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Interleukin 18 (IL-18) and IL-3 in Extracellular Vesicles: Biomarkers for Durable Elite Control of HIV-1.

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Affiliations

  • 1. Group of Virology and Pathogenesis, Galicia Sur Health Research Institute-Complexo Hospitalario Universitario de Vigo, Vigo, Spain.
      Authors
    • Poveda E 1
    • Reglero C 1
    • Pérez-González A 1
    (3 authors)
  • 2. Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
      Authors
    • Fitzgerald W 2
    • Margolis L 2
    (2 authors)
  • 3. Infectious Diseases Unit, Complexo Hospitalario Universitario de Ferrol, A Coruña, Spain.
      Authors
    • Mariño A 3
    • Álvarez H 3
    • Valcarce N 3
    (3 authors)
  • 4. Infectious Diseases Division, University Hospital Germans Trias and Fight Infections Foundation, Badalona, Spain.
      Authors
    • Llibre JM 4
    (1 author)
  • 5. Department of Medicine, University of Alcalá, Alcalá de Henares, Spain.
      Authors
    • Guillén SM 5
    (1 author)
    • Show all (8)

ORCIDs linked to this article

The Journal of Infectious Diseases, 01 Jun 2023, 227(12):1381-1385
https://doi.org/10.1093/infdis/jiad042 PMID: 36790818 PMCID: PMC10266942

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
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Abstract 

Plasma extracellular vesicle (EV)-associated cytokines were quantified in people with HIV (PWH) with different virological control status, including elite controllers (EC) who maintain persistent control (PC) or not (TC). Cytokine signatures and pathways were determined for each group. Median EV-associated cytokine levels were higher among PWH than HIV-uninfected. EC showed the highest levels of EV-associated cytokines among PWH with PC levels higher than TC levels. IL-18 levels best distinguished PWH from uninfected controls, and EC from ART-treated, and IL-3 distinguished PC from TC. The role of EV-cytokines in intercellular communication and endogenous control of HIV expression should be investigated further.

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Logo of jidLink to Publisher's site
J Infect Dis. 2023 Jun 15; 227(12): 1381–1385.
Published online 2023 Feb 15. https://doi.org/10.1093/infdis/jiad042
PMCID: PMC10266942
PMID: 36790818

Interleukin 18 (IL-18) and IL-3 in Extracellular Vesicles: Biomarkers for Durable Elite Control of HIV-1

Eva Poveda,corresponding author Wendy Fitzgerald, Cristina Reglero, Alexandre Pérez-González, Ana Mariño, Hortensia Álvarez, Nieves Valcarce, Josep Maria Llibre, Santiago Moreno Guillén, Maria Angeles Muñoz Fernández, Ezequiel Ruiz-Mateos, Leonid Margolis, Michael M Lederman, and Michael L Freeman, on behalf of ECRIS integrated in the Spanish AIDS Research Network
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Associated Data

Supplementary Materials

Abstract

Plasma extracellular vesicle (EV)-associated cytokines were quantified in people with HIV (PWH) with different virological control status, including elite controllers (EC) who maintain persistent control (PC) or not (TC). Cytokine signatures and pathways were determined for each group. Median EV-associated cytokine levels were higher among PWH than HIV-uninfected. EC showed the highest levels of EV-associated cytokines among PWH with PC levels higher than TC levels. IL-18 levels best distinguished PWH from uninfected controls, and EC from ART-treated, and IL-3 distinguished PC from TC. The role of EV-cytokines in intercellular communication and endogenous control of HIV expression should be investigated further.

Keywords: HIV, cytokines, elite controllers, extracellular vesicles, functional cure

Elite controllers (EC) spontaneously control human immunodeficiency virus-1 (HIV-1) replication without antiretroviral therapy (ART), representing an exceptional model of functional cure. There are currently no reliable biomarkers for predicting EC, nor for distinguishing which EC will be transient controllers (TC) who ultimately lose HIV-1 replication control over time or persistent controllers (PC) who maintain it [1–3].

Previous small studies have found differences in viral (genetic diversity, frequency of viral blips) and host (HIV-1–specific T-cell responses, indices of immune activation) factors between TC and PC [1, 4–6]. However, broader characterization in larger and well-defined cohorts is warranted to address questions such as which EC should be treated with ART and to define distinguishing biomarkers.

