interferon-l restricts west nile virus neuroinvasion by tightening...

R E S EARCH ART I C L E

CORRECTED 27 MAY 2015; SEE ERRATUM

BLOOD -BRA IN BARR I ER

Interferon-l restricts West Nile virus neuroinvasion bytightening the blood-brain barrierHelen M. Lazear,1* Brian P. Daniels,2* Amelia K. Pinto,1 Albert C. Huang,3 Sarah C. Vick,1†

Sean E. Doyle,4 Michael Gale Jr.,3 Robyn S. Klein,1,2,5*‡ Michael S. Diamond1,5,6*‡

http://stD

ownloaded from

Although interferon-l [also known as type III interferon or interleukin-28 (IL-28)/IL-29] restricts infection by severalviruses, its inhibitory mechanism has remained uncertain. We used recombinant interferon-l and mice lacking theinterferon-l receptor (IFNLR1) to evaluate the effect of interferon-l on infectionwithWest Nile virus, an encephaliticflavivirus. Cell culture studies in mouse keratinocytes and dendritic cells showed no direct antiviral effect of exog-enous interferon-l, even though expression of interferon-stimulated genes was induced. We observed no differ-ences inWest Nile virus burden between wild-type and Ifnlr1−/−mice in the draining lymph nodes, spleen, or blood.We detected increased West Nile virus infection in the brain and spinal cord of Ifnlr1−/−mice, yet this was not asso-ciated with a direct antiviral effect in mouse neurons. Instead, we observed an increase in blood-brain barrier per-meability in Ifnlr1−/−mice. Treatment ofmicewithpegylated interferon-l2 resulted in decreasedblood-brainbarrierpermeability, reducedWest Nile virus infection in the brainwithout affecting viremia, and improved survival againstlethal virus challenge. An in vitro model of the blood-brain barrier showed that interferon-l signaling in mousebrain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movementacross the barrier, and modulated tight junction protein localization in a protein synthesis– and signal transducerand activator of transcription 1 (STAT1)–independent manner. Our data establish an indirect antiviral function ofinterferon-l in which noncanonical signaling through IFNLR1 tightens the blood-brain barrier and restricts viralneuroinvasion and pathogenesis.

m.sci

by guest on March 31, 2019

encemag.org/

INTRODUCTION

Interferon-l (IFN-l) belongs to a family of cytokines that signal throughtheheterodimeric IFN-l receptor, IFNLR,which is composedof interleukin-10 receptor b (IL-10Rb) and IFNLR1 (IL-28Ra). Analogous to signalingby IFN-a/b and its heterodimeric receptor, IFNAR, binding of IFN-l toIFNLR initiates a Janus kinase–signal transducer and activator oftranscription (JAK-STAT) signaling cascade that induces expression ofgenes with antiviral and immunomodulatory activities (1). Mice lackingIFNLR1 (Ifnlr1−/−) do not respond to IFN-l but retain relatively normalresponses to IFN-a/b (2). In contrast to IL-10Rb, which is expressedmorewidely, IFNLR1 exhibits a cell type–restricted expression pattern (3, 4).IFNLR1 is expressed preferentially on epithelial cells, and inhibitoryeffects of IFN-l in vivo have been documented against viruses that targetepithelial tissues (4–8). In cell culture, IFN-l reportedly inhibits infectionof a broader panel of viruses, including West Nile virus (WNV) (9).

WNV is an encephalitic flavivirus that cycles in nature between birdsand mosquitoes, with humans becoming infected as incidental dead-end hosts. About 20% of infected humans develop a febrile illness,and a subset of these progress to severe neuroinvasive disease (10).

1Department of Medicine, Washington University School of Medicine, St. Louis, MO63110, USA. 2Department of Anatomy & Neurobiology, Washington University Schoolof Medicine, St. Louis, MO 63110, USA. 3Department of Immunology, University ofWashington School of Medicine, Seattle, WA 98195, USA. 4ZymoGenetics, a Bristol-Myers Squibb Company, Seattle, WA 98102, USA. 5Department of Pathology &Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.6Department of Molecular Microbiology, Washington University School of Medicine,St. Louis, MO 63110, USA.*These authors contributed equally to this work.†Present address: Department of Microbiology and Immunology, University of NorthCarolina, Chapel Hill, NC 27599, USA.‡Corresponding author. E-mail: [email protected] (M.S.D.); [email protected] (R.S.K.)

www.Scie

Among hospitalized patients, the case fatality rate is 5 to 10%, and sur-vivors are commonly left with debilitating neurologic sequelae.

Because WNV is highly neurovirulent, the failure of infections toprogress uniformly to neurologic disease suggests that barriers to neu-roinvasion exist and that dissemination into the central nervous sys-tem (CNS) determines clinical outcome. The precise mechanisms bywhichWNV spreads to the brain and spinal cord remain unclear (11),but neuroinvasion likely occurs by a hematogenous route. Thus, theblood-brain barrier (BBB) represents a key defense against WNV neu-roinvasion (12). The BBB is composed of tight junctions between endo-thelial cells of the CNS microvasculature and the end-feet of astrocytes;the endothelial cells and astrocytes are further separated by two base-mentmembranes and a perivascular space. BBBpermeability is increasedin response to proinflammatory stimuli, including lipopolysaccharide(LPS), tumor necrosis factor–a (TNF-a), and IFN-g, and this openingalso permits trafficking of immune cells into theCNS (13, 14). AlthoughBBB opening may be necessary to resolve certain CNS infections, it isregulated to avoid pathological consequences from infiltrating cells orother substances from the periphery (12).

Although some inflammatory stimuli increase BBB permeability,IFN-a/bmay enhance BBB integrity. IFN-b treatment reduces endo-thelial permeability in an in vitro model of the BBB (14, 15) and is usedtherapeutically to treat multiple sclerosis, which features BBB break-down and trafficking of autoreactive T cells into the CNS as key com-ponents of the disease pathology (16). In tissue culture, IFN-b directlyregulates endothelial permeability and tight junction formation via theactions of the GTPases (guanosine triphosphatases) Rac1 and RhoA(14). Given the analogous JAK-STAT–dependent signaling pathwaysinduced by IFN-a/b and IFN-l, we evaluated the antiviral effects ofIFN-l on WNV infection. Our results suggest that IFN-l signaling in-hibits WNV infection of the CNS by modulating endothelial cell tight

nceTranslationalMedicine.org 22 April 2015 Vol 7 Issue 284 284ra59 1

http://stm.sciencemag.org/content/7/289/289er5.full

http://stm.sciencemag.org/


junction integrity, which decreases BBB per-meability, restricts virus neuroinvasion, andprevents lethal infection.


http://stm.sciencem

ag.org/D

ownloaded from

RESULTS

IFN-l does not inhibit WNV infectionof relevant target cellsGiven that IFN-l reportedly induces an anti-viral gene program similar to IFN-a/b (17–19),we tested whether it would inhibit WNV in-fection in cell culture. We expected IFN-l toexert its greatest effects on epithelial cells be-cause of the reported tissue-specific expressionpattern of Ifnlr1 (3–5). Before assaying for directantiviral effects, we assessed the relative mRNAexpression of the receptors for IFN-a/b (Ifnar1)and IFN-l (Ifnlr1 and Il10rb) in primary cells:cortical neurons and bone marrow–deriveddendritic cells (DCs) and macrophages (Fig. 1,A to D). We also tested a mouse keratinocytecell line because keratinocytes are one of thefew epithelial cells targeted by WNV in vivo(20). All cell types expressed Ifnar1, whereasIl10rb was detected in keratinocytes, DCs,and macrophages, but not cortical neurons.We detected Ifnlr1 expression in keratinocytesand DCs, but expression in macrophages andcortical neurons was near the limit of detection.As expected, Ifnlr1 expression was not detectedin cells from Ifnlr1−/− mice.

On the basis of Ifnlr1 mRNA expression,we chose keratinocytes and DCs for furtherstudies. We treated cells with IFN-l3 or IFN-bfor 6 hours and measured IFN-stimulated gene(ISG) expression by quantitative reverse tran-scription polymerase chain reaction (qRT-PCR).Ifit1 and Rsad2were up-regulated in responseto IFN-l3 or IFN-b (Fig. 1, E and F), althoughinduction was lower with IFN-l than IFN-b.For example, in keratinocytes, IFN-b (0.1 ng/ml)increased Ifit1mRNAexpressionby~1700-fold,whereas IFN-l (100 ng/ml) produced a <150-fold increase (Fig. 1E). The effect of IFN-l onISG expression was less potent in DCs com-pared to keratinocytes and was absent inIfnlr1−/− DCs. Although IFN-l induced ex-pression of the ISGs Ifit1 and Rsad2, unlikeIFN-b, it did not stimulate expression of Irf7(Fig. 1G). An increase in the IFN-l dose to1000 ng/ml failed to increase ISG expressionfurther (Fig. 1H).

We extended these results using RNA-seq(RNA sequencing) analysis to obtain a profile
of the IFN-l gene signature in primary DCs (Fig. 1I). Using a threefoldinduction cutoff (relative to mock) after 6 hours of IFN treatment, wedetected induction of 208 genes in IFN-b–treatedDCs, whereas no genes
www.Scie

were induced to this level in the IFN-l–treated DCs (table S1). We alsodid not detect any ISGs thatwere induced uniquely by IFN-l; the IFN-l–stimulated genes were a subset of those induced by IFN-b.

