Differential partial activation phenotype and production of tumour necrosis factor-α by conventional dendritic cells in response to lipopolysaccharide in HIV⁺ viraemic subjects and HIV⁺ controllers

Abstract

HIV⁺ subjects are reported to have increased soluble CD14 (sCD14) in plasma, an indicator of microbial translocation. We evaluated if microbial translocation has a differential impact on the activation and function of conventional dendritic cells (cDC) from viraemic HIV⁺ subjects and HIV⁺ controllers (CTs). The HIV⁺ subjects were classified into two groups according to their plasma viral load (pVL): CT and viraemic. Subjects without HIV were included as controls (HIV^–). The frequencies and phenotypes of cDC from these subjects were evaluated by multi-parameter flow cytometry. In addition, peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) or single-stranded RNA40 (ssRNA40), the phenotype of the cDC and the intracellular production of tumour necrosis factor (TNF)-α by the cDC were evaluated by flow cytometry. We observed a partial activation phenotype for the cDC in the viraemic subjects and CTs ex vivo and after LPS activation, which showed differences in the expression of CD40 and CD86. Furthermore, in response to LPS the cDC from the viraemic subjects produced more TNF-α compared to the cDC from CTs. Interestingly, the percentage of TNF-α⁺ cDC was found to be correlated positively with the pVL. The partial activation of cDC and the over-production of TNF-α in response to LPS in viraemic HIV⁺ subjects might be related to the increased chronic activation observed in these subjects. In contrast, cDC from CTs seem to have a regulated response to LPS, indicating that they respond differently to chronic immune activation. These results may have implications in the development of HIV therapies and vaccines using DC.

Introduction

Dendritic cells (DC) play a central role in coupling innate to adaptive immune responses by sensing microbes, secreting cytokines, presenting antigens and activating T or B cells 1. All these characteristics make them potential candidates for cell-based therapies and vaccines 2,3. DC can be divided broadly into two groups: conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC); these subsets differ from each other in phenotype, Toll-like receptor (TLR) expression and function 4,5. pDC have been studied extensively in the context of human immunodeficiency virus (HIV) infection due to their association with the control of HIV replication and chronic immune activation, as these cells produce high levels of interferon (IFN)-α in response to HIV 6–11. In contrast, cDC have not been studied widely during HIV infection, especially with respect to their role in the natural control of HIV replication in HIV⁺ controllers (CTs) and their association with chronic immune activation.

cDC are recognized by their high antigen-presenting activity as well as their ability to prime naive T and B cells, facilitate T helper type 1 (Th1) polarization and induce CD8⁺ cytotoxic T cell immunity 4,12. Previous studies of cDC during HIV infection have shown an important reduction in the cDC frequency in the peripheral blood, which starts at the peak viral load and is maintained through the chronic phase of infection 7,13–18, and is not recovered after anti-retroviral treatment (ART) 15,17. Importantly, cDC depletion has been correlated with the HIV plasma viral load (pVL) 14,16,19,20. The cDC of viraemic subjects have been described as partially activated cells, based on their maturation and activation markers. Some studies have shown increased expression of CD40 and low expression of CD86, CD83 and CD80 21,22, while others have shown increased or unchanged expression of CD86 and CD83 ex vivo 14,17. The cDC of viraemic subjects have also been reported to have low stimulatory properties 7,13,18, a characteristic that is related most probably to their partial activation phenotype.

Impaired cytokine production in response to various stimuli, including TLR-7/-8 agonists, attenuated HIV and other HIV strains, has been shown, although some inconsistencies exist between reports 7,18,23–25. While the decreased production of interleukin (IL)-12 and tumour necrosis factor (TNF)-α after TLR-7/-8 stimulation during the primary phase of HIV infection has been reported 7, others have shown that DC are hyperresponsive to stimulation with different TLRs (TLR-4, -7, -8 and -9), with increased production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 18,26, a situation that has been confirmed in a non-human primate model of simian immunodeficiency virus (SIV) 27. A higher percentage of IL-12⁺ cDC is also observed after TLR-7/-8 stimulation 23. This hyperresponse to TLR stimulation suggests a close relationship between cDC and proinflammatory cytokine production. All this evidence supports that cDC are dysfunctional in viraemic HIV⁺ subjects. However, there are only few data on the phenotypical and functional features of cDC in HIV⁺ controllers 28,29.

cDC dysfunction cannot be attributed directly to HIV alone; other factors, including cytokines and other soluble plasma components, such as apoptotic microparticles and lipopolysaccharide (LPS), might also contribute to cDC dysfunction 30. Some of these soluble factors, especially LPS, are associated with the chronic immune activation observed during HIV infection.

Chronic immune activation is a hallmark of progression in HIV infection. Activation markers in T cells [CD38 and human leucocyte antigen D-related (HLA-DR)] correlate with disease progression, and this correlation is independent of other factors, such as the pVL 31–33. One of the major contributors to chronic immune activation is damage to mucosal barriers, which results in increased microbial translocation and can be determined by either the levels of LPS or bacterial DNA in the plasma 34,35. LPS is associated not only with the activation of T cell activation but also of monocytes which, in response, secrete soluble CD14 (sCD14), rendering sCD14 a reliable biomarker of chronic immune activation and mortality in HIV 34,36.

