MAJOR ARTICLE

A Paradoxical Role for Neutrophils

in the Pathogenesis of West Nile Virus
Fengwei Bai,1,4,a Kok-Fai Kong,2,a,b Jianfeng Dai,1 Feng Qian,2 Lin Zhang,2 Charles R. Brown,5 Erol Fikrig,1,3
and Ruth R. Montgomery2
Sections of 1Infectious Diseases and 2Rheumatology, Department of Internal Medicine, and 3The Howard Hughes Medical Institute,
Yale University School of Medicine, and 4L2 Diagnostics, New Haven, Connecticut; and 5Departments of Molecular Microbiology
and Immunology and Veterinary Pathobiology, University of Missouri, Columbia

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Polymorphonuclear leukocytes (PMNs) are key in innate immunity, but their role in viral pathogenesis is
incompletely understood. In infection due to West Nile virus (WNV), we found that expression of 2 PMN-
attracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMNs are
rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted
of PMNs after WNV inoculation developed higher viremia and experienced earlier death, compared with the
control group, which suggest a protective role for PMNs. In contrast, when PMNs were depleted prior to
infection with WNV, and in mice deficient in Cxcr2 (a chemokine receptor gene), viremia was reduced and
survival was enhanced. Collectively, these data suggest that PMNs have a biphasic response to WNV infection,
serving as a reservoir for replication and dissemination in early infection and later contributing to viral
clearance.

West Nile virus (WNV), a mosquito-transmitted single-
stranded RNA (ssRNA) virus, belongs to the family
Flaviviridae, which includes other mosquito-borne hu-
man pathogenic viruses, such as Yellow fever and Den-
gue viruses [1]. WNV has spread rapidly throughout
North America since its first introduction into New
York in 1999 and has caused worldwide morbidity and
mortality. Although infection in humans is typically
asymptomatic, the elderly and immunosuppressed pop-
ulations are particularly susceptible to life-threatening
neurologic disease, including meningitis and enceph-
Received 9 April 2010; accepted 20 July 2010; electronically published 4
November 2010.
a
F.B. and K.-F.K. contributed equally to this article.
b
Present affiliation: Division of Cell Biology, La Jolla Institute for Allergy and
Immunology, La Jolla, California.
Potential conflicts of interest: none reported.
Financial support: National Institutes of Health (AI 50031, AI 070343, AI 079348,
and AI 059292) and The Howard Hughes Medical Institute. FB was supported by
a career development grant (U54-AI057158-Lipkin to F.B.).
Reprints or correspondence: Dr Ruth R. Montgomery, Dept of Internal Medicine,
Yale University School of Medicine, 300 Cedar St/TAC S413, New Haven, CT
06520-8031 (ruth.montgomery@yale.edu).
The Journal of Infectious Diseases 2010; 202(12):1804–1812
 2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20212-0007$15.00
DOI: 10.1086/657416
alitis [1]. An approved therapy for use in humans does
not currently exist, and viral pathogenesis is incom-
pletely understood.
The immune response to WNV is multifactorial and
includes both innate and adaptive immunity. Upon
WNV infection, host innate immune sensors, including
Toll-like receptors, double-stranded RNA-activated
protein kinase, retinoic-acid-inducible protein I, and
melanoma-differentiation-associated gene 5 (MDA-5),
recognize WNV components and initiate anti-viral sig-
naling and production of type I interferon (IFN) and
cytokines [2–4]. Cellular immunity is critical for host
recovery from WNV infection, including contributions
of CD4+, CD8+, and gd T cell responses, as well as
antibody production by B cells [5–8]. The role of nat-
ural killer cells remains unclear [9, 10]. Macrophages
are essential in resistance to WNV infection: macro-
phage depleted mice show accelerated development of
WNV encephalitis and a 50% increase in mortality [11].
Polymorphonuclear leukocytes (PMNs; neutrophils)
are the most abundant type of leukocytes in humans,
a key component of the innate immune response, and
the first immune cells to be recruited to inflammatory
foci [12]. PMNs ingest pathogens, release antimicrobial

