To cite this version: Zhisheng Her, Terk-Shin Teng, Jeslin Tan, Teck-Hui Teo, Yiu-Wing Kam, et al.. Loss of TLR3
aggravates CHIKV replication and pathology due to an altered virus-specific neutralizing antibody
response. EMBO Molecular Medicine, 2015, 7 (1), pp.24-41. 10.15252/emmm.201404459. pasteur-
02391327 HAL Id: pasteur-02391327
https://pasteur.hal.science/pasteur-02391327v1
Submitted on 3 Dec 2019
Loss of TLR3 aggravates CHIKV replication and
pathology due to an altered virus-specific neutralizing
antibody response
Zhisheng Her, Terk-Shin Teng, Jeslin Tan, Teck-Hui Teo, Yiu-Wing Kam,
Fok-Moon Lum, Wendy Wl Lee, Christelle Gabriel, Rossella Melchiotti,
Anand K Andiappan, et al.
To cite this version:
Zhisheng Her, Terk-Shin Teng, Jeslin Tan, Teck-Hui Teo, Yiu-Wing Kam, et al..
Loss of TLR3
aggravates CHIKV replication and pathology due to an altered virus-specific neutralizing antibody
response. EMBO Molecular Medicine, 2015, 7 (1), pp.24-41. 10.15252/emmm.201404459. pasteur-
02391327 Loss of TLR3 aggravates CHIKV replication and
pathology due to an altered virus-specific neutralizing
antibody response Zhisheng Her, Terk-Shin Teng, Jeslin Tan, Teck-Hui Teo, Yiu-Wing Kam,
Fok-Moon Lum, Wendy Wl Lee, Christelle Gabriel, Rossella Melchiotti,
Anand K Andiappan, et al. Zhisheng Her, Terk-Shin Teng, Jeslin Tan, Teck-Hui Teo, Yiu-Wing Kam,
Fok-Moon Lum, Wendy Wl Lee, Christelle Gabriel, Rossella Melchiotti,
Anand K Andiappan, et al. Distributed under a Creative Commons Attribution 4.0 International License 1
Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore, Singapore
2
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
3
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore
4
Doctoral School in Translational and Molecular Medicine (DIMET), University of Milano-Bicocca, Milan, Italy
5
Institute of Technology, University of Tartu, Tartu, Estonia
6
Institute of Infectious Disease and Epidemiology (IIDE), Tan Tock Seng Hospital, Singapore, Singapore
7
Institut Pasteur, Biology of Infection Unit, Paris, France
8
Inserm U1117, Paris, France
9
Paris Descartes University, Sorbonne Paris Cité, Necker-Enfants Malades University Hospital, Institut Imagine, Paris, France
10
Institute of Infection and Global Health, University of Liverpool, Liverpool, UK
*Corresponding author. Tel: +65 6407 0028; Fax: +65 6464 2057; E-mail: lisa_ng@immunol.a-star.edu.sg
†These authors contributed equally to this work
‡Present address: INSERM, U1016, Institut Cochin, 22 Rue Mechain, 75014 Paris, France
§Present address: Laboratory of Microbial Immunity, Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR),
8A Biomedical Grove, #04-06, Singapore, 138648, Singapore Loss of TLR3 aggravates CHIKV replicati
pathology due to an altered virus-speci
neutralizing antibody response Zhisheng Her1,2,†, Terk-Shin Teng1,†, Jeslin JL Tan1,†, Teck-Hui Teo1,3, Yiu-Wing Kam1, Fok-Moon Lum1,2,
Wendy WL Lee1,3, Christelle Gabriel1,‡, Rossella Melchiotti1,4, Anand K Andiappan1, Valeria Lulla5,
Aleksei Lulla5, Mar K Win6, Angela Chow6, Subhra K Biswas1, Yee-Sin Leo6, Marc Lecuit7,8,9,
Andres Merits5, Laurent Rénia1 & Lisa FP Ng1,2,10*,§ Abstract DOI 10.15252/emmm.201404459 | Received 22 July 2014 | Revised 31 October
2014 | Accepted 3 November 2014 | Published online 1 December 2014
EMBO Mol Med (2015) 7: 24–41 DOI 10.15252/emmm.201404459 | Received 22 July 2014 | Revised 31 October
2014 | Accepted 3 November 2014 | Published online 1 December 2014
EMBO Mol Med (2015) 7: 24–41 RNA-sensing toll-like receptors (TLRs) mediate innate immunity
and regulate anti-viral response. We show here that TLR3 regulates
host immunity and the loss of TLR3 aggravates pathology in
Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infec-
tion is markedly increased in human and mouse fibroblasts with
defective TLR3 signaling. Up to 100-fold increase in CHIKV load
was observed in Tlr3/ mice, alongside increased virus dissemina-
tion and pro-inflammatory myeloid cells infiltration. Infection in
bone marrow chimeric mice showed that TLR3-expressing hemato-
poietic cells are required for effective CHIKV clearance. CHIKV-
specific antibodies from Tlr3/ mice exhibited significantly lower
in vitro neutralization capacity, due to altered virus-neutralizing
epitope specificity. Finally, SNP genotyping analysis of CHIKF
patients on TLR3 identified SNP rs6552950 to be associated with
disease severity and CHIKV-specific neutralizing antibody response. These results demonstrate a key role for TLR3-mediated antibody
response to CHIKV infection, virus replication and pathology,
providing a basis for future development of immunotherapeutics
in vaccine development. Research Article
Loss of TLR3 aggravates CHIKV replication and
pathology due to an altered virus-specific
neutralizing antibody response
Zhisheng Her1,2,†, Terk-Shin Teng1,†, Jeslin JL Tan1,†, Teck-Hui Teo1,3, Yiu-Wing Kam1, Fok-Moon Lum1,2,
Wendy WL Lee1,3, Christelle Gabriel1,‡, Rossella Melchiotti1,4, Anand K Andiappan1, Valeria Lulla5,
Aleksei Lulla5, Mar K Win6, Angela Chow6, Subhra K Biswas1, Yee-Sin Leo6, Marc Lecuit7,8,9,
Andres Merits5, Laurent Rénia1 & Lisa FP Ng1,2,10*,§ Research Article  Research Article 8
Inserm U1117, Paris, France HAL Id: pasteur-02391327
https://pasteur.hal.science/pasteur-02391327v1
Submitted on 3 Dec 2019 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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publics ou privés. HAL is a multi-disciplinary open access
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entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Introduction Innate immunity against RNA viruses involves pattern recognition
receptors (PRRs) that recognize structurally conserved molecules
from diverse pathogens known as pathogen-associated molecular
patterns (PAMPs) (Arpaia & Barton, 2011). PRRs include TLRs (par-
ticularly TLR3, TLR7 and TLR8) and members of the cytosolic reti-
noic acid-inducible gene I (RIG-I)-like receptors such as melanoma
differentiation-associated protein 5 (MDA5) and RIG-I that detect
RNA viruses through their genomic RNA or the double-stranded
RNA
(dsRNA)
viral intermediate generated during replication
(Yoneyama et al, 2004, 2005; Gitlin et al, 2006). Activation of these
PRRs induces downstream anti-viral type I IFN response, which can
also occur independently of viral RNA transcription and replication
(Nikonov et al, 2013). The involvement of TLRs in counteracting RNA virus infection is
widely documented (Arpaia & Barton, 2011; Neighbours et al, 2012;
Zhang et al, 2013). TLR3 recognizes dsRNA and can influence
disease outcomes depending on the type of virus and infection
model. TLR3-mediated innate and inflammatory responses were
demonstrated to be protective against HIV, CMV and Dengue virus Keywords Chikungunya virus; innate immunity; joint inflammation;
neutralizing antibodies; TLR3 Subject Categories Immunology; Microbiology, Virology & Host Pathogen
Interaction 24 ª 2014 Agency for Science, Technology and Research (A*STAR). Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine
Vol 7 | No 1 | 2015 Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine EMBO Molecular Medicine was required by hematopoietic cells to direct CHIKV-specific anti-
body response toward important neutralizing linear B-cell epitopes
in the E2 glycoprotein. In the absence of TLR3, high levels of
CHIKV-specific IgG were still generated, but with substantially
diminished neutralizing capacity. The clinical relevance of TLR3
was further investigated in CHIKV-infected patients, where the level
of TLR3 transcripts was increased in PBMCs of CHIKV-infected
patients. Interestingly, SNP genotyping analysis further identified
TLR3 SNPs rs3775292 and rs6552950, whose functional effects
remain unknown, to be associated with prevalence of CHIKV pheno-
types, and in the case of SNP rs6552950, also with disease severity,
CHIKV-specific IgG response and antibody neutralizing capacity. Taken together, these results substantiate a role for TLR3 in the
control of CHIKV replication, immunity and pathology. TLR3 inhibits CHIKV replication To dissect the significance of TLR3-mediated anti-CHIKV response,
primary fibroblasts were isolated from both WT and Tlr3/ mice
and infected with CHIKV ex vivo. The loss of TLR3 significantly
increased susceptibility to CHIKV infection that led to marked virus
replication when compared to WT fibroblasts (Fig 1D–F). To gain
further insights on how TLR3 signaling mediates an anti-CHIKV
state, transcriptional profiles of type I IFNs and related IFN-stimu-
lated genes (ISGs) during infection were analyzed (Supplementary
Fig S1). Results revealed that the induction of type I IFNs were
higher in Tlr3/ fibroblasts, suggesting that the induction of type I
IFN response observed here is independent of TLR3. Introduction infections, while TLR3 stimulation results in detrimental disease
outcomes in Influenza A virus and Punta Toro virus infections
(Tabeta et al, 2004; Goffic et al, 2006; Gowen et al, 2006; Suh et al,
2007; Nasirudeen et al, 2011). TLR3-dependent response has been
shown to be both protective by restricting virus replication in
neurons (Daffis et al, 2008) and also detrimental in West Nile virus
infection by perturbing TNFR1 signaling to promote virus entry into
the brains of mice resulting in lethal encephalitis (Wang et al,
2004). Clinically, patients with impaired TLR3-mediated responses
show an increased susceptibility to HSV-1 encephalitis (Zhang et al,
2007; Pe´rez de Diego et al, 2010; Sancho-Shimizu et al, 2011). Repeated reactivation of HSV-2 that led to the development of Moll-
aret meningitis has also been reported in an individual with TLR3
deficiency (Willmann et al, 2010). The significance of TLR-mediated signaling and how TLR mole-
cules influence clinically important re-emerging viruses such as
CHIKV remains confounding. CHIKV is an ‘Old World’ alphavirus
with a positive sense RNA genome belonging to the Togaviridae
family (Deller & Russell, 1968). CHIKV is the causative agent for
CHIKF, and over the last decade, it has caused simultaneous
outbreaks of unprecedented scale in the Indian Ocean Islands
(Josseran et al, 2006), India (Kaur et al, 2008) and subsequently in
South East Asia (Laras et al, 2005; AbuBakar et al, 2007; Leo et al,
2009) and Europe (Queyriaux et al, 2008). Serious CHIKF outbreaks
have also occurred in Cambodia (Centers for Disease Control &
Prevention, 2012; Duong et al, 2012), Laos (Soulaphy et al, 2013)
and Sierra Leone (Ansumana et al, 2013). Since 2013, it has finally
reached the Americas and triggered ongoing outbreaks in the French
West Indies (Enserink, 2014; Leparc-Goffart et al, 2014). The
clinical presentation of the disease is characterized by flu-like symp-
toms such as fever, rash and muscle aches which subside in
7–10 days (Kam et al, 2009). The hallmark of CHIKV infection is
the incapacitating arthralgia that routinely persists for weeks or
months after resolution of the acute symptoms and has high costs in
terms of both quality of life and healthcare provision/economic loss
(Borgherini et al, 2008). TRIF deficiency increases CHIKV replication Activation of various TLR signaling pathways including TLR2, TLR3
and TLR4 engage the TRIF adaptor protein to induce expression of
downstream anti-viral and pro-inflammatory genes (Yamamoto
et al, 2003). To demonstrate a functional role for TRIF signaling in
anti-CHIKV response, human primary fibroblasts with homozygous
TRIF nonsense mutation (Sancho-Shimizu et al, 2011) were infected
with CHIKV in vitro. CHIKV replication was remarkably higher in
the TRIF/ fibroblasts with a 2-log difference when compared to
healthy control (Fig 1A–C). This observation is in line with previous
findings where TRIF/ fibroblasts were shown to be more suscepti-
ble to infection with HSV-1 and vesicular stomatitis virus (Sancho-
Shimizu et al, 2011) due to a defect in type I interferon induction. This suggested that TLRs could be involved in CHIKV infection. We
focused our study on TLR3 because TLR3-mediated immunity and
polymorphisms to viral infection in human have been demonstrated
to regulate disease progression (Pe´rez de Diego et al, 2010; Reinert
et al, 2012; Zhang et al, 2007b). g
The involvement of TLRs in CHIKV replication was previously