Thus, the continuous stimulation of DC by microbial translocation products may play an important role in their altered cytokine production. To further characterize the role of cDC in HIV immunopathogenesis, specifically their role in chronic immune activation, and to determine if microbial products have an impact on DC activation, we performed a phenotypical and functional characterization of cDC in viraemic subjects and CTs. CTs are a group of HIV⁺ individuals who are able to maintain a low pVL (<2000 RNA copies/ml of plasma) in the absence of ART and have lower levels of chronic immune activation markers than viraemic subjects 37,38, factors that might modulate cDC function and compromise the HIV response. Here, we report that despite a clear reduction in the cDC frequency in both the viraemic subjects and CTs, the cDC from these two groups showed a differential partially activated phenotype. In addition, the cDC from the viraemic subjects over-expressed TNF-α in response to LPS, which might be related to the increased chronic immune activation observed in these subjects. In contrast, the cDC from the CTs seemed to efficiently regulate their production of TNF-α, suggesting that these cells may respond differently to chronic activation.

Materials and methods

Blood samples and study population

This study was conducted at the Departamento de Investigación en Enfermedades Infecciosas of the Instituto Nacional de Enfermedades Respiratorias (INER) ‘Ismael Cosio Villegas’ (México City, Mexico) and was approved by the ethics committee of the INER (B33-10) according to the Declaration of Helsinki.

After written consent was obtained from the study participants, their peripheral blood was collected in acid citrate dextrose (ACD) tubes. The individuals with chronic HIV⁺ infections were divided into two groups according to their pVL: controllers (CTs), untreated HIV⁺ individuals maintaining a pVL of <2000 RNA copies/ml for at least 1 year (n = 18), and viraemic subjects, HIV⁺ individuals without anti-retroviral treatment and a pVL of >10 000 RNA copies/ml (n = 30). Subjects without HIV (HIV⁻) were included as a control group (n = 45). The CD4⁺ T cell counts were significantly lower in the viraemic HIV⁺ subjects than in the CTs (P = 0·0002). Individuals with known hepatitis C or B virus co-infection were excluded from the study.

Plasma HIV viral load and CD4 T cell counts

Routine HIV viral load and CD4⁺ T cell count assays were performed for the HIV⁺ subjects. The plasma viral load was quantified by automated real-time polymerase chain reaction (PCR) using a m2000 system (Abbott Laboratories, Abbot Park, IL, USA). The range of detection for the pVL was 40–10 000 000 copies/ml. The CD4⁺ T cell count was determined using a TruCount kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions and a fluorescence-activated cell sorter (FACS)Canto II cytometer (BD Biosciences).

Determination of cDC frequency and number

Freshly isolated peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation (Lymphoprep; Axis-Shield, Oslo, Norway) and stained with a combination of monoclonal antibodies: anti-CD3 fluorescein isothiocyanate (FITC) (clone UCHT1; eBioscience, San Diego, CA, USA), anti-CD14 FITC (clone 61D3; eBioscience), anti-CD19 FITC (clone HIB19; eBioscience), anti-CD56 FITC (clone B159; BD Biosciences), anti-HLA-DR APC-Cy7 (clone L243; BD Biosciences), anti-CD11c phycoerythrin-cyanin 5 (PE-Cy5) (clone B-ly6; BD Biosciences) and anti-CD123 peridinin chlorophyll (PerCP)-Cy5·5 (clone 7G3; BD Biosciences). Live/Dead Aqua dye (Invitrogen–Life Technologies, Carlsbad, CA, USA) was used to exclude dead cells. The cDC subset was identified as cells being CD3⁻, CD14⁻, CD19⁻ and CD56⁻ (LIN⁻), HLA-DR⁺ and CD11c⁺ cells.

In brief, the PBMCs (2−3 × 10⁶ cells) were washed with phosphate-buffered saline (PBS) and incubated for 20 min at 4°C with Live/Dead Aqua dye. Next, the cells were centrifuged (491 g for 10 min) and labelled for 30 min at 4°C with antibodies diluted in cell staining buffer (BioLegend, San Jose, CA, USA). The cells were then centrifuged (491 g for 10 min), fixed with 1% formalin, and analysed using a FACSAria II flow cytometer (BD Biosciences). A minimum of 2000 events of LIN⁻HLA-DR⁺ cells was acquired. All analyses were performed with FlowJo software version 7·6·2 (Tree Star, Inc., Ashland, OR, USA).

The number of cDC per microlitre of blood was calculated by multiplying the cDC frequency (% of live PBMCs) to the CD45⁺ cell count, which was determined using a TruCount kit (performed independently on whole blood samples using a FACSCanto II flow cytometer; BD Biosciences).

Determination of soluble CD14

Soluble CD14 (sCD14) was evaluated as an indicator of microbial translocation. For this experiment, we used plasma samples from HIV⁻ subjects, CTs and viraemic subjects that were collected and frozen immediately (−80°C) on the same day that the phenotypical or functional assays were performed. For the determination of the sCD14 level, a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) was used following the manufacturer's instructions. The sCD14 measurements were performed in an ELx808 absorbance microplate reader (BioTek, Winooski, VT, USA).

PBMC stimulation with TLR-4 and -8 ligands

Freshly isolated PBMCs from HIV⁻ subjects, CTs or viraemic subjects were suspended in R10 media (RPMI-1640; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Lonza), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at a density of 2 × 10⁶ cells/well. The cells were cultured in the presence or absence of 2 μg/ml LPS (Sigma-Aldrich, St Louis, MO, USA) or 5 μg/ml single-stranded RNA40 (ssRNA40) (InvivoGen, San Diego, CA, USA) for 18 h at 37°C with 5% CO₂. The cells or their supernatants were used for the determination of phenotype or cytokine production, as described in the following methods.

