Cell Paper Influenza Biolipids

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Lipidomic Profiling of Influenza Infection

Identifies Mediators that Induce
and Resolve Inflammation
Vincent C. Tam,1 Oswald Quehenberger,2,4 Christine M. Oshansky,5 Rosa Suen,1 Aaron M. Armando,3,4
Piper M. Treuting,6 Paul G. Thomas,5 Edward A. Dennis,3,4 and Alan Aderem1,*
1Seattle
Biomedical Research Institute, Seattle, WA 98109, USA

of Medicine
3Department of Chemistry and Biochemistry
4Department of Pharmacology
University of California, San Diego, La Jolla, CA 92093, USA
5Department of Immunology, St. Jude Childrens Research Hospital, Memphis, TN 38105, USA
6Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA
*Correspondence: alan.aderem@seattlebiomed.org
http://dx.doi.org/10.1016/j.cell.2013.05.052
2Department
SUMMARY
Bioactive lipid mediators play a crucial role in the

induction and resolution of inflammation. To elucidate their involvement during influenza infection,
liquid chromatography/mass spectrometry lipidomic
profiling of 141 lipid species was performed on a
mouse influenza model using two viruses of significantly different pathogenicity. Infection by the
low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct antiinflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping
pro- and anti-inflammatory states. Integration of the
large-scale lipid measurements with targeted gene
expression data demonstrated that 5-lipoxygenase
metabolites correlated with the pathogenic phase
of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase.
Hydroxylated linoleic acid, specifically the ratio of
13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status
during an active infection. Importantly, some of the
findings from the animal model were recapitulated
in studies of human nasopharyngeal lavages obtained during the 20092011 influenza seasons.
INTRODUCTION
Influenza virus, whether seasonal or pandemic, can cause
severe health conditions, often leading to pneumonia. While
most infections in humans are self-limiting, highly virulent strains
can cause an overexuberant inflammatory response that is detrimental to the host (Kash et al., 2006). Influenza virus is an envel-
oped, negative-sense, single-stranded RNA virus that contains

eight genomic segments that encode up to 13 proteins (Jagger
et al., 2012). One of the hallmarks of influenza virus is its high propensity to acquire mutations, which promotes rapid evolution to
increase fitness and evade pre-existing immunity in a host population. Antigenic drift, or mutations mainly of the segments encoding the surface proteins, fosters evasion of the host immune
system and allows a slightly varied virus to re-infect the population. Antigenic shift, or recombination and exchange of genomic
segments between multiple viruses infecting the same cells,
allows for the generation of emerging viruses with drastic
changes in pathogenicity or host specificity. If a novel virus
with increased fitness and pathogenicity emerges, the herd
immunity of the human population may be insufficient to prevent sustained transmission, creating devastating epidemic or
pandemic events. The most notorious pandemic, caused by
the 1918 H1N1 strain, cost the lives of 40100 million people
worldwide (Taubenberger and Kash, 2010). The latest influenza
pandemic was caused by a swine-origin H1N1 virus, which is a
triple reassortment of human, avian, and swine strains (Neumann
et al., 2009). Although it is well known that the pathogenicity of
influenza strains can vary widely, the precise factors and mechanisms that contribute to the clinical outcome of infection have
not been defined.
Eicosanoids refer to a family of bioactive lipid mediators that
regulate a wide variety of physiological as well as pathophysiological responses and often exhibit potent inflammatory properties (Quehenberger and Dennis, 2011). These mediators are
generated from arachidonic acid and related polyunsaturated
fatty acids after their enzymatic release from membrane phospholipids via complex metabolic mechanisms involving over 50
unique enzymes (Buczynski et al., 2009). The three major metabolic pathways for enzymatic eicosanoid biogenesis are the
cyclooxygenase pathway (COX-1 and COX-2), producing the
prostaglandins and thromboxanes; the lipoxygenase pathway
(5-LOX, 12-LOX, and 15-LOX), producing the leukotrienes as
well as numerous hydroperoxy and hydroxy fatty acids; and
Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. 213
(legend on next page)
214 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.
the cytochrome P450 pathway, producing epoxide and corresponding dihydroxy metabolites of arachidonic acids. Polyunsaturated fatty acids are susceptible to lipid peroxidation,
especially under condition of oxidative stress during inflammation producing isoprostanes as well as other nonenzymatic
oxidation products, including hydroxy fatty acids. In addition to
arachidonic acids, the same enzymes can effectively metabolize
other polyunsaturated fatty acids, such as linoleic and linolenic
acids. Several bioactive lipid mediators have also been appreciated for their anti-inflammatory activity and their participation
in the resolution phase of inflammation (Serhan et al., 2008).
Arachidonic acid-derived lipoxins via the lipoxygenase pathway
and docosahexaenoic acid (DHA)- and eicosapentaenoic acid
(EPA)-derived resolvins, protectins, and maresins have antiinflammatory and proresolution activities (Serhan et al., 2000).
These lipid mediators can prevent further infiltration of immune
cells to the site of infection as well as signaling the nonphlogistic
phagocytosis of apoptotic immune and epithelial cells, therefore
allowing the system to return to homeostasis after microbial
infection. While many of these lipid mediators have been studied
individually in various model systems, only a few studies have
conducted comprehensive quantification of the metabolites in
a natural microbial infection (Chiang et al., 2012; Morita et al.,
2013; Tobin et al., 2012).
We utilized two influenza viruses, PR8/H1N1 a highly pathogenic strain in mice, and X31/H3N2, a low-pathogenicity strain,
and profiled 141 lipid metabolites and bioactive mediators during the course of a mouse influenza infection in vivo. The induction kinetics of the protein levels of cytokines/chemokines in the
bronchoalveolar lavage and the transcript levels of immune
response genes in the host are similar between infections with
the two viruses. In contrast, the lipidomic profile provided a
more dynamic and comprehensive description of the processes
involved in the induction and resolution of inflammation. Metabolites derived from the lipoxygenase and cytochrome P450 pathways and those derived from linoleic acid, DHA, and EPA had
distinct profiles in the two viral infections during the resolution
phase of inflammation. Surprisingly, metabolites of the cyclooxygenase pathway showed similar levels and kinetics across
different conditions. We have further profiled the lipidome of
nasopharyngeal lavages from human influenza clinical samples
obtained during the 20092011 influenza seasons and validated
some of the observations from the animal model. Many of the
metabolites and bioactive mediators induced in the highpathogenicity PR8 mouse influenza infection model were also
produced at significantly higher levels in individuals with
increased clinical symptoms and immune responses.
RESULTS
Mouse Influenza Infection
We used X31 and PR8 to determine how the varying pathogenicity of influenza viral strains affects the transcriptional, proteomic, and lipidomic profiles of the host immune response. These
two strains differ in their hemagglutinin and neuraminidase segments and contain the identical internal genes (PB1, PB2, PA,
NP, NS, and M). PR8 is a high-pathogenicity, mouse-adapted
virus that is lethal even at a low multiplicity of infection (Francis
and De Torregrosa, 1945). In contrast, X31 is a low-pathogenicity
virus that causes a self-limiting infection, even at high doses
(Allan et al., 1990). Three infection conditions were used to determine the lipidomic profiles of the host during influenza infections:
PR8 at a lethal dose (2 3 105 plaque-forming units [pfu];
PR8lethal), PR8 at a sublethal dose (200 pfu; PR8sublethal), and
X31 at a sublethal dose (2 3 105 pfu; X31sublethal). We monitored
the progress of influenza infection by measuring the percentage
of weight loss (Figure 1A). Animals infected with PR8sublethal had
a peak weight loss on day 9 postinfection of 22% and recovered
to their original weights by day 13 postinfection. Animals infected
with X31sublethal differed slightly from animals infected with
PR8sublethal; they had a peak weight loss on day 7 postinfection
of 16% and recovered to their original weights by day 10 postinfection. Finally, animals infected with PR8lethal experienced rapid
weight loss and succumbed 6 days postinfection.
We next determined the viral load in the bronchoalveolar
lavage (BAL) and in separated cell populations by measuring
the levels of the influenza M gene. Cell populations included cells
isolated from the BAL, as well as CD45+ immune cells and
CD45 epithelial cells isolated from dispase-digested lungs
(Figure 1B). Viral loads were measured in cells isolated from sublethally infected animals on days 3, 5, 7, 9, 11, 13, and 19 postinfection, whereas viral loads in lethally infected animals were
measured on days 3 and 5 postinfection (Figure 1B). The viral
load peaked on day 3 postinfection for all three conditions. Not
surprisingly, animals infected with PR8lethal had significantly
higher viral loads in the alveolar epithelium and BAL on day 3
Figure 1. Mouse Influenza Infection Model

