Langerhans Cell Function HIV-1 Susceptibility by Affecting Herpes

Herpes Simplex Virus Type 2 Enhances HIV-1 Susceptibility by Affecting Langerhans Cell Function

This information is current as Marein A. W. P. de Jong, Lot de Witte, Maureen E. Taylor of September 27, 2021. and Teunis B. H. Geijtenbeek J Immunol 2010; 185:1633-1641; Prepublished online 30 June 2010; doi: 10.4049/jimmunol.0904137

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Herpes Simplex Virus Type 2 Enhances HIV-1 Susceptibility by Affecting Langerhans Cell Function

Marein A. W. P. de Jong,*,†, ‡ Lot de Witte,x Maureen E. Taylor,{ and Teunis B. H. Geijtenbeek*,†

Genital herpes is the most prevalent viral sexually transmitted infection worldwide and is mainly caused by HSV type 2 (HSV-2). HSV-2 infection enhances HIV-1 susceptibility, even in the absence of clinical symptoms. In this study, we investigated the effect of HSV-2 on HIV-1 transmission by mucosal Langerhans cells (LCs). LCs are important in heterosexual transmission because they form a barrier against HIV-1 infection; LCs efﬁciently capture and degrade HIV-1 through the C-type lectin langerin, thereby preventing HIV-1 transmission. Notably, our data showed that HSV-2 enhanced HIV-1 infection of LCs and subsequent HIV-1 transmission to T cells. HSV-2 interfered with HIV-1 capture by langerin, which allowed efﬁcient HIV-1 infection of LCs. HSV-2 inhibited the antiviral function of langerin at two levels; HSV-2 decreased langerin expression and competed with HIV-1 for

langerin binding. HSV-2 replication was not required, because both UV-inactivated HSV-2 and TLR-3 agonist polyinosinic:poly- Downloaded from cytidylic acid similarly increased HIV-1 transmission by LCs. Therefore, we identiﬁed a mechanism by which HSV-2 enhances HIV-1 susceptibility, even in the absence of clinical symptoms. Our data demonstrated that viral coinfections, such as HSV-2, breach the protective function of LCs by abrogating langerin function, which increases HIV-1 susceptibility. These data reinforce the importance of preventing sexually transmitted infections, such as HSV-2, to reduce the transmission of HIV-1. The Journal of Immunology, 2010, 185: 1633–1641. http://www.jimmunol.org/

ore than 30 million people worldwide are infected by Notably, HSV-2 can enhance HIV-1 susceptibility in the absence HIV-1, the causative agent of AIDS, and heterosexual of apparent lesions (5). HSV-2 also induces inflammation, thereby M transmission of HIV-1 is the primary route of infection including T cell influx, even in the absence of clinically apparent (1). Numerous studies demonstrated that sexually transmitted lesions. Furthermore, HIV-1–susceptible inflammatory cells can infections (STIs) enhance susceptibility to HIV-1 acquisition and persist for a long period of time after HSV-2 infection has been transmission, even in the absence of clinical symptoms (1–5). The cleared and the lesions have healed (10). most common viral STI acquired during sexual contact in the HSV-2 infection occurs via direct contact with infected lesions or developing world is genital herpes (6), which is primarily caused body fluids and enters the body at mucosal tissues or through small by guest on September 27, 2021 by HSV type 2 (HSV-2), although HSV type 1 infection can also lesions. Epithelial cells and keratinocytes are the primary target cells lead to genital herpes (7). Pre-existing HSV-2 infection is a risk for HSV-2, but it also infects neuronal and immune cells (11). Once factor for acquiring HIV-1 infection through sexual contact (1–4). infected, thevirus causes life-long infection of the host by establishing Although it remains unclear how HSV-2 enhances HIV-1 suscep- latency in the neurons of the sensitive ganglia. Reactivation occurs tibility, several mechanisms have been proposed. The formation of periodically, at times when the immune system is suboptimal, result- pustules and ulcers by genital herpes can facilitate HIV-1 entry ing in the formation of vesicles that break into ulcerations at the into mucosal tissues (8). In addition, mucosal inflammation by genital mucosa (8, 12). The genital mucosa is crucial in sexual trans- HSV-2 can cause an influx of activated CD4+ T cells, which mission of HIV-1, and coinfections with HSV-2 might affect the in- facilitates HIV-1 infection and subsequent dissemination (9). tegrity of the genital mucosa and consequently increase HIV-1 susceptibility. Genital tissues contain Langerhans cells (LCs), the main dendritic cell (DC) subset present in stratified epithelia and *Center of Infection and Immunity Amsterdam and †Center for Experimental and mucosal tissues (13). LCs are involved in HIV-1 dissemination; Molecular Medicine, Academic Medical Center, University of Amsterdam; HIV-1 can infect LCs, and infected LCs subsequently migrate to the ‡Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam; xDepartment of Virology, Erasmus Medical Center, University lymphoid tissues where they transmit the virus efficiently to T cells Medical Center, Rotterdam, The Netherlands; and {Division of Molecular Bioscien- and, thereby, mediate infection of the host (14). However, we recently ces, Department of Life Sciences, Imperial College, London, United Kingdom showed that LCs have an anti–HIV-1 function (15). Under noninflam- Received for publication December 22, 2009. Accepted for publication May 18, matory conditions, LCs prevent HIV-1 transmission by efficient cap- 2010. ture of invading HIV-1 via the C-type lectin langerin, which targets This work was supported by Dutch Scientific Organization Grants 91204025 (to M.d.J.) and 91746367 (to L.d.W.) and Wellcome Trust Grant 075565 (to M.E.T.). the virus for degradation (15). Langerin prevents HIV-1 infection of Address correspondence and reprint requests to Dr. Teunis B.H. Geijtenbeek, Center LCs and, thereby, transmission of the virus to T cells in lymphoid for Experimental and Molecular Medicine, Academic Medical Center, University of tissues. Thus, LCs form a first line of defense against HIV-1infection. Amsterdam, Meibergdreef, 9 1105 AZ, Amsterdam, The Netherlands. E-mail ad- However, inhibition of the antiviral function of langerin or inflamma- dress: [email protected] tory conditions allows infection of LCs by HIV-1, and infected LCs Abbreviations used in this paper: CpG-ODN, CpG-containing oligonucleotides; DC, dendritic cell; eGFP, enhanced GFP; HIV-1–eGFP, HIV-1 virus R5-tropic enhanced efficiently transmit HIV-1 to T cells (15, 16). Therefore, we hypoth- GFP-expressing HIV-1-NL4.3-BaL; HSV-2, HSV type 2; LC, Langerhans cell; MOI, esized that viral STIs, such as HSV-2, increase HIV-1susceptibility by multiplicity of infection; PI, propidium iodide; poly(I:C), polyinosinic:polycytidylic affecting the protective function of LCs. acid; STI, sexually transmitted infection; UV, HSV-2; UV-inactivated HSV type 2. In this article, we show that HSV-2 productively infects LCs Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 and that LC activation by HSV-2 increases HIV-1 transmission by www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904137 1634 HSV-2 INFECTION OF LCs ENHANCES HIV-1 TRANSMISSION

