Immune activation and increased IL-21R expression are associated with the loss of memory B cells during HIV-1 infection

Authors


F. Chiodi, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobelsväg 16. S-17177, Stockholm, Sweden. (fax: 46-8-330498; e-mail: francesca.chiodi@ki.se).

Abstract

Abstract.  Ruffin N, Lantto R, Pensieroso S, Sammicheli S, Hejdeman B, Rethi B, Chiodi F (Karolinska Institutet, Stockholm; and South Hospital, Stockholm, Sweden). Immune activation and increased IL-21R expression are associated with the loss of memory B cells during HIV-1 infection. J Intern Med 2012; 272: 492–503.

Objectives.  Microbial translocation and chronic immune activation were previously shown to be associated with impairment of T cell functions and disease progression during infection with human immunodeficiency virus type-1 (HIV-1); however, their impact on B cell function and number remains unknown. By measuring markers of immune activation and molecules involved in apoptosis regulation, we have evaluated the association between microbial translocation and loss of memory B cells in HIV-1-infected patients.

Methods.  Markers of activation [the interleukin-21 receptor (IL-21R) and CD38] and apoptosis (Bim, Bcl-2 and annexin V) were measured in B cell subpopulations by multicolour flow cytometry. Levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS), measures of microbial translocation, were determined in plasma. Purified B cells were also exposed in vitro to Toll-like receptor (TLR) ligands.

Results.  IL-21R expression was higher in cells from HIV-1-infected patients, compared with control subjects, with the highest levels in nontreated patients. An inverse correlation was observed between IL-21R expression and percentages of circulating resting memory (RM) B cells. IL-21R-positive memory B cells were also more susceptible to spontaneous apoptosis and displayed lower levels of Bcl-2. It is interesting that the levels of sCD14, which are increased during HIV-1 infection, were correlated with decreased percentages of RM B cells and high IL-21R expression. In the plasma of HIV-1-infected individuals, a correlation was found between sCD14 and LPS levels. TLR activation of B cells in vitro resulted in IL-21R up-regulation.

Conclusions.  Microbial translocation and the associated immune activation during HIV-1 infection may lead to high expression levels of the IL-21R activation marker in RM B cells, a feature associated with increased apoptosis and a reduced number of these cells in the circulation.

Introduction

Loss of classical memory (CD27+) B cells and impaired long-term serological memory are major defects that develop during infection with human immunodeficiency virus type-1 (HIV-1) [1]. HIV-1-infected patients present with weakened humoral responses to pathogens and vaccination antigens, which are not fully restored by antiretroviral therapy (ART) [1–3]. In parallel, the B cell compartment of HIV-1-infected patients is characterized by an abnormal activation level, which occurs in association with loss of CD21 in B cells [4] and hypergammaglobulinaemia [5]. Increased B cell susceptibility to apoptosis [6–8] and exhaustion of these cells [9] are also features of HIV-1 infection. HIV-1 replication was shown to induce impaired B cell responses [10] via either polyclonal activation of B cells [11] or inhibition of class–switch recombination through HIV-1 proteins [12]. The mechanisms underlying the loss of memory B cells that occurs during HIV-1 infection need further elucidation to improve therapeutic and vaccine approaches aimed at inducing protective antibody levels.

Abnormally increased immune activation, a driving force of HIV-1 pathogenesis, is thought to arise from HIV-1 viraemia, lymphopenia and microbial translocation [13–15]. Depletion of CD4+ T cells, the major hallmark of HIV-1 immunopathology [16–18], was shown to correlate with T cell activation manifested by high levels of expression of HLA-DR, CD38 and Fas [19–21]. T cell immune activation is reduced, but not normalized, in patients receiving ART [22, 23]. Notably, disease progression during HIV-1 infection was reported to be associated with microbial translocation and immune activation [24, 25].

