Role of MyD88 in Route-Dependent Susceptibility to Vesicular
Stomatitis Virus Infection1
Shenghua Zhou, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Jennifer P. Wang,
Anna M. Cerny, Melvin Chan, and Robert W. Finberg2
TLRs are important components of the innate immune response. The role of the TLR signaling pathway in host defense against
a natural viral infection has been largely unexplored. We found that mice lacking MyD88, an essential adaptor protein in TLR
signaling pathway, were extremely sensitive to intranasal infection with vesicular stomatitis virus, and this susceptibility was dose
dependent. We demonstrated that this increased susceptibility correlates with the impaired production of IFN-? and defective
induction and maintenance of neutralizing Ab. These studies outline the important role of the TLR signaling pathway in nasal
mucosae-respiratory tracts-neuroepithelium environment in the protection against microbial pathogen infections. We believe that
these results explain how the route of infection, probably by virtue of activating different cell populations, can lead to entirely
different outcomes of infection based on the underlying genetics of the host. The Journal of Immunology, 2007, 178: 5173–5181.
sponses are not only essential for limiting the systemic spread
of invading pathogens but also provide critical signals for ac-
tivation of the ensuing specific adaptive immune response (1,
2). The adaptive immune system consists of two arms, the hu-
moral immune response, which produces neutralizing Abs, and
the cellular immune response, through which pathogen-infected
cells are eliminated by CTL or indirectly eliminated via the
release of chemokines or cytokines from activated Th CD4?T
cells or CTL (1–5). A combination of innate and pathogen-
specific adaptive immune responses is required for effective
control of pathogens (1, 2, 4, 5).
TLRs are one of the important participants in the innate immune
response (4–6). The mammalian TLR family is composed of ?12
germline-encoded type I transmembrane receptors which are re-
lated to the Drosophila Toll (6, 7). The engagement of all known
TLRs and their ligands, with the exception of TLR3, activates the
MyD88-IL-1R-associated kinase-TNFR-associated factor 6 signal-
ing pathway, followed by nuclear translocation of NF-?B and ac-
tivation of MAPKs such as JNK and p38, which induce the tran-
scriptional regulation of the proinflammatory cytokines and
effector cytokines (4, 6). MyD88 is also required for the IL-1
family cytokine (IL-1/IL-18)-induced signaling pathways (8).
Cytokines produced as a result of the innate immune response
lead to the activation of adaptive immune responses through the
ammalian hosts have evolutionarily developed the
innate immune system and the adaptive immune
system to combat invading pathogens. Innate re-
up-regulation of MHC class I and II molecules and costimula-
tory molecules on APCs, and direct activation and maturation of
dendritic cells (DCs) and effector T cells (6, 7). The factors that
regulate the development and polarization of either a Th1 or
Th2 immune response are still not fully delineated (9). Increas-
ing evidence has emerged from studies on TLR and MyD88-
deficient mice, suggesting the importance of TLR-MyD88 sig-
naling in host defense and the development of pathogen-specific
adaptive immune responses (10–14).
We have previously demonstrated with a noncytopathic virus,
lymphocytic choriomeningitis virus, that the TLR-MyD88 path-
way plays a critical role in the activation of protective CD8?T
cells and the control of the acute lymphocytic choriomeningitis
virus infection (14). In the present study, we have evaluated the
role of the TLR-MyD88 pathway in the protection of mice from a
cytopathic virus infection, vesicular stomatitis virus (VSV).3VSV
is a member of the Vesiculovirus genus in the Rhabdoviridae fam-
ily. There were two major reasons why VSV was used in this
study. First, the immune responses to VSV have been well char-
acterized (15, 16). VSV infection elicits the production of high
levels of type 1 IFN (IFN-??) and activation of both CD4?and
CD8?T cells. In addition, VSV infection initially induces a Th-
independent IgM; this is followed by a lifelong Th-dependent
IgG response (15). It has been demonstrated that the initial type 1
IFN, Th-independent neutralizing IgM Ab, CD4?T cells, and Th-
dependent neutralizing IgG Abs are all essential for the protection of
mice against VSV infection (16, 17). Secondly, it has recently been
shown in vitro in isolated cell populations that TLR7 and MyD88
participate in the recognition of VSV in the endosomal compartment
and initiate an innate immune response, in particular, the production
of type 1 (IFN-?) from plasmacytoid DCs (pDCs) (18). However, the
involvement of the TLR-MyD88 pathway in the protection of mice
from VSV-induced disease and VSV-induced adaptive immune re-
sponses is unknown.
Department of Medicine, University of Massachusetts Medical Center, Worcester,
Received for publication June 29, 2006. Accepted for publication January
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institute of Allergy and Infectious Diseases
Regional Center of Excellence Grant AI 057159 and National Institutes of Health
Grants R01 AI 49309 (to R.W.F.) and P01 AI 0577484.
