Summary
The objective of this study was to conduct an analysis of peripheral blood Th17 cells with the ability to home to gut mucosa (CD4+Th17+β7+) during recent or chronic human immunodeficiency virus (HIV) infections. The relationship between HIV load and systemic inflammation markers was studied. Twenty‐five patients with recent (n = 10) or chronic (n = 15) untreated HIV infections; 30 treated HIV‐infected patients with undetectable HIV load at the time of inclusion and 30 healthy controls were included. Bacterial translocation markers (16S rDNA), soluble CD14 (sCD14) and interleukin (IL)‐6 monocyte activation parameters, CD4/CD8 ratio and T helper type 17 (Th17) subpopulations [CD4+Th17+ expressing the IL‐23 receptor (IL‐23R) or β7] were analysed at baseline and after 6 and 12 months of anti‐retroviral therapy (ART). 16S rDNA was detected in all patients. Significantly increased serum levels of sCD14 and IL‐6 and a decreased CD4/CD8 ratio were observed in patients. Similar percentages of CD4+IL‐23R+ and CD4+Th17+β7+ cells were observed in healthy controls and patients at baseline. After 12 months of therapy, patients with a recent HIV infection showed significant increases of CD4+IL‐23R+ and CD4+Th17+β7+ cell percentages and a decrease in IL‐6 levels, although 16S rDNA continued to be detectable in all patients. No significant differences were observed in Th17 subpopulations in patients with chronic HIV infection after therapy. Early initiation of ART helps to increase the number of Th17 cells with the ability to home to the intestinal mucosa and to partially restore gut mucosal homeostasis. These results provide a rationale for initiating ART during the acute phase of HIV infection.
Keywords: bacterial translocation, beta7 homing receptor, chronic infection, HIV, intestinal barrier, recent infection, Th17 population
Introduction
T helper type 17 (Th17) cells, a subset of effector CD4+ T cells that promote inflammation, are enriched in the lamina propria of the gastrointestinal tract and play an important role in the defence against microbes at mucosal surfaces by stimulating inflammatory cytokine release, chemokine expression, recruitment of neutrophils and production of anti‐bacterial defensins 1. Because a sizeable portion of Th17 cells express HIV co‐receptor C chemokine receptor type 5 (CCR5) 2, they are HIV target cells. In fact, it has been found that the number of Th17 cells is reduced in the gastrointestinal mucosa of HIV‐infected individuals and simian immunodeficiency virus (SIV)‐infected macaques 3, 4, 5. Depletion of this Th17 subset in the gut mucosa after HIV and pathogenic SIV infections compromises the integrity of the gut mucosal barrier 6. The subsequent translocation of microbial products from the gut lumen into the bloodstream has been associated with systemic inflammation in HIV‐infected individuals and with peripheral blood lymphocyte activation and apoptosis 7. Systemic inflammation parameters including soluble CD14 (sCD14) and interleukin (IL)‐6 are considered to be disease progression markers in HIV‐infected patients 8.
It has been demonstrated that HIV‐infected individuals exhibit a profound loss of peripheral Th17 cells, which may impair mucosal immunity reconstitution 9, 10, 11, 12, 13, 14, although other authors have found opposite results 15. An important question is whether Th17 cell loss during HIV infection can be reversed and an efficient mucosal immune barrier restored. It has been reported 11, 16 that the loss of Th17 cells appears to be recovered upon treatment. However, a low level of immune activation persisted in the gut mucosa and peripheral blood despite long‐term therapy 16, suggesting that HIV replication is suppressed incompletely in the gut despite anti‐retroviral therapy (ART). Alternatively, a lack of recruitment of CD4+ T cells to the gut could also contribute to the incomplete restoration of CD4+ T cells in the gut mucosa 17.
Events occurring during the acute infection period basically dictate the kinetics of viral replication and the rate of disease progression in HIV infection, which suggests that detailed knowledge of changes in Th17 cells during acute or recent HIV infection may provide answers to the questions of how and when immune dysfunction is initiated 18. Consequently, the objectives of this work were: (1) to perform differential analyses of peripheral blood Th17 cells during recent or chronic HIV infections, both with and without ART, as well as study the relationship between HIV load and systemic inflammation markers; (2) to analyse expression of the IL‐23 receptor (IL‐23R) in Th17 cells as a possible contributor to the expansion of Th17 cells. It has been hypothesized that IL‐23 expands differentiated Th17 cells after binding to its receptor IL‐23R 19; IL‐23R expression is up‐regulated remarkably in an early response to IL‐6 20, a cytokine whose serum concentration is elevated in HIV‐infected patients as a result of the systemic proinflammatory state 8, 21; and (3) to analyse peripheral T cells with the ability to repopulate the gut during immune reconstitution in response to ART. In the case of the gut‐homing cells, it has been shown clearly that the α4β7 integrin heterodimer expressed by lymphoid cells and its cognate ligand MAdCAM expressed by gut epithelial cells attract and immobilize cells from the periphery and secondary lymphoid organs to the gut 22, 23, 24. We have therefore investigated peripheral CD4+IL‐17+ T cells, focusing on Th17 β7+ cells as a traceable phenotype for cells that home to the small intestine mucosa 25.
