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Essential Impact of NF-
B Signaling on the H5N1 Influenza A
Virus-Induced Transcriptome
1
Mirco Schmolke,* Dorothee Viemann,
†
Johannes Roth,
†
and Stephan Ludwig
2
*
Systemic infections of humans and birds with highly pathogenic avian influenza A viruses of the H5N1 subtype are characterized
by inner bleedings and a massive overproduction of cytokines known as cytokine storm. Growing evidence supports the role of
endothelial cells in these processes. The aim of this study was to elucidate determinants of this strong response in endothelial cells
with a focus on the transcription factor NF-
B. This factor is known as a major regulator of inflammatory response; however, its
role in influenza virus replication and virus-induced immune responses is controversially discussed. By global mRNA profiling of
infected cells in the presence or absence of a dominant negative mutant of I
B kinase 2 that specifically blocks the pathway, we
could show that almost all H5N1 virus-induced genes depend on functional NF-
B signaling. In particular, activation of NF-
B
is a bottleneck for the expression of IFN-

and thus influences the expression of IFN-dependent genes indirectly in the primary
innate immune response against H5N1 influenza virus. Control experiments with a low pathogenic influenza strain revealed a
much weaker and less NF-
B-dependent host cell response. The Journal of Immunology, 2009, 183: 5180 –5189.
Since 1997, when the first cases of directly transmitted
highly pathogenic avian influenza viruses (HPAIV)
3
from
poultry to humans in East Asia were reported (1), a grow-
ing concern arose that these viruses might become the source of a
new influenza pandemic. So far direct transmissions of HPAIV are
rare events (2), need high doses of infectious particles, and did not
lead to any unlimited spread. In poultry, HPAIV of the H5 subtype
show high transmissibility and mortality accompanied by systemic
infection of the host and severe inner bleedings (3). Infections with
HPAIV are characterized by a hyperreaction of the host immune
response accompanied by massive production of cytokines and
chemokines (4, 5). This overproduction of cytokines and chemo-
kines, known as cytokine storm is a cell intrinsic phenomenon (6).
Growing evidence supports the contribution of endothelial cells in
this process. This cell type is not only a major source of cytokines
and chemokines (7) but is also extremely relevant for systemic
viral dissemination, supported by the fact that endotheliotropsim
has been demonstrated for HPAIV in infected birds (8, 9) and
humans (10, 11). In this study, we used primary human endothelial
cells that are highly permissive for and responsive to influenza A
virus infections and show no defects in the innate immune re-
sponse as widely observed for many transformed cell lines.
The innate host cell response is the first line of defense to viral
infections. In the case of influenza virus infections, it is triggered
by accumulation of viral 5⬘-triphosphate vRNA. This pathogen
pattern is mainly sensed by the cytosolic helicase retinoic acid-
induced gene I (RIG-I) (12). Activation of RIG-I leads to phos-
phorylation and nuclear translocation of the constitutively ex-
pressed transcription factor IFN regulatory factor (IRF) 3 via
TANK-binding kinase 1 (13–15) and activation of NF-
B (p50/
p65) via I
B kinase 2 (IKK2)-mediated phosphorylation/degrada-
tion of the inhibitor of
B(I
B
␣
) (16). NF-
B and IRF3 are
known to regulate the expression of many cytokines and chemo-
kines including IFN-

, a major mediator of the innate antiviral
response. Both factors bind to adjacent promoter regions and as
part of the so-called IFN-

enhanceosome (17, 18). IFN-

is se-
creted by the infected cell and can act in an autocrine or paracrine
fashion by binding to the type 1 IFN receptor (IFNAR). Subse-
quently, activation of the JAK/STAT pathway initiates the forma-
tion of the IFN-stimulated gene factor 3 (ISGF3), consisting of
STAT1, STAT2, and IRF9. ISGF3 binds to IFN-sensitive regula-
tory elements (ISRE) and regulates the expression of IFN-stimu-
lated genes (ISG), including IRF7, myxovirus resistance A (MxA),
2–5-oligoadenylate synthetase 1, RIG-I, and IFN-

itself (19 –23),
which can directly or indirectly interfere with the replication of
viruses.
The function of IRF3 and AP-1 in enhancing type I IFN pro-
duction upon influenza A virus infection are undeniable (24).
However, the role of NF-
B in influenza virus infected cells is the
subject of ongoing discussions. Recent reports either claim no in-
volvement of NF-
B in the antiviral gene expression profile in-
duced by influenza viruses (25) or even present data that support
an inhibitory function of NF-
B in this context (26). This is in
contrast to earlier studies that clearly underline the necessity of
initial NF-
B binding to the IFN-

enhanceosome to allow effi-
cient production of IFN-

and thus promote expression of ISGs
(27).
*Institute of Molecular Virology, Center of Molecular Biology of Inflammation and
Interdisciplinary Center of Medical Research and
†
Institute of Immunology and In-
terdisciplinary Center of Medical Research, Universitaetsklinikum Muenster, Muen-
ster, Germany
Received for publication December 16, 2008. Accepted for publication August
2, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Deutsche Forschungsgemeinschaft
(Lu477-11-2, SFB293 A17), the Interdisciplinary Clinical Research Centre of the
University of Muenster (Grant Lud2/032/06), the “FluResearchNet” funded by the
German Ministry of Education and Research, the European Commission funded Spe-
cific Targeted Research Project EUROFLU, and the VIRGIL European Network of
Excellence on Antiviral Drug Resistance.
2
Address correspondence and reprint requests to Dr. Stephan Ludwig, Institute of
Molecular Virology, von Esmarch Strasse 56, 48149 Muenster, Germany. E-mail
address: ludwigs@uni-muenster.de
3
Abbreviations used in this paper: HPAIV, highly pathogenic avian influenza virus;
IRF, IFN regulatory factor; IKK2, I
B kinase 2; ISRE, IFN-sensitive regulatory el-
ement; ISGF3, IFN-stimulated gene factor 3; ISG, IFN-stimulated gene; MxA, myx-
ovirus resistance A; RIG-I, retinoic acid-induced gene I; EGFP, enhanced GFP; MOI,
multiplicity of infection; GO, Gene Ontology; IP-10, IFN-
␥
-inducible protein 10;
NS1, nonstructural protein 1; HMEC-1, human microvascular endothelial cell 1; p.i.,
postinfection.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804198
To dissect the function of NF-
B signaling in the network of
activated pathways triggering the antiviral response in systemic
infections with H5N1 HPAIV, we specifically interfered with
NF-
B signaling by means of expression of a dominant negative
form of IKK2. This approach has been successfully used previ-
ously to efficiently blunt NF-
B activation (28 –31). By global
gene expression profiling, we demonstrate the essential impact of
NF-
B signaling on the antiviral gene response against HPAIV of
the H5N1 subtype.
Materials and Methods
Reagents and plasmids
Recombinant human IFN-

