recognize native nonoxidized PLs. We
Figure 1. TLR4 Is a Susceptibility Gene for Acute Lung Injury
(A) Changes in lung elastance after acid or saline treatment in WT and tlr4?/?mice. n = 8–10 for acid-treated groups, n = 6 for saline-treated groups. **p < 0.01 for
the whole time course.
(B) Lung edema formation after acid or saline treatment. *p < 0.05.
(C) Lung histopathology. H&E staining. Original magnifications 3 200.
(D) Lung elastance after acid treatment of WT mice transplanted with WT bone marrow (BM) (WT BM / WT), WT mice receiving tlr4?/?BM (tlr4?/?BM / WT),
tlr4?/?mice transplanted with WT BM (WT BM / tlr4?/?), and tlr4?/?mice receiving tlr4?/?BM (tlr4?/?BM / tlr4?/?). n = 6 for each group.
(E) Lung edema formation and (F) lung histopathology 3 hr after acid injury in WT BM / WT, tlr4?/?BM / WT, WT BM / tlr4?/?, and tlr4?/?BM / tlr4?/?
In (D) and (E), *p < 0.05 comparing WT BM / WT or WT BM / tlr4?/?mice. n = 6 for each group. H&E staining. Original magnifications 3 200. Data in (A), (B), (D),
and (E) are mean values ± SEM.
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 237
238 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
observed large amounts of EO6-detectable OxPLs in the BAL
fluid of acid-challenged animals (Figure S5C). In situ immunohis-
tochemistry confirmed the generation of EO6-detectable OxPLs
in alveolar macrophages as well as inflammatory exudates lining
the injured air spaces (Figure 3D). In addition, we observed
formation of the lipid peroxidation breakdown product malon-
dialdehyde detected by the specific mAb MDA2 (Palinski et al.,
1996) (Figure S5D), further confirming that lipid oxidation does
occur in diseased lungs. These data show that acid aspiration
triggers the oxidative stress machinery and the generation of
OxPLs in lung.
OxPLs Induce Cytokine Production and Acute
Lung Injury via TLR4
Minimally modified low-density lipoprotein (LDL) has been previ-
ously shown to mediate macrophage activation (Miller et al.,
2003a, 2005) and to modulate the severity of atherosclerosis
and inflammatory responses via TLR4 (Miller et al., 2003a;
Michelsen et al., 2004). To address whether OxPLs generated
during ALI can activate innate immune responses, we stimulated
WT and tlr4?/?alveolar macrophages with BAL fluid from normal
(BAL control) and diseased lungs (BAL acid). BAL fluid from mice
with ALI, which contains OxPLs (Figure S5C), induced large
amounts of IL-6 production in WT alveolar macrophages. Neu-
tralization of OxPLs by the specific mAb EO6 attenuated BAL
fluid-induced IL-6 production (Figure 3E). BAL fluid did not
trigger IL-6 release in tlr4?/?alveolar macrophages (Figure 3E).
Similarly, BAL fluid from diseased mice induced IL-6 production
in peritoneal (Figure S6A) and bone marrow macrophages (Fig-
ure S6B), which was reverted by EO6 Ab treatment. Induction
of IL-6 was again dependent on TLR4 expression (Figures S6A
and S6B). OxPLs were also observed in acid-treated trl4?/?
mice (Figure S6C).
The major source of PLs in the lung is surfactant, which forms
a film at the alveolar air-water interface and reduces surface ten-
sion. Surfactant contains 80%–90% PLs including unsaturated
phosphatidylcholine that can be oxidized (Rodriguez Capote
et al., 2003; Veldhuizen et al., 1998). In vitro-oxidized surfactant
PLscontain EO6-detectable OxPLs (Figure S6D).Addition of pu-
rified synthetically oxidized surfactant PLs induced IL-6 produc-
tion in a dose-dependent manner in WT peritoneal macrophages
(Figure 3F). IL-6 production in response to surfactant OxPLs was
decreased by the neutralizing mAb EO6 (data not shown) and
dependent on TLR4 expression (Figure 3F). In line with previous
results (Bailey et al., 2004), in vivo administration of nonoxidized
PLs induces slightly impaired lung function (Figure 3G). Intratra-
cheal administration of OxPLs markedly worsened lung function
in normal mice (Figure 3G). In a second experimental model, we
removed endogenous surfactant via saline lavages, which
strongly impairs lung function; wefollowed thiswith intratracheal
replacement with nonoxidized or oxidized surfactant PLs. In this
scenario, challenge with OxPLs resulted in much more severe
impairment of lung function and all experimental animals died
by 2 hr, whereas mice that received control, nonmodified surfac-
tant PLs stabilized their lung function (Figure S7A). Lung function
was comparable among WT and tlr4?/?mice following adminis-
tration of nonoxidized surfactant PLs. Loss of TLR4 expression
markedly alleviated the severe lung failure phenotype following
induced increased levels of IL-6 in the lungs of WT control but
not in those of tlr4?/?mice (Figure S7B). These data indicate
that synthetically oxidized surfactant PLs can trigger cytokine
production in macrophages and, importantly, acute lung injury
in vivo via TLR4.