Recently, interleukin (IL)-21 production from CD4+ T cells of HIV-1-infected patients was found to be associated with control of viral load [26]. Secreted by follicular helper T cells in the lymphoid tissues, IL-21 is also an important cytokine for B cell functions [27, 28]. Naïve B cells constitutively express the IL-21 receptor (IL-21R), whereas memory B cells up-regulate the IL-21R following activation [29]. IL-21 signalling is involved in long-lived antibody responses [30] as suggested by the finding that B cell stimulation in the presence of IL-21 induces plasma cell differentiation [31]. During HIV-1 infection, IL-21 serum levels were decreased and found to be correlated with CD4+ T cell counts [32].

Human immunodeficiency virus type-1-infected patients display high serum levels of lipopolysaccharide (LPS) as the result of microbial translocation [33]; LPS-induced release of soluble CD14 (sCD14) from monocytes makes sCD14 a reliable marker of LPS bioactivity and thus, indirectly, a marker of immune activation induced by microbial translocation. The consequences of microbial translocation for T cell activation have been extensively studied, but the influence of this pathological event on the B cell compartment remains poorly defined. Moreover, the regulation of IL-21R expression in B cells during HIV-1 pathogenesis has not been investigated.

Here, we show that immune activation events that are probably induced by microbial translocation, as measured by increased plasma levels of sCD14 in HIV-1-infected patients, are associated with decreased percentages of circulating resting memory (RM) B cells and increased percentages of activated memory (AM) B cells. Expression levels of both IL-21R and CD38, markers of B cell activation, were higher in memory B cells from HIV-1-infected patients, especially those naïve to treatment, compared with healthy control subjects. Memory B cells expressing the IL-21R were more susceptible to spontaneous apoptosis and expressed lower levels of Bcl-2 expression than cells without this receptor. Of note, the increased IL-21R expression was associated with plasma levels of sCD14, and an inverse correlation was observed between the levels of IL-21R expression and the percentages of circulating RM B cells. The ability of microbial products to increase IL-21R expression was further confirmed in vitro by stimulating purified B cells with toll-like receptor (TLR) ligands. These results show a novel link between immune activation, increased IL-21R expression in memory B cells and loss of this important B cell subpopulation during HIV-1 infection.

Methods

Study participants

Peripheral blood was collected from healthy control subjects (n = 23) and HIV-1-infected individuals (n = 40). Amongst the HIV-1-infected patients, 20 were receiving ART [mean (range) duration of infection: 13.5 (1.5–25) years; duration of treatment: 8.15 (1.5–17) years; CD4+ T cell count: 619 (222–1412) cells mL−1; HIV RNA <50 copies mL−1] and 20 were naïve to treatment [mean (range) duration of infection: 5.4 (1.5–12.5) years; CD4+ T cell count: 600 (278–1357) cells mL−1; HIV RNA: 33 960 (233–132 000) copies mL−1]. ART consisted of the combination of two nucleoside analogues with one nonnucleoside analogue (n = 9) or one protease inhibitor (n = 2) or one integrase inhibitor (n = 7). Two HIV-1-infected individuals were treated with a combination of two nucleoside analogues with one nonnucleoside analogue and one integrase inhibitor. The ethical committee at Karolinska Institutet approved the study involving patient material. All study participants provided informed consent.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation (Lymphoprep; Axis-Shield PoC As, Rodelokka, Norway) and then stored frozen until required for analysis. Plasma was also collected from all donors.

B cell phenotype

Multi-colour flow cytometry was performed on PBMCs. B cell subpopulations were determined using the following fluorochrome-conjugated antibodies: PE-Cy5.5 or AmCyan anti-CD19, PE-Cy7 anti-CD10 (BD Bioscience, San José, CA, USA), V450 anti-CD27 (BD Horizon, San José, CA, USA) and PE-Cy5 anti-CD21 (BD Pharmingen, San Diego, CA, USA) in combination with PerCP anti-CD38 (Biolegend, Atlanta, GA, USA), PE anti-IL-21R and FITC Annexin V (BD Pharmingen).