was purchased from PBL. BAY11-7085 was
purchased from Sigma-Aldrich. Cells were preincubated with 5
Mofthe
incubator for 30 min before infection. The retroviral expression plasmids
pCFG5-IEGZ HA and pCFG5-IEGZ IKK2KD were described previously
(28 –31).
Viruses and cells
The HPAIV strain A/Thailand/KAN-1/2004 (H5N1) isolated form a fatal
human case was used with permission from Dr. P. Puthavathana (Bangkok,
Thailand). The low pathogenic human influenza A virus strain A/WSN/33
was taken from the virus collection of the Institute of Molecular Virology
(Muenster, Germany). Viruses were propagated on Madin-Darby canine
kidney (MDCKII) cells. MDCKII were cultured in MEM (PAA) contain-
ing 10% v/v FCS and 100 U/ml penicillin/0.1 mg/ml streptomycin (1⫻
penicillin/streptomycin; Life Technologies). Primary HUVEC (Cambrex)
were cultured as described previously and used in passages five to seven.
Human microvascular endothelial cell 1 (HMEC-1) were cultured in
MCDB-131 containing 10% FCS, 1
g/ml hydrocortisone, 10 ng/ml re-
combinant human epidermal growth factor (R&D Systems), 10 mM L-
glutamine (Life Technologies), and 50 mg/ml gentamicin. Phoenix pack-
aging cells (Orbigen) and HEK293T (American Type Culture Collection)
were cultured in DMEM (PAA) containing 10% v/v FCS and 1⫻
penicillin/streptomycin.
Plaque assay
PFU of a given virus suspension were determined as described earlier (32).
Retroviral gene transfer
Fifteen micrograms of the empty retroviral pCFG5-IEGZ HA vector or
pCFG5-IEGZ IKK2KD expressing dominant negative IKK2 (29) was
transfected into 3 ⫻10
6
Phoenix packaging cells with polyethyleneimine
as described previously for HEK293 (33) and selected with 250
g/ml
Zeocin (Invitrogen) for 2 wk to gain stable producer cells. Three ⫻10
6
stable producer cells were seeded on 100-mm dishes 48 h before transduc-
tion. Twenty-four hours before transduction, medium was changed to
HUVEC culture medium (6 ml/dish) and retrovirus-containing superna-
tants were harvested 24 h later. HUVEC and HMEC-1 were infected with
retroviral supernatants as described previously (29). Retrovirally trans-
duced HMEC-1 were selected with 250
g/ml Zeocin (Invitrogen) for 2 wk
to gain stable cell lines.
The efficiency of retroviral gene transfer of HUVEC and HMEC-1 was
measured by flow cytometric detection of recombinant enhanced GFP
(EGFP) that was coexpressed with the gene of interest from a bicistronic
mRNA with a FACSCalibur cytometer (BD Biosciences) 48 h after trans-
duction. Transduction rates ranged from 90 to 100%. Expression of trans-
genes was measured by Western blot.
Western blot and flow cytometry
Cells were lysed in radioimmunoprecipitation assay buffer containing pro-
tease and phosphatase inhibitors (34). Radioimmunoprecipitation assay
protein lysates were mixed with 5⫻Laemmli buffer, separated by SDS-
PAGE, and blotted onto nitrocellulose membranes. Antisera directed
against ERK2, I
B
␣
, and IKK2 were purchased form Santa Cruz Biotech-
nology and Abs against STAT1 and phospho-STAT1 Tyr
701
were obtained
from BD Biosciences. A murine mAb against influenza A virus M1 was
purchased from Serotec. A murine mAb against influenza A virus non-
structural protein 1 (NS1) was generated by V. Wixler in the Institute of
Molecular Virology.
Flow cytometric measurement of intracellular cytokines was performed
as described earlier (28). Mouse mAb against human IL-8 and MCP-1 were
purchased from BD Biosciences. Goat anti-mouse Cy5-labeled secondary
Abs was a gift from V. Wixler (Institute of Molecular Virology). At least
10
4
cells were analyzed in a FACSCalibur cytometer (BD Biosciences)
using CellQuest Pro analysis software.
RNA isolation, cDNA synthesis, and real-time PCR
Total RNA was isolated from 100-mm dishes with a 80 –90% confluency-
grown HUVEC monolayer (⬃1.5–2 ⫻10
6
cells) using the RNeasy Kit
(Qiagen) according to the manufacturer’s instruction. One microgram of
total RNA was reverse transcribed with MBI Revert AID Reverse Tran-
scriptase (MBI Fermentas) and oligo(dT) primer according to the manu-
facturer’s instruction. The cDNA was diluted 1/10 and 0.5
l was used for
real-time PCR analysis. Primer sequences are included in the supplemen-
tary material (supplemental Table S1).
4
Real-time PCR was performed
with a Stratagene MX3005P cycler and Brilliant SYBR Green Mastermix
(Stratagene) according to the manufacturer’s instruction. Relative changes
in expression level (n-fold) are calculated according to the 2
⫺⌬⌬CT
method
(35).
DNA microarray and statistical data analysis
Total cellular RNA was isolated from three independent experiments with
wild-type HUVEC that were infected for 5 h with a multiplicity of infec-
tion (MOI) of 5 of the H5N1 virus using a RNeasy kit (Qiagen). Three
additional independent H5N1 infections were performed for microarray
analyses with empty retroviral expression vector- or IKK2KD-expressing
HUVEC. Samples were processed for microarray hybridization using Af-
fymetrix Human Genome 133 Plus 2.0 Gene Arrays according to the man-
ufacturer’s instructions. Fluorescent signals were detected by the GeneChip
Scanner 3000 and recorded and computed by GeneChip Operating Soft-
ware version 1.4 (Affymetrix). Microarray data have been deposited in the
National Center for Biotechnology Information Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/Access code: GSE13637).
For a more sophisticated data analysis, we used the Expressionist Suite
software package from GeneData (Basel, Switzerland) as described else-
where (29). Only genes with a fold change of ⬎2.0 or ⬍2.0 and a pⱕ0.05
(paired ttest) in multiple independent experiments were considered as
regulated genes. “On/off”-regulated genes were evaluated as described pre-
viously (29). We considered genes with on:off ratios of 0:3, 0:2, 1:3, 3:0,
2:0, and 3:1, respectively. From this group of on/off-regulated genes, we
only included regulations with a high fold change of ⱖ5andap⬍0.05 in
the list of regulated genes to differentiate on/off phenomenas occurring
around the background threshold from significant on/off phenomenas. We
applied principal component analyses to reduce mathematically the dimen-
sionality of the entire spectrums of gene expression values of a microarray
experiment to three components (36).
To identify functional categories of genes that are overrepresented in the
data sets of regulated genes, we first assigned Gene Ontology (GO) anno-
tations to every probe set spotted on the Affymetrix 133 Plus 2.0 Array and
compared it with the distribution of GO annotations in the gene group of
interest applying Fisher’s exact test. In the case of genes that are repre-
sented by two or more probe sets, only one transcript was taken into ac-
count to avoid potential bias.
Interaction networks showing direct and indirect relations of given gene
products were generated with the Ingenuity Pathway Analysis 7.1 software
(Ingenuity Systems). The significance of the generated networks was de-
termined by comparison of the interaction of a similar number of random
gene products. The resulting score is a numerical value used to rank net-
works according to their degree of relevance to the network eligible mol-
ecules in your data set. The score takes into account the number of net-
work-eligible molecules in the network and its size, as well as the total
number of network-legible molecules analyzed and the total number of
molecules in Ingenuity’s knowledge base that could potentially be included
in networks. The score is based on the hypergeometric distribution and is
calculated with the right-tailed Fisher’s exact Test. The score is the neg-
ative log of this pvalue. It must be noted that the score is not an indication
of the quality or biological relevance of the network; it simply calculates
the approximate “fit” between each network and your network-eligible
molecules. Networks showing the involvement of a given group of mRNAs
in certain biological processes were generated with Gene Spring GX 10.0
software (Agilent).
Promoter analysis
For the promoter analyses, we took advantage of a computational method
for transcriptional regulatory network inference, CARRIE (Computational
4
The online version of this article contains supplemental material.
5181The Journal of Immunology
Ascertainment of Regulatory Relationships Inferred from Expression) de-
scribed by Haverty et al. (37). Briefly, microarray data and promoter se-
quence data derived from the TRANSFAC database 7.0 are conflated. The
promoter regions of the group of unregulated genes are compared with
the promoters of regulated genes to compute the relative overabundance
of cis-elements in the group of regulated genes. Thereby transcription
factor binding sites are detected which potentially contribute to the
regulation of a given group of genes according to their significance of
overrepresentation.
Results
To gain a global overview on the H5N1 virus-induced gene ex-
pression program in endothelial cells, HUVEC were infected with
a MOI of 5 (MOI ⫽5) to achieve infection rates of ⬎80%, as
detected by immune fluorescence staining of the viral nuclear pro-
tein 8 h after infection (data not shown). HUVEC support efficient
and productive replication of influenza A viruses as shown by
plaque assay of supernatants from infected endothelial cell cultures
in comparison to A549, human lung epithelial cells, which are a
standard cell culture model for influenza A virus infection (sup-
plemental Fig. 1). Before initiation of the microarray analysis, we
tested by real-time RT-PCR at which time point postinfection tran-
scription of mRNAs of prototype genes, such as IFN-

