Regulation of inflammatory gene expression in PBMCs by immunostimulatory botanicals

Abstract

Many hundreds of botanicals are used in complementary and alternative medicine for therapeutic use as antimicrobials and immune stimulators. While there exists many centuries of anecdotal evidence and few clinical studies on the activity and efficacy of these botanicals, limited scientific evidence exists on the ability of these botanicals to modulate the immune and inflammatory responses. Using botanogenomics (or herbogenomics), this study provides novel insight into inflammatory genes which are induced in peripheral blood mononuclear cells following treatment with immunomodulatory botanical extracts. These results may suggest putative genes involved in the physiological responses thought to occur following administration of these botanical extracts. Using extracts from immunostimulatory herbs (Astragalus membranaceus, Sambucus cerulea, Andrographis paniculata) and an immunosuppressive herb (Urtica dioica), the data presented supports previous cytokine studies on these herbs as well as identifying additional genes which may be involved in immune cell activation and migration and various inflammatory responses, including wound healing, angiogenesis, and blood pressure modulation. Additionally, we report the presence of lipopolysaccharide in medicinally prepared extracts of these herbs which is theorized to be a natural and active component of the immunostimulatory herbal extracts. The data presented provides a more extensive picture on how these herbs may be mediating their biological effects on the immune and inflammatory responses.

Figure 1. Scatter plot representation of botanical extract regulation of gene expression.

Microarray analyzed gene data was plotted to compare gene expression differences between botanical and ethanol treatment of PBMCs (Part A). Each spot on the plots represents a specific gene. Only genes with present calls in both treatments are shown. The diagonal lines off the center represent 2-, 3-, 10-, and 30-fold levels of induction or repression of gene expression. Part B illustrates comparative analysis between different botanical treatments of PBMCs.

Figure 2. Host gene expression regulated by Astragalus membranaceus treatment of PBMCs.

Genes were sorted based on a threefold (P<0.01) or greater level of induction for Astragalus treated PBMCs for 18 hours (Astra column). Only genes involved in the immune/inflammatory response are shown. Changes (n-fold) in expression level relative to those of ethanol-treated cells are shown within each box. Red boxes represent genes induced 100-fold or higher, orange boxes represent genes induced 10 to 100-fold, and yellow boxes represent genes induced 3 to 10-fold. Additional botanical treatments include Sambucus cerulea (Sambu column) and Andrographis paniculata (Andro column).

Figure 3. Astragalus membranaceus treatment of PBMCs led to monocyte maturation.

Part A) PBMCs (upper panels) or the monocyte cell line THP-1 (lower panel) were untreated, treated with ethanol (25%), or treated with Astragalus extract for 18 hours. Following treatment, unattached cells were removed and the remaining attached cells photographed. Part B) PBMCs were untreated or treated with Astragalus extract or PMA for 18 hours. After 18 hours, the cell culture media was removed and cells pelleted by centrifugation. The cell-free culture media was added to THP-1 cells for an additional 24 hours. Following treatment, unattached cells were removed and the remaining attached cells photographed. Part C) Cell-free media from mock-, Astragalus, or PMA-treated PBMCs was added to THP-1 cells and incubated in uncoated plastic dishes. After 48 hours, cells were washed and stained with fluorochrome-conjugated antibodies specific for CD14 and CD11b followed by flow cytometry analysis. Part D) Cell-free media obtained from PBMCs isolated from two patients were used to treat THP-1 cells and analyzed for CD14 and CD11b expression as in Part C. Values indicated represent total CD14 cells, total CD11b cells and CD14/CD11b double-positive cells. Data was normalized to mock-treated samples.

Figure 4. Transcriptional profiling of M1/M2 macrophage polarization induced by Astragalus membranaceus.

Human genes involved in defining M1/M2 polarization are shown . The transcriptional regulatory effect of each gene following treatment of PBMCs with Astragalus extract is indicated (‘Expression’ column), with positive values representing increased expression, and negative values representing repressed expression. The ‘X’ (in the ‘M1 like’ or M2 like' column) indicates that the change in gene expression (induced or repressed) following Astragalus treatment was similar to that previously observed following monocyte maturation to either an M1-like or M2-like macrophage polarization.

Figure 5. Different PBMC isolates led to similar changes in gene expression following treatment with Astragalus membranaceus.

Commercially available (PBMC set I) or fresh PBMCs (PBMC set II) were treated with Astragalus extract for 18 hours. Genes were sorted based on a threefold (P<0.01) or greater level of induction or repression relative to ethanol-treatment. Part A represents the overall comparison of genes regulated between PBMC set I and PBMC set II. For Part B, specific immune/inflammatory genes commonly or uniquely altered in expression following Astragalus treatment of PBMC set I and PBMC set II are shown. Changes (n-fold) in expression level relative to those of ethanol-treated cells are shown within each box. Red boxes represent genes induced 100-fold or higher, orange boxes represent genes induced 10-100-fold, and yellow boxes represent genes induced 3-10-fold.

Figure 6. Temporal regulation of gene expression in PBMCs following Astragalus membranaceus treatment.

PBMCs were treated with Astragalus extract for 3, 8 or 18 hours. Microarray analyzed gene data was plotted to compare gene expression differences between botanical and ethanol treatment of PBMCs (Part A). Each spot on the plots represents a specific gene. Only genes with present calls in both treatments are shown. The diagonal lines off the center represent 2-, 3-, 10-, and 30-fold levels of induction or repression of gene expression. Part B represents the roles and overall comparison of genes induced at 3, 8 and 18 hours post Astragalus treatment. Part C lists representative genes induced at 3, 8 and/or 18 hours post Astragalus treatment.

Figure 7. Endotoxin concentration present in botanical extracts.

Endotoxin concentrations in the botanical extracts listed were measured using a modified Limulus Amebocyte Lysate assay. Concentrations (endotoxin units/ml; EU/ml) were determined by comparison to an Escherichia coli standard solution. For Astragalus and Urtica, multiple extract preparations were prepared at different times using multiple lots of plant material obtained from the supplier (Samples I–IV and Samples I–V, respectively).