LCstoT cells.Weidentifiedtwo distincteffects ofHSV-2ontheanti– of LCs. For TNF-a production, cells were incubated with different HIV-1 function of LCs; HSV-2 induced downregulation of langerin concentrations of HSV-2 or UV–HSV-2 for 6, 12, and 24 h or with poly a expression and in addition HSV-2 virions inhibited HIV-1 binding to (I:C). Supernatant was harvested, and TNF- protein production was measured by ELISA (BioSource International, Camarillo, CA). langerin by direct competition with HIV-1. The reduced langerin expression and function, decreased HIV-1 capture by LCs and Fluorescent bead adhesion assay allowed infection of LCs by HIV-1, which increased HIV-1 trans- Streptavidin-coated beads (TransFluorSpheres, Molecular Probes) were coated mission to T cells. Notably, active HSV-2 replication was not re- with HIV-1 gp120, as described previously (21). The adhesion assay (Fig. 3A) quired, because UV-inactivated HSV-2 (UV–HSV-2) similarly was performed as follows (19, 22, 23): 1 3 105 cells were incubated overnight increased HIV-1 transmission by LCs. Furthermore, our data show with TLR agonists or different concentrations of HSV-2 and UV–HSV-2. Cells that TLR-3 agonist polyinosinic:polycytidylic acid [poly(I:C)] also were washed extensively with PBS and stained with CD1a-FITC. Next, cells were washed in Tris buffer (20 mM Tris-HCl [pH 7], 150 mM NaCl, 1 mM increased HIV-1 transmission. Thus, coinfection with HSV-2 is CaCl2,2mMMgCl2 supplemented with 0.5% BSA) before adding HIV-1 a major risk factor that increases HIV-1 susceptibility by affecting gp120 beads for 45 min at 37˚C. Cells were washed and binding was measured the anti–HIV-1 function of LCs. Our data suggest that dsRNA by flow cytometry. Freshly isolated LCs were incubated with HSV-2 or mannan viruses might similarly increase HIV-1 susceptibility. These data for 15 min prior to adding HIV-1 gp120 beads to investigate competition with langerin. After 45 min incubation at 37˚C, cells were washed and binding was further support an important role for LCs in HIV-1 transmission in measured by flow cytometry. the presence of subclinical genital herpes and other viral STIs. Langerin-binding ELISA Materials and Methods Different concentrations of HSV-2 were coated onto ELISA plates overnight at

