Antiretroviral treatment (ART) implementation has greatly reduced mother to child HIV-1 transmission. Despite this, at the end of 2019, 1.7 million people living with HIV-1 were children aged 0 to 14 years old (1). WHO guidelines recommend that ART should be initiated within 1 year of age in all vertically acquired HIV-1 infected children as it is well known that the earlier the ART is initiated after infection, the greater the reduction in the reservoir and disease progression, not only in HIV-1 infected children but also in adults (2–4). Several observations suggested that a late timing of treatment initiation in HIV-1 infected children is associated with the induction of ineffective HIV-specific immune responses (5, 6), altered immune phenotype (7) and a distinct transcriptional signature and protein profile, showing upregulation of immune activation pathways (5, 8), indicated a proinflammatory state that may potentially trigger premature non-AIDS events including atherosclerotic and metabolic diseases. It is still unclear whether the effects of the infection on an immune system under development are reversible later in life, as first children infected since birth are now reaching adulthood. Immunological data on this population remain scarce, but our group along with others has described a premature immune aging profile associated with HIV-1 infection (7, 9).

MicroRNAs (miRNAs) are small noncoding RNAs implicated in the development and progression of multiple diseases and have recently been proposed as therapeutic targets in cancer, cardiovascular and metabolic diseases and infections due to their crucial roles in processes of cell maturation, proliferation, migration, differentiation and function in the immune system (10, 11). Focusing on HIV-1 infection, there is a strong effort centered on determining how HIV-1 can differentially modulate several miRNAs that potentially regulate host cellular pathways such as cell cycle, apoptosis, T-cell signalling and cytokine response (12, 13). Because of that, cellular miRNAs could possibly be involved in establishing HIV-1 latency. Recent strategies for clearing viral reservoir have resulted from studies on the mechanism of how miRNAs influence viral protein expression (14). Up to the moment, most findings related to miRNA in HIV-1-infection have been obtained from patients infected during adulthood focussing on the disease progression and there is no information on the miRNA profile related to an early ART initiation in vertically infected HIV-1-patients that reached adult status. Thus, the objective of the present preliminary study was to investigate the miRNA expression profile from vertically acquired HIV-1-infected young adults based on ART initiation delay as well as its association with inflammatory biomarkers and immune system activation and maturation state.

In this work, for the first time we describe a specific miRNA signature on peripheral blood mononuclear cells of vertically HIV-1-young adults who started an ART during the first year after birth compared to late treated HIV-1-young adults, with the purpose of performing a distinctive screening of miRNAs that potentially control the expression of multiple related genes in the context of HIV-1 control and future delayed progression due to their relevant role in HIV-1 cycle and in the establishment of reservoirs. Besides, the miRNome assay was completed with an in-depth phenotype characterization on innate and adaptive immune system components. The associations found between miRNAs and biochemical inflammatory biomarkers and activation and maturation markers expression highlight the potential role of those miRNAs as biomarkers for immune activation and can be considered good targets for therapies aimed at modulating immune response in HIV-1-young adults.

Our study confirms previous studies in which HIV-1 infection leads to altered miRNome compared to non-HIV-1 infected population. However, all evidences were described on HIV-1 patients infected at adulthood by sexual route among other ways of transmission and tried to find patters related to immune and viral progression (28). Up to the moment, none information had been carried out from vertically acquired HIV-1 infected group that still represents more than 150,000 new infections worldwide every year (1).

Focusing on most relevant identified miRNAs comparing EARLY and LATE treated HIV-1-young adults, miRNA-155-5p has been widely studied due to its role in the functionality of the immune system regulating the immune response and lymphocyte differentiation (29, 30). Besides, miRNA-155 has been highlighted as potential biomarker of long-term nonprogressors (LTNPs) who showed decreased levels compared to viremic naïve HIV-subjects suggesting an important role also in the control and pathogenesis of HIV-1 infection (31). Accordingly to that, but contrary to what we expected due to all well-known benefits associated to an EARLY antiretroviral therapy introduction described up to the moment (2, 4, 32, 33), our group of LATE treated patients would look like LTNPs. In LTNP patients, an increased adaptive immune activation has been described suggesting that spontaneous viral control may imply an immunological cost in those patients who maintain virologic control in the absence of treatment. Similarly, the activated immune phenotype we described herein on LATE treated vertically HIV-1 patients with higher viral reservoir, could reflect the exacerbated immune pressure needed against viral replication on this group. Interestingly, increased levels of miRNA-155 have been correlated with increased immune activation in HIV-scenario (34) accordingly to our results.

Regarding miRNA-18a, despite scarce evidences related to miR-18a-3p, low levels of miRNA family members like miRNA-18a and miR-18b-5p in LTNP compared to typical progressors indicated the potential role of that miRNA in viral replication and disease progression (35) by controlling genes of the inflammatory response as HIF-1α (36). The low levels of miRNA-18a-5p expression observed in EARLY treated vertically acquired HIV-patients studied in this work could explain the low viral reservoir and immune activation that has widely characterized this group of patients (37).

