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AIDS:
doi: 10.1097/QAD.0b013e3282f4de35
Basic Science

Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection

Lester, Richard Ta,b; Yao, Xiao-Danc; Ball, T Blakea; McKinnon, Lyle Ra; Kaul, Rupertd; Wachihi, Charlesb; Jaoko, Walterb; Plummer, Francis Aa; Rosenthal, Kenneth Lc

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Author Information

From the aDepartment of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada

bDepartment of Medical Microbiology, University of Nairobi, Nairobi, Kenya

cDepartment of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

dDepartment of Medicine, University Health Network and University of Toronto, Toronto, Ontario, Canada.

Received 31 August, 2007

Revised 22 November, 2007

Accepted 27 November, 2007

Correspondence to Richard Lester, MD, FRCP(C), PO Box 19676, 00202, University of Nairobi, Nairobi, Kenya. Tel: +254 20 2713139; fax: +254 20 278860; e-mail: rlester@csrtkenya.org

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Abstract

Objectives: Toll-like receptors (TLR) are important in pathogen recognition and may play a role in HIV disease. We evaluated the effect of chronic untreated and treated HIV-1 infection on systemic TLR expression and TLR signalling.

Methods: Two hundred HIV-infected and uninfected women from a Kenya cohort participated in the studies. TLR1 to TLR10 messenger RNA expression was determined by quantitative reverse transcriptase polymerase chain reaction in peripheral blood mononuclear cells (PBMC). TLR ligand responsiveness was determined in or using ex-vivo PBMC by cytokine production in culture supernatants.

Results: Chronic, untreated HIV-1 infection was significantly associated with increased mRNA expression of TLR6, TLR7, and TLR8 and when analysis was limited to those with advanced disease (CD4 cell count < 200 cells/ml) TLR2, TLR3, and TLR4 were additionally elevated. TLR expression correlated with the plasma HIV-RNA load, which was significant for TLR6 and TLR7. In vitro HIV single-stranded RNA alone could enhance TLR mRNA expression. PBMC of HIV-infected subjects also demonstrated profoundly increased proinflammatory responsiveness to TLR ligands, suggesting sensitization of TLR signalling in HIV. Finally, viral suppression by HAART was associated with a normalization of TLR levels.

Conclusion: Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.

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Introduction

Widespread dysregulation of the adaptive immune system has been identified as a hallmark of progressive HIV disease, yet much less is known about the effects of HIV on the innate immune system. In addition to high levels of viraemia, CD4 lymphocyte depletion, and the development of opportunistic disease, hypersystemic immune activation accurately predicts disease progression [1,2] and has therefore been implicated in HIV pathogenesis [3,4]. HIV infection has long been associated with elevated proinflammatory cytokines systemically [5,6], increased T-cell turnover [7], polyclonal B-cell activation [8], the expression of lymphocyte activation markers [9], and more recently the maturation of antigen presenting cells [10–12]. As the innate immune system maintains tight control over inflammation [13], it has recently been implicated as a potential mechanism or driving force of HIV-associated immune activation. In addition, the handling of opportunistic infections and other diseases associated with HIV infection may be influenced by innate immune function [14].

Toll-like receptors (TLR) are prototypical innate sensing molecules, known as pattern-recognition receptors, expressed by immune cells [15] and may thus play a role in HIV disease [14]. They can sense pathogens or ‘danger’, trigger immediate innate antipathogen responses, and guide the development of adaptive immunity [16,17]. There are currently 10 known functional human TLR [18]. Each recognizes broad classes of molecular structures common to groups of microorganisms or endogenous danger signals such as certain lipids or lipopeptides [19], proteins [20], and nucleic acids [21]. HIV may signal directly through TLR that recognize HIV-RNA sequences [22,23], or indirectly by altering the immune milieu and by exposing the host to co-infecting microbes that signal through alternative TLR [24]. ‘Microbial translocation’ of gastroenteric flora or its components such as lipopolysaccharide into the circulation has also been described as a cause of systemic immune activation in HIV [25], and probably signals through TLR. Studies to evaluate TLR signalling specifically in regards to HIV have been limited, but are emerging. One study showed an increased surface expression of TLR2 on monocytes of HIV-infected subjects, and enhanced tumour necrosis factor alpha (TNF-α) responses to TLR2 ligand [26]. Another showed increased TLR3 expression in lymph nodes of SIV-infected macaques [27]. Others have reported altered TLR9 signalling in HIV-infected innate immune cells [28,29] and that stimulation of certain TLR may affect HIV replication in vitro [30–37]. Clinical studies have reported associations between HIV disease progression and polymorphisms in TLR9 in humans [38], and with levels of soluble TLR2 [39].

