Innate Immune Response of Human Alveolar
Macrophages during Influenza A Infection
Jieru Wang1,6*, Mrinalini P. Nikrad1, Emily A. Travanty1, Bin Zhou2, Tzulip Phang3, Bifeng Gao3, Taylor
Alford1, Yoko Ito1, Piruz Nahreini1, Kevan Hartshorn4, David Wentworth2, Charles A. Dinarello5, Robert J.
1Department of Medicine, National Jewish Health, Denver, Colorado, United States of America, 2J. Craig Venter Institute, Rockville, Maryland, United States of America,
3School of Medicine Cancer Center, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado, United States of America, 4Department of Oncology,
School of Medicine, Boston University, Boston, Massachusetts, United States of America, 5Division of Infectious Diseases, University of Colorado at Denver and Health
Sciences Center, Aurora, Colorado, United States of America, 6Department of Medicine, School of Medicine, University of Colorado at Denver and Health Sciences Center,
Aurora, Colorado, United States of America
Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza
virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We
performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips
and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a
contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also
evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated
the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although
macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III
interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly
induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with
the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8.
Inhibition of TNF and/or IL-1b activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and
CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type
lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of
zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local
components of innate immune response might provide a strategy for controlling influenza A infection-induced
proinflammatory response in vivo.
Citation: Wang J, Nikrad MP, Travanty EA, Zhou B, Phang T, et al. (2012) Innate Immune Response of Human Alveolar Macrophages during Influenza A
Infection. PLoS ONE 7(3): e29879. doi:10.1371/journal.pone.0029879
Editor: Samithamby Jeyaseelan, Louisiana State University, United States of America
Received July 19, 2011; Accepted December 6, 2011; Published March 2, 2012
Copyright: ? 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research is supported by the grants from National Institutes of Health AI082982, ExxonMobil Foundation, the Parker B. Francis Foundation, and
Department of Defense USAMRMC:W81XWH-07-1-0550. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: This work was partly supported by ExxonMobil Foundation. This does not alter the authors’ adherence to all the PLoS ONE policies on
sharing data and materials.
* E-mail: email@example.com
Alveolar macrophages (AM) reside at the air-tissue interface in
the lung and are one of the first lines of defense that interact with
inhaled microorganisms and particles . They play a critical role
in homeostasis, host defense, and tissue remodeling , and they
are readily infected by influenza . AM express many pattern
recognition receptors (PRRs) to help recognize the pathogen-
associated molecular patterns (PAMPs) on the surface of
microorganisms [4,5]. They are important in initiating response
to influenza, regulating the inflammatory response, and potentially
limiting secondary bacterial infections .
Influenza A virus causes seasonal and pandemic flu, both of which
pose significant public health burdens. Influenza viral antigens have
been detected in AM from humans and many animal species [7–15],
and AM are critical for controlling viral replication in vivo [11,14].
Recently, several groups have explored the responses of human
monocyte-derived macrophages to avian and/or seasonal flu viral
infection using a genome-wide approach [16–18]. Avian H5N1 and
genes despite the stronger response induced by pathogenic avian
viruses compared to seasonal flu viruses in human monocyte-derived
macrophages [16–19]. However, the genome-wide response of
resident human AM to influenza infection has not been reported.
Our previous study showed that cultured primary human AM
support a productive infection with H5N1 but not H1N1 and H3N2
influenza viruses though AM express both avian and human
influenza receptors [3,19]. However, human monocyte-derived
macrophages support productive infection with both human and
avian viruses [19–21]. These results suggest that the response of
human AM to influenza might be different from the response of
human macrophages derived from peripheral blood .
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e29879
The purpose of our study was to use a genome-wide approach to
define the innate immune response of human AM to influenza.
Using H1N1 influenza virus PR/8, we performed gene profiling of
virus-infected human AM at 4 and 24 h post inoculation (hpi) and
verified the alterations in IFN-related genes by real-time RT-PCR
and cytokine response by ELISA. We investigated the kinetics of
infection-induced cytokine response in human primary AM infected
with both live and UV-inactivated PR/8 and the contemporary
H3N2 virus A/New York/238/2005 (NY/238) . We also
determined if the cytokine response was amplified by paracrine
proinflammatory cytokines, TNF-a and IL-1b. In addition, we
explored whether influenza infection diminishes gene expression of
macrophage scavenger receptors, which could contribute to the
impaired ability of AM to clear other pathogens after influenza.
Overview of global gene expression altered by influenza
Viral infection resulted in significant alterations of mRNA levels in
1,347 transcripts at 4 hpi and 2,152 transcripts at 24 hpi; these
transcripts mapped to 1,077 (4 hpi) and 1,493 (24 hpi) known genes.
Tables 1 and 2 show the top 25 genes that were up-regulated or
down-regulated by influenza virus. The complete list of altered genes
is listed in Data S1. To identify the cellular functions and pathways
affected by the infection, the array data were processed by Ingenuity
Pathway Analysis (IPA) using IPA version 8.0 (IngenuityH Systems,
Redwood City, CA), which associates differentially regulated genes
with known specific biological pathways based on information from
published literature (www.ingenuity.com). The results from IPA
indicate some functional groups of genes were changed at both time
points. These genes are involved in antimicrobial and inflammatory
responses, cell death, cancer, infection mechanisms, cellular growth
and proliferation, cell-mediated immune responses, and immune
cell trafficking. Interferon regulatory factor (IRF) activation and
PRRsignalingwere the mostprominent pathways activatedby viral
infection at both time points. In addition, retinoic acid-induced
gene-1 (RIG-I) and interferon (IFN) signaling were dominant at
4 hpi, whereas homeostasis-related pathways such as IL-10 and IL-
6 were activated at 24 hpi.
Influenza infection triggers an early and strong response
of IFN signaling
As shown in Table 1 at 4 hpi, seven out of the top ten PR/8 up-
regulated genes were type I IFN family members, and another two
up-regulated genes, IFN-stimulated gene (ISG) 20 and CXCL11,
Table 1. The top 25 genes up-regulated or down-regulated by PR/8 infection in human AM at 4 hpi.