Patients and Methods
Patients and controls
After providing informed consent, 55 HIV‐infected adults were recruited: 25 naive patients, including 10 recently infected patients [time since infection, 4 months, interquartile range (IQR) = 2–6] and 15 chronically infected patients (time since infection, 11 years (IQR = 2–21 years), and 30 patients with chronic HIV infection and ART‐induced undetectable HIV viral load (undetectable HIV load for a median of 44 months (range = 15–60 months). Thirty age‐ and gender‐matched healthy volunteer hospital workers were recruited as controls.
Exclusion criteria were as follows: (1) active opportunistic or concomitant infections or neoplasias; (2) active drug use (cocaine, heroin or amphetamines), including significant alcohol ingestion (higher than 50 g/day for at least 5 years); (3) treatments which could interfere with the determination of inflammatory‐related molecules (pentoxifylline, steroidal or non‐steroidal anti‐inflammatory or immunosuppressive drugs); and (4) red blood cell or plasma transfusions in the month before inclusion in the study.
Definitions
Positive serum antibodies against HIV are required for the diagnosis of HIV infection. Patients at risk of HIV infection are tested every 6 months. For this study, recent HIV infection was diagnosed by the presence of anti‐HIV antibodies if the antibodies had been absent in a determination performed in the last 6 months, according to the Spanish Group for AIDS Study guidelines (http://www.gesida.es). Chronic uncontrolled infection was diagnosed if anti‐HIV antibodies were present in a determination performed at least 6 months previously; these patients refused ART when they were diagnosed. Controlled HIV replication was defined as an HIV load less than 50 copies/ml (Abbott RealTime HIV‐1; Abbott Laboratories, Abbott Park, IL, USA).
The start of HIV infection duration was designated the first positive anti‐HIV test.
Patients were classified according to the 1993 Centers for Disease Control and Prevention classification of HIV infection.
Bacterial translocation, as a measure of increased intestinal permeability, is defined as the presence of bacterial products in mesenteric nodes with posterior blood dissemination. The presence of bacterial translocation was evaluated by the qualitative determination of the presence of bacterial 16S ribosomal DNA (16S rDNA) or quantification of lipopolysaccharide‐binding protein (LBP); the sensitivity of these measures has been demonstrated to be higher than measurement of bacterial lipopolysaccharide serum levels 26.
Study protocol
At baseline, bacterial translocation parameters, activation markers and data relative to Th17 cells were obtained from the patients and controls. Patient follow‐up was provided by our hospital, with clinical and analytical revision every 3 months. The Spanish Group for AIDS Study guidelines (http://www.gesida.es) were used to indicate the anti‐retroviral treatment for untreated patients. Analytical parameters were remeasured at 6 and 12 months during patient follow‐up.
Determination of bacterial 16S rDNA and concentrations of LBP and proinflammatory molecules
Blood samples were collected in pyrogen‐free heparinized tubes (Biofreeze; Costar, San Jose, CA, USA) at 8 a.m. to minimize the influence of circadian rhythms. DNA was extracted (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany) and quantified by spectrophotometry (BioRad, Hercules, CA, USA), and 16S rDNA was detected by posterior amplification of the 16S rDNA region of Escherichia coli using the following primers: 16S reverse: 5´‐ACC‐GCC‐ACT‐GCT‐GCT‐GGC‐AC‐3´ and 16S forward: 5´‐AGA‐GTT‐TGA‐TCA‐TGG‐CTG‐AG‐3´ (IDT, Coralville, IA, USA). Polymerase chain reaction (PCR) was performed in a Rotor Gene 6000 instrument (Qiagen), and the size of the band was measured by agarose gel electrophoresis.
Serum LBP was measured by immunometric sandwich assays (Immulite LBP; DPC, Los Angeles, CA, USA). Serum concentrations of soluble CD14 (sCD14) and IL‐6 were analysed using the Quantikine Human Immunoassay kit (R&D, Minneapolis, MN, USA).
Phenotypical studies
Peripheral blood mononuclear cells (PBMC) were isolated from ethylenediamine tetraacetic acid (EDTA) anti‐coagulated fresh whole blood by Ficoll‐Hypaque (Sigma, St Louis, MO, USA), density‐gradient centrifugation within 1 h of collection.
PBMCs were counted and checked for viability and were then resuspended in RPMI‐1640 medium supplemented with 10% heat inactivated fetal bovine serum, 2 mmol/l L‐glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (gibco/Life Technologies, Grand Island, NY, USA).
For analysis of Th17 cells, 1 million fresh PBMC were cultured at 37ºC under a 5% CO2 environment for 5 h in 1 ml of RPMI‐1640 supplemented as above, in the presence of 5 μg/ml of brefeldin A (BD Golgi Plug Transport Inhibitor; BD Bioscience, Franklin Lakes, NJ, USA), 50 ng/ml of phorbol myristate acetate (PMA) (Sigma) and 1 μg/ml of ionomycin (Sigma) before performing intracellular cytokine staining. Cells incubated in media with brefeldin A served as negative control.
Flow cytometry was performed for surface marker expression using antibodies against the following human proteins. Cell suspensions were stained first with blue fluorescent reactive dye (Live/Dead Fixable Dead Cell Stain Kit; Invitrogen, Carlsbad, CA, USA) to exclude dead cells from analysis, then for surface markers, and subsequently for intracellular molecules following fixation and permeabilization with the Intra Stain (Dako A/S, Glostrup, Denmark). PBMC were incubated with combinations of peridinin chlorophyll protein CD4 (PerCP) (clone SK3), phycoerythrin mouse anti‐human CD195 (clone 3A9), phycoerythrin rat anti‐human IL‐23 receptor (clone 3C9), APC anti‐human integrin β (clone FIB504) (Biolegend, San Diego, CA, USA) and fluorescein isothiocyanate (FITC) IL17‐A (clone CZ8‐23G1) (Miltenyi Biotec, Bergisch Gladbach, Germany). Stained cells were washed, acquired and analyzed with two‐colour flow cytometry in a fluorescence‐activated cell sorter (FACS)Calibur cytometer using Cell Quest software (Becton‐Dickinson, San Jose, CA, USA). Isotype controls were used to confirm specificity of staining and to discriminate background staining.
