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Keywords:

  • antiretroviral therapy;
  • CD4 T-cell recovery;
  • chloroquine;
  • HIV;
  • Toll-like receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objectives

Chloroquine (CQ), an anti-inflammatory drug, inhibits Toll-like receptor (TLR) signalling in plasmacytoid dendritic cells (pDCs) and may be beneficial for HIV-infected patients in whom immune activation persists despite effective antiretroviral therapy (ART). The effect of CQ on CD4 T-cell recovery and immune activation in immune nonresponding patients receiving successful ART was therefore studied.

Methods

Nineteen adults on ART with CD4 counts ≤350 cells/μL and undetectable viral load (VL) orally received CQ at 250 mg/day for 24 weeks. Side effects, CD4 and CD8 T-cell counts, VL, T-cell activation, pDC proportion and plasma inflammatory markers were assessed at baseline, at 24 weeks, and at 12 weeks after CQ discontinuation (clinicaltrial.org registration #NCT02004314).

Results

CQ was well tolerated and all patients maintained an undetectable VL. The absolute CD4 and CD8 T-cell counts and their percentages, the pDC proportion, T-cell activation, D-dimer and C-reactive protein (CRP) plasma levels and the kynurenine/tryptophan ratio did not change with CQ treatment. Among nine cytokines/chemokines measured, only levels of interferon (IFN)-α2 were significantly increased by CQ treatment.

Conclusions

CQ was well tolerated in patients with low CD4 T-cell counts despite long-term effective ART; however, 24 weeks of CQ treatment did not improved CD4 T-cell recovery, lymphoid and myeloid immune activation or inflammatory markers.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The chronic phase of HIV infection is characterized by the progressive depletion of CD4 T cells as well as persistent immune activation [1-3]. Antiretroviral treatment (ART) suppresses viral replication and leads to recovery of CD4 T cells in most individuals. However, up to 30% of HIV-infected patients who are receiving long-term ART show a suboptimal CD4 T-cell recovery despite achieving complete suppression of the plasma viral load (VL) [4]. In patients receiving effective ART, the persistence of enhanced T-cell activation [defined by CD38 and human leucocyte antigen (HLA)-DR co-expression on CD8 T cells] and elevated plasma levels of markers of inflammation, such as interleukin (IL)-6, C-reactive protein (CRP) and D-dimers, represent factors associated with low CD4 T-cell recovery [5-9]. The persistence of immune activation and inflammation in treated patients can be related to HIV persistence and/or low levels of viral replication directly, or indirectly through lymph node fibrosis and enhanced translocation of microbial origin products from the gastrointestinal tract to blood [2, 3, 6]. Microbial products from the gut released into systemic circulation trigger Toll-like receptor (TLR) pathways in monocytes and dendritic cells (DCs), resulting in enhanced immune activation [6, 10]. Therefore, supplementary therapeutic strategies targeting immune activation or inflammation should contribute to improving CD4 T-cell recovery. Therapies targeting TLR activation represent an attractive strategy.

Chloroquine (CQ), a quinolone antimalarial drug, has been used for several decades as an antimalarial agent and recently to treat immune-mediated inflammatory disorders such as rheumatoid arthritis [11, 12] and systemic lupus erythematosus [12-15]. CQ treatment was also reported to induce a beneficial response in the context of HIV infection, as it was shown to prevent endosomal acidification and fusion, thus inhibiting the activation mediated by endocytic TLRs 3, 7, 8 and 9 [16, 17]. Consequently, CQ was shown to be able to interfere with TLR7 downstream signalling pathways and consequently prevent the activation of transcription factors, such as interferon regulatory factor (IRF)-7, which regulates the production of interferon-alpha (IFN-α), a potent CD8 T-cell immune activator [12, 17, 18]. This drug might also reduce HIV-1 replication and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN)-mediated transfer of virus from DCs to CD4 T cells [19-21] and also inhibits trans-activator of transcription (Tat)-induced cytokine secretion [22]. Lastly, CQ was shown to block HIV-induced catabolism of tryptophan (Trp) into immunosuppressive kynurenine (Kyn) by inhibition of indoleamine 2,3-dioxygenase (IDO) expressed by DCs [18] (Fig. 1).

