Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

Interleukin-10 Modulation of Virus Clearance and Disease in

Mice with Alphaviral Encephalomyelitis

Nina M. Martin,

Diane E. Griffin

a_{W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg}

School of Public Health, Baltimore, Maryland, USA

ABSTRACT

Alphaviruses are an important cause of mosquito-borne outbreaks of

ar-thritis, rash, and encephalomyelitis. Previous studies in mice with a virulent strain

(neuroadapted SINV [NSV]) of the alphavirus Sindbis virus (SINV) identified a role for

Th17 cells and regulation by interleukin-10 (IL-10) in the pathogenesis of fatal

en-cephalomyelitis (K. A. Kulcsar, V. K. Baxter, I. P. Greene, and D. E. Griffin, Proc Natl

Acad Sci U S A 111:16053–16058, 2014,

https://doi.org/10.1073/pnas.1418966111

). To

determine the role of virus virulence in generation of immune responses, we analyzed

the modulatory effects of IL-10 on disease severity, virus clearance, and the CD4

T cell

response to infection with a recombinant strain of SINV of intermediate virulence

(TE12). The absence of IL-10 during TE12 infection led to longer morbidity, more weight

loss, higher mortality, and slower viral clearance than in wild-type mice. More severe

disease and impaired virus clearance in IL-10

mice were associated with more Th1

cells, fewer Th2 cells, innate lymphoid type 2 cells, regulatory cells, and B cells, and

delayed production of antiviral antibody in the central nervous system (CNS) without an

effect on Th17 cells. Therefore, IL-10 deficiency led to more severe disease in

TE12-infected mice by increasing Th1 cells and by hampering development of the local B cell

responses necessary for rapid production of antiviral antibody and virus clearance from

the CNS. In addition, the shift from Th17 to Th1 responses with decreased virus

virulence indicates that the effects of IL-10 deficiency on immunopathologic responses

in the CNS during alphavirus infection are influenced by virus strain.

IMPORTANCE

Alphaviruses cause mosquito-borne outbreaks of encephalomyelitis,

but determinants of outcome are incompletely understood. We analyzed the effects

of the anti-inflammatory cytokine IL-10 on disease severity and virus clearance after

infection with an alphavirus strain of intermediate virulence. The absence of IL-10

led to longer illness, more weight loss, more death, and slower viral clearance than

in mice that produced IL-10. IL-10 influenced development of disease-causing T cells

and entry into the brain of B cells producing antiviral antibody. The Th1 pathogenic

cell subtype that developed in IL-10-deficient mice infected with a less virulent virus

was distinct from the Th17 subtype that developed in response to a more virulent

virus, indicating a role for virus strain in determining the immune response. Slow

production of antibody in the nervous system led to delayed virus clearance.

There-fore, both the virus strain and the host response to infection are important

determi-nants of outcome.

KEYWORDS

B cells in brain, CD4

T cells, IL-10, Sindbis virus, Th1 cells, antibody in

brain, central nervous system infections, encephalitis, mice

O

utbreaks of mosquito-borne viral diseases are on the rise, with recent extensions

of rash, arthritis and encephalomyelitis to new regions (1, 2). Alphaviruses,

includ-ing chikungunya, eastern equine encephalitis, and Venezuelan equine encephalitis

viruses, are important causes of these outbreaks and can cause encephalomyelitis, a

Received29 August 2017Accepted11 December 2017

Accepted manuscript posted online20 December 2017

CitationMartin NM, Griffin DE. 2018. Interleukin-10 modulation of virus clearance and disease in mice with alphaviral encephalomyelitis. J Virol 92:e01517-17. https://doi.org/10.1128/JVI.01517-17.

EditorTerence S. Dermody, University of Pittsburgh School of Medicine

Copyright© 2018 American Society for Microbiology.All Rights Reserved.

Address correspondence to Diane E. Griffin, dgriffi6@jhu.edu.

crossm

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potentially fatal inflammatory disease of the central nervous system (CNS) with

fre-quent long-term neurological deficits in survivors (3–5).

Sindbis virus (SINV), the prototypical alphavirus, is the most widespread alphavirus

and causes rash and arthritis in humans (6). In mice, SINV causes encephalomyelitis, the

severity of which depends on virus and host factors, including host age and genetic

background and virus strain (7–10). SINV strains with various degrees of pathogenicity

for mice have been developed by passage

in vitro

and

in vivo

and by construction of

recombinant viruses. Neuroadapted SINV (NSV), a strain obtained by passaging the

original isolate AR339 in mouse brain, causes fatal encephalomyelitis in adult C57BL/6

(B6) mice (8, 11), while virus derived from the tissue culture-passaged HRSP clone

Toto1101 causes little disease even in newborn mice (10). TE12 is a recombinant SINV

strain with the E1 and E2 envelope glycoproteins from NSV inserted into the Toto1101

background and has intermediate virulence, with approximately 50% mortality in adult

B6 mice (10). Strains with variable virulence allow for identification of factors associated

with immunopathogenesis and death as well as recovery and virus clearance (7).

Previous studies have shown that the immune response has both positive and

negative effects on disease pathogenesis after SINV infection. In nonfatal infections,

both antibody and interferon gamma (IFN-

␥

) contribute to noncytolytic viral clearance

from neurons (12–16), while in fatal encephalomyelitis, T cell responses regulated by

interleukin-10 (IL-10) are implicated in immunopathogenesis and death (17–21). In

particular, in NSV-infected IL-10-deficient mice, Th17 cells are associated with

acceler-ated morbidity and mortality (19, 20). IL-10 dysregulation has also been implicacceler-ated in

inflammatory disease due to infection with influenza virus and cytomegalovirus (22, 23),

as well as in autoimmune diseases (24–28).

Previous studies of NSV-infected IL-10-deficient mice also indicated a delay in viral

clearance compared to that in wild-type (WT) mice, but rapid death of the mice made

analysis of the mechanism difficult. Therefore, in the current study we analyzed the role

of IL-10 in pathogenesis of disease in mice that survived longer after infection than

NSV-infected mice. IL-10-deficient mice infected with TE12 had longer morbidity, more

weight loss, higher mortality, and slower viral clearance than WT mice. More severe

disease in IL-10

mice was associated with more Th1 cells, fewer Th2 T cells, type 2

innate lymphoid cells, regulatory T cells (Tregs) and B cells (Bregs), and B cells, and

delayed production of antiviral antibody in the CNS after infection without an effect on

Th17 responses. These data demonstrate an important but slightly different role for

IL-10 in regulating pathogenesis during infection with a less virulent strain of SINV than

NSV and identify increased Th1 and reduced Th2 and B cell responses in the CNS that

hamper local antiviral antibody production as reasons for exacerbated disease and

delayed virus clearance.