Abs and reagents room temperature. Nonspecific binding was blocked by incubating the plate Downloaded from with Tris buffer for 1 h at 37˚C. Recombinant human langerin (24) (2 mg/ml) The following Abs were used: PE-conjugated Abs against CD80, CD86 and was added for 1 h at 37˚C. Unbound langerin was washed away, and binding CD4, CD1a-FITC (all from BD Pharmingen, San Diego, CA), CD1a-PE was determined using an anti-langerin Ab (DCGM4; Beckman Coulter), fol- (Abcam, Cambridge, MA), HSV1/2 gpB (Novus Biologicals, Littleton, lowed by peroxidase-conjugated goat anti-mouse IgG Ab (Jackson ImmunoR- CO), goat anti-mouse–FITC (Zymed Laboratories, San Francisco, CA), esearch Laboratories, West Grove, PA). Specificity was determined in the pre- langerin-PE (Beckman Coulter, Fullerton, CA), and CCR5-PE (Invitrogen, sence of mannan (1 mg/ml) or EGTA (10 mM/ml). Tetramethylbenzidine was Breda, The Netherlands). Isotype-control Abs were used against mouse used as a substrate for peroxidase, and absorbance was measured at 450 nm. IgG1 (BD Pharmingen) and rat IgG1 (Invitrogen). The following stimuli http://www.jimmunol.org/ or inhibitors were used: TLR agonists poly(I:C) (10 mg/ml; Sigma-Aldrich, HSV-2 mRNA transcription m St. Louis, MO), imiquimod (10 g/ml; InvivoGen, San Diego, CA), CpG- 3 5 + . containing oligonucleotides (CpG-ODN) 2216 (10 mg/ml; InvivoGen), A total of 1 10 CD1a LCs ( 95% pure) were infected with different Pam3CSK4 (5 mg/ml; InvivoGen), recombinant human TNF-a (0.1 mg/ concentrations of HSV-2 or UV–HSV-2. After 6, 24, or 48 h, the cells were ml; Strathmann Biotec, Dengelsberg, Germany), and anti–TNF-a (20 mg/ washed extensively with PBS, and mRNA was isolated with the mRNA ml; Biovision, Mountain View, CA). Capture Kit (Roche Diagnostic Systems). cDNA was synthesized with the Reverse Transcriptase Kit (Promega, Madison, WI). For quantitative real- Cell lines and viruses time PCR analysis, PCR amplification was performed in the presence of SYBR green, as previously described (16, 25). Specific primers for HSV-2 Jurkat T cells expressing CCR5 were generated by retroviral transduction, thymidine kinase and GAPDH were designed by Primer Express 2.0 as previously described (17, 18). The HSV-2 strain 333 was grown on (Applied Biosystems, Foster City, CA): forward primer sequence, 59-CGA- by guest on September 27, 2021 GMK cells and harvested after 24 h of infection. Supernatant was filtrated GCCGATGACTTACTGGC-39; reverse primer sequence, 59-GCTGCGTGTT- over a 0.22-mm filter, and a plaque-titration assay was performed to de- GTAGATGTTCG-39. Transcription was adjusted for GAPDH transcription. termine viral titers (19). HSV-2 was inactivated by placing a Petri dish containing 3 ml viral suspension under a UV lamp for 10 min on ice. Virus HSV-2 production by LCs 2 inactivation was confirmed, and the virus stocks were stored at 80˚C 3 5 . until further use. Medium from cultured GMK cells was treated similarly A total of 1 10 LCs ( 95% pure) were infected with different concentrations of HSV-2 or UV–HSV-2. After 1 h, cells were washed extensively and used as a mock control. m HIV-1viruses R5-tropic enhanced GFP (eGFP)-expressing HIV-1–NL4.3- and incubated in a total volume of 100 l. After 3, 18, or 40 h, supernatant was harvested and stored at 220˚C until analysis. To measure productive BaL (referred to as HIV-1–eGFP) (20 ng/well) and replication-defective R5- m tropic pseudotyped HIV-1 (HIV-1 BaL-pseudotyped NL4.3-eGFPDENV) infection, supernatant was filtered to remove cell-debris, and 10 l was added to GMK cells. After 24 h, infection of GMK cells was measured by (50 ng/well) were produced in 293T cells, as described previously (16). 3 7 Virus stocks were quantified by p24 ELISA (PerkinElmer, Wellesley, MA) flow cytometry for the expression of HSV-2 gpB. Pure HSV-2 (1 10 PFU/ and titrated using the indicator cells TZM-blue (16, 20). ml) was treated similarly as the supernatant as a control. Isolation of epidermal single-cell suspension and LCs Viability assay 3 5 . Human tissue was obtained from healthy donors undergoing corrective A total of 1 10 LCs ( 95% pure) were infected with HSV-2 or UV– breast or abdominal surgery after informed consent, in accordance with HSV-2 at multiplicity of infection (MOI) of 1. Cells were collected at 3, institutional guidelines, and it was used within 3 h after surgery. An LC- 18, and 40 h postinfection and analyzed for apoptosis using Annexin V and propidium iodide (PI) (BD Pharmingen). Cells were incubated with enriched epidermal single-cell suspension was generated, as described pre- m viously (15, 16). In short, epidermis was separated after dispase II (1 mg/ Annexin V-FITC and 0.1 g PI for 15 min at room temperature in the ml; Roche Diagnostic Systems, Somerville, NJ) treatment and digested in dark. Cells treated with 3% formaldehyde for 30 min on ice were used as a positive control (data not shown). Cells were analyzed by flow cytometry. 0.05% trypsin solution. Cell suspension was purified over Lymphoprep + + gradient. As determined by CD1a/langerin staining, these single-cell sus- PI cells were considered necrotic or late apoptotic; Annexin V cells were pensions generally contained 10–15% LCs. CD1a+ LCs were isolated considered apoptotic. using positive selection for CD1a in MACS, according to the manufac- Immunofluorescence microscopy turer’s protocol (Miltenyi Biotec, Auburn, CA). This yielded a .90% pure CD1a+ LC population. Cells were cultured in IMDM supplemented with CD1a+ LCs (1 3 105; .95% pure) were incubated with 1 3 105 PFU 10% FCS and 10 mg/ml gentamicin. HSV-2 or UV–HSV-2 for 24 h. Cells were washed and stained intracellu- larly for HSV-2–ICP27 or HSV-2–ICP5, in combination with CD1a (all Cell stimulation from Santa Cruz Biotechnology, Santa Cruz, CA). Cells were analyzed by Cells (1 3 105) were preincubated with TLR stimuli, HSV-2 (1 3 105 confocal microscopy (Leica AOBS SP2 CSLM system). 5 PFU), UV–HSV-2 (1 3 10 PFU), or a mock control overnight. Flow HIV-1 infection cytometric analysis for cell-surface markers was performed. To determine productive HSV-2 infection of LCs, 1 3 105 cells were incubated with Cells stimulated overnight were washed extensively and inoculated with R5- different viral input, and expression of HSV-2 gpB was measured by flow tropic HIV-1–NL4.3-BaL (20 ng/well). For mRNA analysis, after 6 and 24 cytometry. A double staining for CD1a was performed to analyze infection h, cells were washed extensively with PBS, and host and viral mRNA were The Journal of Immunology 1635 isolated with the mRNA capture kit (Roche Diagnostic Systems). For real- (20 ng/well). After 24 h, cells were washed extensively and incubated in time PCR analysis, PCR amplification was performed in the presence of 100 ml medium. At day 6, HIV-1 p24 was measured by using retro-tek SYBR green, as previously described (16, 25). HIV-1 Tat/Rev transcripts HIV-1 p24 detection ELISA (Zeptometrix, Buffalo, NY), according to the were measured by using specific primers that detect Tat and Rev mRNA manufacturer’s protocol. across an intron (for sequence see Ref. 16). Transcription was adjusted for GAPDH transcription, and relative mRNA expression of HIV-1–infected HIV-1 transmission control samples was set at 1. For HIV-1 p24 protein production, CD1a+ LCs (1 3 105; .95% pure) Cells were washed extensively and inoculated with HIV-1–eGFP (20 ng/ were incubated with 1 3 105 PFU UV–HSV-2 or poly(I:C) overnight. well) or replication-defective R5-tropic pseudotyped HIV-1–eGFPDENV Cells were washed extensively and inoculated with HIV-1–NL4.3-BaL (50 ng/well). After 2 and 40 h, cells were washed, and CCR5+ Jurkat Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. HSV-2 productively infects LCs. A and B, Epidermal LC fraction was infected with different concentrations of HSV-2 for 18 h. HSV-2 infection was determined by measuring HSV-2 gpB expression on CD1a+ LCs by ﬂow cytometry. A, Numbers represent the percentage of the total population. B, Percentage of LCs that were infected with HSV-2. Error bars represent SDs of duplicates. These results were observed for three donors. C and D, CD1a+ LCs isolated by CD1a MACS were infected with HSV-2 or UV–HSV-2. mRNA expression of HSV-2 thymidine kinase gene was measured at different viral inputs (C) after 24 h or at different time points (D) at MOI of 1. Transcription was adjusted for GAPDH expression. E, CD1a+ LCs were incubated with HSV-2 (MOI 1) for 24 h and stained for HSV-2 ICP5 and ICP27 (red) and CD1a (green). Images were taken using confocal microscopy at 3630 with 34 zoom. F, CD1a+ LCs were incubated with HSV-2 at different concentrations, and supernatant was harvested at different time points. Productive infection was determinedby titrating supernatant on susceptible GMK cells. Infection of GMK cells was analyzed by ﬂow cytometry for HSV-2 gpB expression. As a control, GMK cells were directly infected with HSV-2 (pure HSV-2 condition). G, Viability of LCs infected with HSV-2 or UV–HSV-2 at MOI of 1 was determined 3, 18, and 40 h postinfection by staining for Annexin V and PI. Results are representative of at least two separate experiments. 1636 HSV-2 INFECTION OF LCs ENHANCES HIV-1 TRANSMISSION

