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AIDS:
doi: 10.1097/QAD.0b013e328344cefb
Basic Science

Sigmoid Th17 populations, the HIV latent reservoir, and microbial translocation in men on long-term antiretroviral therapy

Chege, Duncana,*; Sheth, Prameet Ma,*; Kain, Taylora; Kim, Connie Ja; Kovacs, Colinc; Loutfy, Monaa,c,d; Halpenny, Robertad,e,f; Kandel, Gabore; Chun, Tae-Wookg; Ostrowski, Marioa,b,e; Kaul, Ruperta,b,f; the Toronto Mucosal Immunology Group

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Author Information

aDepartment of Medicine, Canada

bDepartment of Immunology, University of Toronto, Canada

cMaple Leaf Medical Clinic, Canada

dDepartment of Medicine, Women's College Hospital, Canada

eSt Michaels Hospital, Canada

fUniversity Health Network, Toronto, Ontario, Canada

gLaboratory of Immunoregulation, National Institute of Allergy and Diseases, National Institutes of Health, Bethesda, Maryland, USA.

*D.C. and P.M.S. contributed equally to the writing of this article.

Received 25 October, 2010

Revised 17 December, 2010

Accepted 19 January, 2011

Correspondence to Duncan Chege, Department of Medicine, University of Toronto, Medical Sciences Building, Room #6356, Toronto, ON M5S 1A8, Canada. Tel: +1 416 946 7054; fax: +1 416 978 8765

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Abstract

Objective: Th17 cells play an important role in mucosal defence and repair and are highly susceptible to infection by HIV. Antiretroviral therapy (ART) suppresses HIV viremia and can restore CD4+ numbers in the blood and gastrointestinal mucosa, but the resolution of systemic inflammation and gut microbial translocation is often incomplete. We hypothesized that this might relate to persistent dysregulation of gut CD4+ Th17 subsets.

Methods: Blood and sigmoid biopsies were collected from HIV-uninfected men, chronically HIV-infected, ART-naive men, and men on effective ART for more than 4 years. Sigmoid provirus levels were assayed blind to participant status, as were CD4+ Th17 subsets, systemic markers of microbial translocation, and cellular immune activation.

Results: There was minimal CD4+ Th17 dysregulation in the blood until later stage HIV infection, but gastrointestinal Th17 depletion was apparent much earlier, along with increased plasma markers of microbial translocation. Plasma lipopolysaccharide (LPS) remained elevated despite overall normalization of sigmoid Th17 populations on long-term ART, although there was considerable interindividual variability in Th17 reconstitution. An inverse correlation was observed between plasma LPS levels and gut Th17 frequencies, and higher plasma LPS levels correlated with an increased gut HIV proviral reservoir.

Conclusion: Sigmoid Th17 populations were preferentially depleted during HIV infection. Despite overall CD4+ T-cell reconstitution, sigmoid Th17 frequencies after long-term ART were heterogeneous and higher frequencies were correlated with reduced microbial translocation.

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Introduction

HIV type 1 and simian immunodeficiency virus (SIV) infections are characterized by a dramatic depletion of CD4+ T cells in the gastrointestinal mucosa (gut) and a more gradual CD4+ T-cell decline in the peripheral blood [1], culminating in the spectrum of opportunistic infections and malignancies that define AIDS. Important predictors of the rate of this CD4+ decline in the peripheral blood are the plasma HIV RNA viral load and the degree of systemic immune activation [2,3]. The early depletion of CD4+ T cells from the gut mucosa may play a role in driving systemic immune activation, as this mucosal immune damage impairs the normal barrier function of the gut and allows increased translocation of pro-inflammatory bacteria from the gut lumen into the systemic circulation [1].

