A Common Polymorphism in TLR3 Confers

Natural Resistance to HIV-1 Infection

This information is current as
of August 21, 2015.
Manuela Sironi, Mara Biasin, Rachele Cagliani, Diego
Forni, Mariacristina De Luca, Irma Saulle, Sergio Lo
Caputo, Francesco Mazzotta, Juan Macías, Juan A. Pineda,
Antonio Caruz and Mario Clerici
J Immunol 2012; 188:818-823; Prepublished online 14
December 2011;
doi: 10.4049/jimmunol.1102179
http://www.jimmunol.org/content/188/2/818

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 The Journal of Immunology

A Common Polymorphism in TLR3 Confers Natural

Resistance to HIV-1 Infection

Manuela Sironi,*,1 Mara Biasin,†,1 Rachele Cagliani,* Diego Forni,* Mariacristina De Luca,†
Irma Saulle,† Sergio Lo Caputo,‡ Francesco Mazzotta,‡ Juan Macı́as,x Juan A. Pineda,x
Antonio Caruz,{ and Mario Clerici‖
TLR3 recognizes dsRNA and activates antiviral immune responses through the production of inflammatory cytokines and type I
IFNs. Genetic association studies have provided evidence concerning the role of a polymorphism in TLR3 (rs3775291, Leu412Phe)
in viral infection susceptibility. We genotyped rs3775291 in a population of Spanish HIV-1–exposed seronegative (HESN) indi-
viduals who remain HIV seronegative despite repeated exposure through i.v. injection drug use (IDU-HESN individuals) as
witnessed by their hepatitis C virus seropositivity. The frequency of individuals carrying at least one 412Phe allele was signifi-
cantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87;

Downloaded from http://www.jimmunol.org/ by guest on August 21, 2015

95% confidence interval, 1.06–3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individ-
uals. Similar results were obtained: the frequency of individuals carrying at least one 412Phe allele was significantly higher
compared with that of a matched control sample (odds ratio, 1.79; 95% confidence interval, 1.05–3.08; p = 0.029). In vitro infection
assays showed that in PBMCs carrying the 412Phe allele, HIV-1Ba-L replication was significantly reduced (p = 0.025) compared
with that of Leu/Leu homozygous samples and was associated with a higher expression of factors suggestive of a state of immune
activation (IL-6, CCL3, CD69). Similarly, stimulation of PBMCs with a TLR3 agonist indicated that the presence of the 412Phe
allele results in a significantly increased expression of CD69 and higher production of proinflammatory cytokines including IL-6
and CCL3. The data of this study indicate that a common TLR3 allele confers immunologically mediated protection from HIV-1
and suggest the potential use of TLR3 triggering in HIV-1 immunotherapy. The Journal of Immunology, 2012, 188: 818–823.

