Are There Any Reviews or Input on the Medicine Moringa
Introduction
MiRNAs are a curt sequence of non-coding pocket-sized RNAs that play a pivotal function in gene regulation at the postal service transcriptional level, in mammals and in plants. It has been demonstrated that a cross-kingdom interaction tin can occur betwixt the plant-derived miRNAs and their "host," through diet (Liang et al., 2015; Teng et al., 2018). In 2012 it was reported for the first time that establish-derived miRNAs from rice passing through the gastrointestinal tract could be absorbed and reach, via the bloodstream, the body organs, thus exerting their biological function (Zhang et al., 2012). Even though it remains an issue still much debated (Campbell, 2020), there is increasing prove on the regulatory role of factor expression machinery in host cell by intake and bioavailability of dietary plant-derived miRNAs (Liang et al., 2015; Zhou et al., 2015; Lukasik and Zielenkiewicz, 2017; Minutolo et al., 2018). Moringa oleifera Lam. (MO) is a medicinal constitute, used for centuries in traditional medicine due to its richness in essential nutrients (Popoola and Obembe, 2013; Matic et al., 2018). Moreover MO-based preparations are scientifically documented as being anti-inflammatory, antihypertensive, antimicrobial, antioxidant, and antidiabetic (Ojewole, 2006; Anwar et al., 2007; Dhongade et al., 2017; Bhattacharya et al., 2018) along with other plants used in traditional medicine (Sagnia et al., 2014). Furthermore, MO improves hepatic and renal functions; it also regulates thyroid hormones, and stimulates the immune organization, protecting against oxidative stress, inflammation, hepatic fibrosis, liver impairment, hypercholesterolemia and cancer (Stohs and Hartman, 2015; Almatrafi et al., 2017). MO bioactivity depends mainly on the presence of different classes of plant secondary metabolites (Kou et al., 2018; Saucedo-Pompa et al., 2018). In 2016 MO miRNome has been sequenced, showing the presence of several conserved miRNAs (Pirrò et al., 2016b). Many of the MO miRNAs are conserved beyond multiple plant species; some of these miRNAs were predicted to interact with multiple target genes in the mammalian cells, with potential therapeutic implication for human diseases (Pirrò et al., 2016a; Minutolo et al., 2018; Potestà et al., 2019). More recently nosotros reported that MO seeds aqueous extract (MOES) comprise p-miRs, that were also characterized (Pirrò et al., 2016b). Moreover, nosotros demonstrated that the MOES was able to modulate proliferation and apoptosis in cancer cells; this power was associated with the presence p-miRs, that were able to modulate these processes at a postal service-transcriptional level (Minutolo et al., 2018; Potestà et al., 2019; Potestà et al., 2020). Because the complex interactions betwixt the immune system and HIV, the present paper aims to investigate the furnishings of MO seed extracts enriched in miRNAs, on the regulation of immune response and on HIV replication and integration on peripheral claret mononuclear cells (PBMCs) from HIV-positive patients.
Materials and Methods
Patients Enrollment and Cohort Characterization
Xxx-v chronic HIV-infected subjects naïve to the combination antiretroviral treatment (cART-naïve) were enrolled in an open up study by the Division of Clinical Infectious Diseases, Department of Organisation Medicine, Academy of Rome "Tor Vergata." Ethical approval for the collection and employ of human samples was obtained in 2014 from the ethics committee of "Tor Vergata" Infirmary, protocol number 15/14 (D.M.08.02.2013 D.G.R.146/2013; D.D.1000.467 del 25.07.2013). The 35 HIV+ subjects were characterized for their immunological and virological status (Table 1 and Supplementary Figure S1) and their PBMCs used for the ex vivo experiments. The PBMCs from thirty good for you donors (HDs) were obtained from thirty individuals attention the local claret transfusion unit of Policlinico "Tor Vergata" in Rome. All HDs provided written informed consent. The PBMCs were separated by centrifugation gradient according to standard methods (Ficoll).
TABLE ane. HIV + individuals enrolled (due north = 35).
Bioinformatics Analysis
The bioinformatic analysis was performed on a list of miRNAs (Supplementary Tabular array S1) found in the MOES (Potestà et al., 2019), derived from MO miRNome, belonging to the most conserved establish families (Pirrò et al., 2016b). A support vector machine (SVM) classifier trained using an experimentally validated set of miRNA-mRNA interactions was used equally a prediction tool. The detailed description of the sklearn. svm.SVC classifier implementation is reported in the Supplementary Cloth (Supplementary Data). For pathways and network assay, the Metascape online tool (http://metascape.org) was used to place the predominant biological processes and networks regulated past MO p-miRs. Briefly, following the identification of each gene, the expression values from the total gene expression data profile were extracted from the repository. The factor list was divided into 2 groups: a list of selected genes involved in immune response and inflammation, and a list of genes down-regulated by the p-miRs. Subsequently, an enrichment analysis was performed to identify the significant biological processes and networks modulated past p-miRs. The DAVID online tool (https://david.ncifcrf.gov/) was used to perform functional annotation clustering, using reference database of human complex diseases and disorders (Table 2) Genetic Association Database (GAD_DISEASE) at p-value cutting of bespeak (p < 0.05).