figure

Figure 1. Anti-inflammatory mechanisms of chloroquine (CQ) in HIV infection. CQ inhibits interferon (IFN) production through endosomal acidification and fusion, thus inhibiting Toll-like receptor (TLR)-associated signalling pathways. CQ also reduces HIV-1 replication as well as trans-activator of transcription (Tat)-induced cytokine secretion. CQ can block HIV-induced catabolism of tryptophan (Trp) into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) expressed by plasmacytoid dendritic cells (pDCs).

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Orally administered, CQ reaches stable serum concentrations [23] and shows an acceptable toxicity profile [11, 23]. Hydroxy-CQ (H-CQ) [24-27] and CQ [28] have been studied in clinical trials with both untreated and ART-treated HIV-infected patients. H-CQ and CQ share a similar chemical structure and mechanism of action. However, their potential benefit in HIV infection remains controversial. In one study, H-CQ did not reduce CD8 T-cell activation [24], while CQ did so in a different study [28]. Similary, the reports on the effect of H-CQ on CD4 T-cell counts in untreated viraemic patients are not consistent. H-CQ in viraemic patients was found to cause CD4 T-cell counts to remain stable [26, 27], decline [24] or increase when used in combination with hydroxyurea and didanosine [25]. In a recent study by Piconi et al., a 24-week treatment with H-CQ at 400 mg/day, in combination with ART, was associated with decreased immune activation and increased CD4 T-cell frequency [29].

In this study, the effect of CQ administration in ART-treated immune nonresponding HIV-infected patients was assessed, with the hypothesis that CQ would reduce T-cell activation and in turn enhance CD4 T-cell recovery. The anti-inflammatory effect of CQ could potentially result in a decrease in the rate of non-AIDS-related events. The effects of CQ on lymphoid and myeloid target cells were also evaluated. CQ was chosen as this drug is universally available, is not expensive and has a good safety profile in patients with rheumatological arthritis and systemic lupus erythematosus [13-15].

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study design

In this single-arm, proof-of-concept pilot study, 20 subjects were recruited from either the McGill University Health Centre, Montreal, or the Ottawa Hospital, Ottawa, Canada. The study lasted 44 weeks, with an initial 8-week observation period on ART alone (pre-CQ) to assess the intra- and inter-person variability of CD4 and CD8 T-cell counts (weeks 0 to 8), followed by 24 weeks of CQ treatment with ART (on-CQ; weeks 8 to 32), and a 12-week follow-up period after discontinuation of CQ while patients remained on ART (post-CQ; weeks 32 to 44) to improve the assessment of the cause−effect relationship for the study intervention and measured outcomes and to determine whether or not discontinuation of CQ was associated with a corresponding increase in immune activation. The primary objective of the study was to evaluate the change in the frequency of immune activation measured using CD38/HLA-DR on CD8 T cells, from baseline to week 24. At each time-point in the pre-CQ phase (weeks −4 and 0), the on-CQ phase (weeks 8, 12, 24 and 32) and the post-CQ phase (week 44), patients underwent a medical visit as well as an evaluation of their viral load (VL), absolute CD4 and CD8 T-cell counts and plasma levels of CRP and the coagulation marker D-dimer. This study is registered as NCT02004314, clinicaltrial.gov.