T
oll-like receptors recognize pathogen-associated molec-
ular patterns and activate innate immune responses. In
humans, four TLR family members (TLR3, TLR7, TLR8,
*Scientific Institute for Recovery and Care E. Medea, 23842 Bosisio Parini, Italy;
†
Department of Clinical Sciences, University of Milan, 20157 Milan, Italy;
‡
S. Maria Annunziata Hospital, 50121 Florence, Italy; xInfectious Diseases and Mi-
crobiology Clinical Unit, Valme Hospital, 41000 Seville, Spain; {Immunogenetics
Unit, University of Jaen, Campus Las Lagunillas, 23001 Jaen, Spain; and ‖Don C.
Gnocchi Foundation, Scientific Institute for Recovery and Care, 20148 Milan, Italy
1
M.S. and M.B. contributed equally to this work.
Received for publication August 15, 2011. Accepted for publication November 10,
2011.
This work was supported by grants from the Istituto Superiore di Sanita “Programma
Nazionale di Ricerca sull’ AIDS”; the European Microbicides Project and Aids
Vaccine Integrated Project European Community WP6 Projects; the nGIN European
Community WP7 Project; the Japan Health Science Foundation; the 2007 Ricerca
Finalizzata (Italian Ministry of Health); the 2007 Ricerca Corrente (Italian Ministry
of Health); Progetto Fondo per gli Investimenti della Ricerca di Base Rete Italiana
Chimica Farmaceutica; Rete Italiana Chimica Farmaceutica CHEM-PROFARMA-
NET (RBPR05NWWC); the Spanish Health Ministry (Fondo de Investigaciones
Sanitarias PI021476, PI051778, PI10/01232, and ISCIII-RETIC RD06/006); Funda-
ció Marató TV3 (020730 and 020732); and Junta de Andalucı́a (PI-0335/2009). J.A.P.
is the recipient of an intensification grant from the Fundación Progreso y Salud of the
Consejerı́a de Salud de la Junta de Andalucı́a (Reference AI-0021).
Address correspondence and reprint requests to Prof. Mario Clerici, Universita degli
Studi di Milano, Facolta di Medicina e Chirugia, DISTeB-LITA Via Flli Cervi, 93,
Segrate-Milano 20090, Italy. E-mail address: mario.clerici@unimi.it
The online version of this article contains supplemental material.
Abbreviations used in this article: CI, confidence interval; HC, healthy control; HCV,
hepatitis C virus; HESN, HIV-1–exposed seronegative; HWE, Hardy–Weinberg equi-
librium; IDU-HESN, HIV-1–exposed seronegative despite repeated exposure through
i.v. injection drug use; LD, linkage disequilibrium; MFI, mean fluorescence intensity;
OR, odds ratio; poly(I:C), polyinosinic-polycytidylic acid; SNP, single nucleotide
polymorphism; WNV, West Nile virus.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
and TLR9) are specialized in sensing viral-derived components
and mainly localize to the endosomes (1). Among these, TLR3
recognizes dsRNA, an almost universal intermediate of viral rep-
lication, and triggers immune responses against both RNA and
DNA viruses (1). A synthetic ligand, polyinosinic-polycytidylic
acid [poly(I:C)], can also mediate immune responses through
activation of TLR3. TLR3 signals through TIR domain-containing
adaptor-inducing INF-b (also known as TICAM1) and modulates
the production of inflammatory cytokines and type I IFNs through
NF-kB activation (1).
The TLR3 ectodomain has a horseshoe-like shape that pos-
sibly increases the available surface for dsRNA binding (2). Pre-
vious studies have described a human polymorphism in TLR3,
Leu412Phe (rs3775291), that changes a highly conserved residue
on the surface of the horseshoe structure. The minor 412Phe allele
occurs with a frequency of ∼30% in populations with European
and Asian ancestry, whereas it is virtually absent in Africans (3).
In vitro analyses have suggested that TLR3 molecules carrying the
412Phe allele display reduced activation after poly(I:C) stimula-
tion and allow increased coxsackievirus replication compared with
412Leu TLR3 receptors (4, 5). Genetic association studies have
provided mixed evidence concerning the role of Leu412Phe in
viral infection susceptibility. In fact, the minor allele has been
shown to predispose to enteroviral myocarditis (5) but to be pro-
tective against tick-borne encephalitis virus infection (6).
Recently, stimulation of distinct TLRs, including TLR3, with
specific agonists was shown to elicit more vigorous immune ac-
tivation in HIV-1–exposed seronegative (HESN) individuals com-
pared with that in healthy controls (7). The notion that a subset of
human subjects remains uninfected despite multiple exposures to
HIV-1 has been known for years (8, 9). Natural resistance to HIV-1

