Immunomodulatory, anti-inflammatory and wound healing properties of Echinacea species

Herbal or botanical supplements have multiple health benefits. Public interest in these supplements has increased greatly in recent years aimed at modulation of the immune system to combat immune-related diseases. Historically, Echinacea has long been used as phytotherapy for wound healing, pain relief and treatment of the common cold. Although the non-specific immunostimulating properties of Echinacea species have been widely investigated, academic knowledge of the adaptive immune-modulating activity, the anti-inflammatory activity and the mechanistic basis for these immunomodulatory properties remain elusive. In this dissertation, we focused our research interest in these less explored fields, with an emphasis on the herbal anti-inflammatory activity in both in vitro macrophage cell line and in vivo oral administration models. In vivo, alcohol extracts of Echinacea exhibited multiple immune-modulating effects. In addition to the non-specific, innate immunomodulation, they enhance B cell and T cell function (determined by increased production of antibodies and cytokines). These modulating effects of Echinacea were more robust when host immune functions were disturbed by mild stress. In vitro, alcohol extracts of Echinacea suppressed activated macrophages (RAW 264.7 cells) so they produced lower amounts of inflammatory mediators including nitric oxide (NO). The decreased NO production might be relative to decreased inducible nitric oxide synthase (iNOS) enzyme expression and increased arginase activity, suggesting that Echinacea could switch macrophage to alternate activation. In a cutaneous wound healing model, we demonstrated that alcohol extract of E. pallida helped improve restraint stress-delayed wound healing. These data provided novel evidence to support the multiple immunomodulatory properties of Echinacea and will help define the mechanisms behind the success of traditional use of Echinacea for pathogenic infections and inflammatory diseases

Inflammation, DNA-centered radicals, and oxidative genotoxicity: The role of HOCl produced by myeloperoxidase in carcinogenesis

Myeloid cells (macrophages and neutrophils) infiltrate and synthesize myeloperoxidase (MPO) in sites of inflammation, producing gentotoxicity. In RAW 264.7 macrophages, bacterial lipopolysaccharide (LPS) induces superoxide radical anion, nuclear deformation (nuclear protuberances), MPO synthesis, biomolecule oxidation and cell death. “Freezing” LPS-triggered macrophage activation with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) prevented cell activation and death. Oxidation of proteins and genomic DNA was also blocked, with formation of protein- and DNA-DMPO nitrone adducts, as analyzed by immuno-spin trapping with a polyclonal anti-DMPO serum. Interestingly, confocal microscopy analysis of these cells showed that MPO, genomic DNA, and DNA-DMPO nitrone adducts co-localized in the nuclear protuberances. These observations, and the fact that DNA is negatively charged and MPO is a cationic protein, suggest a role for uptaken or newly synthesized MPO in oxidative genotoxicity induced by myeloid cells in sites of inflammation. 
In order to understand MPO-induced formation of DNA-centered radicals, we studied DNA-DMPO nitrone adducts in calf thymus DNA treated with micromolar concentrations of hypochlorous acid (HOCl) added as a bolus or generated in situ by the MPO/H2O2/Cl- system in the presence of DMPO. We also investigated DNA-DMPO nitrone adducts inside living cells containing MPO. The cell models we used were: i) human leukemia (HL)-60 cells, which overexpress MPO, ii) RAW 264.7 macrophages activated with LPS (1 ng/ml for 24 h), to induce MPO, and iii) A549 human airway epithelial cells pre-loaded with human MPO. When these cells were activated with the phorbol ester PMA, the number of 6-thioguanine-resistant cells with the hypoxanthine-guanine phosphoribosyl transferase (HRPT) mutation increased. This mutation was prevented by each of the following: the NADPH oxidase inhibitor apocynin; the MPO inhibitors salicylhydroxamic acid and 4-aminobenzoic acid hydrazide; the cell-permeable HOCl scavenger resveratrol; and DMPO, which traps DNA-centered radicals and prevents further oxidation. 
Genomic DNA-centered radicals and further mutagenesis induced by activated myeloid cells in sites of inflammation can be prevented by blocking MPO activity, preventing formation of and/or scavenging HOCl, or trapping DNA-centered radicals. Our findings provide new therapeutic avenues for preventing carcinogenesis induced by infiltration and activation of myeloid cells in sites of inflammation, for example, in the lung exposed to particulate matter. SUPPORTED BY NIEHS 5R00ES015415-03
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The Nitrone Spin Trap 5,5-Dimethyl-1-pyrroline N-oxide Affects Stress Response and Fate of Lipopolysaccharide-Primed RAW 264.7 Macrophage Cells