2Address correspondence and reprint requests to Dr. Robert W. Finberg, Department
of Medicine, University of Massachusetts Medical Center, 364 Plantation Street,
Lazare Research Building, Worcester, MA 01605. E-mail address: Robert.Finberg@
3Abbreviations used in this paper: VSV, vesicular stomatitis virus; DC, dendritic cell;
pDC, plasmacytoid DC; Flt3L, human recombinant fms-related tyrosine kinase 3
ligand; KO, knockout; WT, wild type.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
Our studies reveal that in response to intranasal infection of
VSV, a natural route for VSV infection, MyD88 is essential for the
induction of type 1 IFN, neutralizing Ab production, and protec-
tion of mice from lethal infection. In contrast, although i.v. infec-
tion, which is not a natural route for VSV infection, induced an
impaired adaptive immune response in MyD88 knockout (KO)
mice, MyD88 is not critical for i.v. VSV-induced type 1 IFN, as all
MyD88 KO mice survived this challenge. This suggests that TLR
and MyD88 may be expressed and regulated differently in different
tissues and organs. Together, these studies identify the TLR-
MyD88 pathway as a major component of protective antiviral im-
munity to VSV.
Materials and Methods
MyD88 KO mice were obtained from Dr. S. Akira (Osaka University,
Osaka, Japan; Ref. 2). MyD88 KO mice were backcrossed to C57BL/6
mice for at least six generations. The genotypes of the mice were deter-
mined by PCR of tail DNA, and backcrossing was confirmed by satellite
DNA analysis. Mice were bred and maintained under specific pathogen-
free conditions. Age-matched C57BL/6 mice (wild-type (WT) control),
and TCR-??-deficient mice were purchased from The Jackson Laboratory.
RAG1 KO mice were obtained from Dr. K. L. Rock (University of
Massachusetts Medical School, Worcester, MA). Animals were housed
and experiments were performed in accordance with animal welfare
Virus, virus detection, and experimental infection of mice
The VSV-Indiana (VSV-IND) serotype was used (19). Virus stocks were
prepared on confluent BHK-21 monolayer cells infected at a low multi-
plicity of infection (0.01). Viral titers were determined by plaque assay on
Vero cells (20). To verify VSV authenticity, two approaches were used.
First, VSV-infected cells were stained positive with a mAb against
VSV-GP (P5D4; Sigma-Aldrich); second, PCR and sequence analysis fur-
ther identified that our VSV is an Indiana strain (our VSV strain shared
91–97% nucleotide identity with published VSV-Indiana strain sequence in
GenBank within VSV-GP, M, and NP genes; and within NS gene, shared
92% identity with published VSV-Indiana, but only 54.7% identity with
published VSV-NJ strain). As indicated, mice were either infected i.v. with
200 ?l of 1–2 ? 106PFU of VSV or intranasally with VSV diluted in PBS
buffer. For intranasal infection, mice were lightly anesthetized with Isoflu-
rane and given 10 ?l of various doses of VSV diluted with PBS. Mice were
monitored daily, and the occurrence of hind limb paralysis and death was
recorded. Virus titers of the VSV stock and VSV-infected mouse tissues
were determined by plaque assay on Vero cells.
The VSV-specific CD4 T cell epitope peptides used in this study were
MHC class II-restricted p8 and p17 (21). Peptide was synthesized by the
Tufts University peptide core facility and HPLC purified.
Determination of Ab responses to VSV
ELISAs were used to detect VSV-specific Ab responses. VSV or VSV-
infected BHK-21 cell lysate was used to coat ELISA plates as described
previously (3). Sera were prediluted 1/40 in PBS (pH 7.2) and then 2-fold
diluted with 1% BSA (PBS, 1% BSA) and added to VSV-coated wells.
Each sample was assessed in duplicate. The secondary Abs used were
HRP-conjugated rat anti-mouse IgG and IgM. For specific IgG subclass
analysis, ELISA was conducted using HRP-conjugated rat anti-mouse
IgG1 and IgG2a. All these secondary Abs were purchased from BD Pharm-
ingen and diluted at 1/1000 with PBS, 1% BSA. The substrate was tetra-
methylbenzidine. The OD was read at 450 nm. The results were expressed
as level of titers.
Neutralizing Ab assay
The neutralization activity of VSV-infected mouse serum was determined
by a well-established assay (16). Briefly, sera were prediluted 1/200 in
MEM, 2% FCS medium and heat inactivated for 30 min at 56°C. Fifty
microliters of 2-fold serial dilutions was mixed with equal volume of VSV
(500 PFU/ml) and incubated for 90 min at 37°C. Then the mixture was
transferred onto confluent Vero cell monolayers in 96-well plates and in-
cubated for 60 min at 37°C. The monolayers were overlaid with 100 ?l of
DMEM containing 1% methylcellulose. After incubation for 24 h at 37°C,
the overlay was removed, and the monolayer was fixed with 4% formalin
and stained with 0.5% crystal violet. The highest dilution of serum that
reduced the number of plaques by 50% was taken as titer.
Preparation of GM-CSF or fms-related tyrosine kinase 3 ligand
(Flt3L)-expanded bone marrow (BM) DCs
GM-CSF-stimulated BM conventional DCs were prepared according to a
previously described method (22). Briefly, BM cells were seeded at 2 ?