Cytokines are traditionally measured as soluble factors in plasma, but cytokines are also encapsulated within and embedded on the surface of plasma extracellular vesicles (EVs) [7, 8]. In this context, we performed a detailed evaluation of 39 cytokines associated with plasma EVs to determine if EV-associated cytokine abundance and diversity could distinguish well-characterized cohorts of people with HIV (PWH) with different viroimmunological status.

METHODS

Sample Selection and Preparation

Frozen plasma samples were obtained from 30 participants without HIV and 30 PWH in each of 3 groups: (1) ART-naive; (2) ART-treated with nondetectable viremia for at least 1 year; and (3) EC with <50 HIV-RNA copies/mL in the absence of ART for a median of 14.4 years (detailed demographics in Supplementary Table 1). EC included 15 TC who lost virological control (defined as at least 2 consecutive viral load measurements above the detection limit in 1 year) and 15 PC who sustained viral suppression [1, 9]. Plasma samples for TC were obtained a median of 1.38 years (interquartile range, 0.95–2.88 years) before the loss of control. The study was approved by the regional Ethic Committee (CAEIG), an external scientific and ethics committee of the HIV Biobank (Spanish HIV/AIDS Research Network). Platelet-poor plasma (PPP) was prepared by centrifuging 500 µL plasma at 3000g for 15 minutes at 4°C. PPP was processed using ExoQuick Ultra for plasma (SystemBio) according to the manufacturer's instructions, to obtain EVs.

Cytokine Measurement

Two in-house multiplexed bead-based assays were developed to measure 39 markers using a previously published assay that measured 33 cytokines [7] and another assay to measure an additional 6 markers (Supplementary Methods).

Statistical Analysis

Statistical analyses were performed using Statistical Package for the Social Sciences software (SPSS) 19.0 (IBM) and the packages randomForest, pROC, rpart, and gplots of R software (http://cran.r-project.org). A P < .05 was considered nominally significant (Supplementary Methods).

Ingenuity Pathway Analysis

Comparison analyses on cytokine data (fold change compared to controls, with ± 1.25 cutoff) for each PWH subgroup were performed in ingenuity pathway analysis (IPA) software (Qiagen Bioinformatics). IPA identified canonical signaling pathways, causal networks, and biological functions based on P value of overlap using Fisher exact test, with Benjamini-Hochberg multiple hypothesis correction (P < .01 considered significant) or Z-scores to infer activation (positive z-score) or inhibition (negative z-score), z-score ≥ 2 indicating significance. My Pathway analysis connected significant canonical pathways and biological functions with key cytokines based on the IPA Knowledge Base.

RESULTS

Elevated EV-Associated Cytokine Levels Among PWH, Especially PC

We measured 39 cytokines associated with EVs isolated from PPP from the study populations described in Supplementary Table 1. Overall, the relative median levels of EV cytokines were 1.33-fold higher for the PWH population than for the uninfected control group. EC had higher levels of EV cytokines than did the ART treated (1.11-fold higher) and ART naive (1.32-fold higher). Within EC, the levels of cytokines were 1.36-fold higher for PC than for TC. Figure 1 shows a fold change heat map of the relative concentrations of EV cytokines for each group (see Supplementary Figure 1 for heat map with standardized means).

An external file that holds a picture, illustration, etc. Object name is jiad042f1.jpg

Heap map of the relative cytokine levels associated with extracellular vesicles. The levels of cytokines for each study group are represented as the standardized mean of each group as a comparator for each cytokine. Abbreviations: ART, antiretroviral therapy; EC, elite controller.

Identification of Specific Cytokine Biomarkers for EC and PC

Random forest and principal component analysis (PCA) identified potential EV cytokine signatures for the study groups while area under the curve (AUC) represents the probability of each cytokine to correctly distinguish the study group to which each individual belongs. Overall, higher EV interleukin 18 (IL-18) best distinguished between PWH and uninfected controls with median levels of 1.15 versus 0.26 pg/mL having an AUC of 0.74 by receiver operating characteristic (ROC) analysis. Thus, there is a 74% chance that IL-18 levels distinguish PWH and controls. Sensitivity was 68.9% and specificity 76.7%.

