IKAROS expression drives the aberrant metabolic phenotype of macrophages in chronic HIV infection.

The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by "locking" PLWH monocytes in a pro-inflammatory and activated phenotype.

Mechanisms of miR-3189-3p-mediated inhibition of c-MYC translation in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Our lab previously characterized miR-3189-3p as a microRNA with potent anti-cancer activity against glioblastoma. Here, we hypothesized a similar activity in TNBC cells. As miR-3189-3p is predicted to target a variety of RNA binding proteins, we further hypothesized an inhibitory effect of this miRNA on protein synthesis. METHODS: MDA-MB-231 and MDA-MB-468 cells were used to investigate the effect of miR-3189-3p on cell proliferation, migration, and invasion. TGCA database was used to analyze the expression of miR-3189-3p, c-MYC, 4EPB1, and eIF4E in breast cancer. Western blotting and RT-qPCR assays were used to assess the expression of selected proteins and RNAs after transfections. RESULTS: Although c-MYC is not a predicted gene target for miR-3189-3p, we discovered that c-MYC protein is downregulated in miRNA-treated TNBC cells. We found that the downregulation of c-MYC by miR-3189-3p occurs in both normal growth conditions and in the absence of serum. The mechanism involved the direct inhibition of eIF4EBP1 by miR-3189-3p. Additionally, we found that miR-3189-3p could negatively affect cap-independent translation mediated by internal ribosome entry sites (IRES) or by m6A. Finally, miR-3189-3p sensitized TNBC cells to doxorubicin. CONCLUSION: Overall, results indicated that miR-3189-3p exerts its anti-tumor activity through targeting translational regulatory proteins leading to an impairment in c-MYC translation, and possibly other oncogenic factors, suggesting that miR-3189-3p, alone or in combination, could be a valuable therapeutic approach against a malignancy with few treatment options.

Notch signaling pathway: a comprehensive prognostic and gene expression profile analysis in breast cancer.

Breast cancer is a complex disease exhibiting a great degree of heterogeneity due to different molecular subtypes. Notch signaling regulates the differentiation of breast epithelial cells during normal development and plays a crucial role in breast cancer progression through the abnormal expression of the Notch up-and down-stream effectors. To date, there are only a few patient-centered clinical studies using datasets characterizing the role of Notch signaling pathway regulators in breast cancer; thus, we investigate the role and functionality of these factors in different subtypes using publicly available databases containing records from large studies. High-throughput genomic data and clinical information extracted from TCGA were analyzed. We performed Kaplan-Meier survival and differential gene expression analyses using the HALLMARK_NOTCH_SIGNALING gene set. To determine if epigenetic regulation of the Notch regulators contributes to their expression, we analyzed methylation levels of these factors using the TCGA HumanMethylation450 Array data. Notch receptors and ligands expression is generally associated with the tumor subtype, grade, and stage. Furthermore, we showed gene expression levels of most Notch factors were associated with DNA methylation rate. Modulating the expression levels of Notch receptors and effectors can be a potential therapeutic approach for breast cancer. As we outline herein, elucidating the novel prognostic and regulatory roles of Notch implicate this pathway as an essential mediator controlling breast cancer progression.

Human papillomavirus in spermatozoa is efficiently removed by washing: a suitable approach for assisted reproduction.

RESEARCH QUESTION: Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction? DESIGN: This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA. RESULTS: Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive. CONCLUSIONS: The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used.

Evaluation of salivary and plasma microRNA expression in patients with Sjögren's syndrome, and correlations with clinical and ultrasonographic outcomes.

OBJECTIVES: To correlate the expression of microRNAs (miRNAs) 146a/b, 16, the 17-92 cluster and 181a in salivary and plasma samples taken from primary Sjögren's syndrome (pSS) patients with clinical, laboratory and ultrasound findings. METHODS: Plasma and salivary samples were collected from 28 patients with pSS according to 2012 ACR and/or 2016 ACR/EULAR criteria (27 females, mean age 64.4±10.1 years, mean disease duration 10.7±6.9 years), and from 23 healthy subjects used as controls. The following patient data were recorded: ESSDAI and ESSPRI scores, anti-SSA and anti-SSB antibody status and laboratory data, Schirmer's test, ultrasound scores of the four major salivary glands according to Cornec et al., and concomitant treatments. The retro-transcribed and quantified miRNAs were: miR16-5p, miR17-5p, miR18a-5p, miR19a-5p, miR19b-1-5p, miR20a, miR92-5p, miR146a-5p, miR146b-5p, miR181a-5p. RESULTS: SS patients had higher expression of salivary miR146a than gender- and age-matched controls (p=0.01). Spearman's regression analysis revealed that salivary miR146b was significantly more expressed in the patients with worse ESSPRI scores (p=0.02), whereas salivary miR17 and 146b and plasma miR17 expression was lower in the patients with higher ultrasound scores (respectively p=0.01, p=0.01 and p=0.04). Salivary miR18a expression was significantly increased in the patients who were anti-La/SSB positive (p=0.04). Neither salivary nor plasma miRNAs correlated with disease duration or concomitant therapies. CONCLUSIONS: Our data show that salivary mi146a may represent a marker of the disease, and that the expression of salivary miR17, 18a and 146b may be altered in patients with pSS, and associated with worse ultrasound and ESSPRI scores and anti-La/SSB positivity.

