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* Division of Infectious Diseases and Hospital Epidemiology, Department of Internal Medicine and
Institute of Clinical Immunology, University Hospital of Zurich, Zurich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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receptor, IFN-
, MIP-1, -1
, RANTES, and stromal cell-derived factor-1) did not result in the reversal of R-848’s anti-HIV activity. Thus, the nature of this soluble factor(s) remains unknown. Unlike lymphoid tissue acutely infected with HIV, triggering latently infected promonocytic cells induced the release of HIV virions. The anti-HIV effects of triggering TLR7/8 may inhibit rapid killing, while pro-HIV effects may guarantee a certain replication level. Compounds triggering TLR7/8 may be attractive drug candidates to purge latent HIV while preventing new infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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TLR3, 7, 8, and 9 have been implicated in antiviral defense mechanisms (1). They share an endosomal location and the requirement of endosomal acidification for their triggering (5). Only recently, ssRNA (3) from HIV or influenza was found to trigger TLR7 or 8 (6, 7). In particular, ssRNA-activated dendritic cells (DC),3 released cytokines, and increased NF-
B-driven reporter gene activation in HEK 293 cells by TLR7/8 (6, 7). Notably, TLR7 and 8 are expressed selectively on lymphoid cellular subsets: B cells and monocytes express TLR7 and 8 while plasmacytoid DC (PDC) express only TLR7 and CD11c+ immature DC TLR8 (8, 9, 10).
The main target of HIV is the CD4+ T cell, which does not express TLR7/8. Thus, if TLR7/8 triggering affects HIV replication in CD4+ T cells, this can only happen indirectly through changes in the lymphoid microenvironment. In contrast, DC, monocytes, and macrophages are also targets of HIV, and TLR7/8 triggering in these cells culminates in the activation of various host transcription factors such as NF-B (11). NF-B transcription sites are also present in the long-terminal repeat (LTR) of HIV (11). Ironically, NF-B is critical for the transcription of most immune response genes, including antiviral and proinflammatory cytokines (12).
Thus, the ability of HIV ssRNA to stimulate TLR7/8 raises critical questions about HIV pathogenesis. Is TLR7/8 triggering critical for HIV’s replication or for an effective immune response? What is the role of TLR7/8 triggering in cells of the myeloid-macrophage lineage and in particular latently infected cells?
We examined the role of TLR7/8 triggering in productive HIV infection in PBMC and human aggregate lymphocyte cultures (HLAC) infected ex vivo with HIV. We used the prototype promonocytic cell lines, U1 and OM10, for investigating the effects of TLR7/8 signaling on latent HIV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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PBMC from random donors were isolated by Ficoll-Hypaque density gradient centrifugation (Nycomed) and cultured at 10 x 106 cells/ml in RPMI 1640 (BioWhittaker) with 10% (by volume) FCS (PAA Laboratories), 100 U/ml penicillin and 100 µg/ml streptomycin (P/S; Invitrogen Life Technologies), 2 mM glutamine (Invitrogen Life Technologies), and 10 U/ml IL-2 (National Institutes of Health AIDS Repository). For generating monocyte-derived macrophages (MDM), we isolated monocytes using CD14 microbeads (Miltenyi Biotec). Subsequently, 5 x 104 monocytes/well of a 96-well plate were kept in RPMI 1640 (BE 12-702F; BioWhittaker) with 5% FCS, 5% human AB serum (H1513, Sigma-Aldrich), 1% glutamine, and 1% penicillin-streptomycin for 1 wk until differentiation into MDM. A2.01 cells expressing CD4 (A2.01/CD4+) were cultivated in the same medium as PBMC. U1 and OM10 cells were cultured in RPMI 1640 supplemented with 10% FCS, penicillin, streptomycin, and glutamine. The B cell line Namalwa was cultivated in RPMI 1640, 15% FCS, 1 mM sodium pyruvate, 1 mM nonessential amino acids, and 1% P/S. R-848 (InvivoGen) was used mainly at 1 µg/ml (5, 13, 