Table 2. Disease enriched analysis associated to p-miRs modulated genes (DAVID tool).
MO p-miRs Pool Extraction, Characterization, and Transfection
MO seeds were provided past the Cameroonian Association of Traditional Practitioners. MO p-sR pool was extracted from the aqueous extract of MO seeds past NucleoSpin® miRNA kit (MACHEREY-NAGEL, Frg) as previously described (Potestà et al., 2019). The presence of the nigh conserved p-miRs (Supplementary Table S1) was evaluated by RT-qPCR, as previously reported (Pirrò et al., 2016b; Minutolo et al., 2018); relative quantity of p-miRs was quantified by the two−ΔΔCt method, where 5S rRNA was used as a housekeeping gene. To evaluate the furnishings of p-miRs, PBMCs from HIV+ subjects and from HDs were stimulated by IL2 (twenty U/ml, Sigma-Aldrich, St. louis, MO) for 72 h in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Life Technologies), 2 mM glutamine, 50 IU/ml penicillin, and 50 IU/ml streptomycin (Hyclone, Cramlington, Britain). PBMCs were transfected with the puddle of p-miRs at concentrations of ane μg/ml, this performed according to the lipofectamine (HF) method (Hi-Fect, Qiagen, HF) as previously described (Minutolo et al., 2018). Equally command for the treatment, the PBMCs were treated with the transfection vehicle (HF) alone. Afterward 72 h, cells were harvested, done twice in PBS, and assessments of viability, apoptosis and immunological analysis were performed.
Transfection and Detection of Mimic p-miR858b
The PBMCs from 15 HIV+ participants and 15 HDs were transfected via the lipofectamine method (Hi-Fect, Qiagen German, HF) using v nM synthetic mimic p-miR858b (Invitrogen, United States) following the manufacturer'south instructions. For handling, the mimic p-miR858b, methylated at the iii′ stop (feature of establish-derived miRNAs) was used. The p-miR858b is present in several plant species included Moringa oleifera, Malus domestica, and Arabidopsis thaliana (Supplementary Effigy 2). The mimic p-miR858b FITC-conjugated was used to evaluate the transfection efficacy. The p-miR858b FITC-conjugated positive cells, were observed by fluorescence microscopy, later 72 h from transfection (Evos Floid Cells Imaging Station, ThermoFisher Scientific, United States) and by menses cytometry (CytoFLEX, Beckman Coulter, United States). The presence and quantity of mimic p-miR858b, in PBMCs from HIV+ and HDs, were detected on Bio-Rad thermal cycler (IQ5) according to the instructions of EXIQON pre-designed primers (p-miR858b 5′ UUCGUUGUCUGUUCGACCUGA-three′). For RNA, the isolation was performed using NucleoSpin RNA II (Machenery-Nagel, Dueren, Germany) according to the manufacturer's instructions. Relative quantity was calculated by the 2−ΔΔCt method, using 5S rRNA as housekeeping cistron.
Cell Viability and Apoptosis Assays
Prison cell viability and mortality rates were assessed by a ten% Trypan blue (EuroClone Due south.p.A., Italy) exclusion examination after 72 h of treatment. Apoptosis was assessed by flow cytometry assay, using a CytoFLEX (Beckman Coulter, U.s.), on isolated nuclei stained with Propidium Iodide (PI) (Merck KGaA, Germany). Information conquering and analyses were performed using CytExpert two.0 (Beckman Coulter, United States) on a minimum of 150,000 events for each sample.
Immunostaining and Flow Cytometry Assay of Cellular Proteins
For the analysis of T cells cluster of differentiation markers (CDs), approximately 1 × xvi cells were suspended in 100 µl of PBS, incubated with anti-human CD95 FITC/CD197-CCR7 PE/CD8 pc5.5/CD45RA APC/CD4 APC Alexa700/CD3 APC Alexa750 antibodies (Beckman Coulter, Us) at 4°C for 30 min (Bordoni et al., 2019). In some experiments, the cells were incubated with anti-human FITC CD25-Fas or TNF-blastoff (Beckman Coulter, United States). For Bcl2 intracellular protein expression the transfected PBMCs were harvested, fixed, permeabilized with 70% ethanol and incubated with PE-conjugated anti-human Bcl2 (BD Biosciences, U.s.a.). The intracellular poly peptide expression of HIV-p24 was detected using a PE anti-HIV p24 antibody (Abcam, Britain). All the stained cells were analyzed via CytoFLEX (Beckman Coulter) and the CytExpert ii.0 software (Beckman Coulter).