Lymphocyte populations were gated using forward‐/side‐scatter. The percentage of Th17+ (IL‐17A+CD4+) cells expressing IL‐23R+ and Th17cells (IL‐17A+CD4+) expressing integrin β were detected by flow cytometry using the FACSCalibur cytometer.
Statistical analysis
Data were expressed as the absolute number and percentage or as the median and 25–75 interquartile range (IQR). Categorical variables were compared by 2 tests or Fisher’s exact test when necessary. Quantitative variables from independent groups were compared using the Mann–Whitney U‐test or an analysis of variance (anova) when necessary. Paired analysis of variables was performed using Wilcoxon’s rank test. The Spearman’s correlation test was used to analyse the association between quantitative variables. A P‐value < 0·05 was considered significant. Statistical analyses were carried out using the spss version 18.0 statistical software package (SPSS Inc., Chicago, IL, USA).
Ethics
This study was performed according to the Helsinki Declaration. The project was approved by the hospital ethical research committee. Written informed consent was obtained from each participant.
Results
Characteristics of the untreated patients are shown in Table 1.
Table 1.
General characteristics of the untreated HIV‐infected patients with detectable HIV at baseline (n = 25)
| Healthy controls (n = 30) | Untreated recent HIV infection (n = 10) | Untreated chronic HIV infection (n = 15) | |
|---|---|---|---|
| Age (years) | 36 (25–46) | 31 (24–38) | 43 (33–48) |
| Sex male (n, %) | 24 (80) | 9 (90) | 11 (73) |
| Previous parenteral drug use (n, %) | 0 (0) | 1 (10) | 3 (20) |
| Evolution of the infection (years) | 0.3 (0.2–0.5) | 11 (2–21) | |
| CD4+ T cell/mm3 at diagnosis | 940 (874–1081) | 592 (440–698) | 369 (150–596) |
| CD4+ T cell/mm3 at inclusion | 940 (874–1081) | 592 (440–698) | 385 (335–496) |
| CDC C stage (n, %) | 0 (0) | 6 (40) | |
| HIV load at inclusion (log10 copies/ml) | 4·54 (4·02–4·94) | 4·72 (3·79–5·13) |
Quantitative variables are expressed as median (interquartile range)
Bacterial translocation and activation parameters in untreated HIV‐infected patients
At baseline, all untreated HIV‐infected patients showed evidence of bacterial translocation as detected by the presence of 16S rDNA. Serum levels of LBP were elevated significantly in HIV‐infected patients with recent and chronic HIV infection with reference to healthy controls. Serum concentrations of sCD14 and IL6 were increased in patients with recent or chronic HIV infection. Analysis of CD4+ and CD8+ T lymphocytes revealed a significantly decreased CD4/CD8 ratio in HIV‐infected patients with reference to healthy controls (Table 2).
Table 2.
Soluble markers and T lymphocyte parameters in healthy controls and HIV‐infected untreated patients with detectable HIV at baseline
| Healthy controls (n = 30) | Untreated recent HIV infection (n = 10) | Untreated chronic HIV infection (n = 15) | |
|---|---|---|---|
| Bacterial 16S rDNA (n, %) | 0 (0) | 10 (100) | 15 (100) |
| LBP (ng/ml) | 4 (3–6) | 7 (4–9) b | 7 (6–7) c |
| Soluble CD14 (ng/ml) | 1767 (1154–2219) | 2653 (2299–3094) c | 2940 (2170–3184) c |
| IL‐6 (pg/ml) | 2 (1–3) | 7 (5–9) c | 5 (4–8) c |
| CD4+ T cells (% of total T cells) | 41 (38–47) | 27 (10–36) c | 20 (15–31) c |
| CD8+ T cells (% of total T cells) | 24 (22–27) | 48 (40–60) c | 43 (35–51) c |
| CD4/CD8 ratio | 1·7 (1·3–2·0) | 0·6 (0·4–0·7) c | 0·5 (0·3–0·7) c |
| CD4+IL17+ cells (% of CD4+ cells) | 0·9 (0·4–1·8) | 0·4 (0·2–2·8) | 1·3 (0·4–7·8) a |
| CD4+IL17+IL‐23R+ cells (% of CD4+ IL‐17+ cells) | 38 (17–44) | 20 (17–48) | 34 (28–42) |
| CD4+β7+ cells (% of CD4+ cells) | 10 (8–16) | 6 (4–11) | 7 (6–20) |
| CD4+IL‐17+β7+ cells (% of CD4+ IL‐17+ cells) | 33 (15–42) | 29 (12–65) | 22 (10–38) |
Variables are expressed as median (interquartile range).
P < 0·05 comparing healthy controls and patients;
P < 0·01 comparing healthy controls and patients;
P < 0·001 comparing healthy controls and patients. LBP = lipopolysaccharide‐binding protein; IL = interleukin.