Gene nameSymbolFoldGene nameSymbolFold
interferon, beta 1, fibroblast IFNB16940 ceroid-lipofuscinosis, neuronal 8CLN8* 73
interferon stimulated exonuclease gene 20 kDaISG201916plasminogen activator, urokinasePLAU*23
interferon, alpha 8IFNA8978 Kruppel-like factor 13KLF13*19
chemokine (C-X-C motif) ligand 11CXCL11850 metastasis associated in colon cancer 1MACC119
interferon, alpha 21 IFNA21 737DNA fragmentation factor, 45 kDa, alpha polypeptide DFFA16
interferon, alpha 13IFNA13642dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 DYRK2*16
interferon, alpha 4IFNA4602sorting nexin 12 SNX1215
interferon, alpha 1IFNA1 502zyg-11 homolog B (C. elegans)ZYG11B 15
leprecan-like 1LEPREL1 370 MAX binding proteinMNT14
interferon, alpha 7 IFNA7317 G protein-coupled receptor 157GPR157 14
matrilin 1, cartilage matrix protein MATN1316 X-linked inhibitor of apoptosis XIAP*14
lactate dehydrogenase A-like 6BLDHAL6B 301baculoviral IAP repeat-containing 4 BIRC4*14
BCL2-like 14 (apoptosis facilitator)BCL2L14286potassium voltage-gated channel, Isk-related family, member 3KCNE3 14
Interleukin 29IL 29252slingshot homolog 1 (Drosophila)SSH1* 13
similar to Immune-responsive protein 1LOC730249224tubulin tyrosine ligase-like family, member 4TTLL413
chemokine (C-C motif) ligand 5CCL5223Hypothetical protein LOC158402LOC15840213
glucagonGCG218transforming growth factor, beta receptor 1TGFBR112
hairy and enhancer of split 4 (Drosophila)HES4189SRY (sex determining region Y)-box 4SOX4*11
DKFZp434A119 168tribbles homolog 3 (Drosophila)TRIB311
tumor necrosis factor (ligand) superfamily, member 10TNFSF10126pleckstrin homology-like domain, family A, member 1PHLDA1*11
fibroblast activation protein, alpha FAP125 peptidylprolyl isomerase F (cyclophilin F)PPIF*10
interferon, alpha 17IFNA17122hypothetical protein LOC153346 LOC153346 10
indoleamine-pyrrole 2,3 dioxygenaseINDO 121 ankyrin repeat domain 50ANKRD5010
HESX homeobox 1 HESX192calmodulin regulated spectrin-associated protein 1CAMSAP1*10
chemokine (C-X-C motif) ligand 9CXCL991methyltransferase 10 domain containingMETT10D10
Human AM from 3 non-smoking donors were isolated, cultured, and infected by PR/8 virus at a MOI of 0.5. The gene profiling of infected and non-infected cells at 4 hpi
from each donor was examined by microarray experiments using Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA). The filtered gene list was generated as
described in the Section of Methods. The data show the top 25 genes up-regulated or down-regulated altered by viral infection.
*indicates similar results from multiple probes.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org2 March 2012 | Volume 7 | Issue 3 | e29879
were IFN-stimulated genes . At 24 hpi, IFN-stimulated genes
CXCL9–11, and IFITM1 were among the top ten genes up-
regulated by PR/8 (Table 2). Therefore, we verified the microarray
data with a focus on IFN-associated genes by real-time RT-PCR
(Figure 1). As shown in Figure 1A, PR/8 infection induces an early
response in type I IFN genes IFNA1 and IFNB as well as type III
IFN genes IL-29 and IL-28A, although the degree of increase was
slightly less than that of most type I IFN genes (Tables 1 and 2).
Along with the increased IFN gene expression, the infection also
increased expression of well-known PRR genes associated with IFN
production. mRNA levels of RIG-I and melanoma differentiation
associated protein-5 (MDA-5) were mainly increased at 4 hpi,
whereas TLR3 and 7 were mainly stimulated at 24 hpi (Figure 1B).
The infection also significantly increased mRNA of IFN-stimulated
anti-viral genes myxovirus (influenza virus) resistance 1 (MX1), 2959
oligoadenylate synthase (OAS), and IFN-stimulated gene 56
(ISG56) (Figure 1C and Data S1), as compared to control cells.
Influenza infection induces an extensive cytokine and
In addition to IFN related genes, PR/8 significantly increased
the expression of many cytokine genes and cytokine-regulated
genes. These included the proinflammatory cytokines TNF-a
(7.3-fold at 4 hpi and 25-fold at 24 hpi), IL-1a (6.9-fold at 24 hpi),
and IL-1b (2.3-fold at 4 hpi and 16.3-fold at 24 hpi). We verified
the alteration in IL-1a and IL-1b by real-time RT-PCR (data not
shown). The infection also upregulated expression of TNF-a
induced proteins 2, 3, 6, and 8, as well as TNF receptor family
members 9 and 10. Expression of IL-1 family members inter-
leukin 1 receptor 1 (IL-1R1) and the IL-1 receptor antagonist
(IL-1Ra) was also increased (Data S1). In addition, PR/8 infec-
tion up-regulated mRNA expression of many chemokine genes
including CC chemokines CCL2–5, and CCL20, as well as CXC
chemokines CXCL9–11 (Data S1 and Tables 1 and 2). CXCL9–
11 were markedly increased when compared to controls both in
the microarray studies and in additional verification studies
(Tables 1 and 2 and Figure 1D). CCL5 was the most increased
CC chemokine (232-fold at 4 hpi, 234-fold at 24 hpi) (Tables 1
Besides the increased mRNA expression of proinflammatory
mediators, PR/8 also increased mRNA expression of the anti-
inflammatory cytokine IL-10 (1.9-fold at 4 hpi and 2.8-fold at
24 hpi), its receptor (8-fold at 4 hpi and 6.8-fold at 24 hpi), and
suppressor of cytokine signaling (SOCS)1 (9.4-fold at 4 hpi and
Table 2. The top 25 genes up-regulated or down-regulated by PR/8 infection in human AM at 24 hpi.
Gene nameSymbolFold Gene nameSymbolFold
chemokine (C-X-C motif) ligand 10 CXCL10 2900C-type lectin domain family 7, member A CLEC7A*122
tissue factor pathway inhibitor 2TFPI2 1003lysophospholipase-like 1LYPLAL163
chemokine (C-X-C motif) ligand 11CXCL11 987metastasis associated in colon cancer 1MACC1 63
chromosome 9 open reading frame 152C9ORF152 444progestin and adipoQ receptor family member VPAQR5 58
fibroblast activation protein, alpha FAP384 calcium channel, voltage-dependent, L type,
alpha 1D subunit
hairy and enhancer of split 4 (Drosophila) HES4 368membrane-associated ring finger (C3HC4)* MARCH1 34
interferon induced transmembrane protein 1IFITM1*353solute carrier organic anion transporter family, member 2B1 SLCO2B1*33
DKFZp434A119 339transmembrane 7 superfamily member 4TM7SF4 29
synaptopodin 2 SYNPO2 334inositol(myo)-1(or 4)-monophosphatase 2 IMPA229
chemokine (C-X-C motif) ligand 9 CXCL9305tumor necrosis factor (ligand) superfamily, member 12 TNFSF1228
Interlukin 27 IL27299 cathepsin SCTSS 27
ATPase, Class I, type 8B, member 2ATP8B2236 macrophage scavenger receptor 1MSR1*25
apolipoprotein B mRNA editing enzyme, catalytic
APOBEC3B230mitochondrial antiviral signaling protein MAVS* 25
interleukin 28A (interferon, lambda 2) IL-28A227 prostaglandin F2 receptor negative regulatorPTGFRN25
chemokine (C-C motif) ligand 5CCL5225 solute carrier family 46, member 3SLC46A323
BCL2-like 14 (apoptosis facilitator) BCL2L14195thioesterase superfamily member 2THEM223
bone morphogenetic protein 2BMP2 191 hydroxyprostaglandin dehydrogenase 15-(NAD)HPGD*23
transmembrane protein 47TREM47 170hexokinase 3 (white cell) HK321
somatostatin receptor 2 SSTR2 169choline dehydrogenaseCDH 21
interferon, alpha 1IFNA1 167lung cancer metastasis-associated proteinNAG120
interleukin 29 (interferon, lambda 1) IL-29135 ribulose-5-phosphate-3-epimeraseRPE19
interferon induced transmembrane protein 1 (9–27)IFITM1133 MPN domain containingMPND18
interferon stimulated exonuclease gene 20 kDa ISG20 108 C-type lectin domain family 4, member ACLEC4A* 18
guanylate binding protein 1, interferon-inducible, 67 kDa GBP195deoxyribonuclease II beta DNASE2B18
similar to Immune-responsive protein 1LOC73024991macrophage expressed gene 1MPEG1* 17
Human AM from 3 non-smoking donors were isolated, cultured, and infected by PR/8 virus at a MOI of 0.5. The gene profiling of infected and non-infected cells at
24 hpi from each donor was examined by microarray experiments using Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA). The filtered gene list was
generated as described in the Section of Methods. The data show the top 25 genes up-regulated or down-regulated altered by viral infection.
