Natural Resistance to HIV-1 Infection Confers TLR3 a Common

A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection Manuela Sironi, Mara Biasin, Rachele Cagliani, Diego Forni, Mariacristina De Luca, Irma Saulle, Sergio Lo This information is current as Caputo, Francesco Mazzotta, Juan Macías, Juan A. Pineda, of September 24, 2021. Antonio Caruz and Mario Clerici J Immunol 2012; 188:818-823; Prepublished online 14 December 2011;

doi: 10.4049/jimmunol.1102179 Downloaded from http://www.jimmunol.org/content/188/2/818

Supplementary http://www.jimmunol.org/content/suppl/2011/12/14/jimmunol.110217 Material 9.DC1 http://www.jimmunol.org/ References This article cites 29 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/188/2/818.full#ref-list-1

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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 Macı´as,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) individuals 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 significantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87; Downloaded from 95% confidence interval, 1.06–3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. 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 http://www.jimmunol.org/ 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.

oll-like receptors recognize pathogen-associated molec- and TLR9) are specialized in sensing viral-derived components ular patterns and activate innate immune responses. In and mainly localize to the endosomes (1). Among these, TLR3

T humans, four TLR family members (TLR3, TLR7, TLR8, recognizes dsRNA, an almost universal intermediate of viral rep- by guest on September 24, 2021 lication, and triggers immune responses against both RNA and DNA viruses (1). A synthetic ligand, polyinosinic-polycytidylic *Scientific Institute for Recovery and Care E. Medea, 23842 Bosisio Parini, Italy; acid [poly(I:C)], can also mediate immune responses through †Department of Clinical Sciences, University of Milan, 20157 Milan, Italy; ‡S. Maria Annunziata Hospital, 50121 Florence, Italy; xInfectious Diseases and Mi- activation of TLR3. TLR3 signals through TIR domain-containing crobiology Clinical Unit, Valme Hospital, 41000 Seville, Spain; {Immunogenetics adaptor-inducing INF-b (also known as TICAM1) and modulates ‖ Unit, University of Jaen, Campus Las Lagunillas, 23001 Jaen, Spain; and Don C. the production of inflammatory cytokines and type I IFNs through Gnocchi Foundation, Scientific Institute for Recovery and Care, 20148 Milan, Italy NF-kB activation (1). 1M.S. and M.B. contributed equally to this work. The TLR3 ectodomain has a horseshoe-like shape that pos- Received for publication August 15, 2011. Accepted for publication November 10, 2011. sibly increases the available surface for dsRNA binding (2). Pre- vious studies have described a human polymorphism in TLR3, This work was supported by grants from the Istituto Superiore di Sanita “Programma Nazionale di Ricerca sull’ AIDS”; the European Microbicides Project and Aids Leu412Phe (rs3775291), that changes a highly conserved residue Vaccine Integrated Project European Community WP6 Projects; the nGIN European on the surface of the horseshoe structure. The minor 412Phe allele Community WP7 Project; the Japan Health Science Foundation; the 2007 Ricerca ∼ Finalizzata (Italian Ministry of Health); the 2007 Ricerca Corrente (Italian Ministry occurs with a frequency of 30% in populations with European of Health); Progetto Fondo per gli Investimenti della Ricerca di Base Rete Italiana and Asian ancestry, whereas it is virtually absent in Africans (3). Chimica Farmaceutica; Rete Italiana Chimica Farmaceutica CHEM-PROFARMA- In vitro analyses have suggested that TLR3 molecules carrying the NET (RBPR05NWWC); the Spanish Health Ministry (Fondo de Investigaciones Sanitarias PI021476, PI051778, PI10/01232, and ISCIII-RETIC RD06/006); Funda- 412Phe allele display reduced activation after poly(I:C) stimula- cio´ Marato´ TV3 (020730 and 020732); and Junta de Andalucıá (PI-0335/2009). J.A.P. tion and allow increased coxsackievirus replication compared with is the recipient of an intensification grant from the Fundacioń Progreso y Salud of the Consejerıá de Salud de la Junta de Andalucıá (Reference AI-0021). 