RESULTS

IL-10 is upregulated in response to TE12 infection.

To determine if IL-10 is

upregulated in the CNS after TE12 infection, mRNA expression in the brains and spinal

cords of WT mice was measured and compared to those in uninfected controls. In both

the brain (Fig. 1A) and spinal cord (Fig. 1B),

Il10

gene expression was increased by day

3, peaked at day 10, and remained above baseline through 21 days postinfection.

IL-10 deficiency leads to prolonged morbidity and increased mortality.

To

determine the effect of IL-10 deficiency on TE12-induced morbidity and mortality, 4- to

6-week-old WT and IL-10

mice were intranasally infected and evaluated daily. The

mean days of death were similar, but IL-10-deficient mice had higher mortality (62.5%)

than WT mice (35.0%) (Fig. 2A) (

P

⫽

0.0356). Mice lacking IL-10 also showed signs of

encephalomyelitis earlier (day 3) than WT mice (day 4), and those that survived were

slower to recover from infection (Fig. 2B). IL-10-deficient mice also lost more weight,

and surviving mice recovered weight more slowly than WT mice. These clinical

mani-festations of disease taken together demonstrate an important role for IL-10 in survival

and recovery from TE12 infection.

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IL-10 deficiency leads to delayed clearance of infectious virus and viral RNA.

To

assess the impact of IL-10 deficiency on virus replication and clearance, infectious virus

in brain (Fig. 3A) and spinal cord (Fig. 3B) was measured by plaque assay. Amounts of

infectious virus peaked at day 3 to 5 after infection with similar virus titers in both WT

0 3 5 7 10 14 21 10-1

100 101 102 103 104

Days Post Infection

IL-1

0

m

R

N

A

Fold C

h

a

n

g

e

0 3 5 7 10 14 21 0

20 40 60

Days Post Infection

IL

-10 mRN

A

Fold Cha

n

g

e

A

B

Brain

Spinal Cord

FIG 1IL-10 is upregulated in response to TE12 infection. WT B6 mice were intranasally infected with 105

PFU TE12.Il10mRNA expression was measured by quantitative real-time PCR in the brains (A) and spinal cords (B) of TE12-infected mice.CTvalues were normalized to GAPDH RNA.CTvalues and fold change were calculated relative to uninfected controls (ΔΔCT). Data are averaged from 2 independent experi-ments and represent the mean⫾SEM for 6 individual mice at each time point.

0 3 6 9 12 15 18 21 50

75 100 125

** *

*****

*

%

We

ig

h

t

C

ha

n

g

e

0 4 8 12 16 20

0 1 2 3 4

C

linic

a

l Sc

or

e

**

* ***

*

IL10KO WT

0 5 10 15 20 25 0

20 40 60 80

100 _{p =}

10d

11d

0.0356

P

erce

nt

su

rvi

v

al

A

B

C

Days after infection Days after infection

Days after infection

FIG 2IL-10 deficiency leads to prolonged morbidity and increased mortality after TE12 infection. WT and IL-10⫺/⫺_{B6 mice were intranasally infected with 10}5 _{PFU TE12. (A) Survival was assessed using a}

Kaplan–Meier curve and log rank (Mantel-Cox) test,P⫽0.0356. The median days of death were 10 for WT and 11 for IL-10⫺/⫺_{mice. (B) Signs of disease were monitored daily. The clinical score scale was as}

follows: 0, no signs; 1, abnormal hind-limb and tail posture, ruffled fur, and/or hunched back; 2, unilateral hind-limb paralysis; 3, bilateral hind-limb paralysis and/or moribund; 4, dead. (C) The percent weight change was determined by comparing the weight each day to the original weight of each mouse. Data are pooled from 3 independent experiments for a total of 40 mice for each group and presented as the mean⫾SEM.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001.

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and IL-10

mice. However, viral clearance was delayed in IL-10-deficient mice

com-pared to WT mice in both brain and spinal cord. While infectious virus was cleared from

most WT mice by day 7, it was still detectable in many IL-10

mice at days 7 and 10

after infection.

The amount of viral RNA was also quantified in brain (Fig. 3C) and spinal cord (Fig.

3D). SINV E2 gene RNA was detected by 3 days after infection, and amounts of viral RNA

peaked on day 5 to 7 in brain and spinal cord of both WT and IL-10

mice. However,

IL-10-deficient mice had higher viral RNA levels on days 10 and 14 in brain and on day

10 in spinal cord than WT mice. These results combined show a role for IL-10 in

regulating viral clearance in both the brain and spinal cord.

Effect of IL-10 deficiency on CD3

, CD4

, and CD8

T cells.

To assess the impact

of IL-10 deficiency on T cell responses to TE12 infection, lymphocytes isolated from the

draining cervical lymph nodes (CLNs) and brain tissues from IL-10

and WT mice on

days 5, 7, and 10 after infection were analyzed by flow cytometry (Fig. 4A). IL-10

deficiency did not affect the total number of cells in brain (Fig. 4B) or CLN (Fig. 4E), but

the numbers (Fig. 4C) and percentages (Fig. 4F) of CD3

T cells were higher in the

brains, but not the CLNs (Fig. 4D and G), of IL-10

mice.

To characterize the T cells present, CD4

and CD8

T cells were quantified in brains

and CLNs from WT and IL-10

mice (Fig. 5A and 6A). On days 7 and 10 after infection,

more CD4

T cells were present in the brains of IL-10-deficient mice than in those of

WT mice (Fig. 5B), and the percentage of cells that were CD4

T cells was higher in

IL-10

mice at 5 and 10 days after infection (Fig. 5C). There were no differences in

numbers or percentages of CD4

T cells in CLNs (Fig. 5D and E).