Western Blot Analysis of VAV1
Aliquots of 1 × 10half-dozen cells, subjected to various experimental weather, were lyzed and processed for western absorb analysis, as standard protocol. The master antibodies used were rabbit monoclonal antibodies directed against VAV1 protein and goat monoclonal anti-homo Beta-actin (Santa Cruz Biotechnology, CA United States). The secondary antibodies used were anti-goat and anti-rabbit IgG concatenation specific conjugated to peroxidase for Western Blot detection and anti-rabbit IgG-PE for catamenia cytometry assay (Calbiochem, Merck Millipore, Darmstadt, Germany).
Evaluation of HIV Integration
HIV integration was evaluated as previously described (Matteucci et al., 2015). In summary, Deoxyribonucleic acid isolations were performed using NucleoSpin Tissue XS (Machenery-Nagel, Dueren, Germany) according to the manufacturer'south instructions. The DNA from HIV-infected PBMCs was diluted in Tris one mM at ane,000 cell-equivalents/µl; a qualitative PCR was performed by 100 jail cell-equivalents of genomic DNA in standard conditions: 1 PCR Buffer, 2.5 mM MgCl2, 0.two mM deoxynucleoside triphosphates mix, 1.25 U of Taq (all from Promega) and 10 pmol b-globin-specific primers (b-glob 1 and b-glob2) or ten pmol Alu-gag-specific primers (Alu v-TCCCAGCTACTCGGGAGGCTGAGG-3; gag 5-CTGTGAAGCTTGCTCGGGTC-three). PCR products were used every bit a template for real-time PCR to detect integrated viral Dna by using a specific primer for long terminal repeat (LTR): forward primer (5′-ATACCACACACAAGGCTACTTCC-3′) and reverse primer (5′-GCAGGCTCACAGGGTGTAAC-3′). Each sample was analyzed in triplicate; a negative control (no template reaction) was included in each experiment, to bank check contagion. Each experiment was completed with a melting curve assay and all primer pairs showed a single peak in the melting curve analysis, confirming the specificity of distension and the lack of non-specific products and primer dimers. Real-time PCR results, obtained from five different experiments, were represented as a ratio of treated infected samples vs. infected control samples.
Statistical Assay
All information are presented equally the mean values ± standard deviation (SD) from PBMCs of HDs (north = 30) and HIV+ participants (northward = 35). Information analyses were performed using the SPSS statistical software arrangement (version 17.0 for Windows, The states). Comparisons between treated and untreated cells for the results on the Trypan blue analysis, apoptosis analysis, Bcl2, CD95, TNF-alpha and HIV-p24 intracellular poly peptide expression were all conducted using t-test. For comparison of the ways, the Bonferroni'south post-hoc multiple comparison ANOVA test was utilized. Significant differences are shown every bit *p < 0.05, **p < 0.01 and ***p < 0.001. For non-parametric correlations, a Pearson correlation coefficient was calculated.
Results
Computational Prediction of MO p-miRs Targeting Human Genes, and Network Assay of the Processes Involved in Immune Response, Inflammation Pathways and HIV Replication/Integration past the Most Conserved MO p-miRs
A novel prediction tool obtained by combining different RNA-RNA interaction prediction algorithms was used in club to exam the probability of the most conserved p-miRs to interact with the genes involved in HIV infection. All the conserved p-miRs were predicted to collaborate with a loftier probability for at least iii genes (Supplementary Table S2). Interestingly, 9 of these p-miRs (p-miR160h, p-miR166, p-miR482b, p-miR159c, p-miR395d, p-miR2118a, p-miR393a, p-miR167f-3p, and p-miR858b) were predicted to target BCL2, IL2RA, TNF, and VAV1, all with high affinity (Table iii). To improve understand the pregnant biological processes, and the interconnections among the networks modulated past p-miRs, an analysis was generated by Metascape online tool (Zhou et al., 2019). The p-miRs were capable of binding 45 mRNAs associated with the immune response, 48 mRNAs of cytokines, chemokines, and their receptors. At that place were 94 possible targets of p-miRs associated with p53 and NfKb pathways. The enrichment analysis highlighted how p-miRs were potentially capable of significantly modifying important cellular processes associated with immune response and inflammation (Effigy 1). Treatment with p-miRs showed modulation of genes related to biological processes: immune response, inflammation pathways, and response to infections (Figure two).