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102179
The Journal of Immunology
is thought to result from a favorable genetic and immunologic
milieu that generates systemic and mucosal cell-mediated immune
responses (10–13). In this study, we analyzed the role of the TLR3
Leu412Phe variant in HIV-1 infection. Our data indicate that the
412Phe allele is significantly more common among HESN sub-
jects compared with controls, irrespective of the infection route.
Consistently, cells from individuals carrying at least one 412Phe
allele sustain lower HIV-1 replication compared with that of Leu/
Leu homozygotes and display more vigorous immunological
responses upon TLR3 stimulation.
Materials and Methods
Human subjects
We recruited 102 white males exposed to HIV-1 infection by injection drug
use enrolled in prospective cohort studies in Spain (Valme Hospital, Seville)
who had shared needles for .3 mo. Concurrent markers of hepatitis C virus
(HCV) infection, one of the most prevalent blood-borne viruses, was
present in 100% of individuals who were HIV-1–exposed seronegative
despite repeated exposure through i.v. injection drug use (IDU-HESN).
These values are significantly higher than the reported HCV prevalence
of 1–2% for the general population in Spain. In addition, a group of 124
healthy white males recruited from anonymous blood donors from City
of Jaen Hospital who tested negative for HIV-1 and HCV was used as
a healthy control (HC) sample. The main epidemiological and clinical
characteristics of the series studied were described previously (14). HIV-1
and HCV diagnostics were performed as previously described (14). All
IDU-HESN individuals and their respective controls were Spanish of
Caucasian origin.
The study was designed and performed according to the Declaration of
Helsinki and was approved by the ethics committees of the participating
hospitals and the University of Jaen. All patients and healthy blood donors
provided written informed consent to participate in this study.
As for sexually HESN individuals, inclusion criteria were a history of
multiple unprotected sexual episodes for more than 4 y at the time of the
enrollment, with at least three episodes of at-risk intercourse within 4 mo
prior to study entry and an average of 30 (range, 18 to .100) reported
unprotected sexual contacts per year. These subjects are part of a well-
characterized cohort of serodiscordant heterosexual couples that has been
followed since 1997 (7). HESN and healthy controls were recruited at the
S. Maria Annunziata Hospital (Florence, Italy); all of them were Italian of
Caucasian origin. The study was reviewed and approved by the Institu-
tional Review Board of the S. Maria Annunziata Hospital. Written in-
formed consent was obtained from all subjects.
Genotyping
rs3775291 was genotyped in the HESN and control samples through
PCR amplification and direct sequencing (primer sequences: forward, 59-
TGGAGCACCTTAACATGGAAGA-39; reverse, 59-GTCAGCTGCAGG-
TACTTGTTG-39). PCR products were treated with ExoSAP-IT (USB
Corporation, Cleveland, OH), directly sequenced on both strands with
a Big Dye Terminator sequencing Kit (v3.1; Applied Biosystems), and run
on an Applied Biosystems ABI 3130 XL Genetic Analyzer. Sequences
were assembled using AutoAssembler version 1.4.0 (Applied Biosystems)
and inspected manually by two distinct operators.
HIV infection assay
Whole blood was collected from 46 healthy volunteers by venipuncture in
Vacutainer tubes containing EDTA (Becton Dickinson), and PBMCs were
separated on lymphocyte separation medium (Organon Teknica, Malvern,
PA).
PBMCs (10 3 106 cells/ml) were cultured for 2 d at 37˚C and 5% CO2
in RPMI 1640 containing FBS (20%), PHA (7.5 mg/ml), and IL-2 (15 ng/
ml). After viability assessment, 2.5 3 106 cells were resuspended in
medium containing 1 ng HIV-1Ba-L p24 viral input and incubated for 3 h
at 37˚C. Cells were then washed and resuspended in 3 ml complete
medium with IL-2 (15 ng/ml). Cells were plated in 24-well tissue culture
plates and incubated at 37˚C and 5% CO2. After 3 d, 2.5 3 105 PBMCs
were collected for gene expression analyses. After 5 d, supernatants were
collected for ELISA of p24 Ag. Absolute levels of p24 were measured
using the Alliance HIV-1 p24 ELISA Kit (PerkinElmer). HIV-1Ba-L was