*indicates similar results from multiple probes.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org3March 2012 | Volume 7 | Issue 3 | e29879
12.1-fold at 24 hpi) and SOCS3 (5.4-fold at 4 hpi), which have
been shown to be important in turning off inflammatory responses
and dampening a robust innate immune response . PR/8 also
upregulated expression of IL-6 (36.9-fold at 4 hpi and 66.6-fold at
24 hpi), another important cytokine responsible for homeostasis,
and several cytokines that activate and regulate adaptive immune
response, especially IL-15 and its receptor, IL-23A, and IL-27
[26–28] (Data S1).
We verified the putative increases in secreted cytokines and
chemokines at the protein level by ELISA in 8–14 additional
donors. As shown in Figure 2, PR/8 infection significantly
increased secretion of cytokines of TNF-a, IL-6, IFN-a, and IL-
29, and CXC chemokines CXCL8–11 as well as CC chemokines
CCL2, 4 and 5. Consistent with the mRNA data, CXCL10,
CXCL11 and CCL5 were the main chemokines induced by the
virus, and AM secreted slightly more IFN-a than IL-29 (Figure 2).
Figure 1. Verification of virus-induced increase of mRNAs of IFN and IFN-associated genes by quantitative RT-PCR. Human AM
isolated from donor lungs were cultured and infected with PR/8 at a MOI of 0.5. Total RNA was isolated at 4 and 24 hpi from virus-infected and non-
infected cultures. mRNA expression of IFN and IFN-associated genes were measured by real-time RT-PCR. The data represent mean+SE of the relative
expression levels of each gene in infected cultures to that of non-infected controls after normalization to the level of the constitutive probe
cyclophilin B, N=8. Expression of all the tested genes are significantly different between virus-infected cultures and non-infected cultures, * indicates
that the difference between 4 and 24 hpi is statistically significant (P,0.05).
Figure 2. Verification of virus-induced secretion of chemokines and cytokines by ELISA. Human AM isolated from donor lungs were
cultured and infected by PR/8 at a MOI of 0.5. Secretion of chemokines and cytokines from infected and non-infected cultures was measured by ELISA
at 24 hpi. The data represent mean+SE of each released cytokine and chemokine (pg/ml). The number of individual donors ranged from 8 to 16. *
indicates P,0.05, ** indicates P,0.01, *** indicates P,0.001 vs. non-infected cells.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org4 March 2012 | Volume 7 | Issue 3 | e29879
Secretion of cytokines and chemokines occurs without
the release of a significant amount of infectious viral
From our previous studies we knew that AM do not release a
significant amount of infectious virus particles after infection with
human influenza viruses [3,19]. To investigate whether the
infected macrophages were synthesizing viral proteins, we
performed a time-course infection experiment in AM from an
additional 4 donors using both live (Figure 3A–D) and UV-
inactivated PR/8 viruses (Figure 3E), examined the kinetics of
viral antigen synthesis by staining hemagglutinin (HA) or
nucleoprotein, and measured secretion of selected cytokines by
ELISA. As shown in Figure 3F, there was a slight increase in viral
production at 6 hpi, when about 20% of the cells expressed viral
antigens and then no more net increase in viral release as up to
80% of the cells expressed viral proteins by 48 hpi. The viral
antigen staining was due to viral replication, since there was no
signal with UV-inactivated virus (Figure 3E). Despite the abortive
release of infectious virus, PR/8 infection induced a time
dependent cytokine and chemokine response in human AM
(Figure 3G–K). Viruses triggered an early and rapid secretion of
IFN-a and CXCL10 at 6 hpi. Secretion of CCL5 and CXCL8
followed the pattern of the viral protein synthesis increasing with
time. The virus-induced increase of TNF-a peaked at 24 hpi and
then declined. UV-inactivation abolished the virus-stimulated
TNF-a production, significantly decreased secretion of IFN-a,
CXCL8, and CCL5. However, the inactive virus was able to
stimulate a strong CXCL10 response, although the degree was
slightly smaller than that from live PR/8 (Figure 3I). The different
patterns of the induction suggest that the cytokine response may
involve different regulatory mechanisms. In addition, we com-
pared the alterations in mRNA levels of selected innate immune
response genes at 3 and 24 hpi for both UV-inactivated PR/8 and
live PR/8 infections. Consistent with the protein data, both live
and UV-inactivated PR/8 stimulated a large increase in CXCL10
mRNA at both time points. UV-inactivated PR/8 stimulated an
up to 4 fold increase of CCL5 and IFNA1. UV-inactivated virus
did not alter mRNA levels of RIG-I, TLR7, or ISG56 at either
time point (data not shown). These results indicate that viral
replication is required for most selected innate immune responses
but not required for the CXCL10 response.
Contemporary influenza virus induces a similar response
To investigate whether the results observed with PR/8 can be
extended to contemporary human influenza virus infection, we
performed a time-course experiment using a H3N2 virus NY/238,
a influenza virus rescued by reverse genetics technology based on a
swab sample from a patient from New York during the winter of
2005 . Consistent with the results from PR/8 infection, human
AM do not support a productive NY/238 infection as verified by
no increase in infectious viral particles released from infected
culture as measured by plaque assay (data not shown). As shown in
Figure 4A, NY/238 infection markedly stimulated CXCL10
mRNA, NY/238 virus also triggered an early increase in the
expression of RIG-I and IFNA1 genes and increased mRNA levels
of antiviral gene ISG56 and CCL chemokine CCL5. Inoculation
with the same amount of UV-inactivated NY/238 virus was able
to stimulate an IFNA1 and CXCL10 response. However, the
response was smaller than that observed with live virus. Unlike
PR/8, NY/238 virus did not induce a significant increase in
TLR7 mRNA (Figure 4A). At the protein level, NY/238 virus
induced a similar response as PR/8 virus in terms of cytokine and
IFN production (Figure 4B). Consistent with the finding with PR/
8, viral replication was required for most chemokine and cytokine
response and but was not requisite for CXCL10 release.
Targeting TNF and IL-1 signaling independently reduces
the virus-induced secretion of CXCL8 and CCL5
Because PR/8 infection increased secretion of TNF-a and
increased gene expression of IL-1 family members, well-known
proinflammatory mediators that cause release of inflammatory
chemokines, we were interested in the impact of these proin-
flammatory mediators on the overall chemokine response during
the infection in human AM. Our hypothesis was that TNF and IL-
1 signaling would augment chemokine secretion in a paracrine
manner . As shown in Figure 5, neutralization of TNF
pathway by its soluble receptor significantly decreased secretion of
CXCL8 by 65% (P,0.001) and CCL5 by 53% (P,0.05), but did
not alter secretion of IFNs, CXCL10, or TNF-a itself. Blockade of
the IL-1 receptor by its naturally occurring receptor antagonist IL-
1Ra  had a similar effect. When the activity of both cytokines
was inhibited, there was no further reduction in chemokines
greater than that of a single inhibitor, although there was a slight
decrease in CXCL10 response in the presence of both inhibitors,
the response was not statistically significant.
Influenza infection decreases mRNA expression of
macrophage receptor genes and impairs phagocytosis of
AM are important phagocytes and express many scavenger
receptors. The microarray experiments indicated that PR/8
infection also significantly decreased mRNA levels of many
macrophage receptors especially at 24 hpi (Table 2 and Data
S1). We, therefore, investigated the impact of influenza infection
on expression of scavenger receptors by real-time RT-PCR.