Ifnar1 Ifnlr1 Il10rb

WT0

–2

–3

5

–20

–15

–10

Keratinocytes

-∆C

t (n

orm

aliz

ed t

o 1

8S r

RN

A)

WT Ifnlr1 –/––30

–25

–20

–15

–10

DC

WT Ifnlr1 –/––30

–25

–20

–15

–10

Macrophages

Kerat

inocy

tes

WT

DC

Ifnlr1

–/– DC

0

3

6

9

12

15

Ifit1

Log

2∆∆

Ct

Kerat

inocy

tes

WT

DC

Ifnlr1

–/– DC

0

3

6

9

12

15

Rsad2

Kerat

inocy

tes

WT

DC

Ifnlr1

–/– DC

0

3

6

9

12

15

Irf7

–1 0 1 2 3–18

–16

–14

–12

–10

–8

IFIT1

IFN-λ3 (log10 ng/ml)

- ∆C

t(n

orm

aliz

ed t

o G

apd

h)

Mock

WT–30

–25

–20

–15

–10

10

100

IFN- (ng/ml):

0.11

IFN- (ng/ml):

IFN- IFN-

A

E

H I

F G

CB D Corticalneurons

Fold change (log2)

−2 0 2

Fig. 1. IFN-lproduces cell type–restricted inductionofISGs. (A to D) Expression of Ifnar1, Ifnlr1, and Il10rb wasmeasured by qRT-PCR from uninfected mouse keratino-cytes, DCs,macrophages, or cortical neurons. Gene expres-sion was normalized to 18S ribosomal RNA (rRNA). Dottedlines indicate the sensitivity of the assay. WT, wild type. (E toG) Mouse keratinocytes and DCs were treated with murineIFN-l3 or IFN-b for 6 hours. Expression of Ifit1, Rsad2, and Irf7was measured by qRT-PCR. Gene expression was normal-
ized to 18S rRNA and to mock-treated cells. Results represent means ± SEM of nine samples from threeindependent experiments. Dotted lines indicate gene expression in mock-treated cells. (H) Mousekeratinocytes were treated with IFN-l3 for 6 hours, and expression of Ifit1 was measured by qRT-PCR.Gene expression was normalized to Gapdh. The dotted line represents gene expression in mock-treatedcells. Results represent means ± SEM of 2 to 10 samples from three independent experiments. (I) MouseDCs were treated with IFN-l3 (10 ng/ml) or IFN-b (20 ng/ml) for 6 hours. Total RNAwas analyzed byRNA-seq. Genes that were induced or repressed by more than threefold (relative to mock-treated)are shown in a heatmap representation.




http://stm.sciencem

ag.org/D

ownloaded from

To assess whether the gene program induced by IFN-l inhibitedWNV replication, we treated keratinocytes and DCs with IFN-l3 orIFN-b before infection. In keratinocytes, we observed a dose-dependentinhibition of viral replication by IFN-b (Fig. 2A). In both wild-type andIfnlr1−/− DCs, even the lower dose of IFN-b blocked WNV infection(Fig. 2, B andC).Wedid not observe any inhibition ofWNVreplicationby IFN-l3, even at doses that induced ISGs.We performedmultistep viralgrowth analyses in primary cells from Ifnlr1−/− and wild-type mice andfound no differences in WNV infection between Ifnlr1−/− and wild-typeDCs, macrophages, or cortical neurons (Fig. 2, D to F). Our results suggestthat although IFN-l induced expression of ISGs, the effect was not directlyantiviral. This may be because either the magnitude of the response was in-sufficient or the specific set of genes inducedwas not inhibitory againstWNV.

IFN-l signaling limits WNV neuroinvasionWe assessed a role for IFN-l in restricting WNV infection in vivo.We infected wild-type and Ifnlr1−/−mice and measured viral burdenin tissues. Wild-type and Ifnlr1−/− mice displayed no significant differ-ences in viral loads at any time point in peripheral tissues, including theserum, draining lymph nodes, spleen, and kidney (Fig. 3, A toD). How-ever, Ifnlr1−/− mice sustained higher viral titers in the brain and spinal

www.ScienceTranslationalMedicine.or

cord at days 6 and 8 after infection (Fig. 3, Eand F). We also detected virus in the brainearlier in a subset of Ifnlr1−/−mice comparedto wild-type mice (two of eight Ifnlr1−/−

mice positive at day 4 after infection, com-pared to zero of five wild-type mice).

To determine whether the higher WNVtiters in the CNS of Ifnlr1−/−mice reflected adirect antiviral effect of IFN-l in brain tis-sues, we infected mice by an intracranialroute and measured viral titers 3 and 5 daysafter infection. We found no difference inviral titer between wild-type and Ifnlr1−/−

mice in any of the CNS regions examined(Fig. 3, G to I). Thus, although Ifnlr1−/−micedeveloped enhanced CNS infection afterperipheral WNV infection, no increase wasobserved when the virus was inoculated di-rectly into the brain.

Ifnlr1−/− mice develop normaladaptive immune responses toWNV infectionWe considered whether IFN-lmight restrictWNV neuroinvasion indirectly by regulatingadaptive immune responses, which are re-quired to prevent dissemination to the CNS(21). However, we detected no difference inserum concentrations of anti-WNV immu-noglobulin M (IgM) or IgG at days 4, 6, or8 after WNV infection between wild-typeand Ifnlr1−/−mice (fig. S1). To assess cellularimmune responses, we profiled leukocytesfrom the spleen and brain at 8 days afterWNV infection and found no differences be-tween wild-type and Ifnlr1−/− mice in totalnumbers or percentages of CD11b+, CD11c+,

CD4+, or CD8+ cells (Fig. 4, A to D and K to N). Antigen-specific CD8+

T cell responses were equivalent between wild-type and Ifnlr1−/− mice(Fig. 4, E and O), and effector functions (production of granzyme B,IFN-g, and TNF-a) also were similar between wild-type and Ifnlr1−/−

mice in both the spleen and the brain (Fig. 4, F to J and P to T).

Ifnlr1−/− mice have increased BBB permeability afterWNV infectionBecause IFN-a/b signaling can enhance BBB integrity (14), we hypothe-sized that IFN-lmight exert an analogous effect to prevent virus trans-port across the BBB, which could explain the increased CNS infectionobserved in Ifnlr1−/− mice. We assessed BBB permeability in wild-typeand Ifnlr1−/− mice 4 days after WNV infection. Compared to mock-infected mice, WNV infection resulted in increased extravasationof sodium fluorescein dye from the peripheral circulation into the CNS(Fig. 5, A to C). Although Ifnlr1−/−mice showed no basal differencesin BBB permeability compared to wild-typemice, theWNV-inducedpermeability changewas greater in Ifnlr1−/−mice, suggesting that IFN-lsignalingmay tighten the barrier in response to infection. As an indepen-dent measure of BBB permeability, we assessed leakage of endogenousIgG into the brain parenchyma after WNV infection (14). Although

0 24 481.0

2.0

3.0

4.0

5.0

6.0

Keratinocytes

Hours post-infection

Log

10F

FU

/ml

Mock 10100 IFN- (ng/ml)

0.11

IFN- (ng/ml)

WT Ifnlr1–/–

DC

0 24 48 721.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0


Log

10F

FU

/ml

Macrophages

0 24 48 721.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0


Cortical neurons

0 24 48 721.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0


0 24 481.0

2.0

3.0

4.0

5.0

6.0

WT DC

Hours post-infection0 24 48

1.0

2.0

3.0

4.0

5.0

6.0

Ifnlr1–/– DC


*****

****

**

****

A CB

D FE

Fig. 2. IFN-l does not directly inhibit WNV infection. (A to C) Mouse keratinocytes and DCs were pre-treated with murine IFN-l3 or IFN-b for 6 hours and then infected with WNV at a multiplicity of infection
(MOI) of 0.1. Viral infection was measured by focus-forming assay and expressed as focus-forming units(FFU) permilliliter. (D to F) Primary cells prepared fromWTor Ifnlr1−/−micewere infectedwithWNV. Resultsrepresentmeans ± SEMof nine samples from three independent experiments. Dotted lines indicate thesensitivity of the assay. Viral titers in IFN- treated cells were compared to mock-treated cells [two-way anal-ysis of variance (ANOVA)]. (A) **P=0.0057. (B) ***P=0.0004, ****P<0.0001. (C) **P=0.0075, ****P<0.0001.(D to F) The differences betweenWT and Ifnlr1−/− cells were not statistically significant (two-way ANOVA).
g 22 April 2015 Vol 7 Issue 284 284ra59 3




http://stm.sciencem

ag.org/D

ownloaded from

minimal IgG was detected in the brains of uninfected mice, IgG accu-mulation became apparent at 4 days after infection, and Ifnlr1−/− miceexhibited greater leakage than did wild-type mice (Fig. 5, D and E).Thus, in the context of WNV infection, the BBB of Ifnlr1−/− mice wasmore permeable to both small molecules and proteins. Given that in-flammatory cytokines, including IFN-g and TNF-a, promote BBBopening (14, 22), we considered whether the increased permeabilityin Ifnlr1−/− mice was due to elevated cytokine production. Wemeasured concentrations of 21 cytokines in serum at 3 and 5 days afterinfection and found no differences between wild-type and Ifnlr1−/−mice(table S2). We also assessed IFN-a/b activity in the serum and found nodifferences betweenWNV-infected wild-type and Ifnlr1−/−mice (fig. S2).

www.Scie

IFN-l signaling improves endothelial barrier integrity in vitroTo explore how IFN-l signaling modulatesWNV entry into the CNS,we used an in vitro model of the BBB (14, 23). In this system, brainmicrovasculature endothelial cells (BMECs) were cultured in theupper chamber of a Transwell insert, with supporting astrocytes inthe lower chamber. Transendothelial electrical resistance (TEER)across the BMEC monolayer indicated endothelial monolayer integ-rity, with higher resistance representing a tighter barrier. We firstmeasured expression of IFN receptors and WNV replication in thetwo primary cell types in this system. BMECs and astrocytes expressedabundant Ifnar1 and Il10rbmRNA, whereas Ifnlr1mRNA expressionwas low (Fig. 6, A and B).We found no difference inWNV replication

