Optimized logic rules reveal interferon-γ-induced modes regulated by histone deacetylases and protein tyrosine phosphatases.

The pro-inflammatory cytokine interferon-γ (IFN-γ) is critical for activating innate and adaptive immunity against tumours and intracellular pathogens. Interferon-γ is secreted at the fetal-maternal interface in pregnant women and mice. The outer layer of the placenta in contact with maternal blood is composed of semi-allogeneic trophoblast cells, which constitute the fetal component of the fetal-maternal interface. The simultaneous presence of pro-inflammatory IFN-γ and trophoblast cells at the fetal-maternal interface appears to represent an immunological paradox, for trophoblastic responses to IFN-γ could potentially lead to activation of maternal immunity and subsequent attack of the placenta. However, our previous studies demonstrate that IFN-γ responsive gene (IRG) expression is negatively regulated in human and mouse trophoblast cells. In human cytotrophoblast and trophoblast-derived choriocarcinoma cells, janus kinase signalling is blocked by protein tyrosine phosphatases (PTPs), whereas in mouse trophoblast, histone deacetylases (HDACs) inhibit IRG expression. Here, we used genome-wide transcriptional profiling to investigate the collective roles of PTPs and HDACs on regulation of IRG expression in human choriocarcinoma cells. Logic-rules were optimized to derive regulatory modes governing gene expression patterns observed upon different combinations of treatment with PTP and HDAC inhibitors. The results demonstrate that IRGs can be divided into several categories in human choriocarcinoma cells, each of which is subject to distinct mechanisms of repression. Hence, the regulatory modes identified in this study suggest that human trophoblast and choriocarcinoma cells may evade the potentially deleterious consequences of exposure to IFN-γ by using several overlapping mechanisms to block IRG expression.

Evaluating side effects of nanoparticle-mediated siRNA delivery to mesenchymal stem cells using next generation sequencing and enrichment analysis.

RNA interference has immense potential to modulate cell functions. However, effective delivery of small interfering RNA (siRNA) while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next generation RNA sequencing (RNAseq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a threefold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ∼14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 hr after treatment, while 11% of the treated population was annexin V+-siRNA-. RNAseq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor, RIG-I-like receptor, and nuclear factor-κB signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the hMSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.

Detection of Serum microRNAs From Department of Defense Serum Repository: Correlation With Cotinine, Cytokine, and Polycyclic Aromatic Hydrocarbon Levels.

OBJECTIVE: The aim of this study was to investigate whether serum samples from the Department of Defense Serum Repository (DoDSR) are of sufficient quality to detect microRNAs (miRNAs), cytokines, immunoglobulin E (IgE), and polycyclic aromatic hydrocarbons (PAHs). METHODS: MiRNAs were isolated and quantified by polymerase chain reaction (PCR) array. Cytokines and chemokines related to inflammation were measured using multiplex immunoassays. Cotinine and IgE were detected by enzyme-linked immunoassay (ELISA) and PAHs were detected by Liquid Chromatography/Mass Spectroscopy. RESULTS: We detected miRNAs, cytokines, IgE, and PAHs with high sensitivity. Eleven of 30 samples tested positive for cotinine suggesting tobacco exposure. Significant associations between serum cotinine, cytokine, IgE, PAHs, and miRNA were discovered. CONCLUSION: We successfully quantified over 200 potential biomarkers of occupational exposure from DoDSR samples. The stored serum samples were not affected by hemolysis and represent a powerful tool for biomarker discovery and analysis in retrospective studies.

MicroRNAs as Novel Biomarkers of Deployment Status and Exposure to Polychlorinated Dibenzo-p-Dioxins/Dibenzofurans.

OBJECTIVE: To determine if service members deployed to locations with open air burn pits have different serum microRNA (miRNA) profiles after deployment compared with length-of-service matched, non-deployed individuals. We also tested for correlations between miRNA and serum levels of Polychlorinated Dibenzo-p-Dioxins/Dibenzofurans (PCDD/PCDFs). METHODS: MiRNAs were isolated and quantified by PCR array. Groups were analyzed for differences in miRNA expression. Correlations between serum miRNA and PCDD/PCDFs were assessed with a linear regression model. RESULTS: Several miRNAs were differentially expressed after deployment and a partially overlapping set of miRNAs were identified between deployed and non-deployed individuals. Significant correlations between miRNAs and PCDD/PCDFs were identified. CONCLUSIONS: Serum miRNA levels show a link between deployment to locations with open burn pits and environmental exposures that can take place during deployment.