(A) Weight loss curves of mice infected with PR8sublethal 200 pfu (blue), X31sublethal 2 3 105 pfu (red), or PR8lethal 2 3 105 pfu (black) on day 0. Graphs depict
mean SEM.
(B) Viral loads in BAL (left), CD45+ immune cells (middle), or alveolar epithelium (right) as measured by RT-PCR using Fluidigm. Graphs depict mean SEM.
Mann-Whitney tests were performed to determine statistical significance (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; magenta asterisks denote significance
between PR8lethal and both X31sublethal and PR8sublethal infections; blue and red asterisks denote significance between PR8lethal and PR8sublethal or X31sublethal
infections, respectively; black asterisks denote significance between PR8sublethal and X31sublethal infections).
(C) Representative H&E and anti-influenza antibody-stained sections from PR8 and X31 sublethally infected lungs. Days postinfection, magnifications, terminal
bronchioles (TB), arterioles (a), and alveoli (A) are as indicated. Boxed regions on subgross images (1.25X) correspond to the regions shown at 20X for both H&E
and IHC panels, except for PR8 at d9, where the sections are stepped; note that the same terminal bronchiole and arteriole are present in both panels. Boxed inset
in lower right X31 IHC panel is an example of the control immunoglobulin G-stained tissue. Black bar represents 6 mm.
(D) Representative IHC from PR8 D6- and X31 D5-infected lung sections. Brown staining indicates presence of antigen. Arterioles are indicated (a).
(E) Antiviral response represented as heatmap of RT-PCR data detected in BAL, CD45+, or alveolar epithelial cells. Scale bar represents normalized value to
elongation factor 1 alpha (EF1a).
See also Figure S1.
than animals infected with PR8sublethal or X31subtlethal. Animals infected with X31sublethal had similar viral loads on day 3 and day 5
as animals infected with PR8sublethal but subsequently cleared
the virus after day 7 postinfection. Thereafter, the viral load in
animals infected with PR8sublethal was generally higher than
that found within animals infected with X31sublethal.
In separate experiments, we performed hematoxylin and eosin
(H&E) staining and anti-influenza immunohistochemistry (IHC) on
lung sections to evaluate the histopathologic characteristics,
distribution of changes, and localization of influenza antigens
(Figures 1C and 1D and Figure S1 available online). In order to
compensate for the slightly shifted kinetics in weight loss, we
adjusted the collection times for the X31 and PR8 infections.
Lung tissues were collected during the induction of inflammation
(day 5 for X31; day 6 for PR8), during peak weight loss (day 7 for
X31; day 9 for PR8), and during resolution of inflammation (day
13). The IHC staining indicated that, while PR8 is capable of
spreading into the deeper interstitial lung tissue, X31 is primarily
restricted to the major bronchial region with limited positive
staining in the lung (Figure 1D). H&E staining of PR8-infected
lung tissue indicated polymorphonuclear and mononuclear
leukocyte infiltration in and around major bronchial and lung
interstitial space on day 6 postinfection (Figure 1C). The infiltration spread more extensively in the lung by day 9. On day 13
postinfection, demarcated lymphocytic aggregates were
observed in both infections. For X31 infection, the infiltrating
cells were generally restricted to the major bronchial areas with
lower levels of lymphocyte infiltration at later time points during
the resolution phase than for PR8 infection.
We compared the transcript levels of several interferoninduced genes (Mx1, Rsad2, and Ifit1) and the transcription factor Irf7, all signatures of influenza infection (Sato et al., 2000), to
assess the antiviral response (using Taqman assays listed in
Table S1). The strongest responses were detected in infiltrating
cells isolated from the BAL as well as immune cells from the
lung (CD45+) (Figure 1E). Despite the different kinetics of weight
loss in animals infected with PR8sublethal and X31sublethal, the
abundance of Mx1, Ifit1, Rsad2, and Irf7 transcripts were comparable and reflected the level of viral infection: PR8sublethal
induced a slightly stronger and more sustained response than
X31sublethal, and PR8lethal induced an even more exuberant
response than the sublethal infections.
Cytokine and Chemokine Response
In response to infection of the lung, a concerted signaling cascade
is activated to induce secretion of cytokines and chemokines (as
well as lipid mediators) that promote vasodilation and a massive
infiltration of immune cells to affected tissues. We measured the
cytokine and chemokine levels in the BAL by multiplexed immunoassay (Luminex) to establish the levels of protein mediators
induced during the course of infection (Figures 2 and S2). The shift
in the kinetics of weight loss between PR8sublethal and X31sublethal
was mirrored by the production of interleukin (IL)-5, monocyte
chemoattractant protein (MCP)-1, and tumor necrosis factor
alpha (TNF-a). By contrast, similar levels of IL-6, IL-1b, and
macrophage inflammatory protein-1a (MIP1a) were produced
during infection with PR8sublethal and X31sublethal. Finally, infection
with PR8sublethal was accompanied by higher levels and pro216 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.
longed production of keratinocyte-derived chemokine (KC), interferon-inducible protein-10 (IP-10), granulocyte colony-stimulating factor (G-CSF), regulated on activation, normal T cell
expressed and secreted (RANTES), IL-1a, interferon g, and IL10 compared to infection with X31sublethal (Figures 2 and S2). Interestingly, despite their difference in pathogenicity, PR8lethal and
PR8sublethal produced similar levels of the cytokines and chemokines described above, with the exception of MCP-1, RANTES,
and G-CSF, which were higher during infection with PR8lethal.
Lipidomic and Transcriptomic Profiling
A variety of bioactive lipid mediators play a critical role in the
regulation of inflammation, and we sought to examine their role
in the pathogenesis of influenza. Given the complexity of the
pathways and multiple routes for degradation and other modifications of these mediators, we examined the dynamic lipidome
during influenza infection using liquid chromatography (LC)/
mass spectrometry (MS). Over the course of infection, 141 lipid
species (Extended Experimental Procedures) were profiled and
52 lipid metabolites were detected and quantified. The relative
percentages of metabolites generated through the COX, LOX,
and CYP450 pathways derived from arachidonic acid, as well
as metabolites derived from linoleic acid, linolenic acid, DHA,
and EPA, were determined to assess the composition of bioactive lipids in the BAL (Figures 3A and S3A). For mediators in
the COX pathway, levels of prostaglandins increased relative
to other metabolites during the early inflammatory response
(day 3) but decreased thereafter until returning to the basal level,
regardless of whether the mice were infected with PR8sublethal or
X31sublethal. In contrast, the percentage of prostaglandins relative
to other metabolites was significantly lower during PR8lethal
infection. LOX-derived metabolites also decreased during the
early phase of PR8sublethal and X31sublethal infection, while animals infected with X31sublethal had higher levels of LOX metabolites throughout the resolution phase of inflammation. The
percentage of metabolites of the cytochrome P450 pathway
increased during the induction of inflammation, and the kinetics
were slightly shifted between PR8 and X31, mirroring the kinetics
of weight loss (Figures 1A and S3). In contrast to the COX and
CYP450 pathways, percentages of metabolites derived from
linoleic acid, linolenic acid, DHA, and EPA increased during the
later phase of infection. The concentrations of linoleic acid and
EPA initially decreased during the early phase of inflammation
and subsequently increased throughout the resolution phase.
Moreover, the percentage of linoleic acid metabolites was significantly higher in PR8sublethal than X31sublethal. In contrast, the lowpathogenicity virus, X31sublethal, induced a higher percentage of
both DHA- and EPA-derived metabolites than the high-pathogenicity virus, PR8sublethal (Figures 3 and S3A).
While the lipid class composition during infection offered a
view of the activation kinetics of specific lipid metabolic pathways, it did not shed light on individual mediators, which have
diverse physiological consequences for induction and resolution
of inflammation. The COX pathway generates prostaglandins,
and their levels were surprisingly similar under all three conditions of infection (X31sublethal, PR8sublethal, and PR8lethal) (Figures
3B and 3C). Most of the prostaglandins peaked early and
decreased thereafter (Figure 3B). In general, the messenger
Figure 2. Cytokine and Chemokine Responses in Mouse Influenza Infections