Tabular array 3. MO p-miRs targeting BCL2, IL2RA, TNF and VAV1.
FIGURE 1. Biological processes enrichment analysis of putative p-miRs regulated genes. (A) Enrichment analysis related to the genes associated to immune response and inflammation. (B) Enrichment analysis related to the modulated cistron by the nigh conserved MO p-miRs. Upwardly to the top 20 enriched clusters are shown colored past p-values. All the statistically enriched terms were identified (GO/KEGG terms, canonical pathways, hall mark gene sets, etc.), accumulative hypergeometric p-values and enrichment factors were calculated and used for filtering. Then 0.3 kappa score was applied every bit the threshold to dissever the tree into term clusters. Created past Metascape [http://metascape.org].
Effigy 2. Enrichment network assay for inflammation and immune response. (A) Networks layout of the clusters generated with the list of the genes associated with the inflammation and immune responses. (B) Networks layout of the clusters generated with the list of the genes regulated by p-miRs. Each circumvolve node represents ane enriched term, where its size is proportional to the number of input genes falling into that term, and its color represents its cluster identity (i.east. nodes of the same color vest to the same cluster). All similar terms with a Kappa similarity score >0.three are connected by edges (the thicker the edge higher the similarity). I term from each cluster has been as a label. Created by Metascape [http://metascape.org].
Furnishings of p-miRs on Apoptosis in PBMCs from Naïve HIV+ Individuals and HDs
Through ex vivo experiments performed on the PBMCs from 35 HIV+ subjects and thirty HDs, the effect of p-miR pool transfection on the viability and apoptosis was evaluated, likewise every bit the possible regulation of putative genes target involved in these pathways. 72 h after transfection, the p-miR pool decide a significant decrease in viability of PBMCs from HIV+ subjects analyzed by Trypan bluish exclusion examination, while the same treatment did non modify the viability of PBMCs from HDs (Effigy 3A). To understand whether the result on viability was associated with the apoptosis induction, the cells were stained with PI 72 h after p-miRs transfection and were and then analyzed by catamenia cytometry. As shown in Figure 3B, the p-miRs significantly enhanced the pct of apoptosis in PBMCs from HIV+ subjects and did non bear upon the profile of PBMCs from HDs. The observed apoptosis was associated with both the increase of the percentage of CD4+ lymphocytes expressing CD95 and with the upregulation of its expression (Figures 3C,Due east). Conversely, the percentage of CD4+ lymphocytes expressing Bcl2 decreased following treatment, while the expression of Bcl2 was down-regulated (Figures 3D,E). As expected, PBMCs from HDs showed no susceptibility to p-miRs transfection. Nevertheless, among HIV+ cART-naïve participants, viii (8) out of 35 did non answer to MO p-miRs transfection (HIV+ Not-Responder: NR), while 27 responded with a significant increment in apoptosis later transfection (HIV+ Responder: R). Post-obit stratification of participants equally R vs NR, comparative analysis of absolute number of CD3+CD4+ cells revealed that only R with T cell CD4 counts equal or superior to 200/mmc responded to the MO p-miRs transfection; this is in dissimilarity to the eight NR who had a T cell CD4 count below 200/mmc (Figure 4A). A similar stratification based on CD3+CD4+ cell number (the departure in apoptotic cells, measured every bit fold modify betwixt R vs. NR) was statistically different (Effigy 4B). This analysis was confirmed by a significant correlation between the percentage of hypodiploid nuclei and the expression of CD95 and Bcl2 proteins in R subjects. The correlation was positive in the instance of increase in apoptosis and the number of CD4+ cells expressing CD95. Conversely, the correlation was negative in the case of number of CD4+ cells expressing Bcl2 (Figure 4C). These correlations analyses in NR subjects were not statistically pregnant (Figure 4D). Finally, the apoptosis of PBMCs from HDs, as well equally from HIV+ individuals having CD4 cells lower than 200/mmc, was non influenced by the MO p-miR pool, showing that the treatment was able to induce a CD95-Fas and Bcl2 mediated apoptosis only in HIV+ subjects having higher CD4 T lymphocytes (>200/mmc).