The goal of this study was to assess the effects of HIV infection broadly on systemic TLR expression in a functional context. We compared the expression of all 10 functional human TLR in peripheral blood mononuclear cells (PBMC) in a large cohort of HIV-infected and uninfected individuals. We also evaluated the capacity of TLR ligands to elicit cytokine responses from HIV infected subjects in vitro. We found that chronic, viraemic HIV infection is associated with substantial changes in TLR expression and innate signalling, which may exacerbate immune activation and pathogenesis.

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Methods

Human subjects

HIV-1-infected and HIV-uninfected women (N = 200) were recruited from two clinics in Nairobi, Kenya [40,41] for the various studies. All HIV-infected subjects were in the chronic phase of infection as determined by at least two positive HIV-1 serological tests separated by at least one year. Subjects were defined as ‘chronic’ if their CD4 T-cell counts were greater than 200 cells/μl and having advanced disease or ‘AIDS’ if their CD4 T-cell counts were less than 200 cells/μl. Free antiretroviral therapy had recently been provided and these subjects (N = 19) were analysed separately from other infected subjects. All subjects were sampled during voluntary ‘healthy’ research visits as per the cohort protocol and therefore were not acutely ill at the time of assessment. Each substudy (TLR expression and function) had independent sampling.

Informed consent for collection of demographics, behavioural data, and biological samples was obtained from all study participants. The study was approved by the institutional review boards of the University of Manitoba, and the University of Nairobi through the Kenyatta National Hospital ethical review committee. All clinical investigation was conducted according to the principles of the Helsinki Declaration.

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Sample acquisition and preparation

Peripheral blood was collected by venipuncture into heparin vacutainers for immunology assays, or ethylenediamine tetraacetic acid-containing vacutainers for cytometry and viral load assays. Routine CD4 T-cell assays were performed on all subjects using flow cytometry (FACScan, BD Biosciences, San Jose, California, USA). Plasma viral loads (branched DNA; Roche Diagnostic Systems, Inc., Branchburg, New Jersey, USA) were batch performed on select subjects according to shared cohort research protocols. PBMC were isolated by density gradient centrifugation as previously described [42]. Aliquots of fresh PBMC suspended in RPMI, containing approximately 1 × 106 cells were placed directly in 1.0 ml Trizol and frozen in liquid nitrogen for storage and shipping to laboratories at McMaster University, Canada, where quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) was performed. PBMC were then enumerated and suspended in RPMI media containing 10% heat-inactivated fetal calf serum and penicillin and streptomycin antibiotics for TLR ligand stimulation studies, as described below.

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Quantitative reverse-transcriptase real-time polymerase chain reaction

Total RNA was extracted from Trizol samples as per the manufacturer (Invitrogen, Carlsbad, California, USA) and treated with DNase I, DNA-free (Ambion, Austin, Texas, USA). Reverse transcription (RT) reactions were set up by adding 1 ug DNA-free total RNA to a 40 μl final volume containing 12.5 ng random primers, 0.5 mmol deoxyribonucleotide triphosphate, 1 × first strand buffer, 5 mmol dithiothreitol, 40 units of RNase inhibitor (Ambion) and 200 units of SuperScript III reverse transcriptase (Invitrogen). RT was conducted with incubations of 25°C 5 min, 50°C 50 min and 70°C 15 min. cDNA converted in RT was diluted to fourfold and 400-fold, respectively, for qRT–PCR of expressing genes and internal control 18S ribosomal RNA. TaqMan primers and probes were designed using the program, Primer Express 1.5 (Applied Biosystems, Foster City, California, USA), and selected following the TaqMan rules of Applied Biosystems. qRT–PCR, with one RT sample in duplicate, was performed in 25 μl of reaction containing 5 μl of diluted complimentary DNA, 500 nmol forward primer, 500 nmol reverse primer (Mobix; University of McMaster, Ontario, Canada), 200 nmol probe (5′-6FAM and 3′–MGBNFQ-labelled; Applied Biosystems) and 1 × universal master (AB, Roche) in a 96-well plate with a sequencing detection system 7900HT (Applied Biosystems). The pooled RT from human PBMC and some human cell lines with total RNA extracted in the same way were 10-series of dilution as qRT–PCR standards. The readout of data was a ratio of a gene quantity to its 18S rRNA quantity, defined as relative expression.