IL-17 producing CD4+ T cells (Th17 cells) safeguard the integrity of mucosal surfaces by inducing proliferation of enterocytes, promoting the recruitment of neutrophils to areas of fungal/bacterial infection, and mediating their subsequent activation to produce antimicrobial defensins [4–6]. In keeping with this, autoimmune Th17 deficiency leads to chronic mucosal and cutaneous candidiasis [7]. In this context, they appear to play an opposite role to CD25+FoxP3+ regulatory T cells (Tregs), which dampen potential harmful host immune responses to commensal bacteria [8]. Therefore, the mucosal balance between Treg and Th17 CD4+ subpopulations may facilitate local adaptive effector T-cell responses while allowing for tolerance of normal bowel flora in the gut [9–11]. However, Th17 cells are highly susceptible to HIV/SIV infection and are preferentially depleted during HIV infection [12–14]. This is associated with disease progression in both humans [15] and pathogenic SIV nonhuman primate models, whereas mucosal Th17 numbers are maintained in nonpathogenic SIV infection of African green monkeys and sooty mangabeys [14,16]. Therefore, HIV-associated Th17 depletion in the gut mucosa may be an important cause of increased systemic microbial translocation [13,14,17].

The goal of effective antiretroviral therapy (ART) is an undetectable HIV plasma viral load and normalization of the peripheral blood CD4+ T-cell count. Although the reconstitution of gut CD4+ T-cell numbers is slower [18], these may eventually reach near-normal levels [19]. Nonetheless, a substantial proviral reservoir persists in the gut mucosa [20] and microbial translocation and systemic immune activation may remain increased despite ART [21,22]. The latter may drive adverse health outcomes that persist despite effective ART, such as cardiovascular disease and neurocognitive dysfunction [21,23]. We hypothesized that mucosal Th17 depletion might persist on long-term ART despite overall CD4+ T-cell reconstitution, and that this might allow ongoing microbial translocation. To address this hypothesis, we examined the impact of long-term ART on Th17 populations in blood and the sigmoid colon.

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Participants and methods

Ethics statement

All participants provided written informed consent, and the study protocol was reviewed and approved by the Research Ethics Boards at St Michaels' Hospital, Toronto, and the University of Toronto.

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Participant recruitment and study groups

Participants were enrolled through the Maple Leaf Medical Clinic, Toronto, Canada, and consisted of three groups: therapy-naive men who had been HIV-infected for at least 6 months (chronic infection), HIV-infected men on ART with an undetectable HIV plasma viral load for at least 4 years (long-term suppressed, LTS), and HIV-uninfected men. To further assess the impact of HIV infection on T-cell subsets, HIV-infected participants were defined as having early or advanced HIV infection, based on a cut-off of 350 CD4 T cells per microlitre, the threshold at which ART initiation is currently recommended [24]. All subsequent immune assays were performed by research personnel blinded to participant group.

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Processing of peripheral blood and sigmoid biopsies

Blood was collected by venipuncture into Acid Citrate Dextran solution A (BD Bioscience, La Jolla, California, USA) and peripheral blood mononuclear cells (PBMCs) were then isolated via Ficoll–Hypaque density centrifugation as previously described [25]. Sigmoid pinch biopsies were obtained approximately 25–30 cm from the anal verge, and mononuclear cells were isolated by collagenase type II digestion (Sigma Aldrich St Louise, Michigan, USA) as previously described [19]. Briefly, biopsies were incubated first in a 0.5 μg/ml and then a 1.0 μg/ml collagenase solution on a shaking heating block at 37°C for 30 min each.

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Immune studies

Isolated mononuclear cells from blood and the sigmoid colon were stimulated with staphylococcal enterotoxin B (SEB; 3 μg/ml) (Toxin Technologies, Saratosa, Florida, USA) for 1 h at 37°C and 5% CO2. One microgram per millilitre of Brefeldin A was added, and cells incubated for 5 h at 37°C and 5% CO2. Cells were then permeabilized and stained with combinations of fluorochrome-labelled monoclonal antibodies specific for CD3, CD8, CD4, CD69, HLA DR, CD25, FoxP3, IFNγ and IL17A, (BD BioSciences and eBioscience, San Diego, California, USA). Samples were acquired on a FACSCalibur flow cytometer (BD Systems) and data analysis performed using Flow Jo analytical software version 7.2.4 (Treestar, Ashland, Oregon, USA). Tregs were defined as CD4+ T cells co-expressing CD25 and FoxP3; Th17 cells were defined as CD4+ T cells producing IL17A upon SEB stimulation.