Comparison of these parameters did not reveal significant differences between patients with recent and chronic HIV infections (P > 0·05).
Analysis of the baseline Th17 cell subset in untreated HIV‐infected patient.
The percentage of peripheral blood CD4+IL‐17+ cells was decreased in patients with recent HIV infection with reference to healthy controls, although statistical significance was not reached. Patients with chronic infection showed a significant increase in the percentage of these cells when compared with healthy controls (Table 2 Fig. 1).
Figure 1.
Proportions of (a) CD4+T helper type 17 (Th17)+ cells, (b) CD4+Th17+interleukin (IL)‐23R+ and (c) CD4+Th17+β7+ cells in healthy controls (n = 30) and untreated HIV‐infected patients with recent (n = 10) or chronic (n = 15) infection. Flow cytometry data for representative cases of a healthy control, a patient with recent HIV infection and a patient with chronic HIV infection with detectable HIV load are shown. The grey line in (b) and (c) represents isotype controls, used to confirm specificity of staining and to discriminate background staining.
The expansion of CD4+IL‐17+ T cells is dependent upon the interaction between IL‐23 and its receptor (IL‐23R) on these lymphocytes 19, 20. We analysed the expression of IL‐23R on these cells but did not detect significant differences between healthy controls and HIV‐infected patients (Table 2 Fig. 1).
Next, we evaluated the expression of the intestinal homing marker β in CD4+ cells and in the subgroup of CD4+IL‐17+ cells. The proportions of CD4+ β+ and CD4+IL‐17+β7+ T cells were similar in healthy controls and recently or chronically HIV‐infected patients (Table 2 Fig. 1).
In patients, the percentage of CD4+IL‐17+IL‐23R+ cells was correlated significantly with the percentage of CD4+IL‐17+β7+ cells (r = 0·527, P < 0·001). Additionally, the proportions of both Th17 subpopulations were correlated significantly with the nadir HIV load (CD4+IL‐17+IL‐23R+ cell percentage, r = 0·605, P = 0·001; CD4+IL‐17+β7+ cell percentage, r = 0·433, P = 0·024) but not with nadir or basal CD4+ T cells. No significant correlation was detected between the percentages of CD4+IL‐17+ T cells or their subtypes and markers of intestinal permeability or monocyte activation.
Evolution of intestinal permeability, activation markers and Th17 cells after treatment in patients with a detectable HIV load
Anti‐retroviral therapy was initiated in patients after inclusion in the study. Undetectable HIV load was obtained in every patient at 6 and 12 months. The evolution of intestinal permeability, activation markers and Th17 cells at 6 and 12 months was evaluated.
In all patients with recent infection, bacterial 16S rDNA continued to be detected at 6 and 12 months. However, a decrease of LBP concentration was evident at 12 months with reference to baseline levels, with values similar to those detected in healthy controls (P = 0·269). CD4/CD8 ratios and IL‐6 concentrations showed a similar pattern, but sCD14 levels did not change significantly during evolution. IL‐6 and sCD14 levels continued to be increased significantly. The CD4/CD8 ratio at 12 months of therapy was significantly higher in comparison to those at baseline, although was still minor to those observed in healthy controls. Median CD4+IL‐17+ cell percentages were similar to baseline values at 6 and 12 months. The proportion of lymphocytes expressing IL‐23R increased significantly (Table 3 Fig. 2) and was significantly higher at 12 months than that detected in healthy controls. The number of both CD4+β7+ and CD4+IL‐17+β7+ T cells also increased significantly. After 12 months, the percentage of CD4+IL‐17+β7+ T cells in patients was significantly higher than that observed in healthy controls.
Table 3.
Evolution of soluble markers and T lymphocyte parameters after anti‐retroviral therapy (ART) in recent HIV‐infected patients with detectable HIV at baseline
| Healthy controls (n = 30) | Patients with recent HIV infection | |||
|---|---|---|---|---|
| Baseline (n = 10) | After 6 months of ART (n = 10) | After 12 months of ART (n = 10) | ||
| Undetectable HIV load (n, %) | 0 (0) | 10 (100) | 10 (100) | |
| Bacterial 16S rDNA (n, %) | 0 (0) | 10 (100) | 10 (100) | 10 (100) |
| LBP (ng/ml) | 4 (3–6) |
7 (4–9) n.s. [Link] |
6 (4–8) n.s. n.s. |
5 (4–6) a n.s. |
| Soluble CD14 (ng/ml) | 1767 (1154–2219) |
2653 (2299–3094) n.s. [Link] |
2689 (2290–2983) n.s. [Link] |
2280 (1992–2728) n.s. [Link] |
| IL‐6 (pg/ml) | 2 (1–3) |
7 (5–9) n.s. [Link] |
5 (5–6) a [Link] |
5 (4–5) a [Link] |
| CD4+ T cells/mm3 | 940 (874–1081) |
592 (440–698) n.s. [Link] |
652 (482–775) n.s. [Link] |
689 (526–963) n.s. [Link] |
| CD4+ T cells (% of total T cells) | 41 (38–47) |
27 (10–36) n.s. [Link] |
29 (19–37) n.s. [Link] |
35 (31–39) [Link] [Link] |
| CD8+ T cells (% of total T cells) | 24 (22–27) |
48 (40–60) n.s. [Link] |
44 (33–52) n.s. [Link] |
39 (36–45) a [Link] |
| CD4/CD8 ratio | 1·7 (1·3–2·0) |
0·6 (0·4–0·7) n.s. [Link] |
0·5 (0·3–0·8) n.s. [Link] |
0·9 (0·6–1·1) [Link] [Link] |
| CD4+IL‐17+ cells (% of CD4+ cells) | 0·9 (0·4–1·8) |
0·4 (0·2–2·8) n.s. n.s. |
0·8 (0·3–3·1) n.s. n.s. |
1·1 (0·7–4·1) n.s. n.s. |
| CD4+ IL‐17+IL23+ cells (% of CD4+IL17+ cells) | 38 (17–44) |
20 (17–48) n.s. n.s. |
35 (12–56) n.s. n.s. |
54 (47–70) [Link] [Link] |
| CD4+β7+ cells (% of CD4+ cells) | 10 (8–16) |
6 (4–11) n.s. n.s. |
12 (11–16) [Link] n.s. |
12 (8–14) a n.s. |
| CD4+IL‐17+β7+ cells (% of CD4+IL‐17+ cells) | 33 (15–42) |
29 (12–65) n.s. n.s. |
28 (11–44) n.s. n.s. |
45 (30–61) a [Link] |
Variables are expressed as median (interquartile range).