Effigy 3. Furnishings of MO p-miR pool transfection in 35 HIV + patients and 30 HDs. (A) Number of viable cells analyzed past Trypan bluish analysis after p-miRs transfection in HIV + subjects and HDs. (B) Pct of apoptotic cells afterwards p-miRs transfection in HIV + subjects and HDs, evaluated by propidium iodide staining and flow cytometer analysis. (C) Pct of CD4+ CD95+ cells in command and transfected T cells. (D) Pct of CD4+ Bcl2+ cells in command and transfected T cells. (Due east) MFI of CD4+CD95+ T cells and of CD4+Bcl2+ T cells. Each colored dot, in the scatter plot (D) and (C) represents a unlike subject, command (HF) and treated (p-miRs). ***p < 0.001. All results derived from indistinguishable for each sample (north = 35 HIV+ and n = thirty HDs). Paired sample t-test was performed.
FIGURE iv. p-miRs effects on apoptosis and viability of CD4+ lymphocytes in HIV + subjects. (A) Correlation between apoptosis fold change and the absolute number of CD4+ lymphocytes, for each patient. (B) Apoptosis fold modify in Responder (R) and Non-Responder (NR) subjects. (C) Correlation between Bcl2 MFI expression in CD4+ T cells (left Y axis) or CD95 MFI expression in CD4 T cells (right Y axis), and percentage of hypodiploid nuclei in R HIV + subjects, (D) and in NR HIV + subjects. ***p < 0.001. All results derived from duplicate for each sample (n = 35 HIV+). Nonparametric one-way ANOVA corrected with the Kruskal-Wallis exam, and nonparametric Spearman'south ρ was used.
Effects of MO p-miRs on Activation and Differentiation Markers of CD4+ T Lymphocytes in HIV+ Individuals and HDs
Amidst R HIV+ participants, the MO p-miRs transfection significantly reduced CD4 T cell activation and differentiation. Many phenotypic determinants have been associated with the grade of HIV illness (Corneau et al., 2017), so highlighting the fundamental part of T cell activation in driving the infection (Pardons et al., 2019). CD25 expression, evaluated by flow cytometry in CD3+CD4+ cells 72 h later transfection with the MO p-miRs, revealed that the treatment had a pregnant upshot in reducing of CD4+CD25+ cells (Figure 5A), while no substantial effect was seen in NR subjects (Effigy 5C). In HDs, no substantial change was observed (Figure 5E). Chronic untreated HIV infection has been associated with high T cell turnover and differentiation of T cells from the fundamental memory (CM) to effector memory (EM) phenotype. The transfection of p-miRs modified the T cell differentiation patterns with a reject both in CM and EM cells (CCR7+CD45RA− and CCR7−CD45RA− respectively), and an increase in terminally differentiated effector retention (TEMRA, CCR7−CD45RA+) cells only in the population of R subjects (Figure 5B). In NR and in HDs no modification of differentiation markers was observed (Figures 5D,F).
FIGURE five. Cytofluorimetric analysis of activation and differentiation markers. (A) Percentage of positive CD4+CD25+ lymphocytes in R HIV+. (B) Pct of positive CD3+CD4+ lymphocytes divided as Naïve (CCR7+CD45RA+), CM (CCR7+CD45RA−), EM (CCR7−CD45RA−), TEMRA (CCR7−CD45RA−), in R HIV + subjects. (C) Percent of positive CD4+CD25+ lymphocytes in NR HIV + subjects. (D) Percentage of positive CD3+CD4+ lymphocytes, divided as Naïve, CM, EM, and TEMRA, in NR HIV + subjects. (E) Pct of positive CD4+CD25+ lymphocytes in HDs (F) Pct of positive CD3+CD4+ lymphocytes, divided every bit Naïve, CM, EM, and TEMRA, in HDs. **p < 0.01. All results derived from duplicate for each sample (due north = 35 HIV+ and n = 30 HDs). Paired sample t-exam was performed.
Effects of p-miRs on HIV Replication and Inflammation in PBMCs from R HIV+ Individuals
In R subjects, the ability of the MO p-miRs to interfere with viral replication was evaluated by monitoring intracellular p24. The treatment induced a significant reject in the percent of CD3+CD4+ cells expressing HIV p24 (Figure 6A), and a reduction of the intracellular HIV p24 protein level. (Figure 6B). Additionally, PCR analysis showed that the treatment with MO p-miRs resulted in a pregnant decrease in virus integration (Figure 6C). The consequence of MO p-miR puddle on inflammatory processes revealed a significant reduction of the pct of T cells expressing TNF-alpha, as well every bit a pregnant down regulation of this cytokine's intracellular expression (Figure 6D).