B cell subpopulations were defined according to the expression of the following markers: total (CD19+), naïve (CD19+CD10−CD27−CD21+), classical memory (CD19+CD10−CD27+), resting memory (RM: CD19+CD10−CD27+CD21+), activated memory (AM: CD19+CD10−CD27+CD21−) and tissue-like memory (TLM: CD19+CD10−CD27−CD21−).

Intracellular staining was carried out using the CytoFix/Cytoperm solution kit according to the manufacturer’s instructions (BD Pharmingen) together with either FITC or PE anti-Bcl-2 (BD Pharmingen) and rabbit anti-Bim followed by Alexa-Fluor 667 anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA). Dead cells were excluded using the Live/Death Vivid detection kit labelled with a near-infrared dye (Invitrogen, Paisley, UK). Samples were fixed in 2% formaldehyde. Fluorescence intensities were measured with an LSR II flow cytometer (BD) and analysed using FlowJo version 8.8.7 (Three Star Inc., Ashland, OR, USA). Similar results were obtained in the present study from analyses performed with all markers using paired frozen and fresh samples (n = 5), consistent with a previous study of phenotypic characterization of B cells during HIV-1 infection [34].

Quantification of soluble markers

Plasma samples were assessed by IL-21 enzyme-linked immunosorbent assay (ELISA) using the eBioscience kit (e-Bioscience, San Diego, CA, USA), and sCD14 was measured using the Quantikine Human sCD14 kit (R&D System, Abingdon, UK), according to the manufacturers’ instructions. Plasma LPS levels were quantified using the Limulus Amoebocyte Lysate assay QCL-1000 (Lonza, Basel, Switzerland ).

In vitro B cell activation through BCR and TLRs

B lymphocytes from healthy individuals were separated using the Pan B cell Isolation Kit II (Miltenyi Biotech, Auburn, CA, USA). PBMCs and purified B cells (2 × 105) were cultured in RPMI-1640 containing l-glutamine, 10% foetal calf serum and antibiotics in the presence of 1 μg mL−1 Pam3CSK, 20 μg mL−1 Poly(I:C), 500 ng mL−1 LPS and 1 μg mL−1 CL075 (all from Invivogen, San Diego, CA, USA), as well as 4 μg mL−1 CpG (ODN2006, Invitrogen) or 1 μg mL−1 F(ab’)2 fragment goat anti-human IgA + IgG + IgM (Jackson ImmunoResearch, Suffolk, UK). At 24 h, cells were stained with PerCP-Cy5.5 anti-CD19 (Biolegend), V450 anti-CD27, PE anti-IL21R and PerCP or FITC (BD Pharmingen) anti-CD38 together with the Live/Death Vivid detection kit (Invitrogen). Samples were fixed and fluorescence intensities were measured as described previously.

Statistical analyses

Data from B cell subpopulations and their respective levels of IL-21R, Bim and Bcl-2 expression, as well as serum levels of IL-21 and sCD14, were analysed using Kruskal–Wallis test followed by Dunn’s multiple comparison test. Wilcoxon signed-ranked test was used for analysis between IL-21R-positive and IL-21R-negative B cells. Correlations were evaluated using the Spearman correlation test. A P-value <0.05 was considered significant. Statistical analyses and graphic representation of the results were performed using Prism (v.5.0b; GraphPad, La Jolla, CA, USA).

Results

B cell subpopulations display an activated and pro-apoptotic phenotype

The phenotype of B cell populations was determined by multicolour flow cytometry using the gating system as shown in Fig. S1A. Live B cells were gated on Vivid-CD19+ lymphocytes, and CD19+CD10+ B cells were excluded from the analyses. Consistent with previous work [1, 9, 35], there was a decreased percentage of circulating classical memory (CD27+) B cells and increased percentage of activated (AM) and tissue-like memory (TLM) B cells in HIV-1-infected individuals (Fig. S1B); although AM and TLM levels were normalized in treated patients, the loss of RM B cells was not completely reversed by the introduction of ART.