HIV‐infected patients, comparisons with reference to baseline: n.s. = non‐significant. LBP = lipopolysaccharide‐binding protein; IL = interleukin.
P < 0·05 comparing with baseline values; P < 0·01 comparing with baseline values.
HIV‐infected patients versus healthy controls: n.s. = non‐significant, P < 0·05, P < 0·01, P < 0·001.
Figure 2.
Evolution of cell populations in patients with recent (less than 6 months) (n = 10) and chronic (more than 6 months) (n = 15) HIV infection after anti‐retroviral therapy. (a) CD4+interleukin (IL)‐17+ cells (percentage of CD4+ cells). (b) CD4+IL‐17+IL23R+ cells (percentage of CD4+IL‐17+ cells). (c) CD4+IL‐17+β7+ cells (percentage of CD4+IL‐17+ cells).
In all patients with chronic infection, bacterial 16S rDNA continued to be detected at 6 and 12 months after treatment. In these patients, the evaluated parameters did not show significant differences at 6 or 12 months with reference to baseline values (Table 4 Fig. 2). Serum concentrations of LBP, sCD14 and IL‐6, absolute number of CD4+ T cells and percentages of CD4+ T cells and the CD4/CD8 ratio continue to be decreased significantly with reference to those of healthy controls. CD8+ T cell percentage remain significantly increased. The proportions of CD4+β7+, CD4+IL‐17+β7+ and CD4+IL‐17+IL‐23R+ T cells were similar in healthy controls and chronically HIV‐infected patients after 12 months of therapy. Comparison between parameters obtained after 12 months of ARV in recently and chronically HIV‐infected patients is shown in Table 5.
Table 4.
Soluble markers and T lymphocyte parameters in chronic HIV‐infected, patients with baseline detectable HIV load, after anti‐retroviral therapy (ART)
| Chronic HIV‐infected patients with baseline detectable HIV load (n = 15) | Chronic HIV‐infected patients with baseline undetectable HIV load (n = 30) | |||
|---|---|---|---|---|
| Baseline (n = 15) | After 6 months of ART (n = 15) | After 12 months of ART (n = 15) | ||
| Undetectable HIV load (n, %) | 0 (00) | 15 (100) | 15 (100) | 30 (100) |
| Bacterial DNA (n, %) | 15 (100) | 15 (100) | 15 (100) | 30 (100) |
| LBP (ng/ml) | 7 (6–7) | 6 (6–8) | 6 (5–6) | 6 (5–7) |
| Soluble CD14 (ng/ml) | 2940 (2170–3184) | 2655 (2529–2814) | 2704 (2166–3259) | 2635 (2332–3003) |
| IL‐6 (pg/ml) | 5 (4–8) | 4 (4–7) | 5 (4–6) | 4 (3–6) |
| CD4+ T cells/mm3 | 385 (335–496) | 534 (400–743) | 530 (366–796) | 660 (378–1074) |
| CD4+ T cells (% of total T cells) | 20 (15–31) | 27 (18–32) | 29 (31–35) | 31 (24–35) |
| CD8+ T cells (% of total T cells) | 43 (35–51) | 40 (35–47) | 42 (36–46) | 37 (30–43) |
| CD4/CD8 ratio | 0·5 (0·3–0·7) | 0·6 (0·5–0·8) | 0·6 (0·5–0·9) | 0·8 (0·6–1·0) |
| CD4+IL‐17+ cells (% of CD4+ cells) | 1·3 (0·4–7·8) | 0·9 (0·5–3·1) | 0·8 (0·6–3·8) | 0·9 (0·6–1·1) |
| CD4+ IL‐17+IL23+ cells (% of CD4+IL‐17+ cells) | 34 (28–42) | 36 (21–50) | 35 (31–38) | 36 (11–52) |
| CD4+β7+ cells (% of CD4+ cells) | 7 (6–20) | 8 (5–16) | 10 (6–17) | 13 (9–46) |
| CD4+IL‐17+β7+ cells (% of CD4+IL‐17+ cells) | 22 (10–38) | 27 (24–39) | 28 (23–33) | 23 (9–56) |
Variables are expressed as median (interquartile range). LBP = lipopolysaccharide‐binding protein; IL = interleukin.
Table 5.
