Application of a TLR overexpression cell model in pyrogen detection.

Qing Han, Ruobi Hu, Hui Li, Zhen Lei, Xuehui Zhang, Xiuyan Yu, Qun Zhang, Yiqing Mao, Xi Wang, David M Irwin, Gang Niu, Huanran Tan

Biotechnology and bioengineering 2019 Jun

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Pyrogens are components derived from microorganisms that induce complex inflammatory responses. Current approaches to detect pyrogens are complex and difficult to replicate, thus there is a need for new methods to detect pyrogens. We successfully constructed a pyrogen-sensitive cell model by overexpressing Toll-like receptor (TLR)2, TLR4, MD2, and CD14 in HEK293 cells. Since the cytokine IL-6 is specifically released upon stimulation of the TLR2 and TLR4 signaling pathways in response to pyrogen stimulation, we used it as a read out for our assay. Our results show that IL-6 is released in response to trace amounts of pyrogens in our cell model. Pyrogen incubation times and concentrations were explored to determine the sensitivity of our cell model, and was found to be sensitive to 0.05 EU/ml of LPS and 0.05 ug/ml of LTA after stimulation for 5 hr. Our TLR overexpressing cell model, with IL-6 as readout, could be a new method for in vitro testing of pyrogens and applicable for evaluating the safety of drugs. © 2019 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Citation

Qing Han, Ruobi Hu, Hui Li, Zhen Lei, Xuehui Zhang, Xiuyan Yu, Qun Zhang, Yiqing Mao, Xi Wang, David M Irwin, Gang Niu, Huanran Tan. Application of a TLR overexpression cell model in pyrogen detection. Biotechnology and bioengineering. 2019 Jun;116(6):1269-1279