Edited by: Manfred B. Lutz, Julius Maximilian University of Würzburg, Germany
Reviewed by: Xinjian Chen, The University of Utah, United States; Mohamed Elgazzar, East Tennessee State University, United States
*Correspondence: Yolanda María Pacheco,
This article was submitted to Antigen Presenting Cell Biology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
We have previously observed increased levels of inflammatory biomarkers and Th17 as well as Treg cells, but not other T-cell specific alterations, preceding immunodiscordance of successfully-treated HIV-infected subjects. Our hypothesis is that this could be related with potential alterations in myeloid-derived suppressor cells (MDSCs) and/or monocyte subsets.
We determined the frequencies of MDSCs and monocyte subsets and the expression of several functional markers (CCR2,
Increased frequencies of MDSCs, but normal distribution of monocyte subsets, preceded immunodiscordance. The expression of several functional markers, such as CCR2, CD16, CD11b and PDL1, on MDSCs and monocyte subsets was altered in this scenario. MDSC and monocyte-related functional markers were associated with soluble biomarkers and T-cell parameters. Several of these cellular alterations were not restored after 24 months of suppressive cART.
An early immunosuppressive environment, characterized by the expansion of MDSCs and Tregs, precedes immunodiscordance and is related with a highly inflammatory status.
Although the combined antiretroviral therapy (cART) usually suppresses HIV viremia to lead to a rise in CD4 T-cell counts (
Myeloid-derived suppressor cells (MDSCs) are intermediates of normal myeloid differentiation (
Monocytes are also relevant innate cells able to produce proinflammatory mediators and to impact on differentiation of several T-cell subsets, such as Tregs and Th17 cells (
During the last years, we have described severe immunological alterations preceding immunodiscordance in a deeply characterized cohort of subjects with an incomplete CD4 recovery after treatment. This cohort has been comparatively studied with a control group of adequate CD4 recovery being matched by baseline CD4 T-cell counts (
Samples of HIV-infected subjects were selected from the Spanish AIDS Research Network Cohort (CoRIS) (
Peripheral blood mononuclear cells (PBMCs) were thawed and stained with surface antibodies, followed by fixation and permeabilization according to the manufacturer’s instructions (Foxp3/Transcription Factor Staining Buffer, Ebioscience), and subsequently stained with intracellular antibodies. The antibodies and fluorochromes used are described in
IFN-
Continuous variables are expressed as the median and interquartile range (IQR), whereas categorical variables are expressed as numbers and percentages (%). The Mann–Whitney
Samples from 28 HIV-infected male subjects (13 LR- and 15 HR-subjects) before cART initiation and follow-up samples from 11 of these subjects (six LR- and five HR-subjects) were available for this study. The clinical characterization of the two groups at cART onset showed no differences in age, viral load, sexual transmission or previous C event, whereas a tendency toward lower CD4 T-cell counts was observed in LR-subjects
Clinical Characteristics of the studied HIV-infected subjects before cART onset.
LR-subjects (13) | HR-subjects (15) |
|
|
---|---|---|---|
|
43 [32–57] | 40 [33–49] | 0.5 |
|
70 [53–116] | 136 [72–165] | 0.104 |
|
4.9 [4.1–5.3] | 4.9 [4.4–5.7] | 0.8 |
|
10/13 (77) | 12/15 (80) | 0.8 |
|
4/13 (31) | 3/15 (20) | 0.5 |
Continuous variables are expressed as the median and interquartile range [IQR], whereas categorical variables are expressed as the number and percentage (%). The Mann–Whitney U test was used for comparisons.
We analyzed the frequencies of total-MDSCs, m-MDSCs, and g-MDSCs as well as the expression of different functional markers on MDSCs (
Myeloid-derived suppressive cells (MDSCs) and cellular markers before cART initiation.