Soluble markers and T lymphocyte parameters after anti‐retroviral therapy (ART) in healthy controls and HIV‐infected patients with detectable HIV at baseline
| Healthy controls (n = 30) | Patients with HIV infection and detectable HIV load at baseline (n = 25) after 12 months of ART | ||
|---|---|---|---|
| Recently infected (n = 10) | Chronically infected (n = 15) | ||
| Undetectable HIV load (n, %) | 10 (100) | 15 (100) | |
| Bacterial 16S rDNA (n, %) | 0 (0) | 10 (100) | 15 (100) |
| LBP (ng/ml) | 4 (3–6) |
5 (4–6) n.s. |
6 (5–6) [Link] n.s. |
| Soluble CD14 (ng/ml) | 1767 (1154–2219) |
2280 (1992–2728) [Link] |
2704 (2166–3259) [Link] n.s. |
| IL‐6 (pg/ml) | 2 (1–3) |
5 (4–5) [Link] |
5 (4–6) [Link] n.s. |
| CD4+ T cells/mm3 | 940 (874–1081) |
689 (526–963) [Link] |
530 (366–796) [Link] [Link] |
| CD4+ T cells (% of total T cells) | 41 (38–47) |
35 (31–39) [Link] |
29 (31–35) [Link] n.s. |
| CD8+ T cells (% of total T cells) | 24 (22–27) |
39 (36–45) [Link] |
42 (36–46) [Link] n.s. |
| CD4/CD8 ratio | 1·7 (1·3–2·0) |
0·9 (0·6–1·1) [Link] |
0·6 (0·5–0·9) [Link] [Link] |
| CD4+IL‐17+ cells (% of CD4+ cells) | 0·9 (0·4–1·8) |
1·1 (0·7–4·1) n.s. |
0·8 (0·6–3·8) n.s. n.s. |
| CD4+ IL‐17+IL‐23+ cells (% of CD4+IL‐17+ cells) | 38 (17–44) |
54 (47–70) [Link] |
35 (31–38) n.s. [Link] |
| CD4+β7+ cells (% of CD4+ cells) | 10 (8–16) |
12 (8–14) n.s. |
10 (6–17) n.s. n.s. |
| CD4+IL‐17+β7+ cells (% of CD4+IL‐17+ cells) | 33 (15–42) |
45 (30–61) [Link] |
28 (23–33) n.s. [Link] |
Variables are expressed as median (interquartile range). LBP = lipopolysaccharide‐binding protein; IL = interleukin.
Healthy controls versus HIV‐infected patients: n.s. = non‐significant, P < 0·05, P < 0·01, P < 0·001.
Recently HIV‐infected versus chronically‐infected HIV‐infected patients: n.s. = non‐significant, P < 0·05, P < 0·01, P < 0·001.
To analyse whether the values for bacterial translocation markers, proinflammatory molecules or T lymphocyte populations were modified after prolonged ART, chronic infected patients with detectable HIV loads at baseline were compared with a group of 30 chronic infected patients with undetectable HIV loads at inclusion [age = 46 (39–52) years, male sex 20 (67%), CD4/mm3 at diagnosis 200 (50–498), CD4/mm3 at inclusion 660 (378–1074), undetectable HIV load for 44 (15–60) months]. There were no significant differences with reference to the bacterial translocation (presence of 16S rDNA, LBP) or monocyte activation (sCD14, IL‐6) parameters and CD4/CD8 ratios or percentages of CD4+β7+, CD14+IL‐17+, CD4+IL‐17+CD23R+ or CD4+IL‐17+β7+ cells for chronic HIV‐infected patients with undetectable HIV loads at baseline with reference to those of patients with detectable HIV loads at baseline after 12 months of treatment (P > 0·05 in each case) (Table 4).
Discussion
Th17 cells play a critical role in the immune defence of the gut mucosa. Their depletion in the gut after HIV‐1 infection compromises the integrity of the mucosal barrier 6. This alteration induces bacterial translocation 7, as evidenced by the detection of bacterial 16S rDNA as well as increased LBP levels in patients. Both bacterial translocation and uncontrolled HIV infection stimulate monocyte activation 7, 21, as demonstrated by the increased sCD14 or IL‐6 levels observed in our study.
The main objective of the study was the basal and post‐therapy analysis of peripheral Th17 population characteristics in patients with recent infections and those with chronic infections. We theorized that a difference could be possible when the evolution of the HIV infection was shorter.
At baseline, patients with a recent HIV infection (less than 6 months of evolution) showed a non‐significant decrease of the percentage of peripheral CD4+IL‐17+ T cells. By contrast, the percentage of Th17 in chronic HIV‐infected patients was increased significantly at baseline. To our knowledge, there are no data about Th17 count in recently HIV‐infected patients. In chronic HIV‐infected patients peripheral blood Th17 cells have been described as decreased 9, 10, 27, increased 15 or without differences 28 in relation to those found in healthy controls. A large variation in the Th17 frequencies has been detected for all groups 28. An explanation could be supported by the negative correlation between HIV load and Th17 cell number detected by some authors 27, suggesting that uncontrolled HIV replication is driving Th17 cell depletion. Effectively, Li et al., in a series of patients with an evolution of 17–20 years of HIV infection without ART, have demonstrated that Th17 proportion decreased progressively in HIV‐infected patients 27. In our study, with a shorter evolution of HIV infection, we did not detect correlation between HIV load and Th17 cell proportion. It is possible that the different evolution of chronic HIV infection in the patients could explain the discrepancies of Th17 count in these patients.