NRF2 signaling pathway: A comprehensive prognostic and gene expression profile analysis in breast cancer.

Breast cancer is the most frequently diagnosed malignant tumor in women and a major public health concern. NRF2 axis is a cellular protector signaling pathway protecting both normal and cancer cells from oxidative damage. NRF2 is a transcription factor that binds to the gene promoters containing antioxidant response element-like sequences. In this report, differential expression of NRF2 signaling pathway elements, as well as the correlation of NRF2 pathway mRNAs with various clinicopathologic characteristics, including molecular subtypes, tumor grade, tumor stage, and methylation status, has been investigated in breast cancer using METABRIC and TCGA datasets. In the current report, our findings revealed the deregulation of several NRF2 signaling elements in breast cancer patients. Moreover, there were negative correlations between the methylation of NRF2 genes and mRNA expression. The expression of NRF2 genes significantly varied between different breast cancer subtypes. In conclusion, substantial deregulation of NRF2 signaling components suggests an important role of these genes in breast cancer. Because of the clear associations between mRNA expression and methylation status, DNA methylation could be one of the mechanisms that regulate the NRF2 pathway in breast cancer. Differential expression of Hippo genes among various breast cancer molecular subtypes suggests that NRF2 signaling may function differently in different subtypes of breast cancer. Our data also highlights an interesting link between NRF2 components' transcription and tumor grade/stage in breast cancer.

Hippo signaling pathway: A comprehensive gene expression profile analysis in breast cancer.

Breast cancer (BC) is the most frequently diagnosed malignancy in women and a major public health concern. The Hippo pathway is an evolutionarily conserved signaling pathway that serves as a key regulator for a wide variety of biological processes. Hippo signaling has been shown to have both oncogenic and tumor-suppressive functions in various cancers. Core components of the Hippo pathway consist of various kinases and downstream effectors such as YAP/TAZ. In the current report, differential expression of Hippo pathway elements as well as the correlation of Hippo pathway mRNAs with various clinicopathologic characteristics, including molecular subtypes, receptor status, and methylation status, has been investigated in BC using METABRIC and TCGA datasets. In this review, we note deregulation of several Hippo signaling elements in BC patients. Moreover, we see examples of negative correlations between methylation of Hippo genes and mRNA expression. The expression of Hippo genes significantly varies between different receptor subgroups. Because of the clear associations between mRNA expression and methylation status, DNA methylation may be one of the mechanisms that regulate the Hippo pathway in BC cells. Differential expression of Hippo genes among various BC molecular subtypes suggests that Hippo signaling may function differently in different subtypes of BC. Our data also highlights an interesting link between Hippo components' transcription and ER negativity in BC. In conclusion, substantial deregulation of Hippo signaling components suggests an important role of these genes in breast cancer.

Anti-glioblastoma effects of phenolic variants of benzoylphenoxyacetamide (BPA) with high potential for blood brain barrier penetration.

Glioblastomas are the most aggressive brain tumors for which therapeutic options are limited. Current therapies against glioblastoma include surgical resection, followed by radiotherapy plus concomitant treatment and maintenance with temozolomide (TMZ), however, these standard therapies are often ineffective, and average survival time for glioblastoma patients is between 12 and 18 months. We have previously reported a strong anti-glioblastoma activity of several metabolic compounds, which were synthetized based compounds, which were synthetized based on the chemical structure of a common lipid-lowering drug, fenofibrate, and share a general molecular skeleton of benzoylphenoxyacetamide (BPA). Extensive computational analyses of phenol and naphthol moieties added to the BPA skeleton were performed in this study with the objective of selecting new BPA variants for subsequent compound preparation and anti-glioblastoma testing. Initially, 81 structural variations were considered and their physical properties such as solubility (logS), blood-brain partitioning (logBB), and probability of entering the CNS calculated by the Central Nervous System-Multiparameter Optimization (MPO-CNS) algorithm were evaluated. From this initial list, 18 compounds were further evaluated for anti-glioblastoma activity in vitro. Nine compounds demonstrated desirable glioblastoma cell toxicity in cell culture, and two of them, HR51, and HR59 demonstrated significantly improved capability of crossing the model blood-brain-barrier (BBB) composed of endothelial cells, astrocytes and pericytes.

Attenuated Negative Feedback in Monocyte-Derived Macrophages From Persons Living With HIV: A Role for IKAROS.