investigated in murine models (Schilte et al, 2010, 2012), but their
role in CHIKV pathology and dissemination was not well estab-
lished. We show here that TLR3 signaling plays a critical role in the
control of CHIKV infection, replication, dissemination and pathol-
ogy. Complementing an earlier report where CHIKV-infected Trif/
(Toll/IL-1 resistance domain-containing adaptor inducing IFNb; an
adaptor protein essential for TLR3-mediated signaling) mice showed
more pronounced viremia and joint inflammation compared to WT
mice (Rudd et al, 2012), this study further demonstrated that infec-
tion of cultured primary human TRIF/ and mouse Tlr3/ fibro-
blasts resulted in a significant enhancement of virus replication. Notably, infected Tlr3/ mice developed higher viremia and more
pronounced joint inflammation, associated with a massive infiltra-
tion of myeloid cells such as neutrophils and macrophages when
compared to WT mice. Furthermore, monitoring of virus infection
using a firefly luciferase (FLuc)-tagged recombinant CHIKV (FLuc-
CHIKV) revealed increased CHIKV dissemination in Tlr3/ mice. By infecting bone marrow chimeric mice, we showed that TLR3-
expressing hematopoietic cells were required for effective CHIKV
clearance, but did not directly regulate CHIKV-induced joint inflam-
mation. Mechanistic investigations further demonstrated that TLR3 © 2014 Agency for Science, Technology and Research (A*STAR) Loss of TLR3 leads to more pronounced virus dissemination and
CHIKV-induced pathology A–C
CHIKV replication in TRIF/ primary human fibroblasts was determined by CHIKV Ag detection using flow cytometry (A and B) and viral load quantification using
qRT-PCR (C) after 6 and 12 hpi (MOI 10). Infection was performed in triplicate, and data are representative of two independent experiments and presented as
mean  SD (two-tailed unpaired t-test, ***P = 0.0005 6 hpi CHIKV Ag, ***P = 0.0009 12 hpi CHIKV Ag, **P = 0.0041 6 hpi viral load, **P = 0.0043 12 hpi viral
load). D–F
Primary tail fibroblasts isolated from WT and Tlr3/ mice (n = 3 per group) were infected with CHIKV (MOI 10). CHIKV infectivity was determined by flow
cytometry for CHIKV Ag (D and E) and by qRT-PCR for viral load quantification (F) at 12 hpi. Infection was performed in triplicate, and data are representative of
two independent experiments and presented as mean  SD (two-tailed unpaired t-test, ***P = 0.0001 CHIKV Ag, *P = 0.0368 viral load). monitored daily for survival, viremia and joint inflammation. Although the loss of TLR3 expression did not affect animal survival,
significantly higher viremia was observed throughout the course of
disease in Tlr3/animals (Fig 2A). Strikingly, Tlr3/ mice exhib-
ited a remarkable exacerbation of CHIKV-induced inflammation at
the joint footpad (Fig 2B). Transcriptional analysis on joint footpad
samples harvested at the peak of viremia revealed that the induction
of type I IFNs was differentially induced in Tlr3/ mice as
compared to WT mice (Supplementary Fig S2). bioluminescence signals (Fig 2D) and also from the blood by viral
RNA quantification (Supplementary Fig S3C). The Tlr3/ mice
exhibited delayed virus clearance throughout the disease. On the
contrary, bioluminescence signals were mainly localized at the site
of infection in WT mice (Fig 2C and Supplementary Video S1). Notably, bioluminescence signals peaked at 2–3 dpi in the head
region of Tlr3/ mice and remained detectable until 16 dpi
(Fig 2D). To determine whether the pattern of CHIKV dissemination
was specific to the loss of TLR3 and not due to the site of virus
inoculation, mice were subjected to s.c. inoculation of CHIKV at
the dermis region of the ear prior to live imaging analysis (Supple-
mentary Fig S4 and Supplementary Videos S3 and S4). Similar
patterns of CHIKV dissemination were observed. Loss of TLR3 leads to more pronounced virus dissemination and
CHIKV-induced pathology The importance of TLR3 in CHIKV infection was next examined in
vivo in both WT and Tlr3/ mice via the joint footpad inoculation
route (Gardner et al, 2010; Teng et al, 2012). Animals were EMBO Molecular Medicine
Vol 7 | No 1 | 2015 25 TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al EMBO Molecular Medicine C
A
D
B
F
E
Figure 1. Enhanced CHIKV replication in TRIF/ and Tlr3/ primary fibroblasts from human and mice, respectively. A–C
CHIKV replication in TRIF/ primary human fibroblasts was determined by CHIKV Ag detection using flow cytometry (A and B) and viral load quantification using
qRT-PCR (C) after 6 and 12 hpi (MOI 10). Infection was performed in triplicate, and data are representative of two independent experiments and presented as
mean  SD (two-tailed unpaired t-test, ***P = 0.0005 6 hpi CHIKV Ag, ***P = 0.0009 12 hpi CHIKV Ag, **P = 0.0041 6 hpi viral load, **P = 0.0043 12 hpi viral
load). D–F
Primary tail fibroblasts isolated from WT and Tlr3/ mice (n = 3 per group) were infected with CHIKV (MOI 10). CHIKV infectivity was determined by flow
cytometry for CHIKV Ag (D and E) and by qRT-PCR for viral load quantification (F) at 12 hpi. Infection was performed in triplicate, and data are representative of
two independent experiments and presented as mean  SD (two-tailed unpaired t-test, ***P = 0.0001 CHIKV Ag, *P = 0.0368 viral load). B A C C C B A E F D E F D Figure 1. Enhanced CHIKV replication in TRIF/ and Tlr3/ primary fibroblasts from human and mice, respectively. A–C
CHIKV replication in TRIF/ primary human fibroblasts was determined by CHIKV Ag detection using flow cytometry (A and B) and viral load quantification using
qRT-PCR (C) after 6 and 12 hpi (MOI 10). Infection was performed in triplicate, and data are representative of two independent experiments and presented as
mean  SD (two-tailed unpaired t-test, ***P = 0.0005 6 hpi CHIKV Ag, ***P = 0.0009 12 hpi CHIKV Ag, **P = 0.0041 6 hpi viral load, **P = 0.0043 12 hpi viral Figure 1. Enhanced CHIKV replication in TRIF/ and Tlr3/ primary fibroblasts from human and mice, respectively. Figure 1. Enhanced CHIKV replication in TRIF/ and Tlr3/ primary fibroblasts from human and mice, respectively. © 2014 Agency for Science, Technology and Research (A*STAR) Loss of TLR3 leads to more pronounced virus dissemination and
CHIKV-induced pathology Although the
level of bioluminescence signals measured by the ear inoculation
route was lower, signals detected from the whole body, head
region and inoculated ear in Tlr3/ mice still remained signifi-
cantly higher than that of the WT mice (Supplementary Fig S4B
and Supplementary Videos S3 and S4) and correlated with viremia
(Supplementary Fig S3D). To further study the role of TLR3 on in vivo CHIKV replication
and dissemination, we used a recombinant FLuc-CHIKV to infect
both WT and Tlr3/ mice and tracked the kinetics of CHIKV infec-
tion by live imaging for a duration of 20 dpi. As expected, the loss
of TLR3 once again resulted in more pronounced joint inflammation
and viremia (Supplementary Fig S3A–C). Bioluminescence signals
indicative of virus replication distinctly reveal the differences in the
route of CHIKV dissemination in WT and Tlr3/ mice during the
initial phase of infection (Fig 3C and Supplementary Videos S1 and
S2). From the site of infection (i.e. right joint footpad), signals were
next detected in the posterior regions (tail, lymph node and left foot-
pad) before moving to the anterior regions in the lower abdomen
and head (Supplementary Videos S1 and S2). Notably, CHIKV
dissemination was more rapid and extensive in Tlr3/ mice than in
WT mice (Fig 2C and D and Supplementary Videos S1 and S2). High
level of virus replication was detected from the whole body by the To further evaluate whether a loss of TLR3 expression affects
virus tropism in tissues and deeper organs, mice inoculated via the
joint footpad route were sacrificed at 3 dpi and dissected in order to
quantify bioluminescence signals from the various organs, skeletal
body and skin (Fig 2E). Higher bioluminescence signals were 26 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine detected from the skeletal body, skin, head, lungs, liver, heart,
spleen, kidneys and brain of Tlr3/ mice (Fig 2E), confirming the
role of TLR3 in controlling CHIKV infection and virus dissemination. Interestingly, bioluminescence signals were also detected in the
brain in 40% of Tlr3/ mice (Fig 2E), and positive bioluminescence
signals were still detectable in the body, skin and head of Tlr3/
mice at 6 dpi (Supplementary Fig S5). © 2014 Agency for Science, Technology and Research (A*STAR) Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. A, B
WT and Tlr3/ mice (n = 5 per group) were infected with CHIKV (106 PFU) by joint footpad inoculation. (A) Viremia was determined from blood collected from
tails by viral load quantification. Dotted line indicates the limit of viral load detection. (B) Extent of joint inflammation was measured daily and expressed as
disease score relative to day 0 (pre-infection). Data are representative of three independent experiments and presented as mean  SD (two-tailed Mann–Whitney
U-test, **P = 0.0037 1 dpi viremia, ***P = 0.0006 2 dpi viremia, ***P = 0.0003 3–6 dpi viremia, **P = 0.0012 8 dpi viremia, **P = 0.0012 10 dpi viremia,
***P = 0.0003 12 dpi viremia, ***P = 0.0002 2–9 dpi disease score, ***P = 0.0003 10 dpi disease score, **P = 0.003 11 dpi disease score, *P = 0.014 12 dpi disease
score, **P = 0.0093 13 dpi disease score). C, D
TLR3 modulates CHIKV dissemination in mice. WT and Tlr3/ mice were infected with FLuc-CHIKV (106 PFU) by joint footpad (n = 4–5 per group) inoculation. Bioluminescence signals were measured using an in vivo bioluminescence imaging system. (C) Images of representative 6 dpi in WT and Tlr3/ mice. Color scale
indicates the level of bioluminescence signals detected. (D) Bioluminescence signals of whole body, head region and at site of inoculation were quantified and
expressed as average radiance (p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Data are representative of two independent experiments and presented as
mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 3 hpi whole body, *P = 0.0317 6 hpi whole body, *P = 0.0317 4 dpi whole body, *P = 0.0159 6 dpi
whole body, *P = 0.0159 7 dpi whole body, *P = 0.0317 8 dpi whole body, *P = 0.0195 3 hpi right footpad, *P = 0.0159 3 hpi head, *P = 0.0159 6 hpi head,
*P = 0.0317 5 dpi head, *P = 0.0159 6 dpi head, *P = 0.0317 7 dpi head, *P = 0.0317 8 dpi head, *P = 0.0317 10 dpi head, *P = 0.0476 12 dpi head). / C, D
TLR3 modulates CHIKV dissemination in mice. WT and Tlr3/ mice were infected with FLuc-CHIKV (106 PFU) by joint footpad (n = 4–5 per group) inoculation. Bioluminescence signals were measured using an in vivo bioluminescence imaging system. (C) Images of representative 6 dpi in WT and Tlr3/ mice. Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. Data are representative of two independent experiments and presented as
mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 3 hpi whole body, *P = 0.0317 6 hpi whole body, *P = 0.0317 4 dpi whole body, *P = 0.0159 6 dpi
whole body, *P = 0.0159 7 dpi whole body, *P = 0.0317 8 dpi whole body, *P = 0.0195 3 hpi right footpad, *P = 0.0159 3 hpi head, *P = 0.0159 6 hpi head,
*P = 0.0317 5 dpi head, *P = 0.0159 6 dpi head, *P = 0.0317 7 dpi head, *P = 0.0317 8 dpi head, *P = 0.0317 10 dpi head, *P = 0.0476 12 dpi head). p
p
p
p
p
p
)
E
TLR3 modulates CHIKV tissue tropism in mice. Bioluminescence signals of various organs, body and skin of WT and Tlr3/ mice (n = 10–12 per group) infected
with FLuc-CHIKV (106 PFU) by joint footpad inoculation at 3 dpi were quantified and expressed as average radiance (p/s/cm2/sr). The lowest limit of detection is 0
p/s/cm2/sr. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0206 body,
**P = 0.0034 skin, ***P = 0.0003 head, ***P = 0.0002 lungs, **P = 0.0011 liver, *P = 0.0434 heart, ***P = 0.0003 spleen, *P = 0.0117 kidneys, *P = 0.0213 brain). neutrophils in Tlr3/ mice (Fig 4A and Supplementary Fig S7). This observation was further complemented by transcriptional
analysis of the CHIKV-infected joint footpad that revealed a signifi-
cant induction of neutrophil-associated activation molecules and
chemokines such as defensins Defb4, Defb14, myeloperoxidase
(Mpo), Il-1b and Il-8r in Tlr3/ mice at 6 dpi (Fig 4B). (Supplementary Fig S6) and during the peak of disease severity at