In contrast, a previously non‐reported positive correlation was demonstrated between HIV load and a subpopulation of Th17 cells, the CD4+IL‐17+IL‐23R+ cells, suggesting a role of HIV (directly or indirectly via monocyte activation) in the expansion of this subpopulation. A role for the IL‐23/IL‐23R pathway in the recovery of the CD4+IL‐17 cell population has been postulated. Effectively, a function of IL‐23 is to expand differentiated Th17 cells 19. IL‐23R is not expressed on naive T cells, while IL‐23R is remarkably up‐regulated early in response to IL‐6 20, a cytokine whose plasma concentration was increased significantly. A longer period of actuation of IL‐6 in chronic infected patients could justify the increased percentage of Th17 we observed.
Because peripheral CD4+IL‐17+β7+ cells are those implicated in gut homing and reconstitution of intestinal barrier integrity 22, 23, 24, analysis of this subpopulation was performed. In patients with recent HIV infection, the percentage of this subpopulation in relation to total CD4+Th17+ cells was similar to that observed in healthy controls, implicating a relative diminution in contrast to the expected increase necessary to replenish the depleted gut CD4+Th17 cells. After initiation of therapy and consequent undetectable HIV load, the percentage of cells expressing IL‐23R increased significantly. The increase of CD4+IL17+IL‐23R+ paralleled that of CD4+IL‐17+β7+, reaching a significantly higher level at 12 months of ART than that observed at baseline and that of the healthy controls. The expansion of this population probably contributes to regeneration of intestinal barrier, as supported by decreases in LBP, a marker of intestinal permeability, and IL‐6, a parameter indicative of systemic activation after therapy. However, regeneration of the intestinal barrier was not complete, as has been observed previously 29: 16S rDNA continued to be detectable, IL‐6 levels continued to be significantly higher than those of healthy controls and sCD14 levels persisted unmodified. Persistent enterocyte damage with a reduction in the expression of the ligands for lymphocyte gut‐homing receptors has been described in HIV‐infected patients 25, supporting a possible impairment of Th17 replenishment in the intestine and thus insufficient mucosal reconstitution.
The scenario of chronic HIV‐infected patients with uncontrolled HIV replication is clearly different. After 6 and 12 months of therapy, percentages of CD4+IL‐17+IL‐23R+ and CD4+IL‐17+β7+ populations did not change significantly, even though an undetectable HIV load had been obtained. The absence of modification of peripheral CD4+IL‐17+β7+ cells indicates the continued persistence of microbial translocation and monocyte activation. A more prolonged period of HIV undetectability is unlikely to modify the inability to normalize the Th17 population. Effectively, we have demonstrated that percentages of CD4+IL‐17+IL‐23R+ and CD4+IL‐17+β7+ populations and serum levels of LBP, sCD14 and IL‐6 in chronic HIV‐infected patients with undetectable HIV load for a median of 44 months were similar to those observed in chronic HIV‐infected patients with detectable HIV load at baseline after 12 months of ART.
Patients in which ART initiation is retarded do not attain a normal intestinal lymphoid structure or epithelial barrier integrity 30. CD4+ T cells in the small intestine mucosa remain depleted, despite the subjects being on effective ART for more than 5 years 31, 32, 33. In other words, partial reconstitution of the gut mucosal barrier cannot be obtained if patients begin ART after more than 6 months of evolution of an HIV infection 29, 30, probably because of the deficient expression of intestinal homing markers by Th17 cells, such as those detected in the present work.
In conclusion, our data demonstrate that early initiation of ART helps to increase Th17 cells with ability to homing on the intestinal mucosa and to partially restore gut mucosal homeostasis. These results provide a rationale for initiating ART during the acute phase of HIV infection.
Ethics approval and consent to participate
Approval for the study was granted by the Ethics and Medical Research Committee of the Hospital Universitario ‘Puerta del Mar’, Cádiz, Spain.
Disclosures
The authors declare they have no competing interests.
Acknowledgements
This work was supported by the Instituto de Salud Carlos III, Spain (Grant number PI14/01779).