1804 • JID 2010:202 (15 December) • Bai et al

peptides, and produce reactive oxygen species and extracellular
traps to combat pathogens [13, 14]. Depletion of PMNs or
mutations in key PMN antimicrobial pathways increases sus-
ceptibility to numerous pathogens [15]. PMNs also contribute
to inflammation in a number of disease processes, including a
complex program of apoptosis [16] and an abundant zinc-
chelating bacteriostatic cytoplasmic protein, calprotectin
(S100A8/A9) [17, 18]. PMNs predominate in the cerebrospinal
fluid (CSF) of patients with neuroinvasive WNV meningitis
and encephalitis [19, 20] and are recruited into the brain as
early as day 4 after infection in the rodent model of WNV
infection [21]. We have undertaken the current study to elu-
cidate the role of PMNs in WNV pathogenesis.
MATERIALS AND METHODS
WNV infections and experimental animals. WNV isolate
2471 [22], used in these studies, was propagated 1 time in Vero
cells and titered in a Vero cell plaque-formation assay. All ex-
periments with WNV were performed in a Bio-safety Level 3
animal facility, according to the regulations of Yale University.
C3H/HeJ mice were purchased from the Charles River (Wil-
mington, MA). Cxcr2⫺/⫺ mice were fully backcrossed to C3H/
HeJ background [23]. All infections were performed on age-
and sex-matched 7–8-week-old mice and protocols were ap-
proved by the Yale University Institutional Animal Care and
Use Committee. C3H wild-type and Cxcr2⫺/⫺ mice were in-
oculated intraperitoneally with 100 mL of phosphate-buffered
saline (PBS) with 1% gelatin or WNV (100–10,000 plaque
forming units [pfu], as indicated) in PBS with 1% gelatin [24].
Peritoneal exudates were collected by lavage with 10 mL of cold
PBS. For footpad inoculation, animals were infected with 10
pfu of WNV in 50 mL of PBS containing 1% heat-inactivated
fetal bovine serum (FBS). Animals were monitored twice daily
for morbidity and mortality for up to 21 days after infection
[24].
Preparation of PMNs and macrophages. Mouse peritoneal
PMNs and macrophages were elicited by intraperitoneal injec-
tion of 1 mL of thioglycollate medium (BD BBL). PMN were
collected 6 h after injection, and macrophages were collected
at 3 days after injection [24, 25]. PMNs were isolated by 3%
dextran sedimentation and used for experiments immediately.
Macrophages were cultured overnight at 1 ⫻ 10 6 cells per 35
mm2 dish in 2 mL Dulbecco’s modified eagle medium (DMEM)
containing 10% FBS and 1000 U/mL penicillin and 1 mg/mL
streptomycin (Invitrogen). Nonadherent cells were washed
away prior to infection for 4 h with WNV (multiplicity of
infection [MOI] p 1). Leukocyte populations were isolated
from blood using anti–Gr-1+–conjugated magnetic microbeads
(Miltenyi Biotec).
Heparinized blood from healthy human volunteer donors
was collected in accordance with the regulations of Human
Investigation Committee of Yale University. PMNs were isolated
from blood by 3% dextran sedimentation followed by hypo-
tonic lysis to remove red blood cells [26]. Peripheral blood
mononuclear cells (PBMCs) were separated by Ficoll-Hypaque
density gradient centrifugation and plated at 5 ⫻ 10 6 per 35 mm2
well in Roswell Park Memorial Institute 1640 medium con-
taining 20% human serum (Cambrex) and antibiotics, as above.
PBMCs were washed after 2 h and incubated for 6–8 days to
obtain mature primary macrophages with 99% viability [26].
Fluorescence-activated cell scanning (FACS) analy-
sis. Peritoneal exudate cells were washed twice in PBS con-
taining 1% FBS and stained for 30 min at 4C with antibodies
for PMNs (anti–granulocyte receptor 1 [Gr-1]), macrophages
(F4/80), T cells (CD4+ and CD8+) and B cells (CD19+) (BD
Biosciences). Cells were fixed with 4% paraformaldehyde. FACS