T cells (2 3 104) were added. Transmission to CCR5+ Jurkat T cells was with similar concentrations of UV–HSV-2 (data not shown), dem- measured over time by flow cytometry. onstrating that gpB staining is due to productive infection of LCs Statistical analysis and not to uptake of HSV-2 particles. To confirm productive HSV-2 infection, LCs were purified from One-way ANOVA was used to compare the means of multiple groups. When the overall F-test was significant, differences were investigated the epidermal LC fraction and subsequently infected with HSV-2 or further using a post hoc Bonferroni test using GraphPad Prism software. UV–HSV-2, and expression of HSV-2 thymidine kinase mRNAwas A p value ,0.05 was considered statistically significant. measured by quantitative real-time PCR. In contrast to UV–HSV- 2, HSV-2 infection induced transcription of thymidine kinase, Results which increased over time and was dose dependent (Fig. 1C, HSV-2 productively infects human LCs 1D). In addition, intracellular expression of HSV-2 immediate early HSV-2 is one of the most common viral STIs (7) and is a risk protein ICP27 and late viral protein ICP5 was analyzed by confocal factor for enhanced HIV-1 susceptibility (1–4). Therefore, we microscopy. ICP27 and ICP5 were expressed by LCs after 24 h of investigated whether HSV-2 infects LCs and, thereby, affects infection with HSV-2 but not with UV–HSV-2 (Fig. 1E, data not HIV-1 transmission. Infectious HSV-2 and UV–HSV-2 were used shown). Furthermore, LCs produced infectious HSV-2 particles, to distinguish between effects caused by viral replication or because supernatant from infected LCs caused HSV-2 infection through direct activation by pattern-recognition receptors. An of a susceptible cell line (Fig. 1F). Together, these data demon- epidermal LC fraction, containing 10–15% CD1a+ LCs, was strated that LCs are productively infected by HSV-2. infected with different concentrations of HSV-2 for 18 h, and HSV-2 infection affects the viability of monocyte-derived DCs cell-surface expression of HSV-2 gpB was analyzed to determine (27). Therefore, we investigated the viability of LCs after HSV-2 Downloaded from HSV-2 infection. gpB is an HSV-2 envelope glycoprotein that is or UV–HSV-2 stimulation. LCs were infected with HSV-2 at an expressed as a late viral gene product (26). We used CD1a as an MOI of 1 and analyzed apoptosis and necrosis at 3, 18, and 40 h LC marker to distinguish LCs from other cells in the epidermal postinfection. Annexin V and PI were used to distinguish viable LC fraction [i.e., keratinocytes, T cells (16), and melanocytes]. cells (Annexin V2/PI2), early apoptotic cells (Annexin V+/PI2), 2 LCs and CD1a keratinocytes were productively infected with and late apoptotic cells (Annexin V+/PI+). After 18 h, viability was