Similar analysis of the impact of IL-10 deficiency on CD8

T cells showed more CD8

T cells in the brains of IL-10

mice at 7 and 10 days after infection (Fig. 6B, top panel),

0 3 5 7 10 14 21 0

1 2 3 4 5 6 7 8

***

***

WT IL10KO

Log

(PF

U/

g

)

0 3 5 7 10 14 21 0

1 2 3 4 5

*

Log

(PF

U

/g

)

0 3 5 7 10 14 21

10-1 100 101 102 103 104 105

***

***

SV E2

C

opie

s

/

1

0

^6

Ge

nom

e

C

o

pie

s

0 3 5 7 10 14 21

10-1 100 101 102 103 104 105

***

SV E2

C

opie

s

/

1

0

^6

Ge

nom

e

C

o

pie

s

A

B

C

D

Brain

Spinal Cord

Days after infection

Days after infection

Days after infection

Days after infection

FIG 3IL-10 deficiency leads to delayed clearance of infectious virus and viral RNA. WT and IL-10⫺/⫺_B6

mice were intranasally infected with 105_{PFU TE12. (A and B) Virus titers were determined by plaque assay}

using brain (A) or spinal cord (B) homogenates prepared from WT (filled squares, solid lines) and IL-10⫺/⫺

(open circles, dashed lines) mice. The limit of detection is displayed as a horizontal dotted line. For statistical analysis, mice with no detectable plaques were assigned a value midway between the limit of detection and zero. (C and D) Viral RNA copy numbers were determined by RT-qPCR on RNA isolated from brain (C) or spinal cord (D). Data are pooled from 3 independent experiments and presented as the mean⫾SEM for 6 mice at each time point.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001.

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but the percentages were not different (Fig. 6B, bottom panel). There were no

differ-ences in the numbers or percentages of CD8

T cells in the CLNs (Fig. 6C). Therefore,

IL-10 deficiency generally increases the representation of CD3

, CD4

, and CD8

T cells

in the CNS in response to TE12 infection.

Effect of IL-10 deficiency on Th1 cells and Th1-related cytokines.

Subsets of

CD4

T cells are important for viral clearance but can also be a source of

immuno-pathogenesis. To determine the impact of IL-10 deficiency on production of Th1-related

cytokines, reverse transcription-quantitative PCR (RT-qPCR) was performed on cDNA

from brains of IL-10

or WT mice. Levels of

Il2

mRNA (Fig. 7A) in IL-10

mice

increased similarly to but were higher than those in WT mice at 7 to 14 days after

5 7 10

0 10 20 30 40

** *

#

CD3

+ C

e

ll

s

(x10

4)

WT IL10KO

3 5 7 10

0 10 20 30

WT IL10KO

Ce

ll

C

ount

s

/M

ou

s

e

(x10

5)

5 7 10

0 20 40 60 80 100

# CD3+

Ce

ll

s

(x1

0

5)

3 5 7 10

0 50 100 150 200

C

e

ll C

ount

s

/M

ou

s

e

(x10

5)

5 7 10

0 5 10 15 20

**

** ***

CD3+

T

Cel

ls

(% of L

ive)

5 7 10

0 20 40 60

CD3

+

T

Cel

ls

(% of L

ive)

C

B

D

E

A

F

G

Brain

CLN

ni

ar

B

CLN

A

Days after infection Days after infection Days after infection Days after infection

FIG 4Effect of IL-10 deficiency on CD3⫹_{T cells in the CNS. WT and IL-10}⫺/⫺_{B6 mice were intranasally}

infected with 105_{PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains and CLNs}

of WT (black bars) or IL-10-deficient (white bars) mice at 5 (n⫽6), 7 (n⫽10), and 10 (n⫽10) days after infection is shown. (A) Representative flow cytometry plots of cells isolated from brain at 7 days after infection. (B and E) Total numbers of cells isolated from brains (B) and CLNs (E). (C and F) Number (C) and percentage (F) of CD3⫹_{T cells isolated from brain. (D and G) Number (D) and percentage (G) of CD3}⫹

T cells isolated from CLNs. The data represent the mean⫾SEM from 3 independent experiments.*,P⬍

0.05;**,P⬍0.01;***,P⬍0.001.

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infection. Levels of

Ifn

␥

mRNA (Fig. 7C and D) and IFN-

␥

protein (Fig. 7B) increased more

rapidly in WT mice, but IL-10-deficient mice had higher levels of

Ifn

␥

mRNA and protein

than WT mice at late times after infection.

To identify and quantify Th1 cells during TE12 infection, lymphocytes isolated

from brains and CLNs were stained for CD3, CD4, and T-bet, the master transcription

factor for differentiation and activation of Th1 cells (Fig. 7E). Brains of IL-10

mice

had more Th1 cells at 7 days after infection than those of WT mice (Fig. 7F). There

were no significant differences in numbers or percentages of Th1 cells in the CLNs

(Fig. 7H and I).

C

B

D

E

A

CD3

CD4

42.3

38.8

40.7

41.3

Brain

CLN

Days after infection

Days after infection

Days after infection

Days after infection

FIG 5Effect of IL-10 deficiency on CD4⫹_{T cells in the CNS. WT and IL-10}⫺/⫺_{B6 mice were intranasally}

infected with 105_{PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLNs of}

WT (black bars) or IL-10-deficient (white bars) mice at 5 (n⫽6), 7 (n⫽10), and 10 (n⫽10) days postinfection (dpi) is shown. (A) Representative flow cytometry plots of cells isolated from brain and CLN at 7 days after infection. (B and C) Number (B) and percentage (C) of CD3⫹_{cells that were CD4}⫹_{T cells}

in brain. (D and E) Number (D) and percentage (E) of CD3⫹_{T cells in CLNs that were CD4}⫹_{T cells. The}

data represent the mean⫾SEM from 3 independent experiments.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001.

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Effect of IL-10 deficiency on ILC2 cells, Th2 cells, and Th2-related cytokines.

Th2

cells are important for activation and differentiation of B cells and ultimate production

of antibody. To determine the impact of IL-10 deficiency on expression of genes for

Th2-related proteins, RT-qPCR was performed on cDNA from brains from IL-10

and

WT mice.

Gata3

mRNA was lower in brains of IL-10

mice than in those of WT mice

at all times after infection (Fig. 8A and B). Levels of

Il4

mRNA did not increase in

response to infection in either IL-10

or WT mice (Fig. 8C), consistent with the

posttranscriptionally regulated expression of this cytokine.

Th2 cell frequency in CNS tissues was measured via flow cytometry by quantifying

CD4

T cells that produce IL-4 and express Gata3, a transcription factor important for

the differentiation and activation of Th2 cells (Fig. 8D). Mice deficient in IL-10 had lower

numbers and percentages of CD4

T cells producing IL-4 on days 7 and 10

postinfec-tion than WT mice (Fig. 8E and F). There were no differences in numbers or percentages

in CLNs (Fig. 8G and H). Because IL-10 deficiency had an impact on Th2 responses,

numbers of innate lymphoid cell type 2 (ILC2) cells, early sources of IL-5 and IL-13 and

activators of Th2 and B cell responses (29, 30), were examined 5 days after infection

(Fig. 9A), before entry of substantial numbers of T cells (Fig. 4C). Mice deficient in IL-10

had lower numbers and percentages of ILC2 cells in brains (Fig. 9B and C) and fewer

ILC2 cells in CLNs (Fig. 9D) than WT mice on day 5 after infection.