Persons living with HIV (PLWH) are at higher risk of developing secondary illnesses than their uninfected counterparts, suggestive of a dysfunctional immune system in these individuals. Upon exposure to pathogens, monocytes undergo epigenetic remodeling that results in either a trained or a tolerant phenotype, characterized by hyper-responsiveness or hypo-responsiveness to secondary stimuli, respectively. We utilized CD14+ monocytes from virally suppressed PLWH and healthy controls for in vitro analysis following polarization of these cells toward a pro-inflammatory monocyte-derived macrophage (MDM) phenotype. We found that in PLWH-derived MDMs, pro-inflammatory signals (TNFA, IL6, IL1B, miR-155-5p, and IDO1) dominate over negative feedback signals (NCOR2, GSN, MSC, BIN1, and miR-146a-5p), favoring an abnormally trained phenotype. The mechanism of this reduction in negative feedback involves the attenuated expression of IKZF1, a transcription factor required for de novo synthesis of RELA during LPS-induced inflammatory responses. Furthermore, restoring IKZF1 expression in PLWH-MDMs partially reinstated expression of negative regulators of inflammation and lowered the expression of pro-inflammatory cytokines. Overall, this mechanism may provide a link between dysfunctional immune responses and susceptibility to co-morbidities in PLWH with low or undetectable viral load.

Sterol metabolism modulates susceptibility to HIV-1 Infection.

BACKGROUND: 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG), which catalyzes the synthesis of 25-hydroxycholesterol (25HC). 25HC intervenes in metabolic and infectious processes and controls cholesterol homeostasis and influences viral entry into host cells. We verified whether natural resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) individuals is at least partially mediated by particularities in sterol biosynthesis. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) isolated from 15 sexually exposed HESN and 15 healthy controls were in vitro HIV-1-infected and analyzed for: percentage of IFNα-producing plasmacytoid dendritic cells (pDCs); cholesterol signaling and inflammatory response RNA expression; resistance to HIV-1 infection. MDMs from five healthy controls were in vitro HIV-1-infected in the absence/presence of exogenously added 25HC. RESULTS: IFNα-producing pDCs were augmented in HESN compared with healthy controls both in unstimulated and in in vitro HIV-1-infected PBMCs (P < 0.001). An increased expression of CH25H and of a number of genes involved in cholesterol metabolism (ABCA1, ABCG1, CYP7B1, LXRα, OSBP, PPARγ, SCARB1) was observed as well; this, was associated with a reduced susceptibility to in-vitro HIV-1-infection of PBMCs and MDMs (P < 0.01). Notably, addition of 25HC to MDMs resulted in increased cholesterol efflux and augmented resistance to in-vitro HIV-1-infection. CONCLUSION: Results herein show that in HESN sterol metabolism might be particularly efficient. This could be related to the activation of the IFNα pathway and results into a reduced susceptibility to in-vitro HIV-1 infection. These results suggest a possible basis for therapeutic interventions to modulate HIV-1 infection.

Isolation, Transfection, and Culture of Primary Human Monocytes.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Corrigendum: Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) Is Released in the Secretome of Activated MDMs and Reduces in vitro HIV-1 Infection.

[This corrects the article DOI: 10.3389/fimmu.2019.01648.].

Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) Is Released in the Secretome of Activated MDMs and Reduces in vitro HIV-1 Infection.

Background: Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (HomoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of the antigen processing/presenting machinery. ERAP1 was recently shown to be secreted from the plasma membrane in response to activation; we investigated whether ERAP2 can be released as well and if the secreted form of this enzyme retains its antiviral function. Methods: Human monocyte derived macrophages (MDMs) were differentiated from peripheral blood mononuclear cells (PBMCs) isolated from 6 HomoA healthy controls and stimulated with IFNγ and LPS. ERAP2-FL secretion was evaluated by mass spectrometry. PBMCs (14 HomoA and 16 HomoB) and CD8-depleted PBMCs (CD8-PBMCs) (4 HomoA and 4 HomoB) were in vitro HIV-infected in the absence/presence of recombinant human ERAP2-FL (rhERAP2) protein; p24 viral antigen quantification was used to assess viral replication. IFNγ and CD69 mRNA expression, as well as the percentage of perforin-producing CD8+ T Lymphocytes, were analyzed 3 and 7-days post in vitro HIV-1-infection, respectively. The effect of rhERAP2 addition in cell cultures on T cell apoptosis, proliferation, activation, and maturation was evaluated as well on 24 h-stimulated PBMCs. Results: ERAP2 can be secreted from human MDMs in response to IFNγ/LPS stimulation. Notably, the addition of rhERAP2 to PBMC and CD8-PBMC cultures resulted in the reduction of viral replication, though these differences were statistically significant only in PBMCs (p < 0.05 in both HomoA and HomoB). This protective effect was associated with an increase in IFNγ and CD69 mRNA expression and in the percentage of perforin-expressing CD107+CD8+ cells. RhERAP2 addition also resulted in an increase in CD8+ activated lymphocyte (CD25+HLA-DRII+) and Effector Memory/Terminally differentiated CD8+ T cells ratio. Conclusions: This is the first report providing evidence for the release of ERAP2 in the secretome of immunocompetent cells. Data herein also indicate that exogenous ERAP2-FL exerts its protective function against HIV-1 infection, even in HomoB subjects who do not genetically produce it. Presumably, this defensive extracellular feature is only partially dependent on immune system modulation.