Consistent with the results from microarray experiments, PR/8
infection significantly decreased the mRNA levels of CLEC7A
(Dectin 1), macrophage scavenger receptor 1 (MSR1), CD36, and
the mannose receptor C type 1 (MRC1) but did not change the
expression of MRC2. However, we were not able to confirm the
decrease of MARCO, due to the large variation in responses
among different donors (Figure 6A). To further investigate if the
decrease in macrophage receptor expression was associated with
functional consequences, we evaluated the uptake of zymosan,
which are yeast walls recognized by CLEC7A, and heat-killed S.
aureus. As shown in Figure 6B, PR/8 infection reduced uptake of
zymosan by AM at 24 hpi in a dose dependent manner. We did
not observe a significant cell loss or cytopathic effect at 24 or
48 hpi, although most cells were infected as seen in Figure 3A–C.
In addition, PR/8 infection did not affect uptake of heat-killed S.
aureus until 72 hpi, when the infection induced a significant
cytopathic effect (data not shown).
Alveolar macrophages produce a robust innate immune
response to influenza. This includes a significant induction of
cytokines and chemokines, pathogen recognition, and apoptotic
responses, which are similar to the responses of human monocyte
derived macrophages [16,17]. Consistent with other studies of
avian or human influenza infections in humans and animals
[16,17,31–33], PR/8 stimulated an early and prominent IFN
response in human AM despite of the failure to release infectious
viral particles. Human AM produce both type I and type III
interferons (Figures 1 and 2). In contrast, alveolar epithelial cells
do not produce any type I interferon IFN-a in response to
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org5 March 2012 | Volume 7 | Issue 3 | e29879
Figure 3. Kinetics of influenza infection with live and UV-inactivated PR/8. Primary AM were cultured and infected by live PR/8 at a MOI of
0.5 or the equal amount of UV-inactivated PR/8, and cells were harvested at designated time post inoculation. Panels A–F. Kinetics of viral antigen
synthesis and infectious virus release. Panels A–C show representative immunofluorescence staining for influenza HA from live PR/8-infected AM
culture at 6, 24, and 48 hpi. Panel D shows the quantitation of these experiments. The data represent mean6SE of percentage of positive-stained
cells from 6 donors. Panel E. Representative staining of viral antigen in UV-inactivated PR/8 infection at 48 hpi. Panel F. Representative release of
infectious viral particles from both live and UV-inactivated PR/8-infected AM from 6 donors. Panels G–K. Time course of cytokine and chemokine
response in PR/8-infected AM. The supernatant from cultured cells were collected at 1, 6, 24, and 48 hpi. Secretion of TNF-a (Panel G), IFN-a (Panel H),
CXCL10 (Panel I), CXCL8 (Panel J), and CCL5 (Panel K) was measured by ELISA. Data show representative release of each cytokine from infected AM of
6 donors that all showed similar response.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org6 March 2012 | Volume 7 | Issue 3 | e29879
Figure 4. Innate immune response of both live and UV-inactivated contemporary H3N2 influenza viruses-infected AM. Human AM
isolated from donor lungs were cultured and infected by live NY/238 virus at a MOI of 0.1 or the equal amount of UV-inactivated NY/238. Cells were
harvested at designated times for evaluation of their innate immune response. Panel A. Alterations in mRNAs of innate immune response-related
genes at 3 and 24 hpi by realtime RT-PCR. The data represent mean+SE of the relative expression levels of each gene in infected cultures compared
to that of non-infected controls after normalization to the level of the constitutive probe cyclophilin B, N=4. * indicates P,0.05 and ** indicates
P,0.01 between live and UV-inactivated cells. Panel B. Kinetics of cytokine and chemokine response by ELISA. The data show representative release
of TNF-a, IFN-a, CXCL10, CXCL8, and CCL5 from both live and UV-inactivated NY/238 virus-infected AM from one of 6 donors that all showed similar
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org7 March 2012 | Volume 7 | Issue 3 | e29879
influenza . These results indicate a cell-specific pattern in
producing IFN in response to viral infection. It is well known that
RIG-I like RNA helicases (RLHs) and TLRs are the two main
PRRs responsible for IFN production against RNA viruses
including influenza. RLHs (RIG-I and MDA-5) recognize
cytoplasmic viral double-stranded RNA, whereas TLRs (TLR3
and TLR7) sense viral nucleic acid in the endosomal compartment
[35,36]. In the current study, PR/8 infection up-regulated mRNA
levels of RIG-I and MDA-5 mainly at 4 hpi, but the mRNAs of
TLR3 and 7 mainly at 24 hpi (Figure 1B), which suggests that
RLHs might be the early sensors and TLRs might be the late
sensors for PR/8 in human AM. These results correlate well with
those reported by Takeuchi and Thompson that RLHs were
responsible for local production of IFNs, whereas TLRs were
mainly involved in the late stages of systemic infection [35,36]. At
early times PR/8 triggered mainly pro-inflammatory responses,
whereas at later times PR/8 also activated pathways involved in
the maintenance of homeostasis such as the activation of IL-10
and IL-6, as well as up-regulation of SOCS genes (Data S1).
Therefore, therapeutic regulation of the inflammatory response in
Figure 5. Inhibition of TNF and/or IL-1 pathways decreases release of CXCL8 and CCL5 but not CXCL10 and IFNs induced by
influenza infection. Human AM isolated from donor lungs were cultured and infected by PR/8 at a MOI of 0.5. Soluble TNF p55 receptor and IL-1Ra
were added to the cultures at 10 mg/ml 45 min before the infection and added back to the cultures after viral inoculation. Secretion of chemokines
and cytokines was measured by ELISA at 24 hpi. The data represent mean+SE of each released cytokine and chemokine (pg/ml). N=6.
* indicates P,0.05, ** indicates P,0.01, *** indicates P,0.001 vs. virus-infected cells.
Figure 6. Influenza virus infection decreases CLEC7A (Dectin1) mRNA and reduces phagocytosis of zymosan by AM. Panel A. Human
AM were cultured and infected by PR/8 at a MOI of 0.5. The total RNA from infected and non-infected cells was evaluated for the expression of
macrophage receptor genes by real-time RT-PCR at 24 hpi. The data show the relative expression levels of each gene in virus-infected cells compared
to that of non-infected cells after normalized to the expression of constitutive probe from 4 to 14 donors. Each symbol indicates one donor. *
indicates there was a significant difference between control and virus-infected cells (P,0.05). Panel B. PR/8 infection induced a dose-dependent
decrease of uptake of zymosan. Isolated AM were cultured and infected by PR/8. At 24 hpi, fluorescent FITC-labeled zymosan was added without
serum for 2 h and then the cells were washed and fixed with paraformaldehyde. Uptake of zymosan was measured as percent of cells containing
zymosan evaluated under fluorescent microscopy. The data represent mean+SE of percent cells uptaking zymosan. N=4. ** indicates P,0.01, ***
indicates P,0.001 vs. non-infected cells.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org8 March 2012 | Volume 7 | Issue 3 | e29879
acute lung injury should consider both strategies to inhibit secreted
cytokines but also strategies to dampen the innate immune
response by stimulating IL-10 and SOCS genes. We were able to
confirm the results found with PR/8 in contemporary influenza
virus NY/238-infected human AM with the exception of an
increase in TLR7 mRNA. This might be due to a lower MOI of
virus used in the experiments because of the limitation of the viral
titer, but it could also be due to differences in the natures of these
two viruses or the difference in methods for propagating these two
CXCL9–11 were the most highly induced chemokines by
influenza viruses as verified at both mRNA and protein levels
(Figures 1 and 2). These three chemokines bind to a common
receptor CXCR3, and the importance of CXCR3 signaling has
been shown in the pathogenesis of several viruses including
influenza [32,37–39]. CXCL10 is highly induced in avian flu
(H5N1)-infected ferrets, non-human primates, and human cells
including alveolar epithelial cells and monocyte-derived macro-
phages [16–18,32,33,40], and has been viewed as a prognostic
marker for several viral infections [37,39,41,42]. In mice, the peak
level of CXCL11 mRNA coincides with the peak of the viremia
, and the CXCL11 protein has been reported to have anti-
viral activity . In addition, all three CXCR3 ligands can
induce epithelial cell chemotaxis and proliferation and perhaps
accelerate epithelial wound repair during the resolution of viral
infections [45,46]. The robust induction of CXCL9, 10, and 11 in
both AM (Figures 1 and 2) and human alveolar type II cells  as
well as the distinct CXCL10 response induced by both live and
UV-inactivated influenza virus PR/8 and contemporary virus
NY/238 (Figure 3 and 4) suggest that this family of proteins likely
plays an important role in the human lung alveolar defense against
influenza infection, which will require further study.