LR-subjects (13) | HR-subjects (15) |
|
Univariate AnalysisOR (CI); |
Bivariate AnalysisOR (CI); |
|
---|---|---|---|---|---|
|
0.3 [0.1–0.6] | 0.1 [0.0–0.2] |
|
42.32 (0.76–23158.11); |
21.06 (0.37–1251.30); |
|
0.2 [0.1–0.5] | 0.1 [0.0–0.3] |
|
60.35 (0.53–6831.94); |
26.30 (0.20–3422.43); 0.188 |
|
35.6 [26.3–51.5] | 24.5 [15.2–29.8] |
|
1.07 (0.99–1.16); |
1.06 (0.98–1.15); |
|
53.8 [19.5–73.1] | 54.0 [10.0–88.7] | 0.9 | 1.01 (0.97–1.03); 0.9 | 0.99 (0.97–1.03); 0.9 |
|
2.0 [0.0–33.7] | 3.4 [0.0–69.2] | 0.6 | 0.98 (0.95–1.01); 0.3 | 0.98 (0.95–1.01); 0.3 |
|
42.5 [28.2–66.1] | 16.6 [8.7–36.3] |
|
1.06 (1.00–1.12); |
1.06 (1.00–1.12); |
Variables are expressed as median and interquartile range [IQR]. p, Mann–Whitney U test was used for comparisons. A bivariate regression was performed in order to adjust by CD4 T-cell counts at cART initiation. Results from regression analyses were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). *These comparisons were performed in 21 subjects (LR = 12, HR = 9).
Statistically significant values are highlighted in bold, p < 0.005.
Myeloid-derived suppressor cells (MDSCs) and their expression of functional markers preceding immunodiscordance.
Longitudinal analyses showed that cART increased the frequencies of CCR2+ MDSCs in both groups, although reaching statistical significance only in LR-subjects (
Follow-up analysis of MDSCs and cellular markers (24-months of suppressive cART).
LR-Subjects (6) | HR-Subjects (5) | ||||||
---|---|---|---|---|---|---|---|
Before cART | Post cART |
|
Before cART | Post cART |
|
|
|
|
0.3 [0.1–0.6] | 0.1 [0.1–0-4] | 0.3 | 0.1 [0.0–0.1] | 0.2 [0.0–0.2] |
|
0.9 |
|
0.3 [0.1–0.4] | 0.1 [0.1–0.3] | 0.3 | 0.1 [0.0–0.2] | 0.1 [0.0–0.2] | 0.5 | 0.9 |
|
30.6 [16.4–40.4] | 48.1 [27.5–58.8] |
|
25.1 [23.9–25.1] | 50.3 [40.7–50.3] |
|
0.9 |
|
60.4 [26.0–72.2] | 45.6 [19.5–69.2] | 0.6 | 14.8 [10.3–14.8] | 9.4 [8.5–9.4] |
|
0.5 |
|
2.0 [0.0–28.9] | 3.0 [0.6–7.8] | 0.5 | 3.4 [0.0–3.4] | 4.1 [0.0–4.1] | 0.180 | 0.8 |
|
42.5 [28.7–62.7] | 32.5 [19.6–65.6] | 0.5 | 16.6 [9.2–16.6] | 22.8 [8.9–22.8] | 0.9 | 0.3 |
Variables are expressed as median and interquartile range [IQR]. pW, Wilcoxon rank test was used for longitudinal comparisons. pM-W, Mann–Whitney U test was used for transversal comparisons of post cART data. *The expression of functional molecules was determined in nine subjects (LR = 6 and HR = 3).
Statistically significant values are highlighted in bold, p < 0.005.
We also analyzed the frequencies of monocyte subsets and the expression of different functional molecules. No difference in any monocyte subset was observed between groups before cART onset (
Monocyte subsets and expression of different functional molecules before cART initiation.
