biomolecules
Article
Menthol Pretreatment Alleviates Campylobacter jejuni-Induced
Enterocolitis in Human Gut Microbiota-Associated
IL-10−/− Mice
Markus M. Heimesaat * , Luis Q. Langfeld, Niklas Schabbel, Nizar W. Shayya, Soraya Mousavi and Stefan Bereswill
Citation: Heimesaat, M.M.; Langfeld,
L.Q.; Schabbel, N.; Shayya, N.W.;
Mousavi, S.; Bereswill, S. Menthol
Pretreatment Alleviates Campylobacter
jejuni-Induced Enterocolitis in Human
Gut Microbiota-Associated IL-10−/−
Mice. Biomolecules 2024, 14, 290.
https://doi.org/10.3390/
biom14030290
Academic Editor: Maria Beatrice
Morelli
Received: 23 January 2024
Revised: 21 February 2024
Accepted: 28 February 2024
Published: 29 February 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Biomolecules 2024, 14, 290. https://doi.org/10.3390/biom14030290
Gastrointestinal Microbiology Research Group, Institute of Microbiology, Infectious Diseases and Immunology,
Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu
Berlin, and Berlin Institute of Health, 12203 Berlin, Germany
* Correspondence: markus.heimesaat@charite.de; Tel.: +49-30-450524318
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: menthol; immune-modulatory properties; anti-oxidant effects; human gut microbiota-
associated IL-10−/− mice; campylobacteriosis model; host-pathogen interaction; Campylobacter jejuni;
placebo-controlled preclinical intervention study; pretreatment
1. Introduction
Campylobacter are microaerophilic and rod-shaped Gram-negative bacilli that typically
reside as commensal members of the intestinal tract in warm-blooded vertebrates [1].
Frequently colonized with Campylobacter jejuni, birds, in particular chickens and turkeys,
rarely display clinical signs [2–4]. During the livestock slaughter process, the derived meat
products might become contaminated with bacteria [5]. Upon human consumption of
undercooked or even raw contaminated meat products, unpasteurized milk or surface
water, C. jejuni infection can occur, leading to acute campylobacteriosis [6,7]. After a latency
of 2 to 5 days, infected individual presents symptoms of varying severity, including general
malaise, tenesmus, nausea, vomiting, and fever accompanied by enteritis with watery or
bloody diarrhea accompanied by mucous discharge [8,9]. The severity of the C. jejuni-
induced disease depends on both the arsenal of virulence factors of the enteropathogen and
the immune competence of infected individuals [10,11]. Following successful passage of
the stomach and duodenum, the motile C. jejuni traverse the viscous mucous barrier, reach
the subepithelial layers by invasion of epithelial cells, and initiate macrophage activation,
https://www.mdpi.com/journal/biomolecules
Biomolecules 2024, 14, 290 2 of 15

resulting in the recruitment of monocytes, neutrophilic granulocytes, and leukocytes to