As IL-21R is expressed upon B cell activation and has been shown to be involved in B cell differentiation and survival [28], we measured IL-21R expression in B cells from patients and control subjects (Fig. 1a). In healthy subjects, the IL-21R is present mostly in naïve B cells, whereas classical memory B cells display a moderate expression of this receptor. In HIV-1-infected patients naïve to treatment, the levels of IL-21R were significantly increased in classical memory B cells (25.7 ± 10.3% vs. 15.8 ± 4.8% for control subjects; P < 0.001). Amongst memory B cells, RM cells showed the highest increase in IL-21R expression during HIV-1 infection (21.2 ± 12.3% in patients naïve to treatment, 19.2 ± 8.9% in treated patients and 14.4 ± 4.8% in control subjects); we also observed an increase in IL-21R expression in TLM B cells from viremic patients (67.5 ± 10.0%) compared with treated patients (56.7 ± 11.6%, P < 0.01) and control subjects (60.5 ± 11.9%).

Figure 1.

B cell subpopulations show an activated and pro-apoptotic phenotype. The phenotype of B cells was assessed by multicolour flow cytometry using peripheral blood mononuclear cells (PBMCs). Individual percentages (with means and standard deviations) of IL-21R (a) and CD38 (b) expression in total, naïve, classical memory, resting memory (RM), activated memory (AM) and tissue-like memory (TLM) B cells from healthy subjects (n = 20) and human immunodeficiency virus type-1 (HIV-1)-infected patients under treatment (n = 20) or naïve to treatment (n = 20). Individual levels of Bim expression (mean fluorescence intensity; MFI) (c) and percentages of Bcl-2-negative cells were determined (d) in B cells from healthy control subjects (n = 10) and HIV-1-infected patients under treatment (n = 10) or naïve to treatment (n = 10). Statistical analyses were performed using the Kruskal–Wallis test followed by Dunn’s comparison test: *P < 0.05, **P < 0.01, ***P < 0.001.

IL-21R levels in B cells remained slightly, but not significantly, increased in CD27+ and RM B cells after initiation of ART. No differential expression of IL-21R was observed in naïve and AM B cells in HIV-1-infected and control individuals. We then assessed whether there is a link between IL-21R expression in B cells and IL-21 plasma levels. Consistent with previous results [32, 36], IL-21 levels were lower in HIV-1-infected patients, both treated (77.8 ± 50.4 pg mL−1) and naïve to treatment (58.2 ± 25.8 pgmL−1, P < 0.01), compared with control subjects (99.4 ± 42.3 pg mL−1) (Fig. S2). However, no correlation was found between IL-21 levels and either the percentage of circulating B cells or IL-21R expression.

We also measured the expression of CD38, a known marker of immune activation [37] (Fig. 1b). Total B cells from HIV-1-infected patients naïve to treatment had significantly higher levels of CD38 expression (12.4 ± 7.6) compared with both treated patients and control subjects (5.8 ± 1.9 and 6.2 ± 3.5, respectively, P < 0.001 for both). High CD38 expression was mainly found in classical B cells from viremic patients compared with treated patients (P < 0.05) and control subjects (P < 0.001). A similar trend was observed for RM B cells, although this was not statistically significant. TLM B cells from HIV-1-infected patients had slightly lower CD38 expression levels than control subjects.

These results show that both IL-21R and CD38 expression are increased in memory B cells during HIV-1 infection. However, whereas ART impacts greatly on CD38 expression, IL-21R expression is only partially reduced by treatment. In line with these results, we found that CD38 expression in all B cell subsets, except AM, were negatively correlated with CD4+ T cell counts in the entire group of HIV-1-infected patients but were positively correlated with HIV-1 viral loads in patients naïve to treatment (Fig. S3); this finding suggests a role for HIV-1 viremia and lymphopenia in CD38 expression. Of importance, we could not find a correlation between the levels of IL-21R expression and either viral load or CD4+ T cell counts in HIV-1-infected patients (data not shown).