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Plasma markers of microbial translocation

Lipopolysaccharide (LPS) levels were measured using the limulus amebocyte lysate assay kit (Cambrex; Charles City, Iowa, USA). Plasma samples were diluted 20% in endotoxin-free water (Cambrex), and heated to 80°C for 15 min to inactivate plasma proteins, then LPS levels were assayed using manufacturer's instructions. We also measured levels of soluble CD14 (sCD14) glycoprotein (R&D Systems; Minneapolis, Minnesota, USA) and endotoxin core-binding IgM antibodies (EndoCAb; Hycult Biotech; Plymouth Meeting, Pennsylvania, USA) using commercially available ELISA kits. All samples were run in duplicate and background readings were subtracted from reported values. All assays were run as per manufacturer's instructions.

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Measurement of HIV proviral DNA

CD8+ T cells were depleted from gut mononuclear cells using a column-based cell separation technique (StemCell Technologies, Vancouver, British Columbia, Canada) and proviral DNA levels were assayed as previously described [19]. Briefly, to determine the proviral HIV DNA copies per million CD8+ depleted gut T cells, genomic DNA was isolated using the Puregene DNA isolation kit according to the manufacturer's specifications (Gentra, Minneapolis, Minnesota, USA). One microgram of DNA was then used as template for real-time PCR in an iCycler (Bio-Rad, Hercules, California, USA). The amplification reaction was carried out in triplicate using 0.5 μmol/l primers, 0.2 μmol/l fluorescent probe, 0.8 mmol/l dNTPs, 5 mmol/l MgCl2, and 2.5 U platinum Taq polymerase (Invitrogen, Carlsbad, California, USA) in 50 μl total volume. The following primers were used: 5′GGTCTCTCTGGTTAGACCAGAT-3′ (5′ primer) and 5′-CTGCTAGAGATTTTCCACACTG-3′ (3′ primer) along with the fluorescent probe 5′-6FAM-AGTAGTGTGTGCCCGTCTGTT-TAMRA-3′. PCR conditions consisted of a denaturation step at 95°C for 3 min followed by 45 cycles of 15 s at 95°C and 1 min at 59°C. Standard curves were prepared as previously described [19] and the copy number of HIV DNA per 1 × 106 CD8+ depleted T cells was reported.

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Statistical analysis

Statistical analysis was performed using SPSS 17 (SPSS Chicago, Illinois, USA). Nonparametric analysis (Mann–Whitney with asymptotic two-tailed tests) was used to compare groups, and correlations were examined by Spearman's rank regression analysis.

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Results

Study participants

Forty-one participants were recruited within three groups: chronically HIV-infected, therapy-naive participants (chronic infection; n = 16; Table 1); participants on long-term suppressive ART (LTS; n = 15; Table 2); and HIV-uninfected participants (n = 10). Chronic infection participants had a median absolute CD4+ T-cell count of 325 cells/μl (range 105–990 cells/μl), and a blood viral load of 48 009 (range 2766–500 000) HIV RNA copies/ml. LTS participants had been on therapy for a median of 96 months (8 years; range 51–207 months), with no detectable plasma HIV RNA for 80 months (6.7 years; range 48–129 months). Most LTS participants had initiated therapy with a nadir CD4+ T-cell count less than 350 cells/μl (10/15) or after an AIDS-defining illness (ADI; 2/15); three participants had initiated therapy during earlier stages of HIV infection, with a mean absolute CD4+ T-cell count of 590 cells/μl.

Table 1
Table 1
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Table 2
Table 2
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Impact of untreated HIV infection on blood Th17 subsets and plasma markers of microbial translocation

Overall, chronic untreated HIV infection was not associated with differences in the frequency of blood Th17 cells when compared with HIV-uninfected participants (median 0.28 vs. 1.01%, P = 0.109). However, significantly reduced blood Th17 frequencies were seen in chronic infection participants with a blood CD4+ T-cell count below 350 cells/μl, the threshold at which ART is currently recommended [24] (CD4+ T-cell count <350 cells/μl vs. HIV-uninfected; median Th17 0.12 vs. 1.01%, P = 0.013; Fig. 1a, left three groups). In keeping with the opposing effects of HIV infection on Th17 and Treg subsets, the blood Th17/Treg ratio was reduced almost 20-fold in therapy-naive participants (median HIV-uninfected vs. chronic infection 5.7 vs. 0.3, P = 0.005). Again, this decrease was confined to chronic infection participants with late-stage HIV infection (median Th17/Treg ratio 0.2 vs. 2.3 during untreated early HIV, P = 0.020, and vs. 5.7 in HIV-uninfected, P = 0.003; Fig. 1b, left three groups). The blood Th17/Treg ratio was not associated with HIV viral load (data not shown) and no differences in IFNγ producing CD4+ T cells (Th1 subsets) were seen across study groups (data not shown).