Determination of activation markers and TLRs by flow cytometry

Stimulated or ex-vivo PBMCs were stained with various monoclonal antibodies to identify the cDC subset (LIN⁻ HLA-DR⁺ CD11c⁺ cells) and to analyse the expression of two activation markers (CD86 and CD40) and CD47, a molecule that is expressed in response to IFN, by flow cytometry. The expression of TLR-4 and TLR-8 was also determined. The monoclonal antibodies used in this experiment were: anti-CD3 FITC or biotin (clone UCHT1; eBioscience), anti-CD14 FITC or biotin (clone 61D3; eBioscience), anti-CD19 FITC or biotin (clone HIB19; eBioscience), anti-CD56 FITC or biotin (clone B159; BD Biosciences), streptavidin Alexa Fluor V450 (eBioscience) or streptavidin PE Texas red (BD Biosciences), anti-HLA-DR allophycocyanin (APC)-Cy7 (clone L243; BD Biosciences), anti-CD11c PE-Cy5 (clone B-ly6; BD Biosciences) or Alexa 700 (clone 3·9; eBioscience), anti-CD123 PerCP-Cy5·5 (clone 7G3; BD Biosciences), anti-CD40 PE (clone 5C3; eBioscience), anti-CD86 PE (clone 2331-FUN-1; BD Biosciences) or PE-Cy5 (clone IT2·2; eBioscience), anti-CD47 FITC (clone B6H12; BD Biosciences), anti-TLR-4 Alexa 488 (clone HTA125, eBioscience) and anti-TLR-8 PE (clone 44C143; Imgenex, San Diego, CA, USA). The PBMCs were stained and analysed as described in previous section. TLR-8 expression was determined intracellularly using the BD Biosciences permeabilization kit (BD Biosciences). Fluorescence minus one controls were used to determine the negative/positive gates.

Determination of intracellular cytokines by flow cytometry

For the detection of intracellular cytokines, Golgi Plug (brefeldin A; BD Biosciences) was added to PBMCs after 4 h of stimulation with TLR ligands or to untreated cells. The production of TNF-α, IL-12 and IL-1β was then analysed by flow cytometry. To perform this, the cells were stained with monoclonal antibodies to identify the cDC (LIN⁻ HLA-DR⁺ CD11c⁺), natural killer (NK) cells (CD56⁺), B cells (CD19⁺), T cells (CD3⁺CD4⁺) and monocytes (CD14⁺). The following monoclonal antibodies were used: anti-CD3 biotin (clone UCHT1; eBioscience), anti-CD14 biotin (clone 61D3; eBioscience), anti-CD19 biotin (clone HIB19; eBioscience), anti-CD56 biotin (clone B159; BD Biosciences), streptavidin PE Texas red (BD Biosciences), anti-HLA-DR APC-Cy7 (clone L243; BD Biosciences), anti-CD11c Alexa 700 (clone 3·9; BD Biosciences), anti-CD123 PerCP-Cy5·5 (clone 7G3; BD Biosciences), anti-CD56 PE-CF594 (clone B159; BD Biosciences), anti-CD3 Alexa 700 (clone UCHT1; BD Biosciences), anti-CD16 PerCp Cy 5·5 (clone 3G8; BD Biosciences), anti-CD14 V450 (clone MϕP9; BD Biosciences), anti-CD19 APC (clone HIB19; BD Biosciences), anti-CD4 APC-Cy7 (clone RPA-T4; BD Biosciences), anti-TNFα FITC (clone MAb11; BD Biosciences), anti-IL-12 p40/p70 PE (clone C11·5; BD Biosciences) and anti-IL-1β FITC (clone H1b-98; Biolegend). After surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 30 min at room temperature. They were then washed twice with Perm/Wash solution (BD Biosciences) with centrifugation at 706 g for 10 min and incubated with anti-TNF-α and anti-IL-12, or with anti-IL-1β monoclonal antibodies for 60 min at room temperature. The cells were then washed and centrifuged (706 g for 10 min), fixed with 1% formalin and analysed using a FACSAria II flow cytometer (BD Biosciences).

Quantification of cytokines in culture supernatants

PBMCs were either untreated or stimulated in the absence of GolgiPlug, and their supernatants were collected after 18 h of stimulation with TLR ligands and frozen at −80°C. The supernatants were thawed and used to quantify TNF-α, IL-1β, IL-6 and IL-12 with a multiplex bead assay (Invitrogen–Life Technologies), following the manufacturer's instructions. The samples were analysed using a Luminex 200 xMAP system (Luminex, Austin, TX, USA) with Luminex xPONENT version 3·1 software (Luminex).

Statistical analysis

The data were analysed using the Mann–Whitney U-test for comparisons between groups, and correlations were evaluated with the Spearman test. Additional analysis was performed considering only males among groups; however, the gender was not determinant in the main findings. Differences were considered as statistically significant when P < 0·05. Non-parametric tests were performed using GraphPad Prism 5 software version 5·01 (La Jolla, CA, USA).

Results

Characteristics of the study participants

The demographic details of the subjects included in this study are shown in Table 1. This study included 30 viraemic subjects, 18 CTs and 45 HIV⁻ subjects. The median age was 36 years for the CTs and 30 years for the viraemic and HIV⁻ subjects. The group of HIV⁻ subjects was composed of 31 males and 14 females, the CT group comprised nine males and nine females and the viraemic group contained only males. The median pVLs were 23 106 and 57 RNA copies/ml for the viraemic and CT groups, respectively. The median CD4⁺ T cell count was 529 cells/μl for the viraemic subjects, 781 cells/μl for the CTs and 964 cells/μl for the HIV⁻ subjects.