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In-vitro Toll-like receptor stimulations

Freshly isolated PBMC were cultured at 1 × 106 cells/ml in RPMI and either 0.1 μg/ml lipopolysaccharide (InvivoGen, San Diego, California, USA), 1 Mmol ssRNA40 (InvivoGen), or media alone (RPMI and 10% fetal calf serum). Culture supernatants were diluted 10-fold and assayed for cytokines using cytometric bead array (BD Biosciences) on a FACScan flow cytometer (BD Biosciences) as per the company protocol. Cell pellets were immediately frozen in Trizol for quantitative polymerase chain reaction studies.

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Statistical analysis

Data were plotted and analysed using Microsoft Excel, SPSS, and Prism 4.0 software (GraphPad Software, San Diego, California, USA). Non-parametric tests were used including Mann–Whitney U-tests for unmatched comparisons, Wilcoxon paired t-tests for matched samples, and Spearman's rank test for matched correlations.

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Results

Multiple Toll-like receptors are increased in chronic untreated HIV infection

To determine the influence of untreated chronic HIV infection and disease stage on TLR expression in the systemic immune system, we measured the expression of TLR1–TLR10 by qRT–PCR in PBMC from antiretroviral-naive HIV-infected (n = 47) and uninfected (n = 90) subjects. To assess the disease stage, HIV-infected subjects were divided into those with CD4 T-cell counts greater than 200 cells/μl (chronic) or less than 200 cells/μl (AIDS; Fig. 1). The mRNA expression of TLR1, 5, 9, and 10 were equivalent between HIV-infected and uninfected subjects regardless of disease stage. The expression of TLR6, TLR7, and TLR8 was, however, significantly increased in HIV-infected subjects with CD4 T-cell counts greater than 200 cells/μl compared with uninfected subjects (P = 0.0036, P < 0.0001, and P = 0.0114, respectively). The mRNA of these TLR was elevated in the setting of chronic stable HIV infection. In subjects with AIDS, determined by CD4 T-cell counts less than 200 cells/μl, TLR6, TLR7 and TLR8 were again significantly increased compared with uninfected subjects (P = 0.0005, P = 0.0018, and P = 0.0042, respectively), and also increased compared with subjects with chronic stable infection, which was statistically significant for TLR6 (P = 0.0162). In addition, the expression of TLR2, TLR3, and TLR4 was significantly increased in subjects with AIDS compared both with uninfected subjects (P = 0.0134, P = 0.0012, and P = 0.0178, respectively) and those with chronic stable infection (P = 0.0102, P = 0.0026, and P = 0.0362), but no difference was seen between chronic infection without AIDS and uninfected controls for these TLR. Taken together, increased expression of TLR6, 7, and 8 in PBMC appears to be associated with HIV infection including chronic disease before and including AIDS, whereas increased expression of TLR2, 3, and 4 occurs primarily in advanced stages of infection or AIDS.

Fig. 1
Fig. 1
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Toll-like receptor expression associated with HIV-1 viraemia

We next determined the association between plasma HIV-RNA levels and TLR expression in HIV-infected subjects. Each TLR that was overexpressed in either chronic stable HIV infection or AIDS was correlated with matched plasma viral loads, which were available for 36 study subjects (Fig. 2). Expression levels of TLR6 and TLR7 were positively correlated with viral load (R = 0.3642, P = 0.0290 and R = 0.3577, P = 0.0333), and TLR4 trended towards correlating with viral load (R = 0.2962, P = 0.0794). Viral load correlations with TLR2, 3, and 8 also had positive slopes but did not reach statistical significance (P = 0.17 to P = 0.29).