the infected sites [12,13]. Furthermore, the endotoxin lipo-oligosacchride (LOS) leads to a
Toll-like receptor-4 (TLR-4)-dependent overstimulation of the host immune system [11].
Consequently, pro-inflammatory mediators aim to limit the infection but cause damage to
intestinal tissues and induce even systemic complications [11]. The oxidative stress results
in gut epithelial cell apoptosis, compromised epithelial barrier function, malabsorption and
diarrhea with electrolyte loss (“leaky gut”) [14–16]. Typically, the treatment of symptomatic
C. jejuni-infected individuals involves non-steroidal antiphlogistic compounds and the
substitution of fluids and electrolytes [9]. In rather critical cases, particularly in patients
with immune-compromising co-morbidities, however, antibiotics such as macrolides or
fluoroquinolones may be prescribed [9,17]. Generally, enteritis symptoms completely
resolve within two weeks post-infection (p.i.). However, on rare occasions, autoimmune
disorders might manifest with a latency of a few weeks to months p.i. [18]. These post-
infectious complications of C. jejuni infection can affect the joints (reactive arthritis), the
intestines (irritable bowel syndrome, inflammatory bowel diseases), or the nervous system
(Guillain–Barré syndrome) [19–21] and are known to depend on the sialylation status of the
C. jejuni-LOS [18,22]. Notably, there is a direct correlation between the risk of developing
such autoimmune diseases and the severity of the preceding enteritis [22]. In addition,
given the rising prevalence of multi-drug-resistant C. jejuni strains, which limits effective
antibacterial treatment of severely ill patients [17,23], it is important to identify non-toxic
compounds with potential anti-C. jejuni and anti-inflammatory properties that alleviate
the disease and, in turn, reduce the risk of post-infectious diseases. This urgency is even
further emphasized by the increasing global incidence of campylobacteriosis cases, which
represents the most commonly reported cause of bacterial enteritis, with unreported cases
far exceeding the reported ones [24]. In 2021, more than 127,000 campylobacteriosis cases
were confirmed in the European Union alone, for instance [25], and the associated expenses
for society, including the economies and health care systems, have been shown to be
significant [26–28].
Menthol, a monoterpene with a high abundance of essential oils originating from the
peppermint plant Mentha piperita L. (Lamiaceae), is widely employed as a flavoring agent and
a remedy in traditional folk medicine due to its various health-promoting properties [29].
In fact, menthol has been shown to exhibit potent anti-microbial, anti-inflammatory, anti-
oxidant, and anti-apoptotic effects [30–34], positioning the compound as a promising
candidate for combating C. jejuni-induced morbidities.
Therefore, we tested the potential of prophylactic menthol treatment in experimental
campylobacteriosis, focusing on its pathogen-reducing, immunomodulatory and conse-
quent disease-alleviating effects. To address this, we utilized human microbiota-associated
(hma) IL-10−/− mice. Given that the commensal gut microbiota of murine (as opposed
to human) origin protects against C. jejuni infection [35], we initially depleted the murine
gut microbiota by antibiotic treatment and subsequently introduced a complex human gut
microbiota instead by fecal microbiota transplantation (FMT). Interestingly, mice demon-
strate higher resistance to bacterial TLR-4 ligands, including lipo-polysaccharide (LPS)
and LOS [36]; however, the il10 gene deficiency, encoding the anti-inflammatory cytokine
IL-10, can overcome this LPS and LOS resistance [37]. In fact, microbiota-depleted wild-
type mice were shown not to develop overt C. jejuni-induced enteritis [35]. In contrast,
within less than a week, both microbiota-depleted and hma IL-10−/− mice succumbed
to C. jejuni-induced disease affecting not only the intestinal tract but also extra-intestinal
and systemic organs [38,39]. Hence, the hma IL-10−/− mouse model serves as a valuable
experimental tool to explore the effects of the compound of interest on the interactions
among (i) the enteropathogen (i.e., C. jejuni), (ii) the human gut microbial communities, and
(iii) the vertebrate host immunity during acute campylobacteriosis [39]. This was the major
driver behind subjecting hma IL-10−/− mice to peroral menthol treatment starting a week
before C. jejuni infections to monitor intestinal pathogen burdens, clinical, macroscopic,
Biomolecules 2024, 14, x FOR PEER REVIEW 3 of 16

Biomolecules 2024, 14, 290 3 of 15

macroscopic, and microscopic complications of infection, as well as pro-inflammatory im-

mune responses incomplications
and microscopic the intestinaloftract and beyond.
infection, as well as pro-inflammatory immune responses
in the intestinal tract and beyond.
2. Materials and Methods
2. Materials
2.1. and Methods
Mice, Microbiota Depletion
2.1. In
Mice,
the same unit Depletion
Microbiota of the Forschungseinrichtungen für Experimentelle Medizin (FEM,
In the same unit
Charité-University of the Berlin),
Medicine Forschungseinrichtungen
IL-10−/− mice (of thefür Experimentelle
C57BL/6 background) Medizin
were (FEM,
bred
Charité-University Medicine Berlin), −/− mice (of the C57BL/6 background) were
under specific pathogen-free (SPF) and IL-10
standardized conditions (i.e., 22–24 °C room tem-
bred under
perature, 55 ±specific pathogen-free
15% humidity, and a(SPF)
12 hand standardized
light/12 conditions
dark cycle). (i.e., 22–24 ◦ Csemi-
In an experimental room
temperature, 55 ± 15% humidity, and a 12 h light/12 dark cycle). In
barrier, mice were housed in cages with filter tops covering them and were allowed unre- an experimental
semi-barrier,
stricted access mice were housed
to autoclaved in cages
standard chowwith filterpellets:
(food tops covering them V1534-300,
sniff R/M-H, and were allowed
Sniff,
unrestricted access to autoclaved standard chow (food pellets: sniff
Soest, Germany). After placing 3-week-old littermate animals in sterile cages (two R/M-H, V1534-300,
to four
Sniff,per
mice Soest, Germany).
cage), After
ampicillin placing
and 3-week-old
sulbactam (2 g/Llittermate
and 1 g/L, animals in sterileDr.
respectively; cages (two to
Friedrich
four mice per cage), ampicillin and sulbactam (2 g/L and 1 g/L, respectively;
Eberth Arzneimittel, Ursensollen, Germany) were administered via the drinking water for Dr. Friedrich
Eberth
eight Arzneimittel,
weeks Ursensollen,
(ad libitum) Germany)
as stated earlier wereThe
[38,39]. administered
mice were via the drinking
handled water for
under rigorous
eight weeks (ad libitum) as stated earlier [38,39]. The mice were handled
aseptic conditions to prevent microbial contamination and ensure successful microbiota under rigorous
aseptic conditions to prevent microbial contamination and ensure successful
depletion. Two days before human FMT (day-16; Figure 1A), the antibiotics were replaced microbiota
depletion.
with Twowater
sterile tap days (ad
before human FMT (day-16; Figure 1A), the antibiotics were replaced
libitum).
with sterile tap water (ad libitum).