OxPAPC Triggers Acute Lung Injury
Wenext attempted to identify specific OxPLs thatcan trigger ALI
via TLR4. The mAb EO6 we used for the detection of OxPLs has
been previously reported to specifically bind PC-containing
OxPLs such as oxidized 1-palmitoyl-2-arachidonoyl-phosphati-
dylcholine (OxPAPC) (Figure S7C). We confirmed that the mAB
EO6 indeed detects OxPAPC (Figure S8A). To directly test
whether OxPAPC can trigger cytokine production via TLR4, we
stimulated lung tissue macrophages with OxPAPC and nonoxi-
dized PAPC. Compared to PAPC, OxPAPC induced higher IL-6
levels in WT macrophages, and these levels were attenuated by
duction was decreased in tlr4?/?and trif?/?lung macrophages,
and this residual IL-6 production was not affected by the mAb
EO6 (Figure 4B). Similar to our in vivo experiments, OxPAPC
Figure 2. TRIF-TRAF6-NFkB-Cytokine Signaling Mediates the Severity of Acid Aspiration-Induced Acute Lung Injury
(A) Lung elastance after acid or saline treatment of WT and trif?/?mice. n = 6–10 for acid-treated groups, n = 4 for saline-treated groups. **p < 0.01 for the whole
(B) Reduced inflammation and hyaline membrane formation in acid-treated trif?/?mice. H&E staining. Original magnifications 3 200.
(C) TRAF6 protein expression in bone marrow macrophages isolated from Lys-Cre(?) TRAF6flox/flox(TRAF6MC-WT) and Lys-Cre(+) TRAF6flox/flox(TRAF6MC-KO)
mice. b-actin protein is shown as control. Data from two different mice (1, 2) are shown for each genotype.
(D) Changes in lung elastance in TRAF6MC-WT(n = 6) and TRAF6MC-KOmice (n = 4) following acid aspiration. *p < 0.05 for the whole time course.
(E) Reduced inflammatory cell infiltration, bleeding, and hyaline membrane formation in acid-treated TRAF6MC-KOmice. H&E staining. Original magnifica-
tions 3 200.
(F) Immunolocalization of Ser276-phosphorylated NFkBp65 in lung tissue of saline-treated control WT mice and acid-treated WT, myd88?/?tlr4?/?, trif?/?, and
TRAF6MC-KOmice. Note the accumulation of phosphorylated NFkBp65 in nuclei of macrophages (red arrows) in acid-treated WT and myd88?/?mice, which is
absent in lung macrophages (black arrows) from acid-treated tlr4?/?, trif?/?, and TRAF6MC-KOmice. Original magnifications 3 400.
(G) IL-6 levels in lung tissue after acid treatment in WT Balb/c, WT BL6, control TRAF6MC-WT(wild-type), tlr4?/?(Balb/c background), tlr9?/?(BL6 background),
tlr3?/?(BL6), myd88?/?(Balb/c) trif?/?(BL6), irf3?/?(BL6), and TRAF6MC-KOmice. IL-6 levels were determined by whole lung tissue ELISA. n = 5–8 animals for
each group. *p < 0.05.
(H) Lung elastance after acid treatment in interleukin-6 mutant (il-6?/?) and control WT mice. n = 5–6 each group. *p < 0.05.
(I) Improved lung histopathology in acid-treated il-6?/?mice. H&E staining. Original magnifications 3 200. Lungs were analyzed 3 hr after treatment.
(J) Schematic diagram depicting the role of TLR4 in ALI. Data in (A), (D), (G), and (H) are mean values ± SEM.
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 239
Figure 3. Formation of Oxidized Phospholipids in Acute Lung Injury
(A) Increase in IL-6 production from baseline in WT, tlr4?/?, myd88?/?, and trif?/?alveolar macrophages treated with LPS, BAL fluid from normal control, or BAL
fluid from acid-treated WT mice. **p < 0.01. Data are from four separate experiments.
(B) Increases in ROS expression in alveolar macrophages obtained from WT mice 60 min after treatment with saline (background control, blue) or acid (white).
A representative histogram is shown among five separate experiments.
(C) Increased TLR4 surface expression in alveolar macrophages from WT mice treated with saline (control) or acid. Data are from five separate experiments.
*p < 0.05.
(D) Immunohistochemistry for OxPLs detected by the mAb EO6 in lungs of saline-treated control (upper panel) and acid-treated WT mice (lower panel). OxPLs
werelocalized toinflammatoryexudatesliningtheinjuredalveoli(arrows)inacid-treated lungs.Originalmagnifications3400.Lungswereanalyzed3hrafteracid
(E) BAL fluid from acid-treated mice (BAL acid) induces large amounts of IL-6 in WT but not tlr4?/?alveolar macrophages. BAL fluid plus an isotype-matched
control Ab was compared to BAL fluid plus the mAb EO6. *p < 0.05. Data are from four separate experiments.
