Jia Shao

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights.

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5μM DON for 3, 6 and 24h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4μM DON for 6 and 24h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs.

Toxicogenomics-Based Identification of Mechanisms for Direct Immunotoxicity

Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides, and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T-cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (1) with structural similarities, such as tributyltin chloride and tributyltin oxide that activated the retinoic acid/X receptor signaling pathway and (2) without structural similarities, such as As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9-treated OTA, S9-treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded protein response, and FTY720, lindane, and propanil, which activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the downregulation of acute-phase response genes by OTA. These findings were validated by quantitative real-time PCR analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene, can be used to screen compounds for direct immunotoxicity.

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non‐immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)–PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non‐immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo‐e‐pyrene). Of the four potential (non‐)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non‐immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3‐dichloro‐propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo‐e‐pyrene and anti‐CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT–PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non‐immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity. Copyright

Protein phosphorylation profiling identifies potential mechanisms for direct immunotoxicity

Abstract Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay.