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  (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).
As has been described elsewhere , 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).
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.
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).
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 , 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).
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).
HIV infection is associated with a marked depletion of gastrointestinal CD4+ T cells and with gut-systemic translocation of luminal bacteria . 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 . 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 . 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.
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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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
antiretroviral therapy; microbial translocation; provirus; sigmoid colon; Th17 cells