Effect of IL-10 deficiency on Th17 cells.

Previous studies showed that IL-10

mice had accelerated production of

Il17a

mRNA and pathogenic Th17 cells after NSV

infection (19). However, after TE12 infection,

Il17a

mRNA expression in brains of

A

CL

N

Br

a

in

CD3

CD

8

WT

IL

10

KO

7 dpi

55.4

66.4

C

B

Days after infection Days after infection

FIG 6Effect of IL-10 deficiency on CD8⫹_{T cells in the CNS. WT and IL-10}⫺/⫺_{B6 mice were intranasally}

infected with 105_{PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains or CLNs of}

WT (black bars) or IL-10-deficient (white bars) mice at 5 (n⫽6), 7 (n⫽10), and 10 (n⫽10) days postinfection is shown. (A) Representative flow cytometry plots of cells isolated from brains. (B) Number (top panel) and percentage (bottom panel) of CD3⫹_{brain cells that were CD8}⫹_{T cells. (C) Number (top}

panel) and percentage (bottom panel) of CD3⫹_{cells in CLNs that were CD8}⫹_{T cells. The data represent}

the mean⫾SEM from 3 independent experiments.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001.

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IL-10

and WT mice was not different (Fig. 10A and B), and

Il23

and

Gmcsf

mRNAs

increased more in WT than in IL-10

mice after infection (Fig. 10C to F). We also

assessed Th17 responses after TE12 infection by flow cytometry (Fig. 10G). There were

no differences in the numbers or percentages of IL-17a-producing CD4

T cells in the

brain (Fig. 10H and I) or CLN (Fig. 10J and K). Therefore,

Il17a

mRNA levels and Th17

cells increase in the CNS after TE12 infection, but unlike in NSV infection, IL-10

deficiency did not exacerbate this response.

0 3 5 7 10 14 21

100 101 102 103 104 *** *** * WT IL10KO IL-2 m R N A Fold C ha ng e

0 3 5 7 10 14 21

100 101 102 103 104 105 IFN g m R N A Fold C h a ng e ** * * ***

5 7 10

0 20 40 60 ** WT IL10KO T -b et+ (# of li ve x10 3)

5 7 10

0 2 4 6 8 10 T -b et+ (# o f li v e x1 0 5)

0 3 5 7 10 14 21

100 101 102 103 104 105 *** ****** *** IFN g pg/m L

0 3 5 7 10 14 21

10-1 100 101 102 103 104 105 IFN g m R N A Re la ti ve Ge ne Ex pr e s s io n *** ** ***

5 7 10

0 10 20 30 40 50 T-b e t+ (% o f CD4+ )

5 7 10

0 5 10 15 20 25 T-b e t+ (% o f CD4+ )

A

B

C

D

E

F

Brai

n

CL

N

G

H

Days Post Infection

T-b e t T-b e t CD4 CD4

WT IL10-/- _{WT IL10}

-/-5 dpi 7 dpi

CLN Brain CLN Brai n

I

FIG 7Effect of IL-10 deficiency on Th1 cells and Th1-related cytokines. WT and IL-10⫺/⫺_{B6 mice were}

intranasally infected with 105_{PFU TE12. (A to D) Analysis ofIl2mRNA (A), IFN-}␥_{protein (B), andIfn}␥

mRNA (C and D) in the brains of WT (filled squares, solid lines) and IL-10-deficient (open circles, dashed lines) mice. GeneCTvalues were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A and C) or to uninfected WT mice (D) (ΔΔCT). Data were pooled from three independent experiments and presented as the mean⫾SEM from 6 mice at each time point.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001. (E to I) Flow cytometric analysis of cells isolated from the pooled brains (n⫽6) of WT (black bars) or IL-10⫺/⫺_{(white bars)}

mice at 5, 7, and 10 days postinfection. (E) Representative flow cytometry plots of Th1 cells, defined as CD45hi_CD3⫹_CD4⫹_{T cells expressing T-bet. (F to I) Frequency and percentage of CD4}⫹_{T cells}

expressing T-bet in brain (F and G) and CLN (H and I). The data represent the mean⫾SEM from 2 independent experiments.**,P⬍0.01.

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0 3 5 7 10 14 21 0 10 20 30 WT IL10KO ** *** *** *** *** G A T A3 mRN A Fold C ha ng e

0 3 5 7 10 14 21 0.0 0.5 1.0 1.5 2.0 IL-4 m R N A Fold C ha ng e

5 7 10

0 10 20 30 ** ** WT IL10KO IL -4+ T H 2 Cel ls (x1 0 3)

5 7 10

0 2 4 6 8 10 IL-4 + TH 2 + c e lls (x10 5)

0 3 5 7 10 14 21 0 10 20 30 ** *** *** *** *** G AT A3 mRN A Rel a tiv e Ge ne Ex pr e s s io n ***

5 7 10

0 20 40 60 ** ** IL-4 + TH 2 C e lls (% o f CD4+ )

5 7 10

0 2 4 6 IL-4 + TH 2 Ce lls (% of CD4+ )

A

B

C

D

E

F

Brai

n

CLN

G

H

Days Post Infection

Days after infection

Days after infection

Days after infection

FIG 8Effect of IL-10 deficiency on Th2 cells and Th2-related cytokines. WT and IL-10⫺/⫺_{B6 mice were}

intranasally infected with 105_{PFU TE12. (A to C) Analysis ofGata3(A and B) andIl4(C) mRNA expression}

in the brains of WT (filled squares, solid lines) and IL-10⫺/⫺_{(open circles, dashed lines) mice. GeneC}

T values were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A and C) or to uninfected WT mice (B) (ΔΔCT). Data were pooled from three independent experiments and are presented as the mean⫾SEM from 6 mice at each time point.**,P⬍

0.01;***,P⬍0.001. (D to H) Flow cytometric analysis of isolated cells pooled from the brains of WT (black bars) or IL-10⫺/⫺_{(white bars) mice at 5 (n⫽6), 7 (n⫽10), and 10 (n⫽10) days postinfection. (D)}

Representative flow cytometry plots of Th2 cells, defined as CD45hi_CD3⫹_CD4⫹_Gata3⫹_{T cells producing}

IL-4. (E to H) Frequency and percentage of CD4⫹_{T cells producing IL-4 in brain (E and F) and CLN (G and}

H). The data represent the mean⫾SEM from 3 independent experiments.**,P⬍0.01.

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Effect of IL-10 deficiency on regulatory cells.

