Innate Immune Assay for the Examination of Receptor Activity

  • Fig. 1: Principle of the cell-based test system. After specific ligand binding to the receptor, an intracellular signaling cascade activates NF-ΚB and induces the expression of the reporter gene (secreted alkaline phosphatase SEAP).Fig. 1: Principle of the cell-based test system. After specific ligand binding to the receptor, an intracellular signaling cascade activates NF-ΚB and induces the expression of the reporter gene (secreted alkaline phosphatase SEAP).
  • Fig. 1: Principle of the cell-based test system. After specific ligand binding to the receptor, an intracellular signaling cascade activates NF-ΚB and induces the expression of the reporter gene (secreted alkaline phosphatase SEAP).
  • Fig. 2: Measurement of the sensitivity of the NIH3T3 TLR4/CD14 SEAP cell line against its specific ligand LPS: (A) time response showed an increase in the induced (10 ng/ml LPS) cell line compared to the not induced control cell line. (B) dose–response showed a high sensitivity up to 1 pg/ml LPS.
  • Fig. 3: Measurement of the sensitivity of the NIH3T3 TLR2/6 SEAP cell line against its specific ligand zymosan: (A) time response showed an increase in the induced (4 μg/ml zymosan) cell line compared to the not induced control cell line. (B) dose–response showed a high sensitivity up to 1 μg/ml zymosan.
  • Fig. 4: (A) Comparison of NIH3T3 and HeLa cell lines for unspecific background signal. Both cell lines were stable transfected with the SEAP reporter gene. (B) Analysis of the endogenous receptor activity of the NIH3T3 SEAP cell line after induction (o/n) with the ligands of a TLR1-9. Interrupted line shows the background of the cell line without induction.
  • Dr. Anke Burger-Kentischer

Pattern recognition receptors (PRRs) are the first defense line of the innate immune system. In the last years PRRs had been studied in different ways resulting in a lot of new insights. However, it was not possible to investigate individual combinations of PRRs and their specific ligands because of the complex network in immune signaling. A cell-based assay developed at Fraunhofer IGB allows a direct way to detect the activity of individual human PRR-receptors in a highly specific way.


The innate immune system is the first defense line against pathogenic microbes. Pattern recognition receptors (PRRs), like toll-like receptors (TLRs), Dectins, CD14 or NOD-like receptors (NLRs) are involved in early detection of specific pathogens because they recognize highly conserved and class-specific molecules of bacteria, virus and fungi called the pathogen associated molecular patterns (PAMPs). After binding of these pyrogens intracellular inflammatory pathways are triggered resulting in the activation of specific transcription factors such as the nuclear factor (NF)-κB. These factors induce the transcription of inflammatory cytokines, which activate inflammation cascades and fever [1].The best characterized class of the PRRs is the TLR family. In humans 10 TLRs have been described to date and they differ from each other in expression patterns, ligand specificity and in the target genes they induce [2].

Material and Methods
To study ligand-receptor interaction of individual receptors or receptor dimers, we established a cell-based assay using NIH3T3 mouse fibroblasts stably transfected with a secreted alkaline phosphatase (SEAP) reporter gene plasmid, containing a NF-κB-regulated promoter. To analyze individual PRRs or specific PRR-heterodimers, the cells in addition were stably transfected with the respective specific PRRs or combinations thereof.
Due to the NF-κB-regulated promoter the reporter gene (SEAP) is directly linked to the TLR signaling pathway and the SEAP expression is dependent on the TLR-activation via its ligands (fig. 1). In this article two different NIH3T3 SEAP cell lines, stably transfected with TLR4/CD14 (fig.

2) and TLR2/6 (fig. 3), are described. This activation is easily detected visually or photometrically. The assay can be performed also under GLP conditions in a 96-well format.


Results and discussion
Our experiments showed that the new sensor cell lines are ideally suited for the investigation of individual human PRRs and combinations thereof. This is due to the fact, that the NIH3T3 SEAP cell line has a very low background activity for the NF-κB linked reporter gene SEAP and no or very low endogenous TLR-activity (fig. 4). Therefore, the PRR reporter gene cell lines constructed in this work are activated by their specific ligands in a highly specific manner, as shown in figure 2 and 3. The short detection time of the enzyme substrate reaction of less than 60 min after an overnight induction with the respective ligand-containing sample allows a fast and simple read-out [3]. The new assay can be used in a wide field of applications: It allows the detection of individual ligands or combination of ligands with isolated PRRs. This allows to study in detail the receptor on the one hand and the ligand on the other. Another possible application we envisage is the screening for new agonists and antagonists of the immune receptors, such as TLR-agonists/antagonists which are increasingly used e.g. in dermatology in order to modulate immune reactions. PR- receptors may play an important role especially in the treatment of allergies or pathological changes of the skin such as psoriasis or atopic dermatitis. In the case of allergies erroneous or excessive activation of PRR receptors leads to the release of cytokines, which cause tissue changes, from itching to tissue damage. This cell-based assay system thus allows the screening of defined TLR antagonists, which can be used as immunosuppressants. Furthermore, the assay is also applicable for pyrogen-detection, e.g. in medical devices, such as surgical instruments, catheters, implants and also intravenously administered drugs.


Conclusions
For the first time the new test system enables the investigation of individual human PRRs in defined combinations without the interference of other PRRs, leading to an unbiased analysis of individual PRRs and their response to ligands. In addition this cell line based system does not require the use of animal models like knockout mice and primary cells derived thereof. The reporter cell lines therefore may lead to important, new insights into the innate immune system with a better understanding of the individual receptors roles.
To further develop this modular cell-based assay system we aim at expanding it to all relevant immune receptors until the innate immune system is replicated in a microtiter plate.


References
[1] Kawai T. and Akira S.: Cell Death Differ. 13 (5), 816 (2006)
[2] Medzhitov R. et al.: Nature 388 (6640), 394-397 (1997)
[3] Burger-Kentischer A. et al.: J Immunol Methods 358: 93-103 (2010)


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Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik IGB
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70569 Stuttgart
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