Dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection

Article (PDF Available)inVirology 435(1):92-101 · January 2013with98 Reads
DOI: 10.1016/j.virol.2012.09.039 · Source: PubMed
Abstract
The cytokine storm is an aggressive immune response characterized by the recruitment of inflammatory leukocytes and exaggerated levels of cytokines and chemokines at the site of infection. Here we review evidence that cytokine storm directly contributes to the morbidity and mortality resulting from influenza virus infection and that sphingosine-1-phosphate (S1P) receptor agonists can abort cytokine storms providing significant protection against pathogenic human influenza viral infections. In experiments using murine models and the human pathogenic 2009 influenza viruses, S1P1 receptor agonist alone reduced deaths from influenza virus by over 80% as compared to lesser protection (50%) offered by the antiviral neuraminidase inhibitor oseltamivir. Optimal protection of 96% was achieved by combined therapy with the S1P1 receptor agonist and oseltamivir. The functional mechanism of S1P receptor agonist(s) action and the predominant role played by pulmonary endothelial cells as amplifiers of cytokine storm during influenza infection are described.
Review
Dissecting influenza virus pathogenesis uncovers a novel chemical approach
to combat the infection
Michael B.A. Oldstone
a,
n
, John R. Teijaro
a
, Kevin B. Walsh
1,a
, Hugh Rosen
b,c
a
Viral-Immunobiology Laboratory, Department of Immunology & Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b
Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
c
Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
article info
Keywords:
Cytokine storm
Influenza
Pulmonary endothelial cells
Sphingosine-1-phosphate (S1P) receptor
(rec) signaling
S1P receptor agonist therapy
abstract
The cytokine storm is an aggressive immune response characterized by the recruitment of inflamma-
tory leukocytes and exaggerated levels of cytokines and chemokines at the site of infection. Here we
review evidence that cytokine storm directly contributes to the morbidity and mortality resulting from
influenza virus infection and that sphingosine-1-phosphate (S1P) receptor agonists can abort cytokine
storms providing significant protection against pathogenic human influenza viral infections. In
experiments using murine models and the human pathogenic 2009 influenza viruses, S1P1 receptor
agonist alone reduced deaths from influenza virus by over 80% as compared to lesser protection (50%)
offered by the antiviral neuraminidase inhibitor oseltamivir. Optimal protection of 96% was achieved by
combined therapy with the S1P1 receptor agonist and oseltamivir. The functional mechanism of S1P
receptor agonist(s) action and the predominant role played by pulmonary endothelial cells as
amplifiers of cytokine storm during influenza infection are described.
& 2012 Elsev ier Inc. All rights reserved.
Contents
Introduction. ..........................................................................................................92
Epidemiologic and experimental evidence for cytokine storm ...................................................................93
Sphingosine-1-phosphate (S1P) properties...................................................................................93
Immune virus-specific T cell trafficking in vivo during influenza virus infection. . ...................................................93
S1P receptor signaling system and effect of S1P receptor agonists in modulating the adoptive immune response and clinical course of influenza
virus infection .....................................................................................................94
Specific S1P1 receptor agonists blunt the exaggerated innate immune host reaction ‘‘cytokine storm’’ by modulating S1P1 s ignaling of
pulmonary endothelial cells ..........................................................................................96
Type I interferon signaling is essential for cytokine/chemokine response of cytokine storm but is independent of inn ate inflammatory cell
recruitment into the lung ............................................................................................99
Crystal structure of S1P1 receptor complexed with S1P1 receptor antagonist .......................................................99
Conclusions and future directions . ........................................................................................99
Acknowledgments .....................................................................................................100
References . . .........................................................................................................100
Introduction
Influenza virus infections were responsible for nearly 100
million human deaths in the last century. Further, during the
two-year period of 1918–1919, influenza caused the greatest loss
of life of any infectious disease or medical condition known
(Ahmed et al., 2007; Johnson and Mueller, 2002). During that
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0042-6822/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.virol.2012.09.039
n
Corresponding author. Fax: þ 1 858 784 9981.
E-mail addresses: mbaobo@scripps.edu (M.B.A. Oldstone),
teijaro@scripps.edu (J.R. Teijaro), walsh.kevin@gene.com (K.B. Walsh),
hrosen@scripps.edu (H. Rosen).
1
Current address: Department of Molecular Biology, Genentech Inc., 1 DNA Way,
South San Francisco, CA 94080, USA
Virology 435 (2013) 92–101
period influenza visited roughly 5% of the world’s population,
killing 2%. Since that most virulent episode, several influenza
pandemics have raged, the most recent being the 2009 attack of
swine flu. The 2009 H1N1 influenza viruses rapidly infected
millions of humans worldwide with an estimated 293,500 deaths
of which 201,200 resulted from respiratory failures and 83,300
cardiovascular insults (Dawood et al., 2012).
Susceptibility or resistance to any viral infection is determined
by a balance between the virulence of the microbe, the resistance
of the host including the aggressiveness of its immune response
against the infecting agent. When the immune response is limited
either because of host genetic or acquired defects, or temporarily
due to lack of normal differentiation of the immune system of
newborns and young children; or decreasing immune responses
of the elderly, the advantage goes to the virus. When infection
occurs in those with a fully developed and competent immune
system, the advantage goes to the host unless the infecting virus
overwhelms the individuals immune system or when the
mechanism that modulates the immune response fails resulting
in an over abundant excessive innate and adaptive immune
response termed ‘‘cytokine storm.’’
Vaccination is employed to protect uninfected reservoirs of
individuals and thereby diminishing the spread of infection.