6 dpi (Fig 3). The infiltrated myeloid cells consisted primarily of
F4/80+ macrophages that were localized in the edema region,
while the Ly6G+ neutrophils were localized distinctly in the skeletal
muscles. In addition, cellular infiltrates in the joint of virus-infected
mice were further analyzed during the peak of inflammation at 6
dpi
by
flow
cytometry. Consistent
with
histological
analysis
(Fig 3), the loss of TLR3 resulted in a significant increase in infil-
trating CD11b+ myeloid cells and, in particular, CD11b+Ly6G+ Although both CD4+ and CD8+ T cells were previously demon-
strated to infiltrate the joint footpad, only CD4+ T cells were
responsible for the pathology (Teo et al, 2013). TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. Color scale
indicates the level of bioluminescence signals detected. (D) Bioluminescence signals of whole body, head region and at site of inoculation were quantified and
expressed as average radiance (p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Data are representative of two independent experiments and presented as
mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 3 hpi whole body, *P = 0.0317 6 hpi whole body, *P = 0.0317 4 dpi whole body, *P = 0.0159 6 dpi
whole body, *P = 0.0159 7 dpi whole body, *P = 0.0317 8 dpi whole body, *P = 0.0195 3 hpi right footpad, *P = 0.0159 3 hpi head, *P = 0.0159 6 hpi head,
*P = 0.0317 5 dpi head, *P = 0.0159 6 dpi head, *P = 0.0317 7 dpi head, *P = 0.0317 8 dpi head, *P = 0.0317 10 dpi head, *P = 0.0476 12 dpi head). E
TLR3 modulates CHIKV tissue tropism in mice. Bioluminescence signals of various organs, body and skin of WT and Tlr3/ mice (n = 10–12 per group) infected
with FLuc-CHIKV (106 PFU) by joint footpad inoculation at 3 dpi were quantified and expressed as average radiance (p/s/cm2/sr). The lowest limit of detection is 0
p/s/cm2/sr. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0206 body,
**P = 0.0034 skin, ***P = 0.0003 head, ***P = 0.0002 lungs, **P = 0.0011 liver, *P = 0.0434 heart, ***P = 0.0003 spleen, *P = 0.0117 kidneys, *P = 0.0213 brain). C, D
TLR3 modulates CHIKV dissemination in mice. WT and Tlr3/ mice were infected with FLuc-CHIKV (106 PFU) by joint footpad (n = 4–5 per group) inoculation. Bioluminescence signals were measured using an in vivo bioluminescence imaging system. (C) Images of representative 6 dpi in WT and Tlr3/ mice. Color scale
indicates the level of bioluminescence signals detected. (D) Bioluminescence signals of whole body, head region and at site of inoculation were quantified and
expressed as average radiance (p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Loss of TLR3 leads to more pronounced virus dissemination and
CHIKV-induced pathology Severe joint inflammation in Tlr3/ mice is associated with a
massive infiltration of myeloid cells
In an effort to assess tissue damage in the joints of CHIKV-infec
mice, histological assessments revealed an intensified disease sc
in Tlr3/ mice that correlated with more pronounced subcuta
ous edema and infiltration of CD11b+ myeloid cells at 3
C
D
A
B
E
Figure 2. A B B A  C D D D C detected from the skeletal body, skin, head, lungs, liver, heart,
spleen, kidneys and brain of Tlr3/ mice (Fig 2E), confirming the
role of TLR3 in controlling CHIKV infection and virus dissemination. Interestingly, bioluminescence signals were also detected in the
brain in 40% of Tlr3/ mice (Fig 2E), and positive bioluminescence
signals were still detectable in the body, skin and head of Tlr3/
mice at 6 dpi (Supplementary Fig S5). Severe joint inflammation in Tlr3/ mice is associated with a
massive infiltration of myeloid cells
In an effort to assess tissue damage in the joints of CHIKV-infecte
mice, histological assessments revealed an intensified disease scor
in Tlr3/ mice that correlated with more pronounced subcutane
ous edema and infiltration of CD11b+ myeloid cells at 3 dp
E
Figure 2. E E E E    Severe joint inflammation in Tlr3/ mice is associated with a
massive infiltration of myeloid cells detected from the skeletal body, skin, head, lungs, liver, heart,
spleen, kidneys and brain of Tlr3/ mice (Fig 2E), confirming the
role of TLR3 in controlling CHIKV infection and virus dissemination. Interestingly, bioluminescence signals were also detected in the
brain in 40% of Tlr3/ mice (Fig 2E), and positive bioluminescence
signals were still detectable in the body, skin and head of Tlr3/
mice at 6 dpi (Supplementary Fig S5). In an effort to assess tissue damage in the joints of CHIKV-infected
mice, histological assessments revealed an intensified disease score
in Tlr3/ mice that correlated with more pronounced subcutane-
ous edema and infiltration of CD11b+ myeloid cells at 3 dpi In an effort to assess tissue damage in the joints of CHIKV-infected
mice, histological assessments revealed an intensified disease score
in Tlr3/ mice that correlated with more pronounced subcutane-
ous edema and infiltration of CD11b+ myeloid cells at 3 dpi © 2014 Agency for Science, Technology and Research (A*STAR) EMBO Molecular Medicine
Vol 7 | No 1 | 2015 27 EMBO Molecular Medicine Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 CD3+CD4+ of CD4+ T-cell-
non-depleted WT and Tlr3/ mice, **P = 0.0079 CD3+CD4+ of CD4+ T-cell-depleted and T-cell-non-depleted WT mice, **P = 0.0043 CD3+CD4+ of CD4+ T-cell-
depleted and T-cell-non-depleted Tlr3/ mice, *P = 0.0317 CD3CD11b+Ly6G+ of CD4+ T-cell-non-depleted WT and Tlr3/ mice, **P = 0.0043 CD3CD11b+Ly6G+
of CD4+ T-cell-depleted and T-cell-non-depleted Tlr3/ mice). E
Depletion of CD4+ T cells reduced recruitment of neutrophils in Tlr3/ mice. At 6 dpi, cells from CHIKV-inoculated footpad were harvested and labeled for CD45,
CD3, CD4, CD11b and Ly6G. Absolute cell counts of each immune cell subset were calculated according to the total number of live cells determined before labeling. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 CD3+CD4+ of CD4+ T-cell-
non-depleted WT and Tlr3/ mice, **P = 0.0079 CD3+CD4+ of CD4+ T-cell-depleted and T-cell-non-depleted WT mice, **P = 0.0043 CD3+CD4+ of CD4+ T-cell-
depleted and T-cell-non-depleted Tlr3/ mice, *P = 0.0317 CD3CD11b+Ly6G+ of CD4+ T-cell-non-depleted WT and Tlr3/ mice, **P = 0.0043 CD3CD11b+Ly6G+
of CD4+ T-cell-depleted and T-cell-non-depleted Tlr3/ mice). reduction in Ly6G+ neutrophils infiltration at 6 dpi (Fig 4E),
supporting earlier observations (Figure 3) that neutrophils are a
vital mediator of footpad joint inflammation in Tlr3/ mice. be compelling to assess the number of CD4+ T cells in Tlr3/
mice. Interestingly, the number of infiltrating CD4+ T cells in the
joint footpad was significantly reduced by half (Fig 4A). Joint
inflammation was significantly reduced both in CHIKV-infected WT
and Tlr3/ mice depleted of CD4+ T cells when compared to
controls (Fig 4C), indicating that joint pathology is mediated by
CD4+ T cells in both groups. However, depleting CD4+ T cells did
not modify the viremia in both WT and Tlr3/ mice (Figure 4D),
demonstrating that virus replication is independent of CD4+ T
cells. Furthermore, CD4+ T-cell depletion resulted in a significant Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. Data are representative of one of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney
U-test, **P = 0.0079 total CD3+, **P = 0.0079 CD3+CD4+, *P = 0.0159 CD3CD11b+Ly6G+). B
Increased recruitment of neutrophils in Tlr3/ mice on 6 dpi was associated with induction of neutrophil-associated molecules/chemokines expression. WT and
Tlr3/mice (n = 5–6 per group) were infected with CHIKV (106 PFU) by joint footpad inoculation. The level of gene expression was expressed as fold change
compared to mock-infected mice footpad (n = 5 per group) after normalization to Gapdh. Data are representative of two independent experiments and presented
as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0079 Ccr2, *P = 0.317 Defb4, *P = 0.0159 Defb14, *P = 0.0317 Il-1b, *P = 0.0159 Il-8r, *P = 0.0317 Ip-10,
*P = 0.0159 Mpo, *P = 0.0317 Mip2). p
p )
C, D
Depletion of CD4+ T cells in Tlr3/ mice reduced severity of joint swelling. Tlr3/ mice were injected i.p. with rat anti-mouse CD4 antibody to deplete CD4+ T
cells on 2 and 1 dpi before CHIKV infection (106 PFU) by joint footpad (n = 5–6 per group) inoculation. (C) Extent of joint inflammation was measured daily and
expressed as disease score relative to day 0 (pre-infection), and (D) viremia was determined by viral load quantification. Dotted line indicates the limit of viral load
detection. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0159 6 dpi CD4+
T-cell-depleted and T-cell-non-depleted WT mice, *P = 0.0173 4 dpi CD4+ T-cell-depleted and T-cell-non-depleted Tlr3/ mice, *P = 0.0281 5 dpi CD4+ T-cell-
depleted and T-cell-non-depleted Tlr3/ mice, **P = 0.0087 6 dpi CD4+ T-cell-depleted and T-cell-non-depleted Tlr3/ mice). E
Depletion of CD4+ T cells reduced recruitment of neutrophils in Tlr3/ mice. At 6 dpi, cells from CHIKV-inoculated footpad were harvested and labeled for CD45,
CD3, CD4, CD11b and Ly6G. Absolute cell counts of each immune cell subset were calculated according to the total number of live cells determined before labeling. © 2014 Agency for Science, Technology and Research (A*STAR) Figure 2.
TLR3 modulates CHIKV replication, disease pathology and dissemination in mice. Therefore, it would A 
B 
C 
D 
Figure 3. Loss of TLR3 increases myeloid cells infiltration at the peak of joint swelling. A–D
WT and Tlr3/ mice (n = 5–6 per group) were infected with CHIKV (106 PFU) by joint footpad inoculation. Histological analysis of CHIKV-inoculated joint footpad
samples from WT and Tlr3/ mice by H&E staining (A) and labeling with anti-F4/80 antibody (B), anti-CD11b (C) and anti-Ly6G (D) at 6 dpi. Boxed regions are
shown at higher magnification on the right. * = edema, B = bone, M = muscle, T = tendon. Black arrow heads indicate positively stained cells. Images presented
are from one mouse representative of 3 mice per group from two independent experiments. Scale bars: left, 100 lm; middle, 50 lm; right, 30 lm. B A A B D C C D  Figure 3. Loss of TLR3 increases myeloid cells infiltration at the peak of joint swelling. A–D
WT and Tlr3/ mice (n = 5–6 per group) were infected with CHIKV (106 PFU) by joint footpad inoculation. Histological analysis of CHIKV-inoculated joint footpad
samples from WT and Tlr3/ mice by H&E staining (A) and labeling with anti-F4/80 antibody (B), anti-CD11b (C) and anti-Ly6G (D) at 6 dpi. Boxed regions are
shown at higher magnification on the right. * = edema, B = bone, M = muscle, T = tendon. Black arrow heads indicate positively stained cells. Images presented
are from one mouse representative of 3 mice per group from two independent experiments. Scale bars: left, 100 lm; middle, 50 lm; right, 30 lm. EMBO Molecular Medicine
Vol 7 | No 1 | 2015 28 © 2014 Agency for Science, Technology and Research (A*STAR) Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine A
C
D
E
B
Figure 4. Loss of TLR3 modulates neutrophils recruitment, but not CD4+ T-cell-mediated joint inflammation. A
B B A B A   D C E D E C   Figure 4. Loss of TLR3 modulates neutrophils recruitment, but not CD4+ T-cell-mediated joint inflammation. A
WT and Tlr3/ mice were infected with CHIKV (106 PFU) by joint footpad (n = 5–6 per group) inoculation. At 6 dpi, cells from the CHIKV-inoculated footpad were
harvested and labeled for CD45, CD3, CD4, CD8, CD11b and Ly6G. The absolute cell counts of each immune cell subset were calculated based on the total number
of live cells determined before labeling. Impaired virus clearance in Tlr3/ mice is due to diminished
recognition of CHIKV E2 glycoprotein Chimera and T-cell depletion experiments suggested a role for TLR3
signaling on B cells in virus control. The role of B cells in mediating
virus clearance was demonstrated with CHIKV-specific antibodies
that map to epitopes within the CHIKV E1 and E2 glycoproteins
(Kam et al, 2012b; Lum et al, 2013). These antibodies were demon-
strated to exhibit high neutralizing activities in both macaque and
murine models (Kam et al, 2012b, 2014; Lum et al, 2013). This led
us to further examine whether the impaired virus clearance
observed in Tlr3/ mice (Fig 2A and Supplementary Fig S3C and