(A) Cytokine and chemokine levels detected in BAL by multiplex Luminex represented as a heatmap. Scale represents the amount of protein in pg/ml.
(B) Line graphs of selected representative cytokines/chemokines depicting the mean SEM.
See also Figure S2.
RNA transcripts encoding the enzymes within the cyclooxygenase pathway displayed similar expression kinetics to the specific metabolites that they generate (Figures 3B and S3B). One
striking difference between the PR8sublethal and X31sublethal infections was the significantly higher levels of cytochrome P450 and
linoleic acid-derived metabolites in animals infected with the
high-pathogenicity virus (Figure 3B).
DHA and EPA metabolites have recently been shown to promote the resolution of inflammation (Ariel and Serhan, 2007;
Bannenberg et al., 2005). While 17-HDoHE, a precursor of resolvins and protectins (D series), was found at similar levels in
PR8sublethal and X31sublethal (Figure 3B), two distinct groups of
hydroxylated DHA emerged: 4-, 10-, 13-, and 20-HDoHE were
detected at higher levels in PR8sublethal-infected animals,
while 8-, 14-, and 16-HDoHE were found at higher levels in
X31sublethal-infected animals. Moreover, for lethally infected animals (PR8lethal), 4-, 10-, 13-, and 20-HDoHE had increasing
levels from day 3 to day 5 postinfection, reaching a level similar
to the maximum level found in PR8sublethal-infected mice (Figure 3B). In contrast, 8-, 14-, and 16-HDoHE peaked at day 3
and remained at levels significantly greater than in sublethal infections until day 5 (Figure 3B). Of note, several of these metabolites can be formed either enzymatically or through autoxidation
processes. For example, 17-HDoHE is a putative product of the

mammalian 12/15 LOX activity, which can be further metabolized to protectin D1, a potential suppressor of viral replication
(Morita et al., 2013; OFlaherty et al., 2012). Similarly, other positional isoforms of hydroxylated DHA can be formed via 12/15
LOX (Morgan et al., 2010). Even though the source of these
hydroxylated DHA metabolites are not entirely established, the
profile between PR8 and X31 infection conditions were strikingly
distinct. In general, metabolites of EPA, 12-hydroxyeicosapentaenoic (HEPE), and 15-HEPE were detected at a higher level
in X31sublethal-infected animals than in PR8sublethal-infected animals. The relationship between the level of a metabolite and
that of the transcript of the corresponding enzyme that generates
it can be discerned by comparing Figures 3B and S3B.
Lipidomic Profiling in Human Nasal Washes
The lipidomic profiling in the mouse influenza infection model
allowed us to obtain a comprehensive kinetic view of the lipidome across a well-defined time course. Understanding the
relevance of these results to human infection is crucial. We conducted lipidomic profiling of human nasopharyngeal lavages
obtained from a surveillance study at the St. Jude Childrens
Research Hospital during the 20092011 influenza seasons.
Figure 3. Lipidomic Profiling of Mouse Influenza Infection

(A) Stacked bar graph representing the percentages of arachidonic acid-derived cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450
(CYP450) metabolites, as well as linoleic acid-, linolenic acid-, DHA-, and EPA-derived metabolites. Each vertical line represents data from a single animal/sample
(n = 810).
(B) Quantified lipid mediators represented as a heatmap. Scale bar represents levels measured in pmol/ml.
(C) Line graphs of selected representative lipid mediators depicting the mean SEM.
See also Figure S3.
Study participants were enrolled based on detection of the influenza matrix gene (influenza A) or nonstructural NS1 gene (influenza B) by quantitative real-time PCR. Index cases and their
asymptomatic household contacts were followed for approximately 1 month following enrollment, with contacts sampled as
cases following a positive detection of influenza by quantitative
RT-PCR (qRT-PCR). Based on hierarchical clustering using clinical scores and multiplexed immunoassay of growth factors,
cytokines, and chemokines, we identified three groups within
qRT-PCR-confirmed influenza-infected subjects: (1) patients
with low clinical scores and minimal amounts of cytokines and
chemokines, (2) patients with medium clinical scores and positive for a subset of growth factors and cytokines (epidermal
growth factor, G-CSF, growth-related oncogene, IL1Ra2, IL8,
and IP-10), and (3) patients with high clinical scores and high
levels of a wide panel of cytokines (Figure S4A). A subset of
samples was selected from each group for lipidomic profiling
(n = 911/group, total = 30). While the human nasal cavity is likely
to be a very different physiological environment than the mouse
lung, we detected induced levels of many lipid mediators that
were identified in the mouse influenza infection model. We found
one dramatic difference in the lipid profile between the human
and mouse controls; linoleic acid comprises 68% of the lipidome
in the nasopharyngeal lavage of human low responders (asymptomatic contacts of the index cases), whereas it makes up
33.7% of the lipidome in the BAL of naive mice (Figures 3A
and 4A). High levels of linoleic acid have been detected in
humans in the skin, mucosal surfaces, and nasal fluid and
contribute to antimicrobial activity at these sites (Drake et al.,
2008). The samples from patients with high clinical score and
increased levels of cytokines and chemokines also contained
significantly higher percentages of lipid mediators from the lipoxygenase, CYP450, DHA, and EPA pathways (Figures 4A and
S4B). Amounts of individual lipid mediators (including PGE2,
LTE4, and 17 HDoHE) were also significantly increased within
the high-response group compared to the low- and mediumresponse groups (Figures 4B and 4C).
Proinflammatory and Anti-Inflammatory States during
Influenza Infection
In order to look at global changes in the mouse and human data,
we grouped the bioactive lipids based on their proinflammatory
or anti-inflammatory/proresolution properties and created quantitative indices to characterize the proinflammatory and antiinflammatory contributions of the host response at each stage
of the infection (see Extended Experimental Procedures). These
indices were calculated for each infected mouse or patient at
every time point/condition. To create the index for the mouse
infection model, we first calculated the fold change of individual
lipid mediators in infected animals over naive animals and only
included values greater than or equal to two. The fold changes
for each mediator were then normalized to the maximum value
across all samples, setting the maximum value to one. We separated lipid mediators into groups with pro- or anti-inflammatory
activity and then summed the normalized values of each mediator within the pro- or anti-inflammatory group to define the total
activity. The maximum activity in each group is then equal to the
total number of mediators in the group. To generate the index for
each time point, the percentage of the pro- or anti-inflammatory

mediator activity was calculated by dividing total activity by the
maximum activity in the pro- or anti-inflammatory groups.
As discussed above, the early proinflammatory response was
similar between PR8sublethal and X31sublethal infections, as indicated by levels of mediators such as prostaglandins on day 3
postinfection (Figure 5A). This was reflected in a comparable
increase of proinflammatory indices on day 3 for both sublethal
infections (Figures 5B and S5A). For PR8sublethal infection, a
secondary wave of proinflammatory mediators occurred on day
7 postinfection, including linoleic acid-derived epoxide and dihydroxy linoleic acid. This contributed to a significant increase in the
proinflammatory indices for PR8sublethal, which began to subside
on day 9 postinfection (Figures 5B and S5A). The mediators 9,
10 EpOME; 12, 13 EpOME; 9, 10 diHOME; and 12, 13 diHOME,
which are potent chemoattractants for neutrophils (Totani et al.,
2000), were present at much lower levels in X31sublehtal-infected
animals on day 7 postinfection. For the anti-inflammatory index,
the peak of the response began on day 7 postinfection and was
not significantly different between PR8sublethal and X31sublethal.
The anti-inflammatory indices decreased slightly on day 9 postinfection and sustained these levels until day 13 postinfection. In
contrast to the sublethal infections, PR8lethal induced significant
increases of both pro- and anti-inflammatory indices consecutively on day 3 and day 5 postinfection (Figures 5B and S5A).
Using the same approach to determine the proinflammatory
and anti-inflammatory indices of patient samples (using low clinical symptoms and immune response as the control), the high
response group had significant increases in the pro- and antiinflammatory indices compared to the medium response group
(Figures 5C, S5B, and S5C).
Lipoxygenase Pathway
We next dissected the lipoxygenase pathway in more detail,
since it contains well-defined pro- and anti-inflammatory mediators. Leukotrienes synthesized by 5-lipoxygenase have long
been known as proinflammatory mediators: leukotriene B4
(LTB4) is a potent chemotactic factor for neutrophil recruitment,
and LTC4 and LTE4 are potent mediators for immediate hypersensitivity, bronchoconstriction, smooth muscle contraction,
and increased vascular permeability. Recently, 12-lipoxygenase- and 15-lipoxygenase-derived metabolites, such as 12HETE, hepoxilin, 15-HETE, and lipoxins have been increasingly
appreciated as anti-inflammatory mediators (Kuhn and ODonnell, 2006). Specifically, 12-HETE and 15-HETE are potent activators of peroxisome proliferator-activated receptor gamma
(PPARg) (Kronke et al., 2009), which can dampen inflammation
via inhibition of nuclear factor-kB and modulate macrophage
function (Ricote et al., 1998). Lipoxins can block further recruitment of neutrophils (Serhan et al., 1995) and stimulate nonphlogistic clearance of apoptotic cells (Godson et al., 2000). While
many of these molecules have been studied individually in
various settings, a comprehensive but focused analysis of the
metabolites and mediators within this pathway would yield a
better understanding of the local inflammatory milieu during an
active microbial infection.
The levels of 5-lipoxygenase mediators were similar in the BAL
of sublethally infected mice, with the exception of LTE4, in which
Figure 4. Lipidomic Profiling of Human Nasal Washes