and dif-
ferent ISGs, could be detected. Fig. 1 shows the n-fold mRNA
expression levels normalized to uninfected cells. IFN-

mRNA
expression reaches a maximum after 2 h while prototype ISGs,
such as IFN-
␥
-inducible protein 10 (IP-10), OAS1, or MxA, are
expressed in slightly delayed kinetics as expected (Fig. 1). Proin-
flammatory marker mRNAs like IL-8 or ICAM are induced in a
similar kinetic (data not shown) although to a lesser extent. To
minimize the effects of secondary infections, we decided to use a
time point of 5 h after infection to harvest mRNA, which is well
within the primary replication cycle of influenza viruses. We also
decided to consider only those mRNAs to be regulated that showed
at least a significant ( pⱕ0,05) 2-fold change in expression levels
compared with the control group. In former studies, this threshold
guaranteed reliable results and avoided too much false positive
results due to low signal-to-noise ratio (29, 30).
Analysis of the gene expression profile reveals that within 5 h
after infection of HUVEC with the human H5N1 isolate A/Thai-
land/KAN-1/2004, 111 mRNAs were up-regulated and 38 genes
are switched on compared with mock-infected control cells. Inter-
estingly, ⬎600 mRNAs were down-regulated or switched off upon
H5N1 virus infection (data not shown). Although we cannot rule
out a specific down-regulation of individual host mRNAs, it is
more likely that the majority of mRNAs are down-regulated in
consequence of unspecific 5⬘cap snatching (38) or interference
with processing of cellular RNAs by the viral NS1 (39).
To further characterize the genes induced upon infection with
the H5N1 isolate, we grouped these genes into different functional
clusters according to GO classification (www.geneontology.org/).
About one-third of the depicted mRNAs belong to the GO clusters
of inflammatory/immune response and chemotaxis (Fig. 2). This
pattern matches perfectly with recent findings on infections of hu-
mans with HPAIV of the H5N1 subtype, which are accompanied
by a massive systemic production of cytokines and chemokines,
the so-called cytokine storm. Supplemental Table II shows a rep-
resentative subset of mRNAs induced by H5N1 virus infection
(noncharacterized gene products were not listed). Hits were
grouped according to their GO annotated function and ranked ac-
cording to their fold change expression. Consistently with the GO
clustering, a major subset of H5N1-induced mRNAs are described
to be associated either with the antiviral type 1 IFN response (e.g.,
OAS1, MxA) or inflammatory processes (IL-8, ICAM-1). The cy-
tokine storm is a characteristic feature of systemic infections with
highly pathogenic influenza viruses of the H5 subtype. To test
whether the induction of proinflammatory cytokines and ISGs as
well as the NF-
B dependence of this cellular response is limited
to H5N1 isolates, we analyzed HUVEC infected with a low patho-
genic isolate, WSN. Infection of HUVEC with 5 MOI resulted in
moderate gene induction after 5 h compared with H5N1-infected
HUVEC. Induction of IFN-