412Leu TLR3 receptors (4, 5). Genetic association studies have Address correspondence and reprint requests to Prof. Mario Clerici, Universita degli provided mixed evidence concerning the role of Leu412Phe in Studi di Milano, Facolta di Medicina e Chirugia, DISTeB-LITA Via Flli Cervi, 93, viral infection susceptibility. In fact, the minor allele has been Segrate-Milano 20090, Italy. E-mail address: [email protected] shown to predispose to enteroviral myocarditis (5) but to be pro- The online version of this article contains supplemental material. tective against tick-borne encephalitis virus infection (6). Abbreviations used in this article: CI, confidence interval; HC, healthy control; HCV, Recently, stimulation of distinct TLRs, including TLR3, with hepatitis C virus; HESN, HIV-1–exposed seronegative; HWE, Hardy–Weinberg equilibrium; IDU-HESN, HIV-1–exposed seronegative despite repeated exposure through specific agonists was shown to elicit more vigorous immune ac- i.v. injection drug use; LD, linkage disequilibrium; MFI, mean fluorescence intensity; tivation in HIV-1–exposed seronegative (HESN) individuals com- OR, odds ratio; poly(I:C), polyinosinic-polycytidylic acid; SNP, single nucleotide pared with that in healthy controls (7). The notion that a subset of polymorphism; WNV, West Nile virus. human subjects remains uninfected despite multiple exposures to Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 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 819 is thought to result from a favorable genetic and immunologic Cytokine analyses after HIV infection assay and TLR3 milieu that generates systemic and mucosal cell-mediated immune stimulation responses (10–13). In this study, we analyzed the role of the TLR3 PBMCs (2.5 3 105) freshly isolated from healthy volunteers (six with CC Leu412Phe variant in HIV-1 infection. Our data indicate that the and eight with CT genotype) were incubated for 6 h with medium or 412Phe allele is significantly more common among HESN sub- 10 mg/ml poly(I:C). RNA was extracted from cultured PBMCs and from jects compared with controls, irrespective of the infection route. HIV-infected PBMCs by using the acid guanidium thiocyanate–phenol– Consistently, cells from individuals carrying at least one 412Phe chloroform method. The RNA was dissolved in RNase-free water and puri- fied from genomic DNA with RNase-free DNase (New England Biolabs, allele sustain lower HIV-1 replication compared with that of Leu/ Ipswich, MA). One microgram of RNA was reverse transcribed into first- Leu homozygotes and display more vigorous immunological strand cDNA in a 20-ml final volume containing 1 mM random hex- responses upon TLR3 stimulation. 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, Materials and Methods 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 Human subjects Supplemental Table I. Reactions were performed using a SYBR Green We recruited 102 white males exposed to HIV-1 infection by injection drug PCR mix (5 prime, Gaithersburg, MD). Results were expressed as DDCt use enrolled in prospective cohort studies in Spain (Valme Hospital, Seville) and presented as ratios between the target gene and the GAPDH house- who had shared needles for .3 mo. Concurrent markers of hepatitis C virus keeping mRNA. (HCV) infection, one of the most prevalent blood-borne viruses, was present in 100% of individuals who were HIV-1–exposed seronegative TLR3 expression in basal condition and after specific agonist despite repeated exposure through i.v. injection drug use (IDU-HESN). stimulation Downloaded from These values are significantly higher than the reported HCV prevalence PBMCs isolated from six CC and eight CT subjects were analyzed under of 1–2% for the general population in Spain. In addition, a group of 124 basal conditions and after 24-h stimulation with 10 mg/ml poly(I:C). healthy white males recruited from anonymous blood donors from City PBMCs were resuspended in PBS and stained for surface mAbs of Jaen Hospital who tested negative for HIV-1 and HCV was used as CD14PECY7 