D) was due to a defective CHIKV-specific antibody response. The
levels of CHIKV-specific total IgM and IgG antibodies in sera of
infected mice collected at 0, 3, 6, 9, 12 and 15 dpi were first deter-
mined using virion-based ELISA (Fig 6A and B). CHIKV-specific IgM
antibodies were detected on 3 dpi and peaked at 6 dpi with no differ-
ence between the WT and Tlr3/ mice (Fig 6A). CHIKV-specific
IgGs were detected later, from 6 dpi, and their levels continued to C 
A 
B 
F 
D 
E 
Figure 5. Tlr3/?WT and Tlr3/?Tlr3/ bone marrow chimeras were impaired in CHIKV clearance. C 
A 
B 
F 
D 
E 
Figure 5. Tlr3/?WT and Tlr3/?Tlr3/ bone marrow chimeras were impaired in CHIKV clearance. A, D
WT and Tlr3/ mice (n = 4–7 per group) were lethally irradiated and reconstituted with bone marrow cells from WT or Tlr3/mice. Chimeric mice were infected
with CHIKV (106 PFU) by joint footpad inoculation after 6 wks. Viremia was determined by viral load quantification. Dotted line indicates the limit of viral load
detection. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0095 5–7 dpi viremia
WT recipient chimera, *P = 0.0139 8 dpi viremia WT recipient chimera, **P = 0.0095 10 dpi viremia WT recipient chimera, *P = 0.0350 4 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 5 dpi viremia Tlr3/ recipient chimera, *P = 0.0221 6 dpi viremia Tlr3/ recipient chimera, **P = 0.0012 7 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 8 dpi viremia Tlr3/ recipient chimera). B, E
Extent of joint inflammation was measured daily and expressed as disease score relative to day 0 (pre-infection). TLR3-expressing hematopoietic cells are required for effective
CHIKV clearance It is well established that hematopoietic and non-hematopoietic cells
are engaged in the control of CHIKV infection by the innate immune
system (Her et al, 2010; Schilte et al, 2010). To next assess whether © 2014 Agency for Science, Technology and Research (A*STAR) EMBO Molecular Medicine
Vol 7 | No 1 | 2015 29 TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al EMBO Molecular Medicine displayed a similar level of joint inflammation comparable to the
Tlr3/?Tlr3/ chimeras (Fig 5D–F). effective CHIKV clearance could be mediated directly by TLR3-
expressing hematopoietic cells, bone marrow chimeric mice were
generated by lethal irradiation of WT mice, followed by adoptive
transfer of either WT (referred to as WT?WT) or Tlr3/ (referred
to as Tlr3/?WT) bone marrow cells. Upon successful bone
marrow engraftment after 6 weeks, chimeric mice were infected
with recombinant FLuc-CHIKV by footpad inoculation and moni-
tored for joint inflammation, viremia and CHIKV dissemination. Strikingly, Tlr3/?WT chimeras suffered significantly higher vire-
mia from 5 dpi onward and endured delayed virus clearance till 14
dpi (Fig 5A), but joint inflammation and bioluminescence signals
detected in body and CHIKV-infected footpad were not significantly
different when compared to WT?WT chimeras (Fig 5B and C). The
similar degree of joint inflammation observed between these two
groups further substantiate that the differences observed in joint
inflammation between WT and Tlr3/ mice were not mediated by
TLR3 deficiency in hematopoietic cells. Nonetheless, these results
indicate that TLR3-expressing hematopoietic cells are the main
subsets required for effective CHIKV clearance. This notion was
further supported by the converse bone marrow engraftment experi-
ments, where a significant reduction in viremia was observed in the
WT?Tlr3/ chimeras, even though the WT?Tlr3/ chimeras still Impaired virus clearance in Tlr3/ mice is due to diminished
recognition of CHIKV E2 glycoprotein Data are representative of two independent
experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0117 4 dpi disease score Tlr3/ recipient chimera, *P = 0.0146 5 dpi disease
score Tlr3/ recipient chimera). C, F
CHIKV dissemination is not modulated in WT and Tlr3/ chimeras. Bioluminescence signals of whole body was quantified and expressed as average radiance
(p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Data are representative of one of two independent experiments and presented as mean  SD (two-tailed
Mann–Whitney U-test, *P = 0.0381 1 dpi whole-body WT recipient chimera, **P = 0.0082 1 dpi whole-body Tlr3/ recipient chimera). A B C A C A B B F E D F D E E Figure 5. Tlr3/?WT and Tlr3/?Tlr3/ bone marrow chimeras were impaired in CHIKV clearance. A, D
WT and Tlr3/ mice (n = 4–7 per group) were lethally irradiated and reconstituted with bone marrow cells from WT or Tlr3/mice. Chimeric mice were infected
with CHIKV (106 PFU) by joint footpad inoculation after 6 wks. Viremia was determined by viral load quantification. Dotted line indicates the limit of viral load
detection. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0095 5–7 dpi viremia
WT recipient chimera, *P = 0.0139 8 dpi viremia WT recipient chimera, **P = 0.0095 10 dpi viremia WT recipient chimera, *P = 0.0350 4 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 5 dpi viremia Tlr3/ recipient chimera, *P = 0.0221 6 dpi viremia Tlr3/ recipient chimera, **P = 0.0012 7 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 8 dpi viremia Tlr3/ recipient chimera). B E
E
f j i
i fl
i
d d il
d
d
di
l
i
d
0 (
i f
i
) D
i
f
i d
d A, D
WT and Tlr3/ mice (n = 4–7 per group) were lethally irradiated and reconstituted with bone marrow cells from WT or Tlr3/mice. Chimeric mice were infected
with CHIKV (106 PFU) by joint footpad inoculation after 6 wks. Viremia was determined by viral load quantification. Dotted line indicates the limit of viral load
detection. TLR3 SNP rs6552950 is associated with disease severity and
CHIKV-specific IgG neutralizing antibody capacity in CHIKV-
infected patients To examine the clinical relevance of TLR3-mediated immunity in
CHIKV pathogenesis, transcriptional analysis was carried out in
PBMCs of patients with CHIKV. TLR3 levels were significantly
higher in CHIKV-infected patients when compared to healthy
controls during the acute and early convalescent phases of CHIKV
infection (Fig 7A). These results suggest that TLR3-mediated signal-
ing forms part of the early innate immune response against CHIKV. Studies using plasma samples from CHIKV-infected patients and
sera from CHIKV-infected WT mice have revealed that most of the
linear B-cell epitopes recognized by CHIKV-specific antibodies are
localized in the E2 glycoprotein (Kam et al, 2012a,b; Lum et al,
2013). Based on these observations, linear B-cell epitopes were
screened to assess whether the decreased neutralizing capacity of
anti-CHIKV antibodies from Tlr3/ mice was associated with
diminished epitope recognition. Using the optimized peptide-based
ELISA described (Kam et al, 2012b; Lum et al, 2013), linear
peptides covering the E2 glycoprotein proteome (Supplementary
Table S1) were screened with diluted sera (1:500) from mice
collected at 6 and 15 dpi (Fig 6E and F and Supplementary Fig S8B). Linear peptides were screened individually and only three peptides
were detected to exhibit significant differences toward the sera
between WT and Tlr3/ mice (Fig 6E and F). Recognition of these
three peptides (381, 388 and E2EP3) by CHIKV-specific antibodies
from Tlr3/ mice was significantly diminished compared to WT
mice (Fig 6E–G). Specifically, recognition of an early detection sero-
logical epitope ‘E2EP3’, which is a dominant linear B-cell epitope in
CHIKV-infected patients and animal models (Kam et al, 2012a,b;
Lum et al, 2013), was significantly diminished (Fig 6E–G). More-
over, sera from Tlr3/?Tlr3/ chimeras were also less neutraliz-
ing and similarly associated with a reduced recognition of the same SNP genotyping analysis of 10 TLR3 tagSNPs was performed in
94 CHIKF patients to establish the impact of TLR3 polymorphisms
on
CHIKV-induced
disease
outcome. Association
with
CHIKV
phenotypes revealed that SNP rs3775292 and SNP rs6552950 had
significant
nominal
association
to
disease
occurrence
when
compared to population controls from the 1000 genome project
(Table 1) (1000 Genomes Project Consortium et al, 2010). In both
SNPs, the minor alleles were associated with increased disease
susceptibility as indicated by the odds ratios (OR) (Table 1). Figure 6.
TLR3 modulates early CHIKV-specific IgG response in mice.
/
6 Number corresponds to the region of amino acid sequences in our overlapping 18-mer linear peptides library,
along the CHIKV viral genome. All representations are shown in frontal and back view. G
Localization of identified CHIKV B-cell epitopes (381; blue, 388; green, E2EP3; red) within CHIKV proteome. Epitopes in the E2 glycoprotein were located based on
the structural data obtained from PDB records: 3N42. Number corresponds to the region of amino acid sequences in our overlapping 18-mer linear peptides library,
along the CHIKV viral genome. All representations are shown in frontal and back view. dominant linear E2 epitopes analyzed in Tlr3/ mice (Supplemen-
tary Fig S8B). These observations strongly indicate that the impaired
virus clearance in Tlr3/ mice is related to the diminished recogni-
tion of dominant linear epitopes in the E2 glycoprotein that are func-
tionally important for virus neutralization (Kam et al, 2012b). increase substantially up to 15 dpi (Fig 6B). These results indicated
that the delayed virus clearance observed in Tlr3/ mice was not
due to the inability to produce CHIKV-specific antibodies. Interest-
ingly, the level of CHIKV-specific total IgG antibodies was signifi-
cantly higher in Tlr3/ mice than from WT mice at 6 dpi
(Fig 6B), but this did not translate to a higher CHIKV-neutralizing
capacity
as
demonstrated
by
the
in
vitro
neutralizing
assay
(Kam et al, 2012b) (Fig 6C). Intriguingly, serially diluted pooled
sera taken at 6 dpi from Tlr3/ mice significantly showed in vitro
lower neutralizing activity against CHIKV compared to sera from
WT mice (Fig 6C). Similar observations were obtained using
pooled sera taken during complete virus clearance at 15 dpi from
Tlr3/ mice (Fig 6D) and Tlr3/?Tlr3/chimeras (Supplemen-
tary Fig S8A). Neutralizing activity was significantly reduced
against CHIKV compared to sera from WT and WT?Tlr3/
chimeras, respectively. Figure 6.
TLR3 modulates early CHIKV-specific IgG response in mice.
/
6 Figure 6. TLR3 modulates early CHIKV-specific IgG response in mice. /
6 WT and Tlr3/ mice (n = 6 per group) were infected with CHIKV (106 PFU) by joint footpad inoculation. A, B
Total CHIKV-specific IgM (A) and IgG (B) levels were determined from serum samples collected at 0, 3, 6, 9, 12 and 15 dpi at a dilution of 1:100 and 1:2,000,
respectively, using purified CHIKV virion-based ELISA. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–
Whitney U-test, **P = 0.0087 6 dpi IgG). A, B
Total CHIKV-specific IgM (A) and IgG (B) levels were determined from serum samples collected at 0, 3, 6, 9, 12 and 15 dpi at a dilution of 1:100 and 1:2,000,
respectively, using purified CHIKV virion-based ELISA. Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–
Whitney U-test, **P = 0.0087 6 dpi IgG). C, D
Neutralizing capacity of pooled sera collected at 6 dpi (C) and 15 dpi (D) from WT mice is significantly higher than from Tlr3/ mice. Pooled sera were diluted
1:50–1:5,000 and mixed with CHIKV (MOI 10) for 2 h before infection of HEK 293T cells for 6 h. Assays were performed in quintuplicate, and data are expressed
relative to virus-only-infected samples without sera. Dotted line indicates the detection limit of assay determined from mock-infected samples. Data are
representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0079 6 dpi 1:500 serum dilution,
**P = 0.0079 6 dpi 1:5,000 serum dilution, *P = 0.0286 15 dpi 1:50 serum dilution, *P = 0.0286 15 dpi 1:500 serum dilution, *P = 0.0286 15 dpi 1:5,000 serum
dilution). C, D
Neutralizing capacity of pooled sera collected at 6 dpi (C) and 15 dpi (D) from WT mice is significantly higher than from Tlr3/ mice. Pooled sera were diluted
1:50–1:5,000 and mixed with CHIKV (MOI 10) for 2 h before infection of HEK 293T cells for 6 h. Assays were performed in quintuplicate, and data are expressed
relative to virus-only-infected samples without sera. Dotted line indicates the detection limit of assay determined from mock-infected samples. Figure 6.
TLR3 modulates early CHIKV-specific IgG response in mice.