HSV-2 in a concentration-dependent manner (Fig. 1A,1B). 60% for HSV-2–infected cells compared with 70% for noninfected http://www.jimmunol.org/ HSV-2 infection of LCs increased with the viral load; at 1 3 LCs (Fig. 1G). After 40 h, viability had decreased to ∼40% 105 PFU, ∼10% of LCs were infected, whereas at a higher viral compared with 70% for untreated LCs. These results showed that load (10 3 105 PFU), up to 45% of LCs were infected (Fig. 1B). HSV-2 decreased the viability of LCs after prolonged culture. In We never observed .3% gpB+ cells when LCs were incubated contrast, UV–HSV-2 had no effect on LC viability. by guest on September 27, 2021

FIGURE 2. TLR agonists and HSV-2 induce maturation of LCs. A, The epidermal LC fraction was isolated from human skin, and LCs were analyzed for CD1a and langerin expression by flow cytometry. B, Cells (1 3 105) were stimulated with poly(I:C) (10 mg/ml), CpG-ODN (10 mg/ml), imiquimod (10 mg/ml), HSV-2 (1 3 105 PFU), or UV–HSV-2 (1 3 105 PFU). After 18 h, CD1a+ LCs were analyzed for the expression of costimulatory molecules CD80 and CD86, langerin, and HIV-1Rs CD4 and CCR5 by flow cytometry. Open graphs depict isotype control, and filled graphs represent the depicted molecules. Mean fluorescent intensity is shown in the upper right corner. These results are representative of more than three donors. The Journal of Immunology 1637

HSV-2 and viral TLR agonists induce LC maturation Next, we investigated whether decreased expression of langerin by Next, we investigated whether HSV-2 infection or viral TLR ago- HSV-2 and poly(I:C) decreases HIV-1 capture. Primary LCs were nists induce LC maturation. Viral recognition is mainly mediated treated with HSV-2, UV–HSV-2, and different TLR agonists over- by TLR-3 for dsRNA, TLR-7 and -8 for ssRNA, and TLR-9 for night and washed extensively before binding to HIV-1 gp120 was unmethylated DNA. Immature LCs express TLR-3 and -7 but little determined. As shown previously (15), HIV-1 gp120 interacted effi- TLR-8 and no TLR-9 (28, 29). Epidermal LC fraction was infected ciently with immature LCs, and the interaction was blocked by the with HSV-2/UV–HSV-2 and TLR-3, -7, and -9 agonists poly(I:C), competitive inhibitor of langerin, mannan (Fig. 3A), strongly sug- imiquimod, and bacterial CpG-ODN, respectively (Fig. 2A). LC gesting that langerin is the primary receptor for HIV-1. Notably, maturation was determined by measuring costimulatory molecules HSV-2 infection and incubation with UV–HSV-2 significantly de- CD80 and CD86 after 18 h by flow cytometry. In contrast to a mock creased HIV-1 binding to LCs in a concentration-dependent manner infection control, HSV-2 infection of LCs induced upregulation of (Fig. 3A). Poly(I:C) also decreased HIV-1 binding to LCs in contrast the costimulatory molecules CD80 and CD86 (Fig. 2B, data not to imiquimod or CpG-ODN (Fig. 3A). Subsequent incubation with shown). Notably, UV–HSV-2 induced upregulation of the costimu- mannan reduced the binding to background levels for all conditions latory molecules to a similar extent as infectious HSV-2 (Fig. 2B). (Fig. 3A, data not shown). These data demonstrated that LC matura- Poly(I:C) and, to a lesser extent, imiquimod induced expression tion by HSV-2 infection, UV–HSV-2, and TLR-3 agonists decreased of CD80 and CD86, in contrast to CpG-ODN (Fig. 2B). Thus, HIV-1 capture by LCs, which might be due to downregulation of HSV-2 and viral TLR agonists induce LC maturation. langerin expression. Next, we investigated whether the decreased langerin expression

by HSV-2 and TLR-3 agonist increases HIV-1 infection. LCs were Downloaded from HSV-2 and viral TLR agonists decrease langerin expression incubated overnight with HSV-2, UV–HSV-2, and viral TLR ago- and increase HIV-1 infection nists, washed, and infected with R5-tropic HIV-1. HIV-1 replication HIV-1 receptors CD4 and CCR5 are crucial for the infection of LCs was determined after 6 and 24 h by measuring Tat/Rev transcripts with R5-tropic HIV-1, whereas, in contrast, langerin expression pre- using quantitative real-time PCR (16). Notably, HSV-2, UV–HSV- vents infection of LCs (15). Therefore, we investigated the effect of 2, and poly(I:C) strongly enhanced HIV-1 transcription, in contrast