Figure 1. Timeline of experimental procedures and the microbiota composition of human fecal
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
transplants. (A) Human microbiota-associated (hma) IL-10−/− mice were generated by antibiotic gut
microbialdepletion
microbial depletion(with
(withampicillin
ampicillinplus
plussulbactam)
sulbactam)and andsubsequent
subsequenttriple
triplehuman
humanfecal
fecalmicrobiota
microbiota
transplantation (hFMT). Starting a week before oral Campylobacter jejuni strain 81–176
transplantation (hFMT). Starting a week before oral Campylobacter jejuni strain 81–176 infection, hma infection, hma
mice were subjected to menthol
mice were subjected to menthol treatmenttreatment via the drinking water and followed up until
drinking water and followed up until necropsy necropsy on
on day
day (d)(d) 6 post-infection.
6 post-infection. (B)(B)
GutGut microbial
microbial communities
communities were were quantitated
quantitated in human
in human fecalfecal trans-
transplants
plants
(n = 3(n
per= experiment)
3 per experiment) by culture-independent
by culture-independent (i.e., 16S(i.e., 16S rRNA-based
rRNA-based molecular) molecular)
analyses.analyses.
Box plots
Box plots (25th and 75th percentiles), whiskers (minimum and maximum
(25th and 75th percentiles), whiskers (minimum and maximum values), and medians values), and medians (redin
(red bar
bar in boxes) are given (pooled from three independent experiments). TEL, total
boxes) are given (pooled from three independent experiments). TEL, total eubacterial loads; EB, eubacterial loads;
EB, enterobacteria; EC, enterococci; LB, lactobacilli; BB, bifidobacteria; BP, Bacteroides/Prevotella spe-
enterobacteria; EC, enterococci; LB, lactobacilli; BB, bifidobacteria; BP, Bacteroides/Prevotella species;
cies; CC, Clostridium coccoides group; CL, Clostridium leptum (CL) group; MIB, Mouse Intestinal Bac-
CC, Clostridium coccoides group; CL, Clostridium leptum (CL) group; MIB, Mouse Intestinal Bacteroides.
teroides.
Biomolecules 2024, 14, 290 4 of 15

2.2. Human Fecal Microbiota Transplantation

Fresh fecal samples were voluntarily donated by healthy subjects, suspended in sterile
phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA), aliquoted,
and preserved at −80 ◦ C. Prior to the human FMT, fecal aliquots were thawed and pooled
immediately. To establish hma mice, human fecal microbiota transplantation (hFMT) was
performed over three consecutive days (days-14, -13, and -12; Figure 1A). Therefore, mice
were orally subjected to 0.3 mL of the human fecal donor suspension via orogastric gavage,
as outlined earlier [35].

2.3. Prophylactic Regimen

Commencing seven days prior to C. jejuni infection (day-7; Figure 1A), mice were
treated with synthetic menthol (purchased from Sigma-Alderich, München, Germany).
Menthol was administered to mice by autoclaved tap water (ad libitum). The final menthol
concentration in the drinking solution was 500 mg/L, which meant that 100 mg of treatment
dosage per kilogram of body weight was applied each day. Placebo-control mice received
autoclaved tap water.