1 2 3 4 5 6

0.0

2.0

4.0

6.0

8.0

10.0

Draining lymph node

Days post-infection

Log

10P

FU

equ

ival

ents

/ml

1 2 3 4 5 6–0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Serum

Days post-infection

Log

10P

FU

equ

ival

ents

/ml

WT

Ifnlr1–/–

3 5

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Cortex

Days post-infection

Log

10P

FU

/ml

4 6 8 101.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Brain

Days post-infection

Log

10P

FU

/g

*

**

4 6 8 101.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Spinal cord

Days post-infection

Log

10P

FU

/g

**

3 5

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Spinal cord

Days post-infection

Log

10P

FU

/ml

3 5

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.09.0 9.0

Cerebellum

Days post-infection

Log

10P

FU

/ml

2 4 6 82.0

2.5

3.0

3.5

4.0

4.5

5.0

Spleen

Days post-infection

Log

10P

FU

/g

2 4 6 82.0

2.5

3.0

3.5

4.0

4.5

5.0

Kidney

Days post-infection

Log

10P

FU

/g

E

Intracranial inoculation

D

G H I

E F

BA C

Subcutaneous inoculation

Fig. 3. Viral burden in WT and Ifnlr1−/− mice.(A to I) Nine- to 12-week-oldmice were infectedwith 102 plaque-forming units (PFU) of WNV viasubcutaneous injection (A to F) or 101 PFU via in-tracranial injection (G to I). (A and B) Viral RNAwas measured by qRT-PCR. (C to F) Viral titersweremeasured by plaque assay. Symbols repre-sent individual mice from several independentexperiments; bars indicate the mean of 5 to 15mice per group. (G to I) Viral infection in CNS tis-sues was measured by plaque assay. Bars indi-cate themean of five to six mice per group fromtwo independent experiments. Dotted lines re-

present the limit of sensitivity of the assay. Viral titers inWT and Ifnlr1−/−mice were compared by theMann-Whitney test (A to F) or t test (G to I). (E) *P =

nceTranslationalMedicine

0.0124, **P = 0.0045. (F) **P = 0.0031.

.org 22 April 2015 Vol 7 Issue 284 284ra59 4




http://stm.sciencem

ag.org/D

ownloaded from

in BMECs and astrocytes prepared from wild-type and Ifnlr1−/− mice(Fig. 6, C and D).

We next infected Transwell cultures of BMECs with WNV andmeasuredTEER after 6 hours, a time point that precedes de novo spreadof WNV infection (24). Although wild-type and Ifnlr1−/− BMECs ex-hibited equivalent TEER in the absence of infection, the increase in

www.Scie

TEER after WNV infection was blunted in Ifnlr1−/− cells (Fig. 6E). Theeffect of IFN-l was due to signaling in BMECs rather than astrocytes;in reciprocal culture experiments, only Ifnlr1−/− BMECs exhibited thelower TEER phenotype regardless of the astrocyte genotype (Fig. 6E).Therefore, subsequent Transwell experiments used wild-type astro-cytes paired with wild-type or Ifnlr1−/− BMECs.

WT Ifnlr1–/–0

50

100

150

200

250

NS4b+

WT Ifnlr1–/–0

5000

10000

15000

CD4+

WT Ifnlr1–/–0

2000

4000

6000

8000

CD8+

WT Ifnlr1–/–0

1000

2000

3000

4000

5000

CD11b+N

um

ber

ofc

ells

(x10

3 )

WT Ifnlr1–/–0

500

1000

1500

CD11c+

WT Ifnlr1–/–0

50

100

150

200

GZB+

WT Ifnlr1–/–0

200

400

600

800

NS4b+

WT Ifnlr1–/–0

500

1000

1500

CD4+

WT Ifnlr1–/–0

500

1000

1500

2000

CD8+

WT Ifnlr1–/–0

2000

4000

6000

8000

CD11b+

Nu

mb

ero

fcel

ls(x

102 )

WT Ifnlr1–/–0

1000

2000

3000

4000

5000

CD11c+

WT Ifnlr1–/–0

100

200

300

400

500

GZB+

WT Ifnlr1–/–0

2000

4000

6000

WT Ifnlr1–/–0

5000

10000

15000

GM

FI(

of

CD

8+)

WT0 3.6

96.40

WT0 5.1

94.90

Ifnlr1–/–

0 3.5

96.50

Ifnlr1–/–

0 5.4

94.60

NS

4B

WT WTIfnlr1–/– Ifnlr1–/–

WT Ifnlr1–/–0

10000

20000

30000

WT Ifnlr1–/–0

2000

4000

6000

8000

GM

FI(

of

CD

8+) 0 30.5

69.60

0 31.6

68.40

0 35.3

64.70

0 25.8

74.20

I JH

BA

G

LK M ON P

Q TR S

C D E F

Sp

leen

Bra

in

TNF- +

CD8

CD8

NS

4B

IFN- + TNF- +

IFN- +

IFN

-

IFN

-

Fig. 4. Cellular immune responses in WT and Ifnlr1−/−mice. (A to T) Leu-kocytes were harvested from the spleen (A to J) and brain (K to T) 8 days after

tions are shown. IFN-g and TNF-a production is expressed as geometric meanfluorescence intensity (GMFI). Symbols represent individualmice, and data are

WNV infection. Cellswere stainedwith antibodies against CD45, CD19, CD11b,CD11c, CD3, CD4, CD8, and granzyme B. Cells were either stainedwith a tetra-mer displaying the immunodominant WNV peptide (Db-NS4B) (E and O) orrestimulated with NS4B peptide and then stained with antibodies againstIFN-g and TNF-a (G, H, Q, and R). Total numbers of the indicated cell popula-

combined from two independent experiments. The differences between WTand Ifnlr1−/− mice were not significant (Mann-Whitney test). Representativeflow cytometry plots showing CD8+ and NS4B+ cells (I and S) or CD8+ andIFN-g+ cells (J and T). Numbers indicate the percentage of cells in each quad-rant, and the results are representative of two independent experiments.





http://stm.sciencem

ag.org/D

ownloaded from

We next treated BMECs with recombinantmurine IFNs for 6 hoursand measured TEER. IFN-l3 produced a dose-dependent increase inTEER at 6 hours, and this effect was abolished in Ifnlr1−/− BMECs(Fig. 6F). As observed previously (14, 15), IFN-b treatment producedan increase in TEER. However, this effect was diminished in Ifnlr1−/−

BMECs, suggesting that a portion of the IFN-b effect may be due toIFN-l signaling. To distinguish the contributions of IFN-a/b andIFN-l to the WNV-induced TEER increase, we pretreated BMECswith a blocking monoclonal antibody against IFNAR1 (25). WhenIFN-a/b signaling was blocked, WNV infection did not produce anincrease in TEER in either wild-type or Ifnlr1−/− BMECs (Fig. 6G).These results suggest that IFN-l signaling promotes endothelial bar-rier tightening and that IFN-l production after WNV infection re-quires IFN-a/b signaling, as opposed to being induced directly bypattern recognition receptor signaling pathways.

Because it remained possible that the IFN-l signaling effect onTEER was mediated by an induced cytokine, we tested the requirement

www.ScienceTranslationalMedicine.org

for de novo protein synthesis. BMECs werepretreated with the protein synthesis inhib-itor cycloheximide and then infected withWNV or treated with IFN-b or IFN-l3 for6 hours. When cycloheximide was added,the WNV-induced increase in TEER wasabolished and the IFN-b–induced increasewas diminished (Fig. 6H). Cycloheximidetreatment had no effect on the increaseinTEERproducedby IFN-l3, indicating thatBMECs could form a tighter barrierdownstream of IFN-l–IFNLR signalingwithout requiring de novo protein synthesis.

To define further the signaling pathwayby which IFN-l regulates TEER changes inBMECs, we assessed mRNA expression ofIfnb, Ifna, and Il28b in BMECs after treat-mentwith IFN-b, IFN-l3, orLPS for 6hours.We did not detect expression of these IFNs,although the ISGs Ifit1 and Rsad2 were in-duced (fig. S3). Given the results from thecycloheximide experiments (Fig. 6H), wehypothesized that the IFN-l–inducedTEER response might occur through anoncanonical signaling pathway that didnot require ISG expression. To evaluatethis, we prepared BMECs from wild-typeand Stat1−/−mice andmeasured TEER aftertreatment with IFN-b or IFN-l3. Both IFN-l and IFN-b induced increases in TEERwithin 2 hours of treatment in a STAT1-independent manner (Fig. 6I). At later timepoints (for example, 12 hours), TEERvalues were lower in Stat1−/− cells com-pared to wild-type cells, indicating a subordi-nate role for canonical STAT1 signaling andpossibly ISG expression in barrier tighten-ing. To determine whether IFN-l alsocould induce endothelial barrier tighten-ing in human cells, we treated a humanBMEC cell line (HCMEC/D3) (23) in a

Transwell insert with pegylated human IFN-l1 or human IFN-bandmeasured TEER over time (Fig. 6J). Within 2 hours of IFN treat-ment, TEER increased markedly, demonstrating analogous effects ofIFN-l on mouse and human BMECs. Our experiments suggest amodel in which WNV infection stimulates the production of IFN-l,possibly downstream of IFN-a/b signaling. Along with IFN-a/bsignaling through IFNAR (14), circulating IFN-l would signalthrough IFNLR on BMECs via a noncanonical STAT1-independentand protein synthesis–independent pathway to tighten the endothelialbarrier and limit viral neuroinvasion.