(F) Increase in IL-6 from baseline in peritoneal macrophages isolated from WT and tlr4?/?mice in response to nonoxidized PLs or oxidized PLs. **p < 0.01.
(G) Lung elastance in WT mice following intratracheal administration of saline, nonoxidized PLs, and oxidized PLs. n= 4–6 for each group. **p < 0.01 for the whole
(H) Lung elastance in WT and tlr4?/?mice following treatment with nonoxidized PLs or oxidized PLs. n = 4 for each group. **p < 0.01 for the whole time course.
Data in (A), (C), and (E)–(H) are mean values ± SEM.
240 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
could still trigger IL-6 production in myd88?/?lung tissue macro-
phages. LPS-induced IL-6 production in lung macrophages was
dependent on TLR4, TRIF, and MyD88 (Figure 4B). In addition,
OxPAPC induced higher IL-6 production than nonoxidized
PAPC in alveolar macrophages isolated by lung lavage (Fig-
ure S8B). OxPAPC-induced IL-6 production was decreased in
phages, OxPAPC triggered IL-6 production in peritoneal macro-
phages dependent on TLR4 and TRIF but not MyD88 (Fig-
ure S8D). Thus, OxPAPC can trigger IL-6 production in lung
and peritoneal macrophages via TLR4-TRIF signaling. Impor-
tantly, intratracheal administration of OxPAPC induced marked
deterioration of lung function in WT mice (Figure 4C). In vivo
OxPAPC, but not PAPC, challenges triggered IkBa degradation
indicative of an activated NF-kB pathway (Figure 4D). Moreover,
lung function (Figure 4E) and IL-6 production (Figure S8E) were
markedly alleviated in tlr4?/?and trif?/?but not myd88?/?mice.
These data indicate that OxPAPC can trigger ALI via TLR4
Inactivated H5N1 Avian Influenza Virus Can Induce
OxPL and ALI in Mice
Patients who died of H5N1 avian influenza or Spanish influenza
developed ARDS, and extremely high levels of cytokines have
been observed in patients and animals infected with these vi-
ruses (Beigel et al., 2005; Tumpey et al., 2005). Our data so far
showed that acid aspiration triggers production of OxPLs that
can augment the severity of ALI and cytokine production via
Figure 4. Oxidized PAPC Induces ALI and IL-6 Production via TLR4
(A) Increases in IL-6 production from baseline (unstimulated control) in lung tissue macrophages from WT mice in response to PAPC (10 mg/ml) or OxPAPC
(10 mg/ml) in the presence of an isotype-matched control mAb or the mAb EO6. *p < 0.05 comparing mAb EO6(?). **p < 0.01 comparing OxPAPC EO6(?)
and PAPC EO6(?). Data are from four separate experiments.
(B) Increase in IL-6 from baseline (control) in lung tissue macrophages isolated from WT, tlr4?/?, myd88?/?, and trif?/?mice in response to LPS (1 ng/ml) or
OxPAPC (10 mg/ml). *p < 0.05 comparing OxPAPC-treated WT or myd88?/?macrophages ± EO6. **p < 0.01 compared to OxPAPC EO6(?) treated WT macro-
phages. Data are from three separate experiments.
(C)Lung elastance inWT micefollowing intratracheal administrationof PAPC orOxPAPC(20mg/gbody weight). n=4foreachgroup.**p< 0.01forthewholetime
(D) Western blots of IkBa and b-actin in lungs isolated from mice 90 min after saline treatment (control), OxPAPC, or PAPC (20 mg/g body weight). Blots are
representative of three separate experiments.
(E) Lung elastance at 1.5 hr in WT, tlr4?/?, myd88?/?, and trif?/?mice following OxPAPC challenge (20 mg/g body weight). The correct genetic background
controls are shown. n = 4 for each group. *p < 0.05. Data are mean values ± SEM.
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 241
TLR4. We wanted to extend these findings to H5N1-mediated
ALI. Inactivated influenza A virus (McKinney et al., 2003) or inac-
tivated respiratory syncytial virus (Haeberle et al., 2002) can in-
duce rapid activation of NF-kB and/or proinflammatory cytokine
production in macrophages. The virulence of the 1918 Spanish
influenza virus was largely determined by its hemaglutinin (HA),
high IL-6 production and severe ALI in vivo (Kobasa et al., 2004).
Based on these reports, we tested whether an inactivated H5N1
virus where HA activity is conserved might induce OxPL produc-
tion and ALI in vivo via TLR4. Since H5N1 subtype influenza A vi-
ruses are more potent inducers of cytokines in macrophages
than H1N1 subtype influenza (Cheung et al., 2002), we used
H1N1 as a control.
