Expression of gut-homing β7 receptor on T cells: surrogate marker for microbial translocation in suppressed HIV-1-infected patients?

Authors

  • M Abad-Fernández,

    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
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  • C Gutiérrez,

    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
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  • N Madrid,

    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
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  • B Hernández-Novoa,

    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
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  • L Díaz,

    1. Immunobiology Laboratory, University Hospital Gregorio Marañon, Madrid, Spain
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  • MA Muñoz-Fernández,

    1. Immunobiology Laboratory, University Hospital Gregorio Marañon, Madrid, Spain
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  • S Moreno,

    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
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  • A Vallejo

    Corresponding author
    1. Department of Infectious Diseases, Health Research Institute Ramon y Cajal (IRYCIS), University Hospital Ramon y Cajal, Madrid, Spain
    • Correspondence: Dr Alejandro Vallejo, Department of Infectious Diseases, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Hospital Universitario Ramón y Cajal, Ctra Colmenar Km 9, 28034 Madrid, Spain. Tel: +34 91 336 8711; fax: +34 91 336 8712; e-mail: alejandro.vallejo@salud.madrid.org

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Abstract

Objectives

In view of the fact that mucosal damage associated with HIV-1 infection leads to microbial translocation despite successful antiretroviral treatment, we analysed microbial translocation and expression of the gut-homing β7 receptor on peripheral T cells in HIV-1-infected individuals.

Methods

Fifteen long-term suppressed HIV-1-infected patients, of whom seven had their treatment intensified with maraviroc and eight with raltegravir, were included in the study. Samples at baseline, at week 48 of intensification, and at weeks 12 and 24 after deintensification were analysed for soluble CD14, lipopolysaccharide (LPS), LPS-binding protein, gut-homing β7 receptor and T-cell subsets.

Results

The increases in both microbial translocation and expression of the gut-homing β7 receptor on activated CD8 T cells found during maraviroc intensification were reduced after deintensification. Moreover, the correlations between activated β7+ T cells and LPS levels found during intensification with maraviroc (P = 0.036 and P = 0.010, respectively) were lost during deintensification. In contrast, microbial translocation was stable during raltegravir intensification, with the exception of decreased LPS levels and activated CD4 β7+ T cells, which reverted to baseline values after deintensification.

Conclusions

Microbial translocation is an important factor in gut immune activation and mucosa inflammation, as evidenced by the association between the dynamics of microbial translocation and activated T cells expressing the gut-homing β7 receptor. The recruitment of activated β7+ T cells to the gut tract when alteration of microbial translocation is maximum may be the major mechanism for recovery of mucosal integrity.

Introduction

A high percentage of CCR5+ CD4 T cells, which are susceptible to HIV-1 infection, are localized in the gut mucosa [1, 2], which is believed to be an important viral reservoir in HIV-1-infected individuals [3-7]. Intestinal damage produced in acute HIV-1 infection leads to translocation of microbial products from the intestinal lumen into the systemic circulation, and persists despite successful antiretroviral treatment [8-13], and this is reflected in the detection of increased levels of lipopolysaccharide (LPS) and soluble CD14 (sCD14) in the plasma of HIV-1-infected individuals undergoing antiretroviral therapy [14-17]. Circulating LPS present at elevated levels in HIV-1-infected patients binds the CD14−Toll-like receptor 4 (TLR 4) complex, through the action of the plasma LPS-binding protein (LBP), and this triggers the activation of monocytes and macrophages that increase secretion of sCD14 and proinflammatory cytokines [18-22].

Microbial translocation and persistent low-level viraemia in HIV-1-infected patients despite successful treatment have been proposed as triggering mechanisms of increased immune activation in HIV-1 infection [9, 23-26]. The ongoing and persistent low-level productive viral replication was expected to be reduced using intensification therapy with the new HIV-1 antiviral agents that have emerged, such as CCR5-receptor antagonists (e.g. maraviroc) and integrase inhibitors (e.g. raltegravir). Unfortunately, this strategy failed to show any effect in reducing peripheral low-level HIV-1 viraemia, although it may be efficient in reducing HIV-1 replication in the gut mucosa [27-32].

The severe depletion of CD4 T cells in the gut mucosa during HIV-1 infection is restored much more slowly than in peripheral blood [33-36], and studies using simian models have shown that T-cell restoration in the gut involves trafficking of CD4 T cells from the periphery to the gut mucosa [37, 38]. Our previous studies of treatment intensification showed different behaviours of microbial translocation depending on the drug used; while raltegravir intensification reduced the levels of both microbial translocation and gut-homing β7 receptor expression on peripheral T cells, intensification with maravicoc led to an increase in these two parameters [39, 40].