We questioned whether changes in TEER directly affected transit ofWNV across the endothelial barrier. We treated HCMEC/D3 cells firstwith LPS for 18 hours, and then with IFN-l1 for 6 hours. We thenadded WNV to the upper chamber and, after 6 hours, measured virusthat had crossed into the lower chamber. As expected, we observed de-creased TEER after LPS treatment and an increase in TEER in responseto IFN-l treatment (Fig. 6K). Consistentwith the changes inTEER, LPS

WT Ifnlr1–/–

Mock WNV

Cerebellum

****

Mock WNV0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

Cortex

Rel

ativ

e flu

ore

scei

n p

erm

eatio

n

****

Mock WNV

Spinal cord

****

Cortex

Cereb

ellum

0

10

20

30

40

50

60

70

IgG

inte

nsi

ty

WT

Ifnlr1–/–

* *

A B C E

D

Mock

Cortex Cerebellum

WNV Mock WNV

WT

Ifn

lr1–/

–

100 µm

Fig. 5. BBB permeability in infectedWT and Ifnlr1−/−mice.WT and Ifnlr1−/−mice were infected via asubcutaneous route with WNV or diluent (mock). (A to C) BBB permeability was assessed at 4 days after
infection by measuring the accumulation of sodium fluorescein dye in CNS tissues after intraperitonealadministration. Symbols represent individual animals from two independent experiments. ****P <0.0001 (t test). (D and E) Brain sections were stained to detect endogenous IgG leakage into the CNSparenchyma (shown in green); nuclei are shown in blue. Representative images were taken at ×40mag-nification (scale bars, 100 mm). (E) IgG staining was quantified from two fields from threemice per groupand compared by t test. Cortex: *P = 0.0263; cerebellum: *P = 0.0241.
22 April 2015 Vol 7 Issue 284 284ra59 6




http://stm.sciencem

ag.org/D

ownloaded from

A B C D

E F G

H

J K L

Media CHX

Mock WNV IFN150

175

200

225

250

275

****

*

WT Ifnlr1–/–

Mock 10 10 100150

175

200

225

250

275

*******

(ng/ml)

****

Mock WNV

WT Ifnlr1–/–WT Ifnlr1–/–150

175

200

225

250

275

**** ***

WT Ifnlr1–/–Astrocytes:

BMECs:

Ifnar1 Ifnlr1 Il10rb

WT

Ifnlr1–/–

trocytesAs

0 24 48 721.0

2.0

3.0

4.0

5.0

6.0


BMEC

0 24 48 721.0

2.0

3.0

4.0

5.0

6.0


Log

10F

FU

/ml

Mock WNV

I

WT

–35

–30

–25

–20

–15

–10

BMEC

- C

t (n

orm

aliz

ed t

o 1

8S)

WT

–35

–30

–25

–20

–15

–10

Astrocytes

WT

Stat1–/–Media

WT

Stat1–/–

WT

Stat1–/–

Media

LPS

0

50

100

150

200

PF

U e

qu

ival

ents

/ml

**

0 6 12 18 24 3075

100

125

150

175

200

225

250

275

hours

IFN- WNVLPS RNA

****

****

Media LPS

Media

0 3 6 9 12150

175

200

225

250

275

Hours post-treatment

***

**

****

0 3 6 9 12150

175

200

225

250

275

Hours post-treatment

***

*

*

WT Ifnlr1–/– WT Ifnlr1–/–150

175

200

225

250

275

Isotype Cntrl

****

Fig. 6. IFN-l increases TEER in BMECs. (Aand B) Expression of Ifnar1, Ifnlr1, and Il10rb
wasmeasuredbyqRT-PCR fromWTuninfectedBMECs and astrocytes. Gene expression wasnormalized to 18S rRNA. (C and D) BMECs andastrocytes prepared fromWT and Ifnlr1−/−micewere infected with WNV, and viral replicationwasmeasured by focus-forming assay (expressedas focus-forming units per milliliter). Resultsrepresentmeans ± SEM of nine samples fromthree experiments. Thedifferences betweenWTand Ifnlr1−/− cells were not significant (two-wayANOVA). (E to I) WT or Ifnlr1−/− BMECs werecultured on Transwell inserts with astrocytesprepared from WT (E, left; and F to I) or Ifnlr1−/−
(E, right) mice. Results represent means ± SEMofnine samples from twoexperiments. (E) BMECswere infected with WNV, and TEER wasmeasured6hours later.WNV-infectedWTBMECswere compared to Ifnlr1−/−BMECs (t test). ****P<0.0001, ***P = 0.0006. (F) BMECs were treatedwith murine IFN-b or IFN-l3, and TEER wasmeasured 6 hours later. WT BMECs were com-pared to Ifnlr1−/−BMECs (t test). IFN-b and IFN-l(10 ng/ml): ****P < 0.0001. IFN-l (100 ng/ml):***P = 0.0003. (G) BMECs were pretreated withan IFNAR-blockingor isotypecontrolmonoclonalantibody (MAb) and then infected with WNV.TEER was measured 6 hours after infection. WTBMECswere compared to Ifnlr1−/−BMECs (t test).****P < 0.0001. (H) WT BMECs were pretreatedwith cycloheximide (CHX) (20mg/ml) for 18hoursand then infected with WNV or treated withmurine IFN-b (10 ng/ml) or IFN-l3 (100 ng/ml).TEER was measured 6 hours later. CHX-treatedBMECs were compared to media-treated cells(t test). ****P < 0.0001, *P = 0.009. (I) BMECswereprepared fromWTandStat1−/−miceand treatedwithmurine IFN-b or IFN-l3, and TEERwasmea-sured2, 6, and12hours after treatment. Resultsrepresent means ± SEM of six samples fromtwo experiments. IFN-treated BMECswere com-pared to vehicle-treated cells of the same geno-type at 2 hours after treatment (t test). IFN-b:***P = 0.0005 (WT), ***P < 0.0001 (Stat1−/−).IFN-l: *P = 0.0204 (WT), *P = 0.0347 (Stat1−/−).WT and Stat1−/− cells were compared by two-way ANOVA. IFN-b: *P = 0.0311. (J) HCMEC/D3cells were cultured on Transwell inserts andtreated with human IFN-b (10 ng/ml) or pegy-lated human IFN-l1 (100 ng/ml), and TEERwas measured. Results represent means ±SEM of eight samples from two experiments.IFN-treated cells were compared to vehicle-treated cells at 2 hours after treatment (t test).IFN-b: ****P < 0.0001. IFN-l: **P = 0.0011.
IFN-b– and IFN-l–treated cells were compared by two-way ANOVA. ***P =0.0006. (K and L) HCMEC/D3 cells were pretreated for 18 hours with LPS(100ng/ml)ormediaascontrol, followedbypegylatedhuman IFN-l1 (100ng/ml)for 6 hours (or media). The cells were then infected withWNV at anMOI of0.01 from the upper chamber. TEER was measured over the course of the
www.Scie

experiment (K); at 6 hours after infection, virus crossing into the lowerchamber was measured by qRT-PCR (L). Results represent means ± SEMof 12 samples from three experiments. (K) IFN-l treatment was comparedto media or LPS treatment by two-way ANOVA (K) or t test (L). ****P <0.0001, **P = 0.0047.





http://stm.sciencem

ag.org/D

ownloaded from

treatment resulted in greater crossing ofWNV into the lower chamber,and this effect was diminished by IFN-l treatment (Fig. 6L). Thus,IFN-l signaling promotes endothelial barrier tightening, which restrictsWNV transit.

To define how IFN-lmodulates TEER, we analyzed the location ofthe endothelial cell junction proteins ZO-1 and claudin-5 in BMECsand mouse brain sections after treatment with LPS and IFN-l (Fig. 7).Whereas colocalization of ZO-1 and claudin-5 was observed inmock-treated BMECs, treatment with LPS, which disrupts BBB integrity(13), caused ZO-1 and claudin-5 to dissociate. The effect of LPSon cell junction protein colocalization was reversed by IFN-l3 treat-

www.ScienceTranslationalMedicine.org

ment (Fig. 7, A and B). We observed asimilar effect in the brains of mice treatedwith LPS and pegylated IFN-l2 (Fig. 7C).

IFN-l enhances BBB integrityin vivoGiven that Ifnlr1−/− mice displayed in-creased BBB permeability after WNV in-fection and IFN-l decreased the barrierfunction of BMECs in vitro, we hypothe-sized that administration of IFN-l mightrestrict BBBpermeability in vivo.We treatedwild-type mice with LPS to open the BBBand then administered pegylated murineIFN-l2 or PBS. LPS treatment increasedBBB permeability compared to PBS, butthis effect was diminished in mice receivingIFN-l2 (Fig. 8, A to C).

We next tested whether administrationof IFN-l could restrict WNV neuroinva-sion. Wild-type mice were inoculated sub-cutaneously with WNV, and pegylatedmurine IFN-l2 was administered via anintravenous route on the day of infectionas well as 2 and 4 days afterward. Consist-entwith data from Ifnlr1−/−mice (Fig. 3A),IFN-l2 treatment did not exhibit a directantiviral effect, because viremia remainedunchanged (Fig. 8D). Nonetheless, IFN-l2–treated mice showed improved survivalrates compared to PBS-treated controls(Fig. 8E). To determine whether the im-proved survival observed in IFN-l2–treatedmice correlated with reduced WNV neu-roinvasion, we measured viral burden inthe CNS. Wild-type mice were inoculatedwithWNV and treated with pegylatedmu-rine IFN-l2 at days 2, 3, and 4 after infec-tion.As expected, IFN-l2 treatmentdidnotreduce viremia (Fig. 8F), but at day 8 afterinfection, IFN-l2–treated mice had lowervirus titers in the cerebral cortex comparedto control mice that received PBS (Fig. 8G).This effect was region-specific, because re-ductions in virus titer were not observed inthe cerebellum or spinal cord.