14), polyinosic:polycytidylic acid (poly(I:C); Amersham Biosciences) at 20 µg/ml, flagellin from Salmonella typhimurium (provided by J. C. Sirard (Institut de Biologie de Lille, Université E0364 1, Lille, France)) at 100 µg/ml, peptidoglycan from Staphylococcus aureus (Fluka) at 10 µg/ml and LPS (Escherichia coli K-235; Sigma-Aldrich) at 20 µg/ml. Sequences of phosphothioate-modified ssRNA (i.e., ssRNA40, 41, and 42) were identical to those published by Heil et al. (6); in particular the RNA40 is a 20 mer identical to the U5 region from 108 to 127; RNA41 and 42 are 20 mer where all U or G nucleotides were replaced with adenosine, respectively (IBA). For complexing ssRNA, we used the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) (Roche) as described by Heil et al. (6). Neutralizing Abs (nAbs) against the receptor of IFN- were used at 1 µg/ml (clone: MMHAR-2; PBL Biomedical Laboratories), against IFN- at 100 ng/ml (no. ab9657; Abcam), MIP-1 at 4 µg/ml (no. ab10381; Abcam), MIP-1 at 3 µg/ml (no. PP1051P; Acris), RANTES at 7 µg/ml (no. 9679; Abcam), and stromal cell-derived factor (SDF)-1 at 5 µg/ml (no. PP11067P1; Acris).
Measurement of metabolic activity
The WST-1 kit (Roche Diagnostics) was used according to the manufacturer’s instructions.
Lymphoid tissue
Tonsils from otherwise healthy adult patients were obtained from the Department of the Ear, Nose, and Throat Surgery at the University Hospital of Zurich (Zurich, Switzerland) within 5 h after tonsillectomy. The use of tonsils was approved by the local ethical committee of the USZ. HLAC were prepared by transferring minced tissue into a cell strainer (70 µm; Falcon; BD Biosciences) and grinding the tissue through the sieve with a syringe plunger.
Erythrocytes were lysed with ACK cell-lysing buffer (BioWhittaker). Lymphoid cells were cultured at a concentration of 107 cells/ml in RPMI 1640 containing 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% nonessential amino acids. Viability was assessed by trypan blue exclusion.
Purification of cellular subsets from HLAC and PBMC
We depleted specific cellular subsets from PBMC with Abs coupled to MACS beads, according to the manufacturer’s instructions (CD14–, CD19–, and BDCA-4 Microbeads; Miltenyi Biotec).
Viruses
Viral stocks were obtained by calcium phosphate-mediated transfection (Promega) of 293T cells with pNL4-3, pYU-2, pJR-CSF, and p89.6 (National Institutes of Health AIDS Repository) or p49.5. Virus was harvested 48 h after transfection, filtered (0.22 µm), and frozen at –80°C.
HIV p24 capsid Ag (p24) ELISA
A twin-site sandwich ELISA was performed essentially as described (15). Briefly, a polyclonal Ab was adsorbed to a solid phase to capture p24 Ag from a detergent lysate of virions. Bound p24 was visualized with an alkaline phosphatase-conjugated anti-p24 mAb and luminescent detection system.
Infectivity assays
All infectivity assays with HLAC or PBMC were performed in triplicate in 96-well round-bottom plates. In most assays, R-848 was added 2 days before HIV infection and was partially replenished by substituting 50 µl of medium containing 1 µg/ml R-848 twice a week over the observation period. Cultures were exposed to HIV with p24 Ag in the range of 1–3 ng/sample for 4–6 h. Subsequently, the cultures were washed three times and resuspended in 200 µl of fresh medium containing R-848. In a subset of experiments, R-848 was added only either 2 days before HIV infection, immediately upon infection, or 4 days after infection. Supernatants were tested for p24. In the assays examining the impact of selective cytokines, nAb against either IFN-, , MIP-1, -1, RANTES, or SDF-1 was added simultaneously with R-848 and partially replenished twice a week. For interdonor comparisons, we expressed the p24 values over time as the area under the curve (AUC) representing HIV replication. We calculated the percent inhibition of replication by any given drug and concentration by first expressing the AUC of treated cultures as a percentage of an untreated, infected control culture and then subtracting the percent of AUCtreated samples from 100% (16).