Antiviral drugs are the primary effective therapy used to diminish
ongoing disease. Antiviral drugs are effective, nevertheless, there
are two compelling limitations to their total efficiency and
effectiveness. First, antiviral drugs exert selective pressure on
the virus, resulting in the generation and selection of more fit
viral progeny that per se become resistant to the drug (Nguyen
et al., 2012; Orozovic et al., 2011; Wang et al., 2010). Second, the
pathogenic injury associated with influenza virus infection results
both from the intrinsic virulence of the virus which is attacked by
anti-influenza viral drug therapy and the intensity of the immune
response (cytokine storm and viral-induced immunopathologic
tissue damage) which antiviral drugs do not engage. Here, using a
small animal model, we review the evidence from our labora-
tories that cytokine storm alone plays an important and essential
role in causing significant tissue injury and mortality following
human pathogenic H1N1 2009 influenza virus infection. We
document that dampening the host’s immune response against
influenza virus using specific immunomodulatory sphingosine-1-
phosphate (S1P) receptor (rec) agonists provide significant pro-
tection from mortality over that observed by the neuraminidase
inhibitor oseltamivir. Further, we demonstrate that specific ago-
nists against S1P1 receptor inhibits innate cellular and cytokine/
chemokine responses that limit virus-induced immunopathologic
injury yet still maintain host control and termination of virus
replication by anti-influenza virus cytotoxic T cells and neutraliz-
ing antibodies. Utilizing genetic, molecular, and chemical tools we
locate S1P1 receptor on pulmonary endothelial cells, identify
endothelial cells as the central regulators of cytokine storm and show
a mandatory role for interferon type I signaling in this process. Due to
space limitations we do not discuss the role played by the virus and
its genes as virulence factors in H1N1 influenza virus infection but
point the reader to several publications in this area (Ahmed et al.,
2007; Chou et al., 2011; Fukuyama and Kawaoka, 2011; Hai et al.,
2010; Song et al., 2011; Watanabe et al., 2012).
Epidemiologic and experimental evidence for cytokine storm
When accompanied by manifestations of cyto kine storm or acute
respiratory distress syndrome, infected individuals display high
mortality with elevated cytokines/chemokines, leukocyte inflamma-
tion and edematous lungs during H1N1 1918–1919 and H1N1
2009 pandemic influenza virus infections in experimental animal
models (Baskin et al., 2009; Kobasa et al., 2007; Marcelin et al., 2011;
Zhang et al., 2012)andfor2009infectioninhumans(Arankalle et al.,
2010; Cheng et al., 2011; Lee et al., 2011). Among the reports of H1N1
2009 infection in humans, that of Arankalle et al. (2010)
is illuminat-
ing. These investigators analyzed viral load in lungs of critically ill
patients who died and those who recovered. Both groups showed
roughly equivale nt titers of virus. In contrast, mortality correlated
directly with cytokine storm. Thus, the patients who died had higher
cytokine/chemokine levels but equivalent viral titers in pulmonary
samples when compared to patients having a milder infection course
and recovering.
In summary, both experimental studies and human clinical
observations suggested to us that: (1) cytokine storm is associated
with poor outcome in influenza virus infection, and (2) calming the
host’s aggressive and exaggerated cytokine storm response might
provide the opportunity to decrease morbidity and enhanced survival
to influenza and likely other acute respiratory diseases like SARS,
Hantavirus infection, and pneumococcal pneumonia that manifest
severe cytokine storm. To test this possibility we turned our attention
to the molecule sphingosine-1-phosphate (S1P) and determined if the
harmful immunologic processes accompanying H1N1 influenza virus
infection could be modulated by S1P receptors in the lung. If so,
influenza could be chemically tractable and successfully treated
pharmacologically with therapy directed against the host’s over-
aggressive immune response. Further, this anti-host immune therapy
would be unlikely to generate viral escape variants that is an issue
with antiviral therapy. In addition, such an approach would also
provide new insights into pathogenesis of influenza viral infections
and may uncover surrogate markers useful for identifying those most
susceptible to influenza virus infection.
Sphingosine-1-phosphate (S1P) properties
S1P is a signaling lipid present at a concentration of 1–3
m
Min
plasma and at roughly 100 nM in lymph. The vast majority of S1P
in plasma is bound to high density lipoprotein, leaving a free
concentration between 15 and 45 nM in blood. The metabolism of
S1P is displayed in Fig. 1A. S1P is generated by phosphorylation of
sphingosine by the actions of two intracellular sphingosine
kinases. S1P is degraded either reversible by dephosphorylation
or irreversible by cleavage (reviewed in Rosen et al., 2009, 2007;
Scott, 2011). Physiologically, S1P levels are under tight homeo-
static control and S1P signals through specific S1P receptors of
which there are five: S1P receptor 1–5. These five specific S1P
receptors are coupled to different G proteins in order to regulate a
variety of downstream signaling pathways that are specific for
many cells, tissues, and organs (Rosen et al., 2009, 2007; Scott,
2011). S1P and its analogs have been used clinically to induce
sequestration of lymphocytes in secondary lymphoid organs
(Fig. 1B) and by that means limit the migration of effector
lymphocytes to areas where such cells might mediate immuno-
pathologic injury leading to diseases (Rosen et al., 2009, 2007;
Scott, 2011). Indeed, S1P agonist therapy is currently being
prescribed for treatment of multiple sclerosis (MS) and being
considered for treatment of other inflammatory disorders.
Immune virus-specific T cell trafficking in vivo during
influenza virus infection
Infiltration of lymphoid cells in pulmonary tissues is one of the
signatures of influenza virus infection. To study the kinetics of
influenza virus-specific CD8 and CD4 T cell entry into the lung
and their anatomic distribution during influenza infection we
designed an in vivo model taking advantage of the wealth of reagents
M.B.A. Oldstone et al. / Virology 435 (2013) 92–101 93
we and others have generated to lymphocytic choriomeningitis virus
(LCMV). First, a recombinant influenza virus A/WSN/33 (H1N1 WSN)
was engineered using reverse genetics by our collaborator, Yoshihiro
Kawaoka, to express the immunodominant H-2D
b
(H-2b MHC class I
background) restricted CD8 T (glycoprotein [GP] aa 33–41) and IA
b
(H-2b MHC class II background) CD4 (GP aa 65–77) T cell epitopes for
LCMV into the influenza neuraminidase (NA) stalk (flu/LCMV)
(Marsolais et al., 2009; Neumann et al., 1999). This maneuver still
allowed NA function and influenza replication (Marsolais et al., 2009).