DISCUSSION

Previous studies in Ifnlr1−/− mice revealed a role for IFN-l in con-trolling viral infection in epithelial tissues, although no inhibitory ef-fect was observed with several viruses that disseminate more broadly(2, 4, 5, 7, 8, 26). These observations were consistent with a preferentialepithelial cell expression pattern for IFNLR1 (3, 4) and tissue respon-siveness to treatment with exogenous IFN-l (3, 5–7). AlthoughWNVreplicates in many cell types in vitro, its main targets in vivo are my-eloid cells and neurons (21). Because WNV is not reported to targetepithelial cells other than keratinocytes in the skin (20), a virologic

A Media IFN-

Med

iaL

PS

20µm

B

CPBS LPS LPS + IFN-

Med

ia

IFN

-

LP

S

LP

S +

IFN

-

0.0

0.5

1.0

1.5

Co

loca

lizat

ion

co

effi

cien

t **

**

20 µm

Fig. 7. Cell junction protein localization in endothelial cells. (A and B) WT BMECs were pretreatedfor 18 hours with LPS (100 ng/ml) and subsequently treated withmurine IFN-l3 (100 ng/ml) for 6 hours.
(C) WTmice were treatedwith LPS (3mg/kg via an intraperitoneal route), LPS and pegylatedmurine IFN-l2(25 mg via intravenous route), or phosphate-buffered saline (PBS) alone. (A to C) Cells and brain sectionswere costained for the cell junction proteins ZO-1 (green) and claudin-5 (red); nuclei are shown in blue.Images were taken by confocal microscopy at ×63 magnification (scale bars, 20 mm). Arrows indicateZO-1 and claudin-5 colocalization at intact tight junctions. Results are representative of two independentexperiments. (B) Colocalization of ZO-1 and claudin-5 staining was determined using ImageJ software andcompared by t test. Media versus IFN-l: **P = 0.0033. LPS versus LPS + IFN-l: **P = 0.0014.
22 April 2015 Vol 7 Issue 284 284ra59 8




http://stm.sciencem

ag.org/D

ownloaded from

phenotype for WNV in the CNS of Ifnlr1−/− mice was unexpected.Our experiments show that IFN-l signaling did not directly inhibitWNV infection in vitro or in vivo, but instead indicate that IFN-lsignaling enhances the integrity of the BBB, thereby limiting the neu-roinvasive potential of WNV.

Although IFN-a/b directly stimulates expression of genes that inhib-it WNV and other flaviviruses (27), in our experiments, IFN-l failed todo so in several cell types in culture, even though the receptors were ex-pressed and ISGs were induced. Although a previous study reported anantiviral effect of IFN-l on WNV (9), those observations may not becomparable, because they were based on luciferase production byWNV replicons in human tumor cell lines, as opposed to our study,which measured replication of fully infectious virus in primary mousecells and a mouse cell line.

Why did we not observe a direct inhibitory effect of IFN-l in ourexperiments? On the basis of RNA-seq and qRT-PCR analyses, themagnitude and breadth of the ISG response to IFN-l were reducedcompared to IFN-b. Althoughprevious studies have compared the tran-scriptional signatures induced by IFN-l and IFN-a/b (17–19, 28), thesestudies were performed in human tumor cell lines. Our analysis

www.Scie

compared the transcriptional profiles induced by IFN-l and IFN-bin mouse DCs andmay help to explain the results of other viral patho-genesis studies in Ifnlr1−/− mice. The ability of IFN-l to induce ISGexpression in a more targeted set of cells compared to IFN-a/b suggestsa possible therapeutic application inwhich IFN-lwould provide amorefocused antiviral or immunomodulatory effect with fewer side effects(26, 29).

We observed increasedWNV infection in the brain and spinal cordof Ifnlr1−/− mice. Because our experiments failed to show a direct anti-viral effect of IFNLR1 in neurons, we hypothesized that IFN-l signalingmight minimize WNV spread to the CNS by affecting BBB perme-ability. The following observations support a model in which IFN-lsignaling restricts the neuroinvasive potential ofWNV by improvingBBB function: (i) BBB permeability was greater in WNV-infectedIfnlr1−/− compared to wild-type mice; (ii) wild-type and Ifnlr1−/− micehad equivalent viral burden in peripheral tissues, so the increased in-fection in the brain was not due to antiviral effects of IFN-l in the periph-ery; (iii) proinflammatory cytokine levels were similar in wild-type andIfnlr1−/− mice and thus did not cause differential changes in BBB perme-ability; (iv) TEERwas lower inWNV-infected Ifnlr1−/− BMECs compared

A

D E

C

Cortex Cerebellum Spinal cord0.000

0.001

0.002

0.003

0.004

0.005

0.006

Rel

ativ

e flu

ore

scei

n p

erm

eatio

n

PBS LPS LPS + IFN-

****

****

PBS

LPS

LPS + IFN-

Cortex Cerebellum0

20

40

60

IgG

inte

nsi

ty

*

*B

F G

3 4 5

1.0

2.0

3.0

4.0

Days post-infection

Log

10P

FU

equ

ival

ents

/ml

Cortex Cerebellum Spinal cord

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Log

10P

FU

/ml

***

2 4

1.0

2.0

3.0

4.0

Days post-infection

Log

10P

FU

equ

ival

ents

/ml

0 3 6 9 12 15 18 210

20

40

60

80

100

Days post-infection

Per

cen

tsu

rviv

al

PBS IFN-

Co

rtex

Cer

ebel

lum

PBS LPS LPS + IFN-

100 µm

*

Fig. 8. IFN-l enhances BBB tightness and restrictsWNVneuroinvasion.(A to C) Eight-week-old WT mice were treated with LPS, LPS and pegylated

20 mg of pegylated murine IFN-l2 protein (or PBS) was administered viaan intravenous route. (D) Viral RNA in the plasma was measured on days 2

murine IFN-l2 (25 mg via intravenous route), or PBS alone. BBB permeabilitywas assessed 24 hours later by fluorescein or IgG permeation as describedfor Fig. 5. Symbols represent individual animals from two independentexperiments. Values for LPS-treated mice were compared to mice receivingLPS+ IFN-l (t test). (A) Cortex: ****P<0.0001, cerebellum: **P=0.0096, spinalcord: **P = 0.0082. (C) Cortex: *P = 0.0348, cerebellum: *P = 0.0168. (D and E)Five-week-old WT mice were infected with 102 PFU of WNV via a sub-cutaneous route. On the day of infection and at days 2 and 4 afterward,

and 4 after infection. (E) Survival was monitored for 21 days after infection.n = 22 mice, *P = 0.0264 (log-rank test). (F and G) Nine-week-old WT micewere infected with 102 PFU of WNV via a subcutaneous route. At days 2, 3,and 4, 25 mg of pegylatedmurine IFN-l2 protein (or PBS) was administeredvia an intravenous route. (F) Viral RNA in the plasmawasmeasured on days3, 4, and 5. (G) Viral burden in the CNSwasmeasuredbyplaque assay at day8. Symbols represent individual animals. Dotted lines represent the limit ofsensitivity of the assay. ***P = 0.0009 (Mann-Whitney test).





http://stm.sciencem

ag.org/D

ownloaded from

towild-type cells; (v)TEERwashigher after treatment ofwild-typeBMECsorHCMEC/D3cellswithexogenous IFN-l; (vi) IFN-l treatment restrictedtransit of WNV across HCMEC/D3 monolayers; (vii) treatment of micewith IFN-l minimized BBB permeability changes associated with LPSadministration; and (viii) treatment of mice with IFN-l decreasedWNV infection in the brain without altering viremia, and this was asso-ciated with improved survival rates. We readily observed BBB tighteningeffects of IFN-l even in the context of intact IFN-a/b responses. Circulat-ing IFN-l produced in response to infection may serve a protective roleby limiting virus transit into theCNS and countering the effects of inflam-matory cytokines that increase BBB permeability (for example, TNF-a,IL-1b, or IFN-g) (14, 22, 23).

Howdoes IFN-l signaling affect BBB permeability and limitWNVneuroinvasion? Unlike some viruses, which infect peripheral neuronsand then enter the CNS by retrograde trafficking, WNV likely entersthe CNS via the bloodstream (12). Neuroinvasion by this hematoge-nous route could occur through trafficking of infected cells across theBBB, transcellular or paracellular transit of virus across the cells of theBBB, or direct infection of BBB cells (11, 12). Because we saw no effectof IFNLR1 expression on WNV replication in BMECs or on traffick-ing of immune cells into the CNS, our data suggest a model in whichWNV virions transit through BBB cell junctions and IFN-l signalinglimits this. Indeed, IFN-l treatment inhibited the accumulation of viralRNA on the basal side of anHCMEC/D3monolayer within 6 hours ofexposure to WNV. The effect of IFN-l occurred via a noncanonicalSTAT1- and protein synthesis–independent signaling pathway. IFN-lsignaling enhanced colocalization of the cell junction proteins ZO-1and claudin-5 under conditions of inflammation in vitro and in vivo,which tightened the endothelial barrier. Because the rapid effect ofIFN-l preceded completion of the viral replication cycle (24), the phe-notype was not due to the spread of infection through the cell culture,but rather to cellular responses induced rapidly in response to pathogensensing. Indeed, previous studies demonstrated that active viral repli-cation was not required for endothelial barrier tightening (14).