Regulatory T cells are major

pro-ducers of IL-10 and can affect the function of many immune cells (31). Levels of

foxp3

mRNA transcripts in brain (Fig. 11A and B) were lower in IL-10

mice than in WT mice

from day 5 to 14 after infection. Flow cytometry was performed on lymphocytes

isolated from CLN and brain tissues of IL-10

or WT mice to measure regulatory T

cells (CD3

CD4

CD25

foxP3

) on days 7 and 10 after TE12 infection (Fig. 11C and

D). Staining patterns were distinct, and at day 10 in the CLNs of WT, but not IL-10

,

mice, two populations were evident. IL-10

mice had lower numbers and

percent-ages of Tregs in brains than WT mice on days 7 and 10 (Fig. 11E and F). There were

similar numbers and percentages of Tregs in CLNs (Fig. 11G and H).

Because regulatory B cells (CD19

CD5

CD1d

IgM

), as well as T cells, produce IL-10

and downregulate Th1 and Th17 responses, they were also assessed (Fig. 12A).

IL-10-5 0

10 20

30 ***

WT IL10KO

# I

L

C2

(x10

4)

5 0

2 4 6 8 10

* ***

ILC

2

(%

o

f Li

v

e

)

5 0.0

0.5 1.0

1.5 **

# I

L

C2

(x10

4)

5 0.0

0.5 1.0 1.5 2.0 2.5

ILC

2

(% of L

ive

)

E

D

C

B

Brai

n

CL

N

A

FIG 9Effect of IL-10 deficiency on ILC2 cells. WT and IL-10⫺/⫺_{B6 mice were intranasally infected with 10}5

PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10 WT (black bars) and IL-10⫺/⫺_{(white bars) mice at 5 days after infection is shown. (A) Representative flow cytometry}

plots of ILC2 cells defined as IL-7Ra⫹_lineage⫺_Gata3⫹_{. (B to E) Number and percentage of ILC2 cells in}

brain (B and C) and CLN (D and E). The data are plotted as the mean⫾SEM from 3 independent experiments.**,P⬍0.01;***,P⬍0.001.

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0 3 5 7 10 14 21 0

20 40 60 80 100

IL

-17

a

mRNA

Fold Chang

e WT_IL10KO

0 3 5 7 10 14 21 100

101 102 103 104

IL

-2

3

a

mRNA

Fold Chang

e

*** *** *

* ***

0 3 5 7 10 14 21 100

102 104 106 108

GM-CSF mRNA Fold Chang

e

7 10.

0 5 10 15

# CD4+ IL-17a+ (x10

2)

7 10.

0 10 20 30

# CD4+ IL-17a+ (x10

3)

0 3 5 7 10 14 21 0

20 40 60

IL-17a mRNA

Re

la

tiv

e

Gene Expressio

n

0 3 5 7 10 14 21 100

101 102 103 104

IL-23 mRN

A

Relative

Gene Expressio

n

**

**

0 3 5 7 10 14 21 100

102 104 106 108

GM-CSF mRN

A

Relative

Gene Expressio

n

*

7 10.

0.0 0.5 1.0 1.5

IL-17a+ (% of CD4+

)

7 10.

0.0 0.1 0.2 0.3 0.4

IL-17a+ (% of CD4+

)

B

A

F

E

Brai

n

CLN

G

H

Days Post Infection

D

C

I

J

K

FIG 10Effect of IL-10 deficiency on Th17 cells. WT and IL-10⫺/⫺_{B6 mice were intranasally infected with}

105_{PFU TE12. (A to F) Analysis ofIl17a(A and B),Il23(C and D), andGmcsf(E,F) mRNA expression in the}

brains of WT (filled squares, solid lines) and IL-10⫺/⫺_{(open circles, dashed lines) mice. GeneC}

Tvalues were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A, C, and E) or to uninfected WT mice (B, D, and F) (ΔΔCT). Data are pooled from two independent experiments and presented as the mean⫾SEM from 6 mice at each time point. (G to K) Flow cytometric analysis of cells isolated from the pooled (n⫽10) brains and CLNs of WT (black bars) and IL-10⫺/⫺_{(white bars) mice at 7 and 10 days postinfection. (G) Representative flow cytometry plots}

of Th17 cells, defined as CD3⫹_CD4⫹_{T cells producing IL-17a. (H to K) Number and percentage of Th17}

cells in brain (H and I) and CLN (J and K). The data are plotted as the mean⫾SEM from 3 independent experiments.

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deficient mice also had lower numbers and percentages of regulatory B cells in brains (Fig.

12B and C) and fewer, but similar, percentages in CLNs (Fig. 12D and E) than WT control

mice at 10 days after infection. These data suggest that IL-10 is important for development

of both T and B regulatory cell populations within the infected CNS.

Effect of IL-10 deficiency on CD19

B cells and antibody production in the CNS.

To determine the impact of IL-10 deficiency on B cell entry and antibody production,

CD19

B cells were quantified (Fig. 13A). Mice deficient in IL-10 had lower numbers and

percentages of CD19

B cells in both brains (Fig. 13B and C) and CLNs (Fig. 13D and E).

To determine the effect of fewer B cells on local antibody production, SINV-specific IgM

0 10 20 30 40

WT

IL10KO ***

* **

F

O

X

p

3 mRN

A

Fold C

h

a

n

g

e

7 10

0 10 20 30 40

# TReg

(x10

3)

7 10

0 5 10 15

**

***

WT IL10KO

# TReg

(x1

0

3)

0 3 5 7 10 14 21 0

10 20 30 40

**

FOXp3

m

R

N

A

Re

la

tiv

e

Ge

ne

Ex

pr

e

s

s

io

n

7 10

0 5 10 15

***

***

%

T

Re

g o

f

CD4

7 10

0 10 20 30 40

% T

R

e

g o

f

CD4

A

B

C

D

E

F

Brai

n

CLN

G

H

Days Post Infection

Days after infection

Days after infection

FIG 11Effect of IL-10 deficiency on regulatory T cells. WT and IL-10⫺/⫺_{B6 mice were intranasally infected}

with 105_{PFU TE12. (A and B) Analysis offoxP3mRNA expression in the brains of WT (filled squares, solid}

lines) and IL-10⫺/⫺ _{(open circles, dashed lines) mice during TE12 infection. Gene C}

T values were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A) or to uninfected WT mice (B) (ΔΔCT). Data are pooled from 2 independent experiments and presented as the mean⫾SEM from 6 mice at each time point.*,P⬍0.05;**,P⬍0.01;***,P⬍0.001. (C to H) Flow cytometric analysis of cells isolated from pooled (n⫽10) brains and CLNs from WT (black bars) and IL-10⫺/⫺_{(white bars) mice at 7 and 10 days postinfection. (C and D) Representative flow}

cytometry plots of regulatory T cells, defined as CD3⫹_CD4⫹_CD25⫹_foxP3⫹_{cells. (E to H) Number and}

percentage of regulatory T cells in brain (E and F) and CLN (G and H). The data are plotted as the mean⫾SEM from 3 independent experiments.**,P⬍0.01;***,P⬍0.001.

