Elucidating Mechanisms of Toxicity Using Phenotypic Data from Primary Human Cell Systems—A Chemical Biology Approach for Thrombosis-Related Side Effects

Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo

Regulation of IL-17A production is distinct from IL-17F in a primary human cell co-culture model of T cell-mediated B cell activation.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F

Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction.

<p>Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.</p

SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures.

<p>Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log₁₀ ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F.

<p>Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log₁₀ ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses.

<p>FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).</p

Summary of well-characterized compound targets that preferentially regulated production of both IL-17A and IL-17F, or had selectivity for IL-17A or IL-17F^a.

a<p>Data from a screen of 144 pharmacologic modulators are summarized to show compound classes and targets that regulated production of both IL-17A and IL-17F or had selectivity for IL-17A or IL-17F in BT co-cultures. The number of agents with selectivity for a specific target is shown in parentheses next to the total number of agents in the screen for that target. Class/targets were only included if greater than 50% of agents specific for a class/target had effects on IL-17A and/or IL-17F. Agents that inhibited both IL-17A and IL-17F, or preferentially inhibited IL-17A or IL-17F had readout values less than log₁₀ ratio −0.2 and were observed at 2 or more non-cytotoxic doses. *Agents against these targets selectively inhibited IL-17A or IL-17F at 2 or more relatively low doses and inhibited both IL-17A and IL-17F at 2 or more higher doses.</p

Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters.

<p>Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log₁₀ ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F.

<p>Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log₁₀ ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

IL-17F is the most strongly induced gene in BT co-cultures after three days of stimulation in a model of T cell-dependent B cell activation^a.

a<p>The top ten induced genes as determined by microarray analysis in BT co-cultures stimulated with α-IgM and SAg for three days compared to the same cells co-cultured for three days without stimulation. Data are from 3 independent replicates with 3 different donor pools.</p