The response of alveolar macrophages was different in a several
ways from that reported for human monocyte derived macro-
phages. The major difference is that alveolar macrophages
infected with human influenza viruses do not release much
infectious virus, whereas human monocyte-derived macrophages
do ([19,20] and Figure 3). The mechanism for the non-productive
infection was not investigated in this study and is likely
complicated. One of the possible mechanisms might be related
to the lack of gene expression of transmembrane protease serine
S1 member 2 (TMPRSS2) and human airway trypsin-like protease
(HAT) by human AM (microarray data not shown). Both
TMPRSS2 and HAT are type II transmembrane serine proteases
 possessing trypsin-like activity and are known to be important
for cleaving influenza HA required for productive infection .
In recent studies Bottcher et al suggest that TMPRSS2 is mainly
responsible for cleavage of newly synthesized HA, whereas HAT
cleaves both endocytosed and newly synthesized HA .
Therefore, lack of these two gene products in human AM may
partially explain the lack of released infectious virus by these cells.
In addition, both PR/8 and NY/238 viruses induced an early
activation of type I IFN, especially IFN-a (Table 1 and Figures 1,
3, and 4). The strong anti-viral property of type I IFN  may
also contribute to the non-productive infection in these cells.
Further studies will be required to understand the mechanism for
the failure of release of infectious viral particles by human AM. In
addition, inactivation of influenza by UV did not abolish the
influenza viruses-stimulated CXCL10 secretion by AM (Figures 3
and 4), which is different from studies with human monocyte-
derived macrophages [21,51] and with human alveolar type II
epithelial cells isolated from the same donors ( and data not
shown). In those studies, release of CXCL10 is totally dependent
on viral replication. The mechanism for the distinct CXCL10
response in human AM will require additional and carefully
designed studies. The differences between human AM and
monocyte-derived macrophages indicate the importance of
investigating the response of AM to influenza infection during
the initial phases of infection in the lung because AM are main
targets for both human and avian influenza viruses .
Chemokine and cytokine responses are required for protection
of the host against viral infection. However, an exuberant response
contributes to the influenza-induced morbidity and mortality,
especially in severe pandemic and avian influenza infections
[16,52]. In the current study, PR/8 infection induced an increase
in TNF-a and IL-1b, well-known paracrine proinflammatory
factors. Therefore, we hypothesized that inhibiting these factors
might reduce the influenza-induced-inflammatory response. Since
the contemporary virus NY/238 induced a similar cytokine and
chemokine response as PR/8, it would be reasonable to expect
that the regulation of chemokine and cytokine in contemporary
influenza infection might also be similar to PR/8 infection. As
shown in Figure 5, inhibiting TNF and/or IL-1 decreased more
than 50% of the PR/8-induced secretion of inflammatory
chemokines CXCL8 and CCL5 but did not truly affect type I
interferon or CXCL10 response, although we observed a decrease
of CXCL10 in the presence of both inhibitors (Figure 5). TNF and
IL-1 signaling are known to be regulated by NF-kB and there are
several NF-kB binding sites in the promoter of CXCL10 ,
despite of the fact that CXCL10 is an IFN-induced protein .
This may explain why inhibiting both pathways slightly decreased
the amount of CXCL10 from infected AM. Our results suggest
that short term targeting the critical paracrine factors might be
beneficial for controlling the excessive infiltration of inflammatory
cells and acute lung injury during pandemic or avian flu infection
in vivo. Of course, this would require careful consideration of time
and dose so as not to increase secondary bacterial infections.
Influenza infection significantly decreased mRNA level of
macrophage receptors CLEC7A, MSR1, CD36, and MRC1
(Figure 6A and Table 2). CLEC7A belongs to the C-type lectin
family and functions as a PRR that recognizes a variety of beta-1,
3-linked and beta-1, 6-linked glucans from fungi. A decrease of
CLEC7A in infected AM suggests that these cells might not
efficiently recognize and engulf fungi after influenza infection. As
shown in Figure 6B, the uptake of zymosan, a yeast cell wall
component containing beta-1-3-glycosolic linkeages, was de-
creased in a dose-dependent manner in PR/8-infected human
AM. This effect was not associated with cell loss or cytopathic
effect because we did not observe a significant cytopathic effect
(Figure 3B) even at a MOI of 1 (data not shown). However, the
explanation of the decreased uptake might be more complicated
than simply the loss of this receptor. In addition, other
macrophage receptors MSR1, MARCO, CD36, as well as
mannose receptor MRC1 are important for bacterial and particle
uptake [54–56]. Mice with deletions of MSR1 or CD36 have
increased susceptibilityto pneumococcal
pneumonia [57–59]. Although impairment of macrophage
phagocytosis of bacteria after influenza in mice is well recognized
[60,61] and secondary bacterial infection after influenza is a
common clinical problem, we were not able to detect a significant
decrease in uptake of heat-inactivated S. aureus in human AM until
72 hpi, at which time the cytopathic effect was significant. We did
not observe a consistent decrease of MSR1 protein by flow
cytometry in PR/8-infected AM, which might explain why the
infection did not impair the bacterial uptake (data not shown). We
were also not able to verify the decrease of mRNA level of
MARCO, another important macrophage scavenger receptor for
influenza infections in mice and human cells [54,56,58,62]. Nine
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org9 March 2012 | Volume 7 | Issue 3 | e29879
of 11 donors showed a decrease in mRNA levels of MARCO after
infection with PR/8 (Figure 6A). Two other donors had an
increase in levels of MARCO mRNA. Therefore, changes of
bacteria-related receptors in human AM after influenza require
additional studies, and there may be variations in response among
In summary, we performed a global profiling of innate immune
response and regulation with a focus on chemokine and cytokine
response in influenza-infected human AM. Human AM are
apparently different from human monocyte derived macrophages
in their ability to release infectious virus and the CXCL10
response to UV inactivated virus. Future studies should compare
these responses in peripheral and alveolar macrophages from the
same donors. In addition, during acute lung injury, short term
targeting of paracrine inflammatory factors such as TNF and IL-1
as well as targeting IL-10 and SOCS genes might decrease the
acute injury and allow for better gas exchange.