In the infectivity assays examining R-848’s ability to stimulate the B cell line, Namalwa, and its ability to affect HIV replication when subsequently cocultured with T cells, Namalwa cells at a density of 5 x 104 cells/well of a 96-well plate were pretreated with R-848 for 2 days. Subsequently, the same number of A2.01/CD4+ cells was added to the B cells. Cocultures were infected with NL4-3 for 6 h and thereafter washed three times; HIV replication was quantified by p24 Ag in the supernatant at day 5.
Cytokine ELISA
IL-6, -8, -12, TNF-, RANTES, MIP-1, and - were quantified by enzyme immunoassays (R&D Systems), according to the manufacturer’s instructions. IFN- and - were quantified by enzyme immunoassays from Bender MedSystems Diagnostics and HyCult Biotechnology, respectively.
Immunostaining and flow cytometry
Cells were simultaneously stained with mAbs (BD Biosciences) against the cell surface markers CD4-phycoerytherin, CD8-FITC, or CD4–, CD8–, and CXCR4-Cy or CCR5-allophycocyanin together. For staining B cells, we used the mAb CD20. For staining PDC, we used the lineage mixture 1, CD123-PE, HLA-DR-PerCP and CD11c-allophycocyanin. Flow cytometry was performed on a FACSCalibur (BD Biosciences), and data were analyzed with CellQuest or FlowJo software (Tree Star).
Cell-based fusion assay
We used a cell-based fusion assay with HeLa cells expressing gp140 from LAI and the HIV transactivator tat (HeLa-Env/LAI) (17) and HeLa cells expressing CD4 and the LTR-driven lacZ gene (HeLa SX CCR5) (18). Fusion of HeLa-Env/LAI cells and HeLa SX CCR5 results in transcription of the lacZ gene. The extent of fusion was quantified either by assaying -galactosidase activity in cell lysates (E2000; Promega) or by histochemical staining of cells for -galactosidase activity (Roche Molecular Biochemicals), according to the manufacturer’s instructions. HeLa-Env/LAI cells were treated with R-848 at 1 µg/ml or the fusion inhibitor T20 at 50 µg/ml for 1 h (19). Subsequently, a similar number of HeLa SX CCR5 cells were added to allow fusion to take place. -Galactosidase activity was assessed 12 h later.
Pseudotype virus preparation and challenge of cells with pseudotype viruses
To prepare HIV pseudotype virus packaged by vesicular stomatitis virus (VSV) Env, an HIV proviral construct encoding a luciferase reporter gene (pNL4-3.Luc.R–E–) was cotransfected with a VSV Env expression vector in 293T cells as described (20). To determine permissiveness to entry by pseudotype viruses, HLAC were pretreated with R-848 for 2 days and exposed to pseudotype virus for 6 h. For increased sensitivity of the readout, in six of eight experiments, PHA (Sigma-Aldrich) at 5 µg/ml was added 1 day after infection. PHA acts by immune activation of cells (21) and thereby cooperates with Tat-driven LTR activity resulting in the increased expression of the reporter gene. Luciferase expression was quantified with the luciferase assay system from Promega.
Quantitative PCR (qPCR) for measuring TLR7 and 8 mRNA in various cellular subsets
qPCR was performed as described (22). In brief, for measuring TLR7 and 8 mRNA, we used commercially available primers and probes (Assays-on-demand; Applied Biosystems). Hydroxymethyl bilane synthase (GenBank X04217) was used as a housekeeping gene and designed as a 3' minus groove binder probe (23). Data generated by real-time qPCR were analyzed by determining the mean normalized gene expression for every sample with the software application Q-Gene (calculation procedure 2 for mean normalized gene expression) (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Untreated tonsillar HLAC and IL-2-cultured PBMC uniformly showed vigorous viral replication when exposed to HIV as quantified by p24 Ag in the supernatant (Fig. 1A). Pretreatment of PBMC or HLAC with 10 µg/ml HIV ssRNA 40 complexed to DOTAP or 1 µg/ml R-848 for 1–2 days rendered the cell cultures virtually nonpermissive to HIV (Fig. 1, B and C). ssRNA not complexed or the control ssRNAs 41 or 42 had no antiviral activity (Fig. 1B and data not shown). To compare interindividual experiments, we calculated the percent inhibition of HIV replication over time as compared with untreated HIV-infected cultures. We observed a decrease of HIV replication of >75%, irrespective of HIV coreceptor selectivity (i.e., infection with the primary CCR5-tropic (R5) isolate JR-CSF, the dual-tropic strain 89.6, or the laboratory strains 49.5 (R5) and the CXCR4-tropic (X4) NL4-3).