and CD4PECY7 (Beckman-Coulter, Fullerton, CA). Af- a healthy control (HC) sample. The main epidemiological and clinical ter 15-min incubation at room temperature in the dark, cells were washed characteristics of the series studied were described previously (14). HIV-1 and fixed in 1% paraformaldehyde in PBS. Cells were then permeabilized and HCV diagnostics were performed as previously described (14). All with saponin 0.5% (Sigma-Aldrich, St. Louis, MO) and stained with an http://www.jimmunol.org/ IDU-HESN individuals and their respective controls were Spanish of mAb for TLR3 FITC (1 mg; eBioscience). Cells were incubated for 45 min Caucasian origin. at 4˚C in the dark, washed, and fixed in 1% paraformaldehyde in PBS. The study was designed and performed according to the Declaration of Helsinki and was approved by the ethics committees of the participating Flow cytometric analysis hospitals and the University of Jaen. All patients and healthy blood donors provided written informed consent to participate in this study. All the cytometric analyses were performed using an FC500 flow cytometer As for sexually HESN individuals, inclusion criteria were a history of (Beckman-Coulter, Miami, FL) equipped with a double 15-mW argon ion multiple unprotected sexual episodes for more than 4 y at the time of the laser operating at 456 nm and 488 nm and interfaced with an Intercorp enrollment, with at least three episodes of at-risk intercourse within 4 mo computer. For each analysis, 20,000 events were acquired and gated on prior to study entry and an average of 30 (range, 18 to .100) reported CD4 or CD14 expression and side-scatter properties. Green fluorescence unprotected sexual contacts per year. These subjects are part of a well- from FITC (FL1) was collected through a 525-nm band-pass filter, and far by guest on September 24, 2021 characterized cohort of serodiscordant heterosexual couples that has been red fluorescence from PC7 (FL5) was collected through a 770-nm band- followed since 1997 (7). HESN and healthy controls were recruited at the pass filter. Data were collected using linear amplifiers for forward and side S. Maria Annunziata Hospital (Florence, Italy); all of them were Italian of scatter and logarithmic amplifiers for FL1 and FL5. Caucasian origin. The study was reviewed and approved by the Institu- tional Review Board of the S. Maria Annunziata Hospital. Written in- Statistical analysis formed consent was obtained from all subjects. Genetic association analyses were performed using PLINK (15). Wilcoxon Genotyping rank sum tests were used to evaluate differences in p24 levels, cytokine expression, and cell counts. All tests were two-tailed and were performed rs3775291 was genotyped in the HESN and control samples through with R (http://cran.r-project.org/). PCR amplification and direct sequencing (primer sequences: forward, 59- TGGAGCACCTTAACATGGAAGA-39; reverse, 59-GTCAGCTGCAGG- TACTTGTTG-39). PCR products were treated with ExoSAP-IT (USB Results Corporation, Cleveland, OH), directly sequenced on both strands with Association analysis with HIV-1 infection susceptibility a Big Dye Terminator sequencing Kit (v3.1; Applied Biosystems), and run In populations of European ancestry, rs3775291 (Leu412Phe) is the on an Applied Biosystems ABI 3130 XL Genetic Analyzer. Sequences were assembled using AutoAssembler version 1.4.0 (Applied Biosystems) only non-synonymous variant in TLR3 that segregates at appre- and inspected manually by two distinct operators. ciable frequency (3). This variant was shown to be functional and to be associated with different human phenotypes, including viral HIV infection assay infection susceptibility (5, 6). Thus, we wished to verify whether Whole blood was collected from 46 healthy volunteers by venipuncture in rs3775291 modulates the predisposition to HIV-1 infection. Most Vacutainer tubes containing EDTA (Becton Dickinson), and PBMCs were humans are susceptible to the virus, but a minority of individuals separated on lymphocyte separation medium (Organon Teknica, Malvern, PA). do not seroconvert despite multiple exposures. We genotyped 6 PBMCs (10 3 10 cells/ml) were cultured