contributed by Drs. S. Gartner, M. Popovic, and R. Gallo (courtesy of the
National Institutes of Health AIDS Research and Reference Reagent
Program).
819
Cytokine analyses after HIV infection assay and TLR3
stimulation
PBMCs (2.5 3 105) freshly isolated from healthy volunteers (six with CC
and eight with CT genotype) were incubated for 6 h with medium or
10 mg/ml poly(I:C). RNA was extracted from cultured PBMCs and from
HIV-infected PBMCs by using the acid guanidium thiocyanate–phenol–
chloroform method. The RNA was dissolved in RNase-free water and puri-
fied from genomic DNA with RNase-free DNase (New England Biolabs,
Ipswich, MA). One microgram of RNA was reverse transcribed into first-
strand cDNA in a 20-ml final volume containing 1 mM random hex-
anucleotide primers, 1 mM oligonucleotide, and 200 U Moloney murine
leukemia virus reverse transcriptase (Promega, Madison, WI). cDNA
quantification for IL-1b, IL-6, IL-10, TNF-a, CCL3, RANTES, IFN-g,
IFN-b, CD69, and GAPDH was performed by real-time PCR (DNA En-
gine Opticon 2; MJ Research, Ramsey, NJ). Primer sequences are listed in
Supplemental Table I. Reactions were performed using a SYBR Green
PCR mix (5 prime, Gaithersburg, MD). Results were expressed as DDCt
and presented as ratios between the target gene and the GAPDH house-
keeping mRNA.
TLR3 expression in basal condition and after specific agonist
stimulation
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PBMCs isolated from six CC and eight CT subjects were analyzed under
basal conditions and after 24-h stimulation with 10 mg/ml poly(I:C).
PBMCs were resuspended in PBS and stained for surface mAbs
CD14PECY7 and CD4PECY7 (Beckman-Coulter, Fullerton, CA). Af-
ter 15-min incubation at room temperature in the dark, cells were washed
and fixed in 1% paraformaldehyde in PBS. Cells were then permeabilized
with saponin 0.5% (Sigma-Aldrich, St. Louis, MO) and stained with an
mAb for TLR3 FITC (1 mg; eBioscience). Cells were incubated for 45 min
at 4˚C in the dark, washed, and fixed in 1% paraformaldehyde in PBS.
Flow cytometric analysis
All the cytometric analyses were performed using an FC500 flow cytometer
(Beckman-Coulter, Miami, FL) equipped with a double 15-mW argon ion
laser operating at 456 nm and 488 nm and interfaced with an Intercorp
computer. For each analysis, 20,000 events were acquired and gated on
CD4 or CD14 expression and side-scatter properties. Green fluorescence
from FITC (FL1) was collected through a 525-nm band-pass filter, and far
red fluorescence from PC7 (FL5) was collected through a 770-nm band-
pass filter. Data were collected using linear amplifiers for forward and side
scatter and logarithmic amplifiers for FL1 and FL5.
Statistical analysis
Genetic association analyses were performed using PLINK (15). Wilcoxon
rank sum tests were used to evaluate differences in p24 levels, cytokine
expression, and cell counts. All tests were two-tailed and were performed
with R (http://cran.r-project.org/).
Results
Association analysis with HIV-1 infection susceptibility
In populations of European ancestry, rs3775291 (Leu412Phe) is the
only non-synonymous variant in TLR3 that segregates at appre-
ciable frequency (3). This variant was shown to be functional and
to be associated with different human phenotypes, including viral
infection susceptibility (5, 6). Thus, we wished to verify whether
rs3775291 modulates the predisposition to HIV-1 infection. Most
humans are susceptible to the virus, but a minority of individuals
do not seroconvert despite multiple exposures. We genotyped
rs3775291 in a cohort of 102 Spanish IDU-HESN individuals and
in 131 age- and sex- matched healthy controls (HCs) with the
same geographic origin. We observed a slight deviation from
Hardy–Weinberg equilibrium (HWE) in IDU-HESN individuals
with an excess of heterozygotes (HWE p value = 0.020); HCs
complied with HWE. A significant difference was observed in the
genotypic distribution of rs3775291 in the two cohorts (Table I),
and the frequency of individuals carrying at least one T (412Phe)
allele was significantly higher in IDU-HESN individuals (67.6%)
compared with that in HCs (52.6%). The odds ratio (OR) for
a dominant model with the T allele protecting from HIV-1 in-
820 A TLR3 POLYMORPHISM CONFERS RESISTANCE TO HIV-1 INFECTION