(A) Stacked bar graph represents the percentages of arachidonic acid-derived cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450)
metabolites, as well as linoleic acid-, linolenic acid-, DHA-, and EPA-derived metabolites. Each vertical line represents data from a single patient/sample, which is
categorized into either the low, medium, or high clinical score/immune response group (n = 911).
(B) Quantified lipid mediators represented as a heatmap. Scaled bar represents levels measured in pmol/ml.
(C) Column vertical scatter plot of selected representative lipid mediators depicting the mean SEM.
See also Figure S4.
Figure 5. Pro- and Anti-Inflammatory Indices of Mouse and Human Lipidomic Profiles
(A) Heatmap represents the mean fold change in each lipid mediator (separated by having either pro- or anti-inflammatory activity).
(B) Line graphs of pro- and anti-inflammatory indices depicting the mean SEM.
(C) Column vertical scatter plot of pro- and anti-inflammatory indices for individual patient samples. Horizontal lines indicate the mean SEM.
See also Figure S5.
they were more abundant in PR8sublethal-infected animals on day

7 and day 9 postinfection (Figure 3C). This difference resulted in
higher percentages of 5-lipoxygenase-derived mediators in
PR8sublethal infection (Figures 6A and S6A). In concordance
with its pathogenicity, PR8lethal infection elicited much greater
amounts of LTE4, which contributed to a significant increase in
the percentages of 5-lipoxygenase-derived metabolites compared to the sublethal infections (Figures 3C, 6A, and S6A). Transcription of 5-lipoxygenase (alox5) was downregulated at the
onset of infection (Figure 6B), similar to the regulation observed
in macrophages stimulated in vitro with lipopolysaccharide
Kdo2-lipid A (Dennis et al., 2010). 5-lipoxygenase activating protein, alox5ap, on the other hand, was significantly increased in
the lethal infection, which correlated with the level of the lipid
LTE4 (Figures 3C and 6B).
Mediators of the 12/15-lipoxygenase pathway, 12-HETE, and
hepoxilin B3 (HXB3) had increased levels in the late phase of
X31sublethal infection when compared to PR8sublethal (Figure S6A).

These differences contributed to significantly higher percentages of 12-lipoxygenase-derived mediators for X31sublethal
when compared to PR8sublethal from day 7 to day 13 postinfection
(Figures 6A and S6A). The level of 15-HETE was similar between
the sublethal infections. The transcriptional level of 15-lipoxygenase (alox15) is significantly increased in X31sublethal infection at 9
and 11 days postinfection in comparison to PR8sublethal (Figures
6B and S6B). The discrepancy suggests that the increased
quantity of Alox15 protein may be acting on other substrates,
such as linoleic acid or linolenic acid. 8-lipoxygenase (alox8) exhibited a greater level of expression in PR8sublethal-infected animals than in X31sublethal-infected animals at the late phase of
infection. This transcriptional induction in the epithelium correlated with increased detection of 8-HETE in the BAL fluid (Figures
6B and S6A). In general, a high percentage of proinflammatory
5-lipoxygenase mediators were tightly correlated with the highly
Figure 6. Lipoxygenase Enzymes and Derived Metabolites in Mouse and Human Influenza Infection
Stacked bar graph represents the percentages of lipoxygenase 5-, 15-, 12-, or 8-derived metabolites of all lipoxygenase-derived metabolites in mouse (A)
or human (C) samples. Each vertical line represents data from a single sample (n = 811 per time point). (B) Line graphs of 5-lipoxygenase (alox5),
5-lipoxygenase-activating protein (alox5ap) in CD45+ cells, 15-lipoxygenase (alox15) in BAL, and 8-lipoxygenase (alox8) in alveolar epithelium depicting the
mean SEM.
See also Figure S6.
pathogenic phase of the PR8 virus, whereas an elevated percentage of anti-inflammatory 12/15-lipoxygenase mediators
were associated with the resolution phase of the X31 infection
(Figures 6A and S6A).
This trend was recapitulated in studies of human nasopharyngeal lavage fluid obtained during the 20092011 influenza
seasons. Here, the percentages of 5-lipoxygenase-derived
mediators were strongly correlated with disease symptoms
Figure 7. Hydroxylated Linoleic Acid Metabolites as a Biomarker for Immune Status during Influenza Infection
(A) Pathway for linoleic acid to generate 9-hydroxylated or 13-hydroxylated linoleic acid.
(B) Line graphs of 9-HODE, 13-HODE, and 13-:9-HODE in the mouse lung during influenza infection, depicting mean SEM (red: X31sublethal; blue: PR8sublethal;
black: PR8lethal).
(C) Column vertical scatter plot of 9-HODE, 13-HODE, and 13-:9-HODE in human nasal wash samples. Horizontal lines indicate mean SEM.
See also Figure S7.
from low to medium to high responders (Figures 6C and S6B).

The high response group exhibited significantly elevated levels
of 5-HETE, LTB4, and LTE4 compared to the low and medium responders. In contrast, percentages of 12-lipoxygenase-derived
metabolites were anticorrelated with the level of disease
response (Figures 6C and S6B).
Hydroxylated Linoleic Acid Metabolites as Biomarkers
for the Status of Influenza Infection
While the comprehensive study of bioactive lipid profiles offered
a complete picture of the host response to influenza, the methodology also allowed for identification of useful biomarkers
that indicate the immunological status of an active infection.
Linoleic acid is further metabolized into 9- or 13-hydroxylated
linoleic acid (9-hydroxyoctadecadienoic acid [HODE] and 13HODE). While 13-HODE is generated by 15-lipoxygenase,
9-HODE production can be catalyzed by multiple enzymes and
nonenzymatic reactions (Obinata and Izumi, 2009; Figure 7A).
9-HODE and 13-HODE have previously been proposed as

markers for lipid peroxidation in various chronic diseases
(Spiteller and Spiteller, 1997). More recently, 9-HODE has been
shown to have a proinflammatory role (Hattori et al., 2008;
Obinata and Izumi, 2009), while 13-HODE is anti-inflammatory
(Altmann et al., 2007; Belvisi and Mitchell, 2009). We calculated
the ratio of 13- to 9-HODE to determine whether it accurately describes the pro- versus anti-inflammatory state of an infection.
An advantage of using ratios of mediators is that, while the values
of individual lipid mediators may vary greatly between different
animals and different patients, the ratio between two mediators
is normalized, as they arise from a common precursor. Another
reason for choosing 9- and 13-HODE is that both mediators
were detected robustly across all samples (29/30 clinical samples for 9-HODE and 30/30 clinical samples for 13-HODE). In
contrast, many of the other mediators were undetectable in
either the low or medium response groups, making them less
suitable as biomarkers.
When we compared the levels of 9-HODE, 13-HODE, and the