B cells isolated from HIV-1-infected patients are more susceptible to apoptosis [6, 7]. Bcl-2 and Bim are key molecules involved in cell survival. Bim is a pro-apoptotic molecule that binds Bcl-2 and inhibits its anti-apoptotic function [38]. Additionally, IL-21 has been implicated in Bim regulation [39]. Intracellular Bim expression was significantly higher in total, naïve and CD27+ B cells from HIV-1-infected patients, both treated and naïve to treatment, compared with control subjects (Fig. 1c). The levels of Bcl-2-negative cells were higher in total and naïve B cells from untreated HIV-1-infected patients, compared with both treated patients and control subjects (P < 0.01, Fig. 1d). It is interesting that Bcl-2 expression remained lower in CD27+ B cells from treated HIV-1-infected patients compared with control subjects (13.6 ± 4.8% and 5.7 ± 2.8% negative cells, respectively, P < 0.05, Fig. 1d). Amongst the memory B cell subpopulations, Bim was up-regulated in all subsets (Fig. 1c) during HIV-1 infection. By contrast, ART led to a decrease in Bcl-2-negative cells amongst RM B cells, whereas high percentages of Bcl-2-negative cells were found amongst AM and TLM B cells independent of ART exposure (Fig. 1d). These results show that ART does not impact on Bim expression levels in B cells but leads to a reduction in the percentages of Bcl-2-negative naïve and RM B cells. The cumulative effect of these events may lead to an expanded number of naïve B cells, as observed in treated HIV-1-infected patients (Fig. S1B).

High expression level of the IL-21R in B cells is associated with a decreased percentage of memory B cells during HIV-1 infection

Of note, we found an inverse correlation between the levels of IL-21R expression in classical memory B cells and the levels of these cells in the circulation for the two groups of HIV-1-infected individuals (P < 0.05 and P < 0.01 for treated patients and those naïve to treatment, respectively, Fig. 2a). Similarly, the percentage of RM B cells in HIV-1-infected patients were also inversely correlated with IL-21R expression in these cells (P < 0.05 and P < 0.01 for treated patients and those naïve to treatment, respectively). No correlation was found between the levels of CD38 expression in B cells and the levels of these cells in the circulation either in healthy individuals or in HIV-1-infected patients (Fig. 2b). As more classical and RM B cells expressed IL-21R, lower percentages of these B cell subpopulations were found in the circulation, suggesting a link between IL-21R expression in B cells and cell survival.

Figure 2.

 High expression level of the IL-21R in B cells is associated with reduced percentages of memory B cells during human immunodeficiency virus type-1 (HIV-1) infection. Correlation of IL-21R (a) and CD38 expression (b) in CD27+ (upper panels) and resting memory (RM) (lower panels) B cells and the percentages of these cells in the circulation in control subjects (n = 20) and HIV-1-infected patients treated (n = 20) and naïve to treatment (n = 20). Data were analysed using the Spearman correlation test.

Expression of IL-21R increases the susceptibility of B cells to apoptosis

To determine the possible involvement of IL-21R expression in B cell susceptibility to apoptosis, we measured annexin V in IL-21R-positive and IL-21R-negative B cell subpopulations (Fig. 3a). Apoptosis was not affected in total and naïve B cells by IL-21R expression. However, the ex vivo levels of annexin V were significantly higher in IL-21R-positive memory B cells (CD27+, AM, RM and TLM B cells) compared with B cells lacking the receptor both in patients and control subjects (P < 0.05). We found in addition that cells expressing IL-21R have lower concentrations of Bcl-2 (Fig. 3b). Low levels of Bcl-2 in B cells that express the IL-21R could explain their higher sensitivity to apoptosis.

Figure 3.

