Regulation of Inflammatory Gene Expression in PBMCs by Immunostimulatory Botanicals

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

Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting elect rophoresis mobility shift assay (WEMSA)

Calcitriol (1α, 25-dihydroxyvitamin D3) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position −91 to −79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.Microbiology and Immunology, Department ofScience, Faculty ofNon UBCReviewedFacult

This region spanning 469 bp upstream of the transcriptional start site has been shown to be critical for the induction of CD14 transcription in response to calcitriol

Putative Sp-1-like transcription factor-binding sites in this region were identified using the TFSEARCH program () and are highlighted in boldface, underlined and labeled as (1) 5'-GGGGGGTTGG-3' at position -345 to -336, (2) 5'-GTCCCTCCCCCT-3' at position -159 to -148, (3) 5'-AGGGGGCTGGC-3' at position -113 to -100 and (4) 5'-AGAGGTGGGGAGG-3' at position -91 to -79. <p><b>Copyright information:</b></p><p>Taken from "Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)"</p><p></p><p>Biological Procedures Online 2008;10():29-35.</p><p>Published online Jan 2008</p><p>PMCID:PMC2275043.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2008 Biological Procedures Online.</p

EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter

Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.<p><b>Copyright information:</b></p><p>Taken from "Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)"</p><p></p><p>Biological Procedures Online 2008;10():29-35.</p><p>Published online Jan 2008</p><p>PMCID:PMC2275043.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2008 Biological Procedures Online.</p

Nuclear extract from calcitriol-treated and untreated THP-1 cells were used for WEMSA analysis using non-labeled canonical (A) and (B) the candidate CD14 promoter Sp-1 like oligo (sequence 4 from Fig

).<p><b>Copyright information:</b></p><p>Taken from "Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)"</p><p></p><p>Biological Procedures Online 2008;10():29-35.</p><p>Published online Jan 2008</p><p>PMCID:PMC2275043.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2008 Biological Procedures Online.</p