Table 1.

Demographic details of HIV-1 cohort

Characteristic (n)	HIV− (45)	Controller (18)	Viraemic (30)
HIV plasma viral load; median (range) (RNA copies/ml)	n.a.	57 (<40–668)	23 106 (8373–447 952)*
CD4+ T cell count; median (range) (cells/μl)	964 (249–1493)	781 (417–1394)	529 (16–1149)*,**
Gender, male/female	31/14	9/9	30/0
Age; median (range) (years)	30 (23–52)	36 (25–66)	30 (22–58)
Frequency of cDC within PBMCs; median (range)	0·408 (0·122–0·763)	0·276 (0·167–0·551)**	0·280 (0·032–0·549)**
Absolute numbers of cDC; median (range) (cells/μl)	12·7 (8–18)	7·6 (3–14)**	5·0 (1–18)**

^*P < 0·05 compared to controllers;

^**P < 0·05 compared to HIV⁻ subjects. cDC = conventional dendritic cells; PBMCs = peripheral blood mononuclear cells; n.a. = not applicable.

The cDC number is reduced in HIV⁺ subjects and plasma level of soluble CD14 correlates with the viral load

It has been reported consistently that the DC frequency is reduced in peripheral blood (PB) of HIV⁺ subjects 7,13–18. To determine if there is a difference between the number of cDC in CTs and viraemic subjects, we initially analysed the frequency of cDC in the PB of the CTs and viraemic subjects and compared them with the HIV⁻ subjects. The gating strategies used for the DC analysis as well as representative plots are shown in the Supporting information, Fig. S1. As shown in Table 1 and Fig. 1a, there is a significant decrease in the percentage of cDC in the PBMC populations of the CTs (P = 0·0429) and viraemic subjects (P = 0·0029) when compared with that in the HIV⁻ subjects. These differences were confirmed by evaluating the cDC numbers. A significant reduction was observed for the cDC of the CTs (P = 0·0032) and viraemic subjects (P = 0·0019) compared with the HIV⁻ subjects (Fig. 1b). Notably, no difference was found between the percentage or number of cDC between the CTs and viraemic subjects. To confirm that viraemic subjects have more microbial translocation than CTs, as reported by others 37,38, we evaluated the plasma levels of soluble CD14 (sCD14), a marker of microbial translocation 34, in our study groups. Figure 1c shows an increase in the level of sCD14 in the plasma of the viraemic subjects compared with the HIV⁻ subjects (P = 0·0008); this increase was not observed in the plasma of the CTs. In addition, an association between the pVL and the level of sCD14 was observed (r = 0·4400; P = 0·0191) (Fig. 1d). Overall, these results indicate that although both CTs and viraemic subjects have an important reduction in the number of cDC in their PB compared with HIV⁻ subjects, the difference in the level of sCD14, which correlates with the pVL, suggests a different level of microbial translocation between viraemic subjects and CTs that could impact cDC activation.

Fig. 1.

Reduced frequency of conventional dendritic cells (cDC) in HIV⁺ subjects and correlation of sCD14 with plasma viral load (pVL). Peripheral blood mononuclear cells (PBMCs) were obtained from HIV^– subjects (n = 29), controllers (CT, n = 18) and viraemic subjects (V, n = 21) and were stained ex vivo to determine the frequency (a) and number (b) of cDC. (c) The level of soluble CD14 (sCD14) was determined in plasma from HIV- subjects (n = 12), CTs (n = 14) and V subjects (n = 14). (d) The correlation between the pVL and the plasma sCD14 concentration in CTs (squares) and V subjects (triangles) is shown. Males (closed symbols), females (open symbols). The data were analysed with the Mann–Whitney U-test and Spearman test, respectively, and were considered significantly different when P < 0·05.

Differential expression of activation markers on cDC from viraemic subjects and controllers

Considering that we observed a positive correlation between the level of sCD14 in the plasma and the pVL, we next evaluated if the phenotype of the remaining cDC differs between viraemic individuals and CTs. Figure 2a shows a representative histogram in which a slight increase in the CD40 expression was observed for the cDC of a viraemic subject compared with those for the cDC of a CT and an HIV⁻ subject. When evaluating the median fluorescence intensity (MFI) for CD40 expression, we observed no differences among the three study groups (Fig. 2a). However, a significant increase in the percentage of CD40⁺ cDC was observed in the viraemic subjects when compared to the CTs (P = 0·0478; P = 0·0766 when only males were compared between these groups) (Fig. 2a). CD86 analysis showed that while all the cDC expressed CD86 (Fig. 2b), an increase in the MFI for CD86 was observed in the cDC of the CTs compared with that of the HIV⁻ subjects (P = 0·0011; Fig. 2b). In addition, we observed an increase in the MFI for CD47, a protein that is expressed in response to IFN 39, in the CTs compared with the viraemic (P = 0·0218) and HIV⁻ subjects (P = 0·0013; Fig. 2c). Interestingly, we found a positive correlation between the expression of CD86 and CD47 (r = 0·5644; P = 0·0005), suggesting a relationship between the expression of these two markers on cDC (Supporting information, Fig. S2). Overall, these results strongly suggest a partially activated phenotype for cDC from both CTs and viraemic subjects, with a differential expression of surface markers, showing that cDC from viraemic subjects predominantly express CD40 and that cDC from CTs express CD86 and CD47.

Fig. 2.

