Anti-Infective Mode of Action of EPs® 7630 at the Molecular Level

Abstract

Clinical trials have shown that EPs® 7630, an aqueous ethanolic extract from the roots of Pelargonium sidoides, is an efficacious treatment for respiratory tract infections. A large body of in vitro studies has provided evidence for an anti-infective principle associated with activation of the non-specific immune system. However, the mode of action at the cellular and molecular level is still insufficiently defined. This study, therefore, aimed to provide further insight into the underlying principles of the therapeutic benefits of EPs® 7630 under these conditions. Using BMMΦ experimentally infected with intracellular bacteria, Listeria monocytogenes, incubation with EPs® 7630 (1 - 30 μg/mL) increased release of NO, production of membrane bound/intra- and extracellular IL-1, IL-12 and TNF-α and changed the expressions of the surface markers CD40 and CD119 at an early time point post infection (6 h) in a concentration-dependent manner in most experiments. Compared with non-infected cells, the effects were more pronounced. LPS + IFN-γ served as positive and untreated cells as negative controls. Analyses were carried out at single cell levels using flow cytometry, while ELISA was additionally utilized for monitoring secreted cytokines. Although the current data provide additional valuable information for understanding the anti-infective effects of EPs® 7630, the triggered signalling pathways associated with host immune responses appear even more complex than anticipated and are evidently not shared by ‘classical’ immunomodulators to this extent.

Abbreviations

APC:allophycocyanin

BMMΦ:bone marrow-derived macrophages

DMSO:dimethyl sulfoxide

ELISA:enzyme-linked immunosorbent assay

FACS:fluorescence-activated cell sorter

FCS:foetal calf serum

IL:interleukin

LPS:lipopolysaccaride

NO:nitric oxides

PBS:phosphate buffered saline

PE:R-phycoerythrin

TNF:tumour necrosis factor

Pelargonium sidoides - Geraniaceae - EPs® 7630 - nitric oxides - cytokines - surface markers - macrophages - Listeria monocytogenes - experimental infection model

References

• 1 Kolodziej H, Kiderlen A F. In vitro evaluation of antibacterial and immunomodulatory activities of Pelargonium reniforme, Pelargonium sidoides and the related herbal drug preparation EP® 7630.  Phytomedicine. 2007;  14 (Suppl. VI) 18-26
• 2 Kolodziej H. Aqueous ethanolic extract of the roots of Pelargonium sidoides - new scientific evidence for an old anti-infective phytopharmaceutical. Planta Med 2008, in press
• 3 Heil C, Reitermann U. Atemwegs- und HNO Infektionen: Therapeutische Erfahrungen mit dem Phytotherapeutikum Umckaloabo.  TW Pädiatrie. 1994;  7 523-5
• 4 Haidvogl M, Schuster R, Heger M. Akute Bronchitis im Kindesalter - Multizenter-Studie zur Wirksamkeit und Verträglichkeit des Phytotherapeutikums Umckaloabo.  Z Phytother. 1996;  17 300-13
• 5 Matthys H, Eisebitt R, Seith B, Heger M. Efficacy and safety of an extract of Pelargonium sidoides (EPs® 7630) in adults with acute bronchitis.  Phytomedicine. 2003;  10 (Supp. IV) 7-17
• 6 Matthys H, Heger M. EPs® 7630-solution - an effective therapeutic option in acute and exacerbating bronchitis.  Phytomedicine. 2007;  14 (Suppl. VI) 65-8
• 7 Matthys H, Kamin W, Funk P, Heger M. Pelargonium sidoides preparation (EPs® 7630) in the treatment of acute bronchitis in adults and children.  Phytomedicine. 2007;  14 (Suppl. VI) 69-73
• 8 Kolodziej H. Fascinating metabolic pools of Pelargonium sidoides and Pelargonium reniforme, traditional and phytomedicinal sources of the herbal medicine Umckaloabo®.  Phytomedicine. 2007;  14 (Suppl. VI) 9-17
• 9 North R J, Dunn P L, Conlan J W. Murine listeriosis as a model for antimicrobial defense.  Immunol Rev. 1997;  158 27-36
• 10 Schötz K, Erdelmeier C, Germer S, Hauer H. A detailed view on the constituents of EPs® 7630. Planta Med 2008, in press
• 11 Radtke O A, Kiderlen A F, Kayser O, Kolodziej H. Gene expression profiles of inducible nitric oxide synthase and cytokines in Leishmania-infected macrophage-like RAW 264.7 cells treated with gallic acid.  Planta Med. 2004;  70 924-8
• 12 Prussin C, Metcalfe D D. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.  J Immunol Methods. 1995;  188 117-28
• 13 McMicking J, Xie Q W, Nathan C. Nitric oxide and macrophage function.  Annu Rev Immunol. 1997;  15 323-50
• 14 Nathan C, Shiloh M U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens.  Proc Natl Acad Sci. 2000;  97 8841-8
• 15 Bogdan C, Röllinghoff M, Diefenbach A. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity.  Curr Opin Immunol. 2000;  12 64-76
• 16 Kayser O, Kolodziej H, Kiderlen A F. Immunomodulatory principles of Pelargonium sidoides.  Phytother Res. 2001;  15 122-6
• 17 Dinarello C A. Interleukin-1beta.  Crit Care Med. 2005;  33 460-2
• 18 Feezor R J, Oberholzer C, Baker H V, Nodick D, Rubinstein M. Molecular characterization of the acute inflammatory response to infections with Gram-negative versus Gram-positive bacteria.  Infect Immun. 2003;  71 5803-13
• 19 Watford W T, Moriguchi M, Morinobu A, O′Shea J J. The biology of IL-12: coordinating innate and adaptive immune responses.  Cytokine Growth Factor Rev. 2003;  14 361-8
• 20 Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.  Nat Rev Immunol. 2003;  3 133-46
• 21 Beutler B, Cerami A. The biology of cachectin/TNF: a primary mediator of host response against a human tumour necrosis factor-α receptor.  Annu Rev Immunol. 1989;  7 625-55
• 22 Musicki K, Briscoe H, Tran S, Britton W J, Saunders B M. Differential requirements for soluble and transmembrane tumor necrosis factor in the immunological control of primary and secondary Listeria monocytogenes infection.  Infect Immun. 2006;  74 3180-9
• 23 Van Kooten C, Banchereau J. CD40-CD40 ligand.  J Leukoc Biol. 2000;  67 2-17
• 24 Andrade R M, Portillo J A, Wessendarp M, Subauste C S. CD40 signaling in macrophages induces activity against an intracellular pathogen independently of gamma interferon and reactive nitrogen intermediates.  Infect Immun. 2005;  73 3115-23
• 25 Andrade R M, Wessendar M, Subauste C S. CD154 activates macrophage antimicrobial activity in the absence of IFN-gamma through a TNF-alpha-dependent mechanism.  J Immunol. 2003;  171 6750-6
• 26 Demuth A, Goebel W, Beuscher H U, Kuhn M. Differential regulation of cytokine and cytokine receptor mRNA expression upon infection of bone marrow-derived macrophages with Listeria monocytogenes.  Infect Immun. 1996;  64 3475-83
• 27 Tau G, Rothman P. Biologic functions of the IFN-gamma receptors.  Allergy. 1999;  54 1233-51