Methods

To eliminate the possibility that variation of the parameters studied was attributable solely to the treatment intensification itself, we evaluated these parameters 12 and 24 weeks after discontinuation of drug intensification. Hence, the aim of our work was to determine correlations between different measurements of microbial translocation and the expression of gut-homing receptor β7 on T cells. As a secondary objective, we evaluated the association between both microbial translocation and T cells bearing β7 receptor and the dynamics of T-cell subpopulations.

Patients

This study was performed within two pilot open-label phase II clinical trials (ClinicalTrials.com NCT00795444 and NCT00807443) conducted at the Hospital Ramón y Cajal in Madrid, Spain, in order to evaluate the effect of treatment intensification with a CCR5-receptor antagonist (maraviroc; Pfizer, New York, NY, USA) and an integrase inhibitor (raltegravir; Merck Sharp and Dhome, Whitehouse Station, NJ) on the HIV-1 latent reservoir. The initial intensification trial was amended to include a follow-up period of 24 weeks after deintensification of maraviroc and raltegravir. Immunological and virological parameters were assessed at baseline, after 48 weeks of treatment intensification, and after 12 and 24 weeks of maraviroc and raltegravir deintensification. Results of the intensification trials have been published elsewhere [39, 40]. Here we present findings concerning the effect of deintensification on measurements of microbial translocation, immune activation and the dynamics of T-cell subpopulations.

For this treatment deintensification study, a total of 15 HIV-1-infected adult individuals recruited for the above clinical trials, initially with CD4 T-cell counts > 350 cells/μL and undetectable plasma HIV-1 viral loads (< 40 HIV-1 RNA copies/mL) for at least 2 years, remained in the trials and were included in the analysis. Seven of the patients discontinued maraviroc intensification, while the other eight patients discontinued raltegravir intensification. None of them had previous experience with any of these drugs.

The study was carried out according to the recommendations of the Declaration of Helsinki and current Spanish legislation on clinical trials. It was approved by the Spanish Agency for Medications and Health Products (AEMPS) and our local Independent Ethics Committee (Hospital Ramon y Cajal, Madrid, Spain). All patients provided written informed consent for participation, sample collection and laboratory determinations.

Flow cytometry

Fresh ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood was used to analyse CD4 and CD8 T-cell subpopulations with the following antibody combination: CD3-allophycocyanin (APC)-Cy7, CD4- peridinin chlorophyll protein complex (PerCP), CD8-phycoerythrin (PE)-Cy7, CD38-phycoerythrin (PE), and HLA-DR-allophycocyanin (APC), CD45RA-phycoerythrin (PE), CCR7-allophycocyanin (APC), and β7-APC. Antibodies were from Becton Dickinson (Franklin Lakes, NJ). An isotope and unstained control was performed for all samples. Briefly, 100 μL of blood was lysed with FACS Lysing solution (Becton Dickinson) for 30 min at room temperature, incubated with the antibodies for 20 min at 4°C, washed and resuspended in phosphate-buffered saline (PBS) containing 1% azida. Cells were analysed in a Gallios flow cytometer (Beckman-Coulter, Brea, CA). At least 30 000 CD3 T cells were collected for each sample and analysed with the kaluza software (Beckman-Coulter), initially gating lymphocytes according to morphological parameters. T-cell immune activation was analysed by the co-expression of CD38 and HLA-DR, while T-cell subsets were defined as follows: naïve cells, CD3+CD4+(CD8+)CD45RA+CCR7+; effector memory T cells (TEM), CD3+CD4+(CD8+)CD45RACCR7; central memory T cells (TCM), CD3+CD4+(CD8+)CD45RACCR7+; and transitional memory T cells (TEMRA), CD3+CD4+(CD8+)CD45RA+CCR7. The gating was sustained among different time-points.

Measurement of soluble factors

Microbial translocation was quantified in plasma using three commercial kit assays according to the manufacturers' protocols. Plasma LPS was measured using QCL-1000 Limulus Amebocyte Lysate (Lonza, Basel, Switzerland), plasma sCD14 was quantified using the Quantikine® Human sCD14 Immunoassay (R&D Systems, Minneapolis, MN) and plasma LBP was measured using the LBP Soluble ELISA kit (Enzo Life Sciences, Farmingdale, NY). All the samples were run in duplicate.