 IL-21R-positive B cells are susceptible to apoptosis. (a) Peripheral blood mononuclear cells (PBMCs) from control subjects (n = 10) and human immunodeficiency virus type-1 (HIV-1)-infected individuals treated (n = 8) or naïve to treatment (n = 10) were stainedex vivowith annexin V in combination with staining of the IL-21R and B cell markers. Percentages of annexin V-positive cells were determined in IL-21R-positive and IL-21R-negative total, naïve, classical memory, resting memory (RM), activated memory (AM) and tissue-like memory (TLM) B cells. (b) Levels of mean fluorescence intensity (MFI) for Bcl-2 were determined in IL-21R-negative and IL-21R-positive B cells from healthy control subjects (n = 4) and HIV-1-infected patients under treatment (n = 4) and naïve to treatment (n = 4) in total, naïve, classical memory, RM, AM and TLM B cells. Data are represented by box and whisker plots (minimum to maximum values). + represents the mean value. *P < 0.05, **P < 0.01, Wilcoxon signed rank test.

Plasma sCD14 correlates with loss of memory B cells and expression of the IL-21R and CD38 in memory B cells

To determine whether immune activation induced by microbial translocation could be associated with B cell defects, we measured plasma levels of sCD14. The sCD14 levels were higher in HIV-1-infected patients (Fig. 4a) with the highest levels found in patients naïve to treatment (884 ± 274 ng mL−1), compared with healthy individuals (600 ± 137 ng mL−1, P < 0.001). ART led to a slight decrease in sCD14 levels; treated HIV-1-infected patients, however, still had significantly higher sCD14 levels (746 ± 167 ng mL−1, P < 0.05) than control subjects. It is interest that a positive correlation was found between sCD14 levels and percentage of circulating AM B cells (P < 0.05), whereas a negative correlation was found between the sCD14 levels and RM B cells (P < 0.05, Fig. 4b). There was no correlation between sCD14 levels and percentage of either classical (Fig. 4b) or TLM B cells (data not shown). These results are novel and show an association between immune activation and both the increased percentage of AM B cells and decreased percentage of RM B cells during HIV-1 infection.

Figure 4.

 Plasma soluble CD14 (sCD14) correlates with both activation and loss of memory B cells. Levels of sCD14 were assessed by ELISA in plasma samples from healthy control subjects (n = 19) and human immunodeficiency virus type-1 (HIV-1)-infected patients under treatment (n = 20) or naïve to treatment (n = 19). (a) Data represent individual levels of sCD14 (ng mL−1 of plasma) and the mean and standard deviation. *P < 0.05, **P < 0.01, *P < 0.001, Kruskal–Wallis tests followed by Dunn’s comparison test. (b) The percentages of circulating classical memory (CD27+) B cells (left panels), activated memory (AM, middle panels) and resting memory (RM, right panels) B cells and their respective levels of IL-21R (c) and CD38 expression (d) were correlated with the levels of sCD14 in plasma. Data were analysed using the Spearman correlation test.

Next, we assessed whether the immune activation associated with microbial translocation during HIV-1 infection [33] also has a role in modulation of IL-21R and CD38 expression (Fig. 4c,d). It is interesting that the expression of the IL-21R in classical and RM B cells correlated with sCD14 levels (P < 0.01 and P < 0.05, respectively), whereas the expression levels of CD38 in classical and AM B cells correlated with sCD14 levels (P < 0.001 and P < 0.05, respectively). Furthermore, CD38 but not IL-21R expression in TLM cells was negatively correlated with the percentage of these cells in treated patients (data not shown).

We further evaluated microbial translocation by quantifying levels of LPS in the plasma specimens from the three patient cohorts (control subjects and patients who were treated and naïve to treatment). LPS levels were not significantly different between the groups (0.2082 ± 0.041, 0.2125 ± 0.05 and 0.2122 ± 0.043 EU mL−1 for control and treated and untreated HIV-1-infected individuals, respectively). LPS levels were, however, positively correlated with sCD14 levels measured in plasma from HIV-1-infected individuals (P < 0.05), confirming a possible link between microbial translocation and immune activation (Fig. S4). Of note, IL-21R expression in RM B cells was also positively correlated with LPS levels (P < 0.05).