was ⬃20 times lower after WSN
infection, which consequently results in lower induction of ISGs
like Mx1 and OAS1, as demonstrated by real-time RT-PCR (sup-
plemental Fig. 2). Likewise, production of proinflammatory
cytokines and chemokines or endothelial surface markers was less
pronounced or missing (e.g., IL-6, IL-8, or E-Selectin) in WSN-
infected HUVEC in comparison to H5N1-infected HUVEC.
NF-
B is a crucial factor for expression of antiviral and immune
modulatory cytokines and chemokines that are known to contrib-
ute to the antiviral status of a tissue and are involved in induction
of cell survival/apoptosis (40, 41). To dissect the function of
NF-
B in the primary host gene response, we introduced a dom-
inant negative mutant of the inhibitor of
B kinase 2 (IKK2KD)
into HUVEC by retroviral gene transfer. This tool was already
successfully used for specific and efficient inhibition of stimu-
lus-induced NF-
B signaling in these cells (29, 30). Successful
transduction of the transgene was verified by FACS measure-
ment of coexpressed eGFP that was translated from the same
bicistronic mRNA. Expression of the IKK2KD transgene (Fig.
3A) was further detected in Western blots from total cell lysates
(Fig. 3B). TNF-
␣
-induced I
B
␣
degradation was markedly in-
hibited although not completely abolished in IKK2KD-express-
ing HUVEC (Fig. 3B).
The functional block of NF-
B signaling was verified by im-
paired expression of the NF-
B-dependent gene products IL-8 and
MCP-1 16 h after stimulation with 2 ng/ml TNF-
␣
(Fig. 3C). To
FIGURE 1. Time course of H5N1-induced genes. HUVEC were in-
fected with 5 MOI of H5N1 influenza A virus for the indicated time points.
Expressional changes of mRNAs of IFN-

(⽧), IP-10 (f), ISG15 (Œ),
MxA (✝), and OAS1 (E) were detected by real-time RT-PCR and are
depicted as mean n-fold (⫾SD) of three independent experiments normal-
ized to controls.
FIGURE 2. Clustering of H5N1-induced genes according to their GO
annotated function. H5N1 influenza A virus-induced mRNAs (up-regulated
and switched on) were grouped according to their predominant GO anno-
tation. Relative distribution of mRNAs (percent) per GO cluster is indi-
cated. GO clusters with ⬍5 mRNAs per cluster are not depicted.
5182 H5N1 VIRUS-INDUCED NF-
B-DEPENDENT TRANSCRIPTOME
exclude impaired virus propagation by NF-
B blockage in early
stages of the replication cycle, we determined the expression of the
viral NS1 and the matrix protein M1 5 h after infection, respec-
tively. Viral protein levels were not altered in infected IKK2KD-
expressing HUVEC (Fig. 3D), which is in accordance with earlier
findings in other cell types (31, 42).
To determine the impact of NF-
B on the overall gene expres-
sion program induced by H5N1 infection, we performed mRNA
array analysis of HUVEC infected with the H5N1 virus (MOI ⫽5)
in the presence or absence of IKK2KD. As an initial comparative
analysis of global gene expression profiles in wild-type, vector-
transduced, or IKK2KD-expressing cells, we performed principal
component analysis. This is a bioinformatic tool for efficient data
reduction without loss of information (43). For that purpose, each
gene profile is transformed to a vector according to the expression
data of each gene in all experimental conditions used. This ap-
proach helps to illustrate the global variance by which different
data sets correlate. The principle components of each individual
infection experiment are depicted as vector clouds in a three-di-
mensional vector space (Fig. 4A). The clouds of each group of
up-regulated mRNAs (e.g., control or H5N1 virus infected) per-
fectly localized together, indicating reliable reproduction of data
within the triplicates. Moreover, a clear separation of the groups
representing H5N1 virus-infected cells vs uninfected as well as
IKK2KD-transduced cells is visible. The induced gene entities of
vector-transduced cells perfectly match with the data from wild-
type control cells. Comparison of vector, control, and IKK2KD-
expressing HUVEC clearly shows that blocking of NF-
B signal-
ing reduces the variance of H5N1-mediated expression changes
compared with the gene profiles of uninfected control. How-
ever, inhibition only partially reverts the influenza A virus-in-
duced gene spectrum (Fig. 4A), suggesting that either all genes
are only partially dependent on NF-
B or that there is a more
variable degree of NF-
B dependence, implying that there
might be a distinct gene subset with no or only a slight require-
ment for the factor. A closer look on the gene patterns revealed
that almost 90% of H5N1-induced mRNAs were NF-
B depen-
dent (Fig. 4B), however, to a variable extent. Among these genes,
FIGURE 3. Block of NF-
B signaling by expression of a dominant negative IKK2 mutant. A, Retroviral transduction of HUVEC: eGFP reporter gene
expression was measured by cytometric analysis. Fluorescence histograms of control cells, vector-transduced cells, and IKK2KD-expressing cells are
depicted. Representative data are shown. B, Expression of IKK2KD was confirmed in Western Blot analysis (upper panel,lanes 5 and 6). TNF-
␣
(2 ng/ml)
induced degradation of I
B
␣
(middle panel,lanes 2,4, and 6) is blocked in the presence of IKK2KD. Equal loading was confirmed by detection of ERK2
(lower panel). C, Expression of IKK2KD blocks TNF-
␣
-induced expression of NF-
B-dependent gene products. HUVEC were stimulated with 2 ng/ml
TNF-
␣
for 16 h in the presence of 25 mM monensin for 16 h. Accumulation of IL-8 and MCP-1 was detected by specific Abs and quantified by FACS
analysis. Gray curves indicate untreated cells and black curves indicate TNF-
␣
-treated cells. D, Western blot analysis of viral M1 and NS1 5 h p.i. in control
cells (lanes 1 and 4), vector-transduced cells (lanes 2 and 5), and IKK2KD-expressing cells (lanes 3 and 6). IKK2KD expression was confirmed with
specific Abs and equal loading was verified with Abs against ERK1.
5183The Journal of Immunology
46% were strictly NF-
B dependent (switched off in the presence
of IKK2KD), while 40% were partially NF-
B dependent. In con-
trast, the expression of IKK2KD has no significant effect on the
genes that are down-regulated or switched off upon influenza virus
infection (data not shown). Strikingly, IKK2KD expression most
severely influenced those mRNAs that were massively induced or
switched on by infection (supplemental Table S2). According to
the microarray data, IFN-