GP 33–41 and GP 65–77 incorporated into H1N1 influenza, when
expressed in infected lung cells and bound to H-2D
b
or H-2IA
b
,served
as recognition epitopes for lymphocytes from H-2b transgenic mice
created to express the T cells bearing receptors (TCR) specific for
these two LCMV immunodominant epitopes. By crossing such TCR
mice with H-2b transgeni c mice expressing either GFP or RFP under
transcriptional control of beta actin gene, GFP- or RFP-labeled GP 33
CD8 T cells and GP 65 CD4 T cells allows the isolation of 498% of
GFP- or RFP-labeled LCMV-specific T cells (Marsolais et al., 2009;
McGavern et al., 2002). 2.5 10
4
of these GFP or RFP labeled LCMV-
specific CD8 or CD4 T cells were then adoptively transferred into
H-2b (C57Bl/6) naı
¨
ve adult 8-wk-old mice. One day later 1 10
5
PFU
of flu/LCMV recombinant was administered intranasally (Marsolais
et al., 2009). This protocol allows visualization of influenza virus
replication in the lung by use of immunocytochemistry with specific
fluoroprobe-labeled antibody to influenza, in vivo trafficking and
quantitation of influenza virus-specific CD8 and CD4 T cells in the
lung and visualization of virus-specific T cell/influenza virus-infected
cell interaction (Marsolais et al., 2009; McGavern et al., 2002).
Quantitation of number of CD8 and CD4 virus-specific T cells is also
measured by FACS providing a complimentary assay. Quantitative
differences between in vivo labeling of T cells and FACS identification
assays is less than 10% (McGavern et al., 2002).
Both in vivo trafficking studies and FACS analysis 6 days after
flu/LCMV recombinant virus infection, accumulation of GFP virus-
specific CD8 T cells and CD4 T cells occurred. The plateau reached
in trafficking virus-specific T cells occurred between day 6 and 8
post-infection and then dramatically declined at day 10. When
analyzed at day 7 post-infection viral antigen was found through-
out the lung parenchyma. High resolution light microscopy
identified influenza antigens in epithelial cells lining bronchioles
and alveolar cells. Infiltration of virus-specific RFP-GP 33 CD8 T
and GFP-GP 65 CD4 T cells was evaluated at day 7 following
intranasal inoculation of flu/LCMV recombinant. Numbers of
trafficking CD8 T cells into the lung was always greater than the
trafficking of CD4 T cells. The ratio of virus-specific CD8 T cells to
CD4 T cells was 2.6 to 1. In addition, this model also provided the
opportunity to determine if infiltrating virus-specific T cells could
be modulated by chemical probes that would limit the resultant
pulmonary injury mediated by the adoptive immune response.
Since sphingosine-1-phosphate (S1P) receptor agonist have been
reported earlier to sequester lymphocytes in secondary lymphoid
organs and retard their migration to sites of tissue injury (Rosen
et al., 2009, 2007), we turned our attention to beneficial use
of such probes in terms of altering the infiltration of virus-specific
T cells and the outcome of influenza virus infection.
S1P receptor signaling system and effect of S1P receptor agonists
in modulating the adoptive immune response and clinical course
of influenza virus infection
There are five specific S1P receptors that couple to different G
proteins that regulate multiple downstream signaling pathways
(Rosen et al., 2009, 2007). The biologic functions of S1P are
dependent on the cell/tissue location of these receptors and their
relative expression. We began our study for the in vivo influenza
model using a non-selective S1P receptor pro-drug agonist AAL-R
that signals on receptors S1P1, S1P3, S1P4, and S1P5, but not S1P2
(Fig. 2A) following phosphorylation by sphingosine kinase 2. Local
administration of a single 0.1 mg/kg dose of AAL-R by intratra-
cheal (i.t.) administration when given 2 h (data shown, Fig. 2B,
middle row) or 3 to 4 days (data not shown) after intranasal (i.n.)
infection with 1 10
5
PFU of H1N1 WSN/LCMV significantly
down-modulated virus-specific CD8 T cell accumulation in the
lung whereas administration of vehicle alone (Fig. 2B, top row) or
the chiral enantiomer molecule, AAL-S, that cannot be phosphory-
lated efficiently in vivo was unable to restrict T cell infiltration
(Fig. 2B, bottom row) into the lung. This assay was performed by
adoptively transferring a pure population (4 99%) of 5 10
4
GFP-
labeled GP 33 LCMV-specific T cells 24 h before infection, sacrifi-
cing mice 7 days post-infection, isolating T cells from the lung,
gating on GFP and quantitative analysis by FACS. We then
assessed, in other groups of mice treated the same way, the
ability of the remaining virus-specific T cells to generate a
protective anti-influenza viral immune response that controlled
and terminated the infection. Administration of AAL-R, which
significantly retarded numbers of virus-specific T cells entering
Fig. 1. SIP biochemistry and activity in lymphoid organs. Panel A: Synthesis and
regulation of S1P. Panel B: Cartoon of systemic activity of S1P in secondary
lymphoid tissues (see Rosen et al., 2007, 2009 for details). Figure adapted from
Rosen et al. (2009).
M.B.A. Oldstone et al. / Virology 435 (2013) 92–10194
Fig. 2. The non-selective AAL-R S1P receptor agonist retarded influenza virus specific CD8 T cell expansion in influenza virus infected lungs resulting in significant
protection from pulmonary tissue injury and related mortality. Panel A: AAL-R is a non-selective S1P agonist and signals on S1P1, S1P3, S1P4 and S1P5 receptors but not
the S1P2 receptor. Biologic functions of signaling on S1P1, S1P3, S1P4 and S1P5 receptors and their different G protein couplings are displayed. Panel B: AAL-R therapy
significantly decreased numbers of influenza virus specific CD8 T cells in influenza virus infected lungs (middle row) compared to controls given vehicle (top row) or the
chiral enantiomer of AAL-R, AAL-S (bottom row). Panel C: significant protection from influenza virus induced death of C57Bl/6 mice treated with AAL-R (yellow color),
compared to vehicle (magenta) treatment. Significant protection also followed oseltamivir (blue) therapy when compared to vehicle (magenta). Although AAL-R alone
provided significantly greater protection than oseltamivir, the best protection from influenza infection produced by a lethal challenge with non-mouse passed human
H1N1 2009 (shown) or mouse adapted WSN virus (not shown) came from combined AAL-R with oseltamivir (green) therapy. Panel D complements the mortality graft in
Panel C with histopathologic analysis following treatment with vehicle, AAL-R, oseltamivir or AAL-R combined with oseltamivir. The black bar indicates similar
magnification for each tissue sample. Greatest degree of hemorrhage and loss of alveolar air space were from vehicle o oseltamiviro AAL-Ro AAL-Rþ oseltamivir.