2.4. Campylobacter jejuni Infection

Bacterial cryo stocks of C. jejuni strain 81–176 were preserved at −80 ◦ C. Following
thawing, the bacteria were streaked out and cultured on selective karmali agar plates
(obtained from Oxoid, Wesel, Germany) under microaerophilic conditions for 48 h at
37 ◦ C, as previously reported [35]. The bacteria were then resuspended in sterile PBS
(Thermo Fisher Scientific, Waltham, MA, USA), and the hma IL-10−/− mice (female and
male 3-month-old littermates) were infected with 109 colony-forming units (CFU) of the
enteropathogen on days 0 and 1 via orogastric gavage (Figure 1A).

2.5. Summary of Mouse Cohorts

Naive group (6 female/6 male): human FMT, non-treated, non-infected;
Placebo group (9 female/8 male): human FMT, non-treated, infected;
Menthol group (11 female/10 male): human FMT, verum-treated, infected.

2.6. Measurement of Gastrointestinal C. jejuni Loads

To assess gastrointestinal pathogen levels, the counts of live C. jejuni bacteria were
monitored daily in fecal samples and additionally, upon necropsy in intraluminal gas-
trointestinal biopsies taken from the stomach, duodenum, ileum, and colon lumen that
were homogenized in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). C. jejuni
quantification involved counting CFU after cultivating serial dilutions of intestinal samples
on karmali agar for minimum 48 h at 37 ◦ C under microaerophilic conditions as previously
described in details [35]. The detection limit for viable pathogens was in the range of
100 CFU of C. jejuni bacteria per gram fecal material.

2.7. Fecal Microbiota Composition

For the culture-independent molecular analysis of the fecal microbiota composition,
genomic DNA was isolated from the human fecal transplants and the colonic luminal
murine samples first. In a nutshell, the Quant-iT PicoGreen reagent (Invitrogen, Paisley,
UK) was used to quantify the DNA content, which was then adjusted to 1 ng of DNA
per µL. Using species-, genera-, or group-specific 16S rRNA gene primers (Tib MolBiol,
Berlin, Germany), the most prominent bacterial groups that were abundant in the intestinal
microbiota of the hma mice were evaluated using quantitative real-time polymerase chain
reaction (qRT-PCR), as previously described [40].
Biomolecules 2024, 14, 290 5 of 15

2.8. Clinical Conditions of Mice

We used a standardized cumulative clinical score on a daily basis to record the clin-
ical status of mice before and after enteropathogen application as reported earlier [41]
(Supplementary Table S1).

2.9. Sampling Procedures

Following C. jejuni infection, mice were sacrificed on day 6 p.i. by inhalation of
carbon dioxide. Luminal gastrointestinal samples were obtained under sterile conditions
from the stomach, duodenum, ileum, and colon, in addition to ex vivo biopsies from
the mesenteric lymph nodes (MLN) and the distal colon, for subsequent microbiological,
immunohistopathological, and immunological measurements. Cardiac blood was taken to
generate serum samples.

2.10. Histopathology
Colon explants were promptly fixed in 5% formalin, embedded in paraffin, and 5-µm-
sections were subjected to hematoxylin and eosin (H&E) staining. To evaluate the severity
of histological alterations of the colonic mucosa, the tissue samples were assessed using
light microscopy (100-times magnification), and the histopathological sequelae of infection
were quantified based on an established scoring system [42] (Supplementary Table S2).

2.11. Quantitative In Situ Immunohistochemistry

As previously outlined in detail [43], quantitative in situ immunohistochemistry inves-
tigations were conducted on large intestinal explants that had been promptly subjected to
tissue fixation in 5% formalin paraffin embedding. Subsequently, 5-µm-sections of paraffin-
embedded tissues were stained with distinct primary antibodies (Supplementary Table S3).
A blinded and independent researcher used light microscopy (400-times magnification)
to enumerate the positively stained cells. For each sample, the mean count of specifically
stained cells was determined across a minimum of six high-power fields (HPFs, 0.287 mm2 ,
400-times magnification).