Pulmonary challenge of inactivated H5N1 avian influenza virus
induced rapid impairment of lung function as determined by in-
creased lung elastance (Figure 5A), severe edema formation as
assessed by wet/dry lung weight ratios (Figure 5B), and patho-
logical changes such as alveolar wall thickening, bleeding, or
the accumulation of inflammatory cells (Figure S9A). Administra-
tion of inactivated H1N1 virus had only very mild effects on lung
function and lung histology (Figures 5A,5B, and S9A).In line with
a previous study (van Riel et al., 2006), we observed influenza A
virus nuclear protein-positive cells in the lower respiratory tract
(LRT) in mice treated with inactivated H5N1 virus (Figure 5C).
By contrast, we only found very few virus nuclear protein-posi-
tive cells in the LRT of H1N1-treated mice (Figure 5C). Similar
to acid treatment, we found higher production of ROS in alveolar
Figure 5. Inactivated H5N1 Avian Influenza Virus Can Induce OxPLs and ALI in Mice
(A) Changes in lung elastance in WT mice following intratracheal administration of vehicle (n = 3), inactivated H1N1 (n = 6), or inactivated H5N1 (n = 8) viruses.
*p < 0.05 for the whole time course.
(B) Lung edema formation 5 hr after vehicle, inactivated H1N1, or inactivated H5N1 treatment. **p < 0.01.
(C) Immunohistochemistry for influenza A nucleoprotein in lung of WT mice challenged with vehicle, inactivated H1N1, or inactivated H5N1 viruses. Influ-
enza A protein-positive cells were present in the lower respiratory tract including alveolar macrophage (arrowhead) and pneumocytes (arrows). Original magnifica-
tions 3 400.
(D) ROS expression in alveolar macrophages from WT mice treated with control vehicle (blue), inactivated H1N1 virus (white, upper panel), or inactivated H5N1
virus (white, lower panel).
(E) TLR4 expression in alveolar macrophages from WT mice treated with vehicle (blue), inactivated H1N1 virus (white), or inactivated H5N1 virus (white). Repre-
sentative histograms are shown for five separate experiments. Data in (D) and (E) are at 1 hr after viral challenge.
(F) Immunohistochemistry for influenza A nucleoprotein and EO6-detectable OxPLs in lung of WT mice infected with live H5N1 avian influenza virus. H5N1-in-
fected mice contain large numbers of influenza A protein-positive cells (brown) in the lower respiratory tract (left panels, arrowheads). OxPLs were localized
to inflammatory cells (arrowheads) as well as inflammatory exudates (arrow). Lungs were analyzed 4 days after infection. Original magnifications 3 100 (upper
panels) and 3 200 (lower panels). Data are mean values ± SEM.
242 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
macrophages isolated from mice treated with inactivated H5N1
avian influenza virus (Figure 5D). In addition, TLR4 surface
expression was increased in alveolar macrophages from mice
treated with inactivated H5N1 (Figure 5E). In vivo challenge of
mice with H1N1 did not result in detectable ROS production or
upregulation of TLR4 cell-surface expression (Figures 5D and
5E). Moreover, we detected OxPLs in the BAL fluid of animals
challenged with inactivated H5N1 (Figure S9B). Immunohisto-
chemistry confirmed enhanced generation of EO6-detectable
OxPLs at the sites of injury (Figure S9C). We extended the anal-
(Figure 5C), influenza virus A protein was detected in the LRT of
miceinfected withliveH5N1 avianinfluenza virus(Figure5F).Im-
portantly, infection with live H5N1 avian influenza virus resulted
in the formation of EO6-detectable OxPLs in mouse lung (Fig-
ure 5F). Thus, challenge of mice with H5N1 avian influenza can
trigger OxPLs and rapid onset ALI in vivo.
Inactivated H5N1-Induced ALI Is Attenuated in tlr4
and trif Mutant Mice
affects the severity of ALI via the TLR4-TRIF pathway, we ana-
lyzed ALI in tlr4?/?and trif?/?mice. The tlr4?/?mice, treated
with the inactivated H5N1 virus, showed significantly improved
lung elastance (Figure 6A), pulmonary edema formation (Fig-
ure 6B), and alleviated lung pathologies (Figure 6C) compared
with control WT mice. Moreover, trif?/?mice, treated with the
inactivated H5N1 virus, showed improved lung elastance (Fig-
lung pathologies (Figure 6E). NF-kBp65 activation in macro-
phages was also impaired in lungs from tlr4?/?and trif?/?mice
(Figures 6C and 6E,). In addition, BAL fluid obtained from
H5N1-challenged mice induced high levels of IL-6 in WT but
not tlr4?/?alveolar macrophages. Neutralization of OxPLs using
the mAb EO6 attenuated IL-6 production in WT but not tlr4?/?