106/100-mm dish in 10 ml of RPMI 1640 containing 100 U/ml recombi-
nant mouse GM-CSF (R&D Systems). One-half of the medium was re-
moved every 3 days of culture, and fresh culture medium with GM-CSF
infection. A, Age-matched groups of male mice (WT ? 35, MyD88 KO
mice ? 33, RAG-1 KO mice ? 8, TCR-?? KO ? 4) were intranasally
infected as described in Materials and Methods. The hind limb paralysis
and death were recorded, and data were shown as percentage of survival
per group up to day 20 postintranasal infection. Data are representative of
four experiments for both WT and MyD88 KO mice, two experiments for
RAG1 KO mice and one for TCR-?? KO mice. B, Age-matched groups of
MyD88 KO and WT male mice were intranasally infected with the fol-
lowing doses of VSV: 5 ? 102PFU (n ? 6 for both strains); 5 ? 105PFU
(n ? 6 for WT, n ? 5 for MyD88 KO mice). The hind limb paralysis and
death were recorded, and data were shown as percentage of survival per
group up to day 14 postintranasal infection. Mice were sacrificed on day 9
postinfection for the analysis of the CD4?T cell response and the virus
titers. C, On day 9 postintranasal VSV (5 ? 105PFU) infection, the brains
of both MyD88 KO and WT mice (n ? 4 for both strain) were collected.
The levels of VSV were determined by plaque assay. Results are shown as
PFU per gram of tissue.
MyD88 KO mice were more susceptible to intranasal VSV
5174 MyD88 AND VSV
was added to the cultures. Nonadherent cells were collected at day 9 of
incubation and were examined by staining with Abs specific for CD11c and
CD11b. Nonadherent cells were seeded into 24-well plates at 5 ? 105/well
and were infected with different doses of VSV. The supernatants were
harvested after incubation for 24 h, and the levels of type 1 IFNs were
measured by the bioassay described below.
For the preparation of Flt3L-derived type 1 IFN-producing cells, BM
cells were cultured with human recombinant Flt3L (10 ng/ml; R&D
Systems; Ref. 23). After 7 days, these cultures contained 40%
CD11clowCD11b?B220?pDC. Cells were challenged with medium,
VSV (multiplicity of infection, 5.0), or positive control CpG 2216 (6
?M). The supernatants were harvested after incubation for 40 h. The
levels of type 1 IFNs in culture supernatants were measured using bio-
assay. The bioassay detects both IFN-? and IFN-?.
Bioassay for type 1 IFN activity
To determine the bioactivity of the type 1 IFNs in sera, a biological assay
for protection against VSV was used as previously described (14, 24).
Briefly, serum samples were prediluted 1/5 (culture supernatants) or 1/50
(serum) and exposed to UV light for 30 min to inactivate any potential live
VSV in the samples. NCTC929 cells (provided by Dr. E. Szomolanyi-
Tsuda, University of Massachusetts Medical School) were incubated with
2-fold diluted sera for 18–24 h at 37°C and were challenged with VSV.
The protection of the NCTC929 cells was used as an index of type 1 IFN
activity in the samples. The dilution of sera that resulted in a 50% reduction
of VSV-induced cytopathic effect was defined as 1 U/ml type 1 IFNs. A
recombinant mouse IFN-? (PBL Biomedical Laboratories) was included as
a positive control. Because it has been reported that VSV infection in vivo
dominantly induced IFN-? (25), to further confirm our results, samples
were pretreated with a neutralizing rat mAb against mouse IFN-?
(RMMA-1 clone; PBL Biomedical Laboratories) for 1 h at 37°C before
incubation with NCTC929 cells.
In vivo cytokine responses to VSV infection and ex vivo cytokine
production in response to VSV-specific CD4 peptides and
anti-CD3 plus anti-CD28 stimulation
MyD88-deficient and WT mice were infected intranasally or i.v. with VSV.
Serum samples were collected at indicated time points postinfection using
serum separator tubes. Levels of IFN-? and MCP-1 were determined using
ELISA. Splenocytes isolated from VSV-infected day 7 MyD88-deficient
and WT mice were seeded into 96-well plate at the density of 5 ? 105
cells/well and stimulated with CD4 peptides (p8 and p17) at the final con-
centration of 4.0 ?g/ml. To test whether the MyD88 signaling pathway
affects TCR-dependent CD4 T cell activation, splenocytes were stimulated
with immobilized anti-CD3 (20 ?g/ml) and soluble anti-CD28 (100 ng/
ml). After incubation for 72 h, the levels of IFN-? in the supernatants were
determined using ELISA (BD Pharmingen) according to the manufacturer’s
Intracellular cytokine staining
Intracellular cytokine staining of CD4?T cells for IFN-? or TNF-? was
performed as described previously (26). Briefly, splenocytes were collected
from day 9 intranasal VSV-infected MyD88 KO and WT mice and were
cultured in 96-well flat-bottom plates at a density of 106cells/well in 200
?l of RPMI 1640 supplemented with 10% FCS, recombinant human IL-2
(20 U/ml), and brefeldin A (BD Pharmingen; 1 ?g/ml) in the presence or
absence of VSV-specific CD4 epitope peptide p8 and p17 at a concentra-
tion of 8 ?g/ml. As a positive control, cells were stimulated with PMA (50
ng/ml) plus ionomycin (500 ng/ml). After 5 h of culture, the cells were
harvested, washed once in FACS buffer, and surface stained with allophy-
cocyanin-conjugated monoclonal rat Ab specific to mouse CD4 (clone
RM4-5; BD Pharmingen). After washing, the cells were stained for intra-
cellular cytokines by using the Cytofix/Cytoperm kit (BD Pharmingen)
according to manufacturer’s instructions. PE-conjugated monoclonal rat
1 IFNs induction by i.v. VSV infection is MyD88 independent. A, MyD88-deficient and WT control mice were intranasally infected with 5 ? 105
PFU of VSV. Serum samples were collected at different time points as indicated. The activity of type I IFN in sera was tested by an antiviral bioassay
(inhibition of VSV-induced cytopathic effect in NCTC929 cells), and the level of type 1 IFN in individual mouse was shown (n ? 6 per group) (A).