Isolation and culture of human alveolar macrophages
AM were isolated from deidentified human donor lungs, which
were not suitable for transplantation and donated for medical
research. We obtained the donor lungs through the International
Institute for the Advancement of Medicine (Edison, NJ) and the
National Disease Research Interchange (Philadelphia, PA) .
The Committee for the Protection of Human Subjects at National
Jewish Health approved this research and has designated this
research as non-human project. The isolated AM could be frozen
and recovered in 90% FBS and 10% DMSO. There was no
apparent difference in response with frozen or freshly isolated
macrophages in terms of the level of infection and virus-induced
TNF-a secretion (data not shown). AM were plated in DMEM/
10% FBS with antibiotics, and cultured at 37uC in 10% CO2
overnight. The cells were then washed and cultured for another
day in DMEM and 1% charcoal stripped FBS with antibiotics
before viral infection. Their purity was measured by staining for
CD68 (Dakocytomation, Carpinteria, CA) .
Virus preparation and infection
Influenza A virus A/PR/8/34 (PR/8) was grown in 10-day-old
SPF Premium Eggs (Charles River SPAFAS. North Franklin, CT)
and prepared as described previously . Contemporary influenza
H3N2 virus, A/New York/238/2005 (NY/238), was created by
reverse genetics using plasmids that corresponded to the
consensuses sequence obtained from a human swab specimen
collected in New York State in the winter of 2005 . NY/238
was passaged in Madin-Darby Canine Kidney (MDCK) cells and
the viral titer was measured by plaque assay on MDCK cells as
described previously . Briefly, stocks of purified virus was
serially diluted in DMEM with 1 mg/ml TPCK trypsin (Sigma-
Aldrich, St. Louis, MO) and used to inoculate triplicate wells of
near confluent MDCK cells. After a 1 h inoculation, the inoculum
was removed and the cells were overlaid with MEM with 4% FBS
and 0.5% SeaKem LE agarose (Cambrex, Rockland, ME).
Plaques were stained and counted after 72 h incubation at
37uC, with the agarose overlay medium containing 10% neutral
red (Sigma-Aldrich). For UV-inactivation of PR/8 or NY/238,
500 ml diluted virus was placed in a 35-mm2petri dish on ice and
irradiated twice in a UV Stratalinker (Stratagene, La Jolla, CA) at
a cumulative dose of 120 mJ/cm2. Viral inactivation was
demonstrated by plaque assay on MDCK cells as described above.
On the day of infection, AM were inoculated with live PR/8 at
a designated multiplicity of infection (MOI) or with the same
amount of UV-inactivated PR/8 for 1 h. After inoculation, cells
were washed and then cultured until harvest. Influenza infection
was verified by immuno-fluorescent staining with goat antibody to
the hemagglutinin of PR/8 (kindly provided by BEI Resources,
Manassas, VA). For NY/238 infection, AM was inoculated with a
MOI of 0.1 instead of 0.5 due to the limitation of viral titer and
infection was confirmed by immuno-fluorescent staining with
mouse antibody to influenza nucleoprotein (Millipore, Billerica,
For the inhibition experiments, cells were treated with 10 mg/ml
human IL-1 receptor antagonist (IL-1Ra)  and extracellular
TNF neutralization was achieved by treating cells with 10 mg/ml
recombinant human soluble TNF receptor (sTNFR) . Both IL-
1Ra and/or sTNFR were added to the cells for 45 min before
virus inoculation. DMEM alone was used as vehicle control for
both inhibitors. After inoculation, cells were washed and cultured
with the inhibitors for an additional 24 h.
Affymetrix microarray experiments
At 4 and 24 hpi, total RNA from virus-infected and non-
infected AM from three donors was extracted and purified using
RNeasy kit (QIAGEN, Valencia, CA). The samples were run on
Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA)
and processed as indicated by the manufacturer in the Microarray
Core of the University of Colorado Denver. All data is MIAME
compliant and the raw data had been deposited in a MIAME
compliant database Gene Express Omnibus (GEO). The GEO
accession numbers are GSM762686, GSM762687, GSM762688,
GSM762702, GSM762703, GSM762704, GSM762705. Analyses
of microarray data were performed using R statistical package from
Bioconductor open source software for bioinformatics. Prior to
statistical analyses, raw data from array scans were processed using
the Robust Multi-chip Average (RMA) normalization method to
subtract a background value . After normalization, data were
filtered to exclude all probe sets with an ‘‘absent’’ call in all samples
and to remove transcripts that demonstrated little variation across
all arrays by comparing the variances of the log-intensities for each
gene with the median of all variances for the entire array. The
filtered gene list was generated using the Student’s T test to select
statisticallysignificantgenes and correctedusingthe FalseDiscovery
Rate approach. Genes that had at least a 2-fold change in
comparison to the uninfected controls for all three subjects were
used for further analyses.
mRNA expression of selected genes that were significantly up-
regulated by PR/8 or NY/238 were validated by quantitative real-
time RT-PCR . These genes include IFNs, PRRs, chemo-
kines, and SOCSs. Except for IFN-b and IL-29 genes whose
probes were synthesized in house , the specific probes for other
genes were purchased from Applied Biosystems (Applied Biosys-
tems Inc. Foster City, CA). The expression level of each specific
gene was normalized to the level of a constitutive probe cyclophilin
Enzyme linked immunosorbent assay (ELISA)
Supernatant from PR/8 OR ny/238-infected and non-infected
cells were harvested at designated times after inoculation for the
measurement of chemokine and cytokine secretion by ELISA. The
ELISA kits for human CXCL9, CXCL10, CXCL11, CCL5,
CXCL8, and IL-29 were purchased from ELISA Tech (ELISA
Tech, Aurora, CO). The ELISA kit for IFN-a was purchased from
Invitrogen (Invitrogen, Carlsbad, CA).
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e29879
Uptake of zymosan and heat killed S. aureus
Human AM were cultured and infected with PR/8 at the
designated MOI. Uptake of zymosan or heat-killed S. aureus were
performed according to manufacturer’s instructions. For uptake of
zymosan, cells were incubated with fluorescent-labeled zymosan A
Bioparticles (Invitrogen) at a ratio of 10 particles per cell for 2 h,
then cells were washed and fixed with 4% paraformaldehyde for
10 min. The uptake was analyzed by fluorescent microscopy. For
uptake of heat-killed S. aureus, cells were incubated with pHrodo-
labeled, heat-killed S. aureus (pHrodo-SA) (Invitrogen) at a ratio of
20 particles per cell for 2 h. The cells were then washed to remove
non-internalized particles, collected, and fixed with 4% parafor-
maldehyde. Uptake of the pHrodo-SA was analyzed on the LSR II
flow cytometer (BD Biosciences) in the National Jewish Health
Flow Cytometry Core, and the data were analyzed using FlowJo
software (TreeStar, Ashland, OR). In addition to the PR/8
infected cells, positive control uninfected cells and negative control
paraformaldehyde fixed cells were also used.
Statistical analyses were conducted in GraphPad Prism version
5.0 (GraphPad Software, San Diego, CA). Pair-wise comparisons
were tested for significance using Wilcoxon matched pairs test or
Paired T test. Comparison among three or more groups was
performed using one-way ANOVA with Tukey’s post test analysis.
cultured, and infected by PR/8 virus at MOI of 0.5. The gene
profiling of infected and non-infected cells at 4 and 24 hpi from
each donor was examined by microarray experiments using
Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara,
CA). The filtered gene list was generated as described in the Section
of Methods and Materials. The data show probe ID, gene symbol,
gene name, and fold change at 4 and 24 hpi. Red indicates similar
results from multiple probes for the same gene, and the probe ID is
the representative probe ID from several probes.