Table I. Association of rs3775291 with HIV-1 infection susceptibility

Genotype Counts
Genotype Counts (Dominant)
(TT/CT/CC) (TT+CT/CC)

Sample HESN HC pgenotypica pcombb HESN HC pdominantc pcombb OR (95% CI)d

IDU-HESN (Spain) 9/60/33 16/53/62 0.021 0.0027 69/33 69/62 0.023 0.003 1.87 (1.06–3.34)
Sexually exposed 11/38/34 15/91/132 0.028 49/34 106/132 0.029 1.79 (1.05–3.08)
HESN (Italy)
a
Fisher’s exact test p value for a genotypic model.
b
Combined p value for the two samples.
c
Fisher’s exact test p value for a dominant model.
d
OR for a recessive model with 95% CI.

fection was 1.87 [95% confidence interval (CI), 1.06–3.34, Fish-
er’s exact test, p = 0.023]. To replicate this finding, we analyzed an
HESN population with different geographic origin and exposed to
the virus through a different infection route. Specifically, we
phocytes, was significantly upregulated in CT versus CC indi-
viduals (p = 0.033) (Fig. 2A).
Responsiveness to TLR3 stimulation in PBMCs isolated from
subjects with different rs3775291 genotype

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genotyped rs3775291 in a well-characterized cohort of 83 het-
erosexual Italian subjects who have a history of unprotected sex To determine whether the cytokine profile characterizing hetero-
with their seropositive partners. As a control, 238 Italian healthy zygous individuals after HIV infection could be directly dependent
subjects were genotyped; both samples complied with HWE. on TLR3 activation, we compared the cytokine/chemokine profiles
Again, a significant difference was observed in the genotype in TLR3 agonist-stimulated PBMCs from the same 14 volunteers
distribution of rs3775291 (Fisher’s exact test, p = 0.028) (Table I); described earlier. Stimulation with poly(I:C) increased expression
as in the Spanish sample, subjects carrying at least one 412Phe levels of mRNA specific for IL-1b, IL-6, IL-10, TNF-a, CCL3,
allele were significantly overrepresented in the HESN sample RANTES, IFN-b, and IFN-g in PBMCs from both CC and CT
(59.0%) compared with controls (44.5%). The combined results individuals, but the induction was significantly higher in CT
for the Spanish and Italian samples (Table I) strongly suggest that compared with CC cells (CCL3, p = 0.014; IL-1b, p = 0.007; IL-6,
the 412Phe allele protects from HIV-1 with a dominant effect p = 0.011) (Fig. 2B), resembling the cytokine milieu observed in
(p = 0.003), irrespective of the infection route. A dominant effect response to HIV infection. Similarly, increased expression levels
for the 412Phe allele had previously been noticed (5, 16). of CD69 in response to TLR3 triggering was observed in CT
versus CC individuals (p = 0.028) (Fig. 2B).
HIV-1 infection assay
Basal and stimulated TLR3 expressing CD14+ and CD4+ cells
To verify whether the Leu412Phe variant affects HIV-1 replication in CT versus CC subjects
in vitro, we performed an infection assay. Specifically, PBMCs
from 46 healthy volunteers were cultured and infected with HIV- To determine whether the above-noted increases in cytokine/
1Ba-L. Viral replication was assessed after 5 d by measuring viral chemokine expression levels after TLR3 stimulation of PBMCs
p24 levels produced by the infected cells. Genotyping of these were associated with enhanced TLR3 expression in these sub-
subjects for rs3775291 indicated that most of them were CC populations of cells from subjects carrying a CT genotype for
homozygotes (Leu/Leu, n = 31), and only two had a TT genotype; rs3775291, we analyzed TLR3 expression in CD4+ and CD14+ by
thus, heterozygotes and TT homozygotes were analyzed together
(n = 15), as also suggested by the dominant effect of the 412Phe
allele. Analysis of p24 level as a function of TLR3 genotype in-
dicated that cells from CC individuals sustain higher HIV-1Ba-L
replication compared with that of PBMCs derived from CT plus
TT individuals (two-tailed Wilcoxon rank sum test, p = 0.025)
(Fig. 1).
Cytokine production after HIV infection in PBMCs isolated
from subjects with different rs3775291 genotype
To establish whether the reduced susceptibility to HIV infection
observed in PBMCs from individuals carrying at least one 412Phe
allele could be secondary to a peculiar immunological profile, we
analyzed the cytokine/chemokine expression levels in HIV-infected
PBMCs from 14 healthy volunteers, 8 of them heterozygous for
rs3775291 (CT genotype) and 6 CC homozygotes. Three days
postinfection, expression rates of mRNA specific for IL-6, IL-10,
TNF-a, CCL3, and IFN-g were raised in PBMCs from both CC
and CT individuals. However, the mean responses were higher in
CT compared with CC subjects for all parameters analyzed, and
mainly for CCL3 (p = 0.033) and IL-6 (p = 0.040) (Fig. 2A). FIGURE 1. p24 levels measured from HIV-1Ba-L–infected cells. PBMCs
Besides, after HIV infection, mRNA expression levels of CD69, from healthy volunteers were infected with HIV-1Ba-L; the levels of viral p24
a surface marker specifically induced on early activated T lym- were measured after 5 d and are plotted as a function of rs3775291 genotype.
The Journal of Immunology 821

FIGURE 2. Cytokine and CD69 expression after

HIV infection or stimulation with poly(I:C). IL-1b,
IL-6, IL-10, IFN-b, IFN-g, CCL3, RANTES, TNF-a,
and CD69 expression assessed by real-time quantita-
tive RT-PCR 3 d postinfection (A) or 6 h after poly(I:C)
stimulation (B) of PBMCs isolated from CT hetero-
zygous (black bars) or CC homozygous (white bars)
individuals. Results were calculated relative to a house-
keeping gene and are shown as fold-change expression
from the unstimulated sample. Mean values 6 SEs are
shown.