Higher levels of EV IL-18 also distinguished EC from ART-treated persons with suppressed viremia displaying an AUC of 0.942 and a sensitivity of 73.3% and specificity of 100%. EC also had higher EV IL-18 levels than did the ART naive with a ROC analysis displaying AUC of 0.746 with a sensitivity of 73.3% and specificity of 66.7%. Higher levels of EV IL-3 and EV tumor necrosis factor-related apoptosis inducing ligand (TRAIL) were best at discriminating TC and PC phenotypes with an ROC analysis showing an AUC of 0.824 and 0.814, a sensitivity of 73.3% and 86.7%, and a specificity of the 86.7% and 73.3%, respectively. The figures for all the cytokines identified as signatures for each study group are shown in Supplementary Table 2. See Supplementary Figures 2–5 for random forest, PCA, and ROC curves.

Algorithms designed using Decision Trees analysis showed that among individuals with suppressed viremia (EC and ART treated), 100% of participants with EV IL-18 levels equal to or above 2.23 pg/mL were correctly classified as EC. Within the EC groups, 91% with interferon-γ (IFN-λ) levels equal or greater than 81.41 pg/mL (n = 10) were correctly classified as PC (Supplementary Figure 6).

IPA Analysis of Cytokine Expression by PWH Groups

Comparison analyses examined differential protein expression between EC and ART treated, and between PC and TC, to identify canonical pathways, putative causal networks, and biological functions. In general, all predictions were more significant for EC than ART treated, and more for PC than TC. Top canonical pathways included IL-17 signaling and virus-induced inflammatory pathways (Supplementary Figure 7A). Causal network analysis identified molecules involved in inflammation as potential upstream regulators, particularly nuclear factor κB (NFκB; Supplementary Figure 7B). Biological function analysis predicted more significant antiviral activity, general immune responses, and specific immune cell and endothelial cell responses for EC than for ART treated, and more for PC than TC. (Supplementary Figures 8 and 9). One exception was innate lymphoid cells (ILC), which were significant for TC but not PC.

The IPA Knowledge Base demonstrated the role of IL-18 (distinguishing EC from ART treated) and IL-3 and TRAIL (distinguishing PC from TC) in significant canonical pathways and biological functions. IL-18 was notably associated with natural killer (NK) cell, T-cell, and ILC functions (Figure 2A ), which were more significant in EC. IL-3 and TRAIL were significantly associated with myeloid, especially dendritic cell (DC), and B-cell functions, which were more significant in PC (Figure 2B ).

An external file that holds a picture, illustration, etc. Object name is jiad042f2.jpg

Pathways analysis of key distinguishing cytokines: IL-18 and IL-3/TRAIL. My Pathway analysis in ingenuity pathway analysis was used to link (A) IL-18, the cytokine best distinguishing elite controllers from antiretroviral therapy treated, and (B) IL-3/TRAIL, the cytokines best distinguishing persistent from transient controllers, to top canonical pathways and downstream biological functions. Abbreviations: CP, canonical pathways; Fx, biological functions; IL, interleukin; TRAIL, tumor necrosis factor-related apoptosis inducing ligand.

DISCUSSION

Our study revealed significantly higher levels of plasma EV-associated cytokines among PWH with different virologic control status than among uninfected individuals. EV-association of cytokines may provide advantages, including protection from the action of antagonists, homeostatic systems, or enzymes that limit their biological function, and inclusion of surface receptors that direct cytokines to particular cells or allow them to targeted transportation to distant sites without dilution. EV association of proteins may also confer different or stronger effects compared to soluble proteins, particularly because EVs may carry multiple components (proteins, lipids, nucleic acids) capable of influencing target cells [10, 11].

Among PWH, EC showed the highest levels of EV-associated cytokines, and PC more than TC. Traditionally, only soluble cytokines are measured in plasma, and only a few small studies have compared the inflammatory profiles of TC and PC [1, 12]. Further analyses identified higher levels of EV IL-18 as a biomarker for EC, and higher EV IL-3 and TRAIL best discriminated PC from TC. IPA analysis of EV-associated cytokine expression provided predictions consistent with viral immune defenses and among PWH, but these associations were much more significant for EC than ART treated, and among EC, more significant for PC than TC. IL-18 was linked with biological functions of NK, T-cell subsets, and ILC and IL-3/TRAIL with DC and B-cell functions. Soluble levels of IL-18 and TRAIL have been reported to be increased in the circulation of PWH, but less is known for IL-3, a molecule that supports plasmacytoid DC (pDC) survival, enabling the early innate immune response to HIV infection [13]. Interestingly, it has been observed that pDC from controllers produced higher amounts of type I IFN in response to HIV, likewise higher capacity to induce a reduction of HIV replication and TRAIL-induced apoptosis of HIV-infected CD4+ T-cells than pDC from viremic patients [14, 15].