itself belongs to those mRNAs that
strictly need intact NF-
B signaling for their transcription (sup-
plemental Table S2). To validate the microarray data, we per-
formed real-time RT-PCR analysis for a subset of H5N1-induced
mRNAs in the presence or absence of IKK2KD. All tested H5N1-
induced mRNAs are at least partially down-regulated upon expres-
sion of IKK2KD as shown in supplemental Table S1. In parallel,
we used a chemical inhibitor of the NF-
B pathway to validate the
results obtained with the dominant negative IKK2 mutant in
HUVEC. Like IKK2KD-transduced HUVEC, the BAY11-7085-
treated HUVEC show massively impaired production of proin-
flammatory cytokines and chemokines upon H5N1 infection. Sim-
ilar the expression of IFN-

was reduced 5-fold, which
consequently resulted in impaired ISG levels (supplemental Fig.
3). This further underlined the importance of NF-
B for the pri-
mary gene expression response to influenza virus infection. Al-
though HUVEC are a well-established and widely accepted model
for studies of endothelial cell function, we validated our results in
HMEC-1. In accordance with our findings in HUVEC, infection of
HMEC-1 with H5N1 influenza A virus resulted in massive up-
regulation of proinflammatory cytokines and ISGs (supplemental
Fig. S4, Cand D).
Since we proposed a distinct impact of NF-
B signaling in
H5N1 influenza A virus-infected HUVEC, we were curious about
the effects of IKK2KD expression on the gene profile induced by
the low pathogenic control strain. Interestingly, we observed a re-
duced impact of NF-
B blockade on the already weak response to
WSN infection, either by introduction of the dominant negative
IKK2 or by addition of the pharmacological inhibitor BAY 11-
7085. Although IFN-

mRNA induction was reduced by 5-fold in
the presence of IKK2KD after H5N1 infection, we did not observe
a significant reduction in WSN-infected HUVEC. In HMEC-1, we
saw a similarly reduced dependence of the WSN-induced host re-
sponse on NF-
B signaling (supplemental Figs. S2, Aand B, S4,
Cand D, and S5B). A more direct comparison including statistical
analysis of the two cell models and the NF-
B dependence of the
host responses to the high pathogenic and low pathogenic isolate,
respectively, are shown in supplemental Table IV). By Western
blot, we could show that the low pathogenic WSN seems to rep-
licate slower in HUVEC, which might explain the overall reduced
host response (supplemental Fig. S5A). Taken together, these find-
ings point to specific dependence of the H5N1-induced antiviral
response on NF-
B signaling.
The observation that IFN-

is strongly dependent on NF-
Bin
H5N1-infected HUVEC prompted us to ask whether a portion of
the identified NF-
B-regulated genes may be rather indirectly con-
trolled by the factor via induction through the IFN-

signaling
pathway. STAT1 phosphorylation is an immediate hallmark re-
sponse to type I IFN. To test whether the IFN-

or other STAT1-
activating cytokines are actually released as proteins from infected
cells and reduced in IKK2KD-expressing cells, we performed con-
ditioned medium experiments. Wild-type HUVEC or cells that
were vector transduced or IKK2KD transduced were infected for 5
or 8 h, respectively. Supernatants were sterile filtered and trans-
ferred to untreated HUVEC for 15 min. Fig. 5 shows that the
STAT1 phosphorylation at Tyr
701
can be readily observed in
HUVEC after addition of the conditioned supernatants from
H5N1-infected wild-type cells (Fig. 5, lanes 2,4, and 6,upper
panel). Conditioned supernatants from uninfected HUVEC had no
effect on STAT1 phosphorylation (Fig. 5, lanes 1,3, and 5,upper
panel). The specific inhibition of NF-
B signaling clearly results
in reduced levels of phosphorylated STAT1 (Fig. 5, lane 6), which
suggests a reduced production of type I IFNs in these cells. Thus,
the secondary, autocrine type I IFN response is indirectly influ-
enced by blocking NF-
B signaling.
FIGURE 4. Influence of NF-
B block on H5N1 influenza virus-induced mRNAs: A, Principle component analysis of at least three individual experi-
ments per settings. Vector clouds of control HUVEC, vector-transduced HUVEC, H5N1-infected control HUVEC, H5N1-infected vector-transduced
HUVEC, and H5N1-infected IKK2KD-expressing HUVEC are depicted as indicated. Each vector cloud represents the up-regulated/switched on mRNAs
of an independent experiment. B, Relative distribution of H5N1-induced mRNAs (percent) according to the NF-
B dependence determined by microarray
analysis. Strictly NF-
B-dependent mRNAs show expression levels below 2-fold in presence of IKK2KD. A more detailed analysis is listed in supplemental
Table S1.
FIGURE 5. Influence of NF-
B block on H5N1 influenza virus-induced
IFN expression: Western blot analysis of total lysates of HUVEC treated
with conditioned medium from mock-infected control cells (lanes 1,3, and
5) and H5N1-infected cells (5 MOI, 5 h) (lanes 2,4, and 6). Donor cells
were left untreated (lanes 1 and 2), transduced with empty vector (lanes 3
and 4), or pCFG5-IEGZ IKK2KD (lanes 5 and 6). STAT1 Tyr
701
phos-
phorylation was detected 15 min after treatment with conditioned medium
(upper panel). Equal loading was verified by detection of total STAT1
(lower panel).
5184 H5N1 VIRUS-INDUCED NF-
B-DEPENDENT TRANSCRIPTOME
Next, we addressed the question whether the observed effects of
IKK2KD on ISG expression are exclusively due to indirect mech-
anisms via regulating IFN-