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flow cytometry. The results showed no appreciable differences in
the percentage of CD4+/TLR3+ and CD14+/TLR3+ cells in the
PBMCs of CT compared with CC subjects under basal conditions.
After poly(I:C) stimulation, the percentage of TLR3-expressing
CD4+ and CD14+ cells increased in the PBMCs of both CT and
CC individuals, without showing significant differences between
the two groups.
The mean fluorescence intensity (MFI) indicated that TLR3
densities on CD4+ and CD14+ cells were comparable among the
groups examined (Table II). The similarities observed between the
two groups were maintained even after TLR-specific stimulation
(Table II).
Discussion
The ability of TLR3 to sense dsRNA and stimulate the production
of type I IFNs and inflammatory cytokines suggests that the re-
ceptor plays a central role in antiviral defense. Yet, studies in mice
and humans have indicated that TLR3 may act as a double-edge
sword during viral infection and subsequent pathology. In fact,
Tlr32/2 mice display increased mortality after infection with
murine CMV, encephalomyocarditis virus, coxsackievirus B, and
intracranially inoculated West Nile virus (WNV) (17–20). Con-
versely, animals lacking a functional Tlr3 gene display milder
pathology when challenged with phlebovirus, influenza A virus,
and i.p.-administered WNV (21–23). The reasons for these con-
flicting results are currently unknown, although it has been spec-
ulated that sustained activation of TLR3 might result in increased
permeability of the hematoencephalic barrier to WNV and that
overproduction of proinflammatory cytokines in animals with
functional TLR3 activity might be toxic and eventually lead to
death (21, 23). In humans, rare mutations in TLR3 have been as-
sociated with susceptibility to herpes simplex virus encephalitis
and to enteroviral myocarditis (5, 24). As mentioned earlier, this
latter phenotype has also been shown to occur with increased
frequency in subjects homozygous for the 412Phe allele. Con-
sistently, in an in vitro system, cells expressing TLR3 412Phe
molecules allow increased replication of coxsackievirus B3 com-
pared with 412Leu receptors (5). Similarly, our data are consis-
tent in showing a role for the Leu412Phe variant in HIV-1 in-
fection susceptibility, but the allelic effect is exactly the opposite
of that described in enteroviral infection. We analyzed two pop-
ulations of HESN subjects with different geographic origin and
distinct routes of exposure to the virus. Results were consistent
in showing that the 412Phe allele protects from infection with
a dominant effect. The replication of an association result in
distinct cohorts is regarded as a gold standard for genetic studies;
thus, we consider that, despite the relatively small sample size of
our HESN cohorts, the current data provide strong support to
the role of rs3775291 in HIV-1 infection susceptibility. As a fur-
ther confirmation, infection assays with HIV-1Ba-L indicated that
PBMCs from subjects carrying at least one 412Phe allele sustain
lower viral replication compared with that of cells derived from

Table II. Percentage and MFI of TLR3 expressing CD4+ and CD14+ cells in basal condition and after
stimulation of PBMCs with poly(I:C) for 24 h

Basal Poly(I:C)
(Mean 6 SE) (Mean 6 SE)