Fig. 2
Fig. 2
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HIV single stranded RNA upregulates Toll-like receptor expression

To determine if innate immune signals may be playing a role in increased TLR expression in HIV infection, we evaluated the effects of viral and bacterial TLR ligands on TLR expression in cultured ex-vivo PBMC from HIV-infected (n = 21) and uninfected subjects (n = 71) in an independent study. Two TLR ligands were used: short sequences of single-stranded GU-rich RNA derived from HIV-1 (ssRNA40), which are known to signal through human TLR8 [22]; and lipopolysaccharide, a bacterial cell wall component that is known to signal through TLR4 [43]. Each of these ligands were used to assess their effects on the expression of their cognate TLR (TLR8 and TLR4, respectively) as well as the other (Fig. 3).

Fig. 3
Fig. 3
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The effects of HIV ssRNA stimulation are shown in Fig. 3a. In subjects without HIV infection, stimulation of PBMC with ssRNA led to highly significantly increased expression of its receptor, TLR8 (P < 0.0001), as well as the cross-expression of TLR4 (P = 0.0098). In HIV-infected subjects, in whom a preexisting HIV effect would be expected, ssRNA40 stimulation also significantly elevated TLR8 expression (P = 0.0277); however, TLR4 expression was unchanged (P = 0.7702).

In contrast to HIV ssRNA, stimulation of PBMC with lipopolysaccharide (Fig. 3b) resulted in a significant reduction in TLR4 expression in uninfected subjects (P = 0.0147), supporting previous findings [44]. No change in TLR4 expression was, however, seen after lipopolysaccharide stimulation of PBMC isolated from HIV-infected subjects (P = 0.8512). Interestingly, TLR8 expression was increased by lipopolysaccharide stimulation in HIV-infected subjects (P = 0.0331), further indicating that altered TLR regulation occurs with HIV infection. In summary, our in-vitro studies show that HIV infection is associated with altered regulation of TLR expression, and that HIV ssRNA may itself sensitize TLR signalling by increasing the expression of selective TLR.

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HIV infection enhances Toll-like receptor responsiveness

To assess functionally whether the increases in TLR receptor expression associated with HIV infection are also associated with altered TLR signalling capacity, we compared the induction of cytokines in PBMC from HIV-infected (n = 15) and uninfected (n = 13) subjects by measuring soluble TNF-α and IL-10 from PBMC stimulated with TLR ligands. HIV-1-derived ssRNA40 was again used to stimulate primarily through TLR8, and bacterial lipopolysaccharide was used to stimulate through TLR4. Stimulation with both of these ligands resulted in the significant production of TNF-α and IL-10 compared with media alone (Fig. 4). Significantly higher TNF-α production was, however, seen in HIV-infected subjects compared with uninfected subjects in response to both lipopolysaccharide and ssRNA (P = 0.0448 and P = 0.0018, respectively; Fig. 4a). The production of IL-10, which has a regulatory function and typically appears later in culture than TNF-α, was also elevated but was not significantly different between HIV-infected and uninfected subjects for either ligand (P = 0.1837 for lipopolysaccharide and P = 0.0958 for ssRNA; Fig. 4b). Therefore, HIV-1 infection is not only associated with the increased expression of multiple TLR, but TLR signalling and the production of the proinflammatory cytokine TNF-α are also increased in HIV infection.

Fig. 4
Fig. 4
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Toll-like receptor expression is ‘normalized’ with HAART

Given our findings that increased TLR expression was associated with HIV viral load and that innate signals from HIV may drive increases in TLR levels, we compared TLR expression in untreated subjects (n = 49) and those receiving HAART (n = 19, mean duration 13.4 ± 10.7 months), in whom we expected viral replication and viral load to be reduced. Subjects taking HAART had similar expression of TLR2, 3, 4, 6, and 8 to uninfected controls (Fig. 5a). Although, TLR7 expression remained significantly elevated in subjects taking HAART compared with uninfected subjects (P = 0.0025), it trended towards reduced expression when compared with untreated HIV-infected subjects (P = 0.0617). The effect of HAART on TLR expression was more likely to be related to viral suppression than significant immune reconstitution because HIV-RNA viral loads were clearly suppressed (Fig. 5b) but CD4 cell counts remained low in the group receiving HAART (Fig. 5c), probably because of the relatively short time that antiretroviral drugs have been freely available and the degree of advanced disease at their initiation. Together with the previous findings, these data suggest that untreated viraemic HIV infection is associated with increases in selective TLR in the peripheral blood, but that this effect is reversible when HIV replication is inhibited after antiretroviral therapy. The present fact supports that HIV replication drives the TLR expression increases in vivo.