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and IgG antibodies were quantified via enzyme immunoassay (EIA) (Fig. 14). The

antibody response was delayed in IL-10

mice compared to WT mice, with less

SINV-specific IgM antibody in brains (Fig. 14A) and spinal cords (Fig. 14B) at 7 and 10

days after infection and less SINV-specific IgG in brains (Fig. 14C) and spinal cords (Fig.

14D) on days 10 and 14 after infection.

DISCUSSION

Studies with the intermediate virulent TE12 strain of SINV provided the

oppor-tunity to study mechanisms of immunopathogenesis and viral clearance that are

modulated by IL-10 during alphaviral encephalomyelitis. IL-10 deficiency led to

prolonged morbidity, increased weight loss, increased mortality, and delayed viral

clearance after TE12 infection. IL-10 has an important role in regulating T cell

responses, particularly for subsets of CD4

T cells. More severe disease in

IL-10-deficient mice compared to WT mice was associated with increased numbers of Th1

10 0

10 20 30

40 ***

WT IL10KO

#

B

Reg

s

(x 10

3)

10 0

5 10

15 _***

B

Re

g

s

(% CD19

)

10 0.0

0.5 1.0 1.5

B

Reg

(% CD19

)

10 0

1 2 3 4

***

# B Reg

s

(x

10

4)

B

C

D

E

Brai

n

CL

N

A

FIG 12Effect of IL-10 deficiency on regulatory B cells. WT and IL-10⫺/⫺_{B6 mice were intranasally infected}

with 105_{PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLN of 10 WT}

(black bars) and IL-10⫺/⫺_{(white bars) mice at 10 days postinfection is shown. (A) Representative flow}

cytometry plot of regulatory B cells, defined as CD19⫹_CD1dhi_CD5⫹_IgM⫹_{cells. (B to E) Numbers and}

percentages of Bregs in brain (B and C) and CLN (D and E). The data are plotted as the mean⫾SEM from 3 independent experiments.**,P⬍0.01;***,P⬍0.001.

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cells and decreased numbers of Tregs and Bregs, without a difference in Th17 cells.

IL-10 deficiency also resulted in delayed virus clearance associated with decreased

numbers of Th2 cells, ILC2 cells, and B cells along with lower levels of antiviral

antibody in the CNS. Therefore, in contrast to studies with NSV, IL-10 deficiency

affected outcomes by a combination of increased Th1-mediated rather than

Th17-mediated immunopathogenesis and delayed production of antiviral antibody in the

CNS, which is important for viral clearance.

Many cells can produce IL-10, but regulatory T cells play a major role during viral

infections of multiple organs, including the CNS (21, 32–35), and are important

deter-minants of NSV infection outcome in susceptible and resistant strains of mice (20).

SINV-induced neurologic disease is a manifestation of the inflammatory response to

infection rather than virus replication

per se

(36–38). In both NSV- and TE12-induced

encephalomyelitis, IL-10 deficiency and more severe disease were associated with

fewer Tregs in the CNS than in the CNS of WT mice (19). Furthermore, differences in

staining patterns in the current study suggest that Tregs in IL-10

and WT mice may

also be functionally distinct. Impairment of regulatory cell production of IL-10 also

results in more severe disease during acute encephalitis caused by tick-borne

enceph-alitis virus, Japanese encephenceph-alitis virus, and mouse coronavirus (33, 39) and in the

10 0

10 20 30 40

**

# CD19

+

B cel

ls

(x 10

4)

10 0

5 10 15 20

25 ***

CD19

+

B

cel

ls

(% L

ive

)

10 0

10 20 30 40

**

WT IL10KO

#

CD19

+ B

cel

ls

(x

1

0

5)

10 0

10 20

30 **

CD19

+ B

c

e

ll

s

(%

Li

ve

)

B

C

D

E

Brain

CLN

A

FIG 13Effect of IL-10 deficiency on CD19⫹_{B cells in the CNS. WT and IL-10}⫺/⫺_{B6 mice were intranasally}

infected with 105_{PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10}

WT (black bars) and IL-10⫺/⫺_{(white bars) mice at 10 days postinfection is shown. (A) Representative flow}

cytometry plot. (B to E) Frequency and percentage of CD19⫹_{B cells in brain (B and C) and CLN (D and E). The}

data are plotted as the mean⫾SEM from 3 independent experiments.**,P⬍0.01;***,P⬍0.001.

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autoimmune diseases of the nervous system experimental autoimmune encephalitis

and experimental autoimmune neuritis (40–42). Because IL-10 deficiency did not affect

numbers of Tregs in the CLN, the site of induction of the SINV-specific adaptive immune

response (16, 43), it is likely that IL-10 deficiency affects either recruitment to or

differentiation of Tregs in the CNS.

Both Th1- and Th17-mediated immunopathology can be regulated by IL-10.

Regu-latory T cells decrease Th1 responses primarily by downregulating major

histocompat-ibility complex (MHC) type II on antigen-presenting cells with suppression of

proin-flammatory chemokines and cytokines such as IFN-

␥

(44), while IL-10 directly inhibits

both differentiation and proliferation of Th17 cells (45, 46). IL-10 deficiency led to an

increase in Th17 cells in NSV-infected mice (19), but TE12-infected IL-10-deficient mice

had an increase in CNS Th1 cells without a change in Th17 cells. Early in TE12 infection,

IFN-

␥

was lower in IL-10

mice than in WT mice, but later IFN-

␥

production was

higher and Th1 cells became more abundant. The reason for this change in pattern is

not known, but microglial cells and NK cells can produce IFN-

␥

independently of T cells

in acute infection (47), and in the absence of IL-10, innate factors such as transforming

growth factor

␤

(TGF-

␤

) may suppress IFN-

␥

production prior to arrival of adaptive

components of the immune response (48–52). Because TGF-

␤

can induce the

differen-tiation of Th17 cells (53–58), a potential dose- and cytokine-dependent role for TGF-

␤

in the differential responses of TE12- and NSV-infected IL-10-deficient mice is under

investigation.