?, p ? 0.087. B, MyD88 KO and WT control mice were infected with 1 ? 106PFU of VSV i.v. Sera were collected at different time points as
indicated. The activity of type I IFN in sera was tested by an antiviral bioassay. Data are means ? SD of duplicate wells. Results were shown as
units per milliliter. A comparison of levels of IFN-? between WT and MyD88-deficient mice revealed no significant differences (p ? 0.05).
Representative of two separate experiments (n ? 6 for each group). C, Conventional DCs: recombinant mouse GM-CSF-derived BM DCs were
challenged with different doses of VSV at 37°C for 24 h and type 1 IFN activity was measured by bioassay. D, Plasmacytoid DCs: Flt3L-expanded
BM pDCs were challenged with VSV at a multiplicity of infection of 5 (5 ? 105PFU) for 40 h. The activity of type 1 IFNs in the supernatants was
determined by bioassay. Results are representative of three separate experiments. ?, p ? 0.05.
Intranasal VSV infection induced much lower levels of type 1 IFNs in MyD88-deficient mice compared with WT control, but type
5175The Journal of Immunology
Abs specific to murine IFN-? (clone XMG1.2; BD Pharmingen) or TNF-?
(clone:MP6-XT22), and the isotype control Ab (rat IgG1) were used to
identify cytokine-positive cells. Samples were acquired on a BD-LSR-II
flow cytometer (BD Biosciences). Data were analyzed with FlowJo
Statistical significance was evaluated using Student’s t test in Excel version
5. Results were expressed as means ? SE. Mortality studies were evaluated
using ?2analysis (PRISM software).
MyD88 KO mice were more susceptible to intranasal VSV
VSV is able to access the brain following intranasal infection and
cause encephalitis as well as meningitis, characterized by hind
limb paralysis (3, 27, 28). MyD88, the important adaptor protein
for the TLR signaling pathway, has been reported to be involved
in VSV-induced IFN-? production. To evaluate whether MyD88
KO mice are more susceptible to VSV infection, MyD88 KO mice
were infected by intranasal or i.v. administration of VSV. RAG-1
KO and TCR-?? KO mice, which are deficient of both T and B
cells (RAG-1 KO mice) or T cells (TCR-?? KO mice), were in-
cluded as controls.
Our results demonstrated that ?50% (21 of 33) of MyD88 KO
mice had hind limb paralysis and died after intranasal infection
with 5 ? 105PFU of VSV (Fig. 1A), and furthermore, the intra-
nasal VSV-induced paralysis and death in MyD88 KO mice was
dose dependent (Fig. 1B). In addition, all RAG KO and TCR-??
KO mice died after either intranasal or i.v. infection with VSV
(Fig. 1A) (29). In contrast, very few (3 of 35) WT mice had pa-
ralysis and death after intranasal VSV infection with various doses
of VSV in at least three experiments (Fig. 1, A and B). Interest-
ingly, none of the MyD88 KO mice succumbed to i.v. VSV in-
fection (with ?40 MyD88 KO mice in at least four experiments;
data not shown). Importantly, in one representative experiment, on
day 9 postintranasal infection with 5 ? 105PFU of VSV, 3 of 4
MyD88 KO mice failed to clear VSV infection from the brains
(Fig. 1C). In striking contrast, all 4 WT mice cleared VSV. In
addition, on day 9 postintranasal infection with 5 ? 105PFU of
VSV, the levels of VSV in the spleens, lungs, and livers of both
MyD88 KO and WT mice were below the limit of detection
(?50 PFU/ml). Thus, these results indicate that MyD88 has a
major role in protection against VSV after an intranasal
The initial induction of type 1 IFN, neutralizing Th-independent
IgM, and Th-dependent IgG Abs all contribute to the protection of
mice from VSV-induced encephalitis. Our studies demonstrated
that significantly more MyD88 KO mice were susceptible to in-
tranasal VSV infection, but all MyD88 KO mice survived after i.v.
