Abstract
The exquisite specificity of Toll-like receptors (TLRs) to sense microbial molecular signatures is used as a powerful tool to pinpoint microbial contaminants. Various cellular systems, from native human blood cells to transfected cell lines exploit TLRs as pyrogen detectors in biological preparations. However, slow cellular responses and limited sensitivity have hampered the replacement of animal-based tests such as the rabbit pyrogen test or lipopolysaccharide detection by Limulus amoebocyte lysate. Here, we report a novel human cell-based approach to boost detection of microbial contaminants by TLR-expressing cells. By genetic and pharmacologic elimination of negative control circuits, TLR-initiated cellular responses to bacterial molecular patterns were accelerated and significantly elevated. Combining depletion of protein phosphatase PP2ACA and pharmacological inhibition of PP1 in the optimized reporter cells further enhanced the sensitivity to allow detection of bacterial lipoprotein at 30 picogram/ml. Such next-generation cellular monitoring is poised to replace animal-based testing for microbial contaminants.
Highlights
The exquisite specificity of Toll-like receptors (TLRs) to sense microbial molecular signatures is used as a powerful tool to pinpoint microbial contaminants
The monocyte activation test (MAT) exploits the intrinsic capability of primary human monocytes or monocytoid cell lines to respond to various PAMPS with the expression of immune mediators, such as TNFα or IL-6, which serve as a readout for TLR stimulation[10,11,12,13,14]
In order to generate a cellular reporter system, three constructs containing 0, 4, or 8 disjunct kappa binding sites upstream of the coding sequence for the monomeric blue-fluorescent protein cerulean were stably introduced into monocytic THP-1 cells to allow the monitoring of TLR-induced mCFP expression (Fig. 1A,B)
Summary
A blue fluorescent protein reporter system for NF‐κB activity. The transcription factor NF-κB is a central component in regulating the inflammatory response by inducing genes encoding type I interferon and pro-inflammatory c ytokines[25]. TNF-α (10 ng/mL) induced the highest level of mCFP expression in 8xκB-TAp-mCFP, with ~ 16-fold increase after 24 h (Fig. 1C,D; Supplementary Fig. S1A) These results demonstrate that the magnitude of the reporter gene expression and subsequently the activation of NF-κB in the reporter cells correlates with the number of the kappa binding sites upstream of the reporter gene. In contrast to whole bacterial lysates or isolated bacterial lipoprotein (Pam3CSK4), fungal β-glucan or mycoplasma did not elicit strong reporter activity, pointing to a selective recognition of PAMPs by THP-1 cells (Supplementary Fig. S1B-D) These results indicated that the generated 8xκB-TApmCFP THP-1 cells can serve as a robust NF-κB reporter system, and suggested that the cellular response might be even stronger in individual clones amongst the high tdTomato expressing cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