Study population

Participants were adults (male and female; 18-65 years of age) with HIV infection confirmed by western blot who met the following criteria within 4 weeks of the baseline visit: (1) VL < 50 HIV-1 RNA copies/mL (measured using the Roche Amplicor assay; Roche Diagnostics, Mississauga, Canada) for at least 36 weeks prior to study commencement; (2) CD4 T-cell count ≤ 350 cells/μL; (3) on stable ART for more than 36 weeks; and (4) Karnofsky performance status ≥ 80%. Subjects were excluded from the study if they had concurrent illnesses (including hepatitis B or C virus infection), a history of allergy to CQ, or current use of CQ, cytotoxic chemotherapy agents, systemic corticosteroids or any immuno-modulatory agents; psychiatric or cognitive disturbance or illness that could preclude compliance with the study; a history of retinitis or any retinal condition; G6PD deficiency, porphyria, psoriasis, liver cirrhosis, hearing impairment (including tinnitus), myopathy, cardiomyopathy or any other cardiac condition; pregnancy or breast feeding. Written informed consent was obtained from all study participants. The study and consent form were approved by the Institutional Review Boards of the McGill University Health Centre and the Ottawa Hospital Research Institute.

CQ administration

CQ was administered at an oral dose of 250 mg once daily from week 8 to week 32. The dose used in this study was the same as used for patients with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus [13-15]. An optic grill test was performed at each study visit to screen for potential ocular toxicity [30].

Assessment of T-cell and plasmacytoid dendritic cell (pDC) immune activation in circulating blood

Flow cytometry was performed on peripheral blood mononuclear cells (PBMCs) obtained by Ficoll-Hypaque density gradient centrifugation using a four-laser LSRII flow cytometer (BD Bioscience, Mississauga, ON, Canada) to measure activation marker expression before (week 8) and following (week 32) CQ administration in both CD4 and CD8 T cells as well as pDCs. Data were exported and analysed using FlowJo software v7.6.5 (Tree Star Inc., Ashland, OR, USA). The expression of the activation markers CD38, HLA-DR and programmed-death-1 (PD-1) was determined on CD4 and CD8 T cells. We also evaluated the frequency of CD123+ plasmacytoid DCs (pDCs) and the expression of CD86 and CD83, markers of pDC activation and maturation.

D-dimer and CRP quantification

Plasma levels of D-dimer were assessed using an Innovance D-dimer kit (Siemens, Marburg, Germany) according to the manufacturer's instructions using an automated coagulation analyser, BCS-XP (Siemens). Plasma levels of CRP were evaluated using a highly sensitive near-infrared particle immunoassay rate method employing the Synchron chemistry system (Beckman Coulter Ireland Inc., Galway, Ireland) according to the manufacturer's instructions.

Measurement of the plasma concentration of tryptophan and its catabolites to assess IDO enzymatic activity

Plasma levels of Trp and its catabolite Kyn were measured using an automated on-line solid-phase extraction−liquid chromatography−tandem mass spectrometry (XLC-MS/MS) method as previously reported [31]. Briefly, 250 μL of plasma was mixed with 50 μL of deuterated internal standard working solution (300 μmol/L in diluted acetic acid for Trp and 5 μmol/L for Kyn) and diluted with 200 μL of water. The samples were placed in the autosampler, which picks up 50 μL of the sample and leads it into the solid-phase extraction chromatography (SPE) cartridge. The sample was washed on the SPE cartridge, and the washed cartridge extract was then eluted into the high-performance liquid chromatography (HPLC) column. The binary gradient system consisted of mobile phase A (0.2% formic acid in water) and mobile phase B (acetonitrile). During this step, the chromatography procedure separated Trp and Kyn. The HPLC column effluent was then led into a mass spectrometer operated in positive ionization MRM mode to protonate the ions, and quantitatively detect selected masses. Finally, to quantify the amino acid metabolites, the area of specific mass peaks was measured and related to the concentration of calibration curves of the respective metabolites. The IDO enzymatic activity was determined using the Kyn : Trp ratio [32, 33].