VSV infection (data not shown and Ref. 18). These different out-
comes prompted concern regarding tissue- or organ-specific in-
volvement of MyD88 in the regulation of the protective innate and
adaptive immunity against VSV infection.
Intranasal VSV infection in MyD88 KO mice induced much
lower levels of type 1 IFNs, whereas induction of IFN-? after
i.v. VSV infection occurs independently of MyD88 in vivo
Type 1 IFN is critical for the protection of mice from VSV infec-
tion (30). MyD88 has been shown to participate in type 1 IFN
induction by pDC in response to ssRNA viruses including VSV
(18). To evaluate the systemic role of MyD88 in the production of
type 1 IFN, MyD88-deficient mice were infected intranasally or
i.v. with VSV. Type 1 IFN activity in serum was assessed by
bioassay (14, 24). Surprisingly, intranasal VSV infection did in-
duce systemic type 1 IFNs in MyD88 KO mice, but their levels
were much lower than those in WT mice (p ? 0.087) (Fig. 2A). In
contrast, i.v. VSV infection induced comparable levels of IFN-? in
both MyD88-deficient and WT mice (Fig. 2B). To further dissect
the possible involvement of MyD88 in VSV-induced type 1 IFN,
we also examined type 1 IFN induction in GM-CSF-derived BM
conventional DCs (CD11b?and CD11c?) and Flt3L-expanded
BM-derived pDC. In vitro production of type 1 IFNs in response
to VSV by BM-derived conventional DCs was independent of
MyD88 (Fig. 2C). However, consistent with published data (18),
we found that VSV challenged Flt3L-expanded BM pDC produced
type 1 IFN in a MyD88-dependent manner (Fig. 2D). MyD88 par-
ticipates in the type 1 IFN response of pDC but not conventional
DC after in vitro VSV challenge, but it does not appear to play a
major role for MyD88 after i.v. VSV challenge.
Taken together, these studies demonstrated that despite a clear
role for MyD88 in type 1 IFN production in response to intranasal
VSV infection, MyD88 does not play a significant role in the total
serum type 1 IFN response to i.v. VSV infection in vivo. We
demonstrated that the involvement of MyD88 in VSV-induced
type 1 IFN production is cell type dependent.
MyD88 is critically involved in the regulation of the
VSV-induced humoral immune response
To better understand the involvement of MyD88 in the regulation
of VSV-induced humoral immune response, mice were infected
isotype but comparable levels of total anti-VSV IgG in response to
VSV. Age-matched MyD88-deficient and WT control male mice were
infected i.v. with 1 ? 106PFU of VSV. Serum samples were collected
at different time points as indicated. The VSV-specific total IgG (A),
IgG1 (B), and IgG2a (C) titers were determined by ELISA. Results are
representative of two separate experiments (n ? 4 for each group).
?, p ? 0.05.
MyD88 KO mice produce more IgG1 isotype than IgG2a
5176 MyD88 AND VSV
i.v. with VSV. VSV infection i.v. in WT mice induced IgM (data
not shown) and IgG responses with an isotype bias of more IgG2a
than IgG1, characteristic of a Th1-type immune response (Fig. 3,
A and B). In contrast, MyD88-deficient mice showed much
lower IgM levels on day 4 postintranasal or i.v. VSV infection
(data not shown) and delayed isotype switching (Fig. 3, A and
B). At the peak of the IgG response, MyD88-deficient mice
produced more IgG1 subclass than IgG2a subclass anti-VSV
Abs, a pattern consistent with a Th2 type immune response
(Fig. 3, B and C). Thus, MyD88 affects the anti-VSV humoral
MyD88 is required for the maintenance of neutralizing Ab after
either intranasal or i.v. VSV challenge
The neutralization activity of VSV-specific Abs plays a crucial
role in the protection of mice from VSV-induced CNS disease. To
further examine the bioactivity of these Abs, we determined
whether Ab from WT or MyD88 KO mice could neutralize VSV.
Intranasal VSV infection induced significant lower levels of
neutralizing Ab in MyD88 KO mice than WT mice (Fig. 4A). In
the case of i.v. VSV challenge, neutralizing Ab was detected in
WT mice, increased from day 7 to day 20 postinfection, and
then remained high thereafter (Fig. 4B). In contrast, neutraliz-
ing Abs in MyD88-deficient mice initially increased to a level
comparable with that of WT on day 7, but this was followed by
a rapid decline in titer. Thus, these data demonstrate that
MyD88 is required for the maintenance of neutralizing antiviral
to VSV. Groups of age- and sex (male)-matched MyD88 KO and WT mice
were infected intranasally (A) or i.v. (B) with 5 ? 105PFU or 1 ? 106PFU
of VSV as described in Figs. 2 and 3. Serum samples were collected at
different time points as indicated. The neutralizing Ab titers were measured
by a neutralizing assay described in Materials and Methods.