Figure 6. TLR4- or TRIF-Deficient Mice Show Less Severe ALI to Challenge with Inactivated H5N1 Avian Influenza Virus
(A) Lung elastance in WT and tlr4?/?mice following administration of vehicle or inactivated H5N1 viruses. n = 5 for each group. **p < 0.01 for the whole time
(B) Lung edema formation in WT versus tlr4?/?mice 5 hr after H5N1 challenge. **p < 0.01.
(C) Lung pathology (top; H&E staining) and immunolocalization of NFkBp65 (bottom) in lung tissue of H5N1-challenged WT and tlr4?/?mice. Note nuclear ac-
cumulation of Ser276-phosphorylated NFkBp65 in macrophages from WT lung (red arrows). In H5N1-treated tlr4?/?lungs, nuclear NF-kB accumulation was
mostly absent from macrophages (green arrows) albeit present in a few cells (red arrow). Original magnifications 3 400.
(D) Lung elastance in WT and trif?/?mice after administration of vehicle or inactivated H5N1 viruses. n = 5 for each group. **p < 0.01 for the whole time course.
(E) Lung pathology (top; H&E staining) and immunolocalization of NFkBp65 (bottom) in lung tissue of H5N1-challenged WT and trif?/?mice. Original magnifica-
tions 3 400.
(F) BAL fluid from WT mice 5 hr after a H5N1 challenge (BAL H5N1) triggers IL-6 in WT but not tlr4?/?alveolar macrophages. BAL fluid from H5N1-treated mice
plus an isotype-matched control mAb (?) was compared to BAL fluid from H5N1-treated mice plus the mAb EO6 (+). *p < 0.05. Data are mean values ± SEM.
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 243
Figure 7. The Oxidative Stress Machinery Controls the Severity of ALI in Response to Inactivated H5N1 Avian Influenza Viruses
(A) ROS expression in control PBMCs and PBMCs from the same donor treated with inactivated H1N1 or inactivated H5N1 viruses. Data are from CD14+gated
PBMCs. Representative histograms are shown for six separate donors.
six different donors.
(C) Immunofluorescence staining of TLR4 in PBMCs treated with formulation control or inactivated H5N1. TLR4 was distributed throughout the cytoplasm in
control cells (top). PBMCs treated with inactivated H5N1 showed peripheralization of TLR4 (bottom).
(D) Lung elastance in WT and ncf1?/?mice following intratracheal administration of inactivated H5N1 viruses. n = 4 for each group. **p < 0.01 for the whole time
(E) Lung pathology (top; H&E staining) and immunolocalization of oxidized PLs (bottom) in lung tissue of H5N1-challenged WT and ncf1?/?mice. Original
magnifications 3 200.
(F) Immunohistochemistry of OxPLs in lungs from two patients infected with H5N1 avian influenza virus (top) and two patients infected with SARS-coronavirus
244 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
alveolar macrophages (Figure 6F). Thus, cytokine production in
alveolar macrophages and ALI in vivo are attenuated in tlr4 and
trif mutant mice in response to challenge with inactivated
H5N1 avian influenza virus.
H5N1 Avian Influenza Virus Triggers Oxidative
Stress in Humans
Next, we examined whether inactivated H5N1 avian influenza vi-
ruses can also trigger the oxidative stress machinery in humans.
Stimulation of peripheral blood mononuclear cells (PBMCs) with
inactivated H5N1 viruses indeed resulted in generation of ROS
(Figure 7A). By contrast, challenge with inactivated H1N1 re-
sulted in much less ROS production in human PBMCs. More-
over, FACS staining showed that TLR4 surface expression was
upregulated in PBMCs treated with inactivated H5N1 compared
to H1N1 (Figures 7B and S10A). Whereas TLR4 protein is distrib-
uted throughout the cytoplasm in human PBMCs under control
conditions, challenge of PBMCs with inactivated H5N1 virus in-
duced relocalization and cell membrane association of TLR4
(Figure 7C). Thus, inactivated H5N1 viruses can induce ROS
formation and TLR4 surface expression on primary human
Loss of NCF1 Improves the Severity of ALI
Our data so far showed that inactivated H5N1 viruses can trigger
ROS formation in mouse alveolar macrophages and human
monocytes. We therefore want to test whether such ROS gener-
ation is indeed involved in disease pathogenesis of H5N1 influ-
enza virus-mediated ALI. To address this, we analyzed lung
injury in WT and ncf1 (neutrophil cytosolic factor 1) mutant
mice (Hultqvist et al., 2004). Ncf1 is a key component of the
NADPH oxidase complex required for oxidative burst and ROS
virus showed significantly improved lung elastance (Figure 7D),
pulmonary edema formation (Figure S10B), and alleviated lung
pathologies (Figure 7E) compared with control mice on the
same genetic background. Importantly, in H5N1-challenged
ncf1?/?mice we failed to detect oxidized PL formation (Fig-
ure 7E). Thus, genetic impairment of ROS production results in
reduced formation of OxPLs and significantly improved lung
functions in response to H5N1 challenge.