Differential expression of activation markers on conventional dendritic cells (cDC) from viraemic subjects and controllers. Peripheral blood mononuclear cells (PBMCs) were obtained from HIV^– individuals (n = 22), controllers (CT, n = 16) and viraemic subjects (V, n = 18) and were stained ex vivo to determine the expression of CD40 (a), CD86 (b) and CD47 (c) on the cDC in the samples by flow cytometry. A representative histogram from one individual in each group is shown on the left. The values of the median fluorescence intensity (MFI) are included. The graphs show the MFI and the percentage of positive cells within the cDC population for each marker. Males (closed symbols), females (open symbols). The data were analysed with the Mann–Whitney U-test. FMO = fluorescence minus one. The data were considered significantly different when P < 0·05.

Differential expression of surface markers on cDC in response to LPS between viraemic subjects and controllers

To evaluate if the differential expression of surface markers on cDC observed in the CTs and viraemic subjects is a consequence of their different responses to TLR stimulation, we next analysed surface marker expression after the stimulation of PBMCs from these individuals with LPS or ssRNA40 as ligands of TLR-4 and TLR-8, respectively. Fig. 3a shows an increase in CD40 expression on the cDC from a viraemic subject and a HIV⁻ subject after LPS activation. In contrast, a lesser increase in CD40 expression was observed for the cDC from a CT. When evaluating the fold increase in CD40 expression in the different study groups, we observed that the cDC from the CTs showed a slight increase in the expression of CD40 after LPS activation (Fig. 3b). In addition, we observed a significant difference in the fold increase of CD40 expression between the viraemic subjects and CTs (P = 0·0400; P = 0·3037 when only males were compared between these groups), between the CTs and HIV⁻ subjects (P = 0·0005) and between viraemic and HIV⁻ subjects (P = 0·0451; Fig. 3b). Similar results were observed after stimulation with ssRNA40, although no significant differences were observed between the CTs and viraemic subjects (Fig. 3c). Furthermore, similar to our ex-vivo observations, the cDC of CTs showed high expression of CD86 even without stimulation, but only a slight increase was observed after LPS activation. In contrast, the cDC of viraemic subjects and of HIV⁻ subjects showed a lower expression of CD86 without stimulation, which clearly increased after LPS activation in the HIV⁻ subject but only slightly increased in the viraemic subject (Fig. 3d). When examining the fold increase in CD86 expression in the different subjects, we found that the fold increase in the expression of CD86 after LPS stimulation in the viraemic subjects was significantly lower than the fold increase observed in the HIV⁻ subjects (P = 0·0232; Fig. 3e) and a significant difference between the CTs compared with the HIV⁻ subjects (P = 0·0112); similar results were observed after ssRNA40 activation (Fig. 3f). These results suggest that cDC from viraemic subjects and CTs have a differential expression of surface markers in response to TLR-4/-8 stimulation. The response of cDC from CTs seems to be regulated, as they showed a lower increase in the expression levels of CD40 and CD86 than the viraemic group. In contrast, the response of cDC from viraemic subjects, compared to CTs, is characterized by an increase in the expression of CD40 but not CD86, suggesting that the ex-vivo phenotype observed for the cDC of the viraemic subjects could be related to their chronic immune activation, which is mediated partially by microbial translocation.

Fig. 3.

Differential expression of surface markers on conventional dendritic cells (cDC) after lipopolysaccharide (LPS) or single-stranded RNA40 (ssRNA40) stimulation in viraemic subjects and controllers. Peripheral blood mononuclear cells (PBMCs) were obtained from HIV^– individuals, controllers (CT) and viraemic subjects (V), either left untreated or stimulated with LPS or sRNA40 for 18 h and then stained to determine the expression of CD40 (a) and CD86 (d) on the cDC in the samples by flow cytometry. Representative histograms from each LPS-stimulated group are shown on the left. The values of MFI are included. The fold increase in CD40 (b,c) and CD86 (e,f) expression on cDC from HIV⁻ individuals, CTs and V subjects after 18 h of LPS (b,e) or ssRNA40 (c,f) stimulation was determined. Fold increase was defined as the median fluorescence intensity (MFI) of the stimulated cDC divided by the MFI of the unstimulated cDC. n = 13 for HIV⁻, n = 9 for CT and V. Data are shown as box and whiskers: min to max. The data were analysed with the Mann–Whitney U-test. FMO = fluorescence minus one. The data were considered significantly different when P < 0·05.

TNF-α production by cDC in response to LPS differs between viraemic subjects and controllers and correlates with the viral load

To examine if the differential expression of surface markers in CTs and viraemic subjects both ex vivo and after in-vitro activation is related to functional differences, we next evaluated the intracellular expression of TNF-α by cDC after PBMCs activation with LPS or ssRNA40. Figure 4a shows representative dot-plots of the TNF-α⁺ cDC after LPS activation, with a similar percentage of TNF-α⁺ cDC between the CT and HIV⁻ subject and a higher percentage of TNF-α⁺ cDC in the viraemic subject. When evaluating the different study groups, we observed that the percentage of TNF-α⁺ cDC in the CTs is similar to the percentage of TNF-α⁺ cDC in the HIV⁻ subjects after LPS activation, although the basal percentage of TNF-α⁺ cDC was significantly higher in the CTs compared with the HIV⁻ subjects (P = 0·0113). Notably, we found a significant increase in the percentage of TNF-α⁺ cDC in the viraemic subjects after LPS activation when compared to that in the CTs (P = 0·0005) and HIV⁻ subjects (P = 0·0005; Fig. 4b). After activation with ssRNA40, we found that the percentage of TNF-α⁺ cDC was similar among the different groups (Fig. 4c). To determine if these findings are associated with the clinical parameters of the HIV⁺ subjects, we evaluated the relationship between the percentage of TNF-α⁺ cDC with the HIV pVL and CD4⁺ T cell count. Interestingly, a positive correlation was found between the percentage of TNF-α⁺ cDC and the plasma pVL (r = 0·6421; P = 0·0004; Fig. 4d), whereas no correlation was found between the percentage of TNF-α⁺ cDC and the CD4⁺ T cell count (r = −0·3655; P = 0·0664; Fig. 4e). We did not find any difference in the expression of IL-12 after activation with LPS; however, after ssRNA40 stimulation we found a lower frequency of IL-12⁺ cDC from CTs when compared to HIV⁻ subjects (P = 0·0483; Supporting information, Fig. S3). The strong response to LPS observed in viraemic subjects was not due to a higher expression of TLR-4 on cDC of this group, compared with CTs and HIV^– (Supporting information, Fig. S4). These results indicate that TNF-α expression by cDC in response to LPS differs between viraemic subjects and CTs, suggesting that the cDC from viraemic subjects, but not CTs, are hyperresponsive to LPS. In addition, these results show that the expression of TNF-α by cDC is associated with the pVL.