MyD88 KO mice have defective neutralizing Ab responses
intranasally infected with 5 ? 105PFU of VSV. On day 9 postinfection, splenocytes were restimulated with either VSV-specific CD4 epitope peptides or
PMA plus ionomycin. The CD4?T cell response was quantified by intracellular staining for IFN-? (A) or TNF-? (B). Cells were gated on CD4?T cells.
C and D, MyD88-deficient and WT control mice were infected i.v. with 2 ? 106PFU of VSV. Splenocytes isolated from VSV-infected day 7 MyD88-
deficient and WT mice were seeded into 96-well plate at the density of 5 ? 105cells/well and stimulated with VSV-specific CD4 epitope peptides (p8 and
p17) at a final concentration of 4 ?g/ml (C). In addition, splenocytes were stimulated with immobilized anti-CD3 (20 ?g/ml) and soluble anti-CD28 (100
ng/ml; D). After incubation of 72 h, the levels of IFN-? in culture supernatants were determined by ELISA. Data are means ? SD of duplicate wells. Results
are shown as picograms per milliliter. Results are representative of two separate experiments. ?, p ? 0.05.
MyD88 KO mice have impaired CD4?T cell response to VSV. Age-matched MyD88 KO and WT male mice (n ? 4 for both strains) were
5177 The Journal of Immunology
MyD88 plays a critical role in activation of VSV-specific CD4?
T cell response
CD4?T cells are required for the protection of mice from either
intranasal or i.v. VSV infection. To determine whether VSV-in-
fected MyD88-deficient mice have defective CD4?T cell re-
sponse, the activity of CD4?T cells in the spleens of VSV-in-
fected mice was assessed by either ELISA or intracellular staining
for the expression of IFN-?. Splenocytes were taken from intra-
nasal VSV-infected mice and stimulated in vitro with VSV-spe-
cific CD4 epitope peptides, p8 and p17 (21), or PMA plus iono-
mycin. The expression of IFN-? or TNF-? was determined by
intracellular staining. VSV infection in MyD88 KO mice induced
a significantly lower level of VSV-specific CD4?T cell response
(Fig. 5A). In contrast, both WT and MyD88 KO CD4?T cells
responded equally well to the PMA plus ionomycin (Fig. 5B). Our
results showed MyD88 is also required for i.v. VSV-induced
CD4?T cell function. Although WT mice and MyD88 KO mice
have comparable numbers of CD4?T cells in their spleens 7 days
after infection (data not shown), in the absence of MyD88, CD4?
T cells were impaired in their ability to produce IFN-? in response
to the restimulation with the VSV-specific CD4 epitope peptides
(Fig. 5C), but they responded equally to anti-CD3 and anti-CD28
stimulation (Fig. 5D). Therefore, these studies demonstrated that
MyD88 is required for the functional activation of CD4?T cells in
response to VSV challenge.
MyD88-deficient mice have defective IFN-? and MCP-1
production in response to VSV infection
The initial induction of chemokines and cytokines could play a
role in the determination of the adaptive immunity. To further
study the possible contribution of MyD88 in the initial proinflam-
matory chemokine and cytokine response to VSV infection, we
examined the initial cytokine levels (IFN-? and MCP-1) in the
peripheral blood of VSV-intranasally infected mice. MyD88-defi-
cient mice produced significantly lower levels of MCP-1 (Fig. 6A)
and IFN-? (Fig. 6B) in the early stages of postintranasal VSV
infection than did WT mice. We were unable to detect either IL-4
or IL-10 production from either serum of WT and MyD88 KO
mice. VSV infection i.v. also induced impaired production of both
MCP-1 and IFN-? (data not shown).
The TLR-MyD88 signaling pathway plays a critical role in the
regulation of innate as well as adaptive immunity to protein Ags
(6, 7, 31). In the present study, we have examined the contribution
of the MyD88 signaling pathway to the protection of virus infec-
tion-induced pathogenesis, to in vivo production of type 1 IFN and
to the production of neutralizing Abs in mice infected with a cy-
topathic virus, VSV. We found that VSV-induced mortality is both
MyD88 and route dependent. Moreover, intranasal VSV infection
in MyD88-deficient mice induced encephalitis in a dose-dependent
manner. Second, MyD88 is involved in intranasally VSV-induced
type 1 IFN production, which might account for the highly sus-
ceptible of MyD88 KO mice to intranasal VSV infection. Third,
MyD88 is involved in the activation of the CD4?T cells in re-
sponse to VSV infection. Finally, MyD88 is essential for the initial
IgM induction and for maintenance of the VSV-specific
It has been demonstrated that several factors may contribute to
the protection of mice from intranasal VSV-induced encephalitis,
including initial type 1 IFN production, the early Th cell-indepen-
dent IgM neutralizing Ab, and the later Th-dependent IgG neu-
tralizing Abs (32). The essential role of the initial type 1 IFN in
protection of mice from VSV infection has been clearly docu-
mented in IFN-??R KO mice (30). IFN-??R KO died within 3–6
days after i.v. VSV infection. The important role of CD4?T cells
and CD4?T cell-dependent IgG-neutralizing Ab have been dem-
onstrated in TCR-?? KO mice (Ref. 3 and this study). TCR-?? KO
mice died on days 11–14 after i.v. VSV infection. The early Th-
independent IgM-neutralizing Ab response, although not as im-
portant as the Th-dependent IgG response, plays a role in the initial
restriction of VSV spreading to the brain (32). It has been well
documented that intranasal inoculation of VSV initially leads to
replication in olfactory receptor neurons, followed by the infection
of CNS, which results in encephalitis and death within 6–10 days
postintranasal infection (3, 28). Therefore, consistent with these
observations, with their lack of T cell and long term protective
neutralizing Ab (IgG) responses, all RAG-1 KO and TCR-?? KO
Our studies demonstrated that in response to VSV infection,
MyD88-deficient mice had impaired induction of type 1 IFN, sig-
nificantly delayed or deficient IgM Ab responses, and impaired
CD4?T cell responses, an initial comparable but rapidly de-
creased neutralizing Ab, and ?50% of MyD88 KO mice had the
hind limb paralysis and died after intranasal challenge with a virus
that was not lethal to WT mice. We demonstrated that the TLR
essential adaptor protein, MyD88, is required for the protective
immunity to intranasal VSV infection by both regulating the pro-
duction of type 1 IFN and the activation of CD4?T cells and
regulating the production of neutralizing Abs (early Th-indepen-
dent IgM and later Th-dependent IgG response).