Multiplex quantification of inflammatory plasma cytokines and chemokines

Prior to analysis, 90 μL of each plasma sample was treated with 10 μL of 5% Triton X-100 (to obtain a final concentration of 0.5% of triton) for 1 h at room temperature in order to inactivate HIV-1. Plasma levels of selected inflammatory soluble factors, IFN-α2, IFN-γ, tumour necrosis factor (TNF)-α, IL-1β, IL-6, IL-7, IL-8, IL-12 and IL-13, were measured in duplicate using a MILLIPLEX MAP magnetic bead kit according to the manufacturer's instructions (Millipore, Billerica, MA, USA). A broad range of standards were run in duplicate along with quality controls provided by the manufacturer to ensure the proper functioning of the kit. Mean fluorescence intensities for each analyte in each sample were determined using the MAGPIX instrument (Luminex, Austin, TX, USA) and the results were analysed using analyst software version 3.5.5 (Millipore) to obtain the protein concentration of each soluble factor.

Statistical analysis

Kruskal−Wallis and Wilcoxon matched pairs tests were performed using GraphPad prism software version 5 (GraphPad Software, Inc., La Jolla, CA, USA) to assess the change from baseline (mean of weeks 0 and 8) to follow-up, 24 weeks after introduction of the intervention. Similar comparisons between outcomes at the end of CQ therapy and 12 weeks after stopping therapy were made.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient demographics

Of 20 recruited patients, one declined to continue the study before starting CQ. Baseline characteristics for the 19 other study participants are summarized in Table 1. Eighteen patients were male and the median age was 47. 7 years. All patients maintained a VL < 50 copies/mL during the study. No grade 3 or 4 adverse events occurred, although one subject discontinued because of a grade 2 gastrointestinal side effect during the first week of CQ treatment.

Table 1. Baseline patient characteristics at the time of enrolment in the study
CharacteristicAll subjects (n = 19)
  1. ART, antiretroviral therapy; MSM, men who have sex with men; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor.

Sex male [n (%)]18 (94.7)
Race [n (%)] 
Caucasian11 (57.9)
Black5 (26.3)
Hispanic1 (5.3)
Asian2 (10.5)
Age (years) [median (range)]47.7 (29.7, 62.3)
ART type at baseline [n (%)] 
NRTI(s) only5 (26.3)
NRTI(s) + PI(s)5 (26.3)
Mode of HIV acquisition [n (%)] 
MSM11 (57.9)
Heterosexual contact6 (31.6)
Blood product1 (5.3)
Unknown2 (10.5)
CD4 cell count (cells/μL) [median (range)]203 (41, 394)
CD8 cell count (cells/μL) [median (range)]518 (96, 1667)
Nadir CD4 count (cells/μL) [median (range)]31 (6, 242)
Pre-ART viral load (copies/mL) [median (range)]74084 (1264, >1000000)
Time since HIV diagnosis (years) [median (range)]11.6 (1.0, 24.3)
Time since first ART (years) [ median (range)]4 (1, 20)

Impact of CQ administration on CD4 and CD8 T-cell counts

CD4 and CD8 T-cell counts were evaluated before and during CQ treatment. The median CD4 and CD8 T-cell counts at baseline were 186 [interquartile range (IQR) 127, 278] and 541 (IQR 320, 766) cells/μL, respectively. As shown in Figure 2, no changes were observed in the absolute CD4 and CD8 T-cell counts during the course of the study (P = 0.98 and P = 0.99, respectively). The mean changes (95% CI) for absolute CD4 and CD8 T cells were 16.7 [−6.1, 39.5] and 18.7 [−93.3, 130.8] cells/μL, respectively, for the difference between the pre-CQ and post-CQ phases of the study (Fig. 2). No change was observed in relative CD4 and CD8 T-cell counts or the CD4:CD8 ratio (data not shown).

figure

Figure 2. Absolute (a) CD4 and (b) CD8 T-cell counts at various time-points throughout the study. The Kruskal−Wallis test was used for statistical analysis. W, week; NS, not significant; CQ, chloroquine.