Our findings of IL-21R up-regulation in memory B cells and loss of these cells from the circulation suggest a role for chronic immune activation and microbial translocation in the pathological events during HIV-1 infection.

TLR agonists directly induce IL-21R up-regulation in B cells

To evaluate the direct impact of microbial products on IL-21R and CD38 expression, B cells or PBMCs were incubated with TLR agonists (Fig. 5). The stimulation of TLR-9 and to a lesser extent TLR-4 (by CpG and LPS, respectively) led to increased levels of IL-21R expression in cultures of isolated B cells (Fig. 5a,b). CD38 expression remained unchanged after purified B cells were exposed to TLR ligands. By contrast, incubation of PBMCs with LPS and CpG induced higher levels of both IL-21R and CD38 expression in B cells. Both subsets of naïve and memory B cells similarly increased their IL-21R expression upon TLR stimulation (Fig. 5c); Pam3CSK4 and CpG (acting through TLR-2 and TLR-9, respectively), were the most potent stimuli for IL-21R up-regulation (change in mean fluorescence intensity (MFI) from 180 ± 66 to 304 ± 86 and 339 ± 32 for CD27+ B cells and from 162 ± 30 to 389 ± 104 and 444 ± 60 for CD27− B cells. LPS also induced higher IL-21R expression in isolated B cells or in PBMCs. However, Poly(I:C) and CL075, which bind TLR-3 and TLR-7, did not affect IL-21R expression. BCR triggering, on the other hand, led to higher IL-21R levels in memory (CD27+) B cells (MFI 549 ± 270), compared with CD27− naïve B cells (MFI 244 ± 30).

Figure 5.

 Toll-like receptor (TLR) stimulation induces IL-21R expression in B cells. Peripheral blood mononuclear cells (PBMCs) and purified B cells from three healthy donors were incubated at 1 × 106 cells mL−1 for 24 h in the presence of 1 μg mL−1 Pam3CSK (TLR-2 agonist), 20 μg mL−1 Poly(I:C) (TLR-3 agonist), 500 ng mL−1 lipopolysaccharide (LPS, TLR-4 agonist), 1 μg mL−1 CL075 (TLR-7 agonist), 4 μg mL−1 CpG (ODN2006) or 1 μg mL−1 anti-human IgA + IgG + IgM. A. Representative histograms of IL-21R and CD38 expression in CD19+ cells from purified B cells (a) or from PBMCs cultured in the presence of medium (light grey), LPS (dark grey) or CpG (black) (b). Isotype controls are shown in solid grey. Expression (mean fluorescence intensity; MFI) of IL-21R (c) and CD38 (d) in CD19+CD27+ (upper panels) and CD19+CD27− cells (lower panels) amongst purified B cells (black bars) or PBMCs (white bars) incubated with medium, TLR ligands or BCR agonist (see Methods). Data represent means ± SD of n = 3 independent experiments. Dotted lines represent the mean levels of IL-21R or CD38 expression in B cells cultured with medium.

In contrast to the expression of IL-21R, CD38 expression in both CD27+ and CD27− B cells was not affected by TLR activation of purified B cells (Fig. 5d). BCR stimulation of purified B cells, however, induced a dramatic increase in CD38 expression in memory B cells (MFI from 993 ± 289 to 3160 ± 2772). Only when the activation stimuli were applied to PBMC cultures, B cells (CD27+ and CD27−) showed higher CD38 levels after TLR activation with poly(I:C), LPS, CL075 or CpG, but not with Pam3CSK4. These results show a direct effect of TLR triggering on B cells to induce increased IL-21R expression, but not on CD38 expression, and therefore support the association between sCD14, as a marker of immune activation mediated by microbial translocation, and both high IL-21R expression and loss of memory B cells in HIV-1-infected individuals.