As mosquitoes inoculate WNV beneath the epidermis, the BBBmay represent one of the first physical barriers encountered by WNVas it spreads in a vertebrate host. Modulation of physical barriers toinfection is a previously undescribed antiviral mechanism for IFN-l.Barrier tightening by IFN-l signaling also may restrict disseminationof viruses that transit across epithelial surfaces, including the skin,lungs, and gastrointestinal tract. Because many viruses use cell junc-tion proteins as entry receptors (30), some of the antiviral effects ofIFN-l observed in other viral systems (26) may be explained by relo-calization of viral entry receptors.

Although our experiments demonstrated a BBB tightening effect ofIFN-l on both human andmurine BMECs in culture, one limitation ofthese studies is that we did not analyze the effects of the different IFN-lsubtypes. Mice encode two functional IFN-l genes (IFN-l2 and IFN-l3),and humans have four paralogs (IFN-l1, 2, 3, and 4). Whereas IFN-l2and IFN-l3 are nearly identical (1), IFN-l1 and IFN-l4 may have dis-tinct properties; a polymorphic allele that enables IFN-l4 production isassociated with impaired hepatitis C virus clearance (31). Because of thelimited types of recombinant proteins available, we were unable toassess whether IFN-l subtypes vary in their BBB tightening activity.

In summary, our results suggest a new function of IFN-l, wherebysignaling through IFNLR1 rapidly modulates endothelial cell junctionswithout a requirement for STAT1 signaling or protein synthesis. Thisresults in a tighter BBB, which restricts pathogen entry into the CNS

www.Scien

parenchyma. In humans, multiple polymorphisms in the IFN-l locusare associated with differential clearance of hepatitis B and C viruses(31), as well as the severity of herpesvirus infections (32, 33). Furtheranalysis is warranted to define similar genetic linkages to neuroinvasiveinfections orCNS autoimmune diseases. Such informationmay providenew contexts for therapeutic interventions with IFN-l.

MATERIALS AND METHODS

Experimental designWe initiated this study to determine whether IFN-l controls WNVpathogenesis. Our initial observation was that Ifnlr1−/− mice had in-creased viral burden in the CNS, and subsequent experiments evaluatedwhy this occurred.Wemeasured viral titers in tissues and replication inprimary cells.We assessed immunologic parameters including antibodyand cytokine concentrations in serum and leukocyte phenotypes in thespleen and brain. After excluding antiviral or immunomodulatory effectsof IFN-l, we consideredwhether increased neuroinvasionmight be dueto altered BBB permeability. We used an in vitro model of the BBB, inwhich resistance across a mouse or human endothelial cell monolayercorresponded to barrier tightness. Sample sizes and endpoints wereselected on the basis of our experience with these systems. Mice wereage- and sex-matched between groups. Investigators were not blindedwhen conducting or evaluating the experiments, and no randomizationprocess was performed.

VirusesWNV strain 3000.0259 was isolated in New York in 2000 and passagedonce in C6/36 cells. Virus titers were measured by plaque assay onBHK21-15 cells (34).

Mouse experimentsC57BL/6 wild-type mice were obtained commercially (Jackson Labora-tories) or bred in-house. Stat1−/− mice (35) were provided by H. Virgin(Washington University). Ifnlr1−/−mice (originally called IL-28RA−/−, ob-tained from Bristol-Myers Squibb) lack the entire Ifnlr1 coding sequence(2). Five- to 12-week-old wild-type and Ifnlr1−/− mice were used for invivo studies, as indicated in the figure legends. For peripheral infection,102 PFU of WNV in 50 ml was inoculated subcutaneously into the foot-pad. For intracranial infection, 101 PFUofWNV in 25 ml was injected intothe right cerebral hemisphere. Experiments were approved and performedin accordance withWashington University Animal Studies Guidelines.

CellsMacrophages, DCs, cortical neurons, BMECs, and astrocytes weregenerated from wild-type or Ifnlr1−/− mice as described (14, 23, 36).The human BMEC cell line HCMEC/D3 was cultured as described(23). The mouse keratinocyte cell line PDV was purchased (Cell LinesService) and cultured inDMEM (Dulbecco’smodified Eagle’smedium)supplemented with 10% fetal bovine serum, L-glutamine, and non-essential amino acids.

IFN treatment and viral infection of cellsKeratinocytes and DCs were treated with recombinant murine IFN-l3(0.1 to 1000 ng/ml) or IFN-b (0.1 or 1 ng/ml) (PBL Assay Science) for6 hours and then infected with WNV at an MOI of 0.1. Supernatantswere titered by focus-forming assay (34). Gene expression was analyzed

ceTranslationalMedicine.org 22 April 2015 Vol 7 Issue 284 284ra59 10




http://stm.sciencem

ag.org/D

ownloaded from

6 hours after IFN treatment. Total RNA was isolated using an RNeasyMini Kit (Qiagen), and fluorogenic qRT-PCR was performed usingOne-Step RT-PCRMasterMix and a 7500 Fast Real-Time PCR System(Applied Biosystems). Gene expression was measured using the indi-cated TaqMan reagents (Integrated DNA Technologies) (table S3).Multistep virus growth analysis was performed after infection at anMOI of 0.01 for macrophages, astrocytes, and BMECs, or 0.001 forDCs and cortical neurons. Viral infection was measured by a focus-forming assay (34).

RNA-seq analysisDCs were generated from wild-type mice and treated with serum-freemedium (mock), IFN-b (20 ng/ml), or IFN-l3 (10 ng/ml) for 6 hours.Total RNA was isolated using an RNeasy Kit (Qiagen). Samples weremultiplexed and barcoded for sequencing on the Illumina HiSeq 2000.Aminimum of 25million (50–base pair pair-end) reads were generatedper sample. Sequencing reads were mapped to the mouse genome [Na-tional Center for Biotechnology Information (NCBI) mm10] usingBowtie (2.10) and Tophat (2.010). Transcript assemblies and quantifi-cations (FPKM) were calculated using the Cufflinks package (2.11).Genes with larger than threefold differences with IFN treatment (rela-tive to mock) were retained, sorted, and displayed in a summarizingheatmap using the Matlab software package.

Tissue viral burdenTo monitor viral spread in vivo, mice were infected with 102 PFU ofWNV by subcutaneous inoculation and sacrificed at specified timepoints. To monitor viral replication directly in the CNS, mice wereinfected with 101 PFU of WNV by intracranial injection and sacrificedat day 3 or 5 after infection. After cardiac perfusion with PBS, organswere harvested and virus was titered by plaque assay (34). Viral RNAwas isolated from serum and plasma using a Viral RNA Mini Kit(Qiagen) and from draining lymph nodes using an RNeasy Mini Kit(Qiagen). Viral load was measured by qRT-PCR with the indicatedprimer and probe sequences (table S3).

Antibody responsesWNV-specific IgM and IgG concentrations in serum were determinedusing an enzyme-linked immunosorbent assay (ELISA) against purifiedWNV E protein, as described (37).

Serum type I IFN activityConcentrations of biologically active type I IFN were determined usinga bioassay as described (38).

Cytokine analysisCytokine concentrations in serum were measured 3 and 5 daysafter infection using a Bio-Plex Pro 23-Plex Group I Cytokine Kit(Bio-Rad) and Bio-Plex 200 (Bio-Rad).

Cellular immune responsesWild-type and Ifnlr1−/− mice were infected subcutaneously with 102

PFU of WNV, and at 8 days after infection, spleens and brains wereharvested after cardiac perfusion with PBS (39). Cells were incubatedwith the following antibodies and processed by flow cytometry (LSRII, Becton Dickinson): CD3 (eBioscience, clone 145-2C11), CD4 (Bio-Legend, clone RM4-5), CD8a (BioLegend, clone 53-6.7), CD19 (Invi-trogen), CD45 (BioLegend, clone 30-F11), CD11b (BioLegend, clone

www.Scien

M1/70), CD11c (BioLegend, clone N418), granzyme B (Invitrogen),IFN-g (BioLegend, clone XMG1.2), and TNF-a (BioLegend, cloneMP6-XT22). IFN-g and TNF-a staining was performed after ex vivorestimulation with a Db-restricted NS4B immunodominant peptideusing 1 mMof peptide and brefeldin A (5 mg/ml) (Sigma). Flow cytom-etry data were analyzed using FlowJo software (Tree Star).

BBB permeability measurements and confocal microscopyEight-week-old mice were infected with 102 PFU of WNV or diluent(mock), and BBB permeability was assessed after 4 days. In otherexperiments, mice were treated with LPS (List Biological Laboratories,3 mg/kg via intraperitoneal injection) and pegylated murine IFN-l2(Bristol-Myers Squibb, 25mg via intravenous injection), andBBBperme-ability was assessed 24 hours later. Sodium fluorescein dye (100 mg/ml)was administered via intraperitoneal injection in 100 ml. After 45 min,blood was collected by cardiac puncture into EDTA-coated tubes.Mice were perfused and CNS tissues were harvested, homogenizedinto PBS, clarified by centrifugation, precipitated in 1% trichloroa-cetic acid, and neutralized with borate buffer. Fluorescence emissionat 485 and 528 nm was determined using the microplate reader Syn-ergy H1 and Gen5 software (BioTek Instruments Inc.). Fluoresceinconcentration was calculated from a standard curve, and tissue flu-orescence values were normalized to the plasma fluorescence valuesfrom the same mouse.