Representative tissue samples came from over 10 pulmonary sections from four mice in each group. The graph on the right displays outcomes detected in bronchial
washes (BALF) of influenza virus-infected lung samples after various treatments. Figure adapted from Walsh et al. (2011).
M.B.A. Oldstone et al. / Virology 435 (2013) 92–101 95
influenza virus-infected lungs, did not alter the immune response
sufficiently to alter or raise the viral burden in the lung when
compared to infected mice receiving vehicle or AAL-S. Ten days
after influenza infection AAL-R, AAL-S and vehicle treated mice all
cleared virus from their lungs. Indeed, AAL-R mice sacrificed at
7 days post-influenza virus challenge showed a robust virus-
specific CTL response (
51
Cr release assay) and a vigorous specific
memory T cell response upon virus stimulation occurred at 40
days post-influenza virus challenge. Further, the kinetics, titers,
and immunoglobulin subtypes of neutralizing antibodies in sera
of influenza virus-infected mice treated with AAL-R, AAL-S or
vehicle were equivalent. Taken together these results document
that AAL-R therapy given locally into the respiratory tract down-
modulated the migration of virus-specific CD8 T cells in the lung.
However, neither influenza virus replication nor the generation of
protective neutralizing antibodies was adversely effected. Despite
the reduction in numbers of virus-specific CD8 T cells by AAL-R
activity, influenza viral infection was still controlled.
Associated with the decreased accumulation of virus-specific
T lymphocytes in the lung was enhanced survival of mice
receiving a lethal challenge with H1N1 human pandemic 2009
isolate A/Wisconsin/WSLH3439/09 2 10
5
PFU i.n. (data shown,
Fig. 2C), A/California/04/2009 (data not shown), or WSN mouse
adopted virus (data not shown). Neither human H1N1 viruses
isolated from humans had been passaged before in mice. Thus,
while 6 of 28 mice (21%) receiving only vehicle survived i.n.
infection with 2 10
5
PFU of virus, 23 of 28 mice (82%) receiving
AAL-R were protected (Po 0.001). Susceptibility and death corre-
lated directly with massive lung consolidation, hemorrhage, and
exudate in lungs of mice receiving vehicle or AAL-S. Significantly
less lung pathology occurred in influenza virus infected mice
receiving AAL-R (Fig. 2D, left panel). Tissue histochemistry and
biochemical analysis of pulmonary exudate (Fig. 2) was done 10
days after initiating the influenza infection. Quantitation of lung
exudate was by measuring LDH and total protein in the bronchial
lavage fluid (BALF) (Fig. 2D, right panel).
AAL-R also significantly down-regulated cytokine/chemokine
synthesis in vivo. Upon influenza infection there was significant
up-regulation of IL-1
a
, IL-1
b
, IL-6, IL-10, IL-12, MIP-1, TNF-
a
,
MIP-1
a
, GM-CSF, and RANTES in the BALF. IL-6 and MCP-1 levels
were strikingly enhanced similar to reports in both H5N1 infected
humans (de Jong et al., 2006) and 1918–1919 H1N1 infected
macaques (Kobasa et al., 2007) and mice (Kash et al., 2006).
Treatment with AAL-R significantly inhibited synthesis of IL-1
a
,
IL-1
b
, IL-6, IL-10, MCP-1, TNF-
a
, and GM-CSF in BALF 2 days post-
infection when compared to mice treated with AAL-S or vehicle.
Cytokine/chemokine suppression was associated with a decrease
of GR-1
þ
polymorphic leukocytes and F4/80
þ
macrophages in
the lung which directly correlated with the diminished lung
parenchyma pathology, infiltration, and amount of exudate.
Oseltamivir is a potent anti-influenza drug by its action of
inhibiting the viral neuraminidase. We then tested the therapeutic
potential of an optimal dose of oseltamivir, 5 mg/kg, dissolved in
water administered by gavage for 5 consecutive days starting on
post-infection day 4 alone or in combination with AAL-R to protect
against a lethal challenge of 2 10
5
PFU i.n. of A/Wisconsin/
WSLH34939/09. Vehicle or 0.2 mg/kg of AAL-R was given i.t. 1 h after
virus infection. As shown in Fig. 2C, oseltamivir given alone protected
14 of 28 mice (50%), a result significantly improved over vehicle.
However, AAL-R therapy alone provided significantly greater
protection than that of oseltamivir (82% vs. 50%). Optimal therapy
occurred when both AAL-R and oseltamivir therapy were combined
as 27 of 28 mice (96%) survived the influenza infection (Fig. 2C)
(Walsh et al., 2011). Superior survival observed in treatment of
combined AAL-R and oseltamivir, and of AAL-R over oseltamivir
when administered independently closely mirrored the degree of
histologic evidence of pulmonary injury and degree of exudate in
BALF (Fig. 2D).