ratio of 13-HODE/9-HODE during influenza infection in mice,
PR8sublethal infection induced a significantly increased level of
9-HODE on day 7 postinfection compared to X31sublethal infection. In contrast, both PR8sublethal and X31sublethal induced similar
production of 13-HODE. Interestingly, the ratios of 13-:9-HODE
are increased for both PR8sublethal and X31sublethal during the
resolution phase of the infection, indicating an increasingly
anti-inflammatory state. Moreover, the ratio is significantly
higher in X31sublethal, even at early time points (day 7 and 9 postinfection), which supports the notion that the low-pathogenicity
virus induced a response skewed toward an anti-inflammatory
profile (Figure 7B).
We tested the robustness of these parameters by combining
three additional experiments performed on separate days and
animals from different vendors and harvested at different time
points. While the raw values of 9- and 13-HODE varied between
animals and experiments, the ratio of 13-:9-HODE remained
consistent (Figures S7A and S7B).
In the human nasopharyngeal lavage, levels of 9-HODE were
slightly lower in the high response group, although the difference
was not significant (Figure 7C), while levels of the anti-inflammatory mediator, 13-HODE, were elevated with moderate significance (p < 0.05). When we compared the 13-:9-HODE ratios
between the groups, however, the difference became more dramatic and highly significant (p < 0.001) (Figure 7C). Further measurements on additional samples and clinical data will be
required to determine whether the 13-:9-HODE ratio can be
used to further stratify the severity and/or course of infection.
DISCUSSION
Lipidomic profiling of influenza infection provided a comprehensive map of eicosanoids and other related bioactive lipids during
an active infection of the lung. We detected and quantified
metabolites and mediators within the cyclooxygenase, lipoxygenase, and cytochrome P450 pathways derived from arachidonic
acid, as well as metabolites and mediators derived from linoleic
acid, linolenic acid, DHA, and EPA. The protein levels of cytokines and chemokines and the transcript levels of numerous antiviral genes suggested a straightforward, positive correlation
between the level of immune response and the pathogenicity
of the infection, with the response increasing from infection
with X31, the low-pathogenicity virus, to PR8, the high-pathogenicity virus (PR8sublethal to PR8lethal). In contrast, the lipidomic
profiling uncovered a more complex response, with the levels
of mediators involved in both pro- and anti-inflammatory processes exhibiting more complex dynamics. Surprisingly, the
cyclooxygenase pathway mediators, consisting of the classical
signaling prostaglandins, were produced with similar kinetics
during infections by the different influenza strains. Concordantly,
enzymes within the COX pathway were activated to similar levels
transcriptionally. In contrast to the COX-derived lipid mediators,
metabolites derived from other pathways (LOX and CYP450) and
precursors (linoleic acid, DHA, and EPA) diverged strongly
between the different infections.
Hepoxilin and 12-HETE, mediators derived from the 12/15
lipoxygenase pathway, were detected at a higher level in
X31sublethal than PR8sublethal-infected animals. Hepoxilin has

been suggested to be involved in anti-inflammatory mechanisms, and stable hepoxilin analogs inhibit macrophage influx
as well as fibrosis in the lung (Jankov et al., 2002). 12-HETE
also has anti-inflammatory activities: for example, it is capable
of blocking TNFa-induced IL-6 secretion from macrophages
(Kronke et al., 2009). In contrast, 8-HETE, which is derived
from 8-lipoxygenase, was detected at a higher level in
PR8sublethal- than X31sublethal-infected animals. While the biological activity of 8-HETE within the respiratory system is poorly
characterized, the metabolite has been detected in human lung
vasculature (Kiss et al., 2000). The increased production of
8-HETE late in infection suggests that it plays a role in epithelial
repair after influenza infection. It is important to note that mice do
not have separate 12- and 15-lipoxygenases but rather a combined mouse 12/15-lipoxygenase, which is related in sequence
to the human 12-lipoxygenase as well as human 15-lipoxygenase type I (reticulocyte-type) and type II (epidermis-type) enzymes (Kuhn et al., 2002).
Overall, while metabolites of the LOX pathways displayed a
heterogeneous profile in the mouse infection model, a clear difference between PR8 and X31 infection was found primarily in
metabolites contributing to anti-inflammatory and proresolution
activities. In human samples, however, a general increase was
observed in both the proinflammatory (5-lipoxygenase-derived)
and anti-inflammatory (12-, 15-, and 8-lipoxygenase-derived)
metabolites of the LOX pathway in high responders. Interestingly, when comparing the percentages of mediators derived
from different lipoxygenase enzymes, a consistent pattern
emerged in the mouse and human data. In the mouse model,
the percentage of 5-lipoxygenase metabolites was elevated during the pathogenic phase of the PR8 infection, whereas the
percentage of 12/15-lipoxygenase metabolites was elevated
during the resolution phase of the X31 infection. In the human
samples, increasing percentages of 5-lipoxygenase-derived
metabolites and decreasing percentages of 12-lipoxygenasederived metabolites correlated with increasing clinical symptoms and immune response. However, a direct comparison
between the mouse and human data is difficult, due to the lack
of information on the time course and pathogenicity of the viral
strains in the human infection samples as well as the unknown
contribution of host genetic diversity to susceptibility to viral
infection. When infected with influenza, 12/15-lipoxygenase
gene-deficient mice recovered more slowly than wild-type
mice, similar to the observations of Morita et al. (2013; data not
shown). However, 12/15 gene-deficient mice are of limited utility
in modeling human infections, as the function of this enzyme is
performed in humans by at least four different enzymes that
are expressed in different cell types. Detailed pharmacological
studies using specific inhibitors would be required to determine
the effect of these enzymes at different phases of infection.
Cytochrome P450 enzymes can metabolize arachidonic acid
to produce 5,6- or 14,15-epoxyeicosatrienoic acid (EET) with
subsequent metabolic transformation to diHETrE by the soluble
epoxide hydrolase (sEH). EETs have potent vasodilator activities, and they have been determined to have anti-inflammatory
activities by activating the PPARa pathway (Thomson et al.,
2012; Wray et al., 2009). CypP450 metabolites were detected
at significantly higher levels in PR8sublethal infections than in