This study has some limitations. Despite well-characterized cohorts with different virologic status, the sample size for each group is small, limiting our statistical power. Cytokine concentrations measured by Luminex may differ from those obtained by clinical-use enzyme-linked immunosorbent assay (ELISAs), reducing the clinical applicability of our identified cutoff values. In future studies, inclusion of additional cytokines and signaling molecules would enhance biological interpretation of the data. Finally, the cytokine patterns observed represent a correlation, and may not necessarily contribute causally to the persistent elite control.

In conclusion, higher plasma levels of EV-associated cytokines, specifically IL-18, IL-3, and TRAIL, may represent a new signature for durable ART-free natural control of HIV replication. Protected transport of cytokines in EVs might represent a sophisticated mechanism to deliver targeted signals to host cells selectively maintaining local environments of activation and inflammation that control HIV replication. The balance between endogenous control of HIV replication and the potential long-term harmful health consequences for the EC population should be further evaluated.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Supplementary Material

jiad042_Supplementary_Data

Contributor Information

Eva Poveda, Group of Virology and Pathogenesis, Galicia Sur Health Research Institute-Complexo Hospitalario Universitario de Vigo, Vigo, Spain.

Wendy Fitzgerald, Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

Cristina Reglero, Group of Virology and Pathogenesis, Galicia Sur Health Research Institute-Complexo Hospitalario Universitario de Vigo, Vigo, Spain.

Alexandre Pérez-González, Group of Virology and Pathogenesis, Galicia Sur Health Research Institute-Complexo Hospitalario Universitario de Vigo, Vigo, Spain.

Ana Mariño, Infectious Diseases Unit, Complexo Hospitalario Universitario de Ferrol, A Coruña, Spain.

Hortensia Álvarez, Infectious Diseases Unit, Complexo Hospitalario Universitario de Ferrol, A Coruña, Spain.

Nieves Valcarce, Infectious Diseases Unit, Complexo Hospitalario Universitario de Ferrol, A Coruña, Spain.

Josep Maria Llibre, Infectious Diseases Division, University Hospital Germans Trias and Fight Infections Foundation, Badalona, Spain.

Santiago Moreno Guillén, Department of Medicine, University of Alcalá, Alcalá de Henares, Spain. Ramón y Cajal Institute for Health Research, Madrid, Spain. Department of Infectious Diseases, Ramón y Cajal University Hospital, Madrid, Spain. Infectious Diseases Networking Biomedical Research Centre, Madrid, Spain.

Maria Angeles Muñoz Fernández, Hospital Gregorio Marañón BioBank, Madrid, Spain.

Ezequiel Ruiz-Mateos, Laboratory of Immunovirology, Clinical Unit of Infectious Diseases, Microbiology, and Parasitology, Institute of Biomedicine of Seville, Virgen del Rocío University Hospital, University of Seville, Seville, Spain.

Leonid Margolis, Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

Michael M Lederman, Rustbelt Center for AIDS Research, Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, Ohio, USA.

Michael L Freeman, Rustbelt Center for AIDS Research, Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, Ohio, USA.

Notes

Financial support. This work was supported by Instituto de Salud Carlos III (grant numbers PI16/02159, BA18/00034, PI19/00747, PI19/01127, PI22/01341, PI22/01796, and CM20/00243); European Regional Development Fund, A way to make Europe; Red Española de Investigación en SIDA (grant numbers RD16/0025/0026 and RD16/0025/0020); Fundación Biomédica Galicia Sur; HIV Biobank-Spanish HIV/AIDS Network; Case Western Reserve University Center for AIDS Research; and Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health Intramural Program (grant number ZIA HD008968-05). Funding to pay the Open Access publication charges for this article was provided by Instituto de Salud Carlos III (reference PI19/00747).