/
6 Data are
representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0079 6 dpi 1:500 serum dilution,
**P = 0.0079 6 dpi 1:5,000 serum dilution, *P = 0.0286 15 dpi 1:50 serum dilution, *P = 0.0286 15 dpi 1:500 serum dilution, *P = 0.0286 15 dpi 1:5,000 serum
dilution). E, F
Mapping antibody reactivity to linear B-cell epitopes within CHIKV E2 proteome. CHIKV E2 epitopes recognized at 6 dpi (E) and 15 dpi (F) were determined in
pooled sera collected from infected mice using ELISA specific for overlapping 18-mer linear peptides spanning the CHIKV E2 proteome. The peptide numbers
correspond to the position of the 18-mer linear peptides along the CHIKV E2 proteome. Structural data were retrieved from PDB (id: 3N44 and 2XFB) and visualized
using the software CHIMERA (Pettersen et al, 2004). Assays were performed in triplicate and expressed as relative fold change after normalizing to OD450 from
non-infected sera. Data are representative of two independent experiments and presented as mean  SD (two-tailed unpaired t-test, *P = 0.0191 6 dpi epitope
381, *P = 0.038 6 dpi epitope E2EP3, ***P = 0.0004 15 dpi epitope 381, ***P = 0.0001 15 dpi epitope E2EP3). E, F
Mapping antibody reactivity to linear B-cell epitopes within CHIKV E2 proteome. CHIKV E2 epitopes recognized at 6 dpi (E) and 15 dpi (F) were determined in
pooled sera collected from infected mice using ELISA specific for overlapping 18-mer linear peptides spanning the CHIKV E2 proteome. The peptide numbers
correspond to the position of the 18-mer linear peptides along the CHIKV E2 proteome. Structural data were retrieved from PDB (id: 3N44 and 2XFB) and visualized
using the software CHIMERA (Pettersen et al, 2004). Assays were performed in triplicate and expressed as relative fold change after normalizing to OD450 from
non-infected sera. Data are representative of two independent experiments and presented as mean  SD (two-tailed unpaired t-test, *P = 0.0191 6 dpi epitope
381, *P = 0.038 6 dpi epitope E2EP3, ***P = 0.0004 15 dpi epitope 381, ***P = 0.0001 15 dpi epitope E2EP3). G
Localization of identified CHIKV B-cell epitopes (381; blue, 388; green, E2EP3; red) within CHIKV proteome. Epitopes in the E2 glycoprotein were located based on
the structural data obtained from PDB records: 3N42. © 2014 Agency for Science, Technology and Research (A*STAR) EMBO Molecular Medicine
Vol 7 | No 1 | 2015 Impaired virus clearance in Tlr3/ mice is due to diminished
recognition of CHIKV E2 glycoprotein Data are representative of two independent experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, **P = 0.0095 5–7 dpi viremia
WT recipient chimera, *P = 0.0139 8 dpi viremia WT recipient chimera, **P = 0.0095 10 dpi viremia WT recipient chimera, *P = 0.0350 4 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 5 dpi viremia Tlr3/ recipient chimera, *P = 0.0221 6 dpi viremia Tlr3/ recipient chimera, **P = 0.0012 7 dpi viremia Tlr3/
recipient chimera, **P = 0.0082 8 dpi viremia Tlr3/ recipient chimera). p
p
p
)
B, E
Extent of joint inflammation was measured daily and expressed as disease score relative to day 0 (pre-infection). Data are representative of two independent
experiments and presented as mean  SD (two-tailed Mann–Whitney U-test, *P = 0.0117 4 dpi disease score Tlr3/ recipient chimera, *P = 0.0146 5 dpi disease
score Tlr3/ recipient chimera). C, F
CHIKV dissemination is not modulated in WT and Tlr3/ chimeras. Bioluminescence signals of whole body was quantified and expressed as average radiance
(p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Data are representative of one of two independent experiments and presented as mean  SD (two-tailed
Mann–Whitney U-test, *P = 0.0381 1 dpi whole-body WT recipient chimera, **P = 0.0082 1 dpi whole-body Tlr3/ recipient chimera). C, F
CHIKV dissemination is not modulated in WT and Tlr3/ chimeras. Bioluminescence signals of whole body was quantified and expressed as average radiance
(p/s/cm2/sr). The lowest limit of detection is 0 p/s/cm2/sr. Data are representative of one of two independent experiments and presented as mean  SD (two-tailed
Mann–Whitney U-test, *P = 0.0381 1 dpi whole-body WT recipient chimera, **P = 0.0082 1 dpi whole-body Tlr3/ recipient chimera). 30 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 © 2014 Agency for Science, Technology and Research (A*STAR) Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine Figure 6. © 2014 Agency for Science, Technology and Research (A*STAR) TLR3 SNP rs6552950 is associated with disease severity and
CHIKV-specific IgG neutralizing antibody capacity in CHIKV-
infected patients Strati-
fied analysis of these TLR3 tagSNPs to disease severity revealed that
a non-coding variant SNP rs6552950 had a nominal association with
disease severity with an OR of 2.39 (P < 0.05) in severe CHIKF
patients when compared to mild CHIKF patients (Table 1 and
Fig 7B). In addition, anti-E2EP3 IgG response determined from 69
available patients’ plasma in this study cohort further revealed that
patients with SNP rs6552950 genotype associated with severe
disease outcome and with low anti-E2EP3 IgG response during early
convalescence
phase
(logistic
regression
analysis;
P < 0.05)
(Fig 7C). Complementing the observations in Tlr3/ mice (Fig 6A),
CHIKV-specific antibody neutralizing assays performed from the
plasma of these 69 patients revealed that patients with GG genotype
had significantly less neutralizing antibodies as compared to EMBO Molecular Medicine
Vol 7 | No 1 | 2015 31 TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al EMBO Molecular Medicine A 
B 
C 
D 
E 
F 
G B A B A C D D C E F E F G G Discussion Histogram shows the percentage of patients in each genotype exhibiting
either low or high anti-E2EP3 IgG response. Low and high anti-E2EP3 IgG response were defined as being below or above the mean value of anti-E2EP3 IgG response,
respectively. Association between SNP genotypes (AA, n = 38; AG, n = 20; GG, n = 11) and anti-E2EP3 IgG response (low—AA, n = 16; AG, n = 13; GG, n = 9; high—AA,
n = 22; AG, n = 7; GG, n = 2) is performed using logistic regression analysis; *P = 0.0376. D
TLR3 SNP rs6552950 is associated with the CHIKV-specific antibody neutralizing capacity in 69 CHIKF patients. Plasma collected at early convalescent (median
10 days post-illness onset) was diluted 1:100–1:1,000 and mixed with CHIKV (MOI 10) for 2 h before infection of HEK 293T cells for 6 h. Assays were performed in
quadruplicate, and data are expressed relative to virus-only-infected samples without sera. Data are presented as mean  SEM. Comparison among SNP genotypes
(AA, n = 38; AG, n = 20; GG, n = 11) is performed using one-way ANOVA analysis followed by Tukey’s multiple comparison test. ***P = 0.0001 1:100 serum dilution
GG versus AA, ***P = 0.0001 1:100 serum dilution GG versus AG, *P = 0.0178 1:500 serum dilution GG versus AA, **P = 0.0056 1:500 serum dilution GG versus AG,
**P = 0.0037 1:1,000 serum dilution GG versus AA, **P = 0.0041 1:1,000 serum dilution GG versus AG. D
TLR3 SNP rs6552950 is associated with the CHIKV-specific antibody neutralizing capacity in 69 CHIKF patients. Plasma collected at early convalescent (median
10 days post-illness onset) was diluted 1:100–1:1,000 and mixed with CHIKV (MOI 10) for 2 h before infection of HEK 293T cells for 6 h. Assays were performed in
quadruplicate, and data are expressed relative to virus-only-infected samples without sera. Data are presented as mean  SEM. Comparison among SNP genotypes
(AA, n = 38; AG, n = 20; GG, n = 11) is performed using one-way ANOVA analysis followed by Tukey’s multiple comparison test. ***P = 0.0001 1:100 serum dilution
GG versus AA, ***P = 0.0001 1:100 serum dilution GG versus AG, *P = 0.0178 1:500 serum dilution GG versus AA, **P = 0.0056 1:500 serum dilution GG versus AG,
**P = 0.0037 1:1,000 serum dilution GG versus AA, **P = 0.0041 1:1,000 serum dilution GG versus AG. Discussion TLR3 is highly induced in CHIKV-infected patients during the early disease phase and TLR3 SNP rs6552950 is associated with disease severity and
specific IgG response. A
Transcriptional profiles of TLR3 in PBMCs of CHIKV-infected patients (n = 23) during acute (median 4 days post-illness onset), and early convalescent (median
10 days post-illness onset) disease. The level of TLR3 gene expression was expressed relative to healthy controls (n = 8) after normalization to GAPDH. Data are
presented as mean  SEM (two-tailed Mann–Whitney U-test, ***P = 0.0001 acute versus healthy controls, ***P = 0.0001 early convalescent versus healthy controls). B
TLR3 SNP rs6552950 is associated with disease severity in 94 CHIKV-infected patients. Histogram shows the percentage of patients in each genotype exhibiting either
mild or severe disease phenotype. Severe illness is defined as patients having either a maximum temperature greater than 38.5°C, a maximum pulse rate greater than
100 beats/min or a nadir platelet count less than 100 × 109/l. Mild illness is defined as patients who do not fulfill these criteria. Association between SNP genotype
(AA, n = 43; AG, n = 24; GG, n = 11; unknown, n = 16) and disease phenotype (Mild—AA, n = 29; AG, n = 11; GG, n = 4; Severe—AA, n = 14; AG, n = 13; GG, n = 7) is
performed using logistic regression analysis (Table 1); *P = 0.02. C
TLR3 SNP rs6552950 is associated with the degree of anti-E2EP3 IgG response in 69 CHIKV-infected patients. Plasma collected at early convalescent (median 10 days
post-illness onset) was subjected to E2EP3 peptide-based ELISA at a dilution of 1:2,000. Histogram shows the percentage of patients in each genotype exhibiting
either low or high anti-E2EP3 IgG response. Low and high anti-E2EP3 IgG response were defined as being below or above the mean value of anti-E2EP3 IgG response,
respectively. Association between SNP genotypes (AA, n = 38; AG, n = 20; GG, n = 11) and anti-E2EP3 IgG response (low—AA, n = 16; AG, n = 13; GG, n = 9; high—AA,
n = 22; AG, n = 7; GG, n = 2) is performed using logistic regression analysis; *P = 0.0376. C
TLR3 SNP rs6552950 is associated with the degree of anti-E2EP3 IgG response in 69 CHIKV-infected patients. Plasma collected at early convalescent (median 10 days
post-illness onset) was subjected to E2EP3 peptide-based ELISA at a dilution of 1:2,000. © 2014 Agency for Science, Technology and Research (A*STAR) Discussion patients with AA or AG genotypes (Fig 7D). Taken together, these
results suggest that TLR3 polymorphism in humans could influence
the neutralizing capacity of CHIKV-specific IgGs by modulating the
recognition of CHIKV E2EP3 epitope important for virus neutraliza-
tion. Specifically, SNP rs3775292 and SNP rs6552950 may be
susceptibility factors for CHIKF, with SNP rs6552950 having a possi-
ble role in disease severity due to low anti-E2EP3 IgG response. patients with AA or AG genotypes (Fig 7D). Taken together, these
results suggest that TLR3 polymorphism in humans could influence
the neutralizing capacity of CHIKV-specific IgGs by modulating the
recognition of CHIKV E2EP3 epitope important for virus neutraliza-
tion. Specifically, SNP rs3775292 and SNP rs6552950 may be
susceptibility factors for CHIKF, with SNP rs6552950 having a possi-
ble role in disease severity due to low anti-E2EP3 IgG response. patients with AA or AG genotypes (Fig 7D). Taken together, these
results suggest that TLR3 polymorphism in humans could influence
the neutralizing capacity of CHIKV-specific IgGs by modulating the
recognition of CHIKV E2EP3 epitope important for virus neutraliza-
tion. Specifically, SNP rs3775292 and SNP rs6552950 may be
susceptibility factors for CHIKF, with SNP rs6552950 having a possi-
ble role in disease severity due to low anti-E2EP3 IgG response. The detection of PAMPs by PRRs to elicit an inflammatory response
is an essential process of the host innate immune response against
pathogens (Janeway & Medzhitov, 2002). TLR3-mediated immunity
to natural infection in humans has been demonstrated to both limit
and exacerbate viral disease progression. It has been shown that 32 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 © 2014 Agency for Science, Technology and Research (A*STAR) Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine A
C
D 
B 
TLR3 is highly induced in CHIKV-infected patients during the early disease phase and TLR3 SNP rs6552950 is associated with disease severity and
gG response A B B A D C D D C  Figure 7. TLR3 is highly induced in CHIKV-infected patients during the early disease phase and TLR3 SNP rs6552950 is associated with disease severity and
specific IgG response. TLR3 is highly induced in CHIKV-infected patients during the early disease phase and TLR3 SNP rs6552950 is associated wit
G response. Figure 7. TLR3 is highly induced in CHIKV-infected patients during the early disease phase and TLR3 SNP rs6552950 is associated with disease severity and Figure 7. Discussion NP, Single nucleotide polymorphism from TLR3 gene; SNPs rs5743316 and rs5743310 were excluded from further analysis due t
d being monomorphic in control populations, respectively. #SNP rs13108688 was not in Hardy–Weinberg equilibrium in the p
Chromosome, Position, Chromosome and corresponding position (in base pair) where the SNP is located. nor Allele, The rare allelic form of the SNP variant. hP, P-value obtained for the regression analysis; *Significant P-value < 0.05 is indicated in bold. i hP, P-value obtained for the regression analysis; *Significant P-value < 0.05 is indicated in bold. iOR(L95–U95), Odds ratio for the SNP as estimated from the minor allele with 95% confidence interval limits hP, P-value obtained for the regression analysis; *Significant P-value < 0.05 is indicated in bold. iOR(L95–U95), Odds ratio for the SNP as estimated from the minor allele with 95% confidence interval limits. cells is accompanied by the reduction in CD4+ T-cell infiltration. However, depletion of CD4+ T cells prevented the pathology and