Genotype
Parameter (rs3775291) CD4+ Cells CD14+ Cells CD4+ Cells CD14+ Cells

Percentage CT 50.1 6 2.5 6.0 6 0.8 5.3 6 1.2 6.1 6 1.5

CC 48.3 6 3.9 5.6 6 1.2 4.4 6 0.9 4.0 6 0.7
MFI CT 27.3 6 3.1 25.2 6 3.2 21.3 6 3.1 19.3 6 3.3
CC 26.2 6 1.9 26.3 6 3.1 22.1 6 3.2 18.1 6 2.2
PBMCs were derived from subjects with different genotype for rs3775291.
822 A TLR3 POLYMORPHISM CONFERS RESISTANCE TO HIV-1 INFECTION
Leu/Leu homozygotes. This effect may at least partially be me-
diated by the increased production of proinflammatory cytokines
by innate and activated CD69+ lymphocytes that we observed
in PBMCs derived from individuals carrying the 412Phe allele
after HIV-infection assay. Recently it has been reported that after
HIV exposure in HESN individuals, TLR engagement results in
the induction of enhanced innate and adaptive immune responses,
which in turn interfere with HIV replication and productive in-
fection (7). In line with these observations, the cytokine milieu
derived from TLR3-specific stimulation in PBMCs carrying the
412Phe allele resembles the one detected after HIV infection as-
say. Thus, the huge amount of immunological factors released in
response to HIV-induced TLR3 stimulation may be able to over-
whelm the virus and inhibit the infection process. Again, this latter
finding contrasts with previous data indicating that ectopic ex-
pression of TLR3 412Phe molecules results in blunted immune
responses after agonist stimulation (4, 5). A major difference
between the results we report in this study and previous functional
characterization of TLR3 variants (4, 5) resides in our using an ex
vivo approach rather than relying on cell line transfection ex-
periments. Indeed, previous data on TLR3 activity have been
obtained using HEK293 or COS7 cells that were driven to express
the receptor from an exogenous promoter (4, 5). Thus, some tiers
of physiological regulation might be lost in cells that overexpress
the two TLR3 variants. Another possibility is that one or more
genetic elements in HEK293 and COS7 cells interact with the
TLR3 Leu412Phe polymorphism, resulting in an epistatic effect.
Otherwise, this amplified sensitivity to TLR3 stimulation may
result from an increased expression of TLR3 in cells carrying the
412Phe allele. Indeed, several reports emphasize the relation be-
tween TLR expression and responsiveness in HIV infection (25),
as well as in other physiological or pathological conditions (26–
28). However, our results underscore a comparable TLR3 ex-
pression in CD4+ and CD14+ cells from subjects carrying at least
one 412Phe allele compared with that in cells derived from Leu/
Leu homozygotes, which is maintained even after TLR3 stimu-
lation, ruling out the possibility to establish a correspondence
between TLR3 expression and responsiveness. Finally, the possi-
bility exists that the results we obtained are not directly an effect
of the Leu412Phe single nucleotide polymorphism (SNP) but
of a variant in linkage disequilibrium (LD) with it. We consider
this to be an unlikely explanation for the following reasons: 1)
rs3775291 is the only non-synonymous variant in TLR3 segre-
gating at appreciable frequency in European populations (3); 2)
LD analysis of HapMap data for a 20-kb region surrounding
rs3775291 revealed no SNP in strong LD with it (r2 values ,0.6);
3) resequencing data for TLR3 (3) indicated that only three
variants in TLR3, two noncoding and one synonymous, have a
frequency similar to rs3775291 in Europeans. Although these
variants might in principle represent regulatory SNPs, our data
showing no difference in TLR3-expressing cells depending on
rs3775291 genotype argue against this possibility. Thus, the
results of this study suggest that information obtained through
in vitro expression of antiviral response genes may not be read-
ily generalized to predict viral infection susceptibility in living
organisms. Similar considerations have recently been reported for
another antiviral response gene involved in HIV-1 infection,
namely APOBEC3H (29). Indeed, a haplotype that generates
APOBEC3H products with limited stability and antiviral activity
in vitro was shown to confer resistance against sexually trans-
mitted HIV-1 infection (29). It is also worth noting that the TLR3
412Phe allele has previously been associated with increased re-
sistance against tick-borne encephalitis virus (6), although the
mechanism underlying this observation is currently unknown.
Thus, data in both mouse models and humans suggest that some
clues into TLR3 function and regulation have remained elusive.
Although our data suggest that TLR3 stimulation might be asso-
ciated with an amplified defensive responsiveness in subjects
carrying at least one 412Phe allele, the function of the TLR3
pathway in resistance to HIV infection is still debated. In fact,
TLR3 is not directly implicated in HIV-1 genome detection, but its
triggering has been consistently coupled with the generation of
a robust and defensive anti–HIV-1 response. It is likely that after
HIV-1 exposure, the TLR3 pathway is stimulated through an in-
direct mechanism that is turned into an enhanced and protective
immune response in subjects carrying the 412Phe allele. These
data suggest the potential use of TLR3 triggering in HIV-1 im-
munotherapy.
Acknowledgments
We thank Dr. Montes Trujillo and Dr. Antonio Jose Carrero from the Blood
Bank Unit of City of Jaen Hospital for providing the samples from healthy
blood donors.
Downloaded from http://www.jimmunol.org/ by guest on August 21, 2015
Disclosures
The authors have no financial conflicts of interest.
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