Statistical analysis

Continuous variables were expressed as median and interquartile range (IQR) and categorical variables as percentages. The Mann−Whitney U-test was used to compare medians between groups. Pearson's χ2 was used to determine the significance of differences in proportions between groups, employing the continuity correction or Fisher's exact test, as appropriate. The nonparametric Wilcoxon paired test was used to compare medians during follow-up. Correlations were analysed with Spearman's rank test. Statistical analysis was performed using spss software 21.0 (IBM, Chicago, IL). Graphs were generated using GraphPad Prism 5.01 (Statcon, La Jolla, CA, USA).

Results

Effect of treatment deintensification on microbial translocation

The patients' immunovirological characteristics at baseline (before treatment intensification) and also before treatment deintensification (after 48 weeks of treatment intensification) are shown in Table 1. After 48 weeks of intensification, two patients in the maraviroc clinical trial and one patient in the raltegravir clinical trial were lost to the study. During the treatment deintensification, one patient in the raltegravir clinical trial decided to drop out of the study.

Table 1. Baseline characteristics of the patients
 Baseline of intensification studyDiscontinuation study (48 weeks after intensification)
All patients (n = 18)Maraviroc intensification (n = 9)Raltegravir intensification (n = 9)All patients (n = 15)Maraviroc intensification (n = 7)Raltegravir intensification (n = 8)
  1. Continuous variables are expressed as median (interquartile range) and categorical variables as the percentage of patients.
  2. ART, antiretroviral therapy; LBP, LPS-binding protein; LPS, lipopolysaccharide; sCD14, soluble CD14; MSM, men who have sex with men; IDU, injecting drug user; NRTI, nucleotide/nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; PI, protease inhibitor.
Age (years)46 (41–50)46 (31–48)48 (44–53.5)46 (43–53)46 (32–49)47 (43–54)
Gender (male)88888893.385.7100
Risk factor
MSM50564446.642.850
IDU28332233.342.825
Heterosexual2211332014.225
Time on ART (months)99 (52–146)75 (46–133)144 (71–167)89 (51–144)57 (50–124)127 (61–160)
Antiretroviral regimen
NRTI11.122.2013.328.60
NRTI + NNRTI38.822.255.546.628.662.5
NRTI + PI5055.544.440.042.837.5
Immunological characteristics
CD4 count nadir (cells/μL)202 (62–317)230 (171–416)40 (17–297)230 (70–378)230 (171–416)161 (22–358)
CD4 count (cells/μL)686 (522–897)711 (547–793)655 (417–877)716 (587–847)720 (597–881)669 (491–847)
CD8 count (cells/μL)685 (632–1066)784 (673–1109)636 (367–827)665 (566–1004)803 (575–1138)578 (343–979)
Microbial translocation
sCD14 (μg/mL)1.1 (0.7–1.4)0.8 (0.5–1.4)1.2 (1–1.4)1.3 (1.2–1.5)1.4 (1.2–1.7)1.2 (1.1–1.4)
LPS (pg/mL)23 (17–32.6)22 (13.5–31.5)25 (18.8–41.1)31.8 (18.1–50.4)35.9 (31.8–56.4)18.3 (14.5–37.4)
LBP (ng/mL)34 (19.1–37.1)21.8 (17.3–43.5)35.3 (30.2–36.5)34.8 (30.1–38.1)31.2 (25.9–40.4)34.8 (33.5–37.4)
Gut-homing receptor
Activated CD4 β7+ (%)42.1 (34.5–46.3)40.5 (35.3–51.2)40.7 (28.1–44.5)37.0 (29.1–41.9)37.1 (30.7–42.9)33.1 (27.2–40.8)
Activated CD8 β7+ (%)44.6 (38.4–50.3)41.5 (28.5–47.5)47.5 (39.9–53.2)46.6 (39.7–54.4)52.1 (46.7–59.2)42.2 (35.2–48.4)

The effect of the treatment deintensification on microbial translocation was different in patients who had intensification with maraviroc and those who had intensification with raltegravir. Overall, microbial translocation significantly increased during treatment intensification and then progressively decreased after drug discontinuation in patients who received maraviroc, with levels at the end of the study being similar to those observed at baseline (LPS, P = 0.091; LBP, P = 0.846), except for sCD14 levels, which remained significantly higher (P = 0.043) (Fig. 1a).

Figure 1.

Effect of treatment deintensification on microbial translocation. Levels of microbial translocation in the (a) maraviroc and (b) raltegravir groups during follow-up are shown. Data related to the treatment deintensification part of the study are enclosed in a square. BL, baseline; LBP, LPS-binding protein; LPS, lipopolysaccharide; TI, treatment intensification; TDI, treatment deintensification; sCD14, soluble CD14; 48w, 48 weeks after initiation of treatment intensification; d12w, 12 weeks after drug discontinuation; d24w, 24 weeks after drug discontinuation. P-values were determined using the nonparametric Wilcoxon paired test and only significant values (P < 0.05) are shown.