Fig. 4.

Differential tumour necrosis factor (TNF)-α production by conventional dendritic cells (cDC) in response to lipopolysaccharide (LPS) stimulation between viraemic subjects and controllers and correlation of the plasma viral load (pVL). Peripheral blood mononuclear cells (PBMCs) isolated from HIV⁻ individuals, controllers (CT) and viraemic subjects (V) were stimulated with LPS or single-stranded RNA40 (ssRNA40) for 18 h. Intracellular cytokine production was then analysed in the cDC population. (a) Representative dot-plots of intracellular TNF-α⁺ positive cDC after LPS stimulation. The graphs show the percentage of TNF-α⁺ cDC in the samples from the HIV⁻ individuals (n = 16), CTs (n = 13) and V subjects (n = 11) after LPS (b) or ssRNA40 (c) stimulation. Correlation of the TNF-α⁺ cDC frequency in response to LPS with the pVL (d) and CD4⁺ T cell count (e); CTs (squares) and V subjects (triangles) are shown. Males (closed symbols), females (open symbols). The data were analysed with the Mann–Whitney U-test for comparisons between groups and with the Spearman test for correlations. The data were considered significantly different when P < 0·05.

cDC comprise the main cell population that over-expresses TNF-α in response to LPS in viraemic subjects

Next, we determined the level of TNF-α and other proinflammatory cytokines, such as IL-1β, IL-6 and IL-12, in the supernatants of PBMCs stimulated with LPS or ssRNA40. As shown in Fig. 5a, there is a higher level of TNF-α in the supernatants of the PBMCs from viraemic subjects after LPS activation in comparison with those in the supernatants of the PBMCs from CTs (P = 0·0004) and HIV⁻ subjects (P = 0·0006; Fig. 5a). In addition, we found lower TNF-α in the supernatants of CTs compared with HIV⁻ subjects after ssRNA40 activation (P = 0·0374; Fig. 5b). An increase in the production of IL-1β was also observed in supernatants of the viraemic samples in response to LPS when compared with the CT samples (P = 0·0043; Supporting information, Fig. S5). We did not find differences in the levels of IL-6 among studied groups. Similar to our observations of the intracellular staining for IL-12, we did not observe differences in the secretion of IL-12 from the PBMCs after LPS stimulation (Supporting information, Fig. S5a). Although a significant increase in the production of the different proinflammatory cytokines was observed in all groups after ssRNA40 stimulation compared with unstimulated cells, no significant differences were observed between the different groups (Supporting information, Fig. S5b).

Fig. 5.

Conventional dendritic cells (cDC) compose the cell population that differentially produces tumour necrosis factor (TNF)-α after lipopolysaccharide (LPS) stimulation in viraemic subjects. TNF-α concentration in the culture supernatants of peripheral blood mononuclear cells (PBMCs) that were either left untreated or stimulated with LPS or single-stranded RNA40 (ssRNA40). The production of TNF-α in the HIV⁻ (n = 13), controllers (CT, n = 10) and viraemic (V, n = 11) samples after 18 h of stimulation with LPS (a) or ssRNA40 (b) was determined by LUMINEX assay. (c) Representative dot-plot of TNF-α⁺ natural killer (NK) cells (CD56⁺), B cells (CD19⁺), T cells (CD3⁺CD4⁺) or monocytes (CD14⁺) after 18 h of treatment with or without LPS. (d) Percentage of TNF-α⁺ CD14⁺ cells after LPS or ssRNA40 stimulation (n = 11 for HIV⁻ subjects, n = 6 for CTs and n = 5 for V subjects). Males (closed symbols), females (open symbols).The data were analysed with the Mann–Whitney U-test and were considered significantly different when P < 0·05.

To evaluate the contribution of the different cell populations to the expression of TNF-α and IL-1β, we performed intracellular staining for TNF-α and IL-1β in CD56⁺ (NK cells), CD19⁺ (B cells), CD4⁺ (T cells) and CD14⁺ (monocytes) cells. The gating strategies and representative plots for the IL-1β production are shown in Supporting information, Figs S6 and S7. As shown in Fig. 5c and NK, B and T cells were not an important source of TNF-α after LPS stimulation. In contrast, we found a high percentage of TNF-α⁺ and IL-1β⁺ monocytes (CD14⁺) after LPS and ssRNA40 stimulation (Fig. 5c,d and Supporting information, Fig. S7). However, in contrast to our results for the cDC (Fig 4a,b), there was no significant difference between the percentage of TNF-α⁺ monocytes among the studied groups (Fig. 5d). In addition, we found that cDC from viraemic subjects are also high producers of IL-1β, compared with CT and HIV⁻ subjects (Supporting information, Fig. S7). These results indicate that cDC comprise the primary cell population with a differential expression of TNF-α and probably of IL-1β between viraemic subjects and CTs after TLR-4 stimulation. Together, these results indicate an increase in the production of TNF-α and IL-1β in viraemic subjects compared with CTs after TLR-4 stimulation and that cDC compose the main cell population that over-expresses TNF-α in viraemic subjects.