or additionally controlled by direct
coregulatory functions of NF-
B in ISGF3- mediated transcrip-
tion. In a time course of infection, transcriptional induction of
IFN-

and ISGs is decreased in IKK2KD-expressing cells at any
given time point (Fig. 6). This was a first indication that an initial
block of IFN-

production is the main NF-
B-controlled event.
To further test whether NF-
B mainly functions as the inducer
of IFN-

in concert with IRF3 or directly influences the expression
of ISGs, we stimulated IKK2KD-expressing endothelial cells with
IFN-

for 3 h. This time point was chosen to mimic the peak
activity of virus-induced IFN-

production. IFN-

-induced ex-
pression of MxA and OAS1 mRNA, as prototype ISGs, are not
influenced in the presence of the dominant negative IKK2 mutant,
which means that the induction of distinct genes generally known
as ISGs via IFNAR1 and JAK/STAT signaling does not necessar-
ily depend on NF-
B (Fig. 7A). This experiment however does not
mimic the overall spectrum of cytokines and chemokines that are
released in parallel to IFN-

during an influenza infection and does
not fully exclude a coregulatory role of NF-
B. To simulate a more
realistic stimulation with the complete set of influenza virus-in-
duced cytokines and chemokines, we again performed conditioned
medium experiments. This time the untreated donor cells were
infected for 3 h and conditioned supernatants of mock-treated or
H5N1 influenza virus-infected cells were transferred to the vector-
or IKK2KD-expressing reporter cells for 2 h. A potential transfer
of infectious viruses was monitored by highly sensitive real-time
PCR (44), which did not detect any viral matrix gene vRNA or
mRNA in the reporter cells after 2 h (data not shown). Three hours
after infection, the donor cells clearly produced IFN-

(Fig. 7B)on
mRNA and protein level as shown by real-time PCR and detection
of phosphorylated STAT1 in a Western blot (Fig. 7C) and indi-
cated by the synthesis of ISGs, such as MxA and IP-10 (Fig. 7B).
Interestingly, the reporter cells did not show a drastic increase in
IFN-

mRNA levels (mean n-fold below 2-fold) although ISGs
were readily transcribed. ISGs like Mx1 or OAS1 were induced by
FIGURE 6. Time course of H5N1-induced mRNAs in the presence of
IKK2KD. Induction of (A) IFN-

(⽧and 〫), (B) MxA (⽧and 〫), and
ISG15 (Œand ‚) mRNAs were measured after H5N1 infection in a time-
dependent manner by highly specific real-time RT-PCR. Filled symbols/
constant lines indicate vector-transduced cells and open symbols/dashed
lines indicate IKK2KD-expressing cells. Mean n-fold expression (⫾SD) of
three independent experiments normalized to controls is depicted.
FIGURE 7. Influence of NF-
B block on IFN-

and H5N1-conditioned medium-mediated signaling. A, Vector-transduced HUVEC (columns 1 and 2)
and IKK2KD-expressing HUVEC (columns 3 and 4) were treated for 3 h with 100 U/ml recombinant human IFN-

(f) or left untreated (䡺). Expression
of MxA and OAS1 mRNA was analyzed by real-time RT-PCR. Mean n-fold expression (⫾SD) of three independent experiments normalized to controls
are depicted. B, Donor cells for production of conditioned medium were mock infected (䡺) or infected with 5 MOI of H5N1 for3h(f). Levels of IFN-

,
MxA, and IP-10 mRNA were detected by real-time PCR. Mean n-fold expression (⫾SD) of three independent experiments normalized to controls is
depicted. C, STAT1 Tyr
701
phosphorylation was detected from total lysates of mock-infected donor cells (lane 1,upper panel) or H5N1-infected donor cells
(lane 2, upper panel). Equal loading was verified by detection of total STAT1 (lower panel). D, HUVEC were transduced with empty vector (columns 1
and 2of each panel) or pCFG5-IEGZ IKK2KD (columns 3 and 4of each panel) and treated with conditioned medium of mock-infected cells (䡺)or
H5N1-infected cells (f) for 2 h. Levels of IFN-

, MxA, and OAS mRNAs were detected by real-time RT-PCR. Mean n-fold expression (⫾SD) of three
independent experiments normalized to controls is depicted.
5185The Journal of Immunology
H5N1 virus-conditioned medium (Fig. 7D). However, this induc-
tion was much weaker compared with ISG induction 5 h postin-
fection (p.i.) with influenza A virus. After transfer of H5N1 virus-
conditioned medium, the block of NF-
B signaling did not
significantly alter the expression of OAS1 and Mx1. This under-
lines that NF-
B has no coregulatory function in induced expres-
sion of these prototype ISGs by IFN-

or other STAT-activating
cytokines.
Pathway analysis of the complete H5N1-induced gene profile
(supplemental Fig. 6) using Ingenuity Pathway Analysis 7.1 soft-
ware generated significant interaction networks with pvalues up to
10
⫺59
(only the most significant network is shown). It strength-
ened our initial findings that NF-
B and IFN-

harbor central po-
sitions in the H5N1 influenza virus-induced gene profile. Next, we
wanted to define biological processes that might be differentially
controlled by strictly or partially NF-
B-dependent gene products
induced by H5N1 influenza virus. As shown in supplemental Fig.
7, the strictly NF-
B subset of mRNAs belongs to signaling path-
ways that open up to biological processes like IFN type I produc-
tion (IFN-
␣
and IFN-