data were collected using an LSR II Flow cytometer (BD Bi-
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oscience) and were analyzed using FlowJo software (Tree Star)
[27].
Assessment of Chemokines by quantitative polymerase
chain reaction (Q-PCR) array and enzyme-linked immuno-
sorbent assay (ELISA). Macrophage cultures were washed
with PBS prior to total RNA extraction by Qiagen RNeasy Mini
kit (Valencia) and assayed using RT2Profiler PCR Array kits
(SuperArray Bioscience) according to the user manual. Total
RNA from macrophages (1 mg) was transcribed into first strand
complementary DNA (cDNA) and processed in 96-well PCR
array plates with 25 mL of Q-PCR master mix per well. Thresh-
old cycle values (Ct) were analyzed to compare gene expression
between mock- and WNV-infected macrophages.
Culture supernatants from macrophages were harvested and
chemokines were quantified in duplicate by ELISA according
to the manufacturer’s instructions (R&D Systems).
WNVE and chemokine gene Q-PCR. Total RNA was ex-
tracted from macrophages, PMNs, and blood using the RNeasy
kit (Qiagen) and was used to synthesize cDNA by the
AffinityScript Multi Temperature cDNA synthesis kit (Strata-
gene). WNV-specific RNA was quantified by Q-PCR [24].
TaqMan gene expression assays for Cxcl1, Cxcl2, and Cxcl10
were purchased from Applied Biosystems. For differential RNA
Q-PCR of the WNVE region, primer for positive-sense cDNA
was 5’-TGGGTCAGCACGTTTGTCATTGTG-3’; primer for
negative-sense cDNA was 5’-TTGGAAGGAGTGTCTGGAGC-
AACA-3’. Values for each gene were calculated from the ac-
companying standard curve in each Q-PCR reaction plate, and
copy numbers were expressed as a ratio to cellular b-actin
cDNA copies measured by Q-PCR. Each duplicate measure-
ment was divided by the corresponding measurement for actin
and then averaged to determine the relative levels in each
sample.
PMN depletion in vivo. Circulating PMNs in mice were
depleted using mouse monoclonal antibodies Gr-1 (clone RB6–
PMN in WNV Infection • JID 2010:202 (15 December) • 1805
8C5) [28] or Ly-6G [29]. Gr-1 was obtained from the Brown
laboratory and as a kind gift from Robert L. Coffman (DNAX
Research Institute). Ly-6G (clone 1A8) and isotype-matched
control antibodies were purchased from BD Biosciences. RB6–
8C5 cells were cultured in DMEM with 10% FBS, 10 mM
Hepes, 2 mM L-glutamine, and antibiotics. RB6–8C5 antibody
was affinity purified from cell culture supernatants using a pro-
tein G column (Pharmacia). Seven-week-old female C3H mice
were injected intraperitoneally either with 250 mg of RB6–8C5
or isotype matched IgG2b (Clone MOPC-141; Sigma-Aldrich)
antibodies or with 100 mg Ly-6G and isotype matched IgG2a
in 100 mL of PBS 1 day prior or 1–2 days after infection with
WNV.
Statistical analysis. We calculated standard errors of the
means (SEMs) and analyzed data by nonpaired Student’s t test
for single mean comparisons and one-way analysis of variance
followed by Tukey’s test for assessment of significance in FACS
assays. We performed survival curve comparisons using the
Kaplan-Meier survival and log-rank test (GraphPad Prism soft-
ware, version 4.0). Statistical significance was set at P ! .05 for
all analyses.
RESULTS
Expression of chemokines Cxcl1 and Cxcl2 is rapidly elevated
in macrophages upon WNV infection. To investigate the role
of chemokines in WNV pathogenesis, we infected thioglycol-
late-elicited peritoneal macrophages from C3H mice with WNV
(MOI p 1) and analyzed chemokine and chemokine receptor
gene expression profiles by Q-PCR arrays. Expression of Cxcl1
and Cxcl2, 2 CXC-type chemokines that induce the migration
of PMNs, was dramatically up-regulated by 38-fold over un-