Figure 6. Effect of p-miRs on HIV p24 protein and TNF-blastoff. (A) Percentage of CD3+CD4+ p24+ T cells after p-miRs transfection in all HIV + subjects analyzed. Each colored dot of the scatter plot represents a different discipline, control (HF) and treated (p-miRs). (B) MFI of CD3+CD4+p24+ T cells later transfection with p-miRs. (C) Virus integration detected by PCR products used equally template for real-time PCR to detect integrated viral Deoxyribonucleic acid past using specific primer for LTR. (D) TNF-alpha percentage (left) and MFI (right) of positive T cells. **p < 0.01. All results derived from indistinguishable for each sample (n = 35 HIV+). Paired sample t-exam was performed.
Effects of p-miR858b on VAV1 Expression and HIV Replication
The bioinformatics analysis highlighted that five of the analyzed p-miRs (p-miR160h, p-miR166, p-miR482b, p-miR395d, p-miR393a, and p-miR858b) are predicted to target VAV1. To confirm the p-miR-mRNA interaction, we performed the transfection on PBMCs from HIV+ naïve subjects, with a specific p-miR - the p-miR858b - verifying its ability to regulate the expression of VAV1 gene (Table 3). The p-miR858b was called for 2 reasons: Showtime, amongst all the other p-miRs, it was the i which targeted the fewest genes. Second, it was part of our expertize. The human protein VAV1 is involved in HIV pathogenesis mediated past HIV Nef protein (Rauch et al., 2008), that in the early phase of the viral infection ensures T cell activation and allows the establishment of a persistent state of infection. Nef targets VAV1 and promotes its tyrosine phosphorylation, associated with its nucleus-to-cytoplasm redistribution, determining T lymphocytes activation, thus fostering virus dissemination. Transfection of mimic p-miR858b was performed in xv R HIV+ patients. The transfection efficiency, analyzed by flow cytometry using a fluorescent mimic, was most 30% (Figures 7A–C), with a significant accumulation of p-miR858b detected by RT-PCR (Figure 7D). No specific effect on the proliferation and prison cell death was observed. Transfection of the PBMCs from HIV+ subjects with mimic p-miR858b confirmed the bioinformatics assay, by revealing a meaning subtract in the percent of CD3+VAV1+ cells and VAV1 poly peptide expression level (MFI). Moreover, the western blot assay confirmed that mimic p-miR858b significantly inhibits the expression of VAV1 protein (Figures 8A,B). In these transfected cells, in that location was a significant decrease in both virus replication as demonstrated by the percentage of CD3+CD4+p24+ cells (Figures 8C,D), and in virus integration assay (Figure 8E). This suggests a potential role for p-miR858b in the regulation of infectious mechanism associated with the expression of VAV1 in host cells. Through the RLM-RACE (RNA ligase mediated - Rapid distension of cDNA ends) analysis, no trace of the VAV1 factor transcript was found, suggesting the regulatory action of p-miR858b might be at the post-transcriptional level (Supplementary Figure S2).
Figure 7. Transfection efficacy with mimic p-miR858b-FITC. (A) Representative fluorescent microscopy images of p-miR858b-FITC transfected lymphocytes (right panel) and its control (left console), in xv HIV + subjects. (B) Histogram overlay of transfected cells with mimic p-miR858b-FITC, analyzed in flow cytometry. (C) Percentage of HIV + lymphocytes transfected with mimic p-miR858-FITC. (D) mimic p-miR858b Delta Ct expression in HDs and HIV + subjects' lymphocytes, transfected with mimic p-miR858b-FITC. All results derived from fifteen HDs and fifteen HIV + subjects **p < 0.01. All experiments were performed at least three times.
FIGURE 8. Effect of mimic p-miR858b transfection on VAV1 expression and HIV infection. (A) Representative Western Blot of two HIV+ patient of VAV1 expression level with (+) and without (-) mimic p-miR858b. (B) Densitometry histogram of the hateful ± S.D. of 15 HIV+ subjects, with and without mimic p-miR858b. (C) Flow cytometry analysis of the percentage of FITC-VAV1 positive cells. (D) Pct of p24+ cells, with and without mimic p-miR858b handling. (Due east) Fold change of virus integration level, afterward transfection with p-miRs and mimic p-miR858b. **p < 0.01. All results derived from at least fifteen HIV+ samples. The ANOVA-Bonferroni test was used.