Polarized BMECs on chamber slides were treated first with LPS(100 ng/ml) for 18 hours and then with murine IFN-l3 (100 ng/ml)for 6 hours. Cells were fixed in methanol and blocked with 10% goatserum. Mice were perfused with PBS and then 4% paraformaldehyde.Cryoprotected brains were embedded in O.C.T. medium (Tissue-Tek),and 8-mm frozen sections were mounted on slides. Cells and sectionswere stainedwith primary antibodies against the cell junction proteinsZO-1 (rat monoclonal R40.76, Millipore) and claudin-5 (rabbit poly-clonal, Invitrogen) followed by Alexa Fluor–conjugated secondaryantibodies. Colocalization of ZO-1 and claudin-5 staining was calcu-lated using ImageJ software. Endogenous mouse IgG was detected inbrain sections using an Alexa Fluor 488 anti-mouse IgG antibody.Nuclei were stained with TO-PRO-3. The green channel of individualimages was converted to 8-bit grayscale format, and mean gray values(sum of gray values in all pixels per total number of pixels) were mea-sured using ImageJ software.

Transwell cultures and TEER measurementsBMECs and HCMEC/D3 cells were grown until fully polarized inTranswell cultures (23). BMECs were grown above astrocyte cultures,whereas HCMEC/D3 cells were grown without astrocytes. TEER wasmeasured via chopstick electrode with an EVOM volt meter (WorldPrecision Instruments). Resistance values are reported as W/cm2, withthe resistance value for Transwell inserts with no cells subtracted asbackground. TEERmeasurements were collected at 6 hours after infec-tion withWNV, anMOI of 0.01, or treatment with murine IFN-l3 (10or 100 ng/ml),murine or human IFN-b (10ng/ml), or pegylated humanIFN-l1 (100 ng/ml) (Bristol-Myers Squibb); mock wells were treatedwith culturemedium. To block IFN-a/b signaling, BMEC cultures weretreated with the blocking monoclonal antibody MAR1-5A3 (25 mg/ml)(25) for 1 hour before infection. A nonbinding monoclonal antibody(GIR-208)was used as an isotype control (25). To block de novo proteinsynthesis, BMEC cultures were pretreatedwith cycloheximide (20 mg/ml)for 18 hours before treatment with IFN-b or IFN-l3 or infection with




WNV.Tomeasure virus transit across the endothelial barrier,HCMEC/D3cells in Transwells were treated first with LPS (100 ng/ml) for 18 hours,and then human IFN-b or pegylated human IFN-l1 (100 ng/ml) for6 hours. WNV was added to the upper chamber of the Transwell atan MOI of 0.01. After 6 hours, virus in the lower chamber wasmeasured by qRT-PCR.

Statistical analysisTissue titers and immunophenotyping experiments were analyzed bythe Mann-Whitney test, whereas parametric tests (t test and two-wayANOVA) were used in other experiments. Survival was analyzed by thelog-rank test.

hD

ownloaded from

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/284/284ra59/DC1Fig. S1. Serum antibody responses in wild-type and Ifnlr1−/− mice.Fig. S2. Serum type I IFN activity in wild-type and Ifnlr1−/− mice.Fig. S3. ISG induction in BMECs.Table S1. Transcriptional profiling of DCs treated with IFN-b or IFN-l.Table S2. Serum cytokines after WNV infection.Table S3. Primer and probe sequences used for qRT-PCR.Primary data tables. (Excel)


ttp://stm.sciencem

ag.org/

REFERENCES AND NOTES

1. R. P. Donnelly, S. V. Kotenko, Interferon-lambda: A new addition to an old family. J. InterferonCytokine Res. 30, 555–564 (2010).

2. N. Ank, M. B. Iversen, C. Bartholdy, P. Staeheli, R. Hartmann, U. B. Jensen, F. Dagnaes-Hansen,A. R. Thomsen, Z. Chen, H. Haugen, K. Klucher, S. R. Paludan, An important role for type IIIinterferon (IFN-l/IL-28) in TLR-induced antiviral activity. J. Immunol. 180, 2474–2485 (2008).

3. C. Sommereyns, S. Paul, P. Staeheli, T. Michiels, IFN-lambda (IFN-l) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLOS Pathog. 4, e1000017(2008).

4. M. Mordstein, E. Neugebauer, V. Ditt, B. Jessen, T. Rieger, V. Falcone, F. Sorgeloos, S. Ehl,D. Mayer, G. Kochs, M. Schwemmle, S. Günther, C. Drosten, T. Michiels, P. Staeheli,Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tractsresistant to viral infections. J. Virol. 84, 5670–5677 (2010).

5. J. Pott, T. Mahlakõiv, M. Mordstein, C. U. Duerr, T. Michiels, S. Stockinger, P. Staeheli,M. W. Hornef, IFN-l determines the intestinal epithelial antiviral host defense. Proc. Natl.Acad. Sci. U.S.A. 108, 7944–7949 (2011).

6. N. Ank, H. West, C. Bartholdy, K. Eriksson, A. R. Thomsen, S. R. Paludan, Lambda interferon(IFN-l), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activityagainst select virus infections in vivo. J. Virol. 80, 4501–4509 (2006).

7. M. Mordstein, G. Kochs, L. Dumoutier, J. C. Renauld, S. R. Paludan, K. Klucher, P. Staeheli,Interferon-l contributes to innate immunity of mice against influenza A virus but notagainst hepatotropic viruses. PLOS Pathog. 4, e1000151 (2008).

8. T. J. Nice, M. T. Baldridge, B. T. McCune, J. M. Norman, H. M. Lazear, M. Artyomov, M. S. Diamond,H. W. Virgin, Interferon-l cures persistent murine norovirus infection in the absence of adaptiveimmunity. Science 347, 269–273 (2015).

9. D. Ma, D. Jiang, M. Qing, J. M. Weidner, X. Qu, H. Guo, J. Chang, B. Gu, P. Y. Shi, T. M. Block,J. T. Guo, Antiviral effect of interferon lambda against West Nile virus. Antiviral Res. 83, 53–60(2009).

10. J. J. Sejvar, Clinical manifestations and outcomes of West Nile virus infection. Viruses 6,606–623 (2014).

11. W. W. Suen, N. A. Prow, R. A. Hall, H. Bielefeldt-Ohmann, Mechanism of West Nile virusneuroinvasion: A critical appraisal. Viruses 6, 2796–2825 (2014).

12. O. O. Koyuncu, I. B. Hogue, L. W. Enquist, Virus infections in the nervous system. Cell HostMicrobe 13, 379–393 (2013).

13. S. Lustig, H. D. Danenberg, Y. Kafri, D. Kobiler, D. Ben-Nathan, Viral neuroinvasion andencephalitis induced by lipopolysaccharide and its mediators. J. Exp. Med. 176, 707–712 (1992).

14. B. P. Daniels, D. W. Holman, L. Cruz-Orengo, H. Jujjavarapu, D. M. Durrant, R. S. Klein, Viralpathogen-associated molecular patterns regulate blood brain barrier integrity viacompeting innate cytokine signals. mBio 5, e01476-14 (2014).

www.Scien

15. J. Kraus, K. Voigt, A. M. Schuller, M. Scholz, K. S. Kim, M. Schilling, W. R. Schäbitz, P. Oschmann,B. Engelhardt, Interferon-b stabilizes barrier characteristics of the blood–brain barrier in fourdifferent species in vitro. Mult. Scler. 14, 843–852 (2008).

16. C. E. Markowitz, Interferon-beta: Mechanism of action and dosing issues. Neurology 68, S8–S11(2007).

17. S. E. Doyle, H. Schreckhise, K. Khuu-Duong, K. Henderson, R. Rosler, H. Storey, L. Yao, H. Liu,F. Barahmand-pour, P. Sivakumar, C. Chan, C. Birks, D. Foster, C. H. Clegg, P. Wietzke-Braun,S. Mihm, K. M. Klucher, Interleukin-29 uses a type 1 interferon-like program to promoteantiviral responses in human hepatocytes. Hepatology 44, 896–906 (2006).

18. Z. Zhou, O. J. Hamming, N. Ank, S. R. Paludan, A. L. Nielsen, R. Hartmann, Type III interferon(IFN) induces a type I IFN-like response in a restricted subset of cells through signalingpathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases.J. Virol. 81, 7749–7758 (2007).

19. T. Marcello, A. Grakoui, G. Barba-Spaeth, E. S. Machlin, S. V. Kotenko, M. R. MacDonald,C. M. Rice, Interferons a and l inhibit hepatitis C virus replication with distinct signaltransduction and gene regulation kinetics. Gastroenterology 131, 1887–1898 (2006).

20. P. Y. Lim, M. J. Behr, C. M. Chadwick, P. Y. Shi, K. A. Bernard, Keratinocytes are cell targets ofWest Nile virus in vivo. J. Virol. 85, 5197–5201 (2011).

21. M. S. Suthar, M. S. Diamond, M. Gale Jr., West Nile virus infection and immunity. Nat. Rev.Microbiol. 11, 115–128 (2013).

22. T. Wang, T. Town, L. Alexopoulou, J. F. Anderson, E. Fikrig, R. A. Flavell, Toll-like receptor 3mediates West Nile virus entry into the brain causing lethal encephalitis. Nat. Med. 10,1366–1373 (2004).

23. B. P. Daniels, L. Cruz-Orengo, T. J. Pasieka, P. O. Couraud, I. A. Romero, B. Weksler, J. A. Cooper,T. L. Doering, R. S. Klein, Immortalized human cerebral microvascular endothelial cells maintainthe properties of primary cells in an in vitro model of immune migration across the bloodbrain barrier. J. Neurosci. Methods 212, 173–179 (2013).