Multiple Lethal Lung Pathogens Trigger Formation
of OxPLs in Different Species
Finally, to test whether OxPLs are generated during actual H5N1
influenza virus infections in humans, we analyzed lung samples
from two patients who had developed ARDS following H5N1
avian influenza virus infections. Consistent with our mouse
acid aspiration and H5N1 models, we detected massive forma-
tion of OxPLs localized at the inflammatory exudates lining the
injured air spaces as well as alveolar macrophages in the lungs
of both H5N1-infected patients (Figure 7F). Lungs from patients
that died of extrapulmonary diseases did not show any detect-
able OxPL formation (Figure S10C). Thus, H5N1 infections in
humans result in the local activation of the oxidative stress
machinery and OxPL formation in the lung.
To examine whether OxPL production is a general feature of
lethal lunginjurythatcan beseenin differentpathogen infections
and across wider species barriers, we analyzed lung samples
from nine individuals who had developed ARDS following
SARS infections. In all SARS-infected human cases tested, we
observed marked production of OxPLs in the inflammatory
exudates lining the injured air spaces, pneumocytes, as well as
these findings to additional species and pathogens, we analyzed
lung samples from animals infected with potential bioterrorism
agents. We observed formation of OxPLs in Anthrax-infected
Rhesus monkeys, Anthrax-infected rabbits, Monkey Pox-in-
fected Cynomolgus monkeys (Macaca fascicularis), as well as
Yersinia pestis-infected Cynomolgus monkeys that developed
lung plague (Figure 7G). By contrast, we could not detect ele-
vated OxPLs in Smallpox orMarburg virus-infected Cynomolgus
monkeys, nor in monkeys infected with Western Equine Enceph-
alitis (WEE) or Eastern Equine Encephalitis (EEE) viruses, dis-
eases that did not cause severe lung injury (data not shown).
Thus,inmultiple species,infections withvariouslethallungpath-
ogens such as H5N1 avian influenza virus, SARS-coronavirus,
Anthrax, Y. pestis, or Monkey Pox virus can trigger formation
of OxPLs in the lung. These results should be extended to
additional pathogens and species.
signaling pathways: MyD88-dependent or TRIF-dependent
pathways (Akira et al., 2006). In the classical pathway, MyD88
recruits IL-1R-associated kinase (IRAK) and TRAF6 leading to
activation of IKKa/b/g complexes. TRIF signals through IKK-3
leading to activation of IRF3 and the expression of IFN-inducible
genes. It has been shown that TRAF6 binds to TRIF though
TRAF6-binding motifs in the N-terminal portion of TRIF and is in-
volved in TRIF-mediated activation of NF-kB in vitro (Sato et al.,
2003). Importantly, our study shows that innate immune signal-
ing via TLR4-TRIF-TRAF6 is a key genetic pathway that deter-
mines the susceptibility to acute lung failure in vivo. Moreover,
our bone marrow transplantation experiments and our LysCre
TFAF6 mutant mice show that this pathway operates in myeloid
cells, in particular lung macrophages. Since macrophages can
utilize both TRIF- and MyD88-dependent pathways for cytokine
induction, our data might provide an opportunity to minimize ALI
via TRIF inhibition while not affecting MyD88-dependent innate
vironment and thus is a highly susceptible site for oxidative
strate for such lipid modifications. Recently oxidized moieties,
(G) Immunohistochemistry of OxPLs in rhesus monkeys and rabbits infected with Bacillus anthracis (left panels) and Cynomolgus monkeys infected with Monkey
Pox or Yersinia pestis (right). In (F) and (G), OxPLs detected by the mAb EO6 were seen in inflammatory exudates lining the injured alveoli (arrows) and
macrophages (triangles). Original magnifications 3 400. Data are mean values ± SEM.
Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc. 245
including OxPLs, have been implicated as pathogen-associated
molecular patterns that are detected by several conserved pat-
tern-recognition receptors (Binder et al., 2002; Miller et al.,
2003b). In particular, minimally oxidized LDL has been found to
activate TLR4 on macrophages (Miller et al., 2003a, 2005),
though the exact mechanism of this activation remains elusive.
However, this connection has been controversial (Bochkov
et al., 2002). We therefore used a genetic approach to examine
whether TLR4 expression is indeed required for OxPL-induced
cytokine production in macrophages in vitro or OxPL-induced
ALI in vivo. Our genetic data show that OxPL-mediated cytokine
release and OxPL-induced ALI depend on TLR4 expression.
Thus, OxPLs can either directly or indirectly activate the TLR4
signaling cascade during the course of lung injury. Consistent
with previous reports (Powers et al., 2006; Kurt-Jones et al.,
2000), we also found that chemical injury and inactivated H5N1
virus upregulate TLR4 surface expression in macrophages.