X31sublethal-infected animals. Similar to arachidonic acid-derived
EET and diHETrE, cytochrome P450 enzymes can metabolize
linoleic acid to produce 9,10 or 12,13 EpOME, and sEH further
converts them to 9,10- or 12, 13-diHOME. The epoxides of
linoleic acid are leukotoxins produced by activated neutrophils
and macrophages. Elevated levels are associated with acute
respiratory distress syndrome (ARDS) and cause pulmonary
edema, vasodilation, and cardiac failure in animal models (Ishizaki et al., 1995, 1999). These EpOME mediators and their
sEH-derived mediators, diHOME, are chemotactic to neutrophils
and exert their toxicity by disrupting mitochondrial functions
(Sisemore et al., 2001; Totani et al., 2000). The elevated levels
of these mediators in animals infected with the highly pathogenic
influenza strain may explain the severe damage occurring in the
lung during infection.
Recently, a new class of endogenous lipid mediators (derived
from DHA and EPA) that promote resolution of inflammation provides great promise to treat inflammatory diseases (Ji et al.,
2011). Although assays for resolvins and protectins were
included in the panel, these anti-inflammatory/ proresolution
molecules were not detected in the animal model or human clinical samples. This is interesting because the production of PD1 is
suppressed in lung tissues of influenza-infected mice at early
time points (Morita et al., 2013). Additionally, PD1 and 17-HDoHE
were detected at higher levels in exhaled breath condensate
from healthy individuals compared to asthma patients (Levy
et al., 2007). Further studies will determine whether these differences arise from the different sampling methods employed (BAL
versus lung tissue; nasopharyngeal lavage versus exhaled
breath condensate). Aside from different tissue, the infection
model and the time frame of sampling is quite different between
our study and previous reports. Even though our profiling
approach was comprehensive, the biological activities of several
of the metabolites detected in our screens are unknown, and
further systematic studies will be necessary to determine their
involvement in the inflammatory process and return to homeostasis within the respiratory system.
By creating quantitative pro- and anti-inflammatory indices,
we defined lipid profiles that readily distinguished infections by
the low-pathogenicity strain, X31, and the highly pathogenic
strain, PR8. X31sublethal induced a normal sequence of events,
with induction of a proinflammatory state on day 3 after infection,
followed by induction of an anti-inflammatory state on day 7,
which restored homeostasis to the system. Interestingly, while
PR8sublethal also induced a proinflammatory state on day 3, there
was a second, more robust induction of proinflammatory mediators on day 7, occurring simultaneously with the induction of
anti-inflammatory mediators. This overlap of pro- and antiinflammatory states was also quite apparent in the PR8lethal
infection, in which both indices were significantly higher than
the sublethal infections on day 3 and 5 postinfection. The clinical
data were similar to the mouse data, suggesting that pro- and
anti-inflammatory states overlap within infected humans as
well. Further studies are warranted to determine whether this
simultaneous pro- and anti-inflammatory lipid response is beneficial or detrimental to the host during an infection with a highly
pathogenic influenza strain.
From the mouse lipidome, we have identified 9-HODE, 13HODE, and their ratio as potential biomarkers for immune status
during an infection. While there are numerous parameters to
assess the proinflammatory status of an infection, parameters
characterizing the resolution of inflammation are lacking.
Because these metabolites can be robustly detected by ELISA
and are derived from a common precursor, making their abundance ratio self-normalizing, they offer great potential as a
biomarker of immune status during influenza infection. A larger
cohort of patients and samples is required to confirm the correlation between the ratio of 13-:9-HODE and the status of disease
and recovery.
Lipidomic profiling provides an additional parameter for understanding the host response during a microbial infection.
Although transcriptomic analysis of the response to infection
has been conducted routinely, our data suggest that, while
some transcript levels of the enzymes correlate with the lipid
levels, the majority of the changes seen in the lipid response
were regulated posttranscriptionally. It is likely that the response
to infection is accelerated by the uncoupling of metabolite production from the transcriptional regulation of the specific upstream enzymes.
EXPERIMENTAL PROCEDURES
Mouse Influenza Infection
Female C57Bl/6 animals (812 weeks) (Jackson Laboratory and Charles River)
were kept in specific pathogen-free conditions. Experiments conducted were
approved by the Institute for Systems Biology Institutional Animal Care and
Use Committee. Animals were anesthetized with a ketamine/xylazine mixture
and infected intranasally with 200 or 2 3 105 pfu of influenza virus strain PR8 or
2 3 105 pfu of X31 in 30 ml sterile PBS. Mock-infected animals were inoculated
with 30 ml sterile PBS. Mice were monitored and weighed daily.
Human Clinical Samples
This study was carried out in accordance with Good Clinical Practice (GCP) as
required by the U.S. Code of Federal Regulations applicable to clinical studies
(45 CFR 46), the International Commission on Harmonization guidelines for
GCP E6, and the National Institutes of Health (NIH) Clinical Terms of Award.
Children and adults with influenza-like illness receiving medical counsel
were approached by study personnel to participate. Study participants were
enrolled based on detection of the influenza matrix gene (influenza A) or
nonstructural NS1 gene (influenza B) by quantitative real-time PCR. Individuals
with acute respiratory illness meeting the case definition for influenza (fever or
feverishness accompanied by cough or sore throat) who were symptomatic for
96 hr or less and their household or family contacts (symptomatic or asymptomatic) were eligible to participate in the study and followed for approximately
1 month postenrollment. Contacts that became influenza-positive were
sampled cases after conversion. Nasopharyngeal swabs and lavages were
collected upon enrollment and at each subsequent study visit.
Lipidomic Profiling by LC Mass Spectrometry
Lipid mediators were analyzed by LC and MS essentially as described previously (Dumlao et al., 2011; Quehenberger et al., 2010). Briefly, 0.9 ml of BAL
or nasal washes were supplemented with a mix consisting of 25 deuterated internal standards (Cayman Chemical) and lipid metabolites isolated by solid
phase extraction on a Strata X column (Phenomenex). The extracted samples
were evaporated, reconstituted in a small volume, and the eicosanoids were
separated by reverse phase LC using a Synergy C18 column (Phenomenex).
The eicosanoids were analyzed by tandem quadrupole MS (MDS SCIEX
4000 QTRAP, Applied Biosystems) operated in the negative-ionization mode
via multiple-reaction monitoring (MRM) using transitions that were optimized
for selectivity and sensitivity. Authentic standards were analyzed under
identical conditions, and eicosanoid quantitation was achieved by the stable

isotope dilution method and comparison with 141 quantitation standards.
Data analysis was performed using the MultiQuant 2.1 software (Applied Biosystems). A complete list of all metabolites included in the MRM profile is given
in Table S2.
Statistical Analysis
The two-tailed Mann-Whitney test was used to determine statistical significance between population means (for mouse infections, red: X31sublethal;
blue: PR8sublethal; black: PR8lethal. Magenta asterisks denote significance
between PR8lethal and both X31sublethal and PR8sublethal infections; blue and
red asterisks denote significance between PR8lethal and PR8sublethal or
X31sublethal infections, respectively; and black asterisks denote significance
between PR8sublethal and X31sublethal infections). Statistical significance:
*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. For correlation studies, the
two-tailed Spearman test was used to calculate correlation significance.
Heatmaps and clustering analysis were generated using Genedata and Java
TreeView.
Data Dissemination
Raw files and processed data for lipidomics profilings are available at the
Systems Influenza Website (http://www.systemsinfluenza.org) and at Metabolights (http://www.ebi.ac.uk/metabolights/; MTBLS31).
ACCESSION NUMBERS
The Metabolights accession number for the lipidomics profilings reported in
this paper is MTBLS31.
SUPPLEMENTAL INFORMATION
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antibiotic requirements. Nature 484, 524528.
Dennis, E.A., Deems, R.A., Harkewicz, R., Quehenberger, O., Brown, H.A.,
Milne, S.B., Myers, D.S., Glass, C.K., Hardiman, G., Reichart, D., et al.
(2010). A mouse macrophage lipidome. J. Biol. Chem. 285, 3997639985.
Drake, D.R., Brogden, K.A., Dawson, D.V., and Wertz, P.W. (2008). Thematic
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(2011). High-throughput lipidomic analysis of fatty acid derived eicosanoids
and N-acylethanolamines. Biochim. Biophys. Acta 1811, 724736.
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T. (2008). G2A plays proinflammatory roles in human keratinocytes under
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Supplemental Information includes Extended Experimental Procedures, seven