The last series in this category investigated the mec-
hanism(s) of how AAL-R impaired the number of virus-specific
CD8 T cells that trafficked to and deposited in the lung parench-
yma. Upon pulmonary infection, influenza virus-specific T cells
are induced and proliferate in mediastinal lymph nodes (MLNs),
then migrate to infected sites in the lung (Baumgarth and Kelso,
1996; Belz et al., 2004). AAL-R significantly reduced the numbers
of virus-specific CD8 and CD4 T cells in MLNs at days 5 and 6
post-infection (Marsolais et al., 2009) and in the lung days 6–8
post-infection indicating that this S1P receptor agonist inhibited
clonal expansion of T cells. Analysis for dead cells (annexin V-
positive virus-specific T cells) in MLNs and lungs indicated that
these cells were not killed by local respiratory tract therapy with
AAL-R. This result suggested that AAL-R likely altered T cell
stimulation by influenza virus antigen presenting dendritic cells
(DC) (Allan et al., 2006) rather than by T cell deletion. Study of
AAL-R effect on DCs found that AAL-R did not reduce the numbers
of DCs or their specific subsets (Marsolais et al., 2009). However,
AAL-R suppressed influenza virus-induced DC activation in the
lungs and MLNs as measured by reduction in surface expression
of MHC-I, MHC-II, and B7.2 molecules on the DC surfaces. AAL-R
treatment also impaired the stimulatory capacity of DCs as
confirmed by inefficient induction of virus-specific CD8 T cell
proliferation in vitro (Marsolais et al., 2009). Thus, AAL-R locally
administered in the respiratory tract during influenza infection
disrupts the antigen-presenting DC network (Steinman and
Hemmi, 2006) by blocking DC-mediated signal transmission from
the infected site to MLNs, leading to a dramatic decrease in T cell
expansion in MLNs and in lungs.
Specific S1P1 receptor agonists blunt the exaggerated innate immune
host reaction ‘‘cytokine storm’’ by modulating S1P1 signaling
of pulmonary endothelial cells
The non-selective agonist AAL-R reacts by modulating S1P1, 3,
4, 5, and not S1P2 receptors (Rosen et al., 2009, 2007). To
determine if a single or multiple receptors were involved in the
initial cytokine storm and the later immunopathologic adoptive
immune response, discovery and optimization of S1P receptor
subtype selective agonists and antagonists were begun by Hugh
Rosen and colleagues. Further, a variety of genetically engineered
S1P1 receptor knock-out and S1P1 receptor eGFP knock-in mice
were utilized that were physiologically and pharmacologically
normal when compared to wild-type controls. Initially, a series of
well-characterized S1P1 selective agonists were administered
i.t. (CYM-5442: 2 mg/kg; RP-002: 3 mg/kg), or orally (RP-002:
6 mg/kg) (Teijaro et al., 2011). All S1P1 selective agonists provide
protection against a lethal i.n. challenge with human H1N1
A/Wisconsin/WSLH34939 or A/California/04/209 (Fig. 3A) and
blunted cytokine storm (Fig. 3B and C) to a degree equivalent to
that observed earlier with non-selective S1P agonist AAL-R
(Fig. 3D and E). The S1P1 receptor agonists significantly inhibited
secretion of cytokines and chemokines associated with influenza
infection including IFN-
a
,CCL-2,IL-6,TNF-
a
, CCL-3, CCL-5, CXCL-2,
IL-1
a
,andIFN-
g
. While most of these cytokines/chemo kines were
inhibited to a similar degree as with AAL-R therapy, suppression of
CXCL-2, TNF-
a
, and IFN-
g
was not as effective, suggesting,
perhaps, a role for other S1P receptor subtypes in modulating
these cytokine/chemokines. In addition, the S1P1 selective ago-
nists significantly blunted the accumulation of innate infiltration
of macrophages/monocytes (CD11b
þ
, F480
þ
, LyG6
), neutrophils
(CD11b
þ
, LyG6
þ
, F480
), and natural killer (NK) cells (NK1.1
þ
,
CD3
). Further, the expression of the activation marker CD69 was
significantly reduced following S1P1 agonist treatment. As with
M.B.A. Oldstone et al. / Virology 435 (2013) 92–10196
earlier results with selective S1P receptor agonist AAL-R, there
was no increase in viral titers following chemical treatment.
Further, viral infection was effectively terminated and both
humoral (antibody) and cell-mediated (CD8 T cell) arms of the
immune response were generated during S1P1rec agonist ther-
apy. Since cytokine storm, pathologic injury to the lung parench-
yma, and survival of influenza virus infection were all achieved
with S1P1 agonist therapy, our results implicated S1P1 receptor
signaling as the essential player in the initiation of cytokine storm
and resultant immune-mediated injury. Importantly, our results
also indicated that a severe pulmonary disease associated with
cytokine storm was chemically tractable with a single chemical
molecule, S1P1 agonist that avoided signaling through S1P2, 3, 4,
5 receptors.
Having identified S1P1rec signaling as the primary pathway
for the initiation of cytokine storm we sought to identify the cell
or cells in the lung that expressed the S1P1 receptor. Since
epithelial cells are the primary cell infected by influenza virus
we suspected that S1P1 receptor was located on that cell. To
determine the pulmonary cell(s) bearing the S1P1 receptor, we
took advantage of eGFP-S1P1 receptor knock-in mice made by
Stuart Cahalan in the Rosen laboratory (Cahalan et al., 2011). In
this mouse, the native S1P1 receptor was homologously replaced
with a functional fused eGFP-tagged S1P1 receptor (Cahalan et al.,
2011). Utilizing this mouse model we could directly detect eGFP-
S1P1 receptor protein expression of pulmonary cells which we could
then identify by antibodies to specific pulmonary cell markers and
flow cytometry. Additional conformation was achieved by biochem-
ical analysis of purified pulmonary cells. High levels of S1P1 -eGFP
receptor expression was found on lung lymphatic (CD45
,CD31
þ
,
GP38
þ
) and vascular (CD45
,CD31
þ
,GP38
) endothelial cells but
surprisingly was absent on pulmonary epithelial cells (CD45
,
CD31
,EpCAM
þ
)(Fig. 4A, top panel). These results were confirmed
by doing Western blots on 498.5% pure populations of pulmonary
endothelial and epithelial cells (Fig. 4C). As expected and previously
reported (19,20), CD4 T cells (CD4
þ
,CD3
þ
), CD8 T cells (CD8
þ
,
CD3
þ
), and B cells (B200
þ
,CD19
þ
) also expressed S1P1-eGFP
receptor (Fig. 4A). In contrast, pulmonary leukocytes, including
macrophages/monocytes (CD11c
þ
, CD11b
, F480
þ
), DCs (CD11c
þ
,
IA-IE
þ
,CD205
þ
,F480
), neutrophils, NK cells (NK1.1
þ
,CD3
)
(Fig. 4B), and immature lymphoid cells (LIN
,SCA-1
þ
)failedto
express significant levels of eGFP-S1P1 receptor protein. S1P1-eGFP
receptor expression was similar whether cells were harvested from
mice that were uninfected or infected with influenza virus. Other
experiments in infected mice (Fig. 4C) indicated that S1P1-eGFP
receptor expression is not altered during influenza virus infection.