In contrast, in individuals who had raltegravir intensification, levels of microbial translocation remained stable during intensification and after its discontinuation, except for LPS levels, which significantly increased after 12 and 24 weeks of discontinuation compared with week 48 of intensification (P = 0.025 and P = 0.036, respectively), reversing the significant decrease observed during treatment intensification compared with baseline (P = 0.008) (Fig. 1b).

Expression of gut-homing β7 receptor on peripheral activated T cells during treatment deintensification

The effect of the discontinuation of drug intensification on the expression of the gut-homing β7 receptor was also different according to whether patients had intensification with maraviroc or raltegravir. In patients who received maraviroc, activated CD8 β7+ T cells significantly increased during treatment intensification and then significantly decreased after 24 weeks of drug discontinuation (P = 0.028) compared with 48 weeks, but with levels at the end of the study being significantly higher than those observed at baseline (P = 0.043). No changes in activated CD4 β7+ T cells were observed during the analysed follow-up period (Fig. 2a). In individuals who had intensification with raltegravir, levels of activated CD8 β7+ T cells remained stable during intensification and after its discontinuation, with levels at the end of the study being similar to those at baseline (Fig. 2b). However, activated CD4 β7+ T cells significantly increased after 12 and 24 weeks of discontinuation compared with week 48 of intensification (P = 0.012 and P = 0.017, respectively), reversing the decrease observed during treatment intensification, although not significantly (P = 0.066) (Fig. 2b).

Figure 2.

Effect of treatment deintensification on the expression of the gut-homing β7 receptor on activated T cells. Percentages of activated CD4 and CD8 T cells expressing the β7 receptor during the follow-up period in the maraviroc (a) and raltegravir (b) groups are shown. Data related to the treatment deintensification part of the study are enclosed in a square. BL, baseline; TI, treatment intensification; TDI, treatment deintensification; 48w, 48 weeks after initiation of treatment intensification; d12w, 12 weeks after drug discontinuation; d24w, 24 weeks after drug discontinuation. 48w is also the baseline point of the discontinuation study. P-values were determined using the nonparametric Wilcoxon paired test and only significant values (P < 0.05) are shown.

Association between microbial translocation and gut-homing β7 receptor

No correlation was observed between microbial translocation and the gut-homing β7 receptor at baseline, i.e. before intensification. However, in patients who received maraviroc, a correlation was found during the treatment intensification (week 48) between both activated CD4 β7+ and CD8 β7+ T cells and microbial translocation which was significant when LPS levels were analysed (Fig. 3). Of note, this correlation was lost after drug deintensification. In contrast, those who received raltegravir as treatment intensification showed no correlation between either activated CD4 β7+ or CD8 β7+ T cells and microbial translocation.

Figure 3.

Association of activated β7+ T cells with lipopolysaccharide (LPS) levels. A positive correlation between LPS levels and both activated CD4 β7+ and CD8 β7+ T cells was found after 48 weeks of maraviroc intensification. These correlations were lost after the drug deintensification. Data related to the treatment deintensification part of the study are enclosed in a square. TI, treatment intensification; TDI, treatment deintensification; 48w, 48 weeks after initiation of treatment intensification; d12w, 12 weeks after drug discontinuation; d24w, 24 weeks after drug discontinuation. 48w is also the baseline point of the discontinuation study. P-values were determined using Spearman's rank test and only significant values (P < 0.05) are shown.

Dynamics of T-cell subpopulations during treatment deintensification

CD4 or CD8 T-cell counts were stable during the treatment deintensification regardless of the drug of intensification. Moreover, naïve CD4 and CD8 T cells also remained stable at the time-points studied, regardless of the drug used for treatment intensification, and no differences were found in CD4 or CD8 TCM, TEM or TEMRA (data not shown). T-cell subpopulations did not correlate with microbial translocation (data not shown).

Discussion

We found that the effects of drug intensification on microbial translocation during treatment intensification depended on the drug used, and were reversed when the intensified drugs were discontinued. Regardless of the drug used, the dynamics of microbial translocation during the follow-up period were associated with the expression of the gut-homing β7 receptor on T cells.