References
- 1. Aujla SJ, Dubin PJ, Kolls JK. Th17 cells and mucosal host defense. Semin Immunol 2007;19:377–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Berlin C, Berg EL, Briskin MJ et al Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM‐1. Cell 1993;74:185–195. [DOI] [PubMed] [Google Scholar]
- 3. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 2005;434:1093–1097. [DOI] [PubMed] [Google Scholar]
- 4. Brenchley JM, Paiardini M, Knox KS et al Differential Th17 CD4 T‐cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 2008;112:2826–2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Cecchinato V, Trindade CJ, Laurence A et al Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus‐infected macaques. Mucosal Immunol 2008;1:279–288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Raffatellu M, Santos RL, Verhoeven DE et al Simian immunodeficiency virus‐induced mucosal interleukin‐17 deficiency promotes Salmonella dissemination from the gut. Nat Med 2008;14:421–428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Brenchley JM, Price DA, Schacker TW et al Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006;12:1365–1371. [DOI] [PubMed] [Google Scholar]
- 8. Sandler NG, Wand H, Roque A et al Plasma levels of soluble CD14 independently predict mortality in HIV infection. J Infect Dis 2011;203:780–790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Ndhlovu LC, Chapman JM, Jha AR et al Suppression of HIV‐1 plasma viral load below detection preserves IL‐17 producing T cells in HIV‐1 infection. AIDS 2008;22:990–992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Prendergast A, Prado JG, Kang YH et al HIV‐1 infection is characterized by profound depletion of CD161+ Th17 cells and gradual decline in regulatory T cells. AIDS 2010;24:491–502. [DOI] [PubMed] [Google Scholar]
- 11. He Y, Li J, Zheng Y et al A randomized case‐control study of dynamic changes in peripheral blood Th17/Treg cell balance and interleukin‐17 levels in highly active antiretroviral‐treated HIV type 1/AIDS patients. AIDS Res Hum Retroviruses 2012;28:339–345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Jenabian M‐A, Patel M, Kema I et al Distinct tryptophan catabolism and Th17/Treg balance in HIV progressors and elite controllers. PLOS ONE 2013;8:e78146. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. El Hed A, Khaitan A, Kozhaya L et al Susceptibility of human Th17 cells to human immunodeficiency virus and their perturbation during infection. J Infect Dis 2010;201:843–854. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Salgado M, Rallón NI, Rodés B, López M, Soriano V, Benito JM. Long‐term non‐progressors display a greater number of Th17 cells than HIV‐infected typical progressors. Clin Immunol 2011;139:110–114. [DOI] [PubMed] [Google Scholar]
- 15. Maek ANW, Buranapraditkun S, Klaewsongkram J, Ruxrungtham K. Increased interleukin‐17 production both in helper T cell subset Th17 and CD4‐negative T cells in human immunodeficiency virus infection. Viral Immunol 2007;20:66–75. [DOI] [PubMed] [Google Scholar]
- 16. Macal M, Sankaran S, Chun TW et al Effective CD4+ T‐cell restoration in gut‐associated lymphoid tissue of HIV‐infected patients is associated with enhanced Th17 cells and polyfunctional HIV‐specific T‐cell responses. Mucosal Immunol 2008;1:475–88. [DOI] [PubMed] [Google Scholar]
- 17. Mehandru S, Poles MA, Tenner‐Racz K et al Lack of mucosal immune reconstitution during prolonged treatment of acute and early HIV‐1 infection. PLOS Med 2006;3:e484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Haase AT. Targeting early infection to prevent HIV‐1 mucosal transmission. Nature 2010;464:217–223. [DOI] [PubMed] [Google Scholar]
- 19. Toussirot E. The IL23/Th17 pathway as a therapeutic target in chronic inflammatory diseases. Inflamm Allergy Drug Targets 2012;11:159–68. [DOI] [PubMed] [Google Scholar]
- 20. Zhou L, Ivanov II, Spolski R et al IL‐6 programs T(H)‐17 cell differentiation by promoting sequential engagement of the IL‐21 and IL‐23 pathways. Nat Immunol 2007;8:967–974. [DOI] [PubMed] [Google Scholar]
- 21. de Oca Arjona MM, Marquez M, Soto MJ et al Bacterial translocation in HIV‐infected patients with HCV cirrhosis: implication in hemodynamic alterations and mortality. J Acquir Immune Defic Syndr 2011;56:420–427. [DOI] [PubMed] [Google Scholar]
- 22. Ansari AA, Reimann KA, Mayne AE et al Blocking of a4b7 gut‐homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus‐infected rhesus macaques. J Immunol 2011;186:1044–1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Acosta‐Rodriguez EV, Rivino L, Geginat J et al Surface phenotype and antigenic specificity of human interleukin 17‐producing T helper memory cells. Nat Immunol 2007;8:639–646. [DOI] [PubMed] [Google Scholar]
- 24. Arthos J, Cicala C, Martinelli E et al HIV‐1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 2008;9:301–309. [DOI] [PubMed] [Google Scholar]
- 25. Mavigner M, Cazabat M, Dubois M et al Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV‐infected individuals. J Clin Invest 2012;122:62–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Abad‐Fernández M, Vallejo A, Hernández‐Novoa B et al Correlation between different methods to measure microbial translocation and its association with immune activation in long‐term suppressed HIV‐1‐infected individuals. J AIDS 2013;64:149–53. [DOI] [PubMed] [Google Scholar]
- 27. Li D, Chen J, Jia M et al Loss of balance between T helper type 17 and regulatory T cells in chronic human immunodeficiency virus infection. Clin Exp Immunol 2011;165:363–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Brandt L, Benfield T, Mens H et al Low level of regulatory T cells and maintenance of balance between regulatory T cells and TH17 cells in HIV‐1‐infected elite controllers. J AIDS 2011;57:101–8. [DOI] [PubMed] [Google Scholar]
- 29. Guadalupe M, Reay E, Sankaran S et al Severe CD4 T‐cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 2003;77:11708–11717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Kök A, Hocqueloux L, Hocini H et al Early initiation of combined antiretroviral therapy preserves immune function in the gut of HIV‐infected patients. Mucosal Immunol 2015;8:127–40. [DOI] [PubMed] [Google Scholar]
- 31. Brenchley JM, Schacker TW, Ruff LE et al CD4 T‐cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004;200:749–759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Mehandru S, Poles MA, Tenner‐Racz K et al Primary HIV‐1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004;200:761–770. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Abad‐Fernández M, Gutiérrez C, Madrid N et al Expression of gut‐homing β7 receptor on T cells: surrogate marker for microbial translocation in suppressed HIV‐1‐infected patients? HIV Med 2015;16:15–23. [DOI] [PubMed] [Google Scholar]