LR-subjects (13) | HR-subjects (15) |
|
Univariate AnalysisOR (CI); |
Bivariate AnalysisOR (CI); |
|
---|---|---|---|---|---|
|
79.6 [65.6–88.1] | 69.7 [62.0–84.9] | 0.3 | 1.03 (0.98–1.03); 0.2 | 1.03 (0.97–1.09); 0.4 |
|
2,189 [1,910–2,469] | 1,521 [1,228–2,399] |
|
1.01 (1.00–1.00); |
1.01 (1.00–1.00); |
|
82.8 [74.4–90.8] | 84.2 [78.2–89.9] | 0.9 | 0.98 (0.93–1.05); 0.6 | 0.97 (0.90–1.0); 0.3 |
|
9.1 [4.1–26.2] | 16.7 [2.6–26.9] | 0.9 | 0.99 (0.96–1.03); 0.6 | 0.98 (0.94–1.02); 0.3 |
|
1.4 [0.6–3.4] | 1.0 [0.7–4.6] | 0.9 | 1.05 (0.83–1.22); 0.9 | 0.98 (0.98–1.20); 0.8 |
|
23.9 [12.3–42.3] | 13.7 [7.0–31.6] |
|
1.04 (0.99–0.11); |
1.03 (0.99–1.08); 0.163 |
|
10.8 [6.6–13.7] | 15.4 [9.1–26.1] | 0.3 | 0.97 (0.91–1.04); 0.4 | 0.97 (0.90–1.04); 0.4 |
|
1,090 [511–1,607] | 544 [429–845] | 0.088 | 1.02 (1.00–1.00); |
1.02 (1.00–1.00); |
|
2,281 [1,944–3,052] | 2,395 [1,324–2,956] | 0.4 | 1.00 (0.99–1.00); 0.4 | 1.00 (0.99–1.00); 0.4 |
|
51.9 [44.3–61.4] | 64.8 [45.9–80.0] | 0.189 | 0.97 (0.93–1.02); 0.189 | 0.94 (0.91–1.01); |
|
9.1 [2.5–16.4] | 6.9 [2.5–24.3] | 0.7 | 0.98 (0.93–1.03); 0.4 | 0.96 (0.91–1.01); |
|
3.5 [1.0–7.8] | 1.9 [0.7–6.8] | 0.5 | 1.03 (0.92–1.17); 0.6 | 1.02 (0.89–1.15); 0.8 |
|
47.7 [31.0–69.5] | 37.4 [23.6–58.7] | 0.2 | 1.02 (0.98–1.05); 0.3 | 1.01 (0.98–1.05); 0.5 |
|
2.8 [1.9–8.9] | 4.1 [2.4–7.5] | 0.6 | 1.01 (0.86–1.16); 0.9 | 1.09 (0.91–1.31); 0.4 |
|
2,372 [1,307–3,253] | 1,248 [872–2,513] |
|
1.00 (1.00–1.00); 0.3 | 1.00 (1.00–1.01); 0.3 |
|
827 [612–1,029] | 734 [512–1,007] | 0.6 | 1.00 (0.99–1.00); 0.8 | 0.99 (0.99–1.00); 0.4 |
|
9.6 [6.2–15.9] | 20.6 [10.4–30.6] |
|
0.98 (0.94–1.02); 0.3 | 0.97 (0.93–1.02); 0.189 |
|
5.4 [4.1–10.4] | 8.8 [4.9–27.3] | 0.2 | 0.96 (0.90–1.02); 0.2 | 0.94 (0.88–1.01); |
|
0.4 [0.1–1.4] | 0.5 [0.3–1.4] | 0.7 | 1.08 (0.74–1.59); 0.7 | 1.04 (0.70–1.53); 0.9 |
|
69.3 [42.9–83.9] | 46.5 [25.6–64.9] |
|
1.04 (0.99–1.08); |
1.03 (0.99–1.08); 0.166 |
|
4.3 [2.5–5.2] | 5.9 [3.7–9.9] |
|
0.69 (0.48–1.01); |
0.71 (0.48–1.05); |
Variables are expressed as median and interquartile range [IQR]. p, Mann–Whitney U test was used for comparisons. A bivariate regression was performed in order to adjust by CD4 T-cell counts at cART initiation. Results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). *Comparisons of the expression of molecules were performed in 26 subjects (LR = 12, HR = 14).
Statistically significant values are highlighted in bold, p < 0.005.
Monocyte subsets and their expression of functional markers preceding immunodiscordance.
Longitudinal analysis showed no reduction of CD11b levels in LR-subjects, but a tendency toward a decrease in HR-subjects (
Follow-up analysis of monocyte subsets and functional molecules (24-months of suppressive cART).