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Figure 1. Cxcl1 and Cxcl2 expression is elevated in vitro at early stage of West Nile virus (WNV) infection (A–G). Murine thioglycollate-elicited
peritoneal macrophages from strain C3H mice were infected with WNV (multiplicity of infection [MOI] of 0, 0.1, 0.5 or 1). A, Macrophages were
infected with WNV (MOI p 1) for 4 h, and total RNA was extracted for chemokine quantitative PCR (Q-PCR) array analysis. The data are normalized
to mouse b-actin messenger RNS (mRNA) and are expressed as the relative fold increase over normalized RNA from mock controls (n p 3 ). B and
C, Cxcl1 mRNA was measured by Q-PCR and normalized to mouse b-actin (Actb; unitless ratio  1 standard error of the mean [SEM]) and enzyme-
linked immunosorbent assay (ELISA) results are shown for Cxcl1 protein (pg/mL in the media. D and E, Q-PCR and ELISA results of Cxcl2 expression.
F and G, Q-PCR and ELISA results of Cxcl10 expression. H, Human monocyte–derived macrophages were infected with WNV (MOI p 1 and n p 6).
Cxcl1, Cxcl2, and Cxcl10 mRNA was measured by Q-PCR. The data are normalized to human b-actin mRNA and are expressed as the relative fold
increase over normalized RNA from time 0.