Fig. 5
Fig. 5
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Discussion

This study, to our knowledge, represents the first broad analysis of how HIV-1 infection influences systemic innate immunity by evaluating basal levels of all functional human TLR. We found that untreated HIV infection has a profound impact on TLR expression and signalling in circulating PBMC. The cumulative changes we observed in TLR expression parallels to an extent the disease progression, which is classically marked by CD4 T-cell depletion, and there was also a positive association between TLR expression and HIV viral load. In our study, TLR6, 7 and 8 mRNA levels were significantly increased in subjects with chronic HIV-1 infection whose CD4 cell counts were above 200 cells/μl. In subjects with more advanced disease, as determined by CD4 cell counts less than 200 cells/ml, these increases were even more pronounced, and TLR2, 3, and 4 mRNA increases were also seen. In-vitro, HIV-1 ssRNA could alone increase TLR expression, suggesting a direct virus-mediated immunomodulatory pathway is involved in regulating TLR expression. As those subjects whose viral loads were suppressed by HAART, but whose CD4 T-cell counts remained very low, had relatively normal TLR expression, this also suggests that viral products are important in regulating TLR expression in vivo. Finally, heightened proinflammatory responsiveness to TLR ligands in the PBMC of HIV-infected subjects, as demonstrated by the greater production of TNF-α responses to TLR ligands, suggests that the immune cells of HIV-infected individuals may be hypersensitized to TLR-mediated innate immune activation.

HIV itself, or its components, may provide a significant source of innate stimulus for chronic innate immune activation. Two of the TLR that were shown to be elevated in the current study, TLR7 and TLR8, are known to recognize GU-rich ssRNA sequences that are common to certain viruses including HIV-1 [22,45]. It was ssRNA derived from HIV-1 sequences (RNA40) that demonstrated TNF-α hyperresponsiveness in the in-vitro studies. Multiple sequences of HIV ssRNA were also recently shown to induce prominent T-cell activation markers [46]. Dendritic cell maturation and natural killer cell activation are also known to occur in response to HIV ssRNA [47–49]. Although signalling through TLR7 and TLR8, when used as vaccine adjuvant, have been shown to improve HIV-specific CD4 and CD8 T-cell responses [17,50–52], it remains unclear whether the recognition of HIV ssRNA by these TLR induces protective immune responses against actual infection [37]. Interestingly, our findings that TLR7 and TLR8 expression increased further in advanced HIV disease suggests that innate recognition of HIV may be incrementally hypersensitized during the development of AIDS.

Other TLR were also increased in our study. The expression of TLR2, 3, 4, and 6 mRNA were increased in subjects with advanced HIV disease and TLR6 was increased regardless of disease stage. TLR2, 4, and 6 are expressed on immune cell surfaces and primarily recognize bacterial or fungal molecular patterns. Bacterial immunodeficiency in both early and advanced HIV disease has been proposed to be related to phagocytic cell and TLR signalling changes [14]. TLR3, which was also increased in subjects with advanced disease in our study, is known to recognize double-stranded RNA from the life cycle of certain viruses [53]. As with TLR recognition of HIV, it is unknown whether altered expression is a protective response to pathogen exposure or simply adds to the level of immune activation.