24. M. A. Brinton, Replication cycle and molecular biology of the West Nile virus. Viruses 6, 13–53(2014).

25. K. C. Sheehan, K. S. Lai, G. P. Dunn, A. T. Bruce, M. S. Diamond, J. D. Heutel, C. Dungo-Arthur,J. A. Carrero, J. M. White, P. J. Hertzog, R. D. Schreiber, Blocking monoclonal antibodies spe-cific for mouse IFN-a/b receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hy-drodynamic transfection. J. Interferon Cytokine Res. 26, 804–819 (2006).

26. P. Hermant, T. Michiels, Interferon-l in the context of viral infections: Production, responseand therapeutic implications. J. Innate Immun. 6, 563–574 (2014).

27. J. W. Schoggins, Interferon-stimulated genes: Roles in viral pathogenesis. Curr. Opin. Virol.6, 40–46 (2014).

28. H. Shindo, S. Maekawa, K. Komase, M. Miura, M. Kadokura, R. Sueki, N. Komatsu, K. Shindo,F. Amemiya, Y. Nakayama, T. Inoue, M. Sakamoto, A. Yamashita, K. Moriishi, N. Enomoto, IL-28B(IFN-l3) and IFN-a synergistically inhibit HCV replication. J. Viral Hepat. 20, 281–289 (2013).

29. A. J. Muir, S. Arora, G. Everson, R. Flisiak, J. George, R. Ghalib, S. C. Gordon, T. Gray, S. Greenbloom,T. Hassanein, J. Hillson, M. A. Horga, I. M. Jacobson, L. Jeffers, K. V. Kowdley, E. Lawitz, S. Lueth,M. Rodriguez-Torres, V. Rustgi, L. Shemanski, M. L. Shiffman, S. Srinivasan, H. E. Vargas,J. M. Vierling, D. Xu, J. C. Lopez-Talavera, S. Zeuzem; EMERGE Study Group, A randomizedphase 2b study of peginterferon lambda-1a for the treatment of chronic HCV infection.J. Hepatol. 61, 1238–1246 (2014).

30. J. Grove, M. Marsh, The cell biology of receptor-mediated virus entry. J. Cell Biol. 195,1071–1082 (2011).

31. T. R. O’Brien, L. Prokunina-Olsson, R. P. Donnelly, IFN-l4: The paradoxical new member ofthe interferon lambda family. J Interferon Cytokine Res. 34, 829–838 (2014).

32. S. J. Griffiths, M. Koegl, C. Boutell, H. L. Zenner, C. M. Crump, F. Pica, O. Gonzalez, C. C. Friedel,G. Barry, K. Martin, M. H. Craigon, R. Chen, L. N. Kaza, E. Fossum, J. K. Fazakerley, S. Efstathiou,A. Volpi, R. Zimmer, P. Ghazal, J. Haas, A systematic analysis of host factors reveals a Med23-interferon-l regulatory axis against herpes simplex virus type 1 replication. PLOS Pathog. 9,e1003514 (2013).

33. A. Egli, A. Levin, D. M. Santer, M. Joyce, D. O’Shea, B. Thomas, L. F. Lisboa, K. Barakat, R. Bhat,K. P. Fischer, M. Houghton, D. L. Tyrrell, D. Kumar, A. Humar, Immunomodulatory function ofinterleukin 28B during primary infection with cytomegalovirus. J. Infect. Dis. 210, 717–727(2014).

34. J. D. Brien, H. M. Lazear, M. S. Diamond, Propagation, quantification, detection, and storageof West Nile virus. Curr. Protoc. Microbiol. 31, 15D.3.1–15D.3.18 (2013).

35. J. E. Durbin, R. Hackenmiller, M. C. Simon, D. E. Levy, Targeted disruption of the mouseStat1 gene results in compromised innate immunity to viral disease. Cell 84, 443–450(1996).

36. M. A. Samuel, K. Whitby, B. C. Keller, A. Marri, W. Barchet, B. R. Williams, R. H. Silverman, M. Gale Jr.,M. S. Diamond, PKR and RNase L contribute to protection against lethal West Nile virus infectionby controlling early viral spread in the periphery and replication in neurons. J. Virol. 80,7009–7019 (2006).

37. E. Mehlhop, M. S. Diamond, Protective immune responses against West Nile virus areprimed by distinct complement activation pathways. J. Exp. Med. 203, 1371–1381(2006).




38. S. Daffis, M. A. Samuel, B. C. Keller, M. Gale Jr., M. S. Diamond, Cell-specific IRF-3 responsesprotect against West Nile virus infection by interferon-dependent and -independent mechanisms.PLOS Pathog. 3, e106 (2007).

39. H. M. Lazear, A. K. Pinto, H. J. Ramos, S. C. Vick, B. Shrestha, M. S. Suthar, M. Gale Jr.,M. S. Diamond, Pattern recognition receptor MDA5 modulates CD8+ T cell-dependentclearance of West Nile virus from the central nervous system. J. Virol. 87, 11401–11415(2013).

Acknowledgments: We thank J. Brien for 18S qRT-PCR reagents and D. Dorsey for technicalassistance. Funding: This work was supported by NIH grants U19 AI083019 (M.S.D., R.S.K., andM.G.), PCTAS AI083019-02S1 and T32-AI007172 (H.M.L.), R01 AI074973 (M.S.D. and M.G.), and R01NS052632 (R.S.K.). B.P.D. was supported by NSF (DGE-1143954) and NIH (F31-NS07866-01)Fellowships. Author contributions: Study concept and design: H.M.L., B.P.D., R.S.K., and M.S.D.Data acquisition: H.M.L. (qRT-PCR, viral tissue titers, primary cell infections, serum antibodyELISA, BBB permeability measurements, and in vivo treatment with IFN-l), B.P.D. (endothelialcell culture, TEER measurements, BBB permeability measurements, and confocal microscopy),

www.Scien

A.K.P. (immunophenotyping), A.C.H. (RNA-seq), and S.C.V. (viral titers and cytokine assays).Analysis and interpretation of data: H.M.L., B.P.D., A.K.P., A.C.H., M.G., R.S.K., and M.S.D. Providedcritical reagents: S.E.D. Initial drafting of the manuscript: H.M.L. and M.S.D. Critical revision of themanuscript: H.M.L., B.P.D., M.G., R.S.K., and M.S.D. Competing interests: S.E.D. is an employee ofZymoGenetics. Data and materials availability: RNA-seq data are deposited in the GEO (GeneExpression Omnibus) archive, accession number GSE64735. Ifnlr1−/− mice and pegylated IFN-lproteins can be obtained from Bristol-Myers Squibb with a material transfer agreement.

Submitted 5 December 2014Accepted 2 April 2015Published 22 April 201510.1126/scitranslmed.aaa4304

Citation: H. M. Lazear, B. P. Daniels, A. K. Pinto, A. C. Huang, S. C. Vick, S. E. Doyle, M. Gale Jr.,R. S. Klein, M. S. Diamond, Interferon-l restricts West Nile virus neuroinvasion by tightening theblood-brain barrier. Sci. Transl. Med. 7, 284ra59 (2015).



http://stm.sciencem

ag.org/D

ownloaded from


restricts West Nile virus neuroinvasion by tightening the blood-brain barrierλInterferon-

Robyn S. Klein and Michael S. DiamondHelen M. Lazear, Brian P. Daniels, Amelia K. Pinto, Albert C. Huang, Sarah C. Vick, Sean E. Doyle, Michael Gale, Jr.,

DOI: 10.1126/scitranslmed.aaa4304, 284ra59284ra59.7Sci Transl Med

contributes to maintaining tissue barriers that restrict viral pathogenesis.λinterferon- protected mice from West Nile virus infection in the brain and subsequent death. Thus,λof exogenous interferon-

signaling tightened the blood-brain barrier and limited West Nile virus dissemination into the brain. Administration λ did not inhibit viral replication directly. Instead, interferon-λthe brain and spinal cord, even though interferon-

signaling sustained greater West Nile virus infection inλ observed that mice with genetic defects in interferon-et al. is among many secreted host proteins that activate an antiviral response. In new work, Lazear λInterferon-

Interfering with viral neuroinvasion

ARTICLE TOOLS http://stm.sciencemag.org/content/7/284/284ra59

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2015/04/20/7.284.284ra59.DC1

CONTENTRELATED

http://stm.sciencemag.org/content/scitransmed/10/440/eaao0052.fullhttp://stke.sciencemag.org/content/sigtrans/10/466/eaaf5185.fullhttp://stm.sciencemag.org/content/scitransmed/7/289/289er5.fullhttp://stke.sciencemag.org/content/sigtrans/8/374/ec111.abstract

REFERENCES

http://stm.sciencemag.org/content/7/284/284ra59#BIBLThis article cites 39 articles, 13 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science Translational Medicinetitle licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. TheScience, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine


http://stm.sciencem

ag.org/D

ownloaded from

http://stm.sciencemag.org/content/7/284/284ra59

http://stm.sciencemag.org/content/suppl/2015/04/20/7.284.284ra59.DC1

http://stke.sciencemag.org/content/sigtrans/8/374/ec111.abstract

http://stm.sciencemag.org/content/scitransmed/7/289/289er5.full

http://stke.sciencemag.org/content/sigtrans/10/466/eaaf5185.full

http://stm.sciencemag.org/content/scitransmed/10/440/eaao0052.full

http://stm.sciencemag.org/content/7/284/284ra59#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


interferon-l restricts west nile virus neuroinvasion by tightening...

Documents