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly used to study free radicals. Due to its free radical trapping properties, DMPO is thought to reduce free radial-mediated oxidative damage and other related cellular responses. The purpose of this study was to assess the effect of DMPO on lipopolysaccharide (LPS)-induced inflammation, endoplasmic reticulum (ER) stress, and apoptosis in RAW 264.7 cells. The results showed that DMPO at 50 mM inhibited inducible nitric oxide synthase expression when added shortly after LPS treatment (≤3 h). Interestingly, DMPO increased anti-inflammatory heme oxygenase-1 (HO-1) expression and reversed LPS-induced decrease in HO-1 expression. LPS could increase cellular ER stress as indicated by C/EBP homologous protein (CHOP) induction; DMPO reduced LPS effect on CHOP expression. Unexpectedly, DMPO had a synergistic effect with LPS on increased caspase-3 activity. Overall, DMPO harbors multiple modulating effects but may induce apoptosis in LPS-stressed cells when given at 50 mM, an effective dose for its anti-inflammatory activity in vitro. Our data provide clues for further understanding of the nitrone spin trap with therapeutic potential.Fil: Zhai, Zili. University of Chicago. Department of Medicine. Section of Gastroenterology; Estados UnidosFil: Ramirez, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Gomez-Mejiba, Sandra Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; Argentin

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells indicative of alternative macrophage activation

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity. The alcohol extracts of three medicinal Echinacea species, E. angustifolia, E. pallida and E. purpurea, significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line, among them E. pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of E. pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, L-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8- bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression. These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation

Echinacea tennesseensis ethanol tinctures harbor cytokine- and proliferation-enhancing capacities

Background—Members of the genus Echinacea are used medicinally to treat upper respiratory infections such as colds and influenza. The aim of the present investigation was to characterize the phytomedicinal properties of the American federally endangered species Echinacea tennesseensis. Methods—Fifty-percent ethanol tinctures were prepared from roots, stems, leaves, and flowers and tested separately for their ability to influence production of IL-1β, IL-2, IL-10, and TNF-α as well as proliferation by young human adult peripheral blood mononuclear cells (PMBC) in vitro. Tincture aliquots were stored at three different temperatures (4°, −20°C, and −80°C) for 21 h before testing. At one-month post-extraction, tinctures stored at −20°C were tested again for cytokine modulation. Phytochemical analyses were performed using HPLC. Results—Fresh root, leaf, and flower tinctures stimulated PBMC proliferation. Fresh root tinctures alone stimulated IL-1β, IL-10, and TNF-α production. No tinctures modulated IL-2 production. Stem tinctures showed no activity. Storage temperature did not influence any outcomes. Root tinctures maintained their ability to modulate IL-1β, IL-10, and TNF-α production after one month of storage at −20°C. Conclusions—These results suggest E. tennesseensis harbors phytomedicinal properties that vary by plant organ, with roots demonstrating the strongest activities

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concominantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling

CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies

Immunomodulatory, anti-inflammatory and wound healing properties of Echinacea species

Herbal or botanical supplements have multiple health benefits. Public interest in these supplements has increased greatly in recent years aimed at modulation of the immune system to combat immune-related diseases. Historically, Echinacea has long been used as phytotherapy for wound healing, pain relief and treatment of the common cold. Although the non-specific immunostimulating properties of Echinacea species have been widely investigated, academic knowledge of the adaptive immune-modulating activity, the anti-inflammatory activity and the mechanistic basis for these immunomodulatory properties remain elusive. In this dissertation, we focused our research interest in these less explored fields, with an emphasis on the herbal anti-inflammatory activity in both in vitro macrophage cell line and in vivo oral administration models. In vivo, alcohol extracts of Echinacea exhibited multiple immune-modulating effects. In addition to the non-specific, innate immunomodulation, they enhance B cell and T cell function (determined by increased production of antibodies and cytokines). These modulating effects of Echinacea were more robust when host immune functions were disturbed by mild stress. In vitro, alcohol extracts of Echinacea suppressed activated macrophages (RAW 264.7 cells) so they produced lower amounts of inflammatory mediators including nitric oxide (NO). The decreased NO production might be relative to decreased inducible nitric oxide synthase (iNOS) enzyme expression and increased arginase activity, suggesting that Echinacea could switch macrophage to alternate activation. In a cutaneous wound healing model, we demonstrated that alcohol extract of E. pallida helped improve restraint stress-delayed wound healing. These data provided novel evidence to support the multiple immunomodulatory properties of Echinacea and will help define the mechanisms behind the success of traditional use of Echinacea for pathogenic infections and inflammatory diseases.</p