1806 • JID 2010:202 (15 December) • Bai et al


Figure 2. Neutrophils are rapidly recruited to the peritoneal cavity after
intraperiotoneal inoculation of West Nile virus (WNV). C3H wild-type or
Cxcr2⫺/⫺ mice were infected intraperitoneally with 10,000 plaque-forming
units of WNV, and mice were euthanized at the selected time points up
to 72 h to collect total cells infiltrating into the peritoneal cavity. A,
Lavaged cells were stained with granulocyte receptor 1 (Gr-1) (neutrophil
[neu] marker), F4/80 (macrophage [mac] marker), CD8+, CD4+, and CD19+
(B cell marker) and differentiated by fluorescence-activated cell scanning
(FACS) (n p 2–3 per group). *P ! .05 , **P ! .01, and ***P ! .001 when
compared with Gr-1+ cell group at each time point. Data were analyzed
with use of one-way analysis of variance followed by Tukey’s test. B,
FACS results of Cxcr2⫺/⫺ mice infiltrating cells (n p 2 per group).
infected macrophages at 4 h after infection, and another CXC-
type chemokine, Cxcl10, was slightly elevated (2-fold) at the
same time point (Figure 1A). The expression of the remaining
81 chemokine and receptor genes in the array, including other
CXC-type and CC-type chemokines, did not show up-regu-
lation after 4 h of infection with WNV. This is consistent with
reports showing that CXC type chemokine interleukin 8 (IL-
8) is among the first cytokines produced in response to infection
with another related flavivirus, dengue virus [30].
The findings from the array were confirmed by both Q-PCR
and ELISA when we assessed expression of Cxcl1, Cxcl2, and
Cxcl10 messenger RNA (mRNA) over a time course of infection
by reverse transcription Q-PCR and secreted protein levels by
ELISA from the same cells. Expression of Cxcl1 and Cxcl2 was
markedly up-regulated after WNV infection in vitro (Figure
1B–E) at 4 h. In contrast, up-regulation of Cxcl10 expression
was lower and developed relatively later, with elevation ob-
served at 48 h after infection (Figure 1F and 1G). Consistent
with murine macrophage results, primary human macrophages
infected with WNV (MOI p 1) showed time-dependent in-
creases in mRNA expression of Cxcl1, Cxcl2, and Cxcl10 (Figure
1H), with expression of mRNA for Cxcl1 and Cxcl2 increasing
as early as 3 h after infection and decreasing by 24 h, whereas
up-regulation of Cxcl10 only began at 8 h and peaked at 24 h.
Elevated levels of Cxcl1 and Cxcl2 suggest that PMNs may be
recruited rapidly to sites of infection with WNV.
PMNs are rapidly recruited to the site of WNV infection.
To assess the kinetics of recruitment of PMNs, we infected mice
with WNV and assessed peritoneal exudate cells with cell lin-
eage markers for flow cytometry (FACS). Analysis of the com-
position of the exudates at time points up to 72 h showed that
PMNs (Gr-1+ cells) were the most abundant cell type (1.5 
0.3 ⫻ 10 6 cells/mouse; ∼62%) in the peritoneal cavity as early
as 12 h after WNV inoculation (Figure 2A) with a peak abun-
dance at 24 h that began to decrease at 72 h after infection.
Of the other immune cells detected, only CD8+ T cells followed
similar kinetics, but at only ∼50% (7.0  1.7 ⫻ 10 5 cells/mouse)
of the PMN number (Figure 2A). These data suggest that PMNs
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are the predominant immune cells that are initially recruited
to sites of WNV infection.
The efficient accumulation of PMN depends on recruitment
through the chemokines Cxcl1 and Cxcl2. When we conducted
infections in mice deficient in Cxcr2 (Cxcr2⫺/⫺), a receptor gene
for chemokines Cxcl1 and Cxcl2, there was a reduction of up
to 85% (2.4 ⫻ 10 5 cells) and 64% (4.5  0.003 ⫻ 10 5 cells) of
PMNs in peritoneal exudates at 24 h and 48 h, respectively
(Figure 2B), and levels of CD8+ T cells were also reduced, albeit
at a lower level (loss of 35% [4.5  0.6 ⫻ 10 5] and 40% of
CD8+ T [2.7  0.08 ⫻ 10 5] cells/mouse at 24 h and 48 h, re-
spectively). Lower CD8+ cell counts may be attributable to re-
duced recruitment through their Cxcr2 [31] or to an unknown
role of PMNs in modulating recruitment of adaptive cells.
Depletion of PMNs prior to but not after WNV challenge
increases survival. To investigate the role of PMNs in WNV
pathogenesis, we infected mice depleted of PMNs by treatment
with the monoclonal antibodies Gr-1 (clone RB6–8C5), which
selectively binds and depletes PMNs and eosinophils [28], or
Ly-6G (clone 1A8), which specifically binds and depletes PMNs
[29]. Gr-1 (250 mg) or Ly-6G (100 mg) in 100 mL of PBS reduced
levels of PMNs to 50% of levels in mice injected with isotype-
matched control antibodies (Figure 3A;11% vs 22%; P ! .05;
Ly-6G data not shown). When viral load in the blood was
quantified on day 2 or 3 after infection by Q-PCR for WNV
envelope protein gene (WNVE), the level of WNV was signif-
icantly reduced in animals that had been depleted of PMNs 1
day prior to inoculation (Figure 3B and 3C; P ! .05). Further-
more, PMN-depleted mice by Gr-1 showed dramatically in-
creased survival (Figure 3D; P ! .001, by log-rank test). The
effect of PMN depletion by Gr-1 prior to challenge was also
noted in an alternate route of infection, when mice were in-
fected with WNV by footpad injection (10 pfu). These PMN-
depleted animals had lower viral loads and significantly in-
creased survival (Figure 3E; 40% vs 0%; P ! .01), suggesting
PMN in WNV Infection • JID 2010:202 (15 December) • 1807
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Figure 3. Neutrophil depletion prior to but not after West Nile virus (WNV) challenge increases mouse resistance (A–D). C3H mice were injected
with neutrophil-depleting antibodies (intraperitoneal injection with a single 250-mg dose of granulocyte receptor 1 (Gr-1) or 100 mg of Ly-6G in 100
mL of phosphate-buffered saline [PBS]) or isotype-matched control antibodies 1 day prior to WNV infection (intraperitoneal injection of 100 plaque-
forming units [pfu]). A, Polymorphonuclear leukocyte (PMN) percentage in blood smears on day 1 after Gr-1 treatment (isotype control, n p 5 ; Gr-1,
n p 4). B, Mice were treated with Gr-1 and WNVE was measured by quantitative polymerase chain reaction (Q-PCR) in blood at day 3 after infection
(isotype control, n p 10; Gr-1, n p 7). C, Mice were treated with Ly-6G, and WNVE was measured by Q-PCR in blood at day 2 after infection
(n p 10 per group). These data are representative of 2 independent experiments. D, Kaplan-Meier survival analysis of isotype-matched control and
Gr-1 antibody–treated mice after intraperitoneal inoculation of WNV (n p 10 per group). E, Survival curves of C3H mice that were injected with Gr-
1 (intraperitoneal injection with 1 dose of 250 mg in 100 mL PBS) 1 day before WNV infection by footpad inoculation (10 pfu; n p 10 per group). F–
H, C3H mice were injected with 2 doses of neutrophil-depleting antibodies (intraperitoneal injection with 250 mg Gr-1 or 100 mg Ly-6G in 100 mL
PBS) on days 1 and 2 after WNV infection (intraperitoneal injection of 100 pfu). F and G, WNVE was measured by Q-PCR in blood specimens at day
3 after infection (n p 10 per group). H, Survival analysis of isotype-matched control and Gr-1 antibody–treated mice after intraperitoneal inoculation
of WNV (n p 10 per group). Survival data shown are representative of at least 2 independent experiments. *P ! .05 and **P ! .001.