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We wondered whether triggering other TLR would also block HIV replication in HLAC. Peptidoglycan (TLR2), poly(I:C) (TLR3), LPS (TLR4), and flagellin (TLR5) had only modest anti-HIV activities when HLAC were exposed to the R5-tropic strain 49.5 (Fig. 1D). Poly(I:C) and flagellin also inhibited the X4-tropic strain. However, peptidoglycan and LPS showed no effect. Thus, the robust anti-HIV activity is specific for TLR7/8 and not a uniform phenomenon of TLR triggering.
TLR7/8 triggering causes no adverse metabolic effects
To exclude a cytopathic effect of TLR7/8 triggering that could explain the observed anti-HIV activity, we examined the effects of R-848 treatment on the metabolic activity of HLAC. For this purpose, we used a colorimetric assay (WST-1 kit), which is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. On day 7, no differences in metabolic activity were observed between cultures either treated with R-848 or not (mean ± SEM of untreated vs R-848-treated, respectively, 0.27 ± 0.04 and 0.28 ± 0.07, n = 6; NS, Wilcoxon signed rank test).
B cells are key to the anti-HIV activity of TLR7/8 triggering
We wondered what cellular population from primary lymphoid tissue mediates the TLR7/8-dependent anti-HIV effects. For this purpose, we selectively depleted B cells, monocytes, or PDC from PBMC. As measured by flow cytometry, the numbers of these cells were reduced by >95%. Depleting PDC or monocytes did not reverse the observed anti-HIV effects; however, depleting B cells did (Fig. 2, A–C).
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Soluble factors may mediate the anti-HIV activity of triggering TLR7/8
To explore whether soluble factors were responsible for the anti-HIV effects observed, HeLa CD4/R5 cells were infected with the R5-tropic HIV strain 49.5 and subsequently treated with R-848-conditioned medium from HLAC; indeed, this conditioned medium inhibited HIV replication substantially (Fig. 3A). R-848 added directly to HeLa-CD4/R5 cells after infection had no antiviral activity.
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To determine the effects of triggering TLR7/8 on the cellular microenvironment, the cytokine expression patterns in HLAC were examined at days 1 and 2 after R-848 treatment (Table I). The cytokine profile changed substantially: increases were noted in the levels of secretion of the proinflammatory cytokines IL-6 and TNF-, of the innate immunity marker IFN-, and of Th1 cytokines IFN- and IL-12. Levels of the chemotactic cytokines RANTES and MIP-1 and -1 were also increased.
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receptor nor against IFN-, MIP-1, MIP-1, RANTES, or SDF-1
To explore whether the anti-HIV effects by triggering TLR7/8 is due to the release of IFN-, IFN-, MIP-1, -1, RANTES, or SDF-1, we added nAb against the IFN- receptor or those cytokines/chemokines. Blocking of the IFN- receptor site or the cytokines/chemokines did not reverse the effects subsequent to TLR7/8 triggering (Fig. 3B). As expected, blocking IFN- by adding nAb against its receptor, without triggering TLR7/8 simultaneously, resulted in increased HIV replication; a similar but less prominent stimulation of HIV replication was observed when nAb against RANTES was added (Fig. 3C).