Fig. 1
Fig. 1
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As has been described elsewhere [26], HIV-infected ART-naive participants had increased plasma levels of LPS compared with uninfected controls (median 1.29 vs. 1.18 EU/ml, P = 0.007), and soluble CD14 (sCD14) levels were also elevated (median 1.40 vs. 0.71 μg/ml in HIV-uninfected, P = 0.002). No differences were evident in endotoxin core IgM antibodies (EndoCAb IgM) titres (both P > 0.15), and no associations with disease stage or Th17 subsets were seen. Plasma LPS was not correlated with these immune subsets but was associated with an increased blood HIV viral load (Spearman's rho = 0.615, P = 0.044; Fig. 1d). T-cell activation was apparent in chronic infection participants (HLA-DR+CD4+, 1.73 vs. 0.56% in HIV-uninfected, P = 0.003; HLA-DR+CD8+, 7.37 vs. 0.99%, P = 0.003; CD69+CD4+, 1.74 vs. 0.39%, P = 0.010).

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Impact of antiretroviral therapy on blood Th17 subsets and markers of microbial translocation

After long-term ART, blood Th17 frequencies remained reduced compared to HIV-uninfected controls (median 0.22 vs. 1.01% in HIV-uninfected, P = 0.020). This reduction was limited to participants who had initiated ART during advanced infection (CD4 <350 cells/μl; median 0.17 vs. 1.01% in HIV-uninfected; P = 0.008), and in the three participants in whom ART had been initiated early, the blood Th17 frequencies were similar to HIV-uninfected controls (median 0.80 vs. 1.01%, P = 0.732; Fig. 1a, two right panels). Although the Th17/Treg ratio in the LTS group was comparable to that in the HIV-uninfected participants, overall (1.8 vs. 5.7, P = 0.176), there was a trend to a lower ratio in participants who had initiated ART during late-stage infection (CD4 <350 cells/μl; median 0.68 vs. 5.7, P = 0.081; Fig. 1b, two right panels), but not those in whom treatment was started earlier (CD4 >350 cells/μl; median 8.0 vs. 5.7, P = 0.909; Fig. 1b).

LPS levels remained elevated in LTS compared with HIV-uninfected participants (median 1.45 vs. 1.18 EU/ml, P = 0.002) and were comparable to ART-naive individuals (median 1.45 vs. 1.29 EU/ml in chronic infection, P = 0.106). EndoCAb IgM titres were lower in the LTS group than in either HIV-uninfected (median 26.25 vs. 88.5 MMU/ml, P = 0.017) or chronic infection participants (median 26.25 vs. 77.0 MMU/ml, P = 0.035). sCD14 levels were reduced in the LTS group compared with the chronic infection group (median 1.06 vs. 1.40 μg/ml, P = 0.039), but remained higher than that in uninfected controls (median LTS vs. HIV-uninfected, 1.06 vs. 0.71 μg/ml, P = 0.047). Markers of microbial translocation were not directly correlated with systemic immune activation in the LTS group (all P > 0.2), and CD4+ T-cell expression of HLA-DR and CD69 were similar in LTS and HIV-uninfected participants (HLA-DR, 0.49 vs. 0.56% in HIV-uninfected, P = 0.953; CD69, 1.03 vs. 0.39% in HIV-uninfected, P = 0.266).

Overall, plasma LPS levels remained elevated and blood Th17 frequencies depressed despite ART. Therefore, we hypothesized that Th17 dysregulation might persist in the gut mucosa despite ART, allowing for ongoing microbial translocation.