Word
There is growing involvement in the office of plant-derived miRNA in the command of several diseases, including viral infections such as HIV. This required a deeper understanding of factor networks contributing to the etiology of complex diseases. In this regard, the generated list of total genes, modulated by a pool of MO p-miRs to perform a functional annotation clustering, enabled us to draw the potential importance of p-miRs treatment by modulating a span of cistron sets dysregulated in several other diseases. The broad range of disorders evidenced by the affliction enrichment analysis highlighted HIV/AIDS among diseases with major burden, thereby underscoring the relevance in assessing such regulatory pathways during HIV infection. There is an urgent demand to better understand such a regulatory mechanism, and so unlocking its potential for enhancing HIV infection management through the use of plant-derived miRNAs. Of note, miRNAs are an important regulator of prison cell functions, allowing a fine-tuning at a post-transcriptional level (Bartel, 2004). These brusk sequences of nucleic acids are conserved across the species, and foreign miRNAs take modulatory activity in host cells, as demonstrated by interactions between plant-derived miRNAs and fauna mRNAs (Zhou et al., 2015; Chin et al., 2016; Hou et al., 2018; Teng et al., 2018; Potestà et al., 2020). Information technology is well established that establish-derived miRNAs are commonly taken through the nutrition and are able to cantankerous the gastrointestinal barrier, exerting a biological effect in the host. In a recent work we demonstrated that these miRNAs are vehiculated through microvesicles, assuasive their delivery in the host cells (Potestà et al., 2020). These characteristics make them perfect candidates for possible therapeutic developments, compared to other pocket-sized RNAs already used for the treatment of diverse diseases. We, therefore, focus on this Cross-Kingdom interaction to identify plant miRNA that tin modulate different pathways involved in HIV pathogenesis, using PBMCs from HIV+ subjects and treated with the enriched extract of p-miRs from MO seeds to verify its properties. Worth noting, this cohort of HIV-individuals was chosen prior to cART initiation and within viremic individuals, in society to mitigate bias related to Art when assessing viral replication and integration mediated past p-miRs and specific mimics. Thus, the effects observed in the report are derived from interventions following treatment with the p-miRs. The putative target genes have been identified for all the well-nigh abundant p-miRs from the p-sR pool through bioinformatics analysis. This assay supports the validation that has been highlighted in the ex vivo experiments, which also suggest the targets may be dissimilar, such equally VAV1, TNF, BCL2, and IL2RA. Therefore, in that location would be diverse pathways in which these genes are involved. This underscoring the multiple regulatory capacities of the MO p-miR pool, since immunodeficiency in HIV cART-naïve individuals is largely due to apoptosis of CD4 T cells and to the chronic activation of the allowed system. For this reason, the goals of antiviral handling are not only to reduce viral load, but as well to limit chronic activation of the immune response and lymphocytes apoptosis (Cummins and Badley, 2010). All the same, the reduction of apoptosis may also come at the cost of preserving the latent viral reservoirs and its increment in HIV-infected cells is desirable in this context. Our results show that the MO p-miRs have the ability to induce apoptosis (on both Fas- and Bcl2-mediated apoptosis) and to reduce both lymphocytes viability and viral replication. In item, the handling with the p-miR puddle induces an increment of the apoptosis in lymphocytes, equally demonstrated past an increment of the hypodiploid nuclei, and evidenced past an increment in Fas expression and a decrement of Bcl2 expression, compared to their control every bit we demonstrated earlier (Potestà et al., 2020). Still, a broad distribution of apoptosis level has been observed among the HIV + individuals: the MO p-miR pool induced its furnishings, in term of apoptosis and Fas and Bcl2 expression, only in HIV + subjects with a CD4 T cell count above 200/mmc. Conversely, in HIV + subjects with a CD4 T cell count below 200/mmc no significant result was evidenced, as well as in PBMCs from HDs. Therefore, it becomes essential to further investigate the immune-modulatory effects of p-miRs treatment in the 2 major populations of HIV+ cART-naïve individuals (Responder or Non-Responder) in real-life (Supplementary Table S2). The flow cytometry analysis of differentiation and activation markers of T lymphocytes, routinely used both in diagnosis and in the monitoring of HIV infection, highlights how the treatment modifies the differentiative design of CD4 T lymphocytes. Of note, declining percentage of CD4 T cell with CM (CD45RA−CCR7+) and EM (CD45RA−CCR7-) phenotype toward the TEMRA (CD45RA+CCR7-) phenotype were observed together with a decrease of activated CD25+ CD4+ lymphocytes. Similar to apoptosis, the modulation of the CD4 T prison cell sub-population is effective only in Responder HIV+ individuals, resulting from the long-lived resting retention CD4+ T cells (CM) serving as major reservoir of latent HIV infection (Zhang et al., 2019). In this context, apoptosis induced by the MO p-miRs in Responder HIV+ individuals may represent a powerful arrangement to selectively