) and activation of leukocytes and mono-
cytes. In contrast, the remaining partially dependent mRNAs
control biological processes like T cell activation. However, there
is some overlap of biological functions belonging to the innate
antiviral response in which both groups participate (supplemental
Fig. 7). This is not surprising, since the initial antiviral response is
a highly cross-linked process, with several self-enhancing signal-
ing loops (45, 46).
Finally, we asked which transcription factors are additionally
involved in regulation of H5N1-induced genes that were only par-
tially blocked by IKK2KD expression. The promoter regions of
up-regulated genes were examined by CARRIE analysis as de-
scribed in Materials and Methods. Supplemental Table S3 depicts
only those transcription factor binding sites which were found in at
least four of six analyses with independent control groups of pro-
moter regions of unregulated genes. Strikingly, the transcription
factors that regulate type I IFN gene expression and that are sub-
sequently involved in expression of ISGs (IRF1, IRF7, IRF8 (also
known as ICSBP) and IRF9 (component of the ISRE-binding com-
plex)), NF-
B, and to a lesser extent AP-1 ( p⫽0.03) are over-
represented in this set. It should be noted that IRF3 itself is con-
sidered within the group of the general IRF binding site, because
the consensus site is overlapping to that of other IRF members.
Although this in silico approach does not clarify which of these
binding sites are functionally active, it provides a good overview
of factors that may principally be involved.
The expression of several influenza virus-induced mRNAs was
only partially blocked by expression of IKK2KD. This gave rise to
the question which other factors besides NF-
B are relevant for the
H5N1 virus-induced gene spectrum. AP-1, IRF3, and IRF7 also
have been shown to be components of the IFN-

enhanceosome
(18). IRF3 is constitutively present in the cytosol and migrates to
the nucleus upon TBK/IKK-dependent activation (15) while
IRF7 is induced in response to IFN1R activation via the JAK/
STAT pathway (47). Computational analysis of the promoter re-
gions of partially NF-
B-dependent genes unraveled that binding
sites for members of the IRF family, namely, IRF1, 7, 8 (ICSBP),
9 (enclosed in the ISRE element), and a general IRF binding site
covering IRF3 are highly overrepresented (supplemental Table
S2). This indicates that IRFs may drive the residual virus-induced
gene expression observed upon blockade of NF-
B activity.
Discussion
Systemic infections with HPAIV of the H5N1 subtype are asso-
ciated with a massive production of cytokines and chemokines.
Endothelial cells have been shown to contribute to this hyperacti-
vation of the immune system in human cases (11). It is still un-
certain whether this so-called cytokine storm contributes to the
pathogenesis of the virus (48) and so far it remains unsolved why
H5N1 influenza A viruses allow the production of such a broad
spectrum of antiviral-acting ISGs despite their efficient antagoniz-
ing strategies. Moreover, it is still an open question which signal-
ing pathways are involved in the regulation of the H5N1 influenza
virus-induced overexpression of cytokine and chemokine genes.
By global mRNA profiling, we could show here that infection of
primary endothelial cells with highly pathogenic influenza A vi-
ruses of the H5N1 subtype has a severe impact on the transcrip-
tome of the host cell. This is to our knowledge the first report on
H5N1-induced gene expression in a human primary cell culture
model. Moreover, it is the first study of highly pathogenic influ-
enza A virus-infected endothelial cells. A recent study could show
virus replication with a H3N2 influenza A virus isolate in HUVEC
(49). Since endothelial cells line all blood vessels in mammalian
organisms and are a major source of cytokines and chemokines, it
is highly likely that these cells are involved in a systemic infection
with HPAIV and contribute to the global host response. Case re-
ports from human and animal infections with HPAIV strengthen
this hypothesis (11, 50). These cells replicate the virus efficiently
in cell culture and show a fast and profound host response upon
infection. About 10 times more mRNAs were down than up-reg-
ulated. It is highly likely that the massive down-regulation of
mRNAs is at least partially initiated by 5⬘cap snatching and sub-
sequent degradation of cellular mRNAs (38). Although further
studies have to be performed to examine whether there is a specific
down-regulation of antiviral-acting genes by H5N1 viruses, in this
study we exclusively focused on the up-regulated mRNAs. The
induced profile indeed shows a broad overlap to mRNA profiles
from IFN-

-stimulated HUVEC (51). However, there are certain
published ISGs which were not induced upon IFN type I stimula-
tion in the study by Indraccolo et al., (51) but are expressed in the
case of influenza A virus infection (e.g., IP-10, IP9, Mig, RAN-
TES). A recent study underlined that certain genes, classified as
ISGs, are expressed independently of, although often in parallel to
type I IFNs (52).
In comparison to a previously performed transcriptional pro-
file of an influenza A virus-infected lung epithelial cell line
(53), we detect a much broader spectrum of induced mRNAs in
endothelial cells including a broad spectrum of cytokines and
chemokines. Interestingly, Geiss et al (53) did not detect IFN-

mRNA itself among the up-regulated mRNAs at 4 or 8 h after
infection. This implicates a cell-type-specific response to influ-
enza virus infection. Infection of HUVEC with a low patho-
genic control isolate (WSN) resulted in a less pronounced an-
tiviral and proinflammatory response.
It is well known that influenza virus infection induces activation
of NF-
B signaling via different mechanisms (54, 55), although
the function of NF-
B activation in the signaling network is dis-
cussed highly controversially. We and others could show that in-
fluenza A virus infection is associated with expression of NF-
B-
dependent gene products such as proinflammatory cytokines in cell
culture (6, 53, 56 –58) and in vivo models (59 – 61). Work from our
group and others revealed that influenza A virus replication in-
duces and depends on activation of NF-
B (31, 42, 62, 63), most
presumably by subsequent activation of caspases that enhance the
passive release of viral ribonucleoproteins from the nucleus in late
phases of viral replication. During preparation of this manuscript,
Kumar et al. (64) additionally claimed that NF-
B signaling is
important for the synthesis of viral RNA in influenza virus-in-
fected cells. In contrast, studies in NF-
B knockout cells in recent
5186 H5N1 VIRUS-INDUCED NF-
B-DEPENDENT TRANSCRIPTOME
years showed that activation of NF-
B is either obsolete for pro-
duction of ISGs upon virus infection (25) or even negatively reg-
ulates production of ISGs and suppresses viral replication (26). In
contrast, the importance of NF-
B for the formation of the IFN-