that PMN may play a negative role in the host immune response
against WNV infection.
To investigate whether PMNs play a detrimental role in the
defense against WNV, we further dissected the kinetics of the
PMN response to infection with WNV. Remarkably, when mice
were infected with WNV first and then depleted of PMNs on
days 1 and 2 after infection, treated animals showed higher
levels of viral burden (Figure 3F and 3G; P ! .05 ) and reduced
survival rate, compared with the mice that were administrated
control antibody (Figure 3H; 0% vs 30%; P ! .001; Ly-6G data
not shown). Taken together, these data suggest, paradoxically,
that PMN may enhance WNV infection at the early stages of
infection, whereas in the later course of infection, PMNs appear
to contribute to control of infection.
PMNs are an important reservoir for WNV replication. To
further dissect the antiviral mechanisms, we infected murine
PMNs and macrophages with WNV ex vivo at a range of MOIs
and quantified viral load by Q-PCR at 8 h after infection [24,
25, 27] and found that PMNs were infected by WNV to a
significantly higher level than were macrophages (eg, 55-fold

1808 • JID 2010:202 (15 December) • Bai et al

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Figure 4. Neutrophils are an important reservoir for West Nile virus (WNV) replication. A, Thioglycollate elicited peritoneal macrophages and
neutrophils from C3H mice were infected with WNV or mock (phosphate-buffered saline [PBS]) for 8 h. Quantitative polymerase chain reaction (Q-
PCR) was performed for WNVE to measure WNV replication (macrophages, n p 18 ; neutrophils, n p 6 ). B, Q-PCR results of WNV-infected human
monocyte–derived macrophages and neutrophils at 8 h (macrophages, n p 5 ; neutrophils, n p 12 ). C, Differential Q-PCR results of WNV-infected
mouse peritoneal macrophages and neutrophils at 8 h. D, C3H mice were infected via intraperitoneal injection with 10,000 plaque-forming units (pfu)
of WNV, and mice were euthanized at day 3 after infection to collect blood (n p 8 per group). Neutrophils were isolated using anti-Gr-1 Macs beads,
and Q-PCR was performed for WNVE in the neutrophils and the Gr-1⫺ cells. *P ! .05, **P ! .01, *** P ! .001, and ****P ! .0001.