2.12. Pro-Inflammatory Mediators

Terminal ileal and colonic explants (approximately 1 cm2 each) were longitudinally
cut and the tissues cleansed in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA).
Then, in addition to the intestinal samples, ex vivo biopsies derived from the MLN
(3 nodes) were transferred to 24-flat-bottom well culture plates (Thermo Fisher Scien-
tific, Waltham, MA, USA) containing 500 µL serum-free RPMI 1640 medium (Thermo
Fisher Scientific, Waltham, MA, USA), penicillin (100 µg/mL; Biochrom, Berlin, Germany)
and streptomycin (100 µg/mL; Biochrom, Berlin, Germany). Following an 18 h incubation
period at 37 ◦ C, culture supernatants were tested for monocyte chemoattractant protein-1
(MCP-1) by the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Heidelberg,
Germany) in a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide (NO) levels
were determined by the Griess reaction [44].

2.13. Statistics
GraphPad Prism (version 9; San Diego, CA, USA) was used to compute medians and
significance levels following the pooling of data from three independent experiments. The
normal distribution of the data was evaluated using the Anderson-Darling test. Pairwise
comparisons of regularly distributed and non-normally distributed data were performed
using the Student’s t-test and the Mann–Whitney test, respectively. The one-way ANOVA
with Tukey post hoc test (for regularly distributed data) and the Kruskal–Wallis test with
Dunn’s post hoc test (for non-normally distributed data) were used for multiple compar-
isons. Significant two-sided probability (p) values were defined as <0.05. Significant outliers
were determined by using the Grubb’s test (α = 0.001).
Biomolecules 2024, 14, x FOR PEER REVIEW 6 of 16

Biomolecules 2024, 14, 290 comparisons. Significant two-sided probability (p) values were defined as <0.05. Signifi-
6 of 15
cant outliers were determined by using the Grubb’s test (α = 0.001).

3. Results
3. Results
3.1. Clinical Conditions over Time following C. jejuni Infection of hma IL-10−/− Mice with Men-
3.1. Clinical Conditions over Time following C. jejuni Infection of hma IL-10−/− Mice with
thol Pretreatment
Menthol Pretreatment
First,
First,we
wetested
testedwhether
whethermenthol
mentholpretreatment
pretreatmentwould
wouldimprove
improvethe theclinical
clinicalconditions
conditions
in
inhma
hmaIL10
IL10−mice
−/− following
/− mice C. jejuni
following infection.
C. jejuni Our daily
infection. assessments
Our daily revealed
assessments that mice
revealed that
from the menthol and placebo cohorts presented with comparable campylobacteriosis-
mice from the menthol and placebo cohorts presented with comparable campylobacteriosis-
related
relatedconditions
conditionsat atindividual
individual days
days p.i.
p.i.(not
(notsignificant
significant(n.s.);
(n.s.);Figure
Figure 2).
2).This
Thiswas
wasalso
alsothe
the
case
case when focusing on clinical scores specifying wasting symptoms, diarrhea, andfecal
when focusing on clinical scores specifying wasting symptoms, diarrhea, and fecal
flood
flood(n.s.;
(n.s.;Supplementary
SupplementaryFigure FigureS1).
S1).Notably,
Notably,thethestandard
standarddeviations
deviationsof ofthe
therespective
respective
clinical
clinicalscores
scoreswerewerevery
veryhigh
highwithin
withinboth
bothinfected
infectedgroups.
groups.Hence,
Hence,menthol
mentholpretreatment
pretreatment
did
didnot
notalleviate
alleviatecampylobacteriosis
campylobacteriosissymptoms.
symptoms.

Clinicalconditions
Figure2.2.Clinical conditions over time following C. jejuni infection of hma IL-10 −/− mice with
Figure over time following C. jejuni infection of hma IL-10 −/− mice with men-
thol pretreatment.
menthol HmaHma
pretreatment. micemice
werewere
orally pretreated
orally with
pretreated menthol
with menthol(MEN,
(MEN, white
whitebars)
bars)ororplacebo
placebo
(PLC,
(PLC,grey
greybars)
bars)and
andinfected
infectedwith
withC.C.jejuni
jejunion
onday
day00(d0)
(d0)and
andd1d1by
by orogastric
orogastric gavage.
gavage.The Theclinical
clinical
conditions
conditionsofofmice
micewere
weresurveyed
surveyeddaily
dailyuntil
untilnecropsy
necropsyby byusing
usingaaclinical
clinicalcampylobacteriosis
campylobacteriosisscorescore
recording
recordingwasting,
wasting,diarrhea,
diarrhea,and
andfecal
fecalblood
blood(see
(seemethods).
methods).Box Boxplots
plots(25th
(25thand
and75th
75thpercentiles),
percentiles),
whiskers (minimum and maximum values), medians (red line in boxes), and numbers of analyzed
whiskers (minimum and maximum values), medians (red line in boxes), and numbers of analyzed
mice (in parentheses) from three experiments are given.
mice (in parentheses) from three experiments are given.