In addition to everything described so far related to miRNA-155-5p, that miRNA and miRNA-1248, which is associated with good survival prognoses in cancer (38), both upregulated on EARLY treated HIV-1 patients, strongly correlated with the activation marker HLA-DR, the natural cytotoxicity family activation receptor NKp30 and the inhibiting receptor NKG2A expressed on NK cells and more specifically on the main cytotoxic NK subset, CD16^high and the activated and mature CD56^negCD16+ NK cells, described as a subset with impaired effector functions in HIV-1 infection (39). Despite the limitation that miRNA profile was determined in the pool of PBMCs without isolated different cell subsets, our results are in agreement with recent insights that have highlighted the role of miRNAs in maintaining killing effect of NK cells in cancer scenario via activation markers modulation (40–42), in promoting antiviral immunity in the scenario of viral infection (43) and altogether suggests the possible role of both, miRNA-155-5p and miRNA-1248 in regulating inflammatory cytokine production in NK cells, potentially balancing disease progression during chronic HIV infection. Higher TIM-3 expression in NK cells has been considered a marker of better prognosis during HIV infection since its down regulation is indicative of IFN-γ deficient response (44) and interestingly, strong and direct correlations were also found between miRNA-155-5p levels, miRNA upregulated on EARLY treated HIV-1 young adults, and MFI expression of TIM-3 in CD56^high NK cells.

MiRNAs have also arisen as critical regulators of lipidic metabolism controlling genes involved in HDL and LDL secretion, the most important risk factors of atherosclerosis, cholesterol biosynthesis and hepatic LDL receptor expression (45, 46). In our study EARLY treated HIV patients had decreased HDL levels traditionally associated with risk for developing cardiovascular diseases (CVD) and, while no differences were found in ratio LDL/HDL comparing groups, both biochemical parameters correlated with miRNA-501-3p and miRNA-155-5p relative levels. In the case of miRNA-501-3p, downregulated on EARLY treated HIV-1 young adults, that miRNA levels correlated directly with soluble HDL and inversely with ratio LDL/HDL and the opposite correlations were observed for miRNA-155-5p, upregulated on EARLY treated group.

Multiple miRNAs are being also evaluated as novel plasma biomarkers that out-perform or add value to the conventional liver injury biomarkers ALT, AST among others (47). Interestingly, miRNA-296-5p and miRNA-501-3p levels, both downregulated on EARLY group compared with LATE group, also correlated directly with AST levels. Recent evidences indicate that miRNA-296-5p regulates liver PIN1 and PLK1 genes that encode peptidylprolyl Cis/Trans isomerase NIMA-Interacting-1 and polo-like kinase 1 respectively, both involved in HIV-1 replication cycle stages (18, 19), suggesting that miRNA-296-5p may have a regulatory activity on virus replication in HIV pathogenesis and contributing to immune activation. These indicators could be potential biomarkers for disease progression and, in agreement with previous observations (48), good targets for therapies aimed at modulating the immune response in HIV infection. All explain herein, reinforce the idea that those distinct miRNAs could be considered regulators of cholesterol metabolism, hepatic lipoprotein synthesis, potential liver damage biomarkers and therapeutic targets for the treatment of CVD. However, genetic ablation of other miRNAs in the context of CVD development have led also to contradictory effects depending on animal model raising concerns about the use of therapies based on miRNAs (49).

There is unclear information on the risk of developing CVDs among other pathologies as our study is based on the time of treatment introduction and the cross-sectional design of our study as main limitation. In addition, the scarce number of participants and the low statistical power do not allow us to find differences in the immunological comparisons and results obtained here should be validated in a larger cohort of patients. Moreover, the in-depth pairing between groups in terms of CD4, CD8-T cell counts, time since ART initiation and since virological control can also explain the lack of immune phenotype differences on T- and NK cells. The panel used for miRNAs profile had limited number of them. However, samples of vertically infected patients reaching adulthood are scarce and mentioned limitations are also counterbalanced by the exhaustive follow-up of the participants during a median of 21 years since HIV diagnosis and during that period none of them has developed any inflammatory disease or non-AIDS-event. Further longitudinal observation would be necessary to determined clinical consequences that could be explained by the EARLY start of treatment.

In conclusion, our finding demonstrates that there is a miRNA signature that discriminates LATE treated from EARLY treated vertically acquired HIV-1-infected young adults. The role of those distinct miRNAs target genes in the modulation of HIV-1 replication and latency reveals new host targets that may be modulated in future antiviral strategies. Moreover, we found strong correlations between the most significant miRNAs and soluble inflammatory biomarkers and NK and T-cell activation and maturation suggesting that those miRNAs may be potential biomarkers for immune activation and progression disease in vertically acquired HIV-1-infected young adults who initiated a late ART and in general HIV-1-infected patients.

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 Ética de Investigación con Medicamentos. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

MAM-F designed the research study. LT-D, IC, EV-A designed and performed the research studies. RR contributed essential tools. LT-D and EV-A analyzed the data. LT-D wrote the paper. JTR, MLN, and MAM-F critically discussed the paper. All authors have read and approved the final manuscript.

LT-D is supported by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CD20/00025” through the Sara Borrell Program and by GeSIDA through the “Premio para Jóvenes Investigadores 2021”. EV-A was supported by Comunidad de Madrid, Programa de Empleo Joven “PEJ-2017 AI_BMD-7446”. This work has been (partially) supported by the RD16/0025/0019 projects as part of Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (2020–2022) and co-financed by Instituto de Salud Carlos III (Subdirección General de Evaluación) and Fondo Europeo de Desarrollo Regional (FEDER), RETIC PT17/0015/0042, Fondo de Investigación Sanitaria (FIS) 2020-2022 (grant number PI19/01638) and EPIICAL Project. Moreover, this work has been supported partially by a EUROPARTNER: Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection for interdisciplinary research and innovation of the University of Lodz Programme: NAWA International Academic Partnership Programme. This article/publication is based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology) 2018-2022.

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.

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