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Impact of CQ treatment on T-cell and pDC activation

The co-expression of activation markers CD38 and HLA-DR on CD4 and CD8 T cells was evaluated, comparing samples collected in the pre-CQ phase (week 8) and at the end of CQ treatment (week 32). Previous observations showed that CQ and H-CQ were able to decrease immune activation in simian immunodeficiency virus (SIV)-infected Chinese rhesus macaques and HIV-infected patients [28, 29, 34]. In the present study, we found no difference in T-cell activation following CQ administration. The percentage of CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ cells assessed prior to and after CQ treatment was not changed (Fig. 3a,b). No effect of CQ treatment on PD-1 expression in T cells was observed following CQ treatment (data not shown). As CQ treatment has been reported to inhibit the HIV-induced activation/maturation of pDCs in vitro [18], the impact of CQ on the expression of activation and maturation markers CD83 and CD86 on pDCs was also evaluated in the studied patients. The results demonstrated that pDC frequency and the expression of CD86 remained unchanged (Fig. 3c,e), whereas a moderate but significant decrease in the expression of the maturation marker CD83 following CQ treatment was observed (89% before treatment vs. 84% after treatment; P = 0.042; Fig. 3d).

figure

Figure 3. Levels of lymphoid and myeloid immune activation following chloroquine (CQ) treatment. Expression of lymphoid immune activation markers CD38/HLA-DR on (a) CD4 and (b) CD8 T cells as well as (c) plasmacytoid dendritic cell (pDC) frequency and expression of (d) maturation marker CD83 and (e) activation marker CD86 on pDCs were evaluated in the pre-CQ (week 8) and post-CQ (week 32) phases of the study. The Wilcoxon matched pairs test was used for statistical analysis. *P < 0.05. NS, not significant; PBMCs, peripheral blood mononuclear cells; W, week.

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Impact of CQ administration on plasma levels of inflammatory and coagulation markers

Plasma levels of CRP and D-dimer were evaluated at weeks −4, 0, 8, 12, 24, 32 and 44. As shown in Figure 4, plasma levels of D-dimer and CRP remained unchanged during the entire study. It was previously shown in vitro that CQ inhibited HIV-induced immunosuppressive IDO enzymatic activity measured using the Trp : Kyn ratio [18]. We therefore evaluated Trp catabolism in the present study, comparing the pre-CQ phase (week 8) and the end of CQ treatment (week 32). Plasma levels of Trp remained unchanged by CQ treatment [median ± standard deviation (SD) 48.36 ± 10.65 pg/mL at week 8 vs. 46.22 ± 12.33 pg/mL at week 32; Wilcoxon matched P = 0.22; data not shown]. Despite a modest, nonsignificant decrease in plasma levels of Kyn (median ± SD 1.86 ± 0.94 at week 8 vs. 1.81 ± 0.91 pg/mL at week 32; Wilcoxon matched P = 0.061; data not shown), the IDO enzymatic activity (Kyn : Trp ratio) remained unchanged during the study (0.039 ± 0.006 at week 8 vs. 0.038 ± 0.005 at week 32; Wilcoxon matched P = 0.929; data not shown). The impact of CQ treatment on the plasma levels of inflammatory cytokines and IFN-α was also evaluated. Our results demonstrated that the addition of 0.5% triton had no impact on the concentration of soluble inflammatory markers measured by Multiplex (data not shown). Among the nine cytokines/chemokines measured, only the levels of IFN-α2 were significantly increased following CQ treatment, while the levels of the others remained unchanged (Table 2). To evaluate whether the increase in IFN-α2 was attributable to CQ administration, we also measured IFN-α2 levels at 12 weeks following CQ discontinuation (week 44). We observed that the IFN-α2 levels became comparable to pre-CQ levels following drug discontinuation, indicating that the IFN-α2 increase was caused by CQ [median ± standard error (SE) 0.19 ± 1.88 pg/mL; Wilcoxon matched P = 0.23 for the comparison of week 8 and week 44; data not shown].

figure

Figure 4. Impact of chloroquine (CQ) treatment on plasma levels of (a) d-dimer and (b) C-reactive protein (CRP) at various time-points throughout the study. The Kruskal−Wallis test was used for statistical analysis. NS, not significant; W, week.