LR-Subjects (6) | HR-Subjects (5) | ||||||
---|---|---|---|---|---|---|---|
Before cART | Post cART |
|
Before cART | Post cART |
|
|
|
|
85.3 [74.5–91.8] | 81.1 [60.0–91.8] | 0.3 | 78.8 [69.0–85.4] | 80.1 [72.2–87.5] | 0.5 | 0.9 |
|
2,285 [2,154–2,806] | 2298 [2,028–2,785] | 0.5 | 2,399 [1,371–2,883] | 1,797 [1,142–1,977] |
|
|
|
82.7 [66.6–92.7] | 93.4 [86.3–97.5] |
|
81.4 [78.9–84.9] | 93.1 [90.8–95.1] |
|
0.7 |
|
18.3 [4.3–32.2] | 13.8 [4.2–25.4] | 0.6 | 4.8 [2.1–52.1] | 9.4 [1.8–37.1] | 0.5 | 0.9 |
|
1.0 [0.7–4.1] | 0.2 [0.1–1.4] |
|
1.0 [0.9–7.2] | 0.4 [0.1–6.3] |
|
0.8 |
|
26.5 [12.8–47.3] | 16.1 [5.9–54.2] | 0.5 | 13.7 [4.1–47.0] | 5.3 [2.8–22.4] |
|
0.2 |
|
8.8 [4.1–13.3] | 7.0 [1.7–19.9] | 0.9 | 11.5 [6.0–19.9] | 5.1 [3.5–13.6] |
|
0.9 |
|
1,441 [700–1,677] | 1,298 [730–1,653] | 0.8 | 536 [462–1,007] | 1,043 [504–1,227] |
|
0.2 |
|
2,792 [2,369–3,178] | 3,112 [2,475–4,311] | 0.3 | 2,836 [2,031–3,019] | 2,031 [1,484–2,570] |
|
|
|
53.9 [41.6–75.2] | 63.2 [61.2–75.4] | 0.2 | 57.9 [44.7–64.8] | 60.3 [51.4–86.4] | 0.3 | 0.4 |
|
13.2 [2.8–19.8] | 7.3 [2.7–19.7] | 0.5 | 6.7 [1.9–41.1] | 3.4 [1.5–18.0] | 0.3 | 0.6 |
|
3.6 [2.0–9.7] | 0.5 [0.3–3.3] |
|
1.9 [1.6–11.8] | 0.7 [0.0–9.1] |
|
0.9 |
|
46.3 [39.0–62.9] | 38.3 [24.0–68.1] | 0.2 | 24.5 [22.3–81.5] | 17.9 [9.6–37.2] |
|
0.2 |
|
2.8 [0.8–8.2] | 7.0 [1.6–15.8] | 0.2 | 2.4 [1.2–9.0] | 7.7 [2.4–10.0] | 0.5 | 0.9 |
|
2,926 [,2037–4,878] | 3,122 [,2842–3,531] | 0.9 | 1,677 [1,160–4,545] | 2,128 [1,630–3,170] | 0.9 |
|
|
980 [708–1,256] | 803 [635–1,026] | 0.6 | 795 [520–972] | 582 [484–682] |
|
|
|
9.6 [5.3–31.5] | 12.7 [6.5–20.2] | 0.6 | 20.6 [7.5–22.4] | 8.3 [4.1–20.6] | 0.7 | 0.7 |
|
6.9 [4.8–17.0] | 4.7 [4.2–8.1] |
|
6.9 [6.0–38.6] | 5.5 [4.2–17.2] | 0.7 | 0.5 |
|
0.5 [0.3–1.7] | 0.2 [0.1–0.6] |
|
0.4 [0.3–2.7] | 0.4 [0.2–1.5] | 0.2 | 0.5 |
|
74.1 [61.8–86.2] | 42.6 [28.7–57.0] |
|
47.4 [29.2–65.8] | 18.3 [10.7–35.9] |
|
|
|
3.5 [1.3–5.3] | 5.0 [4.2–5.7] |
|
4.8 [3.8–7.1] | 5.1 [4.3–7.6] | 0.5 | 0.8 |
Variables are expressed as median and interquartile range [IQR]. pW, Wilcoxon rank test was used to analyze longitudinal comparisons. pM-W, Mann–Whitney U test was used for transversal comparisons of post cART data.
Statistically significant values are highlighted in bold, p < 0.005.
We also explored potential correlations between MDSCs and monocyte subsets, with the levels of soluble inflammatory biomarkers, including IL-6, hsCRP, D-dimers, sCD14, LPS, IP-10, and hyaluronan. The cellular Th17/Treg ratio and the anti-CMV antibodies titers were also tested for potential associations with innate subsets. The LR-subjects included herein showed significantly higher IL-6 levels (
Relationships between MDSCs and monocytes with proinflammatory biomarkers. Correlations using the Spearman rank test were explored between
We observed increased frequencies of total-MDSCs and m-MDSCs, although no differences in the distribution of monocyte subsets, preceding the poor CD4 T-cell recovery of immunodiscordant subjects. An altered expression of several functional markers, such as CCR2, CD16, CD11b, and PDL1, on MDSCs and monocyte subsets also preceded such anomalous response to cART. Remarkably, these alterations were associated with the levels of inflammatory biomarkers and the Th17/Treg ratio. Finally, longitudinal assessment showed only slight modifications on these parameters after 24 months of suppressive cART.