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Effect of HIV infection and therapy on Th17 CD4 subsets in the sigmoid colon

We collected sigmoid biopsies from a subset of recruited participants in whom blood samples were also obtained; HIV-uninfected (n = 5), chronic infection (n = 7), and LTS (n = 8). Sigmoid Th17 frequencies were reduced during untreated HIV infection (median chronic infected vs. HIV-uninfected, 0.25 vs. 0.74%, P = 0.042; Fig. 2a, centre group), with a dramatic reduction in the Th17/Treg ratio (median ratio 0.2 vs. 1.7 in HIV-uninfected, P = 0.004; Fig. 2b, centre group). In contrast to blood, Th17 dysregulation in the sigmoid colon was apparent during early as well as later HIV stages (median Th17/Treg ratio, 0.1 in chronic infection early stage vs. 0.2 in chronic infection late stage, P = NS). There was no direct correlation between these immune perturbations and direct or indirect markers of microbial translocation (all P > 0.2).

Fig. 2
Fig. 2
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In the long-term ART group no differences were apparent between HIV-infected and HIV-uninfected participants in either the mucosal Th17 frequency (Th17 median 1.40 vs. 0.74% in HIV-uninfected, P = 0.558; Fig. 2a, right) or the sigmoid Th17/Treg ratio (median 2.4 vs. 1.7 in HIV-uninfected, P = 0.884; Fig. 2b, right). However, there was substantial interindividual heterogeneity within the Th17 subset: sigmoid Th17 frequencies in four of eight ART-treated participants were equal to or higher than uninfected participants, whereas the remaining four of eight participants had relatively reduced Th17 frequencies (Fig. 2a). The subgroup with higher sigmoid Th17 frequencies tended to have higher blood CD4+ T-cell counts (715 vs. 545 CD4 counts/μl, P = 0.144) and to have been on ART longer (159 vs. 99 months, P = 0.149). No differences were apparent in nadir CD4+ T-cell counts prior to starting ART (P = 0.564), duration of prior HIV infection (P = 0.480), blood Th17 frequencies (P = 0.564) or overall CD4+ T-cell proportion in the sigmoid (P = 0.773). In addition, levels of immune activation markers, HLADR/CD69, in blood and sigmoid CD4+ and CD8+ T cells did not differ between the two groups (all P > 0.05). Participants with enhanced Th17 reconstitution also demonstrated increased IFNγ production by sigmoid CD4+ T cells (median 1.22 vs. 0.19%, P = 0.021). However, co-production of IL-17 and IFNγ was uncommon, as previously described [14], with sigmoid CD4+ T cells from only two participants co-producing significant amounts of both IL17 and IFNγ; both were within the Th17 ‘high’ group. Sigmoid T-cell subsets in LTS participants were not associated with plasma markers of microbial translocation, although there was a weak inverse relationship between Th17 frequencies and plasma LPS levels (Spearman's rho = −0.476, P = 0.233).

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Associations of HIV proviral DNA in the sigmoid colon after long-term therapy

The HIV proviral load was assayed in the sigmoid colon of all participants on long-term ART (n = 8). Gut provirus was detectable in all participants (median 240.95 HIV DNA copies/106 CD8 depleted cells, range 9.1–899.0), although provirus levels tended to decrease with the duration of ART (Spearman's rho = −0.65, P = 0.058). Two major associations were apparent. First, the sigmoid HIV proviral load was directly correlated with the plasma LPS level (Spearman's rho = 0.762, P = 0.028; Fig. 3a) and tended to be inversely associated with the plasma EndoCAb IgM titre (Spearman's rho = −0.515, P = 0.192). There was no association with plasma sCD14 levels (Spearman's rho = −0.167, P = 0.693). Second, the size of the sigmoid provirus reservoir was inversely correlated with the sigmoid Th17 frequency (Spearman's rho = −0.762, P = 0.028; Fig. 3b). This was specific to the Th17 subset, as there was no association with the degree of overall CD4+ reconstitution (Spearman's rho = −0.286, P = 0.493; Fig. 3c). LTS participants with sigmoid Th17 frequencies above the median had a reduced sigmoid HIV proviral load (665.1 vs. 82.0/106 CD8+ depleted cells, P = 0.043; Fig. 3d). No significant associations were seen with gut Treg frequencies, markers of inflammation, IFNγ production by sigmoid CD4+ T cells, or with any blood T-cell subsets (all P > 0.05).