abrogated neutrophils without interfering with the development of
the viremia. This demonstrated that CD4+ T cells are essential for
the pathology in WT and Tlr3/ mice. One plausible explanation
for the exacerbation of joint footpad inflammation in Tlr3/ mice is
that the loss of TLR3 may reduce the number of anti-inflammatory
CD4+ T regulatory cells present in the infiltrate. However, this is
highly unlikely as demonstrated by bone marrow reconstitution
experiments. If TLR3 negatively controlled the number of CD4+ T
regulatory cells, the WT mice reconstituted with Tlr3/ bone
marrow would have less CD4+ T regulatory T cells. This was not
case since WT mice reconstituted with Tlr3/ bone marrow had
the same level of joint inflammation as the WT mice reconstituted
with WT bone marrow. et al, 2014), the precise roles of TLRs as PRRs for CHIKV remain
unknown. In this study, TLR3 was demonstrated to be a critical PRR
in the control of CHIKV replication, immunity and pathology in
humans and mice. The increased susceptibility of both TRIF/ and
Tlr3/ primary fibroblasts to CHIKV infection demonstrates a role
of TLR3 in controlling CHIKV replication. Furthermore, the loss of
TLR3 expression in CHIKV-infected mice significantly increased
viremia and exacerbated CHIKV-induced inflammation. However,
these findings are in contrast to an earlier report where TLR3 was
reported to play a modest role in controlling CHIKV infection in
young mice (Schilte et al, 2010). © 2014 Agency for Science, Technology and Research (A*STAR) Discussion This disparity could be due to the
age of the animals and the different inoculation routes used. When
inoculated intradermally, CHIKV triggered a strong local type I IFN
response that was sufficient to locally control virus replication in
WT mice and hence no effect on TLR3 signaling (Schilte et al,
2010). However, in this study, the effect of TLR3 signaling is appar-
ent because the type I IFN response is independent of TLR3 and
therefore insufficient to locally control CHIKV replication during the
early phase of the infection. The dissemination of CHIKV into the brains of Tlr3/ mice is of
particular interest because, unlike other organs, the brains of WT
mice were not infected. CHIKV is not commonly neurotropic, but
the occurrence of CHIKV-associated neurological complications has
been increasingly reported in patients since the 2006 Indian Ocean
outbreak (Rampal et al, 2007; Wielanek et al, 2007; Das et al, 2010;
Kashyap et al, 2010). Although previous studies in mice have
shown that CHIKV dissemination into the central nervous system is
dependent on type I IFN signaling (Couderc et al, 2008; Abraham
et al, 2013), it remains to be elucidated whether this is a bystander
effect due to overwhelming CHIKV replication or whether the
blood–brain barrier has been breached due to the absence of TLR3,
permitting virus entry into the brain. By tracking virus dissemination using a recombinant FLuc-
CHIKV, the loss of TLR3 clearly resulted in exacerbated CHIKV-
induced pathology in mice. Moreover, a marked tropism was
observed for skeletal muscle, joints and skin in WT and Tlr3/
mice. The increased pathology in Tlr3/ mice was mediated by an
increased infiltration of Ly6G+ neutrophils into the infected joint
footpad with F4/80+ macrophages, which were previously shown
to be the primary cellular infiltrates (Gardner et al, 2010; Rudd et al,
2012; Teng et al, 2012). While the exact role of neutrophils in the
development of CHIKV-induced inflammation remains unclear,
neutrophils were recently implicated in the control of CHIKV infec-
tion in mouse (Dhanwani et al, 2014; Poo et al, 2014) and zebrafish
(Palha et al, 2013) models. The increased infiltration of myeloid Infection in bone marrow chimeric mice provided evidence on
how TLR3-expressing hematopoietic cells are required to elicit an
anti-CHIKV innate immune response and regulate CHIKV infection
and pathology. Discussion di
’ i d fi
d
ti
t
h h d ith
i
t
t
t
th
38 5°C
i
l
t p
J
q
y
j
b‘Severe disease’ is defined as patients who had either a maximum temperature greater than 38.5°C, a maximum pulse rate greater than 100 beats/min or a
nadir platelet count less than 100 × 109/l. ‘Mild disease’ is referred to patients who do not fulfill these criteria. b‘Severe disease’ is defined as patients who had either a maximum temperature greater than 38.5°C, a maximum pulse rate
nadir platelet count less than 100 × 109/l. ‘Mild disease’ is referred to patients who do not fulfill these criteria. b‘Severe disease’ is defined as patients who had either a maximum temperature greater than 38.5°C, a maximum pulse rate greater than 100 beats/min or a
nadir platelet count less than 100 × 109/l. ‘Mild disease’ is referred to patients who do not fulfill these criteria. cSNP, Single nucleotide polymorphism from TLR3 gene; SNPs rs5743316 and rs5743310 were excluded from further analysis due to failed Sequenom assay design
and being monomorphic in control populations, respectively. #SNP rs13108688 was not in Hardy–Weinberg equilibrium in the population. d,eChromosome, Position, Chromosome and corresponding position (in base pair) where the SNP is located. fMinor Allele, The rare allelic form of the SNP variant. gSTAT, Statistic obtained for the regression analysis for the SNP. hP, P-value obtained for the regression analysis; *Significant P-value < 0.05 is indicated in bold. p
p
cSNP, Single nucleotide polymorphism from TLR3 gene; SNPs rs5743316 and rs5743310 were excluded from further analysis due to failed Sequenom assay design
and being monomorphic in control populations, respectively. #SNP rs13108688 was not in Hardy–Weinberg equilibrium in the population. d,eChromosome, Position, Chromosome and corresponding position (in base pair) where the SNP is located. fMinor Allele, The rare allelic form of the SNP variant. cSNP, Single nucleotide polymorphism from TLR3 gene; SNPs rs5743316 and rs5743310 were excluded from further analysis due to failed Sequenom assay design
and being monomorphic in control populations, respectively. #SNP rs13108688 was not in Hardy–Weinberg equilibrium in the population. d e NP, Single nucleotide polymorphism from TLR3 gene; SNPs rs5743316 and rs5743310 were excluded from further analysis due t
d being monomorphic in control populations, respectively. #SNP rs13108688 was not in Hardy–Weinberg equilibrium in the p
Chromosome, Position, Chromosome and corresponding position (in base pair) where the SNP is located. Discussion defects in TLR3 signaling axis rendered both humans and mice
permissive to HSV-1 encephalitis infection (Zhang et al, 2007; Pe´rez
de Diego et al, 2010; Reinert et al, 2012). The immunological
control of HSV in the central nervous system was proposed to be
mediated through astrocytes which sense HSV-2 in a TLR3-
dependent manner and restricts virus replication by inducing
anti-viral IFNb response (Reinert et al, 2012). In the case of West Nile virus infection, TLR3 was either protective or deleterious to the
host by limiting virus replication in neurons or promoting efficient
virus entry into the brain, respectively, depending on the route of
inoculation (Wang et al, 2004; Daffis et al, 2008). Although studies have emerged demonstrating the functions of
RIG-I/MDA5-mediated
signaling
in
anti-CHIKV
host
response
(Schilte et al, 2010; White et al, 2011; Rudd et al, 2012; Olagnier EMBO Molecular Medicine
Vol 7 | No 1 | 2015 33 TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al EMBO Molecular Medicine Table 1. Association of TLR3 SNPs to CHIKV phenotype and severity. Information of TLR3 SNPs
Comparison of SNP allele frequency
to population controlsa
Association of SNP with CHIKV
disease severityb
SNPc
Chromosomed
Positione
Minor Allelef
STATg
P-valueh
OR (L95–U95)i
STAT
P-value
OR (L95–U95)
rs3775292
4
187003025
C
3.06
0.002*
2.16 (1.31–3.42)
0.63
0.53
0.80 (0.4–1.6)
rs6552950
4
186994856
G
2.11
0.03*
1.54 (1.03–2.29)
2.39
0.02*
2.31 (1.16–4.57)
rs7657186
4
186994039
A
1.62
0.11
1.43 (0.93–2.21)
1.32
0.19
0.61 (0.29–1.28)
rs11721827
4
186991137
C
1.22
0.22
0.70 (0.39–1.24)
0.20
0.84
0.90 (0.33–2.46)
rs5743312
4
187000256
T
1.08
0.28
1.28 (0.82–2.00)
0.91
0.36
0.71 (0.33–1.5)
rs7668666
4
187001292
A
0.92
0.36
1.20 (0.81–1.78)
0.12
0.91
1.04 (0.54–2.01)
rs3775291
4
187004074
T
0.82
0.41
1.18 (0.79–1.76)
0.47
0.64
1.15 (0.64–2.09)
rs13108688#
4
186994832
A
0.42
0.68
0.92 (0.60–1.39)
0.83
0.41
0.72 (0.33–1.56)
rs3775296
4
186997767
A
0.34
0.73
1.08 (0.71–1.63)
0.89
0.37
0.74 (0.38–1.45) Table 1. Association of TLR3 SNPs to CHIKV phenotype and severity. opulation controls consist of 60 CEU, 60 CHBJPT and 59 YRI individuals sequenced by the 1000 Genomes Pilot Project. Discussion Since the role of CD4+ T cells was shown to be EMBO Molecular Medicine
Vol 7 | No 1 | 2015 34 Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine EMBO Molecular Medicine Transcriptional
analysis
of
PBMCs
isolated
from
CHIKV-
infected patients revealed that TLR3 expression was up-regulated
during the acute and early convalescent phase of the disease. Transcriptional analysis of other PRRs such as RIG-I, MDA5 and
IPS-1 (adaptor molecule involved in RIG-I and MDA5 mediated
signaling) has shown that these molecules were similarly signifi-
cantly up-regulated and associated with viral load during acute
CHIKV infection (Teng et al, 2012), suggesting that the enhanced
TLR3 expression is part of a general innate immunity against
CHIKV. Previous genetic epidemiological studies have implicated
inborn errors in TLR3 immunity and polymorphism in the patho-
genesis of HSV-1 encephalitis (Zhang et al, 2007; Herman et al,
2012; Lafaille et al, 2012) and Influenza virus infection (Esposito
et al, 2012). While the number of CHIKF patients in our study
is too small to demonstrate any direct functional link between
TLR3 gene polymorphisms and CHIKV disease outcomes, our
findings suggest a possible relationship between the presence of
TLR3 SNPs, rs3775292 and rs6552950 with an increased risk of
CHIKV
disease
occurrence. In
particular,
the
SNP
rs6552950 important for the pathology in the WT and Tlr3/ mice via the
control
of
neutrophils
recruitment,
this
suggested that
B-cell
responses could be defective in Tlr3/ mice. Growing evidence has
also shown that activation of TLRs promotes B-cell proliferation and
IgG isotype class switch (Ruprecht & Lanzavecchia, 2006; Xu et al,
2008; Sariol et al, 2011). Here, the production of CHIKV-specific
IgM and IgG antibodies was not disrupted by the loss of TLR3, and
the IgG response was even higher at 6 dpi. Rather, the reduced
CHIKV-neutralizing capacity was due to diminished recognition for
neutralizing linear B-cell epitopes located in the CHIKV E2 glycopro-
tein (Kam et al, 2012a,b; Lum et al, 2013). The loss of TLR3 expres-
sion
could
modulate
CHIKV
antigen
(Ag)
processing
and
presentation by APCs, particularly the immunodominant ‘E2EP3’
epitope, to result in an immunodominance shift (Siddiqui & Basta,
2011). Discussion Other virus infection models have demonstrated that TLR3
stimulation in dendritic cells decreases production of nitric oxide
which in turn increases proteasomal activity and consequently
increases viral antigen processing and presentation (Schwarz et al,
2000; Siddiqui et al, 2011). Figure 8. Proposed model for TLR3-mediated anti-CHIKV response during infection. Upon CHIKV infection of the target cell, TLR3 functions (blue arrows) in synergy with multiple host cytosolic RNA sensors such as RIG-I and MDA5, to vanguard the coordinated
endosomal and cytoplasmic recognition of CHIKV RNA, triggering anti-viral type I IFN response and expression of pro-inflammatory genes. This concerted activation acts to
restrict CHIKV replication (red arrows). In parallel, the stimulation of cells will up-regulate TLR3 expression and enhance the anti-CHIKV response. Consequently, activation of
TLR3 signaling mediates protection from CHIKV-induced pathology by preventing rapid CHIKV dissemination and restricting tissue tropism (red arrows) tied in with assistance
from chemokine regulation (blue arrows). TLR3 signaling can also modulate CHIKV immune recognition during the adaptive immune response to promote effective CHIKV
clearance through the production of CHIKV-specific immunodominant antibodies by B cells. Therefore, a loss of TLR3 would lead to a reduction in CHIKV antibody neutralizing
capacity due to an impaired production of such antibodies, resulting in the persistence of CHIKV viremia in vivo and enhanced/increased disease severity. Figure 8. Proposed model for TLR3-mediated anti-CHIKV response during infection. model for TLR3-mediated anti-CHIKV response during infection Upon CHIKV infection of the target cell, TLR3 functions (blue arrows) in synergy with multiple host cytosolic RNA sensors such as RIG-I and MDA5, to vanguard the coordinated
endosomal and cytoplasmic recognition of CHIKV RNA, triggering anti-viral type I IFN response and expression of pro-inflammatory genes. This concerted activation acts to
restrict CHIKV replication (red arrows). In parallel, the stimulation of cells will up-regulate TLR3 expression and enhance the anti-CHIKV response. Consequently, activation of
TLR3 signaling mediates protection from CHIKV-induced pathology by preventing rapid CHIKV dissemination and restricting tissue tropism (red arrows) tied in with assistance
from chemokine regulation (blue arrows). TLR3 signaling can also modulate CHIKV immune recognition during the adaptive immune response to promote effective CHIKV