Triggering TLR7/8 blocks HIV after entry and before integration
We next sought to identify the step at which TLR7/8 triggering interferes with the HIV replication cycle. First, we investigated the expression level of the viral receptor complex of the cell surface molecules CD4 and either CCR5 or CXCR4. Untreated cultures contained 2.7 ± 0.8% of CD4+/CCR5+ cells, while R-848-treated cultures had 0.7 ± 0.4% of CD4+/CCR5+ cells (mean + STD; n = 4). The corresponding values for CXCR4 are 93.1 ± 2.7% CD4+/CXCR4+ cells in untreated, and 91 ± 3.1% CD4+/CXCR4+ cells in R-848-treated HLAC.
Next, we looked at the fusion of HIV and the cell membrane with a cell-cell fusion-based assay. As determined either histochemically or by measuring -galactosidase activity in cell lysates, R-848 did not affect syncytium formation (Fig. 4, A and B). The fusion inhibitor Fuzeon was used as a positive control for blocking syncytium formation.
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Dose- and time-dependent inhibition of HIV replication
In most experiments, R-848 levels were maintained in the culture medium throughout the entire observation period. However, we wondered whether R-848’s anti-HIV effect relies on an altered microenvironment or direct viral inhibition. To answer this question, we compared the results of adding R-848 at different time points. Adding R-848 only before or after exposing HLAC to HIV as well as continuous treatment inhibited viral replication (Fig. 5A). In contrast, R-848 added 4 days after HIV infection tended to be somewhat less effective. Our results indicate that an altered microenvironment is critical for the anti-HIV effects observed.
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Triggering TLR7/8 induces the release of HIV virions from latently infected monocytoid cell lines
TLR7/8 triggering culminates in the activation of NF-B (6). Because the HIV LTR contains NF-B transcription sites, activation of NF-B results in increased LTR-dependent transcription and increased HIV replication (25). Thus, we wondered whether TLR7/8 triggering reactivates latent HIV infection. We examined this question in the promonocytic latently infected cell lines U1 and OM10 (26, 27, 28, 29). Both cell lines express TLR7 and 8 as quantified by qPCR (Fig. 6A). Primary cells of myeloid-monocytic origin such as monocytes and MDM have even higher levels of TLR7/8 mRNA levels (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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HIV ssRNA rendered human primary lymphoid tissue barely permissive to HIV replication. The guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides, ssRNA40, derived from HIV (6), which induced the release of cytokines, was the most potent ssRNA. In contrast, the ssRNA41 and ssRNA42, in which all U’s or G’s were replaced with adenosine, were not or were only marginally anti-HIV. Consistent with published work, only ssRNA complexed to cationic lipids, which facilitate its cellular uptake, was anti-HIV active (6).
R-848 also triggers TLR7/8, and for practical reasons, we used it instead of ssRNA in the subsequent experiments (6). R-848 reduced HIV replication in a manner similar to HIV ssRNA. The antiviral state observed was independent of the origin of the lymphoid tissue or coreceptor preference of the virus. We have previously demonstrated that triggering TLR9 was also anti-HIV active (30). In contrast, we found that other TLR ligands, such as peptidoglycan (TLR2), poly(I:C) (TLR3), LPS (TLR4), or flagellin (TLR5), had only modest anti-HIV effects at best. Thus, triggering TLR7/8 or 9 unfavorably changes the microenvironment for HIV replication; the failure to induce an antiviral state after stimulation with other TLR ligands points to specific anti-HIV effects by triggering TLR7/8 or 9 and rules out a common biologic property of TLR triggering.
We wondered what cellular subset was responsible for the antiviral state induced. PDC are the most potent IFN--producing cells (31), and IFN- has strong antiviral activity (32). However, depleting PDC from lymphoid tissue did not reverse the anti-HIV effects observed. Similarly, depleting monocytes did not change the anti-HIV activity of TLR7/8 triggering. In contrast, depleting B cells resulted in significant loss of anti-HIV activity. The critical role of B cells was corroborated by data showing that treatment of the B cell line, Namalwa, with R-848 prevented HIV replication in CD4+ T cell lines. These data unequivocally indicate that B cells have an unexpected role in the innate immunity for constraining viral infection. The variability in these experiments may be explained by differences in genetic host factors. Alternatively, we have to consider that the induction of antiviral effects observed requires the cooperative action of more than one cell type which shows distinct responsiveness to TLR7/8 triggering. For example, a two-step recognition mechanism by TLR involving epithelial cells and DC is critical for the induction of effector T cells in a mouse model infected by HSV (33). Certainly, additional studies of this intriguing observation are warranted.