Fig. 3
Fig. 3
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Discussion

HIV infection is associated with a marked depletion of gastrointestinal CD4+ T cells and with gut-systemic translocation of luminal bacteria [26]. The latter may be an important driver of systemic immune activation and HIV immunopathogenesis [26–29], and in some studies has been shown to persist despite effective HIV therapy [30,31]. The cause of persistent microbial translocation in participants on long term ART is not clear, and we hypothesized that this might relate to persistent dysregulation of the Th17 subset in the gut mucosa. Our studies confirm that untreated HIV infection is associated with mucosal Th17 dysregulation and gut microbial translocation [26,28,29], and found that there was complete overall restoration of HIV-associated defects in sigmoid Th17 and Treg subsets in our long-term ART (LTS) group. However, this overall restoration masked substantial intra-group heterogeneity, and within the LTS group incomplete restoration of gut Th17 frequencies (but not overall CD4+ T-cell restoration) was directly associated with higher sigmoid provirus levels, and the latter was also correlated with plasma LPS levels.

Recent studies suggest that direct effects of the virus on gut epithelial cells and the mucosal basement membrane may be an important factor contributing to microbial translocation [32]. We only saw a weak direct association between gut Th17 frequencies and microbial translocation, but our sample size was small and this may be an important area for future research. The reasons underlying heterogeneity in gut Th17 reconstitution in our cohort were not clear, although incomplete reconstitution tended to be associated with a shorter duration of ART and a lower blood CD4+ T-cell count at the time of sampling. While incomplete Th17 reconstitution was also associated with reduced IFNγ production by sigmoid CD4+ T cells, possibly implying a broader problem with functional subset reconstitution in the gut, only Th17 cells were linked to a reduced sigmoid HIV proviral load.

Significant differences were apparent in the effects of HIV infection and therapy on T-cell subsets in the blood and gut. Both Th17 frequencies and Th17/Treg ratios were normal in the blood during the early stage HIV, but were dysregulated in participants with a CD4+ T-cell count below 350 cells/μl and remained low in the blood of such participants even after long-term ART. In contrast, a dramatic reduction in the sigmoid Th17/Treg ratio was apparent at all stages of chronic untreated HIV infection, but there was near-complete resolution of these defects on therapy (albeit with the significant interindividual heterogeneity discussed above). The clinical implications of persistent blood Th17/Treg dysregulation in the context of delayed ART initiation are not clear, and this may represent another important area for future research. These data suggest that early ART initiation may result in more complete reconstitution of the systemic CD4+ T-cell functional repertoire. Furthermore, as these blood CD4+ T-cell subsets were only altered during relatively advanced HIV infection this may explain, at least in part, the discrepancy in findings of previously published studies regarding the impact of HIV infection on these CD4+ functional subsets [14,33–35].

Our finding that there was no reduction in LPS levels, a plasma marker of microbial translocation despite effective ART was unexpected, and is at odds with some [22,26,36,37] but not all [30,31] prior studies. Elevated LPS levels in this cohort were confirmed by a second blinded ELISA and were also associated with a significant depletion of EndoCAb titres. The latter may result from saturation and/or depletion of endotoxin-specific antibodies and is also thought to indicate increased microbial translocation [38]. In addition, the associations of plasma LPS levels with both the sigmoid provirus reservoir and mucosal Th17 frequencies strongly suggests that this was a true observation, although clearly not one that is applicable to all treated cohorts [22,26,36,37].

There are several limitations to our study. Our sample size was relatively small, and so these observations need to be confirmed by larger studies. The flow cytometric studies were run on a four-colour FACSCalibur, which limited the number of surface markers that could be examined and meant that we could not ascertain whether alterations in specific T-cell memory subsets were associated with the differences in Th17 frequency and Th17/Treg ratio. Our FoxP3 staining was dimmer than has been reported elsewhere (data not shown), likely because specialized FoxP3 staining buffers were not used. However, as the same protocol was used throughout and all data were acquired and analysed blindly, our results and conclusions should not be affected. Nonetheless, for this reason, Treg subset-specific data are not presented, except in the context of Th17/Treg ratios.