Discussion

Chronic activation of the immune system has been reported to play a pivotal role in the pathogenesis of HIV infection 31,33,36,37,40,41. In addition, it is known that innate immune cells can also be activated by TLRs, with the consequent over-production of proinflammatory cytokines such as IL-6 and TNF-α 26,29,42. However, it is still uncertain if cDC also participate in the chronic immune activation observed in HIV⁺ individuals as well as in the pathogenesis of this disease. In this report, we show a differential phenotype and expression of TNF-α by cDC in response to LPS between viraemic HIV⁺ subjects and HIV⁺ CTs, which may have implications in the pathogenesis of HIV infection. Our results suggest that cDC from viraemic subjects become hyperresponsive to TLR-4 activation, while cDC from HIV⁺ CTs have a more controlled response to TLR-4 stimulation, which might be related to their reduced chronic immune activation.

Initially, the cDC frequencies in the PB samples from our study group of HIV-infected subjects were evaluated and found to be similar to those in other reports 15,17,18,20. Notably, we did not find any differences in this parameter between the viraemic subjects and CTs, indicating that the reduction in cDC that has been reported to occur during the early stages of HIV infection 7,18 is maintained during chronic infection independently of the pVL. In addition, we found an increase in the levels of sCD14 in the plasma of the viraemic HIV⁺ subjects, as reported by others 36,43. This finding led us to suggest that the presence of LPS, as a result of microbial translocation 34, might have an impact on the activation of the remaining cDC. As reported previously, 17,21,22,28,44, we found that the cDC of the viraemic HIV⁺ subjects showed a partially activated phenotype compared to the cDC of the HIV⁻ individuals and HIV⁺ CTs. Notably, a different partially activated phenotype was observed in the cDC from the CTs, which showed increased expression of CD86 and CD47 but not CD40. The phenotypical ex-vivo staining of the cDC also indicated that the differential expression of surface markers between the CTs and viraemic subjects could be the consequence of different activation pathways. The ex-vivo phenotype observed for the cDC of the CTs suggests that cDC activation in CTs might result from a regulated activity of type I IFN, as it has been reported that IFN plays an important role in HIV pathogenesis during the early phase of HIV/SIV infection 45–49. Signatures of IFN activity, such as IFN-inducible proteins and genes, have been reported as markers of HIV infection 48,50,51. Accordingly, we found an increase in the expression of CD47, a protein that is expressed in response to IFN 39 in the cDC of the CTs. Furthermore, a positive correlation was observed between the expression of CD86 and CD47. It has been demonstrated in mice that CD47 expression in cDC is essential for their migration to LNs and to sites where T cell priming occurs 52 and that its expression in DC limit proinflammatory cytokine production 53. The consequences of the differential partially activated phenotype observed here between cDC from viraemic subjects and CTs on the activation of T cells required further detailed investigation, as it is known that T cells from HIV CTs have polyfunctional and strong responses against HIV 54–57.

In this study, we approached this question by evaluating the differences in the phenotype and cytokine production of cDC of HIV⁺ viraemic subjects and CTs in response to TLR stimulation with LPS or ssRNA40. Importantly, it has been suggested that phenotypical maturation involving the up-regulation of co-stimulatory molecules, rather than the production of cytokines, plays a major role in the activation of memory T cells 42. After in-vitro TLR-4 or TLR-8 stimulation, we showed different patterns of phenotypical maturation markers in the cDC. We observed that the cDC from the viraemic subjects had increased expression of CD40 but not of CD86. In contrast, the cDC from the CTs showed only a slight increase in both CD40 and CD86 expression. It is known that the expression of CD40, CD86 and CD80 is needed for the expansion of antigen-specific T cells, CD40 expression in DC is required for CD40L ligation and maturation of T cells, while CD86 and CD80 expression is required for CD4⁺ T cell proliferation upon antigen stimulation 21. However, the relevance of expressing one molecule versus another in a DC and its interaction with or activation of a T cell is still not known. Furthermore, our results suggest that the expression of surface markers after activation might be related to gender, as some significant differences were lost when only males were compared among groups; therefore, further studies are necessary to address these points.