3.2.
3.2.Pathogen
PathogenNumbers
Numbers in
in Gastrointestinal
Gastrointestinal Luminal Samples from
Luminal Samples from C.
C. jejuni
jejuniInfected
Infectedhma IL-10−−/−
hmaIL-10 /−
Micewith
Mice withMenthol
MentholPretreatment
Pretreatment
Next,we
Next, weaddressed
addressedwhether
whetheroral
oralmenthol
mentholwould
wouldinterfere
interferewith
withintestinal
intestinalC.C. jejuni
jejuni
colonization.Results
colonization. Resultsfromfromdaily
dailycultural
culturalanalyses
analysesindicated
indicatedcomparably
comparablyhigh highC. C. jejuni
jejuni
numbers in fecal samples over time following infection of mice from both
numbers in fecal samples over time following infection of mice from both cohorts (n.s.; cohorts (n.s.;
SupplementaryFigure
Supplementary FigureS2).
S2).On
Onthetheday
dayofofnecropsy
necropsy(i.e.,
(i.e.,day
day66p.i.),
p.i.),luminal
luminalC.C.jejuni
jejuniloads
loads
didnot
did notdiffer
differin
in the
the stomach,
stomach, duodenum,
duodenum,and andileum
ileumof of menthol
menthol andand placebo-pretreated
placebo-pretreated
mice(n.s.),
mice (n.s.),whereas
whereasless lessthan
thanone
one order
order of
of magnitude
magnitude lowerlower median
median pathogen
pathogen numbers
numbers
were detected in the colonic lumen of the former vs. the latter (p < 0.01;
were detected in the colonic lumen of the former versus the latter (p < 0.01; Figure Figure 3). Hence,
3).
menthol pretreatment slightly but significantly lowered colonic C. jejuni numbers.
Hence, menthol pretreatment slightly but significantly lowered colonic C. jejuni numbers.

3.3. Inflammatory Changes in the Colon of C. jejuni Infected hma IL-10−/− Mice with
Menthol Pretreatment
Further, we tested whether menthol pretreatment could mitigate C. jejuni-induced
macroscopic and microscopic inflammatory changes. As a macroscopic inflammatory
parameter, we recorded the absolute lengths of the large intestines upon necropsy. On
day 6 p.i., we found shorter colons in mice from the placebo (p < 0.01) as opposed to the
menthol cohort (n.s.) when compared to naive (i.e., non-treated and non-infected) control
Biomolecules 2024, 14, 290 7 of 15

animals (Figure 4A), indicative of mitigated macroscopic inflammatory signs of C. jejuni-

infection. When addressing the severity of microscopic inflammatory sequelae in the colon,
we revealed comparable histopathological scores in mice from both treatment cohorts but
observed a trend towards lower values in the menthol group as compared to the placebo
group that did not reach statistical significance due to high standard deviations in the former
(Figure 4B). We further investigated the extent of C. jejuni-induced intestinal apoptosis and
found increased numbers of apoptotic cells in colonic epithelia in both infected groups
Biomolecules 2024, 14, x FOR PEER REVIEW 7 of 16
(p < 0.01–0.001), but considerably fewer apoptotic cell counts in menthol as compared to
placebo-treated mice on day 6 p.i. (p < 0.001; Figure 4C). Hence, menthol pretreatment
alleviated both macroscopic and microscopic complications of the C. jejuni infection.

Pathogen numbers C. jejuni −/−

Figure3.3.Pathogen
Figure numbers in in gastrointestinal
gastrointestinal luminal
luminal samples
samples from
from C. jejuni infected
infected hmahma IL-10
IL-10 −/−

