Dirk Weisensee

Assessment of network perturbation amplitudes by applying high-throughput data to causal biological networks

BackgroundHigh-throughput measurement technologies produce data sets that have the potential to elucidate the biological impact of disease, drug treatment, and environmental agents on humans. The scientific community faces an ongoing challenge in the analysis of these rich data sources to more accurately characterize biological processes that have been perturbed at the mechanistic level. Here, a new approach is built on previous methodologies in which high-throughput data was interpreted using prior biological knowledge of cause and effect relationships. These relationships are structured into network models that describe specific biological processes, such as inflammatory signaling or cell cycle progression. This enables quantitative assessment of network perturbation in response to a given stimulus.ResultsFour complementary methods were devised to quantify treatment-induced activity changes in processes described by network models. In addition, companion statistics were developed to qualify significance and specificity of the results. This approach is called Network Perturbation Amplitude (NPA) scoring because the amplitudes of treatment-induced perturbations are computed for biological network models. The NPA methods were tested on two transcriptomic data sets: normal human bronchial epithelial (NHBE) cells treated with the pro-inflammatory signaling mediator TNFα, and HCT116 colon cancer cells treated with the CDK cell cycle inhibitor R547. Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach. The NPA scoring method successfully quantified the amplitude of TNFα-induced perturbation for each network model when compared against NF-κB nuclear localization and cell number. In addition, the degree and specificity to which CDK-inhibition affected cell cycle and inflammatory signaling were meaningfully determined.ConclusionsThe NPA scoring method leverages high-throughput measurements and a priori literature-derived knowledge in the form of network models to characterize the activity change for a broad collection of biological processes at high-resolution. Applications of this framework include comparative assessment of the biological impact caused by environmental factors, toxic substances, or drug treatments.

A Modular Cell-Type Focused Inflammatory Process Network Model for Non-Diseased Pulmonary Tissue

Exposure to environmental stressors such as cigarette smoke (CS) elicits a variety of biological responses in humans, including the induction of inflammatory responses. These responses are especially pronounced in the lung, where pulmonary cells sit at the interface between the bodys internal and external environments. We combined a literature survey with a computational analysis of multiple transcriptomic data sets to construct a computable causal network model (the Inflammatory Process Network (IPN)) of the main pulmonary inflammatory processes. The IPN model predicted decreased epithelial cell barrier defenses and increased mucus hypersecretion in human bronchial epithelial cells, and an attenuated pro-inflammatory (M1) profile in alveolar macrophages following exposure to CS, consistent with prior results. The IPN provides a comprehensive framework of experimentally supported pathways related to CS-induced pulmonary inflammation. The IPN is freely available to the scientific community as a resource with broad applicability to study the pathogenesis of pulmonary disease.

A Systems Biology Approach Reveals the Dose- and Time-Dependent Effect of Primary Human Airway Epithelium Tissue Culture After Exposure to Cigarette Smoke In Vitro

To establish a relevant in vitro model for systems toxicology-based mechanistic assessment of environmental stressors such as cigarette smoke (CS), we exposed human organotypic bronchial epithelial tissue cultures at the air liquid interface (ALI) to various CS doses. Previously, we compared in vitro gene expression changes with published human airway epithelia in vivo data to assess their similarities. Here, we present a follow-up evaluation of these in vitro transcriptomics data, using complementary computational approaches and an integrated mRNA-microRNA (miRNA) analysis. The main cellular pathways perturbed by CS exposure were related to stress responses (oxidative stress and xenobiotic metabolism), inflammation (inhibition of nuclear factor-kB and the interferon gamma-dependent pathway), and proliferation/differentiation. Within post-exposure periods up to 48 hours, a transient kinetic response was observed at lower CS doses, whereas higher doses resulted in more sustained responses. In conclusion, this systems toxicology approach has potential for product testing according to “21st Century Toxicology”.

Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.

Potency ranking of dermal sensitizing chemicals using the IVSA and epiCS ® skin tissues