A major effect of IL-10 deficiency on TE12 pathogenesis was a reduction in CNS Th2

and B cell responses compared to those in WT mice. Because Th1 cells and IFN-

␥

suppress Th2 cells and production of IL-4, increases in IL-10

mice may contribute to

this defect. IL-10, along with IL-4, also plays an important role in isotype switching to

IgG and B cell survival (31, 59, 60). Antibody is essential to viral clearance during

alphaviral encephalomyelitis (12–16), and without Th2 cell activation, a mature B cell

and antibody response cannot be induced. Furthermore, a role for early production of

CNS antiviral antibody in determining outcome from arboviral encephalitis is

recog-nized for infections of both mice and humans (20, 61, 62). Decreased B cell infiltration

into the CNS was likely responsible for the delay in local production of antiviral IgM and

IgG, a delay in viral clearance, and higher mortality.

0 3 5 7 10 14 21 0.0

0.5 1.0 1.5

*** ***

O

D,

450n

m

0 3 5 7 10 14 21 0.0

0.1 0.2 0.3 0.4

** ***

WT IL10KO

O

D

, 450n

m

0 3 5 7 10 14 21 0.0

0.2 0.4 0.6 0.8

*** ***

O

D

, 450n

m

0 3 5 7 10 14 21 0.00

0.05 0.10 0.15 0.20

*** ***

OD,

450n

m

SINV-specific IgG

SINV-Specific IgM

Brai

n

S

p

in

al

Co

rd

A

C

B

D

Days after infection Days after infection Days after infection Days after infection

FIG 14Effect of IL-10 deficiency on antibody production in the CNS. WT and IL-10⫺/⫺_{B6 mice were}

intranasally infected with 105_{PFU TE12. EIA measurement of SINV-specific IgM (A and B) and IgG (C and}

D) antibodies in the brains (A and C) and spinal cords (B and D) of WT (filled squares, solid lines) and IL-10⫺/⫺_{(open circles, dashed lines) mice is shown. Data were pooled from 2 independent experiments}

and are plotted as the means⫾SEM for 6 mice at each time point.***,P⬍0.001.

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Reasons for the differences in the characteristics of NSV- and TE12-induced

immu-nopathogenesis and disease are not clear. The sequences of the surface glycoproteins

are identical, so this difference in virulence is likely the result of differences in the

nonstructural region of the virus genome that could lead to differences in peptide

epitopes recognized for induction of effector T cells (35). Another potentially important

difference is nucleotide 5 in the 5

=

untranslated region (UTR) (10). NSV has a G, while

Toto1101/TE12 has an A. The UTRs of alphaviral genomes play critical roles in the

regulation of RNA replication and translation, as well as interaction with the host

cellular machinery and immune response (63). This G/A polymorphism affects

interac-tion of the viral genomic RNA with the viral restricinterac-tion factor IFIT1/ISG56 (63), an

interferon-stimulated protein that blocks viral replication by interacting with the 5

=

UTRs of RNA viruses (63–66). IFIT1 usually distinguishes host mRNA from viral RNA that

is unmethylated at the 2

=

O position of the cap (64). Alphaviruses lack this modification

but evade IFIT1 inhibition by altering the structure of the 5

=

UTR with a G at nucleotide

5 (63).

Finally, there is interest in host viral replication blockers to identify new classes

of drugs that activate these genes. Antiviral drugs that target host proteins instead

of viral proteins could in theory minimize the emergence of resistance (66). IL-10

immune therapy is the focus of many clinical trials for the treatment of

inflamma-tory diseases (67–70). Because there are no therapeutic interventions for viral

encephalomyelitis, understanding the potential of IL-10 as a therapeutic agent

remains important.

MATERIALS AND METHODS

Animals and virus.C57BL/6J (WT) and B6.129P2-Il10tm1Cgn/J (C57BL/6 [backcrossed 13 times] IL-10⫺/⫺_{) mice were purchased from Jackson Laboratories and bred in-house. Mice in all experiments}

were sex and age (4 to 6 weeks) matched. The TE12 strain of SINV was grown and assayed by plaque formation in BHK cells (10), and mice were infected intranasally with 1⫻105_{PFU in phosphate-buffered}

saline (PBS). Morbidity and mortality were assessed as previously described (19) using the following scoring system: 0, no clinical signs observed; 1, abnormal hind-limb and tail posture, ruffled fur, and/or hunched back; 2, unilateral hind-limb paralysis; 3, bilateral hind-limb or full-body paralysis; and 4, dead. For collection of cervical lymph nodes (CLNs), brains, and spinal cords, mice were first deeply anesthe-tized with isoflurane and perfused with ice-cold PBS. Tissues were used fresh or were snap-frozen and stored at⫺80°C. All experiments were performed according to guidelines and protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee.

Quantification of infectious virus.Tissue homogenates (20%, wt/vol) were made by homoge-nizing frozen tissues in PBS. Homogenates were serially diluted in Dulbecco modified Eagle medium (DMEM) plus 1% fetal bovine serum (FBS), plated on BHK-21 cell monolayers, incubated for 1 h, overlaid with 1.2% Bacto agar, and incubated at 37°C with 5% CO2for 48 h. Cells were stained with

neutral red and plaques counted. The result for each time point is plotted as the geometric mean of the log10value of PFU per gram⫾standard error of the mean (SEM). Samples in which no virus

was detected at a 1:10 dilution were given a value midway between the lower limit of detection and zero for statistical analyses.

Mononuclear cell isolation.Mononuclear cells were isolated from brains and CLNs collected from WT and IL-10⫺/⫺_{mice at 5, 7, and 10 days postinfection. CLNs were pooled from 6 to 10 mice}

per group per time point in 10 ml of RPMI plus 1% FBS and homogenized in C tubes using the GentleMACS system (Miltenyi) spleen program 1 for 2 cycles. Brain tissue was collected from 6 to 10 mice per group per time point in ice cold Hanks balanced salt solution. For cell isolation, 2 brains were pooled per C tube containing 4 ml of enzyme digest mix (RPMI–1% FBS, 1 mg/ml collagenase [Roche], 0.1 mg/ml DNase) and dissociated with scissors. Further dissociation was performed via GentleMACS (Miltenyi) brain program 3 (total of 5 times with two 15-min 37°C incubations with gentle rocking). All suspensions were filtered through 70-␮m cell strainers and centrifuged. Red blood cells were lysed using 2 ml of red blood cell lysis buffer for 3 min. Cells were washed with PBS and resuspended in 30% Percoll, underlaid with 70% Percoll for a 30/70% discontinuous gradient, and centrifuged for 30 min at 850⫻gat 4°C. The top layer was removed, mononuclear cells at the interface were collected, washed, and resuspended in PBS plus 2 mM EDTA, and live cells were counted using trypan blue exclusion.