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Table 2. Inflammatory cytokine changes in the pre-chloroquine (CQ) (week 8) and on-CQ (week 32) phases of the study (median ± SE). Wilcoxon matched pairs test was used for statistical analysis
CytokineConcentration (pg/mL) (median ± SE)P-value
Week 8Week 32
  1. IFN, interferon; IL, interleukin; TNF, tumour necrosis factor.

IFN-α20.19 ± 0.310.66 ± 1.80.011
IFN-γ0.80 ± 0.772.20 ± 1.180.135
IL-120.25 ± 0.080.27 ± 1.280.109
IL-130.03 ± 0.090.03 ± 0.331
IL-1β0.21 ± 1.630.21 ± 0.011
IL-60.08 ± 0.110.08 ± 0.210.125
IL-73.1 ± 1.053.63 ± 0.770.602
IL-85.35 ± 3.266.33 ± 1.020.695
TNF-α7.90 ± 1.988.51 ± 1.850.947

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Herein we assessed whether CQ administration may have a beneficial effect in reducing T-cell activation and enhancing CD4 T-cell recovery in 19 aviraemic HIV-infected ART-treated subjects. To our knowledge, this is the first study investigating the immune mechanisms by which CQ might reduce persistent T-cell immune activation in ART-treated HIV-infected patients, while a similar study has been conducted with H-CQ [29]. CQ is an inexpensive, universally available and relatively well-tolerated drug initially developed for the treatment of malaria in the 1930s [35]. CQ is also used as an anti-inflammatory drug in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus [11-15, 35]. In addition, CQ inhibits in vitro HIV-induced inflammation via various mechanisms. However, its role in HIV infection remains controversial. In the present study, CQ was administrated at a single oral dose of 250 mg once daily for 24 weeks, a similar dosage to that used in autoimmune diseases [13-15]. In our study population, CQ was well tolerated and no grade 3 or 4 side effects were observed during the study. Contrary to our original hypothesis, the results clearly demonstrate that CQ treatment, in combination with ART, was not able to reduce HIV-induced immune activation or improve CD4 T-cell counts after 24 weeks of therapy. Indeed, no changes were observed in CD4 or CD8 T-cell counts after CQ administration. In contrast, Piconi et al. reported that H-CQ at a dose of 400 mg/day in combination with ART for 6 months increased only the percentage of CD4 T cells but not the absolute number of cells in immunologically nonresponding patients [29]. This moderate increase in CD4 T-cell percentage might be explained by the expected changes induced by ART alone, as patients included in the Piconi et al. study had a very low average CD4 count compared with our patients (134 ± 13 cells/mL vs. 212 ± 24 cells/mL, respectively). Furthermore, patients in the Piconi et al. study were treated with ART for an average of only 4.4 years prior to H-CQ treatment, while our patients were treated with ART for an average of 7.5 years prior to receiving CQ. In addition, the H-CQ daily dose used in their study (400 mg daily) was higher compared with the CQ dose used in our trial (250 mg daily). Of note, our study and the Piconi et al. study enrolled the same number of patients (n = 20), giving similar statistical power. Other clinical trials using either CQ or H-CQ in the absence of ART demonstrated an inconsistent impact on CD4 T-cell recovery. Indeed, these studies showed either an absence of change [26, 27], a decrease in a large randomized study [24] or an increase in CD4 T-cell recovery when combined with hydroxyurea and didanosine [25]. More recently, CQ was administrated during the acute phase of SIVmac251 infection in rhesus macaques and resulted in a transient increase in the expression of IFN-stimulating genes and reduced recovery of CD4 T-cell counts [36].

