Molecular Mechanism behind the Safe Immunostimulatory Effect of Withania somnifera.

Withania somnifera (L.) Dunal (family Solanaceae) is a medicinal plant known for, among many pharmacological properties, an immune boosting effect. Our recent study revealed that its key immunostimulatory factor is lipopolysaccharide of plant-associated bacteria. This is peculiar, because, although LPS can elicit protective immunity, it is an extremely potent pro-inflammatory toxin (endotoxin). However, W. somnifera is not associated with such toxicity. In fact, despite the presence of LPS, it does not trigger massive inflammatory responses in macrophages. To gain insights into the safe immunostimulatory effect of W. somnifera, we conducted a mechanistic study on its major phytochemical constituent, withaferin A, which is known for anti-inflammatory activity. Endotoxin-triggered immunological responses in the presence and absence of withaferin A were characterized by both in vitro macrophage-based assay and in vivo cytokine profiling in mice. Collectively, our results demonstrate that withaferin A selectively attenuates the pro-inflammatory signaling triggered by endotoxin without impairing other immunological pathways. This finding provides a new conceptual framework to understand the safe immune-boosting effect of W. somnifera and possibly other medicinal plants. Furthermore, the finding opens a new opportunity to facilitate the development of safe immunotherapeutic agents, such as vaccine adjuvants.

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

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.

Abundance of Plant-Associated Gammaproteobacteria Correlates with Immunostimulatory Activity of Angelica sinensis.

Background: Angelica sinensis is a medicinal plant known for a variety of biological effects, including its ability to stimulate innate immune cells in humans. Recent studies indicate that the immunostimulatory activity of A. sinensis arises from microbe-associated molecular patterns (MAMPs) of plant-associated bacteria. However, it is unknown which bacterial taxa in A. sinensis are responsible for the production of immunostimulatory MAMPs. Methods: Samples of A. sinensis were subjected to a cell-based assay to detect monocyte-stimulation and 16S ribosomal RNA amplicon sequencing, which revealed their immunostimulatory activity and microbial communities. The resulting data were analyzed by Linear discriminant analysis effect size (LEfSe), an online biostatistical tool for metagenomic biomarker discovery, to identify the bacterial taxonomical features correlated with the immunostimulatory activity. Results: A series of bacterial taxa under Gammaproteobacteria correlated positively with the immunostimulatory activity, whereas several Gram-positive taxa and Betaproteobacteria correlated negatively with the activity. Conclusions: The identified bacterial taxa set a new stage to characterize immunostimulatory MAMPs in plants.

Biomarker-guided screening of Juzen-taiho-to, an oriental herbal formulation for immunostimulation.

Juzen-taiho-to is an immunostimulatory herbal formulation that is clinically used in East Asia for cancer patients undergoing chemotherapy and radiation. The formulation stimulates various leukocytes, including T, B, and NK cells and macrophages. Although Juzen-taiho-to is known to contain numerous compounds with various pharmacological activities, it is not clear which compounds are responsible for the stimulation of individual cell types. Here, we conducted what we call "biomarker-guided screening" to purify compounds responsible for the macrophages stimulatory activity. To this end, gene expression was analyzed by a DNA array for macrophages treated with Juzen-taiho-to and DMSO (vehicle control), which identified intercellular adhesion molecule 1 as a biomarker of macrophage stimulation by Juzen-taiho-to. A quantitative reverse transcription polymerase chain reaction assay of intercellular adhesion molecule 1 was then used to guide the purification of active compounds. The screening resulted in the purification of a glycolipid mixture, containing ß-glucosylceramides. The glycolipid mixture potently stimulated intercellular adhesion molecule 1 expression in primary dendritic cells as well as in primary CD14+ (macrophages) cells. The identification of this glycolipid mixture opens up an opportunity for further studies to understand how plant-derived glycolipids stimulate macrophages and dendritic cells in a safe and effective manner as demonstrated by Juzen-taiho-to.