Persistent immune activation in HIV is thought to contribute to pathogenesis by progressively disturbing cytokine expression, functional organization of the immune system, and by increasing cell turnover, dysfunction and cell death[4]. It is logical that TLR-mediated signalling could play a role, given that TLR pathways initiate and maintain inflammatory responses [54]. The production of TNF-α is known to increase rapidly in primary HIV infection [55], and has been reported to associate with HIV replication [56]. Our findings suggest that both HIV-specific and non-specific increases in TLR expression are associated with enhanced proinflammatory signalling because TNF-α hyperresponsiveness was demonstrated to both HIV ssRNA and to bacterial lipopolysaccharide. Importantly, subclinical endotoxaemia (plasma lipopolysaccharide) was recently associated with systemic immune activation in HIV infection and was implicated as a cause of systemic immune activation [25]. The source of endotoxin was presumed to be from the leakage of bacterial products across HIV-induced mucosal disturbances, termed ‘microbial translocation’, because the immune activation was reversible in SIV-infected macaques given broad-spectrum antibiotics to reduce bowel flora, and also that significant pathology of the gastrointestinal mucosa is associated with HIV [57–59]. As TLR4 expression and lipopolysaccharide responsiveness were both increased in HIV infection in our study, hypersensitized TLR signalling in HIV could actually exacerbate the relationship between microbial translocation and immune activation. Although with lipopolysaccharide, downregulation and tolerance might be expected to protect from overstimulation and toxic shock [44,60–62], proinflammatory priming of TLR responsiveness has previously been reported with malaria [63] and tuberculosis [64], both, interestingly, conditions that exacerbate HIV viraemia [65].

The mechanisms by which HIV increases TLR expression and signalling need to be further elucidated. Changing proportions of TLR high and TLR low-expressing cells is one potential mechanism [66,67]. The association between TLR expression and plasma HIV viral load, and more importantly, the apparent ‘normalization’ of TLR expression in subjects with viral loads that are suppressed by HAART, despite low CD4 cell counts, point towards the regulation of expression by viral products rather than large shifts in T-cell populations. In addition, HIV ssRNA was able to increase TLR expression in PBMC overnight culture, a timeframe unlikely to result in significant cell turnover, but certainly within the timeframe of altered transcriptional and even protein expression. It will still be important, however, to know the cell subsets that are most responsible for TLR expression and functional changes, and these studies are underway.

In summary, we have identified marked changes in TLR expression and function as a consequence of HIV-1 infection. By increasing TLR expression and signalling in the blood, HIV-1 infection may progressively disturb the immune response and control processes that normally protect individuals from opportunistic infection and disease. On the basis of our results, increased TLR expression in HIV infection leads to increased innate sensing and responsiveness of the immune system that may also serve as a primary driver for immune activation and thus HIV progression. By sending innate molecular signals through TLR ligands such as HIV ssRNA or lipopolysaccharide, in addition to enhancing their receptor expression and responsiveness, HIV appears to be sending the immune system a ‘double hit’ that could mark the difference between pathogenesis and tolerance induced by HIV signals. Therapies that intervene in these processes may therefore provide additional opportunities to interrupt HIV disease.

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Acknowledgements

The authors would like to thank longstanding participants of the Pumwani Sex-worker Cohort and dedicated clinic and laboratory staff.

Contributions: R.T.L. and X.D.Y. contributed equally to experimentation and analysis; C.W. and W.J. were instrumental in the recruitment of human subjects and provision of samples; R.K. provided viral load data. R.T.L. wrote the paper and all authors contributed to the final manuscript; K.L.R and F.A.P. co-supervised the project.

Sponsorship: This work is supported by a grant from the Bill and Melinda Gates Foundation and the Canadian Institutes of Health Research through the Grand Challenges in Global Health Initiative. Viral loads were provided with support from CIHR (HOP-75350) (R.K.). R.T.L. has an unrestricted research award through AMMI Bristol-Myers-Squibb (Canada), L.R.M has a CIHR training award and R.T.L. and L.R.M. have additional awards from the University of Manitoba hosted CIHR/ICID National Training Programme in Infectious Diseases. K.L.R. is supported by a Career Scientist Award from the Ontario HIV Treatment Network. F.A.P is the Canadian research Chair in Resistance and Susceptibility to Infections.

Conflicts of interest: None.

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Keywords:

HIV/AIDS; immune activation; innate immunity; lipopolysaccharide; pathogenesis; ssRNA; Toll-like receptor

© 2008 Lippincott Williams & Wilkins, Inc.

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