Abstract
Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.
Highlights
A recurrent problem in biomedical research is the presence of microbial contaminants in biological samples
The human monocytic cell line THP-1 [24], the human myelogenous leukaemia cell line K562 [25] and HEK293 hTLR4A-MD2-CD14 (Invivogen, San Diego, CA) were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 μg/mL streptomycin and 100 U/mL penicillin
We have previously described the generation of Jurkat reporter cell lines based on a set of highly sensitive and selective fluorescent transcriptional reporter constructs for the activity of the transcription factors activator protein 1 (AP-1), NFAT and nuclear factor-κB (NF-κB) [26, 31]
Summary
A recurrent problem in biomedical research is the presence of microbial contaminants in biological samples. Unchecked contaminations with bacterial products seriously impact on experimental research and can render data unusable. Sensitive detection methods for the presence of microbial products are of vital importance. Various test systems are currently in routine use: The Limulus amebocyte lysate (LAL) test for endotoxin and various PCR-based or enzymatic tests for mycoplasma detection [1, 2]. Most of these assays are time intensive and require additional non-standard reagents and equipment. For the current study we aimed to exploit the exquisite sensitivity of evolutionary conserved pattern recognition receptors (PRRs) for the generation of a sensitive cellular reporter platform
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