induce elimination of HIV-infected CM CD4 T jail cell, thereby avoiding lymphocyte activation. As TNF is among the putative target of MO p-miRs, we as well postulated that diverse cellular processes, such equally inflammation, immune regulation or apoptosis, are mediated through binding of TNF to its receptors (Zelová and Hošek, 2013; Aquilano et al., 2019), all these processes generally existence involved in HIV pathogenesis (Kumar et al., 2015; Pasquereau et al., 2017) and characterized by a high level of TNF-alpha expression (Keating et al., 2012; Paiardini and Müller-Trutwin, 2013; Planès et al., 2018). Putting this into context, our ex vivo system reveals that exposure to the MO p-miRs reduces TNF-alpha expression, thus supporting the power of the p-miRs to downregulate the allowed system activation likewise through this pathway. Assay of the Alu sequence as well shows that the handling with both the mimic p-miR858b and with the MO p-miR pool substantially reduces viral integration. This is consistent with turn down in HIV infected cells, in the frequency of p24-positive CD4 T lymphocytes, too as reduction of their intracellular p24 protein level. Regarding the use of a specific plant miRNA, p-miR858b has VAV1 cistron amidst its putative targets, so highlighting this gene's function in T cell antigen receptor (TCR) signaling for the activation of dissimilar pathways (Tybulewicz, 2005; Ksionda et al., 2012), and for the modulation of different targets in the host cells (Abraham and Fackler, 2012; del Río-Iñiguez et al., 2018). In a context where PBMCs from HIV+ ART-naïve subjects are transfected with the mimic p-miR858b, the declining tendency of VAV1 expression infers a decline of both viral replication and HIV DNA integration in the host cells. This regulatory power shown past the p-miR858b might exist due to the interaction of this miRNA with VAV1 mRNA (Supplementary Figure S2). Interestingly, MO p-miR puddle can reduce HIV-infected cells, albeit the machinery behind this ability is yet unclear. We therefore postulate that treatment-induced apoptosis mainly affects HIV-infected cells, reducing their number, a hypothesis reinforced by the p-miRs-induced decrease of CM T cells (main reservoir of latent HIV). The p-miRs described hither exhibit a biological activeness similar to that of synthetic p-miR858b. This evidence underscores the concept of Cross-Kingdom with specific human mRNAs and the restoration of expression in allowed response and HIV control. This putative machinery could be considered an active component in regulating the prepare of genes involved in the immune response against HIV. Current antiretroviral therapy recommended for HIV-infected subjects requires daily and indefinitely medication that is stressful and non without side effects, especially for younger patients. Moreover, the life-long need for therapy adherence implies high costs of chronic treatment and encourage the exploration of alternative approaches such as immunotherapy that could potentially exist complementary to long-term management of HIV infection (Palma et al., 2013; Ward et al., 2020). To conclude, plant pocket-sized RNAs from Moringa oleifera may restore normalcy in immune system and reduce replication of HIV-infection, besides at the level of cellular reservoir. This suggests a role for MO p-miRs in standard HIV treatment, then contributing to the long-term control of the affliction.
Information Availability Argument
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries tin be directed to the corresponding authors.
Ethics Statement
Ethical approval for the drove and use of human samples was obtained in 2014 from the ethical board of "Tor Vergata" Hospital, protocol number 15/fourteen (D.M.08.02.2013 D.G.R.146/2013; D.D.G.467 del 25.07.2013). The patients/participants provided their written informed consent to participate in this study.
Author Contributions
Conceptualization, VC, CM, AM, MP, RM, and RC; methodology, MP, VR and AM; software, FI, and MC; validation, AM, MP, VR, MC, and CC; writing—original typhoon preparation, AM, MP, VR; writing—review and editing, CM, AM, MP, VR, RM, MC, JF, SG, VC and MA; supervision, CM, RM, AD, MA, SG, and VC. All authors have read and agreed to the published version of the manuscript.
Funding
The present study was supported by the STARBIOS2 European Union's Horizon 2020 enquiry and innovation program under grant agreement no. 709517 oriented to promote Responsible Research and Innovation in biosciences.
Conflict of Interest
The authors declare that the research was conducted in the absence of whatever commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
AM and MP received fiscal support for the grant from the STARBIOS2 Horizon 2020 European Project - Enquiry and Innovation program under grant agreement no. 709517. FI was supported by an AIRC fellowship for Italy. The computing resources and the related technical back up used for this work have been provided by CRESCO/ENEAGRID Loftier Performance Computing infrastructure and its staff funded by ENEA and by Italian and European inquiry programmers. Nosotros wish to thank Martin Bennett for the linguistic aid.
Supplementary Material
The Supplementary Textile for this article can exist constitute online at: https://www.frontiersin.org/articles/10.3389/fphar.2020.620038/total#supplementary-material.
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Source: https://www.frontiersin.org/articles/10.3389/fphar.2020.620038/full
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