The antiviral activity is, at least partially, due to soluble factors. R-848-conditioned medium from HLAC decreased HIV replication in indicator cells and induced an array of cytokines. Some of these cytokines, such as IFN-, have antiviral activity. Others, such as TNF- and IL-6, stimulate HIV activity (22, 34). Thus, it is highly unlikely that one antiviral cytokine is responsible for these effects. Indeed, we previously showed that rIFN- only modestly affected HIV replication in this experimental setting and, thus, was insufficient to explain the striking effects of TLR7/8 triggering (30). Moreover, nAb against the IFN- receptor or IFN-, MIP-1, -1, RANTES, or SDF-1 did not reverse the observed anti-HIV effect. Therefore, the sum of cytokine changes or a yet unidentified factor is likely responsible for the potent anti-HIV activity observed.
Triggering TLR7/8 blocks HIV infection after entry. In particular, TLR7/8 triggering resulted in a consistent reduction of the expression of HIV coreceptors CCR5 and CXCR4 on CD4+ T cells. Because the basal expression level of CCR5 is low, its relative reduction was more pronounced than for CXCR4. Chemokine receptor expression level, and in particular expression of CCR5, is a determinant of HIV infectability (35). To further explore the block in HIV replication, we used VSV Env-pseudotyped HIV strains encoding a luciferase-reporter gene. VSV Env-pseudotyped viruses circumvent the HIV entry step. We found that luciferase expression was decreased when HLAC were pretreated with R-848. Thus, while reduced CCR5 expression may contribute to R-848’s anti-HIV activity, the experiment using VSV-pseudotype viruses clearly demonstrate that TLR7/8 triggering interferes with HIV replication after virus-cell fusion but before viral integration into the host gene. We excluded a nonspecific blocking of the fusion process by R-848 as a mechanism for the antiviral state using a cell-cell fusion-based assay.
To more carefully examine R-848’s potency, we titrated it by its anti-HIV activity. Although there was a dose-dependent loss of R-848’s anti-HIV activity, a marked decrease in its activity was only observed when it was used at <0.1 µg/ml. The lowest concentration at which R-848 displays its anti-HIV activity is similar to the concentration at which it induces the release of cytokines (36). In animal models, 0.1 µg/ml R-848 protected against established infection with Herpes simplex (37) and enhanced clearance of Mycobacterium bovis (38). Thus, taking into account the wide therapeutic activity it displays in vitro against HIV, R-848 likely displays anti-HIV effects in vivo at similar concentrations.
R-848 added before HIV infection was only slightly less active against HIV than when it was added throughout the entire culture period. Thus, triggering TLR7/8 seems to induce an antiviral state that remains even after limited exposure of tissue to R-848. In contrast, triggering TLR7/8 with R-848 4 days after HIV infection resulted in only modest anti-HIV effects. The subsequent observation period may be too short to appreciate the full biologic effects of TLR7/8 activity, or key cells responding to R-848 may have lost their reactivity over time in this in vitro setting. Alternatively, we speculate that triggering TLR7/8 of HIV-infected cells of the myeloid-macrophage lineage activates HIV and thus masks the anti-HIV effects.
To further explore the effects on viral latency, we used the latently infected cell lines U1 and OM10. These cell lines express TLR7 and to a lesser extent TLR8. Triggering TLR7/8 resulted in a dramatic increase of HIV replication. Monocytes and MDM have a higher expression of TLR7 and 8 than the U1 or OM-10 cells; thus, it is highly likely that TLR7/8 triggering of latently infected primary cells of monocytic-myeloid origin will result in the increased release of HIV in vivo. Thus, TLR7/8 triggering may have a dichotomous effect in HIV infection: it may prevent infection of CD4+ T cells while it activates HIV replication in cells of the myeloid-macrophage lineage. Although at first seeming contradictory, these two opposite activities may be beneficial to HIV. They may block overwhelming infection and destruction of the host while simultaneously guaranteeing continuous HIV replication from cells of the myeloid-macrophage lineage. Notably, cells of the myeloid-macrophage lineage are less susceptible to the direct cytopathic effects of HIV.