Human AM from 3 non-smoking donors were isolated,
The authors would like to thank Mitch White from Boston University for
preparing the virus. We appreciate support from Ivana Yang from
National Jewish Health for using the Ingenuity Pathway Analysis software.
The authors would also like to thank Karen E. Edeen, Elise Messier, and
Beata Kosmider for their help in the isolation of human lung cells. We also
thank Jennifer Kemp, Lydia Orth and Teneke M. Warren for their help in
editing and submitting this manuscript.
Conceived and designed the experiments: JW RJM. Performed the
experiments: JW MN EAT YI PN TA BZ. Analyzed the data: JW TP BG.
Contributed reagents/materials/analysis tools: KH CAD BZ DW. Wrote
the paper: JW RJM.
1. Sibille Y, Reynolds HY (1990) Macrophages and polymorphonuclear neutro-
phils in lung defense and injury. Am Rev Respir Dis 141: 471–501.
Lambrecht BN (2006) Alveolar macrophage in the driver’s seat. Immunity 24:
Wang J, Oberley-Deegan R, Wang S, Nikrad M, Funk CJ, et al. (2009)
Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda
1) in response to influenza A infection. J Immunol 182: 1296–1304.
Stafford JL, Neumann NF, Belosevic M (2002) Macrophage-mediated innate
host defense against protozoan parasites. Crit Rev Microbiol 28: 187–248.
Krutzik SR, Modlin RL (2004) The role of Toll-like receptors in combating
mycobacteria. Semin Immunol 16: 35–41.
McGill J, Heusel JW, Legge KL (2009) Innate immune control and regulation of
influenza virus infections. J Leukoc Biol 86: 803–812.
Castleman WL, Powe JR, Crawford PC, Gibbs EP, Dubovi EJ, et al. (2010)
Canine H3N8 Influenza Virus Infection in Dogs and Mice. Vet Pathol.
Chen Y, Deng W, Jia C, Dai X, Zhu H, et al. (2009) Pathological lesions and
viral localization of influenza A (H5N1) virus in experimentally infected Chinese
rhesus macaques: implications for pathogenesis and viral transmission. Arch
Virol 154: 227–233.
Coleman JR (2007) The PB1-F2 protein of Influenza A virus: increasing
pathogenicity by disrupting alveolar macrophages. Virol J 4: 9.
10. Guarner J, Falcon-Escobedo R (2009) Comparison of the pathology caused by
H1N1, H5N1, and H3N2 influenza viruses. Arch Med Res 40: 655–661.
11. Kim HM, Lee YW, Lee KJ, Kim HS, Cho SW, et al. (2008) Alveolar
macrophages are indispensable for controlling influenza viruses in lungs of pigs.
J Virol 82: 4265–4274.
12. Lohr CV, Debess EE, Baker RJ, Hiett SL, Hoffman KA, et al. (2010) Pathology
and Viral Antigen Distribution of Lethal Pneumonia in Domestic Cats Due to
Pandemic (H1N1) 2009 Influenza A Virus. Vet Pathol.
13. Powe JR, Castleman WL (2009) Canine influenza virus replicates in alveolar
macrophages and induces TNF-alpha. Vet Pathol 46: 1187–1196.
14. Tumpey TM, Garcia-Sastre A, Taubenberger JK, Palese P, Swayne DE, et al.
(2005) Pathogenicity of influenza viruses with genes from the 1918 pandemic
virus: functional roles of alveolar macrophages and neutrophils in limiting virus
replication and mortality in mice. J Virol 79: 14933–14944.
15. Gill JR, Sheng ZM, Ely SF, Guinee DG, Beasley MB, et al. (2010) Pulmonary
pathologic findings of fatal 2009 pandemic influenza A/H1N1 viral infections.
Arch Pathol Lab Med 134: 235–243.
16. Cheung CY, Poon LL, Lau AS, Luk W, Lau YL, et al. (2002) Induction of
proinflammatory cytokines in human macrophages by influenza A (H5N1)
viruses: a mechanism for the unusual severity of human disease? Lancet 360:
17. Lee SM, Gardy JL, Cheung CY, Cheung TK, Hui KP, et al. (2009) Systems-
level comparison of host-responses elicited by avian H5N1 and seasonal H1N1
influenza viruses in primary human macrophages. PLoS One 4: e8072.
18. Zhou J, Law HK, Cheung CY, Ng IH, Peiris JS, et al. (2006) Differential
expression of chemokines and their receptors in adult and neonatal macrophages
infected with human or avian influenza viruses. J Infect Dis 194: 61–70.
19. Yu WC, Chan RW, Wang J, Travanty EA, Nicholls JM, et al. (2011) Viral
replication and innate host responses in primary human alveolar epithelial cells
and alveolar macrophages infected with influenza H5N1 and H1N1 viruses.
J Virol 85: 6844–6855.
20. Hofmann P, Sprenger H, Kaufmann A, Bender A, Hasse C, et al. (1997)
Susceptibility of mononuclear phagocytes to influenza A virus infection and
possible role in the antiviral response. J Leukoc Biol 61: 408–414.
21. Sakabe S, Iwatsuki-Horimoto K, Takano R, Nidom CA, Le MQ, et al. (2011)
Cytokine production by primary human macrophages infected with highly
pathogenic H5N1 or pandemic H1N1 2009 influenza viruses. J Gen Virol 92:
22. van Riel D, Leijten LM, van der Eerden M, Hoogsteden HC, Boven LA, et al.
(2011) Highly pathogenic avian influenza virus H5N1 infects alveolar
macrophages without virus production or excessive TNF-alpha induction. PLoS
Pathog 7: e1002099.
23. Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, et al. (2009) Single-
reaction genomic amplification accelerates sequencing and vaccine production for
classical and Swine origin human influenza a viruses. J Virol 83: 10309–10313.
24. Groom JR, Luster AD (2011) CXCR3 ligands: redundant, collaborative and
antagonistic functions. Immunol Cell Biol 89: 207–215.
25. Cassel SL, Rothman PB (2009) Chapter 3: Role of SOCS in allergic and innate
immune responses. Advances in immunology 103: 49–76.
26. Gordy LE, Bezbradica JS, Flyak AI, Spencer CT, Dunkle A, et al. (2011) IL-15
Regulates Homeostasis and Terminal Maturation of NKT Cells. J Immunol.
27. McAleer JP, Kolls JK (2011) Mechanisms controlling Th17 cytokine expression
and host defense. J Leukoc Biol 90: 263–270.
28. Jankowski M, Kopinski P, Goc A (2010) Interleukin-27: biological properties and
clinical application. Arch Immunol Ther Exp (Warsz) 58: 417–425.
29. Miura TA, Wang J, Holmes KV, Mason RJ (2007) Rat coronaviruses infect rat
alveolar type I epithelial cells and induce expression of CXC chemokines.
Virology 369: 288–298.
30. Dinarello CA (2010) Anti-inflammatory Agents: Present and Future. Cell 140:
31. Cilloniz C, Shinya K, Peng X, Korth MJ, Proll SC, et al. (2009) Lethal influenza
virus infection in macaques is associated with early dysregulation of
inflammatory related genes. PLoS Pathog 5: e1000604 p.
32. Baskin CR, Bielefeldt-Ohmann H, Tumpey TM, Sabourin PJ, Long JP, et al.
(2009) Early and sustained innate immune response defines pathology and death
in nonhuman primates infected by highly pathogenic influenza virus. Proc Natl
Acad Sci U S A 106: 3455–3460.