Importantly, S1P1 agonist treatment of infected eGFP-S1P1 receptor
knock-in mice did not lessen expression of S1P1-eGFP receptor
indicating that administration of specific S1P1 agonist does not
degrade the endothelial S1P1 receptor. These results indicated that
functional agonism of S1P1 and not antagonism effect of receptor
degradation is the mechanism by which S1P1 receptor blocking
molecules CYM-5442 and RP-002 suppressed cytokine storm.
T and B lymphocytes as well as pulmonary endothelial cells
are the only ones within the lung that express measurable
amounts of S1P1-eGFP protein (Fig. 4). We therefore determined
whether lymphocytes expressing S1P1 receptor were involved in
S1P1 agonist inhibition of cytokine storm or were merely bystan-
der cells accompanying the innate immune response to influenza
virus infection. Rag2
/
mice are deficient in lymphocytes and
we reasoned that if such influenza virus infected Rag2
/
mice
generated a cytokine storm that could be blocked by S1P1 agonist
then lymphocytes did not contribute to initiation of cytokine
storm and could be excluded as key regulators of influenza
Fig. 3. Severe influenza respiratory disease is blunted by S1P1 receptor agonist. Protection by S1P1 specific receptor agonist is comparable to protection provided by AAL-R
S1P1, S1P3, S1P4, S1P5 receptor agonist. Panels A, B, C: Significant protection and ablation of cytokine storm by S1P1 receptor agonist RP-002. Panels D and E: Significant
protection by S1P1 receptor agonist CYM-5442 and its comparison to the protection provided by AAL-R agonist to S1P1, S1P3, S1P4 and S1P5 receptor signaling. Figure
adapted from Teijaro et al. (2011), with permission from Elsevier.
M.B.A. Oldstone et al. / Virology 435 (2013) 92–101 97
virus-induced cytokine storm. Fig. 4D documents that cytokine
storm occurred in Rag2
/
mice infected with influenza virus and
that treatment with S1P1 agonist CYM-5442 significantly reduced
cytokines/chemokines (shown IFN-
a
, CCL-2, IL-6, TNF-
a
, IFN-
g
)
and innate infiltration of macrophages/monocytes and NK
cells. A further series of experiments conclusively documented
Fig. 5. Interferon
a
production is essential to yield the cytokine/chemokine component of cytokine storm. Regulation of innate inflammatory cell recruitment into the lung
is also mediated by endothelial cells but is independent of type 1 interferon signaling. Eliminating interferon
a
occurred by use of influenza virus infected interferon
a
b
receptor knock-out mice. Panel A shows that chemokines/cytokines were significantly reduced when interferon
a
b
sufficient or interferon
a
b
deficient mice were
treated with S1P1 receptor agonist. In contrast, inflammatory cell infiltration of macrophages/monocytes, neutrophils and NK cells following S1P1 receptor agonist therapy
were down-regulated only in interferon
a
b
sufficient mice but not their interferon
a
b
receptor knock-out counterparts. Figure adapted from Teijaro et al. (2011), with
permission from Elsevier.
Fig. 4. S1P1 receptor is present on pulmonary endothelial cells but not on pulmonary epithelial cells. Panel A identifies S1P1 receptor primarily on pulmonary endothelial
cells, and modestly on CD4 T cells as well as CD8 T cells but not on pulmonary epithelial cells. Panel B displays absence of S1P1 receptor on pulmonary macrophages,
monocytes, dendritic cells, neutrophils or NK cells. Data represented in Panels A and B used eGFP-S1P1 receptor knock in mice, antibodies to specific markers of various cell
populations and FACS. Cell populations were 4 98.5% pure. Panel C: Western blotting shows S1P1 receptor primarily on pulmonary endothelial cells but none on
pulmonary epithelial cells. Panel D: Rag2
/
knock-out mice do not contain T cells. S1P1 specific agonist C
g
M5442 blocked cytokine storm (chemokines/cytokines in
bronchial wash: left panel; and migration of innate inflammatory cells: right panel) indicating that the S1P1 receptor agonist acted on pulmonary endothelial cells, not
lymphoid cells to dampen the cytokine storm. Figure adapted from Teijaro et al. (2011), with permission from Elsevier.
M.B.A. Oldstone et al. / Virology 435 (2013) 92–10198
(Teijaro et al., 2011) that cytokine/chemokine production and innate
inflammatory cell recruitment were independent events that
occurred after influenza virus infection. However, both cytokine/
chemokine production and innate inflammatory cell recruitment
were inhibited by S1P1 receptor agonism of pulmonary endothelial
cells. An important observation is that initial inflammatory cell
infiltration into the lungs was not dependent or required for
cytokine/chemokine production (Teijaro et al., 2011).
Type I interferon signaling is essential for cytokine/chemokine
response of cytokine storm but is independent of innate inflammatory
cell recruitment into the lung
Our observations with influenza and those of others have
repeatedly documented that type I interferon, predominantly
the IFN-
a
species, are elevated early after acute respiratory viral
infection. The release and action of type I interferon is believed
crucial for the production of proinflammatory cytokines/chemo-
kines. We found that S1P1 receptor agonists inhibited the pro-
duction of IFN-
a
in the pulmonary parenchyma early after
influenza infection (Figs. 3 and 4)(Teijaro et al., 2011). To prove
that blunting of IFN-
a
production was a mechanism by which
S1P1 receptor agonist inhibited cytokine storm, IFN
a
-
b
rec knock-
out and IFN
a
-
b
rec sufficient mice infected with 1 10
5
PFU i.n. of
H1N1 virus were treated with S1P1 receptor agonist CYM-5442 or
vehicle and both cytokine/chemokine proteins and innate inflam-
matory cell recruitment measured in the bronchial lavage fluid at
48 h post-infection. Cytokines/chemokines IFN-
a
, CCL-2, IL-6,
IFN-
g
(shown), CCL-5, and CXCL-10 in the bronchial lavage fluid
were significantly reduced in IFN
a
-
b
rec sufficient and knock-out
mice (Fig. 5A) but inflammatory cell infiltration of macrophages/
monocytes, neutrophils and NK cells following S1P1 receptor
agonist therapy were only down-regulated in IFN
a
-
b
sufficient
and not IFN
a
-
b
knock-out mice (Fig. 5B). Thus, regulation of
innate inflammatory cell recruitment into lung was mediated by
endothelial cells and was independent of type I interferon
signaling. Cytokine/chemokine production in the lung was also
mediated by endothelial cells but, in contrast, S1P1 receptor
agonism of endothelial cells inhibited IFN-
a
production leading
to dampening of production of inflammatory cytokine/chemokine
responses.