Discussion

Understanding the mechanisms leading to loss of memory B cells and serological memory during HIV-1 infection could potentially lead to new approaches for therapy and/or vaccination [40]. Microbial translocation, which occurs through the damaged gut epithelium, and the associated immune activation have been shown to play a major role in HIV-1 pathogenesis [13]. Furthermore, chronic immune activation was found to be a better predictor of disease progression than CD4+ T cell count [24]. Here, we have shown a novel link between immune activation in HIV-1-infected patients and the loss of RM B cells. Indeed, the level of plasma sCD14 was associated with both lower levels of RM and higher levels of AM B cells amongst circulating B cells.

As previously reported, the levels of sCD14 and LPS were correlated with the plasma of HIV-1-infected patients [13]. Of note, we also observed a correlation between the levels of both sCD14 and LPS and IL-21R expression in memory B cells, consistent with our finding of a direct induction of IL-21R expression in B cells by TLR stimulation. We found that TLR agonists induced up-regulation of the IL-21R but not CD38 in purified B cells, showing a direct impact of microbial products on levels of IL-21R in B cells. By contrast, CD38 expression in B cells was induced by TLR ligands only in PBMC cultures, probably due to the presence of other cell types, particularly macrophages.

Our results also show an association between IL-21R expression and the loss of memory B cells during HIV-1 infection. IL-21R expression was higher in both classical and RM B cell subsets, which correlated with loss of these cells in blood of both treated and nontreated HIV-1-infected patients. The higher susceptibility of IL-21R-positive B cells to apoptosis was confirmed by annexin V staining and the decreased expression of the anti-apoptotic molecule Bcl-2 in these cells. van Grevenynghe et al. [41] recently described Foxo3a as an important transcriptional factor for memory B cell survival during HIV-1 infection. These authors also showed an association between the levels of Foxo3a and Bim expression. Consistent with these results, we found a higher Bim expression in memory B cells from HIV-1-infected patients.

It is important to note that no link was found between the expression of CD38, which is marker of B cell activation, and loss of RM B cells. CD38 expression was highest in B cells from HIV-1-infected patients naïve to treatment; CD38 expression in B cells was also inversely correlated with CD4+ T cell count and positively correlated with viraemia in patients naïve to treatment. Whereas IL-21R expression appears to depend on microbial translocation, CD38 expression in B cells is most probably driven by HIV-1 and lymphopenia.

We and others have shown that IL-21 serum levels are decreased in HIV-1-infected patients [36], possibly leading to impaired B cell differentiation towards plasma cells. Indeed, it has been questioned whether long-lived plasma cells, responsible for antibody secretion several years after vaccination and natural infection, could be established in HIV-1-infected patients [42, 43]. IL-21 induces B cell differentiation towards plasma cells from both naïve and memory B cells [44] and is crucial in the formation of germinal centres [45]. During the course of HIV-1 infection, a high level of viral replication takes place in lymphoid organs, eventually leading to loss of follicular dendritic cells and CD4+ T cells in this compartment [46]. It remains to be established whether and how follicular helper T cells, the main producers of IL-21 [27], are affected by HIV-1 infection and whether IL-21 could be used as therapy to improve B cell immune responses.

In the present study, we have demonstrated a potential role of immune activation and microbial products in IL-21R expression in memory B cells. IL-21R-positive memory B cells express lower levels of Bcl-2 and are more susceptible to spontaneous apoptosis than cells without this receptor. Both immune activation, measured by serum sCD14 levels, and IL-21R expression in B cells were correlated with lower levels of circulating memory B cells. Therefore, in addition to its association with T cell defects observed during HIV-1 pathogenesis [33], microbial translocation and the consequent chronic immune activation may also result in impairment of the B cell compartment.

Acknowledgements

We thank Maarit Maliniemi and her colleagues at Venhälsan for their valuable help with the collection of samples from patients. This study was supported by grants received from the Swedish MRC, the Swedish International Development Agency (SIDA-SAREC) and the Fp6 EU Europrise Network of Excellence. Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. NR was supported through the Europrise network (PhD scholarship).

Conflict of interest statement

No conflict of interest to declare.

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