RT Journal Article
SR Electronic
T1 Visualization and Modeling of Inhibition of IL-1β and TNF-α mRNA Transcription at the Single-Cell Level
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.10.16.342576
DO 10.1101/2020.10.16.342576
A1 Daniel Kalb
A1 Huy D. Vo
A1 Samantha Adikari
A1 Elizabeth Hong-Geller
A1 Brian Munsky
A1 James Werner
YR 2021
UL http://biorxiv.org/content/early/2021/06/10/2020.10.16.342576.abstract
AB IL-1β and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNF-α at the single-cell level. The amount of IL-1β and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental modeling efforts shows the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.Competing Interest StatementThe authors have declared no competing interest.