and 69-fold higher when infected with MOIs of 1 and 10,
respectively) (Figure 4A). A similar enhancement of infection
and replication of WNV in PMNs was noted in human mac-
rophages and PMNs (eg, 17-fold and 13-fold; Figure 4B). These
data suggest that PMNs may act as a reservoir of virus in early
infection.
We used a differential Q-PCR method to demonstrate active
replication of WNV by distinguishing the positive- and nega-
tive-sense RNA in WNV-infected cells [32]. Because viral RNA
polymerase generates negative-sense RNA from the positive-
sense stranded genomic RNA that serves as a template for
replication, accumulation of negative-sense RNA indicates that
active replication is occurring. In both human and murine
PMNs, differential Q-PCR detected both positive- and negative-
sensed viral RNA, suggesting that the higher levels of WNVE
in PMN may be attributed to active replication and not only
to increased permissiveness (Figure 4C).
To determine whether PMN serve as a reservoir for WNV
in vivo, we infected mice with 10,000 pfu of WNV intraperi-
toneally and quantified viral load in blood cells at day 3 after
infection. Q-PCR for WNVE measurement in PMN (Gr-1+
cells) versus other cell types (Gr-1⫺ cells) showed that PMNs,
which accounted for only ∼25% of the leukocytes in mouse
blood, harbored ∼8-fold more WNVE than the other cell types
combined (Figure 4D; P ! .05). These results suggest that PMNs
may play a critical role in vivo for WNV replication and dis-
semination, especially in humans, where PMNs are the pre-
dominant cell type in blood.
Cxcr25/5 mice show delayed death after lethal WNV
infection. We assessed the course of WNV infection in