Crystal structure of S1P1 receptor complexed with S1P1
receptor antagonist
Recently, Hugh Rosen and Ray Stevens’ laboratories solved the
crystal structure of the lyso-phospholipid sphingosine-1-
phosphate-1 receptor fused to T4 lysozyme in complex with an
antagonist sphingolipid analog ML-056 (W-146) (Hanson et al.,
2012)(Fig. 6). Unique features of the S1P1 receptor were not
predictable from the extensive and rigorous analysis by mutagen-
esis previously performed (Fujiwara et al., 2007, 2005; Inagaki
et al., 2005; Jo et al., 2005; Parrill et al., 2000a, 2000b; Schurer
et al., 2008; Wang et al., 2001). The extracellular face of S1P1
receptor is tightly structured (Hanson et al., 2012) where the
N-terminus a-helix folds over the top of the receptor to block
access to the binding pocket from the aqueous phase. This static
view of the receptor suggests that S1P might require an alternate
route into the binding pocket.
Shown in Fig. 6 is the alpha-carbon tracing of S1P1 receptor at
2.8
˚
A resolution. The orthosteric antagonist ML-056 is fully
visualized within the binding pocket immediately above the
rhodopsin GPCR family conserved W269, which serves as a
rotamer toggle switch in rhodopsin, but in S1P1 serves as the
binding point of contact for alternate binding mode agonists like
CYM-5442. By virtue of these unique interactions with the
receptor, these molecules no longer require the zwitterionic
amino-phosphate headgroup and thus have enhanced physico-
chemical properties that allow delivery to the lung for the
influenza studies. The crystal structure provides the detailed
insight into molecular interaction that may rationally enhance
the properties of S1P1 agonists for the relief of cytokine storm.
Conclusions and future directions
Conclusions reached indicate the following four major points
displayed in tabular form at:
1. Cytokine storm plays an essential role in the pathogenesis and
clinical outcome of influenza virus infection.
i. Blockade of cytokine storm provides greater protection than
antiviral therapy to inhibit virus replication and does so
without compromising the host’s ability to control and termi-
nate influenza virus infection.
ii. Observations made with human pathogenic H1N1 Swine 09
influenza virus isolates and mouse adapted H1N1 influenza
virus.
2. Sphingosine-1-phosphate (S1P) receptor agonists blunts
cytokine storm thus cytokine storm is chemical tractable.
i. Blunting of cytokine storm is mediated by just one of the five
S1P receptors: S1P1.
3. Molecular mechanism: S1P1 receptor signaling occurs on
pulmonary endothelial cells and not influenza virus infected
epithelial cells.
i. S1P1 receptor is located on pulmonary endothelial cells and
not on pulmonary epithelial cells.
ii. S1P1 agonism suppresses cytokine and innate immune cell
recruitment.
4. Immune cell infiltration and cytokine production are distinct
events both orchestrated by pulmonary endothelial cells.
Fig. 6. The alpha-carbon tracing of the S1P1 receptor at 2.8
˚
A resolution. The
orthosteric antagonist ML-056 is fully visualized within the binding pocket
immediately above the rhodopsin GPCR family conserved W269, that serves as a
rotamer toggle switch in rhodopsin, but in S1P1 serves as the binding point of
contact for alternative binding mode agonists like CYM-5442. Extracellular loops
(ECL) 1, 2 and 3 are shown. There are 7
TM
domains and N-term, I, 1st ICL, II, 1st ECL,
III, 2nd ICL, IV, 2 ECL, V, 3rd ICL, VI, 3rd ECL, VII, C-term are shown. V and VI are not
marked in this pose. See Figs. 3–5 for biologic action of the S1P1 agonist CYM-
5442 and Hanson et al. (2012), for additional structural details and amino acid
alignments. Figure adapted from Hanson et al. (2012), reprinted with permission
from AAAS.
M.B.A. Oldstone et al. / Virology 435 (2013) 92–101 99
i. Proinflammatory cytokine responses depend on type I inter-
feron signaling.
A kinetic outline of events of molecular pathogenesis of
influenza virus infection in the lung leading to cytokine storm
and its resultant morbidity and mortality is sketched as given
below.
Cytokine storm has an early and late stage. The early stage is
reflective of a pulmonary endothelial amplification network
taking place within the first few days of infection. This phase is
mediated by S1P1 signaling on pulmonary endothelial cells. The
second phase of immunopathologic injury and activity occurs
later during influenza virus infection, days 6–8 post-infection, is
T cell-mediated and signals likely through S1P3, 4, 5rec signaling,
with S1P3rec and S1P4rec the most likely culprits.
Step 1: Influenza virus enters the upper respiratory tract and
infects primary epithelial cells in the lung parenchyma.
Step 2: Infected epithelial cells produce and release a signaling
molecule(s), not yet defined, that cross-talk with and activate
endothelial cells.
Step 3: Pulmonary endothelial cells augment influenza virus-
induced cytokine storm by two distinct mechanisms. First,
such non-influenza virus-infected endothelial cells release a
currently unidentified molecule(s) that activate primarily
plasmacytoid
2
DCs to produce IFN-
a
.
Step 4: IFN-
a
production stimulates the expression of proin-
flammatory molecules leading to the initiation of cytokine
storm.
Step 5: The second mechanism by which pulmonary endothe-
lial cells augment influenza virus-induced cytokine storm is by
attracting the recruitment of innate inflammatory cells into
the lung. Such innate inflammation infiltrates exacerbate
cytokine storm by producing additional proinflammatory
molecules.