It has been reported that neither i.v. nor i.p. infection with VSV
causes morbidity in immunocompetent mice and that VSV does
not replicate to detectable levels. However, if VSV is given intra-
nasally, virus can gain access to the brain and survival depends on
the host’s natural resistance (28, 33), i.e., the production of type 1
production in response to in vivo VSV infection. Age-matched MyD88 KO
mice and WT male mice were intranasally infected with VSV. Serum sam-
ples were collected at the indicated time points. The levels of MCP-1 (A)
and IFN-? (B) in serum were measured by ELISA. Results are shown as
picograms per milliliter (n ? 4–6 per group). Results are representative of
two separate experiments. ?, p ? 0.05.
MyD88-deficient mice have defective MCP-1 and IFN-?
5178 MyD88 AND VSV
IFN as well as the levels of IgM- and IgG-neutralizing Abs. The
initial impaired induction of type 1 IFNs together with impaired
induction of neutralizing IgM and IgG Abs in intranasal VSV-
infected MyD88 KO mice may be relevant to this failure of
MyD88 KO to control VSV spread into brain and cause enceph-
alitis (Ref. 33 and Fig. 1 and Table I).
How the MyD88 signaling is involved in the regulation of VSV-
induced type 1 IFN production, the activation of both CD4?T
cells and B cells, as well as the induction and maintenance of the
neutralizing Ab is currently not clear.
The mechanism responsible for VSV-induced type 1 IFN is
complex. Several distinctive key molecules and signaling path-
ways have been reported to be involved in the VSV-induced type
1 IFN production, such as TLR7 and MyD88 (18), TLR4/CD14
(34), protein kinase R (33), RIG-1 (35), IRF7 (36), TANK-binding
kinase 1 and I?B kinase-? (37), and recently TNFR-associated
factor 3 (38). These studies suggested that VSV might interact
with multiple molecules including TLRs and through both
MyD88-dependent and –independent mechanisms to activate
the innate as well as adaptive immunity (38, 39).
With respect to the involvement of TLR signaling in the induc-
tion of type 1 IFN, Lund et al. (18) first reported that VSV interacts
with TLR7 to initiate a MyD88-dependent type 1 IFN. However,
we have demonstrated in the present study that the involvement of
MyD88 in VSV-induced in vitro type 1 IFN is cell type dependent.
Most importantly, our in vivo experiments revealed that i.v. VSV-
induced type 1 IFN was MyD88 independent, and interestingly,
intranasal VSV-induced type 1 IFN was MyD88 dependent. Many
cell types besides pDC are capable of production of type 1 IFN
(36, 40). Like Lund et al. (18), we found that Flt3L-derived pDC
produce type 1 IFN in response to VSV via MyD88-dependent
pathways. Yet we also found that GM-CSF-derived BM conven-
tional DCs produce type 1 IFNs in response to VSV largely inde-
pendent of MyD88. Interestingly, a recent study has suggested that
pDC are not the major type 1 IFN producer in response to VSV
(41). Lund et al. used ELISA to quantify the levels of type 1 IFN.
In this study, we predominantly used the bioassay to measure the
activities of type 1 IFN. The bioassay is a well-defined classical
and sensitive assay for the determination of the bioactivity of all
types/subspecies of type 1 IFN (24, 42), whereas ELISA may not
be able to detect all subtypes of type 1 IFN. The IFN-? activity in
VSV-infected MyD88-deficient and WT murine sera could be
completely blocked by a neutralizing Ab specific for mouse IFN-?,
which verified the specificity of the type 1 IFN detection bioassay.
Moreover, the bioactivity of type 1 IFN produced from BM pDC
(Fig. 2D) is comparable with the protein levels measured by
ELISA (data not shown and Ref. 3). We used a natural VSV-
Indiana strain, whereas Lund et al. used a recombinant VSV strain.