micewith
mice withmenthol
menthol pretreatment.
pretreatment. Hma Hma
micemice
werewere orally
orally pretreated
pretreated with with menthol
menthol (MEN, (MEN,
white white
bars) bars)
ororplacebo
placebo(PLC,
(PLC, grey
grey bars)
bars) and and infected
infected with
with C. jejuni
C. jejuni on days
on days 0 and0 and
1 by 1orogastric
by orogastric gavage.
gavage. The The
luminal
luminalC.C.jejuni numbers
jejuni numberswere
weredetermined
determinedin gastrointestinal samples
in gastrointestinal takentaken
samples on dayon6day
post-infection
6 post-infection
by
byculture.
culture.Box
Boxplots (25th
plots and
(25th and 75th percentiles),
75th whiskers
percentiles), whiskers(minimum
(minimum andand
maximum
maximum values), medi-
values), medians
ans (red line in boxes), significance levels (p values) determined by the Mann–Whitney test, and
(red
Biomolecules 2024, 14, x FOR PEER REVIEW line in boxes), significance levels (p values) determined by the Mann–Whitney test, and numbers
8 of 16
numbers of analyzed mice (in parentheses) from three experiments are given. CFU, colony-forming
of analyzed mice (in parentheses) from three experiments are given. CFU, colony-forming units.
units.

3.3. Inflammatory Changes in the Colon of C. jejuni Infected hma IL-10−/− Mice with Menthol
Pretreatment
Further, we tested whether menthol pretreatment could mitigate C. jejuni-induced
macroscopic and microscopic inflammatory changes. As a macroscopic inflammatory pa-
rameter, we recorded the absolute lengths of the large intestines upon necropsy. On day
6 p.i., we found shorter colons in mice from the placebo (p < 0.01) as opposed to the men-
thol cohort (n.s.) when compared to naive (i.e., non-treated and non-infected) control an-
imals (Figure 4A), indicative of mitigated macroscopic inflammatory signs of C. jejuni-
infection. When addressing the severity of microscopic inflammatory sequelae in the co-
lon, we revealed comparable histopathological scores in mice from both treatment cohorts
but observed a trend towards lower values in the menthol group as compared to the pla-
−/− mice with menthol
Figure
cebo
Figure Inflammatory
4.4.Inflammatory
group that did not changes in the
reachinstatistical
changes the colon
colon C.C.
of of jejuni
significance
jejuni infected
due
infectedto hmahma
high IL-10
standard
IL-10 −/− micedeviations
with menthol in
the former (Figure 4B). We − / − further investigated the extent of C. jejuni-induced
pretreatment.
pretreatment. Hma
Hma IL-10IL-10−/− mice mice were
were orally orally pretreated
pretreated with with menthol
menthol (MEN, white bars) intestinal
(MEN, white bars) or
or placebo placebo
apoptosis
(PLC, greyand
(PLC,grey bars) found
bars) and increased
andinfected
infected numbers
C. C.
with
with jejuni of on
jejuni
on apoptotic
daysdays
0 and cells
0 and in
bycolonic
1 by1orogastric epithelia
orogastric Onin
gavage.
gavage. both
On
day in-6 post-
6 day
post-
fected groups
infection,
infection, (A)
(A)the(pcolonic
the <colonic
0.01–0.001),
lengths
lengths but
were
wereconsiderably
measured.
measured. fewer apoptotic
Furthermore,
Furthermore, (B) the cell counts in menthol
(B)histopathological as and
scoresscores
the histopathological and
(C)
(C)numbers
compared
numbers toofofapoptotic
apoptotic epithelial
placebo-treated cells
mice
epithelial positive
on
cells day 6for
positive cleaved
p.i.
for (p caspase-3
< 0.001;
cleaved (Casp3
Figure
caspase-3
+) were
4C).
(Casp3 determined
Hence,
+ ) were menthol in
determined in
colonic paraffin
pretreatment sections.
alleviated Naive mice (non-infected without pretreatment) served as negative con-
colonic paraffin sections. both
Naivemacroscopic and microscopic
mice (non-infected complications
without pretreatment) servedofasthe C. jejuni
negative controls.
trols. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians
infection.
Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (red line
(red line in boxes), significance levels (p values) determined by the one-way ANOVA test with Tukey
in boxes),
post significance
hoc test levels (p values)
(A,C) and Kruskal–Wallis determined
test by post
with Dunn’s the one-way ANOVA
hoc test (B), test with
and numbers ofTukey post hoc
analyzed
test (A,C)
mice and Kruskal–Wallis
(in parentheses) from three test with Dunn’s
experiments post hoc
are given. test
HPF, (B),
high and numbers
power field. of analyzed mice (in
parentheses) from three experiments are given. HPF, high power field.
3.4. Colonic Immune Cells in C. jejuni Infected hma IL-10−/− Mice with Menthol Pretreatment
Potential menthol-dependent immune cell responses upon C. jejuni infection were
investigated by in situ immunohistochemical analyses in colonic paraffin sections. Results
demonstrated that C. jejuni infection was accompanied by increases in innate immune cell
populations such as F4/80+ macrophages and monocytes (p < 0.05–0.001; Figure 5A) and
 Biomolecules 2024, 14, 290 8 of 15