In recent years, MDSCs have been explored in the context of viral infections, and increased frequencies have been described in HIV-infected subjects (
MDSCs could contribute to changes in CD4 T cells and Tregs by different mechanisms. Among others, they express several enzymes and molecules with suppressive function, such as IDO, and ARG1 (both depriving key nutrition factors for T-cells from the microenvironment as L-tryptophan and L-arginine, respectively); MDSCs also produce reactive nitrogen and oxygen species and upregulate immune checkpoints, as PD-Ll and galectin-9 (
The potential alterations of monocyte subsets in immunodiscordant subjects have been scarcely investigated and never before the onset of cART (
Monocyte and MDSC alterations contribute to HIV pathogenesis (
Our study has limitations. First, this is an exploratory and descriptive analysis of multiple variables using small-sized groups, but our rough observations raise interesting new questions about immunodiscordance-related mechanisms that need to be addressed in higher cohorts. Indeed, the information here provided only shows associations but not a causal relationship of MDSCs with recovery. Second, although baseline CD4 T-cell counts were matched in initial groups of study, a trend toward lower CD4 T-cell counts at cART initiation was found in the restricted group of immunodiscordant subjects herein included, as this is a potent risk factor for immunodiscordance (
Importantly, before the onset of cART, subjects with subsequent poor CD4-recovery showed expanded MDSCs and both innate subsets, MDSCs and monocytes showed altered expression of functional markers as CCR2, CD16, CD11b, and PDL1. Moreover, two years of suppressive cART scarcely modified these parameters in the groups of study. Innate subsets correlated with soluble biomarkers of inflammation and bacterial translocation with the Th17/Treg ratio and anti-CMV titers. Our study contributes to the knowledge of the main immune alterations preceding poor CD4-recovery. Such knowledge can be useful in the search for new therapeutic targets for this clinical situation.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by Comité de Etica de la Investigación de los Hospitales Universitarios Virgen Macarena-Virgen del Rocío. The patients/participants provided their written informed consent to participate in this study.
IR-S designed the study, performed experiments, data analysis and interpretation, and wrote the draft. RP-B contributed to the design of the study and revised the draft for important intellectual content. JG, SM, DV, and VE provided samples and critically revised the draft. MM-F, AR, FV, and ML contributed to the study design and critically revised the draft. YP conceived and designed the study, contributed to data analysis and interpretation and to the writing. All authors contributed to the article and approved the submitted version.
This study was funded by grants from the Instituto de Salud Carlos III, Fondo de Investigación Sanitaria [FIS; PI16/00503, PI18/01216, PI19/01337; PI20/00326] and contratos para la intensificación de la actividad investigadora en el SNS [INT20/00031], co-funded by European Regional Development Fund/European Social Fund; “A way to make Europe”/”Investing in your future”, by the Programa de Suport als Grups de Recerca AGAUR (2017SGR948). The Spanish AIDS Research Network of Excellence also supported this study (RD16/0025/0019; RD16/0025/0006). YP was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the ‘‘Nicolás Monardes’’ programme [C-0013-2017]. AR is supported by IISPV through the project “2019/IISPV/05” (Boosting Young Talent), by GeSIDA through the “III Premio a Jóvenes Investigadores” and by ISCIII through the Miguel Servet Program “CP19/00146”. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We want to particularly acknowledge the patients in this study for their participation and to the HIV BioBank integrated in the Spanish AIDS Research Network and collaborating Centres for the generous gifts of clinical samples used in this work. The HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by Instituto de Salud Carlos III, Spanish Health Ministry (Grant n° RD06/0006/0035, RD12/0017/0037 and RD16/0025/0019) as part of the Plan Nacional R + D + I and cofinanced by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). This study would not have been possible without the collaboration of all the patients, medical and nursery staff and data managers who have taken part in the project. The RIS Cohort (CoRIS) is funded by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en SIDA (RIS C03/173, RD12/0017/0018 and RD16/0002/0006) as part of the Plan Nacional R+D+I and cofinanced by ISCIII-Subdirección General de Evaluacion y Fondo Europeo de Desarrollo Regional (FEDER).
The Supplementary Material for this article can be found online at:
Representative gating strategy of MDSCs and monocytes subsets.
Associations between CMV titers and patrolling monocytes. CMV titters were strongly associated with homing and activation markers of patrolling monocytes, but only in LR-subjects. Correlations were performed in 11 LR-subjects and 14 HR-subjects.
cART, combined antiretroviral therapy; CMV, Cytomegalovirus; HIV, Human Immunodeficiency Virus; HP, homeostatic proliferation; hsCRP, high-sensitivity C-reactive protein; MDSC, myeloid-derived suppressor cells; PBMC, peripheral blood mononuclear cells; Treg, regulatory T-cells.