Human 3D reconstructed skin epidermal equivalents have been shown to release IL-18 in response to a wide range of dermal sensitizing chemicals. The concentration of these chemicals that produce greater than a threshold positive response (Stimulation Index, SI ≥ 2.0) is correlated to their potency or strength in an In Vitro Sensitization Assay (IVSA). In our experiments, 4-Nitrobenzyl bromide (NBB) and 2,4-Dinitrochlorobenzene (DNCB) were strong inducers of IL-18 secretion into the culture medium (SI-2 = 0.02% and 0.03%, respectively). The strong sensitizer p-Phenylenediamine (PPD) had an SI-2 of 0.13%. Cinnamaldehyde (CA) (SI-2 = 0.33%) and Isoeugenol (IE) (SI-2 = 0.56%) were moderate sensitizers, while Eugenol (EU) (SI-2 = 0.75), Resorcinol (RES) (SI-2 = 2.9%) and Hexylcinnamaldehyde (HCA) (SI-2 = 8.08%) were weak sensitizers. Sensitizer potency ranked using an SI-2 as follows: NBB > DNCB > PPD > CA > IE > EU > RES > HCA, with NBB, DNCB and PPD classified as strong, CA, IE and EU as moderate, and RES and HCA classified as weak sensitizers. Of the total of 18 chemicals tested, seven were irritants and two were non-sensitizers (Glycerol and Isopropanol); of these, only Chlorobenzene (50%) was incorrectly predicted as a sensitizer. epiCS gave an Accuracy of 89% and Sensitivity of 89%, and all other Cooper Statistics (Specificity, Negative and Positive Predictivity) values were 89%. In summary, measuring IL-18 release from 3D tissues allows for highly accurate and sensitive identification of dermal sensitizers. Also, the ability to rank-order potency of these chemicals based on SI-2.0 values of IL-18 secretion is a powerful tool for further classification into potency categories. MB Research Laboratories CellSystems® Biotechnologie Vertrieb GmbH Potency Ranking of Dermal Sensitizing Chemicals Using the IVSA and epiCS® Skin Tissues SOT 2015 MB Research Labs +1-215-536-4110 clientservices@mbresearch.com 2 INTRODUCTION The CDC estimates more than 13 million individuals in the US are likely exposed to chemicals that cause skin diseases. Many of these skin diseases are caused by contact dermatitis, a common type of illness associated with many occupational hazards, costing an estimated one billion dollars annually in healthcare and lost productivity (http://www.cdc.gov/niosh/topics/skin/). Currently there are no fully validated and regulatory accepted animal-alternative methods available to assess for Allergic Contact Dermatitis (ACD) / sensitization. In ACD, keratinocytes in the skin are the first to contact and elicit a response to allergens during an exposure. The 3D human epidermis equivalent epiCS is reconstructed from normal human primary epidermal keratinocytes. Keratinocytes have been shown to secrete a wide range of cytokines. Evidence demonstrates that cytokine IL-18 is an essential component of dermal sensitization. Most notable it has been shown using IL-18-deficient mice that IL-18 is not required for irritation contact dermatitis, but is required for an optimal ACD response (Antonopoulos et al., 2008. Journal of Leukocyte Biology 83: 361-7). An IL-18 endpoint has been used to predict sensitization in tissue models using filter paper applications (Gibbs et al., 2013, Toxicology and Applied Pharmacology 272(2):529-41). To identify sensitizing compounds, we measured IL-18 secretion from epiCS after treatment with pure chemicals via direct chemical application. MATERIALS AND METHODS Sensitizers and irritants/non-sensitizers were topically applied directly to the stratum corneum of epiCS tissues. The primary vehicles used were Ethanol and Acetone:Olive Oil (AOO). At 24 hours post-chemical application, media was sampled and analyzed by ELISA (MBL, Nagano, Japan) for secreted IL-18. IL-18 responses were measured as a Stimulation Index (SI), a fold increase above vehicle control. Tissues were washed and then tissue viability was measured by the MTT assay. The chemical concentration that results in 50% loss in tissue viability (TV50) was calculated when possible. At least two independent experiments were performed for each chemical. An SI of 2.0 was chosen as a cut-off for a positive response. A material was considered positive if at least one concentration of the material tested positive in at least two independent experiments. RESULTS Table 1. Contingency Table (SI=2 of IL-18) Known + Known – Total Tested + 8 1 9 Tested – 1 8 9 Total 9 9 18 Accuracy 89% (16/18) Sensitivity 89% (8/9) Specificity 89% (8/9) Positive Predictivity 89% (8/9) Negative Predictivity 89% (8/9) MB Research Laboratories CellSystems® Biotechnologie Vertrieb GmbH Potency Ranking of Dermal Sensitizing Chemicals Using the IVSA and epiCS® Skin Tissues SOT 2015 MB Research Labs +1-215-536-4110 clientservices@mbresearch.com 3 RESULTS (cont’d) Table 2. Chemicals Tested in epiCS IVSA Chemicals IL-18 a Result SI-2 % TV50 c LLNA d EC3 % Human DSA05 (μg/cm) Sensitizer 1* 4-Nitrobenzylbromide (NBB) + 0.02 0.04 0.05 — 2 1-Chloro-2,4-Dinitrobenzene (DNCB) + 0.03 0.05 0.03 3.80 3 p-Phenylenediamine (PPD) + 0.13 0.67 1.31 176 4 Cinnamaldehyde + 0.33 0.48 1.71 634 5 IsoEugenol + 0.56 0.78 1.71 1,054 6 Eugenol + 0.75 1.09 11.73 5,926 7 Resorcinol + 2.90 2.82 5.5 — 8 α-HCA + 8.08 20.91 9.37 23,622 Cinnamyl Alcohol (Cinn-OH) – na 1.40 21.0 13,747 Irritants Lactic Acid – na 3.11 na na Phenol – na 2.22 na na Sodium Dodecyl Sulfate (SDS) – na — na na Methyl Salicylate – na 3.16 na na Chlorobenzene + na 16.40 na na Salicylic Acid – na 2.5 na na Tween 20 – na — na na Non-Toxic Glycerol – na — na na Isopropanol – na — na na Using a SI-2 as the cutoff for a positive response with IL-18 in epiCS tissues The chemical concentration (%) that achieved an SI-2 with IL-18 in epiCS tissues The chemical concentration (%) that achieved 50% loss is tissue viability (TV50) in epiCS LLNA EC3(%) Human DSA05 (μg/cm) data as reported in Gibbs et al., 2013, Toxicology and Applied Pharmacology 272(2):529-41 na = not applicable *see Figure 1 Figure 1. Correlation of TV50 (EC50) and IL-18 SI-2 1 3 4 2 5 7 6 8 R2 = 0.94