PMN in WNV Infection • JID 2010:202 (15 December) • 1809


Figure 5. Cxcr2⫺/⫺ mice show delayed death after lethal West Nile
virus (WNV) infection. Cxcr2⫺/⫺ mice and wild-type (WT; C3H) mice were
challenged with WNV by intraperitoneal injection (100 plaque-forming
units). A, quantitative polymerase chain reaction (Q-PCR) was performed
for WNVE in peripheral blood on days 1, 2, and 3 (n p 10 per group).
B, Survival analysis of WT and Cxcr2⫺/⫺ mice after intraperitoneal in-
oculation of WNV (WT, n p 43 ; Cxcr2⫺/⫺ mice, n p 34). Data are pooled
from 4 independent experiments. *P ! .05 and **P ! .01.
Cxcr2⫺/⫺ mice, which have equivalent numbers of PMN as wild-
type mice but whose PMNs have reduced chemotactic re-
sponses. Consistent with our previous data, the Q-PCR results
on day 1 after infection showed that viremia in blood was
significantly lower in Cxcr2⫺/⫺ mice than in wild-type mice
(Figure 5A; P ! .05), again suggesting that PMN may support
WNV replication in early WNV infection. However, as infec-
tion progressed, we detected similar levels of viremia in both
Cxcr2⫺/⫺ and wild-type mice at day 2 and a modest but sta-
tistically higher viral burden on day 3 after infection in blood
specimens from Cxcr2⫺/⫺ mice, compared with blood speci-
mens from wild-type mice, which argued in favor of an antiviral
role for PMN at this later time point (Figure 5A; 2.7-fold higher;
P ! .01). Analysis of survival of infected animals, which encom-
passes both early and later responses, showed delayed death of
Cxcr2⫺/⫺ mice, compared with wild-type mice (median dura-
tion of survival, 11 vs 9 days for Cxcr2⫺/⫺ vs wild-type mice;
P p .01; Figure 5B). Collectively, these data support the hy-
pothesis that, although PMNs may be permissive in the early
1810 • JID 2010:202 (15 December) • Bai et al
phase for WNV replication and dissemination, they later con-
tribute to limiting WNV infectivity.
Effects of the cellular milieu may contribute to the shift in
PMN from the early replication-permissive state to the later
antiviral state. In the first 24 h after WNV infection, macro-
phages initiate a robust antiviral program including abundant
type I IFN [27, 33]. When PMN in vitro were treated with IFN
(2000–10,000 IU) for 1 h prior to infection with WNV
(MOI p 1), WNVE gene levels at 20 h were reduced (0.83-
fold; n p 9; P ! .016). Although not comprehensive, this re-
duction suggests that the antiviral responses of surrounding
immune cells, such as macrophages, may promote the antiviral
state of PMNs in vivo.
DISCUSSION
WNV is a neuroinvasive virus and lethality is caused by viral
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invasion of the brain and subsequent encephalitis [7, 24, 25].
Early control of WNV, which relies on innate immune re-
sponses, is crucial to restrict WNV dissemination, contain in-
flammation, and prevent permeabilization of the blood-brain-
barrier leading to neural infection and death [33]. Although
immune responses are generally successful at combating infec-
tion due to WNV in young immunocompetent individuals,
WNV attenuates activation of macrophages [34], and increased
susceptibility in the elderly population may be attributable to
dysregulation of innate responses in that population [27, 35].
Activation of PMNs in infectious or inflammatory foci leads
to the release of powerful antimicrobial products and is crucial
for effective innate immunity [12]. Evidence supporting a role
for PMNs in response to flaviviral infections includes the pre-
dominance of PMN noted in infiltrates of cerebrospinal fluid
from human patients with WNV meningitis and encephalitis
[19, 20]; the enhanced binding of PMNs to dengue virus–
infected endothelial cells [36], suggesting a preferential recruit-
ment for PMNs; and the increased number of immature PMNs
in the blood of patients at an early stage (5–6 days after onset)
of dengue hemorrhagic fever [37].
In our examination of the initial chemokine responses to
WNV infection, we noted that expression of PMN-recruiting
chemokines was dramatically elevated and that PMN were
quickly recruited to sites of WNV infection in vivo. Our results
showing up-regulation of Cxcl10 somewhat later are consistent
with a role in recruitment of CD8+ T cells, which are critical
to clear viral infection in the central nervous system [38, 39].
The predominance and early arrival of PMN supports a crit-
ical role in response to WNV infection, but depletion of PMNs
prior to WNV challenge (or studies in Cxcr2⫺/⫺ mice) para-
doxically lowered viremia and enhanced survival, suggesting
that PMNs initially have an enhancing role in WNV infection.
Mechanisms underlying PMN facilitation of early WNV infec-
tivity may include: (1) abundant reservoir, because PMNs are
the predominant cell type recruited to the site of infection; (2)
efficient replication of WNV in PMN; and (3) higher levels of
viral load in PMNs than in other cells circulating in WNV-
infected mice. In support of our finding, elevated replication
rates in PMNs may contribute to efficient dissemination of
virus in other systems, as has been shown previously for cy-
tomegalovirus and human immunodeficiency virus [40–42].
We noted a biphasic effect of the absence of PMNs on the
course of WNV infection. Initially, the absence of PMNs led
to lower viremia and delayed death but a later depletion of
PMN led to increased viral burden. Collectively, these data
indicate a complex and previously unappreciated role for PMN
in infection with WNV, facilitating WNV infection initially
through acting as a reservoir for viral replication and allowing
dissemination, and only subsequently serving a protective func-
tion. Studies on the effects of macrophage-derived type I IFN
suggest that the cytokine milieu in vivo may influence the shift
in PMN antiviral activity. This finding reflects the well-de-
scribed cooperativity of PMNs and macrophages in immune
responses [43] and, in particular, a role for type I IFN, which
has recently been shown to modulate PMN recruitment and
turnover in response to infection with the protozoan parasite
Leishmania [44].
Interestingly, a role for PMNs as a reservoir for pathogens
as has been described previously for PMN infection by Sta-
phylocccus aureus [45, 46] and may be relevant also in infection
with H5N1 Avian influenza [47]. Imaging studies in the skin
of sand fly–mediated infections with the intracellular protozoan
Leishmania reveal that PMNs initially capture the parasites and
contribute to establishment of infection [48]. In addition, as a
vector-transmitted infection, WNV enters the host with vector
saliva, which has been shown to have immunosuppressive ac-
tivities, including the inhibition of PMN functions [49, 50].
These reports suggest that there may be a more general role of
PMNs facilitating initial pathogen infections, which may be an
important target for early anti-microbial interventions.
Acknowledgments
We thank Deborah Beck and Xianzhong Liu.
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