Thus, local lung injury not only triggers activation of the oxidative
stress machinery but also induces upregulation of TLR4 involved
in the OxPL-induced acute lung injury response.
We have identified OxPAPC as an important trigger of ALI. In
fact, OxPAPC has been known to be generated at the sites of in-
flammation and is found in membranes of apoptotic cells (Chang
et al., 2004). Furthermore, OxPAPC can play a proinflammatory
role in atherosclerosis (Berliner and Watson, 2005; Furnkranz
et al., 2005) and impairs the outcome of gram-negative sepsis
in vivo (Knapp et al., 2007). Interestingly, in the E. coli-induced
peritonitis model, coadministration of OxPAPC also increased
the local production of cytokines, e.g., IL-6 (Knapp et al.,
2007). On the other hand, anti-inflammatory properties of Ox-
PAPC have been reported in LPS-induced sepsis or ALI, which
may be explained by direct antagonism of LPS recognition
(Bochkov et al., 2002; Nonas et al., 2006). Besides OxPAPC,
other OxPLs might also trigger ALI. Moreover, additional TLR4
ligands are also likely to be present, e.g., modified matrix com-
ponents, dying cells, hyaluronan, or HMGB1 (Miyake, 2007).
involved in disease pathogenesis of ALI. Our data showed that
mation in mouse alveolar macrophages and human monocytes.
in all species tested. Of note, gene expression analysis of mice
infected with recombinant viruses expressing the 1918 viral HA
revealed a high upregulation of ROS-related genes (Kash et al.,
2004). We therefore tested H5N1-induced lung injury in ncf1
mutant mice. Ncf1 encodes one of the activating proteins in the
phagocytic NADPH oxidase complex. Importantly, this genetic
defect in ROS production resulted in a significantly reduced
lung injury in response to challenge with our inactivated H5N1 vi-
observe EO6-detectable oxidized PLs confirming that Ncf1-reg-
activation of the oxidative stress machinery is not simply a
by-product of the disease process but controls disease severity.
Our results in two experimental ALI models, acid aspiration
and inactivated H5N1-induced ALI, indicate that chemical as
well as viral lung pathogens trigger the oxidative stress machin-
ery resulting in ROS generation and the local production of
OxPLs. Massive formation of OxPLs occurred in all severe cases
of acute lung failure we have analyzed including humans who
died of H5N1 or SARS infections and animals that developed
lethal lung failure due to Y. pestis, Monkey Pox, pulmonary An-
thrax, or H5N1 infections. OxPLs such as OxPAPC can directly
trigger cytokine production in macrophages and modulate the
severity of acute lung injury in vivo dependent on TLR4-TRIF-
TRAF6 expression. Thus, the acute onset of proinflammatory
immune responses and severe lung injury caused by different
machinery that couples to innate immunity (Figure S11). Modula-
tion of this injury pathway, therefore, could be utilized to protect
patients infected with H5N1 avian influenza virus, SARS-corona-
virus, Anthrax, or other as yet unknown lethal lung pathogens
from developing acute severe lung failure.
For detailed Experimental Procedures see the Supplemental Data.
Clinical Patient Profiles
Lung samples from two H5N1- and nine SARS-infected humans were used.
C57BL6 mice were infected intranasally with live H5N1 avian influenza virus
(strain A/HK/483/97) and euthanized for tissue sampling on day 4. Lethal lung
infections with Anthrax, Y. pestis, and Monkey Pox virus, as well as infections
with Smallpox virus, Marburg virus, WEE, and EEE and lung sampling were
performed at the US Army Medical Research Institute of Infectious Diseases.
tlr4, tlr9, myd88, tlr3, trif, irf3, il-6, and ncf1 mutant mice have been previously
described. Mice harboring a floxed TRAF6 allele will be reported in the future
background-matched mice were used. Basal lung function and lung structure
were comparable among all the mice tested.
Acid Aspiration-Induced ALI in Mice
for 3 hr. Total positive end expiratory pressure (PEEPt) and plateau pressure
(Pplat) were measured at the end of expiratory and inspiratory occlusion.
Elastance was calculated as (Pplat ? PEEPt) divided by tidal volume (VT)
every 30 min during the ventilation periods.
Bone Marrow Transplantation
Recipient mice were irradiated and 1 3 106donor bone marrow cells were de-
livered i.v. through the tail vein. Chimeras were used for experiments 12 weeks
Preparation of Oxidized Surfactant PLs and In Vivo Treatments
In vitro oxidation of surfactant PLs was performed according to previously
established conditions (Rodriguez Capote et al., 2003). The in vivo impact of
nonoxidized surfactant PLs and oxidized surfactant PLs was tested in normal,
healthy lung as well as saline-lavaged lungs to remove surfactant.
Polymyxin B, LPS, and OxPAPC Treatment in Mice In Vivo
LPS (E. coli O111:B4; 0.5 mg/g body weight), OxPAPC (20 mg/g body weight),
or control PAPC (20 mg/g body weight) were instilled intratracheally. Animals
were then ventilated for 3 hr and pulmonary elastance determined.