clearance through the production of CHIKV-specific immunodominant antibodies by B cells. Therefore, a loss of TLR3 would lead to a reduction in CHIKV antibody neutralizing
capacity due to an impaired production of such antibodies, resulting in the persistence of CHIKV viremia in vivo and enhanced/increased disease severity. © 2014 Agency for Science, Technology and Research (A*STAR) Study approval Blood samples from CHIKF patients used in this study were
collected by Institute of Infectious Disease and Epidemiology at Tan
Tock Seng Hospital from 1 August through September 23, 2008 with
approval from the National Healthcare Group’s domain-specific
ethics review board (DSRB Reference no. B/08/026). Written
informed consent was obtained from all participants in accordance
with the Declaration of Helsinki principles. All animal protocols
were approved by the Institutional Animal Care and Use Committee
(IACUC no. 120714) at the Biological Resource Center at Biopolis,
Singapore. All studies involving animals are reported in accordance
with the ARRIVE guidelines for reporting experiments involving
animals (Kilkenny et al, 2010). Genotyping and association analysis A total of 11 tagSNPs were identified to cover the linkage
disequilibrium block for TLR3 gene. Genotyping was performed
using matrix-assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-TOF MS) for the determination of
allele-specific primer extension products using Sequenom’s Mass-
ARRAY system and iPLEX technology (Sequenom Inc). One SNP
(rs5743316) failed Sequenom assay design. The design of oligo-
nucleotides was carried out according to the guidelines of Seque-
nom and performed using MassARRAY Assay Design software. Multiplex PCR amplification of amplicons containing the SNPs of
interest was performed using QIAGEN HotStart Taq Polymerase
using 5 ng of genomic DNA. Primer extension reactions were
carried out according to manufacturer’s instructions for iPLEX
chemistry. Assay data were analyzed using the Sequenom TYPER
software. Clustering of genotype calls was evaluated to deter-
mine that the clustering was sufficient for inclusion in the statis-
tical
analysis. All
SNPs
were
tested
for
Hardy–Weinberg
equilibrium for quality control and subjected to further statistical
analysis. Population controls used for association analysis were
the 179 samples sequenced by the 1000 Genomes Pilot Project
(60 CEU, 60 CHB+JPT, 59 YRI) (1000 Genomes Project Consor-
tium et al, 2010). Sequenom genotypes for SNPs in reverse
strand notation (SNPs rs3775291 and rs3775292) were converted
to a forward strand notation using the option –flip provided by
the software PLINK v1.07 (Purcell et al, 2007). One of the 10
TLR3 SNPs (rs5743310) was monomorphic in two of the three
control
populations
(CEU
and
CHBJPT)
and
was
therefore
excluded from further analysis. Association between the other 9
TLR3 SNPs and the CHIKV infection phenotype was computed
using a logistic regression model. Association between TLR3
SNPs and severity (mild/severe) was computed using a logistic
regression model that included gender and age as covariates. One SNP rs13108688 was not in Hardy–Weinberg equilibrium
(P < 0.005) in the population; however, this SNP was not signifi-
cant for association. Both association analyses were performed
using the software PLINK v1.07 (Purcell et al, 2007). P-values Cell culture African green monkey kidney epithelial cells (Vero-E6), HEK293T,
and a mouse hepatocyte cell line (Hepa 1–6) were cultured in
DMEM supplemented with 10% FBS (Gibco). Mouse tail fibroblasts
were cultured in DMEM supplemented with 10% FBS and 1%
penicillin–streptomycin. Aedes albopictus mosquito cell line (C6/36)
was cultured in Leibovitz’s L-15 medium (Life Technologies)
supplemented with 10% FBS. All cells were maintained at 37°C with
5% CO2, except for the C6/36 cell line which was maintained at
28°C without CO2 supplementation. Collectively, we provided clear evidences for TLR3 in the control
of CHIKV infection. The synergy of TLR3 with multiple host RNA
sensors such as RIG-I and MDA5 is indispensable for specific inter-
actions with viral dsRNA to restrict CHIKV replication by inducing a
rapid anti-viral type I IFN response together with pro-inflammatory
gene
expression
(Fig 8). Essentially,
TLR3-mediated
immunity
controls
CHIKV-induced
pathology
by
preventing
rapid
virus
dissemination. The observations reported here also provide further
insights on how TLR3-mediated innate responses against CHIKV
infection can influence the adaptive immune response, as well as
the mechanisms by which TLR3 modulates in vivo CHIKV immune
recognition. These findings provide a better understanding on one
of the key components that induce protective anti-CHIKV responses
and will have critical implications in the future development of
novel therapeutic strategies against CHIKV and other clinically
important alphaviruses that have serious global health impacts. Discussion 35 © 2014 Agency for Science, Technology and Research (A*STAR) © 2014 Agency for Science, Technology and Research (A*STAR) EMBO Molecular Medicine
Vol 7 | No 1 | 2015 TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al EMBO Molecular Medicine EMBO Molecular Medicine polymorphism was demonstrated to be associated with severe
CHIKV disease outcome in patients from the Singapore 2008
CHIKV cohort. Antibody response studies in these patients have
demonstrated that naturally acquired neutralizing IgG response is
dominated by anti-E2EP3 IgG antibodies (Kam et al, 2012b). Furthermore,
the
low
anti-E2EP3
IgG
response
in
the
SNP
rs6552950
GG
genotype
patients
was
demonstrated
to
have
reduced neutralizing antibodies, which in turn influenced the
severity of disease outcomes in these patients. SNP rs3775292
was previously reported to associate with persisting IgG concen-
tration and serum bactericidal antibody following serogroup C
meningococcal polysaccharide–protein conjugate vaccination and
an increased risk of colon cancer development (Moore et al,
2011; Slattery et al, 2011). It will be insightful to assess whether
these SNPs would lead to a loss of TLR3 function in larger
patient cohorts, including the emergent CHIKV outbreaks in the
Caribbean islands (Leparc-Goffart et al, 2014). clinical parameters defined in Chow et al (2011) and Ng et al
(2009), these individuals were further classified into 53 mild and 41
severe cases. Illness was defined as ‘severe’, if a patient had either a
maximum temperature greater than 38.5°C, a maximum pulse rate
greater than 100 beats/min or a nadir platelet count less than
100 × 109/l. Patients who do not fulfill these criteria are classified
as ‘mild’ (Ng et al, 2009; Chow et al, 2011). Virus infections Three-week-old WT or Tlr3/ C57/BL6 female mice were inocu-
lated s.c. in the ventral side of the right-hind footpad toward the
ankle with 106 PFU CHIKV in 25 ll PBS in a non-randomized and
non-blinded fashion. Viral RNA extraction was performed from
10 ll of blood collected from the tail, and viremia was determined
by qRT-PCR as described (Teng et al, 2012; Lum et al, 2013; Teo
et al, 2013). For joint (footpad) inoculated mice, joint inflammation
by the measurement of the height (thickness) and the breadth of the
footpad using a vernier caliper was calculated as [height × breadth]. The degree of inflammation was expressed as relative increase
compared to pre-infection (day 0; d 0) with the following formula:
[(x  d 0)/d 0] where x is footpad size measurements for respective
dpi as described (Kam et al, 2012b). For joint footpad extraction,
mice
were
sacrificed
by
terminal
anesthesia
with
ketamine
[150 mg/kg]/xylazin [10 mg/kg] followed by intra-cardial perfusion
with PBS. Joint footpads were removed and preserved in Trizol (In-
vitrogen) at 80°C. Tissues were homogenized using a rotor–stator
homogenizer (Xiril Dispomix) at 500 g for 15 s. Homogenized
tissues were transferred to clean tubes and mixed with 230 ll of
chloroform. Following 2-min incubation, tissue mixtures were
centrifuged at 13,523 g for 15 min at 4°C. The aqueous phase was
collected, and total RNA isolated as described (Teng et al, 2012). For bone marrow chimera, 6-week-old recipient mice were irradi-
ated twice with 600Rad (3 h apart) and i.v. injected with 106 donor
bone marrow cells. Absolute CD45+ leukocyte blood count was
determined at week 4–6 post-reconstitution using flow cytometry
and compared to non-chimeric mice of same age. To test for
successful adoptive bone marrow cell transfer, Tlr3 expression in
peripheral blood leukocytes from reconstituted mice was deter-
mined 6 weeks after reconstitution using quantitative real-time PCR
analysis before proceeding with infection. Depletion of CD4+ T cells
was performed as described (Teo et al, 2013). Each mouse was i.p. injected with 500 lg of either CD4-depleting antibody or rat IgG2b
isotype control on 2, 1 and 4 dpi. Complete CD4+ T-cell
depletion was assessed before CHIKV inoculation (day 0). CHIKV infections on primary fibroblasts and continuous cell lines
from mouse and human were performed at multiplicity of infection
(MOI) 10. Each infection mix consisted of virus suspension prepared
in serum-free medium. Total RNA extraction and gene expression analysis CHIKV-IMT isolate used for in vitro infections of human cells was
isolated from Reunion Island during the 2006 CHIKF outbreak
(Bessaud et al, 2006). Virus stocks were prepared in Vero-E6
cultures, washed and pre-cleared by centrifugation before storing at
80°C. CHIKV-SGP011 isolate used for in vitro and in vivo infections
in mouse studies was isolated from an outbreak in Singapore in
2008 at the National University Hospital (Her et al, 2010) and prop-
agated in Hepa 1–6 and C6/36 cultures, respectively. CHIKV vari-
ants expressing FLuc was constructed using a full-length infectious
cDNA clone of CHIKV LR2006-OPY1 isolate as described (Tsetsarkin
et al, 2006; Pohjala et al, 2011; Teng et al, 2012). Infectious viruses
were propagated in C6/36 cultures, washed and pre-cleared by
ultracentrifugation before storing at 80°C. Virus titer of all virus
stocks used was determined using standard plaque assays with
Vero-E6 cells (Her et al, 2010; Kam et al, 2012b). Total
RNA
was
extracted
using
RNeasy
Mini
Kit
(QIAGEN)
according to manufacturer’s instructions. Quantification of total
RNA was performed using NanoDrop 1000 Spectrophotometer
(Thermo Scientific), and RNA samples were further diluted to
10 ng/ll. qRT-PCR
was
performed
using
QuantiFast
TM
SYBR
Green RT-PCR Kit (QIAGEN) according to manufacturer’s recom-
mendations in 12.5 ll reaction vol. All reactions were performed
using 7900HT Fast Real-Time PCR System machine (Applied
Biosciences) with thermal cycling conditions as described (Teng
et al, 2012). The fold change for each gene between CHIKV-
infected and mock-infected was calculated as 2DDCt (Teng et al,
2012). The primer sequences of the mouse genes analyzed are
listed in Supplementary Table S3. Study population A total of 94 PCR-confirmed CHIKV-positive individuals were
included in this study. There were 48 Chinese, 26 Malay, 10 Indians
and 10 of other ethnicity. The median age was 38 with a range of 21
to 67 years of age with 77.7% male and 22.3% female. Based on the © 2014 Agency for Science, Technology and Research (A*STAR) 36 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis Zhisheng Her et al
TLR3-mediated immunity controls CHIKV pathogenesis EMBO Molecular Medicine EMBO Molecular Medicine lower than 0.05 were considered significant. The distribution of
each TLR3 SNP genotype and disease severity for each SNP
genotype is listed in Supplementary Table S2. vol. All reactions were performed using 7900HT Fast Real-Time PCR
System machine (Applied Biosciences) with thermal cycling condi-
tions as described previously (Teng et al, 2012). The limit of detec-
tion was 10 RNA copies/ll. Flow cytometry and antibodies Detection of CHIKV Ag was carried out in a two-step indirect intra-
cellular labeling process (Her et al, 2010; Teng et al, 2012). Data
were acquired in BD FACSCanto
TM II (BD Bioscience) using BD
FACSDiva
TM software. Dead cells and duplets were excluded in all
analysis with FSC/SSC gating. Results were analyzed with FlowJo
version 7.5 software (Tree Star, Inc). Antibodies against CHIKV Ag
were purchased from Santa Cruz Biotechnology. Antibodies against
mouse CD45 (cat# 557659) and CD8 (cat# 553035) were purchased
from
BD
Bioscience. Antibodies
against
mouse
CD11b
(cat#
12-0112-82) was purchased from eBioscience. Antibodies against
mouse CD3 (cat# 100200), CD4 (cat# 100531) and Ly6G (cat#
127612) were purchased from Biolegend. Mouse CD4-depleting anti-
body (cat# BE0003-1) and rat IgG2b isotype control (cat# BE0090)
were purchased from Bio X cell. Virus infections Viruses were incubated at 37°C and allowed
to adsorb for 1.5 h with intermittent shaking before virus inoculum
was removed and replaced with complete medium. Cells were incu-
bated at 37°C until harvest at different hpi. Mock infections (medium
only) were performed in parallel as controls. © 2014 Agency for Science, Technology and Research (A*STAR) Impact p
This is the first direct evidence on how TLR3-mediated innate
responses against CHIKV infection can influence the adaptive immune
response, as well as the mechanisms by which TLR3 modulates in vivo
CHIKV immune recognition. It is timely, relevant and significant to the
field. Mice were terminally anesthetized with ketamine [150 mg/kg]/xyla-
zin [10 mg/kg] and perfused with PBS by intra-cardial injection. Tissues were fixed in 4% paraformaldehyde (PFA), decalcified and
embedded in paraffin wax before 5-lm-thick sections were cut and
underwent H&E or immunohistochemical (IHC) staining against
mouse Ly6G (Biolegend, cat# 127602), CD11B (AbCam, cat#