Different strains/isolates of VSV could have different properties
(43), including induction of type 1 IFN and replication in different
type of murine cells.
Recent studies have revealed that TLR-MyD88 pathways are
involved in the activation of CD4?T cell and humoral immune
responses to model Ags (7, 44, 45). Our study demonstrates that,
in response to either intranasal or i.v. VSV infection, MyD88-
deficient mice had a negligible IgM response and delayed isotype
switching. Analysis of the IgG isotypes revealed that VSV infec-
tion induced more IgG1 than IgG2a in MyD88-deficient mice,
characteristic of a Th2 type immune response, whereas VSV in-
fection in WT mice induced more IgG2a than IgG1 which is char-
acteristic of a Th1-type immune response. Thus, based on analysis
of Ab switching and formation of IgG subclasses, our study dem-
onstrates that the MyD88 signaling is required for the regulation of
the balance of Th1- and Th2-type immune responses and in the
absence of MyD88, the immune response to VSV infection is
skewed toward a Th2-type immune response. This has physiolog-
ical significance because the neutralization activity of VSV-spe-
cific Ab in MyD88-deficient mice decreased rapidly after infec-
tion, while neutralizing titers in WT mice remained stable over
time. Moreover, although the initial levels of total IgG Abs in both
WT and MyD88-deficient mice were comparable, the neutraliza-
tion activity and concentration of the Abs in WT mice were much
higher than those in MyD88-deficient mice after day 7 post either
i.v. or intranasal infection with VSV (Fig. 4). In addition, the sim-
ilar physiological role of MyD88 in the regulation of Ab responses
to microbes has also been demonstrated in Borrelia burgdorferi-
infected MyD88 KO mice (46, 47). B. burgdorferi infection in
MyD88 KO mice induced comparable levels of total IgG Ab but
induced significantly lower levels of IgG2a isotype Ab in MyD88
KO mice than in WT mice.
It has been suggested that several factors may be involved in the
regulation of the polarization of naive CD4?T cells. These include
the dose of Ag, the type and activation status of APC, the costimu-
latory molecules, and the local cytokine environment (31, 48). It
has been proposed that IL-12, together with IFN-?, positively reg-
ulates the Th1 polarization of naive CD4?T cells through up-
regulation of IL-12R?2 and IL-18R expression, whereas IL-4
negatively regulates IL-18R expression to polarize naive CD4?
T cell toward Th2 development (49). VSV infection induces a
normal Th1 type immune response in IL-12-deficient mice, and
IL-12 is not required for the protection of mice from VSV in-
fection (50). IFN-? has been found to promote Ab response
switching to IgG2a isotype cells (3, 51, 52). Thus, the initial
MyD88-dependent production of chemokines, including IFN-?
production in response to VSV infection, may affect the acti-
vation and differentiation of the naive CD4?T cells as well as
the activation of the B cells and the production of neutralizing
antiviral Abs (3, 51, 52).
Table I. Neutralizing Ab production is MyD88 dependent, but the mortality is both MyD88 and route
CD4 T Cell
p vs WT
aBoth intranasal (i.n.) and i.v. VSV infections in WT mice induce normal levels of type 1 IFN as well as neutralizing Ab.
In marked contrast, intranasal VSV infection in MyD88 KO mice induces both impaired type 1 IFN and CD4 T cell response
as well as neutralizing Ab responses, which result in ?50% of the MyD88 KO death (p ? 0.005). Alternatively, i.v. VSV
infection in MyD88 KO mice induces a normal level of type 1 IFN response but an impaired neutralizing Ab; all mice survive.
5179The Journal of Immunology
The MyD88 adaptor protein is also required for IL-1 and IL-18
signaling pathways (53), and IL-1 and IL-18 have been proposed
to contribute to the balance of Th1 and Th2 type immune re-
sponses (54, 55). However, our study (unpublished observations)
and the study of Hodges et al. (56) have demonstrated that IL-18
does not contribute to intranasal VSV-induced neuropathogenesis.
A role for IL-1 in the defective antiviral response of MyD88 KO
mice cannot be ruled out. We are conducting studies to address that
In conclusion, by using VSV as a model, we have demonstrated
that the MyD88 signaling pathway plays an important role in the
regulation of innate immunity (type 1 IFN) as well as adaptive
immunity (CD4?T cells and neutralizing Abs) to viral infection.
Significantly more MyD88-deficient mice succumbed to intranasal
VSV infection (Fig. 1 and Table I). These findings indicate that
VSV inoculated via different routes may interact with different
types of cells to make type 1 IFN, and these studies further suggest
that MyD88 is more critically involved in type 1 IFN induction
from cells in nasal-respiratory tracts-neuroepithelium environment
after intranasal VSV infection. These studies provide further evi-
dence that the TLR-MyD88 pathway is critically involved in the
regulation of the protective antiviral immune response. They also
emphasize the importance of different routes of infection on acti-
vation of innate immunity and its consequences in terms of viral
We thank Junko Kato for excellent secretarial assistance.
The authors have no financial conflict of interest.
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