Menthol Pretreatment Alleviates Campylobacter jejuni-Induced Enterocolitis in Human Gut Microbiota-Associated IL-10-/- Mice

Abstract

Human Campylobacter jejuni infections are of worldwide importance and represent the most commonly reported bacterial enteritis cases in middle- and high-income countries. Since antibiotics are usually not indicated and the severity of campylobacteriosis is directly linked to the risk of developing post-infectious complications, non-toxic antibiotic-independent treatment approaches are highly desirable. Given its health-promoting properties, including anti-microbial and anti-inflammatory activities, we tested the disease-alleviating effects of oral menthol in murine campylobacteriosis. Therefore, human gut microbiota-associated IL-10^-/- mice were orally subjected to synthetic menthol starting a week before C. jejuni infection and followed up until day 6 post-infection. Whereas menthol pretreatment did not improve campylobacteriosis symptoms, it resulted in reduced colonic C. jejuni numbers and alleviated both macroscopic and microscopic aspects of C. jejuni infection in pretreated mice vs. controls. Menthol pretreatment dampened the recruitment of macrophages, monocytes, and T lymphocytes to colonic sites of infection, which was accompanied by mitigated intestinal nitric oxide secretion. Furthermore, menthol pretreatment had only marginal effects on the human fecal gut microbiota composition during the C. jejuni infection. In conclusion, the results of this preclinical placebo-controlled intervention study provide evidence that menthol application constitutes a promising way to tackle acute campylobacteriosis, thereby reducing the risk for post-infectious complications.

Keywords: Campylobacter jejuni; anti-oxidant effects; campylobacteriosis model; host-pathogen interaction; human gut microbiota-associated IL-10−/− mice; immune-modulatory properties; menthol; placebo-controlled preclinical intervention study; pretreatment.

Figure 1

Timeline of experimental procedures and the microbiota composition of human fecal transplants. (A) Human microbiota-associated (hma) IL-10^−/− mice were generated by antibiotic gut microbial depletion (with ampicillin plus sulbactam) and subsequent triple human fecal microbiota transplantation (hFMT). Starting a week before oral Campylobacter jejuni strain 81–176 infection, hma mice were subjected to menthol treatment via the drinking water and followed up until necropsy on day (d) 6 post-infection. (B) Gut microbial communities were quantitated in human fecal transplants (n = 3 per experiment) by culture-independent (i.e., 16S rRNA-based molecular) analyses. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), and medians (red bar in boxes) are given (pooled from three independent experiments). TEL, total eubacterial loads; EB, enterobacteria; EC, enterococci; LB, lactobacilli; BB, bifidobacteria; BP, Bacteroides/Prevotella species; CC, Clostridium coccoides group; CL, Clostridium leptum (CL) group; MIB, Mouse Intestinal Bacteroides.

Figure 2

Clinical conditions over time following C. jejuni infection of hma IL-10^−/− mice with menthol pretreatment. Hma mice were orally pretreated with menthol (MEN, white bars) or placebo (PLC, grey bars) and infected with C. jejuni on day 0 (d0) and d1 by orogastric gavage. The clinical conditions of mice were surveyed daily until necropsy by using a clinical campylobacteriosis score recording wasting, diarrhea, and fecal blood (see methods). Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), and numbers of analyzed mice (in parentheses) from three experiments are given.

Figure 3

Pathogen numbers in gastrointestinal luminal samples from C. jejuni infected hma IL-10^−/− mice with menthol pretreatment. Hma mice were orally pretreated with menthol (MEN, white bars) or placebo (PLC, grey bars) and infected with C. jejuni on days 0 and 1 by orogastric gavage. The luminal C. jejuni numbers were determined in gastrointestinal samples taken on day 6 post-infection by culture. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), significance levels (p values) determined by the Mann–Whitney test, and numbers of analyzed mice (in parentheses) from three experiments are given. CFU, colony-forming units.

Figure 4

Inflammatory changes in the colon of C. jejuni infected hma IL-10^−/− mice with menthol pretreatment. Hma IL-10^−/− mice were orally pretreated with menthol (MEN, white bars) or placebo (PLC, grey bars) and infected with C. jejuni on days 0 and 1 by orogastric gavage. On day 6 post-infection, (A) the colonic lengths were measured. Furthermore, (B) the histopathological scores and (C) numbers of apoptotic epithelial cells positive for cleaved caspase-3 (Casp3⁺) were determined in colonic paraffin sections. Naive mice (non-infected without pretreatment) served as negative controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), significance levels (p values) determined by the one-way ANOVA test with Tukey post hoc test (A,C) and Kruskal–Wallis test with Dunn’s post hoc test (B), and numbers of analyzed mice (in parentheses) from three experiments are given. HPF, high power field.

Figure 5

Colonic immune cells in C. jejuni infected hma IL-10^−/− mice with menthol pretreatment. Hma IL-10^−/− mice were orally pretreated with menthol (MEN, white bars) or placebo (PLC, grey bars) and infected with C. jejuni on days 0 and 1 by orogastric gavage. On day 6 post-infection, numbers of (A) macrophages and monocytes (F4/80⁺), (B) neutrophils (MPO7⁺), (C) T lymphocytes (CD3⁺), and (D) B lymphocytes (B220⁺) were determined in the colonic lamina propria (immunohistochemically stained paraffin sections). Naive mice (non-infected without pretreatment) served as negative controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), significance levels (p values) determined by the one-way ANOVA test with Tukey post hoc test (A) and Kruskal–Wallis test with Dunn’s post hoc test (B–D), and numbers of analyzed mice (in parentheses) from three experiments are given. HPF, high power field.

Figure 6

Intestinal nitric oxide secretion in C. jejuni infected hma IL-10^−/− mice with menthol pretreatment. Hma IL-10^−/− mice were orally pretreated with menthol (MEN, white bars) or placebo (PLC, grey bars) and infected with C. jejuni on days 0 and 1 by orogastric gavage. On day 6 post-infection, nitric oxide concentrations were measured in ex vivo biopsies sampled from the (A) colon, (B) ileum, and (C) mesenteric lymph nodes (MLN). Naive mice (non-infected without pretreatment) served as negative controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), significance levels (p values) determined by the Kruskal–Wallis test with Dunn’s post hoc test, and numbers of analyzed mice (in parentheses) from three experiments are given. The Grubb’s test was used to identify definite outliers.

Figure 7

Fecal microbiota during C. jejuni infection of hma IL-10^−/− mice with menthol pretreatment. Immediately before (i.e., day (d)0) and on d6 post C. jejuni infection, the fecal microbiota compositions were assessed in hma IL-10^−/− mice with menthol (MEN; white bars) or placebo (PLC; grey bars) prophylaxis by culture-independent methods. The (A) total eubacterial loads, (B) enterobacteria, (C) enterococci, (D) lactobacilli, (E) bifidobacteria, (F) Bacteroides/Prevotella species, (G) Clostridium coccoides and (H) Clostridium leptum groups, and (I) Mouse Intestinal Bacteroides are expressed as copies per ng DNA. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line in boxes), significance levels (p values) determined by the one-way ANOVA test with Tukey post hoc test (A) and the Kruskal–Wallis test with Dunn’s post hoc test (B–I), and numbers of analyzed mice (in parentheses) from three experiments are given.