for 2 d at 37˚C and 5% CO2 rs3775291 in a cohort of 102 Spanish IDU-HESN individuals and in RPMI 1640 containing FBS (20%), PHA (7.5 mg/ml), and IL-2 (15 ng/ in 131 age- and sex- matched healthy controls (HCs) with the ml). After viability assessment, 2.5 3 106 cells were resuspended in same geographic origin. We observed a slight deviation from medium containing 1 ng HIV-1Ba-L p24 viral input and incubated for 3 h Hardy–Weinberg equilibrium (HWE) in IDU-HESN individuals 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 with an excess of heterozygotes (HWE p value = 0.020); HCs 5 plates and incubated at 37˚C and 5% CO2.After3d,2.53 10 PBMCs complied with HWE. A significant difference was observed in the were collected for gene expression analyses. After 5 d, supernatants were genotypic distribution of rs3775291 in the two cohorts (Table I), collected for ELISA of p24 Ag. Absolute levels of p24 were measured and the frequency of individuals carrying at least one T (412Phe) 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 allele was significantly higher in IDU-HESN individuals (67.6%) National Institutes of Health AIDS Research and Reference Reagent compared with that in HCs (52.6%). The odds ratio (OR) for Program). 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)

a b c b d Sample HESN HC pgenotypic pcomb HESN HC pdominant pcomb OR (95% CI) 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) aFisher’s exact test p value for a genotypic model. bCombined p value for the two samples. cFisher’s exact test p value for a dominant model. dOR for a recessive model with 95% CI.

fection was 1.87 [95% confidence interval (CI), 1.06–3.34, Fish- phocytes, was significantly upregulated in CT versus CC indi- er’s exact test, p = 0.023]. To replicate this finding, we analyzed an viduals (p = 0.033) (Fig. 2A). HESN population with different geographic origin and exposed to Responsiveness to TLR3 stimulation in PBMCs isolated from the virus through a different infection route. Specifically, we Downloaded from subjects with different rs3775291 genotype genotyped rs3775291 in a well-characterized cohort of 83 heterosexual 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 http://www.jimmunol.org/ 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 by guest on September 24, 2021 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/ chemokine expression levels after TLR3 stimulation of PBMCs 1Ba-L. Viral replication was assessed after 5 d by measuring viral p24 levels produced by the infected cells. Genotyping of these were associated with enhanced TLR3 expression in these sub- populations of cells from subjects carrying a CT genotype for subjects for rs3775291 indicated that most of them were CC + + 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 indicated 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 heterozygous (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. Downloaded from

flow cytometry. The results showed no appreciable differences in ulated that sustained activation of TLR3 might result in increased http://www.jimmunol.org/ the percentage of CD4+/TLR3+ and CD14+/TLR3+ cells in the permeability of the hematoencephalic barrier to WNV and that PBMCs of CT compared with CC subjects under basal conditions. overproduction of proinflammatory cytokines in animals with After poly(I:C) stimulation, the percentage of TLR3-expressing functional TLR3 activity might be toxic and eventually lead to CD4+ and CD14+ cells increased in the PBMCs of both CT and death (21, 23). In humans, rare mutations in TLR3 have been as- CC individuals, without showing significant differences between sociated with susceptibility to herpes simplex virus encephalitis the two groups. and to enteroviral myocarditis (5, 24). As mentioned earlier, this The mean fluorescence intensity (MFI) indicated that TLR3 latter phenotype has also been shown to occur with increased densities on CD4+ and CD14+ cells were comparable among the frequency in subjects homozygous for the 412Phe allele. Con- by guest on September 24, 2021 groups examined (Table II). The similarities observed between the sistently, in an in vitro