H5N1Avian Influenza IsolatesandH5N1-InducedLungInjury inMice
Inactivated avian influenza subtypes (H1N1 and A/ck/Yamaguchi/7/04 H5N1)
were obtained from the Istituto Zooprofilattico Sperimentale delle Venezie.
246 Cell 133, 235–249, April 18, 2008 ª2008 Elsevier Inc.
Vehicle and inactivated H1N1 or H5N1 viruses were instilled intratracheally.
Animals were then ventilated for 5 hr and elastance was calculated every
Assessment of Blood Oxygenation and Pulmonary Edema
Partial pressure of arterial oxygen (PaO2) was measured to assess arterial
blood oxygenation as an indicator for respiratory failure. To assess pulmonary
edemas, the lung wet/dry weight ratios were calculated.
Histology, In Situ NF-kB Detection, and Immunohistochemistry
Lung tissue was fixed in 4% buffered formalin. For histological analysis,
sections were stained with hematoxylin and eosin (H&E). For detection of ac-
tivated NFkB, paraffin sections were incubated with a polyclonal anti-phos-
pho-NFkBp65 (Ser276) antibody. Immunohistochemistry to detect oxidized
phospholipids (OxPLs) was performed using the mAb EO6 (kindly provided
by J.L. Witztum). Immunohistochemistry for influenza A virus nucleoprotein
was performed using the HB 65 (EVL anti-influenza NP, subtype A) antibody.
Multiple Cytokine Expression Analyses and Cytokine ELISA
Frozen lung tissues were homogenized in cell lysis buffer, and supernatants
were assayed using multiplex cytokine arrays. For ELISA analyses, superna-
tants of homogenized lung tissue were assayed using specific kits.
Isolation and Activation of Macrophages
Bone marrow macrophages were isolated from femurs of 8- to 10-week-old
mice. Peritoneal macrophages were obtained by injecting mice with thiogly-
collate, followed by peritoneal lavage. Alveolar macrophages were obtained
from mice by bronchoalveolar lavage (BAL). Lung tissue macrophages were
prepared using collagenase and DNase. For measurement of IL-6, macro-
phages were plated on 96-well plates and stimulated in vitro for 24 hr.
Detection of ROS and TLR4
Mouse alveolar macrophages were obtained from mice treated with vehicle,
acid, or inactivated H5N1. Human PBMC were isolated with Ficoll-Hypaque
and treated with inactivated H5N1 or H1N1 virus. For ROS detection, cells
were incubated with DCF. For mouse TLR4 surface staining, cells were double
stained for CD11c and TLR4. For human TLR4 detection, cells were double
stained for TLR4 and CD14. ROS and TLR4 expression were assessed by
FACS. For TLR4 immunolocalization, PBMCs were fixed, permeabilized, and
incubated with anti-TLR4.
All data are shown as mean ± standard error of the mean (SEM). Measure-
ments at single time points were analyzed by ANOVA and, if significant, further
analyzed by a two-tailed t test. Time courses were analyzed by repeated-
measurements (mixed model) ANOVA with Bonferroni post-tests.
Supplemental Data include eleven figures, Supplemental Experimental Proce-
dures, and Supplemental References and can be found with this article online
We thank all members of our laboratories for helpful discussions and critical
reading of the paper. We thank Tada Taniguchi for providing IRF3 mutant
mice and V. Komnenovic for immunostaining. We are particularly grateful to
Fred Possmeyer, Ernst Malle, Juergen Arnold, and Holger Spalteholz for key
advice and cooperation. We also thank Hongliang Wang, Haolin Liu, Kangtai
Liu, Shunxin Wang, Yang Sun, Ping Ma, Shuan Rao, Feng Guo, and Peng
Yang for help with cloning of the recombinant H5 protein and human PBMC
work. In addition, we thank LL Pitt and USAMRIID pathology for providing
specimens for this study and DTRA as S.B.’s funding agency. J.M.P. is sup-
ported by IMBA, the Austrian National Bank, and the Austrian Ministry of
Science and Education. Y.I. and K.K. are supported by an EU network grant
(EuGenHeart). K.K. and G.v.L. were supported by Marie-Curie Fellowships.
Work in the lab of M.P. was supported by EMBL and by grants from the Euro-
pean Union (LSHG-CT-2005-005203 and CT04-005632). M.K. is supported by
SNF grant # 3100A0-100233/1. A.S. is supported by the Canadian Institutes of
Health Research (CIHR) and the Canada Foundation for Innovation (CFI). This
work was also funded by National Natural Science Foundation of China
(30421003, 30528002, 30625013, and 30623009) and Ministry of Science
and Technology of China (2006AA02Z152 and 2005CB523000).
Received: April 25, 2007
Revised: December 10, 2007
Accepted: February 29, 2008
Published: April 17, 2008
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