When microbial translocation levels increased during maraviroc intensification, levels of activated T cells bearing β7 also increased, supporting the idea of cell recruitment from peripheral blood to the gut tract as the major mechanism for recovery of mucosal integrity. Hence, microbial translocation and systemic inflammation would be reduced consequently [36, 38, 39, 41]. Interestingly, this cell recruitment was reversed when microbial translocation was no longer increasing after maraviroc discontinuation. In accordance with these findings, in the maraviroc group there was a strong correlation between both activated CD4 β7+ and CD8 β7+ T cells and LPS levels at the end of the intensification treatment, when the alteration of microbial translocation was maximal. We do not have a good explanation as to why maraviroc induced an increase in microbial translocation.

The mechanisms by which maraviroc produces such effects remain unclear. CCR5 plays an important role in the inflammatory function of several mucosal immune cell types (T cells, macrophage and dendritic cell subpopulations, innate lymphoid cells, etc). Blocking of this role is expected to interfere with mucosal immune cell functions required to control microbial intrusion and, indeed, these findings demonstrate microbial translocation, and β7 T-cell activation, presumably in response to inflammatory activation in response to the intrusion. CCR5 blockade increases the expression of CCR5 on the cell membrane, increasing the rate of cell death and damaging the gut-associated lymphoid tissue, and also resulting in an increase in the production of ligands such as macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) that could signal through other receptors, which eventually could increase gut immune activation. All these processes together could therefore increase the level of microbial translocation and the expression of the gut-homing β7 receptor. It is also possible that CCR5 blockade impairs T-cell migration to gut-associated lymphoid tissue, where its natural ligands are being produced, increasing the levels of CCR5+ T cells that co-express the β7 receptor (in particular CD8 T cells which are much more likely than CD4 T cells to express CCR5) in the peripheral blood. Consistent with this hypothesis is the fact that there was no relationship between β7 expression and microbial translocation markers at baseline or after deintensification. However, there was a positive correlation among maraviroc-treated patients at week 48 when the levels of microbial translocation markers were highest. This is consistent with the hypothesis that maraviroc increased β7 expression and microbial translocation through independent mechanisms.

In contrast, raltegravir intensification showed a significant decrease in microbial translocation (measured by LPS levels) that was related to a significant decrease in CD4 β7+ cells. This effect was reversed during treatment deintensification. These findings are consistent with those of one study of intensification with raltegravir in HIV-1-infected adults with CD4 T-cell counts > 200 cells/μL, in which intestinal tissue biopsies were included [5]. No differences were found in gut immune activation, also consistent with our findings in peripheral blood. Raltegravir behaved as expected for an antiretroviral drug, i.e. it did not appear to alter microbial translocation or the level of the gut-homing β7 receptor on T cells, suggesting no alteration in the level of gut immune activation.

However, CD4 and CD8 T-cell counts in peripheral blood did not change during the entire follow-up period in patients who had intensification with either maraviroc or raltegravir, showing no correlations with microbial translocation; these findings are supported by those of another study that failed to show such a correlation during raltegravir intensification [42]. The fact that the CD4 T-cell count in peripheral blood was constant during the follow-up period demonstrates a delayed recovery of gut CD4 T cells compared with CD4 T-cell restoration in peripheral blood [24]. In addition, no significant changes were observed in naïve CD4 or CD8 T cells, TCM, TEM or TEMRA during the intensification period with maraviroc or raltegravir, or during deintensification, with no correlation with microbial translocation or gut-homing receptor expression.

Microbial translocation in well-suppressed HIV-1-infected patients indicated that the mucosal damage associated with HIV-1 infection persisted despite successful treatment [43-46]. Translocation of microbial products to the lumen has been proposed as a possible driver mechanism of immune activation in HIV-1 infection. We have been able to demonstrate, on the one hand, that microbial translocation is an important factor in gut inflammation, as shown by the observed association between the dynamics of microbial translocation and activated T cells expressing the gut-homing receptor, and, on the other hand, that recruitment of activated β7+ T cells in well-suppressed HIV-1-infected individuals from peripheral blood to the gut tract seems to be the major mechanism for recovery of mucosal integrity.

Acknowledgements

We would like to thank Carmen Page, Raquel Lorente and Ester Domínguez for their excellent technical assistance. We especially thank all of our patients and their families for their participation in the study.

Conflicts of interest: The authors declare no financial or commercial conflicts of interest.

Financial disclosure: This work was supported in part by the Spanish AIDS Network Red Temática Cooperativa de Investigación en SIDA (RD06/0006), the Spanish National Institute of Health, Instituto de Salud Carlos III (grants FIS-PI080958 and CP08/00046), and the Foundation of Investigation and Prevention of AIDS (FIPSE-36-0844/09).

Author contributions: MAF and AV performed the laboratory work. SM designed and coordinated the clinical trials. CG, NM, BHN, LD and MAMF helped with the analysis of the data, and approved the manuscript in its final form.

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