In conclusion, TLR7/8 triggering inhibits HIV replication in unfractionated lymphoid tissue. In contrast, it induces the release of HIV virions in latently infected cells. These surprisingly different actions may play a critical role in acute and chronic HIV infections. In acute HIV infection, the antiviral state induced by TLR7/8 may restrict overwhelming HIV replication until the generation of an adaptive immune response. In chronic HIV infection, the anti-HIV effect of triggering TLR7/8 may still restrict overwhelming replication, whereas the pro-HIV effect guarantees a certain level of replication that does not kill the host too rapidly. Thus, HIV is able to exploit TLR7/8 triggering for its own purposes. However, the dichotomous effects of TLR7/8 signaling may be of therapeutic value. It may be possible to purge latently infected cells while preventing new infections (39). Notably, TLR signaling links the innate and adaptive immune response (40). Thus, similar to protocols investigating GM-CSF (41), we propose to exploit immune response modifiers targeting TLR7/8 in HIV-infected patients (42).
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the "Stiftung für wissenschaftliche Forschung an der Universität Zürich." 
2 Address correspondence and reprint requests to Dr. Roberto F. Speck, Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, Raemistrasse 100, Rae U 76, CH-8091 Zurich, Switzerland. E-mail address: roberto.speck{at}usz.ch
3 Abbreviations used in this paper: DC, dendritic cell; PDC, plasmacytoid DC; LTR, long terminal repeat; HLAC, human aggregate lymphocyte culture; MDM, monocyte-derived macrophage; poly(I:C), polyinosic:polycytidylic acid; nAb, neutralizing Ab; SDF, stromal cell-derived factor; AUC, area under the curve; VSV, vesicular stomatitis virus; qPCR, quantitative PCR; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate.
Received for publication September 14, 2005. Accepted for publication December 15, 2005.
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References |
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 K. Hattermann, S. Picard, M. Borgeat, P. Leclerc, M. Pouliot, and P. Borgeat The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis FASEB J, May 1, 2007; 21(7): 1575 - 1585. [Abstract] [Full Text] [PDF]
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, untreated; 
, R-848-treated lymphoid tissue). B, Human PBMC were treated with ssRNA40, -41, or -42 complexed to the cationic lipid DOTAP and subsequently infected with NL4-3 (n = 3). HIV replication was monitored twice weekly by quantifying p24 in the supernatant. C, HLAC of tonsillar origin (
) or PBMC (
) were treated with R-848 and subsequently infected with HIV strains with distinct coreceptor selectivity (HLAC infected with 49.5 (n = 12), NL4-3 (n = 15), JR-CSF (n = 2), or 89.6 (n = 2); PBMC infected with 49.5 (n = 5) or NL4-3 (n = 4). 49.5 and JR-CSF are CCR5-tropic, NL4-3 is CXCR4-tropic and 89.6 dual-tropic). D, HLAC were treated with peptidoglycan (TLR2), poly(I:C) (TLR3), LPS (TLR4), or flagellin (TLR5), and subsequently infected with 49.5 or NL4-3 (n = 6 for poly(I:C), n = 2 for peptidoglycan, LPS and flagellin). B–D, Data are represented as HIV replication over time after R-848 treatment as related to untreated and infected control culture. Data are presented as mean ± SD.
) was measured by qPCR. Data presented as mean normalized gene expression (MNE). B–E, R-848 was added to U1 (B and C) or OM10 cells (D and E), and HIV replication was monitored either by quantifying p24 in the supernatant (B and D; n = 3 for U1; n = 4 for OM10) or by staining for intracellular p24 by mAb and subsequently analysis by flow cytometry (C and E; n = 2). (R-848