Interestingly, our results also showed that the partially activated phenotypes observed in the HIV⁺ CTs and viraemic HIV⁺ subjects had functional differences, mainly in response to LPS. The cDC from the viraemic HIV⁺ subjects showed an increased production of TNF-α compared with those from the CTs and HIV⁻ individuals, indicating a hyperresponse to LPS. Although the CT and viraemic cDC showed a high basal expression of TNF-α compared with the HIV⁻ subjects, the expression of TNF-α after LPS stimulation was similar between the CTs and HIV⁻ subjects, which was lower compared to the viraemic subjects, suggesting that CTs employ a regulatory mechanism that avoids the increased production of TNF-α. Interestingly, a positive correlation was found between the percentage of TNF-α⁺ cDC in response to LPS and the pVL and no correlation was found between this percentage and the CD4⁺ T cell count, indicating that the high frequency of TNF-α⁺ cells could be explained in part by the pVL and thus by the consequent TLR activation pathways. Consistent with this finding, it has been reported that high levels of TNF-α⁺ could have a deleterious impact on the number of CD4⁺ T cells 58–61 and that TNF-α induces viral replication in the CD4⁺ T cell lymphocytes of HIV-infected subjects by activating the nuclear factor (NF)-κB pathway 62,63, favouring the high pVL observed in viraemic individuals. We could not find any differences in the production of IL-12 when the cDC of the viraemic subjects and CTs were stimulated with either LPS or ssRNA40. IL-12p70 has been demonstrated to be optimally expressed only after simultaneous stimulation with IL-1β, TNF-α and IFN-γ 25, suggesting that IL-12 production is a consequence of the prolonged stimulation of multiple proinflammatory cytokines, a condition that was not tested in this study. The fact that the levels of IL-12 were not increased in the cDC from the viraemic HIV⁺ subjects after TLR-4 stimulation suggests that the hyperresponse to LPS is caused mainly by the over-production of TNF-α and possibly of IL-1β, as we also observed an increase of this cytokine in cDC from viraemic subjects.

In this study, we found that the culture supernatants of LPS-treated PBMCs showed a significant increase in TNF-α, but only for the samples from the viraemic subjects. It has been reported that large amounts of TNF-α participate in the destruction of intestinal epithelial cells 64,65, leading to the increase in microbial translocation observed in HIV⁺ viraemic individuals. Our results showed that monocytes (CD14⁺ cells) also produced high amounts of TNF-α and IL-1β in response to LPS; however, we did not find any differences in the production of these cytokines by the monocytes from the viraemic, CT and HIV⁻ subjects. It was reported previously that MDC-8⁺ monocytes are increased in viraemic HIV⁺ subjects and that this population is responsible for the increased production of TNF-α⁺ after LPS activation 66. However, we did not evaluate the production of TNF-α by MDC-8⁺ cells in particular. Therefore, it is possible than in addition to cDC, this population of monocytes or monocyte-derived inflammatory DC also contributes to the over-production of TNF-α in viraemic HIV⁺ subjects.

Our results suggest that despite the fact that we did not find any significant difference in the levels of sCD14 in CTs versus viraemic HIV⁺ subjects, it is possible that even in the presence of LPS, cDC from HIV⁺ CT subjects may have a regulated response that avoids their exacerbated activation. The combination of more than one factor that favours chronic immune activation, such as the high pVL and high microbial translocation of viraemic individuals, might be responsible for the hyperresponse of cDC to LPS that is found only in viraemic subjects and not in CTs. A previous study showed that if PBMCs from viraemic subjects were incubated with ssRNA40 and LPS, TNF-α production was higher than if the cells were incubated with LPS or ssRNA separately 67. It has been shown that TLR-4 expression is correlated positively with the pVL but not with LPS in plasma 26,67. Thus, as CTs have a low pVL, they are expected to have a lower expression level of TLR-4 compared to viraemic subjects; however, we did not find differences in TLR-4 expression on cDC between CTs and viraemic HIV subjects. Therefore, the controlled response of cDC from CTs might be related to the TLR signalling pathway.

Overall, our results suggest that the partial activation of cDC and the over-production of TNF-α in response to LPS in viraemic HIV⁺ subjects might be related to the increased chronic immune activation observed in these subjects. In contrast, cDC from HIV⁺ CTs seem to have a regulated response to LPS. It is well known that TLR activation induces a proinflammatory response against microbes, and after elimination the system must return to a homeostatic state 68. Proper control and inhibition of the TLR signal is important for limiting excessive inflammation during immune responses against microorganisms 69,70. The results shown here suggest that cDC from CTs are TLR-responsive, i.e. they are able to control the production of inflammatory mediators, avoiding damage to the immunological system. The homeostatic control of TLR signalling involves regulation of TLR trafficking within the cell, degradation of TLR molecules and sequestration of adapter proteins, among others 61,70. Further experiments are required to describe the exact mechanism by which TLR-4 signalling is controlled. Thus, considering the use of cDC in alternative therapies or vaccines for controlling HIV replication, it would be ideal to find a controlled response to TLR stimulation that is similar to that observed in the cDC of HIV⁺ CTs.

Disclosure

The authors declare that they have no conflicts of interest.

Author contributions

P. M. D. R. E., G. R. T. and L. B. conceived and directed the project. L. B., R. C. and P. M. D. R. E. designed the experiments. R. C. and A. R. A. performed the experiments. R. C. analysed the data. G. R. T., P. M. D. R. E. and L. B. A. contributed reagents/materials/analysis tools. P. M. D. R. E., R. C. and L. B. A. wrote the paper.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. Gating strategy to identify dendritic cell (DC) subsets.

Fig. S2. CD86 and CD47 expression are correlated positively.

Fig. S3. Frequency of IL-12 + conventional dendritic cells (cDC) in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMCs) is similar between viraemic subjects and controls.

Fig. S4. Toll-like receptor (TLR)-4 and TLR-8 expression in conventional dendritic cells (cDC).

Fig. S5. Interleukin (IL)-1β cytokine production by peripheral blood mononuclear cells (PBMCs) after lipopolysaccharide (LPS) stimulation differs between controls and viraemic subjects.

Fig. S6. Gating strategy to identify B cells, natural killer (NK) cells, CD4⁺ T cells and monocytes.

Fig. S7. Differential frequency of interleukin (IL)-1β in conventional dendritic cells (cDC) after lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMCs) between viraemic subjects and controllers.