In summary, we found that the sigmoid provirus reservoir remained high despite long-term suppressive ART and was associated with persistently elevated gut-systemic microbial translocation and with impaired restoration of sigmoid Th17 populations. Starting ART with a blood CD4+ T-cell count above 350 cells/μl was associated with improved restoration of sigmoid Th17 subsets, and therefore this might be an important clinical rationale for early initiation of therapy.

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Acknowledgements

We would like to thank Dr Lyle McKinnon, Dr David Willer, Dr Ali Sakhdari, and Ms Bahareh Vali for helpful proofing of this manuscript. This work was supported in part by the Ontario HIV Treatment Network (R.K., ROGB-G123; P.M.S., salary award); the Ontario Graduate Student Science & Technology Scholarships/Canadian Institutes of Health Research – Banting and Best Scholarship (D.C. salary) and the Canadian Research Chair Program (R.K., salary support). Study sponsors played no role in study design, collection or analysis of data, interpretation of results, writing of the manuscript or decision to submit for publication.

The Toronto Mucosal Immunology Group consists of: Tae J. Yi, Department of Immunology, University of Toronto, Toronto, Ontario, Canada; Sanja Huibner, Maple Leaf Medical Clinic, Toronto, Ontario, Canada; Shariq Mujib, Department of Medicine, University of Toronto, Toronto, Ontario, Canada; Desmond Persad, Maple Leaf Medical Clinic, Toronto, Ontario, Canada; Erika Benko, Maple Leaf Medical Clinic, Toronto, Ontario, Canada.

These data were presented in part at the 2009 Keystone Conference on HIV Pathogenesis (Keystone, Clorado, USA) and the 2009 Ontario HIV Treatment Network conference (OHTN; Toronto, Ontario, Canada).

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References

1. Brenchley JM, Price DA, Douek DC. HIV disease: fallout from a mucosal catastrophe? Nat Immunol 2006; 7:235–239.

2. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983; 220:868–871.

3. Gallo RC, Sarin PS, Gelmann EP, Robert-Guroff M, Richardson E, Kalyanaraman VS, et al. Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science 1983; 220:865–867.

4. Aujla SJ, Dubin PJ, Kolls JK. Th17 cells and mucosal host defense. Semin Immunol 2007; 19:377–382.

5. Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006; 203:2271–2279.

6. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 2008; 28:454–467.

7. Kisand K, Boe Wolff AS, Podkrajsek KT, Tserel L, Link M, Kisand KV, et al. Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines. J Exp Med 2010.

8. Round JL, Mazmanian SK. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A 2010; 107:12204–12209.

9. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 2006; 24:677–688.

10. Ivanov II, Frutos Rde L, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 2008; 4:337–349.

11. Kanwar B, Favre D, McCune JM. Th17 and regulatory T cells: implications for AIDS pathogenesis. Curr Opin HIV AIDS 2010; 5:151–157.

12. Kader M, Wang X, Piatak M, Lifson J, Roederer M, Veazey R, et al. Alpha4(+)beta7(hi)CD4(+) memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection. Mucosal Immunol 2009; 2:439–449.

13. Cecchinato V, Trindade CJ, Laurence A, Heraud JM, Brenchley JM, Ferrari MG, et al. Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques. Mucosal Immunol 2008; 1:279–288.

14. Brenchley JM, Paiardini M, Knox KS, Asher AI, Cervasi B, Asher TE, et al. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 2008; 112:2826–2835.

15. Favre D, Mold J, Hunt PW, Kanwar B, Loke P, Seu L, et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med 2010; 2:32ra36.

16. Favre D, Lederer S, Kanwar B, Ma ZM, Proll S, Kasakow Z, et al. Critical loss of the balance between Th17 and T regulatory cell populations in pathogenic SIV infection. PLoS Pathog 2009; 5:e1000295.

17. Cecchinato V, Franchini G. Th17 cells in pathogenic simian immunodeficiency virus infection of macaques. Curr Opin HIV AIDS 2010; 5:141–145.

18. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, et al. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 2003; 77:11708–11717.