33. Cameron CM, Cameron MJ, Bermejo-Martin JF, Ran L, Xu L, et al. (2008)
Gene expression analysis of host innate immune responses during Lethal H5N1
infection in ferrets. J Virol 82: 11308–11317.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org11 March 2012 | Volume 7 | Issue 3 | e29879
34. Wang J, Nikrad MP, Phang T, Gao B, Alford T, et al. (2011) Innate immune Download full-text
response to influenza A virus in differentiated human alveolar type II cells.
Am J Respir Cell Mol Biol 45: 582–591.
35. Takeuchi O, Akira S (2007) Recognition of viruses by innate immunity.
Immunol Rev 220: 214–224.
36. Thompson AJ, Locarnini SA (2007) Toll-like receptors, RIG-I-like RNA
helicases and the antiviral innate immune response. Immunol Cell Biol 85:
37. Zeremski M, Dimova R, Brown Q, Jacobson IM, Markatou M, et al. (2009)
Peripheral CXCR3-associated chemokines as biomarkers of fibrosis in chronic
hepatitis C virus infection. J Infect Dis 200: 1774–1780.
38. Fadel SA, Bromley SK, Medoff BD, Luster AD (2008) CXCR3-deficiency
protects influenza-infected CCR5-deficient mice from mortality. Eur J Immunol
39. Thapa M, Carr DJ (2009) CXCR3 deficiency increases susceptibility to genital
herpes simplex virus type 2 infection: Uncoupling of CD8+ T-cell effector
function but not migration. J Virol 83: 9486–9501.
40. Chan MC, Cheung CY, Chui WH, Tsao SW, Nicholls JM, et al. (2005)
Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in
primary human alveolar and bronchial epithelial cells. Respir Res 6: 135.
41. Fife BT, Kennedy KJ, Paniagua MC, Lukacs NW, Kunkel SL, et al. (2001)
CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+
T cell accumulation in the central nervous system during experimental
autoimmune encephalomyelitis. J Immunol 166: 7617–7624.
42. Larrubia JR, Benito-Martinez S, Calvino M, Sanz-de-Villalobos E, Parra-Cid T
(2008) Role of chemokines and their receptors in viral persistence and liver
damage during chronic hepatitis C virus infection. World J Gastroenterol 14:
43. Hamilton NH, Mahalingam S, Banyer JL, Ramshaw IA, Thomson SA (2004) A
recombinant vaccinia virus encoding the interferon-inducible T-cell alpha
chemoattractant is attenuated in vivo. Scand J Immunol 59: 246–254.
44. Harvey SA, Romanowski EG, Yates KA, Gordon YJ (2005) Adenovirus-directed
ocular innate immunity: the role of conjunctival defensin-like chemokines (IP-10,
I-TAC) and phagocytic human defensin-alpha. Invest Ophthalmol Vis Sci 46:
45. Ji R, Lee CM, Gonzales LW, Yang Y, Aksoy MO, et al. (2008) Human type II
pneumocyte chemotactic responses to CXCR3 activation are mediated by splice
variant A. Am J Physiol Lung Cell Mol Physiol 294: L1187–1196.
46. Yates CC, Whaley D, A YC, Kulesekaran P, Hebda PA, et al. (2008) ELR-
negative CXC chemokine CXCL11 (IP-9/I-TAC) facilitates dermal and
epidermal maturation during wound repair. Am J Pathol 173: 643–652.
47. Hooper JD, Clements JA, Quigley JP, Antalis TM (2001) Type II
transmembrane serine proteases. Insights into an emerging class of cell surface
proteolytic enzymes. J Biol Chem 276: 857–860.
48. Bottcher E, Matrosovich T, Beyerle M, Klenk HD, Garten W, et al. (2006)
Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and
HAT from human airway epithelium. J Virol 80: 9896–9898.
49. Bottcher-Friebertshauser E, Freuer C, Sielaff F, Schmidt S, Eickmann M, et al.
(2010) Cleavage of influenza virus hemagglutinin by airway proteases
TMPRSS2 and HAT differs in subcellular localization and susceptibility to
protease inhibitors. J Virol 84: 5605–5614.
50. Meager A, Visvalingam K, Dilger P, Bryan D, Wadhwa M (2005) Biological
activity of interleukins-28 and -29: comparison with type I interferons. Cytokine
51. Bussfeld D, Kaufmann A, Meyer RG, Gemsa D, Sprenger H (1998) Differential
mononuclear leukocyte attracting chemokine production after stimulation with
active and inactivated influenza A virus. Cell Immunol 186: 1–7.
52. Maines TR, Szretter KJ, Perrone L, Belser JA, Bright RA, et al. (2008)
Pathogenesis of emerging avian influenza viruses in mammals and the host
innate immune response. Immunol Rev 225: 68–84.
53. Elsharkawy AM, Oakley F, Lin F, Packham G, Mann DA, et al. (2010) The NF-
kappaB p50:p50:HDAC-1 repressor complex orchestrates transcriptional
inhibition of multiple pro-inflammatory genes. Journal of hepatology 53:
54. Sun K, Metzger DW (2008) Inhibition of pulmonary antibacterial defense by
interferon-gamma during recovery from influenza infection. Nat Med 14:
55. Peiser L, Mukhopadhyay S, Gordon S (2002) Scavenger receptors in innate
immunity. Curr Opin Immunol 14: 123–128.
56. Arredouani M, Yang Z, Ning Y, Qin G, Soininen R, et al. (2004) The scavenger
receptor MARCO is required for lung defense against pneumococcal
pneumonia and inhaled particles. J Exp Med 200: 267–272.
57. Arredouani MS, Yang Z, Imrich A, Ning Y, Qin G, et al. (2006) The
macrophage scavenger receptor SR-AI/II and lung defense against pneumo-
cocci and particles. Am J Respir Cell Mol Biol 35: 474–478.
58. Zhou H, Imrich A, Kobzik L (2008) Characterization of immortalized MARCO
and SR-AI/II-deficient murine alveolar macrophage cell lines. Part Fibre
Toxicol 5: 7.
59. Thomas CA, Li Y, Kodama T, Suzuki H, Silverstein SC, et al. (2000) Protection
from lethal gram-positive infection by macrophage scavenger receptor-
dependent phagocytosis. J Exp Med 191: 147–156.
60. Nugent KM, Pesanti EL (1982) Staphylococcal clearance and pulmonary
macrophage function during influenza infection. Infect Immun 38: 1256–1262.
61. Jakab GJ (1982) Immune impairment of alveolar macrophage phagocytosis
during influenza virus pneumonia. Am Rev Respir Dis 126: 778–782.
62. Ghosh S, Gregory D, Smith A, Kobzik L (2011) MARCO Regulates Early
Inflammatory Responses against Influenza: A Useful Macrophage Function with
Adverse Outcome. Am J Respir Cell Mol Biol 45: 1036–1044.
63. Dinarello CA (2005) Blocking IL-1 in systemic inflammation. J Exp Med 201:
64. Vazquez-Torres A, Fantuzzi G, Edwards CK, 3rd, Dinarello CA, Fang FC
(2001) Defective localization of the NADPH phagocyte oxidase to Salmonella-
containing phagosomes in tumor necrosis factor p55 receptor-deficient
macrophages. Proc Natl Acad Sci U S A 98: 2561–2565.
Influenza Infection and Human Alveolar Macrophages
PLoS ONE | www.plosone.org12March 2012 | Volume 7 | Issue 3 | e29879