Step 6: The late stage occurs by day 6–8. Influenza virus-
specific T cells activated and expanded in MLN and in pul-
monary tissues produce additional inflammatory molecules,
lyse virus-infected epithelial cells and thereby augmenting
cytokine storm and immune-mediated injury.
Future directions include investigating IFN-I as to its cellular
source, species and signaling pathways while dissecting molecu-
lar cross-talk and signaling between pulmonary endothelial cells
and other pulmonary cells, especially influenza virus infected
epithelial cells and plasmacytoid dendritic cells.
Expansion and generalities of our findings for other acute
respiratory infections, infectious and autoimmune disorders in
which cytokine storm is a major component. Indeed, preliminary
investigations with Matthew Friedman at the University of Mary-
land Medical School suggest a similar scenario for SARS. Studies
by Kevin Walsh from our laboratory recently found that S1P
agonist successfully aborts pneumonia virus of mice, a murine
model of human respiratory syncytial virus (RSV) disease.
3
Additionally, in collaboration with the NIH and Battelle group,
and with Yoshi Kawaoka, we have been studying human patho-
genic H1N1 2009 virus in ferrets, a host whose infection with
influenza virus is more akin to humans than the mouse. Samples
from infected ferrets not treated; or treated with S1P agonists
only, oseltamivir alone, or S1P agonist combined with oseltamivir
are currently under analysis. Lastly, it is likely that genetic
aberrations in the S1P pathway being uncovered may prove to
be of use for screening and identifying those humans who would
be most susceptible to severe cytokine storm during an influenza
viral infection. We are currently exploring such genetic profiling.
Acknowledgments
Parts of the work were done with the active collaboration and
consultation with Yoshihiro Kawaoka, Stuart Cahalan, David
Marsolais, Ray Stevens, Fiona Scott, Edward Roberts, and Robert
Peach. The research was supported by NIH Grants AI074564
(MBAO, HR), AI009484 (MBAO), AI005509 (HR), MH084512
(HR), and NIH Training Grants NS041219 and AI007244
(K. Walsh), NIH Training Grant AI007364 and American Heart
Association Fellowship 11POST7430106 (J. Teijaro). We thank
Marcus Boehm, Liming Huang, and Bryan Clemons (Receptos,
Inc.) for helping provide RP-002 as a chemical tool. HR is a
founder of Receptos, Inc.
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    • "As to whether this is relevant in the course of the Ebola virus, especially following increased gut permeability and the induction of sepsis following the crossing of gut gram-negative bacteria to the circulation is unknown. However, recent data showing the efficacy of S1P1r agonists in the treatment of a number of viruses [Oldstone et al., 2013], suggests that a relative increase in S1P1r activation may play some role, including in the regulation of gut and endothelial cell permeability [Singleton et al., 2009] and the subsequent effects of emergent sepsis. S1P is a significant regulator of many aspects of the immune response, including the activation of dendritic cells and levels of pro-inflammatory cytokine produced [Arlt et al., 2014]. "
    [Show abstract] [Hide abstract] ABSTRACT: There is currently an urgent need for a viable, cheap, and readily available treatment for the Ebola virus outbreak in West Africa. Here, it is proposed that melatonin may have significant utility in helping the management of this outbreak. Optimizing natural killer (NK) cell responses seems crucial to surviving Ebola virus infection. Melatonin increases NK cell cytotoxicity significantly, suggesting efficacy in managing the Ebola virus. Under conditions of challenge, melatonin increases heme oxygenase-1 (HO-1), which inhibits Ebola virus replication. Melatonin also has protective effects in cases of septic shock, which, although bacterial, has similar end-point presentations involving blood vessel leakage. Melatonin's effects on haemorrhage are mediated primarily by a decrease in pro-inflammatory cytokines. By optimizing the appropriate immune response, melatonin is likely to afford protection to those at high risk of Ebola viral infection, as well as having direct impacts on the course of infection per se. Although no direct data pertain to the utility of melatonin in the management of the Ebola virus, convergent bodies of data suggest its utility, which is reviewed in this article. J. Med. Virol. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2015
    • "As to whether this is relevant in the course of the Ebola virus, especially following increased gut permeability and the induction of sepsis following the crossing of gut gram-negative bacteria to the circulation is unknown. However, recent data showing the efficacy of S1P1r agonists in the treatment of a number of viruses [Oldstone et al., 2013], suggests that a relative increase in S1P1r activation may play some role, including in the regulation of gut and endothelial cell permeability [Singleton et al., 2009] and the subsequent effects of emergent sepsis. S1P is a significant regulator of many aspects of the immune response, including the activation of dendritic cells and levels of pro-inflammatory cytokine produced [Arlt et al., 2014]. "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract There is currently an urgent need for a viable, cheap and readily available treatment for the Ebola virus outbreak in West Africa. Here we propose that melatonin may have significant utility in helping the management of this outbreak. Optimizing natural killer (NK) cell responses seems crucial to surviving the Ebola virus. Melatonin significantly increases NK cell cytotoxicity, suggesting efficacy in managing the Ebola virus. Under conditions of challenge, melatonin increases heme oxygenase-1 (HO-1), which inhibits Ebola virus replication. Melatonin also has protective effects in cases of septic shock, which, although bacterial, has similar endpoint presentations involving blood vessel leakage. Melatonin's effects on haemorrhage are primarily mediated by a decrease in pro-inflammatory cytokines. By optimizing the appropriate immune response, melatonin is likely to afford protection to those at high risk of Ebola viral infection, as well as having direct impacts on the course of infection per se. Although no direct data pertain to the utility of melatonin in the management of the Ebola virus, convergent bodies of data suggest its utility, which we review in this article.
    Full-text · Article · Jan 2015
    • "Entry process is attractive as targets to block infection efficiently as it is the first essential step for virus replication. The acute nature of influenza virus infection and the accompanying inflammatory disease also make an intervention strategy by blocking the early viral entry process particularly favorable [35]. This is consistent with our previous study that the Cryptoporus volvatus extract blocks PRRSV entry into cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.
    Full-text · Article · Dec 2014
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