figures, and two tables and can be found with this article online at http://dx.doi.
org/10.1016/j.cell.2013.05.052.
Ishizaki, T., Shigemori, K., Nakai, T., Miyabo, S., Ozawa, T., Chang, S.W., and
Voelkel, N.F. (1995). Leukotoxin, 9,10-epoxy-12-octadecenoate causes
edematous lung injury via activation of vascular nitric oxide synthase. Am. J.
Physiol. 269, L65L70.
ACKNOWLEDGMENTS
Ishizaki, T., Ozawa, T., and Voelkel, N.F. (1999). Leukotoxins and the lung.
Pulm. Pharmacol. Ther. 12, 145155.
We thank Alan Diercks and Peter Askovich for helpful advice and critical
reading of the manuscript. This work was supported by National Institute of
Allergy and Infectious Diseases Contract no. HHSN272200800058C, A Systems Biology Approach to Infectious Disease Research, the American Lebanese Syrian Associated Charities (ALSAC), the NIH/NIAID Center of Excellence
for Influenza Research and Surveillance (HHSN266200700005C), and NIGMS
U54 GM069338 (to E.A.D.).
Received: March 7, 2013
Revised: April 8, 2013
Accepted: May 20, 2013
Published: July 3, 2013
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Cells and Bronchoalveolar Lavage Isolation
Mice were euthanized by CO2 inhalation. Broncho-alveolar lavage (BAL) was collected in 2 3 1 ml washes with HBSS buffer. Cells
from the BAL were collected by light centrifugation. Distinct cell types from the dissected lungs were isolated as described (Gereke
et al., 2007; Marsh et al., 2009; Snelgrove et al., 2008). Dispase digested lung tissue was disrupted into single-cell suspensions by
passage through 100-mm, 70-mm, and 40-mm filters. Cells were incubated with anti-mouse CD16/32 (UCSF Monoclonal Antibody
Core) and anti-mouse CD45 microbeads (Milteny) and separated by magnetic separation. RNA was extracted from distinct cell populations using TRIzol reagent (Invitrogen).
Proteomic Quantitation by Luminex
Multiplex quantitation of chemokines and cytokines was conducted using the Luminex xMAP system with a MILLIPLEX MAP human
cytokine/chemokine immunoassay panel or Mouse Cytokine/Chemokine panel as described by manufacturer (Millipore).
Histological and Immunohistochemical Analysis
Dissected mouse lungs (left lobes) were fixed in 10% neutral-buffered formalin, processed routinely into paraffin and stained with
hematoxylin and eosin (H&E) or anti-influenza antibody (Meridian Life Science) followed by a rabbit anti Goat IgG secondary antibody
(Jackson ImmunoResearch). The isotype control antibody was purified Goat IgG (Invitrogen). Staining was performed using the Leica
Bond Automated Immunostainer and signal detected with 3,3-Diaminobenzidine (DAB, brown) and counterstained with Harris hematoxylin. H&E and IHC-stained slides were digitized with the Olympus Nanozoomer and images captured with Nikon Digital Pathology
viewing software.
Transcriptional Profiling Using Fluidigm RT-PCR
RNA sample was treated with TURBO DNase (Ambion) before reverse transcription with Superscript II Reverse Transcriptase
(Invitrogen). For pre-amplification, 20x TaqMan Gene Expression Assays of selected genes (Table S1) were mixed together to a final
of concentration of 0.2x for each gene. This mix was used to set up amplification reactions of the reverse-transcribed samples. Each
Fluidigm 48.48 Dynamic array was first primed with Fluidigms IFC Controller MX, loaded with 48 pre-amplified samples, and the
primer/probe pairs for the selected genes described above and in the Extended Experimental Procedures. The array was run in
the BioMark HD System and data were collected using the BioMark Data Collection Software. Subsequent analysis was performed
with the Fluidigm Real-Time PCR Analysis software.
Calculation of Pro- and Anti-Inflammatory Indices
Fold change was calculated using the median of mock-infected animals (in the mouse model) or of the low response group (in human
samples). Only values that were greater than or equal to two were included. The fold changes were then normalized to the maximum
value across all samples, setting the maximum value to one. Mediators were divided into two groups based on having either pro- or
anti-inflammatory activity. Pro-inflammatory mediators include 6k PGF1a, PGF2a, PGE2, PGD2, PGF1a, 6, 15 dk dh PGF1a, 15k
PGF1a, 15k PGF2a, 15k PGE2, dhk, PGE2, dhk PGD2, LTB4, LTE4, 5 HETE, 9 HODE, 9 10 EpOME, 12 13 EpOME, 9 10 diHOME,
12 13 diHOME. Anti-inflammatory mediators include PGA2, PGB2, tetranor 12 R HETE, PGE1, 15 HETE, 8 HETE, 12 HETE, HXB3, 16
HETE, 5 6 EET, 14 15 EET, 11 12 diHETrE, 14 15 diHETrE, 13 HODE, 13 HOTrE, 15 HETrE, 13 HDoHE, 8 HDoHE, 16 HDoHE, 20
HDoHE, 7 HDoHE, 4 HDoHE, 17 HDoHE, 10 HDoHE, 14 HDoHE, 19 20 EpDPE, 19 20 DiHDPA, 15 HEPE, 12 HEPE. We then added
together the normalized value of each sample within the pro- and anti-inflammatory groups to define the total activity. The maximum
activity in each group is equal to the total number of mediators in that group. To generate the index for each time point, the percentage
of pro- and anti-inflammatory mediator activity was calculated by dividing the measured activity by maximum possible activity in the
each group.
We included most of the prostaglandins in having pro-inflammatory activities because of their vasomodulatory activities (Holtzman, 1992; Sheibanie et al., 2007). Leukotrienes have chemoattractant activities and are potent mediators for immediate hypersensitivity, bronchoconstriction, smooth muscle contraction, and increased vascular permeability (Peters-Golden et al., 2006). 5 HETE
has been shown to induce airway contraction and potentiate neutrophil transcellular migration (Bittleman and Casale, 1995). 9 HODE
has been attributed to have a proinflammatory role, by activating G2A, a G protein-coupled receptor, which mediates intracellular
calcium mobilization and JNK activation (Hattori et al., 2008; Obinata and Izumi, 2009). The epoxides of linoleic acid (9,10 EpOME
and 12,13 EpOME) are leukotoxins produced by activated neutrophils and macrophages. Elevated levels are associated with
ARDS (acute respiratory distress syndrome) and caused pulmonary edema, vasodilation, and cardiac failure in animal models
(Ishizaki et al., 1999, 1995). These metabolites and their soluble epoxide hydrolase metabolites (DiHOME) are chemotactic to neutrophils and exert their toxicity by disrupting mitochondrial functions (Sisemore et al., 2001; Totani et al., 2000).
PGA2 and PGB2, non-enzymatic derivatives of PGE2, were included to having anti-inflammatory activities. PGA2 is known to suppress activation of microglia and astrocytes in the experimental autoimmune encephalomyelitis (EAE) model and activates the
intrinsic apoptotic pathway (Lee et al., 2010; Storer et al., 2005). PGB2 has been shown to induce pulmonary hypertension, an opposite effect of PGE2 (Liu et al., 1994). PGE1 has been shown to inhibit the acute inflammatory edema response by activating the EP3
Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. S1
receptor (Ahluwalia and Perretti, 1994). 12- and 15-HETE also have anti-inflammatory activities by blocking TNFa-induced IL-6
secretion from macrophages (Kronke et al., 2009). 16-HETE has been shown to inhibit human polymorphonuclear cells adhesion
and aggregation, as well as decreasing LTB4 synthesis (Bednar et al., 2000). HXB3 and stable hepoxilin analogs inhibit macrophage
influx as well as fibrosis in the lung (Jankov et al., 2002). EET and diHETrE have been determined to have anti-inflammatory activities
by activating the peroxisome proliferator-activated receptor alpha (PPARa) pathway (Thomson et al., 2012; Wray et al., 2009); (Deng
et al., 2010). Specifically, 5,6 EET and 11,12 diHETrE have been shown to prevent leukocyte adhesion to the vascular wall
{{Node:1999wp}}. 14,15 EET has an anti-inflammatory role by preventing TNFa-induced IkBa degradation in human airway smooth
muscle cells (Morin et al., 2008). 14,15 diHETrE is a potent activator of PPARa that contribute to its anti-inflammatory activities (Fang
et al., 2006). 13 HODE is an agonist for PPARg and having an anti-inflammatory role (Altmann et al., 2007; Belvisi and Mitchell, 2009;
Emerson and LeVine, 2004; Fritsche, 2008; Stoll et al., 1994). Lastly, we included metabolites derived from DHA and EPA into having
anti-inflammatory activities. Some of these characterized mediators have direct and potent effects on the anti-inflammatory and proresolution processes (Groeger et al., 2010; Lima-Garcia et al., 2011). While other metabolites are generated by auto-oxidation or
other non-enzymatic routes, they can also be enzymatically metabolized into generating potent mediators, such as resolvins, protectins, and maresins.
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S2 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.
Figure S1. Histological Analysis Score, Related to Figure 1