References

1. Pernas M, Tarancón-Diez L, Rodríguez-Gallego E, et al. . Factors leading to the loss of natural elite control of HIV-1 infection. J Virol 2018; 92:e01805-17. [Europe PMC free article] [Abstract] [Google Scholar]
2. Poveda E, Tabernilla A, Fitzgerald W, et al. . Massive release of CD9+ microvesicles in human immunodeficiency virus infection, regardless of virologic control. J Infect Dis 2022; 225:1040–9. [Europe PMC free article] [Abstract] [Google Scholar]
3. Ruiz-Mateos E, Poveda E, Lederman MM. Antiretroviral treatment for HIV elite controllers? Pathog Immun 2020; 5:121. [Europe PMC free article] [Abstract] [Google Scholar]
4. Tarancon-Diez L, Rodríguez-Gallego E, Rull A, et al. . Immunometabolism is a key factor for the persistent spontaneous elite control of HIV-1 infection. EBioMedicine 2019; 42:86–96. [Europe PMC free article] [Abstract] [Google Scholar]
5. Rodríguez-Gallego E, Tarancón-Diez L, García F, et al. . Proteomic profile associated with loss of spontaneous human immunodeficiency virus type 1 elite control. J Infect Dis 2019; 219:867–76. [Abstract] [Google Scholar]
6. Canouï E, Lécuroux C, Avettand-Fenoël V, et al. . A subset of extreme human immunodeficiency virus (HIV) controllers is characterized by a small HIV blood reservoir and a weak T-cell activation level. Open Forum Infect Dis 2017; 4:ofx064. [Europe PMC free article] [Abstract] [Google Scholar]
7. Fitzgerald W, Freeman ML, Lederman MM, Vasilieva E, Romero R, Margolis L. A system of cytokines encapsulated in extracellular vesicles. Sci Rep 2018; 8:8973. [Europe PMC free article] [Abstract] [Google Scholar]
8. Konadu KA, Chu J, Huang MB, et al. . Association of cytokines with exosomes in the plasma of HIV-1–seropositive individuals. J Infect Dis 2015; 211:1712–6. [Europe PMC free article] [Abstract] [Google Scholar]
9. Dominguez-Molina B, Leon A, Rodriguez C, et al. . Analysis of non-AIDS-defining events in HIV controllers. Clin Infect Dis 2016; 62:1304–9. [Abstract] [Google Scholar]
10. Shi C, Ulke-Lemée A, Deng J, Batulan Z, O’Brien ER. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy. FASEB J 2019; 33:1617–30. [Abstract] [Google Scholar]
11. Álvarez V, Sánchez-Margallo FM, Macías-García B, et al. . The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta. J Tissue Eng Regen Med 2018; 12:2088–98. [Abstract] [Google Scholar]
12. Côrtes FH, de Paula HHS, Bello G, et al. . Plasmatic levels of IL-18, IP-10, and activated CD8+ T cells are potential biomarkers to identify HIV-1 elite controllers with a true functional cure profile. Front Immunol 2018; 9:1576. [Europe PMC free article] [Abstract] [Google Scholar]
13. Mitchell JL, Takata H, Muir R, et al. . Plasmacytoid dendritic cells sense HIV replication before detectable viremia following treatment interruption. J Clin Invest 2020; 130:2845–58. [Europe PMC free article] [Abstract] [Google Scholar]
14. Barblu L, Machmach K, Gras C, et al. . Plasmacytoid dendritic cells (pDCs) from HIV controllers produce interferon-α and differentiate into functional killer pDCs under HIV activation. J Infect Dis 2012; 206:790–801. [Abstract] [Google Scholar]
15. Machmach K, Leal M, Gras C, et al. . Plasmacytoid dendritic cells reduce HIV production in elite controllers. J Virol 2012; 86:4245–52. [Europe PMC free article] [Abstract] [Google Scholar]
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Funding 

Funders who supported this work.

Case Western Reserve University Center for AIDS Research

    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (1)

    European Regional Development Fund

      Fundación Biomédica Galicia Sur

        HIV Biobank-Spanish HIV/AIDS Network

          Instituto de Salud Carlos III (7)

          Intramural NIH HHS (1)

          Red Española de Investigación en SIDA (2)