system, cells expressing TLR3 412Phe two groups were maintained even after TLR-specific stimulation molecules allow increased replication of coxsackievirus B3 com- (Table II). pared with 412Leu receptors (5). Similarly, our data are consistent in showing a role for the Leu412Phe variant in HIV-1 in- Discussion fection susceptibility, but the allelic effect is exactly the opposite The ability of TLR3 to sense dsRNA and stimulate the production of that described in enteroviral infection. We analyzed two pop- of type I IFNs and inflammatory cytokines suggests that the re- ulations of HESN subjects with different geographic origin and ceptor plays a central role in antiviral defense. Yet, studies in mice distinct routes of exposure to the virus. Results were consistent and humans have indicated that TLR3 may act as a double-edge in showing that the 412Phe allele protects from infection with sword during viral infection and subsequent pathology. In fact, a dominant effect. The replication of an association result in Tlr32/2 mice display increased mortality after infection with distinct cohorts is regarded as a gold standard for genetic studies; murine CMV, encephalomyocarditis virus, coxsackievirus B, and thus, we consider that, despite the relatively small sample size of intracranially inoculated West Nile virus (WNV) (17–20). Con- our HESN cohorts, the current data provide strong support to versely, animals lacking a functional Tlr3 gene display milder the role of rs3775291 in HIV-1 infection susceptibility. As a fur- pathology when challenged with phlebovirus, influenza A virus, ther confirmation, infection assays with HIV-1Ba-L indicated that and i.p.-administered WNV (21–23). The reasons for these con- PBMCs from subjects carrying at least one 412Phe allele sustain flicting results are currently unknown, although it has been spec- 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- Thus, data in both mouse models and humans suggest that some diated by the increased production of proinflammatory cytokines clues into TLR3 function and regulation have remained elusive. by innate and activated CD69+ lymphocytes that we observed Although our data suggest that TLR3 stimulation might be asso- in PBMCs derived from individuals carrying the 412Phe allele ciated with an amplified defensive responsiveness in subjects after HIV-infection assay. Recently it has been reported that after carrying at least one 412Phe allele, the function of the TLR3 HIV exposure in HESN individuals, TLR engagement results in pathway in resistance to HIV infection is still debated. In fact, the induction of enhanced innate and adaptive immune responses, TLR3 is not directly implicated in HIV-1 genome detection, but its which in turn interfere with HIV replication and productive in- triggering has been consistently coupled with the generation of fection (7). In line with these observations, the cytokine milieu a robust and defensive anti–HIV-1 response. It is likely that after derived from TLR3-specific stimulation in PBMCs carrying the HIV-1 exposure, the TLR3 pathway is stimulated through an in- 412Phe allele resembles the one detected after HIV infection as- direct mechanism that is turned into an enhanced and protective say. Thus, the huge amount of immunological factors released in immune response in subjects carrying the 412Phe allele. These response to HIV-induced TLR3 stimulation may be able to over- data suggest the potential use of TLR3 triggering in HIV-1 im- whelm the virus and inhibit the infection process. Again, this latter munotherapy. finding contrasts with previous data indicating that ectopic expression of TLR3 412Phe molecules results in blunted immune Acknowledgments responses after agonist stimulation (4, 5). A major difference We thank Dr. Montes Trujillo and Dr. Antonio Jose Carrero from the Blood between the results we report in this study and previous functional Bank Unit of City of Jaen Hospital for providing the samples from healthy characterization of TLR3 variants (4, 5) resides in our using an ex blood donors. Downloaded from vivo approach rather than relying on cell line transfection ex- periments. Indeed, previous data on TLR3 activity have been Disclosures obtained using HEK293 or COS7 cells that were driven to express The authors have no financial conflicts of interest. the receptor from an exogenous promoter (4, 5). Thus, some tiers of physiological regulation might be lost in cells that overexpress References the two TLR3 variants. Another possibility is that one or more http://www.jimmunol.org/ genetic elements in HEK293 and COS7 cells interact with the 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783–801. TLR3 Leu412Phe polymorphism, resulting in an epistatic effect. 