The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes

Abstract

Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

Fig 1. EPs 7630 stimulates human blood immune cells.

(A, B) PBMCs, isolated from the blood of healthy donors, were treated with EPs 7630 as indicated, TLR3 and TLR4 ligands (T3L, T4L), a cytokine mixture (IL-1β, IL-2, IL-12), or anti-CD3 and anti-CD28 antibodies for 4 h and 24 h. TNF-α (4 h), IL-6 (24 h), and IL-10 (24 h) were quantified in respective culture supernatants by ELISA. Mean (± SEM) data from 6 donors are given as cytokine concentrations (A) or TNF-α/IL-10 ratio (B). (C) Healthy donor PBMCs were cultured in the presence of different concentrations of EPs 7630 as indicated for 48 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 12 donors are given. (D) Healthy donor PBMCs were stimulated with EPs 7630 and TLR4 ligand as indicated for 4 to 72 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 4 donors (except TNF-α 48h: n = 3) are given. (A, B) Significant differences compared to control group are indicated (* p<0.05, ** p<0.01, Wilcoxon matched-pairs signed-rank test).

Fig 2. CD4⁺ memory T cells are not the main responders to EPs 7630.

(A) Memory T-helper cells (CD4⁺ CD45RO⁺) were isolated from healthy donor PBMCs by magnetic labeling-based cell sorting. Representative dot plots of the cells (live gate cells with CD4⁺ lymphocytes marked in blue), stained and analyzed by flow-cytometry before and after sorting, are given (left panel). Mean (± SEM) purity of isolated cells was 97.68 ± 0.4%. Isolated cells were then treated with different concentrations of EPs 7630 as indicated for 48 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given (right panel). (B) Memory T-helper cells, isolated as in (A), were treated with different concentrations of EPs 7630 as indicated for 24 h. Subsequently, cells were additionally stimulated with anti-CD3/anti-CD28 antibodies for another 24 h. Quantification of TNF-α, IL-6 and IL-10 in respective culture supernatants was carried out by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given as percent of 0 μg/ml EPs 7630 group (control). Cytokine concentrations in the absence of EPs 7630 were: 4753±608 pg/ml (TNF-α), 3.4±0.9 pg/ml (IL-6), 737±103 pg/ml (IL-10). (C) Healthy donor PBMCs were stimulated with different concentrations of EPs 7630 as indicated for 4 to 5 h. Brefeldin A was added for the last 3 to 4 h of culture, followed by antibody-based staining of intracellular TNF-α and surface markers and subsequent flow-cytometric analysis. Representative dot plots showing the proportions of TNF-α⁺ cells out of all CD4⁺ cells of one out of 3 independent experiments are given.

Fig 3. EPs 7630 mediates its effects via specific activation of MAPKs.

(A, B) Healthy donor PBMCs were treated with different concentrations of EPs 7630 as indicated for 4 to 5 h. Brefeldin A was added for the last 3 to 4 h of culture, followed by antibody-based staining of surface markers and intracellular TNF-α and subsequent analysis by flow-cytometry. (A) Representative dot plots of CD14⁺ cells (monocytes) from one representative experiment are given, showing proportions of TNF-α⁺ cells out of all CD14⁺ cells. (B) Mean (± SEM) data of the proportions of TNF-α⁺ cells out of all CD14⁺ cells from 3 donors are given. Significant differences compared to control group (0 μg/ml EPs 7630) are indicated (* p<0.05, Wilcoxon matched-pairs signed-rank test). (C) Healthy donor PBMCs, starved for 3.5 h, were treated or not (control) with different concentrations of EPs 7630, TLR3 and TLR4 ligands (T3L, T4L), cytokine mixture (IL-1β, IL-2 and IL-12), and anti-CD3/anti-CD28 coated Dynabeads for 10 and 30 min as indicated. Activation of the phosphorylated signaling molecules JNK1/2, p38, Erk1/2, Akt, STAT5, and p65 as well as of the standardization marker GAPDH was assessed by Western blot analysis. (D) Healthy donor PBMCs were pretreated with inhibitors targeting MAP kinase and NF-κB signaling pathways [SP600125 (JNK1/2/3), PD98059 (MEK), SB202190 (p38α/β), wedelolactone (IκB kinase)] or a respective control solvent for 45 min, followed by addition of 10 μg/ml EPs 7630 and culture for further 4 h. Brefeldin A was added for the last 3 h of culture, followed by antibody-based staining of surface markers and intracellular TNF-α, and analysis by flow-cytometry. Mean fluorescence intensity (MFI) data of TNF-α signal in CD14⁺ cells (monocytes) are given from 5 donors as mean ±SEM. Significant differences compared to control group are indicated (* p<0.05, Wilcoxon matched-pairs signed-rank test).

Fig 4. TLR3- and TLR4-induced responses in immune cells are modulated by EPs 7630 pretreatment.

Healthy donor PBMCs were treated with different concentrations of EPs 7630 as indicated for 24 h. Afterwards, TLR3 or TLR4 ligands (T3L, T4L) were added for additional 24 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 12 donors are given as percent of 0 μg/ml EPs 7630 group (control). Cytokine concentrations in the absence of EPs 7630 in TL3L and TL4L groups were: 1267±414 and 1534±324 pg/ml (TNF-α), 1278±369 and 2135±650 pg/ml (IL-6), 1011±127 and 1430±140 pg/ml (IL-10), respectively. Significant differences compared to 0 μg/ml EPs 7630 group are indicated (* p<0.05, ** p<0.01, Wilcoxon matched-pairs signed-rank test).