HSV-2 and viral TLR agonists on the expression of CD4, CCR5, to imiquimod and CpG-ODN (Fig 3B–D). In addition, UV–HSV-2 http://www.jimmunol.org/ and langerin. The expression of HIV-1Rs CD4 and CCR5 was or poly(I:C) stimulation of CD1a+ LCs increased HIV-1 p24 protein neither affected by HSV-2 nor any of the TLR agonists (Fig. 2B). production (Fig. 3E). Thus, HSV-2 infection, as well as stimulation Notably, HSV-2 infection, UV–HSV-2, and poly(I:C) strongly de- with UV–HSV-2 and viral TLR-3 agonists, enhances HIV-1 infec- creased langerin expression, whereas imiquimod caused only a mar- tion of LCs, which might be due to LC maturation and downregu- ginal decrease (Fig. 2B). lation of langerin expression. by guest on September 27, 2021

FIGURE 3. HSV-2 and TLR stimulation decrease HIV-1 capture and enhance HIV-1 infection of LCs. A, The epidermal LC fraction was stimulated for 18 h with different stimuli, and LCs were analyzed for HIV-1 gp120 binding. Speciﬁcity was determined by inhibiting langerin using mannan and EGTA. Bar graph depicts percentages of LCs that captured HIV-1 gp120. Error bars represent SDs of triplicates. These results are representative of three donors. The epidermal LC fraction (B, C) or CD1a+ LCs (D) were stimulated for 20 h with different stimuli. Next, stimulated cells were incubated with R5-tropic HIV-1-NL4.3-BaL for 6 h (B–D)or24h(C, D). HIV-1 replication was determined by measuring Tat/Rev transcripts by quantitative real-time PCR and normalized for GAPDH expression. Medium condition was set at 1. Error bars represent SDs of duplicates. These results were observed for at least two donors. E, CD1a+ LCs were stimulated for 20 h with different stimuli. Next, stimulated cells were incubated with R5-tropic HIV-1-NL4.3-BaL for 24 h. Cells were washed extensively; at day 6, HIV-1 p24 was determined in the cultures by ELISA. Results are representative of two donors. pp , 0.05; ppp , 0.01; pppp , 0.001. 1638 HSV-2 INFECTION OF LCs ENHANCES HIV-1 TRANSMISSION

FIGURE 4. HSV-2 competes with HIV-1 for langerin binding. A, HSV-2 viral particles were coated onto ELISA plates, and binding was detected using recombinant langerin. Mannan and EGTA preincubation demonstrate the speciﬁcity for langerin. Error bars represent SDs of triplicates. These results were observed in three individual experiments. B and C, LCs were preincubated with different concentrations of HSV-2 for 15 min before HIV-1 gp120 beads were added. The percentages of LCs that captured HIV-1 gp120 are depicted. Error bars represent SDs of duplicates. The results are representative of three donors.

HSV-2 competes with HIV-1 for langerin binding HSV-2 and TLR-3 agonist poly(I:C) increased HIV-1 infection of Downloaded from Langerin interacts with different pathogens, such as HIV-1, Candida LCs by inducing LC maturation and affecting langerin function. albicans,andMycobacterium (15, 30–33). Therefore, we investigated Therefore, we investigated whether HSV-2 and TLR agonists in- whether HSV-2 interacts with langerin and, thereby, competes with creased HIV-1 transmission to T cells. Infectious HSV-2 could not HIV-1 for the carbohydrate-recognition domain of langerin. Recombi- be used in the transmission assays because it also infects T cells and nant langerin strongly interacted with HSV-2 in a concentration- induces cell death in the coculture after several days, even in the dependent manner (Fig. 4A), as determined by the langerin-binding presence of HSV-2 inhibitor acyclovir (data not shown). The epi- ELISA (24). The binding was specific for the carbohydrate- dermal LC fraction was treated with viral TLR agonists and UV– http://www.jimmunol.org/ HSV-2 for 20 h, washed extensively, pulsed with R5-tropic HIV-1– recognition domain of langerin, because it was blocked by mannan + and calcium-chelator EGTA. These data indicated that HSV-2 com- eGFP for 2 h, and subsequently cocultured with CCR5 Jurkat petes with HIV-1 for the langerin-binding site. To investigate this fur- T cells (16). Viral transmission was assessed over time by flow ther, LCs were preincubated with HSV-2 for 15 min, and HIV-1 gp120 cytometry (16). LCs did not efficiently transmit HIV-1 to T cells capture was measured by flow cytometry. The short incubation time (Fig. 5A,5B) (15, 16). Strikingly, UV–HSV-2 and poly(I:C) signif- excludes LC maturation and infection. Notably, preincubation of LCs icantly enhanced HIV-1 transmission by LCs obtained from differ- with HSV-2 strongly reduced binding of HIV-1 gp120 to LCs (Fig. 4B, ent donors (Fig. 5A,5B). Imiquimod marginally enhanced HIV-1 4C), demonstrating that HSV-2 blocks HIV-1 capture by directly com- transmission, but this was not statistically significant, whereas by guest on September 27, 2021 peting with HIV-1 for langerin. Thus, these data showed that HSV-2 CpG-ODN did not affect transmission. prevents HIV-1 capture by langerinbydownregulationoflangerin To investigate whether the increased transmission by HSV-2 and expression, as well as by competition for the langerin-binding site. poly(I:C) was a direct effect on LC function, a bystander effect through activation of keratinocytes or caused by contaminating T cells, LCs were purified from the epidermal single-cell suspen- HSV-2 and poly(I:C) enhance HIV-1 transmission by LCs to sion by CD1a isolation. CD1a+ LCs and the CD1a2 fraction were T cells stimulated with UV–HSV-2 or poly(I:C) overnight and subse- Under steady-state conditions, LCs are refractory to HIV-1 infection quently infected with R5-tropic HIV-1–eGFP for 40 h. Cells through the protective function of langerin (15). However, were washed extensively, CCR5+ Jurkat T cells were added, and