ab75476) and F4/80 (DAKO, cat# K3468) using established proto-
cols (Teng et al, 2012). diluted (1:50–1:5,000), heat-inactivated human plasma or pooled
mouse sera and incubated for 2 h at 37°C with gentle agitation
(350 rpm). Virus–antibody mixtures were then added to HEK 293T
cells seeded on fibronectin-coated 96-well plates and incubated for
1.5 h at 37°C. Virus inoculum was removed and replaced with
DMEM supplemented with 10% FBS and incubated for 6 h at 37°C
before being fixed with 4% PFA and permeabilized in PBS contain-
ing 0.2% Tween 20. Cells were incubated with mouse anti-alphavirus
mAb
(Santa
Cruz,
cat#
sc-58088)
at
1:500
dilution
in
PBS
followed by incubation with Alexa Fluor 488-conjugated goat
anti-mouse IgG F(ab’)2 ab (Invitrogen, cat# A11017) at 1:500
dilution in PBS. Nuclei were stained with DAPI (1 lg/ll stock) at
1:10,000 dilution in PBS followed by immunofluorescence quanti-
fication using the Cellomics ArrayScan high content analysis
reader (Thermo Scientific). Viral RNA extraction and viral load analysis Viral RNA was extracted using QIAamp Viral RNA Mini Kit
(QIAGEN) according to manufacturer’s instructions. Quantification
of CHIKV non-structural protein (nsP) 1 negative-sense RNA was
determined according to a quantitative real-time PCR (qRT-PCR)
TaqMan assay adapted from Plaskon et al (Plaskon et al, 2009)
using QuantiTect Probe RT-PCR Kit (QIAGEN) in 12.5 ll reaction 37 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 © 2014 Agency for Science, Technology and Research (A*STAR) EMBO Molecular Medicine TLR3-mediated immunity controls CHIKV pathogenesis
Zhisheng Her et al Peptide-based ELISA Streptavidin-coated polystyrene 96-well microtiter plates (Pierce,
Thermo Scientific) were blocked with 0.1% PBST supplemented
with 1% w/v sodium caseinate (Sigma-Aldrich) for 1 h at RT before
being coated with biotinylated 18-mer overlapping peptides synthe-
sized based on consensus E2 glycoprotein sequence (Kam et al,
2012b) for 1 h at RT. Mouse serum diluted 1:500 (mouse) or human
plasma diluted 1:2,000 in 0.1% PBST and 0.1% w/v sodium
caseinate were added and incubated for 1 h at RT followed by the Virion-based ELISA CHIKV-specific antibody titers were assessed by virion-based ELISA
(Kam et al, 2012c). Polystyrene 96-well microtiter plates (MaxiSorp,
Nunc) were coated with purified CHIKV (106 infectious units per
well). Wells were blocked with PBS containing 0.05% Tween 20
and 5% non-fat milk (0.05% PBST + 5% milk) for 1.5 h at 37°C. Serum samples were diluted in 0.05% PBST + 2.5% milk before
adding to wells and incubating for 1 h at 37°C. Separately, HRP-
conjugated goat anti-mouse IgM (Santa Crus, cat# sc-2064) or IgG
(Santa Cruz, cat# sc-2005) were used to detect mouse antibodies
bound to virus-coated wells. Reactions were developed using
3,30,5,50-tetramethylbenzidine substrate (Sigma-Aldrich) and termi-
nated by Stop reagent (Sigma-Aldrich). Absorbance at 450 nm was
measured using a TECAN Infinite M200 microplate reader and
analyzed using Magellan
TM software. Results The susceptibility to CHIKV infection is markedly increased in both
human TRIF-deficient and mouse TLR3-deficient fibroblasts with
defective TLR3 signaling. The absence of TLR3 expression in Tlr3/
mice resulted in higher viremia and more pronounced joint inflamma-
tion due to increased pro-inflammatory myeloid cells infiltration. Mechanistically, infection in bone marrow chimeric mice showed that
TLR3-expressing hematopoietic cells are required for effective CHIKV
clearance and pointed toward a role for B cells. Tlr3/ mice’s
impaired ability to effectively clear CHIKV was due to a shift in anti-
virus antibody specificity that led to a reduced recognition of virus-
neutralizing B-cell epitopes by anti-CHIKV IgG. The clinical relevance
of TLR3 was further investigated in CHIKV-infected patients, where
the level of TLR3 transcripts was increased in PBMCs of patients. Single nucleotide polymorphism (SNP) genotyping analysis on TLR3
from 94 patients identified SNP rs6552950 as associated with disease
severity and as in mice, a reduced antibody response. The paper explained Bioluminescence signals were assessed daily from 1 to 8 dpi and
subsequently on every alternate day until 20 dpi using an in vivo
bioluminescence imaging system (IVIS Spectrum, Xenogen) as
described (Teo et al, 2013). Luciferin solution containing the lucifer-
ase substrate, D-luciferin potassium salt (Caliper Life sciences), was
prepared by dissolving in PBS at a concentration of 5 mg/ml. Mice
were shaved and anesthetized in an oxygen-rich induction chamber
with 2% isoflurane. Bioluminescence signals were measured 2 min
after s.c. injection of 100 ll of luciferin solution. Whole-body imag-
ing was performed with the animals in a ventral position, while the
feet and head were imaged with the animals in a dorsal position. Bioluminescence imaging was acquired with a field of view (FOV)
of 21.7 cm for whole body (FOV-D) and 13.1 cm for foot and head
(FOV-C). The mice were exposed for an initial 60 s, followed by a
4-min delay before another exposure at 60 s. When luminescence
readings were above the upper detection limit of machine, the expo-
sure time was reduced and kept consistent across groups. Biolumi-
nescence signals of the region of interest were quantified using
the Living Image 3.0 software (Caliper Life sciences) and expressed
as average radiance (p/s/cm2/sr). The lowest detection limit is
0 p/s/cm2/sr. Problem Chikungunya fever has re-emerged as an important human arboviral
infection of global significance, but the factors determining host
immunity and pathology are still largely unknown. Toll-like receptors
(TLRs) are crucial sensors of virus infection mediated through the
recognition of viral nucleic acids, although until now, their role in
Chikungunya virus (CHIKV) infection has not been clearly established. © 2014 Agency for Science, Technology and Research (A*STAR) genome variation from population-scale sequencing. Nature 467: addition of relevant HRP-conjugated goat secondary IgG (Santa
Cruz, cat# sc-2005) to detect bound antibodies. Reactions were
developed using 3,30,5,50-tetramethylbenzidine substrate (Sigma-
Aldrich) and terminated by Stop reagent (Sigma-Aldrich). Absor-
bance at 450 nm was measured using TECAN Infinite M200 micro-
plate reader and analyzed using Magellan
TM software. 1061 – 1073 Abraham R, Mudaliar P, Padmanabhan A, Sreekumar E (2013) Induction of
cytopathogenicity in human glioblastoma cells by Chikungunya virus. PLoS
One 8: e75854 AbuBakar S, Sam IC, Wong PF, MatRahim N, Hooi PS, Roslan N (2007) Reemergence of endemic Chikungunya, Malaysia. Emerg Infect Dis 13:
147 – 149 Author contributions ZH, T-ST, JJLT, T-HT, F-ML, WWLL, CG, RM, AKA, Y-WK, VL, AL, MKW, AC, SKB,
Y-SL and AM performed the experiments, provided the clinical and experimen-
tal reagents and analyzed the data. ZH, T-ST, JJLT, T-HT, ML, LR and LFPN
conceptualized the study, analyzed the data and wrote the manuscript. All
authors read and approved the manuscript. Enserink M (2014) Crippling virus set to conquer Western Hemisphere. Science 344: 678 – 679 Esposito S, Molteni C, Giliani S, Mazza C, Scala A, Tagliaferri L, Pelucchi C,
Fossali E, Plebani A, Principi N (2012) Toll-like receptor 3 gene
polymorphisms and severity of pandemic A/H1N1/2009 influenza in
otherwise healthy children. Virol J 9: 270 Esposito S, Molteni C, Giliani S, Mazza C, Scala A, Tagliaferri L, Pelucchi C,
Fossali E, Plebani A, Principi N (2012) Toll-like receptor 3 gene
polymorphisms and severity of pandemic A/H1N1/2009 influenza in
otherwise healthy children. Virol J 9: 270
Gardner J, Anraku I, Le TT, Larcher T, Major L, Roques P, Schroder WA, Higgs
S, Suhrbier A (2010) Chikungunya virus arthritis in adult wild-type mice. J
Virol 84: 8021 – 8032
Gitlin L, Barchet W, Gilfillan S, Cella M, Beutler B, Flavell RA, Diamond MS,
Colonna M (2006) Essential role of MDA-5 in type I IFN responses to
polyriboinosinic:polyribocytidylic acid and encephalomyocarditis
picornavirus. Proc Natl Acad Sci USA 103: 8459 – 8464 Conflict of interest The authors declare that they have no conflict of interest. Gardner J, Anraku I, Le TT, Larcher T, Major L, Roques P, Schroder WA, Higgs
S, Suhrbier A (2010) Chikungunya virus arthritis in adult wild-type mice. J
Virol 84: 8021 – 8032 Acknowledgements Am J Trop Med Hyg 17:
107 – 111 Dhanwani R, Khan M, Lomash V, Rao PVL, Ly H, Parida M (2014) Dhanwani R, Khan M, Lomash V, Rao PVL, Ly H, Parida M (2014) Characterization of Chikungunya virus induced host response in a mouse
model of viral myositis. PLoS One 9: e92813 Characterization of Chikungunya virus induced host response in a mouse
model of viral myositis PLoS One 9 e92813 Characterization of Chikungunya virus induced host response in a mouse Duong V, Andries AC, Ngan C, Sok T, Richner B, Asgari-Jirhandeh N, Bjorge S,
Huy R, Ly S, Laurent D et al (2012) Reemergence of Chikungunya virus in
Cambodia. Emerg Infect Dis 18: 2066 – 2069 Statistical analysis Ansumana R, Jacobsen KH, Leski TA, Covington AL, Bangura U, Hodges MH,
Lin B, Bockarie AS, Lamin JM, Bockarie MJ et al (2013) Reemergence of
Chikungunya virus in Bo, Sierra Leone. Emerg Infect Dis 19:
1108 – 1110 Statistics were performed using Prism 5.01 (GraphPad Software). Pairwise comparison between infected and mock-infected primary
fibroblasts from human and mice was performed using two-tailed
unpaired t-test. Pairwise comparison between WT and Tlr3/ mice
in animal studies was performed using two-tailed Mann–Whitney
U-test. TLR3 SNPs association analysis (disease severity and anti-
E2EP3 IgG response) was performed using logistic regression analysis,
while CHIKV-neutralizing capacity association was performed using
one-way ANOVA analysis followed by Tukey’s multiple comparison
test. P-values less than 0.05 are considered statistically significant. Arpaia N, Barton GM (2011) Toll-like receptors: key players in antiviral
immunity. Curr Opin Virol 1: 447 – 454 Bessaud M, Peyrefitte CN, Pastorino BA, Tock F, Merle O, Colpart JJ, Dehecq JS,
Girod R, Jaffar-Bandjee MC, Glass PJ et al (2006) Chikungunya virus
strains, Reunion Island outbreak. Emerg Infect Dis 12: 1604 – 1606 Borgherini G, Poubeau P, Jossaume A, Gouix A, Cotte L, Michault A,
Arvin-Berod C, Paganin F (2008) Persistent arthralgia associated with
Chikungunya virus: a study of 88 adult patients on Reunion Island. Clin
Infect Dis 47: 469 – 475 Supplementary information for this article is available online:
http://embomolmed.embopress.org Centers for Disease Control and Prevention (2012) Chikungunya outbreak— Centers for Disease Control and Prevention (2012) Chikungunya outbreak—
Cambodia, February-March 2012. Morb Mortal Wkly Rep 61: 737 – 740
Chow A, Her Z, Ong EKS, Chen JM, Dimatatac F, Kwek DJC, Barkham T, Yang H, Centers for Disease Control and Prevention (2012) Chikungunya outbreak—
Cambodia, February-March 2012. Morb Mortal Wkly Rep 61: 737 – 740
Chow A, Her Z, Ong EKS, Chen JM, Dimatatac F, Kwek DJC, Barkham T, Yang H,
Renia L, Leo YS et al (2011) Persistent arthralgia induced by Chikungunya
virus infection is associated with IL-6 and GM-CSF. J Infect Dis 203: 149 – 157 Acknowledgements We thank Jean-Laurent Casanova and his team (Shen-Ying Zhang, Vanessa
Sancho-Shimizu, Michael J. Ciancanelli) from Rockefeller University for provid-
ing the human primary TRIF/ fibroblasts. We thank Anis Larbi and the SIgN
Flow Cytometry core for assistance with cytometry analysis. We are grateful to
Michael Poidinger for assistance with genotyping analysis and to Ruo-Yan
Ong, Irina Shalova and Hong-Rong Loh from SIgN for technical assistance. We
also thank the Advanced Molecular Pathology Laboratory (IMCB, A*STAR) for
performing the histology work. We acknowledge Kai-Er Eng from SIgN and
Lucy Robinson of Insight Editing London for assistance with manuscript edit-
ing. This research was funded by SIgN, A*STAR and supported by the Biomedi-
cal Research Council, A*STAR, and European Union FP7 project ‘Integrated
Chikungunya Research’ (ICRES; Grant no. 261202). Zhisheng Her and Fok-Moon
Lum are supported by the President’s Graduate Fellowship from the Yong Loo
Lin School of Medicine, National University of Singapore. Teck-Hui Teo is
supported by the A*STAR postgraduate scholarship. Wendy W.L. Lee is
supported by the postgraduate scholarship from the NUS Graduate School for
Integrative Science and Engineering. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript. Renia L, Leo YS et al (2011) Persistent arthralgia induced by Chikungunya
virus infection is associated with IL-6 and GM-CSF. J Infect Dis 203: 149 – 157 Couderc T, Chrétien F, Schilte C, Disson O, Brigitte M, Guivel Benhassine F, Touret Y, Barau G, Cayet N, Schuffenecker I et al (2008) A mouse model
for Chikungunya: young age and inefficient Type I interferon signaling are
risk factors for severe disease. PLoS Pathog 4: e29 Daffis S, Samuel MA, Suthar MS, Gale MJ, Diamond MS (2008) Toll-like
receptor 3 has a protective role against West Nile virus infection. J Virol
82: 10349 – 10358 Das T, Jaffar-Bandjee MC, Hoarau JJ, Krejbich Trotot P, Denizot M,
Lee-Pat-Yuen G, Sahoo R, Guiraud P, Ramful D, Robin S et al (2010)
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107 – 111 Deller JJ, Russell PK (1968) Chikungunya disease. Sero-neutralization Neutralizing capacity of antibodies from CHIKV-infected mice was
analyzed by immunofluorescence-based cell infection assays using
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virus required to achieve an infection of MOI 10 were mixed with © 2014 Agency for Science, Technology and Research (A*STAR) 38 EMBO Molecular Medicine
Vol 7 | No 1 | 2015 Zhisheng Her et al
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