enhanceosome is undeniable (24). These contradictory findings
prompted us to examine which role NF-
B signaling plays in in-
fluenza A virus-induced cellular gene expression responses, with a
focus on the induction by HPAIV of the H5N1 subtype, that are
characterized by massive induction of cytokines and chemokines
in vivo.
To block NF-
B signaling, we used a dominant negative mutant
of IKK2. This approach has been successfully used previously to
efficiently block NF-
B signaling for gene expression analysis
(28 –30). Selective inhibition of NF-
B signaling by the dominant
negative mutant IKK2KD reverts at least partially the H5N1 virus-
induced gene expression profile. The expression of 46% of the
H5N1-induced mRNAs was completely blocked in the presence of
the dominant negative IKK2 mutant, among them IFN-

mRNA
itself. Among all microarray studies performed so far in this field,
to our knowledge our analysis shows for the first time directly the
obligatory dependence of influenza A virus-induced IFN-

pro-
duction on functional NF-
B signaling. Approximately 43% of
H5N1-induced mRNAs were partially regulated by NF-
B.
Surprisingly, the antiviral host response induced by the low
pathogenic influenza isolate WSN did not show a similar depen-
dence on NF-
B. This points to a distinct function of NF-
B sig-
naling in the antiviral response against highly pathogenic influenza
strains. Ongoing studies address the question if this could be a
molecular reason for the observed cytokine storm in vivo.
In case of the H5N1-induced gene profile, almost all genes that
were previously described to be ISGs were found among NF-
B-
regulated genes. This suggests, that the inhibitory function of
IKK2KD on the H5N1 virus-induced gene profile is to a large
extent mediated indirectly via reduced expression levels of IFN-

.
By analyzing the nature of the regulatory binding sites within the
promoter regions of virus-induced genes, we could show that
NF-
B is less significantly overrepresented than different IRFs. In
combination with the drastic effect of IKK2KD expression exhib-
ited on the overall gene expression profile, it is highly likely that
NF-
B is essential for the direct regulation of a few crucial sig-
naling molecules regulating the antiviral gene program. We could
strengthen this hypothesis by analyzing the time course of IFN-

and ISG induction in the presence of IKK2KD. At any time point
of the kinetic up to8hofinfection, the H5N1 virus-induced pro-
duction of IFN-

was reduced in cells expressing the dominant
negative IKK2. In consequence, the production of ISGs was de-
layed in a similar fashion. Moreover, stimulation of IKK2KD-ex-
pressing HUVEC with IFN-

or treatment with virus-conditioned
medium did not reveal a direct NF-
B dependence in ISG expres-
sion. Thus, functional NF-
B signaling in HUVEC is a critical
bottleneck for an efficient antiviral response against H5N1 influ-
enza viruses and indirectly affects ISG expression by initially con-
trolling expression of IFN-

. This most likely occurs by enhancing
the accessibility of the IFN-

promoter for IRF3 and AP-1 as the
initial step for formation of the enhanceosome and subsequent
IFN-

transcription (65). In support of that hypothesis, we could
demonstrate by conditioned medium transfer experiments that the
initial gene products induced by virus infection are capable of
mediating an IKK2-independent ISG response. Furthermore, re-
lease of the factors appeared to be time dependent and to boost the
antiviral response in cells infected for 5 h, in comparison to cells
only treated for 2 h with conditioned medium (harvested 3 h p.i.
from infected cells).
In contrast to our findings, studies in cells of NF-
B-deficient
mice revealed an inhibitory function of NF-
B in IFN-mediated
signaling (26). This would implicate that ISGs are expressed to a
higher extent in IKK2KD-expressing cells compared with vector-
transduced cells upon stimulation. With our approach, we could
neither confirm this for IFN-

-induced ISGs nor for influenza A
virus-induced ISGs. One reason for this discrepancy might be that
there are compensatory effects in the knockout situation of mouse
cells. In knockout cells, NF-
B signaling is completely blunted,
while in our approach it is not. We manipulated the NF-
B path-
way by introduction of a dominant negative isoform of IKK2,
which allows residual signaling via the remaining endogenous
IKK2 to a certain extent.
In contrast to NF-
B, IRFs were shown to be essential and suf-
ficient for activation of the IFN-

promoter. Previous studies by
Grandvoux et al. (66) used a constitutively active IRF3 mutant
(IRF3–5D) to induce certain ISGs, such as OAS1 or ISG15. It
would be of great interest to see to what extent the coexpression of
a dominant negative IKK2 mutant reverts the effects. Our results
would imply that indeed a basal activity of NF-
B is essential for
IFN-

expression and consequently for the IRF3–5D-induced gene
profile. This cooperation of NF-
B and IRF3 was described in a
virus-independent model by Schafer et al. (67).
Recently, Lee et al. (6) showed in a macrophage model that p38
MAPK signaling is of major importance for the hypercytokinemia
in H5N1 influenza virus-infected individuals (6). The p38 MAPK
signaling pathway triggers a broad spectrum of transcription fac-
tors, among them activating transcription factor 2. Together with
c-Jun, this transcription factor forms the AP-1 that binds to the
IFN-

enhanceosome. By computational analysis, we found that
AP-1 binding sites in H5N1-induced genes are much less promi-
nent compared with the representation of IRF or NF-
B binding
sites within the total spectrum of H5N1-activated genes. More-
over, the presence of AP-1 binding sites is enhanced in partially
NF-
B-dependent mRNAs, indicating an NF-
B-independent reg-
ulatory function of AP-1.
Experimental approaches have to reveal whether p38 MAPK
signaling like NF-
B is a bottleneck for the expression of essential
regulatory molecules like AP-1 in the primary innate immune re-
sponse against highly pathogenic influenza viruses, which would
mean that NF-
B and AP-1 are essential and equivalent partners in
backing up the IRF-mediated production of IFN-

.
In conclusion we could demonstrate here by pathway specific
transcriptome analysis that the gene profile induced by highly
pathogenic influenza A viruses largely depends on functional
NF-
B signaling. Future studies will have to reveal whether this
dependence on NF-
B signaling is the molecular reason for the
massive production of cytokines and chemokines in vivo.
Acknowledgments
We thank Ulla Nordhues for excellent technical assistance in sample prep-
aration for the microarray experiments and Edith Willscher from the Inte-
grated Functional Genomic (Muenster, Germany) for the bioinformatics
support and Desiree Spiering for critically reading this manuscript.
Disclosures
The authors have no financial conflict of interest.
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5189The Journal of Immunology