19. Sheth PM, Chege D, Shin LY, Huibner S, Yue FY, Loutfy M, et al. Immune reconstitution in the sigmoid colon after long-term HIV therapy. Mucosal Immunol 2008; 1:382–388.

20. Chun TW, Nickle DC, Justement JS, Meyers JH, Roby G, Hallahan CW, et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. J Infect Dis 2008; 197:714–720.

21. Deeks SG. Immune dysfunction, inflammation, and accelerated aging in patients on antiretroviral therapy. Top HIV Med 2009; 17:118–123.

22. Jiang W, Lederman MM, Hunt P, Sieg SF, Haley K, Rodriguez B, et al. Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection. J Infect Dis 2009; 199:1177–1185.

23. McDonald CL, Kaltman JR. Cardiovascular disease in adult and pediatric HIV/AIDS. J Am Coll Cardiol 2009; 54:1185–1188.

24. Hammer SM, Eron JJ Jr, Reiss P, Schooley RT, Thompson MA, Walmsley S, et al. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society – USA panel. JAMA 2008; 300:555–570.

25. Sheth PM, Danesh A, Shahabi K, Rebbapragada A, Kovacs C, Dimayuga R, et al. HIV-specific CD8+ lymphocytes in semen are not associated with reduced HIV shedding. J Immunol 2005; 175:4789–4796.

26. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006; 12:1365–1371.

27. Picker LJ, Hagen SI, Lum R, Reed-Inderbitzin EF, Daly LM, Sylwester AW, et al. Insufficient production and tissue delivery of CD4+ memory T cells in rapidly progressive simian immunodeficiency virus infection. J Exp Med 2004; 200:1299–1314.

28. Kotler DP, Reka S, Clayton F. Intestinal mucosal inflammation associated with human immunodeficiency virus infection. Dig Dis Sci 1993; 38:1119–1127.

29. Ullrich R, Zeitz M, Riecken EO. Enteric immunologic abnormalities in human immunodeficiency virus infection. Semin Liver Dis 1992; 12:167–174.

30. Lester RT, Yao XD, Ball TB, McKinnon LR, Omange WR, Kaul R, et al. HIV-1 RNA dysregulates the natural TLR response to subclinical endotoxemia in Kenyan female sex-workers. PLoS One 2009; 4:e5644.

31. Wallet MA, Rodriguez CA, Yin L, Saporta S, Chinratanapisit S, Hou W, et al. Microbial translocation induces persistent macrophage activation unrelated to HIV-1 levels or T-cell activation following therapy. AIDS 2010; 24:1281–1290.

32. Nazli A, Chan O, Dobson-Belaire WN, Ouellet M, Tremblay MJ, Gray-Owen SD, et al. Exposure to HIV-1 directly impairs mucosal epithelial barrier integrity allowing microbial translocation. PLoS Pathog 2010; 6:e1000852.

33. Macal M, Sankaran S, Chun TW, Reay E, Flamm J, Prindiville TJ, et al. Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses. Mucosal Immunol 2008; 1:475.

34. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol 2004; 78:2454–2459.

35. Andersson J, Boasso A, Nilsson J, Zhang R, Shire NJ, Lindback S, et al. The prevalence of regulatory T cells in lymphoid tissue is correlated with viral load in HIV-infected patients. J Immunol 2005; 174:3143–3147.

36. Marchetti G, Bellistri GM, Borghi E, Tincati C, Ferramosca S, La Francesca M, et al. Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy. AIDS 2008; 22:2035–2038.

37. Troseid M, Nowak P, Nystrom J, Lindkvist A, Abdurahman S, Sonnerborg A. Elevated plasma levels of lipopolysaccharide and high mobility group box-1 protein are associated with high viral load in HIV-1 infection: reduction by 2-year antiretroviral therapy. AIDS 2010; 24:1733–1737.

38. Strutz F, Heller G, Krasemann K, Krone B, Muller GA. Relationship of antibodies to endotoxin core to mortality in medical patients with sepsis syndrome. Intensive Care Med 1999; 25:435–444.

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Keywords:

antiretroviral therapy; microbial translocation; provirus; sigmoid colon; Th17 cells

© 2011 Lippincott Williams & Wilkins, Inc.

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