Therapeutic Effects of Oral Application of Menthol and Extracts from Tormentil (Potentilla erecta), Raspberry Leaves (Rubus idaeus), and Loosestrife (Lythrum salicaria) during Acute Murine Campylobacteriosis

Abstract

Human food-borne infections with the enteropathogen Campylobacter jejuni are becoming increasingly prevalent worldwide. Since antibiotics are usually not indicated in campylobacteriosis, alternative treatment regimens are important. We here investigated potential disease-alleviating effects of menthol and of extracts from tormentil, raspberry leaves, and loosestrife in acute murine campylobacteriosis. Therefore, C. jejuni-infected microbiota-depleted IL-10^-/- mice were orally treated with the compounds alone or all in combination from day 2 until day 6 post-infection. Whereas neither treatment regimen affected gastrointestinal pathogen loads, the combination of compounds alleviated C. jejuni-induced diarrheal symptoms in diseased mice on day 6 post-infection. Furthermore, the therapeutic application of tormentil and menthol alone and the combination of the four compounds resulted in lower colonic T cell numbers in infected mice when compared to placebo counterparts. Notably, pro-inflammatory cytokines measured in mesenteric lymph nodes taken from C. jejuni-infected mice following tormentil, menthol, and combination treatment did not differ from basal concentrations. However, neither treatment regimen could dampen extra-intestinal immune responses, including systemic pro-inflammatory cytokine secretion on day 6 post-infection. In conclusion, the combination of menthol and of extracts from tormentil, raspberry leaves, and loosestrife constitutes an antibiotic-independent approach to alleviate campylobacteriosis symptoms.

Keywords: Campylobacter jejuni; acute campylobacteriosis; immune modulation; loosestrife (Lythrum salicaria); menthol; microbiota-depleted IL-10−/− mice; plant-derived compounds; raspberry leaves (Rubus idaeus); tormentil (Potentilla erecta).

Figure 1

Fecal C. jejuni numbers over time following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with menthol and extracts derived from tormentil, raspberry leaves, and loosestrife alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC). Pathogen loads were determined in fecal samples collected (A) immediately before and (B–F) at defined time points after infection by culture (in colony-forming units per gram; CFU/g). Medians (black bars) and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 2

Gastrointestinal C. jejuni numbers following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Gastrointestinal pathogen loads were determined in luminal samples collected from the (A) stomach, (B) duodenum, (C) ileum, and (D) colon on day 6 post-infection by culture (indicated as colony-forming units per gram; CFU/g). Medians (black bars) and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 3

Clinical conditions over time following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on day (d)0 and d1. From d2 until d6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO) or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. The clinical conditions of mice were determined (A) immediately before and (B–F) at defined time points after infection by a clinical scoring scheme. Medians (black bars), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s multiple comparison test, and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 4

Diarrheal symptoms over time following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on day (d)0 and d1. From d2 until d6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO) or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. (A–C) The severity of diarrheal symptoms was determined at defined time points post-infection by a clinical scoring scheme. (D–F) The frequencies of diarrheal mice are shown (in %). Medians (black bars), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s multiple comparison test, and numbers of diarrheal mice out of the total number of animals included from three independent experiments (in parentheses) are indicated.

Figure 5

Macroscopic and microscopic inflammatory signs following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO) or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. On day 6 post-infection, (A) the colonic lengths were measured (in cm) and (B) the histopathological changes in the colon were quantified with a histopathological scoring scheme. Furthermore, (C) the apoptotic colonic epithelial cells were counted in paraffin sections of large intestinal explants stained with cleaved caspase-3 (Casp3) and indicated as average numbers out of six representative high-power fields (HPF, 400-times magnification). Medians (black bars), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s multiple comparison test, and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 6

Colonic immune cell responses following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. On day 6 post-infection, (A) neutrophils (MPO7⁺), (B) T lymphocytes (CD3⁺), (C) regulatory T cells (FOXP3⁺), and (D) B lymphocytes (B220⁺) were counted in paraffin sections of large intestinal explants stained with respective antibodies and indicated as average numbers out of six representative high-power fields (HPF, 400-times magnification). Medians (black bars), significance levels (p values) determined by the one-way ANOVA test with Tukey’s post-correction (A,B) and the Kruskal–Wallis test with Dunn’s multiple comparison test (C,D), and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 7

Pro-inflammatory cytokine secretion in the colon following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. On day 6 post-infection, (A) TNF-α, (B) IFN-γ, and (C) IL-6 concentrations were measured in colonic explants. Medians (black bars), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s multiple comparison test, and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 8

Pro-inflammatory cytokine secretion in mesenteric lymph nodes following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC), whereas naive mice (N) served as uninfected and untreated controls. On day 6 post-infection, (A) TNF-α, (B) IFN-γ, and (C) IL-6 concentrations were measured in mesenteric lymph nodes (MLN) explants. Medians (black bars), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s multiple comparison test (A,B), and the one-way ANOVA test with Tukey’s post-correction (C), and numbers of mice included from three independent experiments (in parentheses) are indicated.

Figure 9

C. jejuni translocation to mesenteric lymph nodes following oral treatment of infected gut microbiota-depleted IL-10^−/− mice with distinct natural compounds alone or in combination. Mice were orally infected with C. jejuni 81-176 strain on days 0 and 1. From day 2 until day 6, mice were treated with either tormentil (TOR), raspberry leaves (RAS), loosestrife (LOO), or menthol (MEN) alone or with a combination of all four compounds (COM) via the drinking water. Placebo control mice received vehicle only (PLC). On day 6 post-infection, C. jejuni loads were determined in mesenteric lymph nodes (MLN) by culture. Medians (black bars) and numbers of mice included from three independent experiments (in parentheses) are indicated.