FIGURE 5. HSV-2 and poly(I:C) stimulation of LCs enhances HIV-1 transmission. A and B, The epidermal LC fraction was stimulated for 18 h. Cells were washed extensively and incubated with R5- tropic HIV-1–eGFP for 2 h. Cells were washed, and CCR5+ Jurkat cells were added. Transmission of HIV-1 was assessed over time by flow cytometry. A, Time course of HIV-1 transmission for two different donors. B, Combined results for five donors are depicted. C, CD1a+ LCs and CD1a2 fraction were stimulated for 18 h with poly(I:C) or UV–HSV-2. Cells were washed and incubated with R5-tropic HIV-1–eGFP for 40 h. CCR5+ Jurkat cells were added, and transmission of HIV-1 was assessed over time by flow cytometry. Results are representative for two independent donors. Error bars represent SDs of duplicates. ppp , 0.01; pppp , 0.001. The Journal of Immunology 1639

HIV-1 transmission to T cells was measured at day 6. Strikingly, poly(I:C) and UV–HSV-2 strongly enhanced HIV-1 transmission by CD1a+ LCs, whereas no transmission was observed with the CD1a2 fraction containing keratinocytes and T cells (Fig. 5C). These data strongly suggest that LCs, but neither keratinocytes nor T cells, mediate HIV-1 transmission (16) and that HSV-2 and TLR3 agonist poly(I:C) affect LC function and, thereby, increase HIV-1 transmission to T cells. TNF-a production and trans-infection are not involved in enhanced HIV-1 transmission by HSV-2 and poly(I:C) The proinflammatory cytokine TNF-a enhances HIV-1 replication (16, 34, 35). Therefore, we investigated whether TNF-a production was involved in the enhanced HIV-1 infection of LCs after HSV-2 and poly(I:C) incubation. The epidermal LC fraction was stimulated with HSV-2, UV–HSV-2, or poly(I:C), and the production of TNF-a was measured at different time points. HSV-2 and UV– HSV-2 produced low levels of TNF-a, which increased over time and was concentration dependent. Poly(I:C) induced low levels of Downloaded from TNF-a production (Fig. 6A). To investigate whether TNF-a production was involved in the enhanced HIV-1 transmission by HSV-2 and poly(I:C), a neutralizing Ab against TNF-a was added during the transmission assay (16). Anti–TNF-a Ab did not significantly affect HIV-1 transmission induced by UV–HSV-2 and poly(I:C), a whereas it abrogated TNF- –induced HIV-1 transmission (Fig. 6B). http://www.jimmunol.org/ These data show that TNF-a is not responsible for the observed enhanced HIV-1 transmission by LCs after poly(I:C) or HSV-2 stimulation. Our data strongly suggest that HSV-2 and TLR3 ligands increase transmission of HIV-1 by de novo production of HIV-1 by LCs. It was demonstrated previously that Pam3CSK4-activated LCs (16) and LPS/TNF-a–activated LC-like cells (36) enhance HIV-1 transmission without LC infection, a mechanism known as trans- infection. Although we observed an enhanced replication of HIV-1 in LCs in response to HSV-2 and poly(I:C) (Fig. 3B), we also by guest on September 27, 2021 investigated whether HSV-2 or poly(I:C) could enhance HIV-1 FIGURE 6. Direct HIV-1 infection of LCs, but neither TNF-a production trans-infection. Replication-defective R5-tropic pseudotyped HIV- nor trans-infection, contribute to enhanced HIV-1 transmission by HSV-2 1DENV-eGFP was used to monitor HIV-1 trans-infection. In con- and poly(I:C). A, The epidermal LC fraction was incubated with different trast to Pam3CSK4, neither HSV-2 nor poly(I:C) transmitted concentrations of HSV-2, UV-HSV-2, and poly(I:C) for 6, 12, and 24 h. replication-defective HIV-1 to T cells (Fig. 6C). These data suggest Supernatants were harvested and TNF-a protein production was measured that HSV-2 and TLR-3 agonist poly(I:C) enhance HIV-1 transmis- by ELISA. Error bars represent SDs of duplicates. These results were sion by increasing HIV-1 infection of LCs. observed for three donors. B, The epidermal LC fraction was stimulated for 18 h. Cells were washed extensively and preincubated with anti– TNF-a, followed by incubation with R5-tropic HIV-1–eGFP for 2 h. Discussion CCR5+ Jurkat cells were added, and transmission of HIV-1 was assessed Genital HSV-2infection is highly prevalent in sexually active people over time by flow cytometry. Addition of anti–TNF-a did not alter the worldwide, with a prevalence in the general population ∼20% in the enhanced transmission by HSV-2– and poly(I:C)-stimulated LCs. Error bars United States and 60% in sub-Saharan Africa (37). Epidemiologi- represent SDs of duplicates. C, Replication-defective R5-tropic HIV-1–eGFP cal studies demonstrated that HSV-2 infection increases the risk for was used in the transmission assay. HSV-2 and poly(I:C), in contrast to HIV-1 acquisition, even in the absence of clinical symptoms (1–5). PAM3CSK4, do not transmit HIV-1 via trans-infection. LCs were isolated In this study, we demonstrated that HSV-2 strongly increases from three donors (A, B, and C). Error bars represent SDs of duplicates. pp , ppp , HIV-1 transmission by LCs. Immature LCs form a protective bar- These results were observed for three donors. p 0.01; p 0.001. rier against HIV-1 infection through the C-type lectin langerin (15, 30–32). However, our data suggest that HSV-2 interferes with this anti–HIV-1 function by decreasing langerin expression and saturat- interact with TLR-2, which is expressed by LCs and may be in- ing langerin activity, leading to increased HIV-1 infection of LCs volved in LC maturation (38). However, we previously showed that and, consequently, increased transmission to