Cell‐line‐based assay for the toxicity/benefit analysis of lipopolysaccharides in plants

Abstract

There are many immune‐boosting medicinal plants that can potently activate innate immune cells. Recent studies indicate that the active factors of some immune‐boosting plants are lipopolysaccharides (LPSs) of plant‐associated bacteria. However, little is currently known about the potential risk and benefit of LPSs in medicinal plants. To facilitate their characterization, we established a simple cell‐line‐based assay that can be used to screen the toxicity and benefit of LPSs in medicinal plants. The assay can distinguish endotoxic diphosphoryl lipid A (DPL) from beneficial monophosphoryl lipid A (MPL), which is a clinically used immunological adjuvant for vaccines. The established assay was used to characterize commercial supplements of Ashwagandha, which was shown to contain immunostimulatory LPSs. The study revealed that Ashwagandha activates macrophages in a manner similar to MPL. The current finding underscores the importance of further studies to characterize the LPSs in immune‐boosting medicinal plants.

Molecular Mechanism behind the Safe Immunostimulatory Effect of Withania somnifera

“…The detailed protocol for cell treatment and lysis has been published previously [28]. Briefly, human monocytic THP-1 cells were plated in a 12-well plate at 200,000 cells/mL of RPMI-1640 media, to which 25 nM PMA was added to differentiate the cells to macrophage phenotype.…”

“…RNA purification, cDNA synthesis, and qPCR on an Applied Biosystems 7500 Real-Time PCR system were carried out as described previously [28]. The qPCR experiments used pre-optimized assays for IL-6 (FAM, ThermoFisher Assay Id: Hs00985639_m1), CCL5 (FAM, ThermoFisher Assay Id: Hs00982282_m1), and GAPDH endogenous control (Ther-moFisher Catalog Number: 4325792).…”

“…MPL is clinically used as an immunological adjuvant for various vaccines [31,32]. W. somnifera, despite the presence of LPS, also elicits MPL-like balanced TLR4 activation (Figure 1C) [28]. The lack of MYD88 bias suggests an as-yet uncharacterized mechanism by which the proinflammatory toxicity of LPS is attenuated in this plant.…”

“…Our recent study indicates that W. somnifera stimulates APCs in a manner similar to monophosphoryl lipid A (MPL) [28], which is a detoxified analog of DPL (Figure 1B) [29,30]. Unlike DPL, MPL exhibits much more attenuated activation of the MYD88 pathway, while retaining the ability to activate the TRIF pathway (Figure 1C).…”

“…The finding of LPS in Echinacea and Juzen-taiho-to opened a possibility that APCstimulatory effect of many other medicinal plants, including W. somnifera, could also arise from bacterial LPSs. In fact, when W. somnifera extracts were treated with Detoxi-Gel™, which removes LPS [26,27], the resulting samples no longer exhibited the APC-stimulatory effect [28]. Thus, LPS is indeed additionally a key APC-stimulatory factor in W. somnifera.…”