Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

Kata Tombácz, Duncan Mwangi, Dirk Werling, Amanda J. Gibson

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Abstract

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-B-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-B reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Original languageEnglish
Pages (from-to)329-335
Number of pages7
JournalInnate Immunity
Volume23
Issue number4
Early online date27 Feb 2017
DOIs
Publication statusPublished - 01 May 2017

Keywords

  • Bovine TLR2
  • IL-8
  • luciferase assay
  • reporter assay
  • SEAP

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