T cells. These data TLR-1/-2 ligand Pam3CSK4 does not affect langerin expression on show that, in addition to the previously described mechanisms, viral LCs (16). Thus, HSV-2 might also trigger other receptors that in- STIs enhance HIV-1 susceptibility by altering LC function. duce LC maturation. Although epithelial cells and keratinocytes are the primary target A comparison of different viral TLR agonists showed that cells for HSV-2, we demonstrate that LCs are productively infected TLR-3 agonist poly(I:C) was the most potent in inducing LC mat- by HSV-2. Infection with HSV-2 induced LC maturation, with in- uration, as well as decreasing langerin expression, whereas TLR- creased expression of costimulatory molecules but decreased ex- 7/-8 agonist imiquimod had a marginal effect on LC maturation pression of langerin, whereas expression of CD4 and CCR5 was not and langerin expression. As expected, TLR-9 agonist CpG did affected. Maturation was independent of viral replication because not affect LC function because TLR-9 is not expressed by LCs UV–HSV-2 induced a similar maturation. HSV-2 was shown to (29). These data strongly suggest that HSV-2, as well as other 1640 HSV-2 INFECTION OF LCs ENHANCES HIV-1 TRANSMISSION viruses containing TLR-3 ligands, induce LC maturation and affect pathogens or viral TLR agonists hamper langerin expression and langerin expression. function, which increases HIV-1 infection of LCs (15, 16, 36). A Notably, our data show that HSV-2 interacts with langerin. These recent study demonstrated that immune infiltrates persist well after data support a function for langerin as a pattern-recognition receptor the HSV-2 lesion has healed. The increased expression of DC-SIGN (39) for different pathogens, including viruses, such as HIV-1, HSV (DC-specific ICAM-3 grabbing non-integrin) on DCs in these infil- type 1 (data not shown), and HSV-2, fungi, and bacteria (15, 30– trates and T cell influx might contribute to HIV-1 susceptibility (10). 33). The function of langerin as a pattern-recognition receptor is Further studies are necessary to investigate the expression of lan- unclear. HSV binding to langerin might result in more efficient Ag gerin on LCs present in the healed HSV-2 lesions, because pro- processing and presentation (31, 40). Previously, we showed that longed immune activation might lead to a decrease in the antiviral langerin prevents HIV-1 infection of LCs by capturing HIV-1 for function of LCs. degradation (15). Thus, langerin might perform a similar function Collectively, our data support a role for LCs in the enhanced for HSV-2. However, LCs express high levels of proteoglycans, susceptibility to HIV-1 in the presence of HSV-2. Infectious such as syndecan-3 (41), which function as attachment receptors HSV-2 and noninfectious particles abrogate the anti–HIV-1 function for HSV-2 and might facilitate HSV-2 infection, even in the pres- of LCs by inducing LC activation and decreasing langerin expres- ence of langerin. Further studies are necessary to identify the func- sion as well as function. Continuous HSV-2 shedding by mucosal tion of langerin in HSV infection. tissue maintains the presence of HSV-2, which further blocks lan- Langerin is an important C-type lectin on LCs that prevents gerin function and induces the activation of LCs. Furthermore, HIV-1 infection by degrading HIV-1 (15, 42). Previous studies dsRNA viruses that are mimicked by poly(I:C) might also increase showed that inhibition of langerin by a blocking Ab or HIV-1 susceptibility. Our data emphasize the importance of STI Downloaded from a carbohydrate ligand increased HIV-1 infection of LCs (15). Nota- prevention, because it is a risk factor for the acquisition of HIV-1, bly, our data show that HSV-2 and TLR-3 agonist poly(I:C) de- even in the absence of clinical symptoms. creased langerin expression, which prevented efficient HIV-1 binding by LCs. Moreover, HSV-2 competed with HIV-1 for lan- Acknowledgments gerin binding, further hampering the protective function of langerin. We thank the Boerhaave Medisch Centrum (Amsterdam) and Dr. A. Knot- Thus, HSV-2 affects langerin function at different levels, and this tenbelt (Flevoclinic, Almere, The Netherlands) for valuable support. We http://www.jimmunol.org/ prevents HIV-1 capture, leading to increased HIV-1 infection of LCs also thank Dr. G.M. Verjans (Erasmus Medical Center, Rotterdam, The and, consequently, enhanced transmission to T cells. Notably, Netherlands) for HSV strains. We also thank the members of the Host De- HSV-2 replication is not required for the observed increase in fense Group (Academic Medical Center, Amsterdam, The Netherlands) for HIV-1 transmission, suggesting that virus particles present at lesions valuable input. might already affect LC function. Various studies showed that HSV can increase HIV-1 replication at several levels (43–45), and this Disclosures might also affect HIV-1 infection of LCs. However, our data showed The authors have no financial conflicts of interest. that HSV-2 replication is not required, because UV–HSV-2 induced by guest on September 27, 2021 HIV-1 infection and transmission to a similar level as replication- References competent HSV-2. These data suggest that aside from competition 1. Royce, R. A., A. Senã, W. Cates, Jr., and M. S. Cohen. 1997. Sexual transmission of HSV-2 with HIV-1 for langerin binding, innate signaling by pat- of HIV. N. Engl. J. 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