Identification of cells by flow cytometry.For cell typing, 106_{cells were used for each panel. Cells}

were stained with a violet live/dead stain (Invitrogen), blocked with rat anti-mouse CD16/CD32 Block (BD), surface stained for phenotyping, and fixed using a 1:1 dilution of 4% paraformaldehyde and fluorescence-activated cell sorter (FACS) buffer, and data were acquired. The surface marker anti-bodies (all from BD or eBioscience) used were anti-CD45–fluorescein isothiocyanate (FITC), CD3-allophycocyanin (APC), CD4 –peridinin chlorophyll protein (PerCP)-Cy5.5, CD8 –phycoerythrin (PE)-Cy7, CD25-PE, Lineage-FITC, IL-7Ra–PE-(PE)-Cy7, Gata3–PerCP-Cy5.5, CD19 –PerCP-Cy5.5, CD5–PE-(PE)-Cy7,

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CD1d-APC, and IgM-FITC. Cell types were defined as follows: T cells (CD3⫹_{), CD4 T cells (CD3}⫹_CD4⫹_),

CD8 T cells (CD3⫹_CD8⫹_{), regulatory B cells (CD19}⫹_CD5⫹_CD1dhi_IgM⫹_{), and ILC2 cells (lineage}⫺

IL-7Ra⫹_Gata3⫹_).

For determination of T cell differentiation, transcription factor expression was assessed using the Foxp3 buffer set (eBioscience). After surface staining, cells were fixed, permeabilized, and stained (eBioscience) for Gata3, foxP3, ROR␥t, or Tbet. Cell types were defined as follows: Th1 cells (CD3⫹CD4⫹

Tbet⫹_{), Th2 cells (CD3}⫹_CD4⫹_Gata3⫹_{), Th17 cells (CD3}⫹_CD4⫹_ROR␥t⫹_{), and regulatory T cells (CD3}⫹

CD4⫹_CD25⫹_foxp3⫹_).

For determination of CD4⫹_{T cell cytokine production, 2⫻10}6_{to 3⫻10}6_{cells were stimulated with}

RPMI plus 1% FBS containing 50 ng/ml of phorbol-12-myristate 13-acetate (PMA) and 1 ␮g/ml of ionomycin in the presence of GolgiPlug (BD) for 4 h. After surface staining with CD3-APC (BD) and CD4-FITC (BD), cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD). Cytokine staining was performed alone or in conjunction with transcription factor staining. Cells were stained (BD) for IFN-␥–PE, IL-4 –PE, or IL-17a–PE. All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

Gene expression and viral RNA analysis using real-time PCR.RNA was extracted from frozen tissues using the RNeasy lipid mini-RNA isolation kit (Qiagen). RNA was quantified with a NanoDrop instrument, and 500 ng was used to synthesize cDNA with a high-capacity cDNA reverse transcription kit (Life Technologies). Quantification of viral RNA in brains and spinal cords was performed using 2.5␮l of cDNA, TaqMan Universal PCR master mix (Roche), TaqMan probe (5=-6-carboxyfluorescein [FAM]-CGCA TACAGACTTCCGCCCAGT-6-carboxytetramethylrhodamine [TAMRA]-3=(Applied Biosystems), and primers to the SINV E2 gene (forward, 5=-TGGGACGAAGCGGACGATAA-3=; reverse, 5=-CTGCTCCGCTTTGGTCGTA T-3=) on a 7500 Fast real-time PCR system for 50 cycles. Results were analyzed using Sequence Detector software, version 1.4, using a standard curve made of 10-fold dilutions of the SINV subgenomic region derived from a pGEM-3Z plasmid and normalized to endogenous mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA.

For cytokine gene expression, quantitative real-time PCR was performed using 2.5␮l of cDNA and TaqMan gene expression arrays forIfn␥,Il2,Il10,Il17a,Il4,gata3, andfoxp3in 2⫻Universal PCR Mastermix (Applied Biosystems). GAPDH mRNA levels were determined using the rodent primer and probe set (Applied Biosystems). All reactions were run on an Applied Biosystems 7500 real-time PCR machine. Transcript levels were determined by normalizing the target gene threshold cycle (CT) value to theCT value of the endogenous GAPDH housekeeping gene. This normalized value was used to calculate the fold change relative to uninfected mice of the same strain and, for IL-10⫺/⫺_{mice, also gene expression}

relative to that in uninfected WT mice (ΔΔCTmethod).

Quantification of protein via EIA.IFN-␥protein levels in brains from WT and IL-10⫺/⫺_{mice infected}

with TE12 were measured by enzyme immunoassay (EIA) (R & D Systems) according to the manufac-turer’s instructions. Brain homogenates (10%, wt/vol) from 6 mice per time point were pooled and tested in duplicate.

EIA was also used to quantify SINV-specific IgM and IgG antibodies in brains and spinal cords. Ninety-six-well MaxiSorp plates (Nalgene Nunc) were coated with 106_{PFU/well of polyethylene}

glycol-precipitated SINV in 50 mM NaHCO3(pH 9.6) at 4°C overnight. Blocking buffer (10% FBS and 0.05%

Tween 20 in PBS) was added and left for 2 h at 37°C. Tissue homogenates (10%, wt/vol) diluted 1:2 in blocking buffer were added to the wells and incubated at 4°C overnight. Bound antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgM or IgG (Southern Biotech) diluted 1:1,000 in blocking buffer and incubated at room temperature for 2 h. Plates were developed using the BD OptEIA tetramethylbenzidine (TMB) substrate reagent kit with H2SO4as a stop solution.

Absorbance was read at 450 nm. Average optical density (OD) values for tissues from uninfected mice were subtracted from the OD values for tissues from infected mice.

Statistical analysis.Data were from three independent experiments or at least 6 mice per group. All statistical analysis was done using GraphPad Prism 5 (v5.01). Survival was calculated using Kaplan-Meier survival curves (log rank [Mantel-Cox] test). Differences between groups during the course of infection were determined using a 2-way analysis of variance (ANOVA) and Bonferroni posttests to compare the differences between groups at each time point (morbidity, cellular infiltrates, cytokine analysis, and viral titers).

ACKNOWLEDGMENTS

This work was supported by a research grant from the National Institutes of Health

(R01 NS87539) to D.E.G. and a training grant (T32 AI007417) to N.M.M.

We thank Johns Hopkins School of Public Health animal facility technicians Mary

Archer and D. J. Johnson for help with animal maintenance and Victoria K. Baxter and

Elizabeth Troisi for animal breeding assistance.

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