Lung sections stained with hematoxylin and eosin (H&E) were scored for the levels of interstitial pneumonia/alveolitis (IP), bronchial epithelium necrosis (BN) and
hyperplasia (BH), alveolar hyperplasia(AH), lymphoid aggregates (LA), perivascular cuffing by mononuclear cells (PC-M) and neutrophils (PC-N), perivascular or
alveolar edema(PE, E), hemorrhage (H), vasculitis (V) and percent of section affected in any manner (E1) or in most severe manner (E2). Lesions were scored 0 =
normal or no change; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. Extent scores were applied as: 0 = no lesions; 1 = < 5%; 2 = 6%30%; 3 = 31%60%; 4 >
61%. Significant histological disease is reflected by summed score > 15.
(A) Total histological scores plotted as scattered plot of uninfected control (PBS, green circle), PR8 (blue square), or X31 (red triangle) infected tissues. Plot
represents mean +/ SEM. Mann-Whitney tests were performed to determine statistical significance between PR8sublethal D6 p.i. versus X31sublethal D5 p.i.,
PR8sublethal D9 p.i. versus X31sublethal D7 p.i., and PR8sublethal D13 p.i. versus X31sublethal D13 p.i. (N.S.; not statistically significant).
(B) Mean and standard deviation of individual scores (n = 4).
(C) Representative examples of histological lesions in influenza infected lungs. A. Normal lung. Terminal bronchiole (TB), pulmonary arteriole (a) and alveolus (A)
are indicated. B. Early lesions are principally focused on the pulmonary arteriole and terminal bronchiole anatomical unit. The arteriole wall is thickened and there
are abundant intravascular marginating leukocytes and at higher magnification, reactive endothelial cells are discernable (scored collectively as vascular lesions;
V). The perivascular space (PV) is moderately edematous (scored as perivascular edema; PE) with inflammatory cells extending mildly into the adjacent interstitium (scored as interstitial pneumonia; IP); D6 p.i. PR8. C. Necrosis of the bronchiolar epithelium (scored a bronchiolar necrosis; BN) lining the terminal
bronchioles (TB) is also noted during early infection. The epithelium of the terminal bronchiole (TB) is multifocally denuded and necrotic cells are sloughed into the
airway. There are moderate perivascular inflammatory cells that at higher power can be discerned into either mononuclear (lymphocytes and macrophages) or
neutrophilic (scored as PV-M and PV-N respectively) There are moderate numbers of peribronchiolar inflammatory cells and increased interstitial pneumonia and
alveolar inflammatory cells and fibrin; D6 p.i. PR8.
(D) At D9 (nadir weight loss), the lung is consolidated with severe interstitial pneumonia and the terminal bronchiole is multifocally reepithelized with deeply
basophilic and plump regenerative epithelium (scored as bronchiolar hyperplasia; BH); D9 p.i. PR8.
(E) Severe interstitial pneumonia with dense mononuclear perivascular inflammation, sloughed cells and debris within the TB and there is moderate alveolar
hyperplasia (white A) characterized by alveoli lined with cuboidal pneumocytes (scored as alveolar hyperplasia; AH); D13 p.i. PR8.
(F) Moderate peribronchiolar lymphoid aggregate (scored as lymphoid aggregate; LA) with mild perivascular edema and inflammation and relatively normal
terminal bronchioles; D13 X31. Histological lesions scored but not shown in C include alveolar edema (E) characterized by flooding of alveolar spaces with
proteinaceous fluid and hemorrhage (H) characterized by extravascular erythrocytes within airways. All panels original magnification 20 3, bar in A equals
300 mm.
Figure S2. Cytokine and Chemokine Levels Detected in BAL by Multiplex Immunoassay Represented as Line Graphs, Related to Figure 2
y axis represents the amount of protein in pg/ml. Line graphs of selected representative cytokines/chemokines depicting the mean +/ SEM (red: X31sublethal;
blue: PR8sublethal; black: PR8lethal). Statistical significance: * 0.01 < p < 0.05; ** 0.001 < p < 0.01; ***p < 0.001; magenta asterisks denote significance between
PR8lethal and both X31sublethal and PR8sublethal infections; blue and red asterisks denote significance between PR8lethal and PR8sublethal or X31sublethal infections,
respectively; black asterisks denote significance between PR8sublethal and X31sublethal infections).
Figure S3. Percentages of Lipid Mediator Classes and Transcriptomic Analysis in Mouse Influenza Infection, Related to Figure 3
(A) Line graphs of percentages of cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), linoleic acid, linolenic acid, DHA, and EPA derived
metabolites. Also depicted is the line graph of total lipid mediators (without arachidonic acid) as measured in BAL (pmol/ml). Line graphs represent the
mean +/ SEM (red: X31sublethal; blue: PR8sublethal; black: PR8lethal). Mann-Whitney tests were performed to determine statistical significance (* 0.01 < p < 0.05;
** 0.001 < p < 0.01; ***p < 0.001; magenta asterisks denote significance between PR8lethal and both X31sublethal and PR8sublethal infections; blue and red asterisks
denote significance between PR8lethal and PR8sublethal or X31sublethal infections, respectively; black asterisks denote significance between PR8sublethal and
X31sublethal infections).
(B) Heat map represents transcript levels of gene involved in lipid metabolism as measured using Fluidigm RT-PCR. Scale bar represents values normalized
to EF1a.
Figure S4. Clustering Analysis and Percent of Lipid Mediator Classes in Human Clinical Nasal Washes Samples, Related to Figure 4
(A) Hierarchical clustering or clinical symptoms and cytokine/chemokine levels measured in human nasal washes. Three separate groups were identified: low,
medium, and high degree of symptoms and immune responses.
(B) Column vertical scatter plots of the percentages of cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), linoleic acid, linolenic acid, DHA,
and EPA derived metabolites in nasal wash samples, mean +/ SEM. Mann-Whitney tests were performed to determine statistical significance (* 0.01 < p < 0.05;
** 0.001 < p < 0.01; ***p < 0.001).
Figure S5. Pro- and Anti-Inflammatory Indices of Mouse and Human Lipidomic Profiles, Related to Figure 5
(A) XY scatter plots represent the pro- versus anti-inflammatory indices of individual mice infected with PR8sublethal (blue), X31sublethal (red), or PR8lethal (black).
(B) Heatmap represents the fold change of medium or high responders over low responders for each lipid mediator (separated by pro- or anti-inflammatory
activity).
(C) XY scatter plot representing the pro- versus anti-inflammatory indices of individual clinical sample (green: medium responders; red: high responders).
Figure S6. Lipid Mediator Levels within the Lipoxygenase Pathway, Related to Figure 6
Line graphs and column vertical scatter plots of percentages or measured levels of metabolites within the 5-, 15-, 12-, and 8-lipoxygenase subpathways in mouse
(A) and human (B) samples, respectively. Graphs represent the mean with SEM as error bars (in A, red: X31sublethal; blue: PR8sublethal; black: PR8lethal). MannWhitney tests were performed to determine statistical significance (* 0.01 < p < 0.05; ** 0.001 < p < 0.01; ***p < 0.001; magenta asterisks denote significance
between PR8lethal and both X31sublethal and PR8sublethal infections; blue and red asterisks denote significance between PR8lethal and PR8sublethal or X31sublethal
infections, respectively; black asterisks denote significance between PR8sublethal and X31sublethal infections). Graphs represent mean +/ SEM.
Figure S7. Linoleic Acid Metabolites in a Compilation of Three Additional Experiments, Related to Figure 7
(A) Heatmap represents the level of linoleic acid derived metabolites measured in BAL of animals infected with PR8sublethal or X31sublethal. Data represents the
compilation of three additional experiments performed on separate days and on animals obtained from a separate vendor (Jackson Laboratory). Scale bar
represents levels measured in pmol/ml.
(B) Line graphs of 13:9 HODE ratios from these data, mean +/ SEM.

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Lipidomic Profiling of Influenza Infection

Biomedical Research Institute, Seattle, WA 98109, USA

Bioactive lipid mediators play a crucial role in the

oped, negative-sense, single-stranded RNA virus that contains

(legend on next page)

214 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Figure 1. Mouse Influenza Infection Model

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Figure 2. Cytokine and Chemokine Responses in Mouse Influenza Infections

processes. For example, 17-HDoHE is a putative product of the

Figure 3. Lipidomic Profiling of Mouse Influenza Infection

218 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

each time point, the percentage of the pro- or anti-inflammatory

Figure 4. Lipidomic Profiling of Human Nasal Washes

220 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

they were more abundant in PR8sublethal-infected animals on day

X31sublethal infection when compared to PR8sublethal (Figure S6A).

from low to medium to high responders (Figures 6C and S6B).

9-HODE and 13-HODE have previously been proposed as

When we compared the levels of 9-HODE, 13-HODE, and the

X31sublethal than PR8sublethal-infected animals. Hepoxilin has

at significantly higher levels in PR8sublethal infections than in

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. 225

identical conditions, and eicosanoid quantitation was achieved by the stable

Supplemental Information includes Extended Experimental Procedures, seven

226 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Serhan, C.N., Maddox, J.F., Petasis, N.A., Akritopoulou-Zanze, I., Papayianni,

Thomson, S.J., Askari, A., and Bishop-Bailey, D. (2012). Anti-inflammatory

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. 227

S2 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Figure S1. Histological Analysis Score, Related to Figure 1

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. S3

S4 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. S5

S6 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. S7

S8 Cell 154, 213227, July 3, 2013 2013 Elsevier Inc.

Cell 154, 213227, July 3, 2013 2013 Elsevier Inc. S9

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