As already mentioned, the main purpose of CQ administration in ART-treated patients was to improve CD4 T-cell recovery by reducing persistent immune activation. The results here clearly demonstrate no change in T-cell activation markers as defined by the co-expression of CD38 and HLA-DR on both CD4 and CD8 T cells. In the study of Piconi et al. using H-CQ in combination with ART, a decrease in HLA-DR+ CD8 T cells was only observed 2 months after H-CQ interruption. In contrast, despite a nonsignificant decreasing trend in CD38 and CD69 activation markers on CD8 T cells following H-CQ treatment, their levels became identical to baseline 2 months after H-CQ interruption [29]. Importantly, in untreated HIV viraemic patients, the co-expression of CD8 T-cell activation markers CD38/HLA-DR was not changed by H-CQ treatment [24]. As CQ is an inhibitor of TLR7/9 signalling pathways [12, 18, 34] we therefore also evaluated its impact on pDCs. We found no change in pDC frequency or on the expression of the activation marker CD86. However, we observed decreased expression of the maturation marker CD83 on pDCs following CQ administration, which could potentially contribute to a decrease in DC-mediated inflammation.

CRP and the coagulation marker D-dimer are predictors of overall mortality and cardiovascular events in HIV-infected patients [7-9]. The results of the present study demonstrate that CQ has no impact on the plasma levels of CRP and D-dimer. The catabolism of Trp into Kyn via the IDO enzyme expressed by DCs reduced mucosal immune defence in HIV-infected patients, contributing to microbial translocation [32, 33]. CQ has been shown to inhibit HIV-induced IDO enzyme activity in vitro [18]. However, in this study, despite a trend towards lower plasma levels of immunosuppressive Kyn, the Kyn : Trp ratio, a marker of IDO activity, did not change with CQ treatment. Furthermore, the absence of a reduction in inflammatory cytokines and chemokines after CQ treatment demonstrates the lack of CQ efficacy in reducing HIV-mediated generalized inflammation. The unexpected increase in IFN-α2 produced by CQ treatment and its normalization following CQ discontinuation could be explained by the antimicrobial properties of CQ [37]. Indeed, in HIV-infected patients, changes in gut microbial flora contribute to a generalized immune activation [6]. We hypothesize that the antimicrobial effect of CQ could potentially contribute to changes in the gut flora, resulting in stimulation of DCs to produce IFN-α2, and this effect is greater than the inhibitory effect of CQ on IFN production. In addition, the unexpected increase in IFN-α2 produced by CQ treatment could explain, in part, the negative impact of H-CQ on HIV VL and CD4 T-cell lymphopenia observed in nontreated chronically HIV-infected patients [24]. Further investigations are needed to evaluate the impact of CQ on gut microbial flora in HIV infection.

Taken together, the results of this study show that daily oral administration of 250 mg of CQ for 24 weeks was safe for patients with low CD4 T-cell counts who were concomitantly receiving ART. In contrast to our hypothesis, no improvements in CD4 T-cell recovery, lymphoid or myeloid cellular immune activation or inflammatory markers were observed during CQ therapy in immune nonresponding patients receiving ART. Initiatives to decrease immune activation in combination with ART should, therefore, involve alternative immunomodulatory interventions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are grateful to Dr Mohamed-Rachid Boulassel for his helpful contribution to the study design and his technical assistance and advice. We also thank Helen Preziosi for her invaluable assistance in patient recruitment and nursing; Johanna Spaans, Dr Gina Graziani, Angie Massicotte and Kishanda Vyboh for clerical and coordination assistance; Ryhan Pineda for coordination and blood banking; and Benoit Lemire for pharmacy support. We are grateful to Jacquie Sas and Jim Pankovich from the CIHR Canadian HIV Trials Network (CTN) for study implementation and coordination and Dr Terry Lee for his input in clinical data collection and statistical analysis. The authors acknowledge Dr Dominique Gauchat and Annie Gosselin from the flow cytometry department of the CHUM-Research Centre, Saint-Luc Hospital, Montréal, QC, Canada, for technical assistance. This work was supported by the CIHR Canadian HIV Trials Network (CTN 246) and the Research Institute of the McGill University Health Centre. JPR is a holder of the Louis Lowenstein Chair in Hematology & Oncology, McGill University. JBA is supported by an Ontario HIV Treatment Network Career Scientist Award and MAJ by a CANFAR/CTN Postdoctoral Fellowship Award.

Conflicts of interest: The authors have no competing interests to declare.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References