2. Choe, J., M. S. Kelker, and I. A. Wilson. 2005. Crystal structure of human toll- Otherwise, this amplified sensitivity to TLR3 stimulation may like receptor 3 (TLR3) ectodomain. Science 309: 581–585. 3. Barreiro, L. B., M. Ben-Ali, H. Quach, G. Laval, E. Patin, J. K. Pickrell, result from an increased expression of TLR3 in cells carrying the C. Bouchier, M. Tichit, O. Neyrolles, B. Gicquel, et al. 2009. Evolutionary 412Phe allele. Indeed, several reports emphasize the relation be- dynamics of human Toll-like receptors and their different contributions to host tween TLR expression and responsiveness in HIV infection (25), defense. PLoS Genet. 5: e1000562. 4. Ranjith-Kumar, C. T., W. Miller, J. Sun, J. Xiong, J. Santos, I. Yarbrough, as well as in other physiological or pathological conditions (26– R. J. Lamb, J. Mills, K. E. Duffy, S. Hoose, et al. 2007. Effects of single nu- 28). However, our results underscore a comparable TLR3 ex- cleotide polymorphisms on Toll-like receptor 3 activity and expression in cul- pression in CD4+ and CD14+ cells from subjects carrying at least tured cells. J. Biol. Chem. 282: 17696–17705. by guest on September 24, 2021 5. Gorbea, C., K. A. Makar, M. Pauschinger, G. Pratt, J. L. Bersola, J. Varela, one 412Phe allele compared with that in cells derived from Leu/ R. M. David, L. Banks, C. H. Huang, H. Li, et al. 2010. A role for Toll-like Leu homozygotes, which is maintained even after TLR3 stimu- receptor 3 variants in host susceptibility to enteroviral myocarditis and dilated lation, ruling out the possibility to establish a correspondence cardiomyopathy. J. Biol. Chem. 285: 23208–23223. 6. Kindberg, E., S. Vene, A. Mickiene, A. Lundkvist, L. Lindquist, and between TLR3 expression and responsiveness. Finally, the possi- L. Svensson. 2011. A functional Toll-like receptor 3 gene (TLR3) may be a risk bility exists that the results we obtained are not directly an effect factor for tick-borne encephalitis virus (TBEV) infection. J. Infect. Dis. 203: of the Leu412Phe single nucleotide polymorphism (SNP) but 523–528. 7. Biasin, M., L. Piacentini, S. Lo Caputo, V. Naddeo, P. Pierotti, M. Borelli, of a variant in linkage disequilibrium (LD) with it. We consider D. Trabattoni, F. Mazzotta, G. M. Shearer, and M. Clerici. 2010. TLR activation this to be an unlikely explanation for the following reasons: 1) pathways in HIV-1-exposed seronegative individuals. J. Immunol. 184: 2710– 2717. rs3775291 is the only non-synonymous variant in TLR3 segre- 8. Clerici, M., J. A. Berzofsky, G. M. Shearer, and C. O. Tackett. 1991. Exposure to gating at appreciable frequency in European populations (3); 2) human immunodeficiency virus (HIV) type I indicated by HIV-specific T helper LD analysis of HapMap data for a 20-kb region surrounding cell responses before detection of infection by polymerase chain reaction and 2 , serum antibodies [corrected]. J. Infect. Dis. 164: 178–182. rs3775291 revealed no SNP in strong LD with it (r values 0.6); 9. Clerici, M., J. V. Giorgi, C. C. Chou, V. K. Gudeman, J. A. Zack, P. Gupta, 3) resequencing data for TLR3 (3) indicated that only three H-N.Ho,P.G.Nishanian,J.A.Berzofsky,andG.M.Shearer.1992.Cell- variants in TLR3, two noncoding and one synonymous, have a mediated immune response to human immunodeficiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1. frequency similar to rs3775291 in Europeans. Although these J. Infect. Dis. 165: 1012–1019. variants might in principle represent regulatory SNPs, our data 10. Miyazawa, M., L. 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