3.4. Colonic Immune Cells in C. jejuni Infected hma IL-10−/− Mice with Menthol Pretreatment
Potential menthol-dependent immune cell responses upon C. jejuni infection were
investigated by in situ immunohistochemical analyses in colonic paraffin sections. Results
demonstrated that C. jejuni infection was accompanied by increases in innate immune
cell populations such as F4/80+ macrophages and monocytes (p < 0.05–0.001; Figure 5A)
and MPO7+ neutrophilic granulocytes (p < 0.001; Figure 5B) in the colonic mucosa and
lamina propria. The increase in the former was, however, less pronounced in menthol as
compared to placebo-pretreated mice (p < 0.001; Figure 5A). In the case of the neutrophils,
at least a trend towards fewer cell counts could be observed upon menthol challenge on
day 6 p.i. (n.s. vs. placebo; Figure 5B). When assessing C. jejuni-induced adaptive immune
cell responses, we detected increased colonic numbers of CD3+ T lymphocytes in placebo
(p < 0.01 vs. naive) but not in menthol-pretreated mice (p < 0.05 vs. placebo; n.s. vs. naive)
on day 6 p.i. (Figure 5C). Like neutrophils, C. jejuni-infected mice displayed elevated
B220+ B lymphocyte numbers in their colonic mucosa and lamina propria (p < 0.05–0.001;
Figure 5D), whereas a trend towards lower counts was detected in menthol-treated mice
Biomolecules 2024, 14, x FOR PEER REVIEW
as compared to placebo-treated mice on day 6 p.i. (n.s.; Figure 5D). Hence, menthol 9 of 16
pretreatment resulted in attenuated recruitment of macrophages/monocytes and of T
lymphocytes into the colon upon C. jejuni infection.

Figure 5. Colonic immune cells in C. jejuni infected hma IL-10−/− mice with menthol pretreatment.
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
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,
bars) and infected with C. jejuni on days 0 and 1 by orogastric gavage. On day 6 post-infection,
numbersofof(A)
numbers (A)macrophages
macrophagesand andmonocytes (F4/80+),+ ),(B)
monocytes (F4/80 (B)neutrophils (MPO7+),+ ),(C)
neutrophils(MPO7 (C)TTlymphocytes
lymphocytes
(CD3 +
+), ), and(D)
(D)BBlymphocytes
lymphocytes (B220 +
(B220+) )were
were determined
determined in in the
(CD3 and the colonic
colonic lamina
laminapropria
propria(immunohis-
(immuno-
tochemically stained
histochemically stainedparaffin
paraffinsections).
sections).Naive
Naivemice
mice(non-infected
(non-infectedwithout
withoutpretreatment)
pretreatment)servedservedas
asnegative
negativecontrols.
controls.Box
Boxplots
plots(25th
(25thand
and 75th
75th percentiles),
percentiles), whiskers
whiskers (minimum
(minimum andand maximum
maximum val-
values),
ues), medians
medians (red(red
line line in boxes),
in boxes), significance
significance levels
levels (p values)
(p values) determined
determined by the
by the one-way
one-way ANOVA
ANOVA test
test
withwith
Tukey Tukey post
post hochoc
testtest
(A)(A)
andand Kruskal–Wallis
Kruskal–Wallis testtest
with with Dunn’s
Dunn’s post
post hochoc test
test (B–D),
(B–D), andand num-
numbers
bers of analyzed mice (in parentheses) from three experiments are given. HPF, high power field.
of analyzed mice (in parentheses) from three experiments are given. HPF, high power field.

3.5. Intestinal Proinflammatory Mediator Secretion in C. jejuni Infected hma IL-10−/− Mice with
Menthol Pretreatment
Next, we determined NO concentrations in distinct intestinal organs. Whereas en-
hanced NO secretion could be detected in ex vivo biopsies derived from the colon, ileum,
and MLN of placebo control animals on day 6 p.i. (p < 0.005 versus naive), menthol-pre-
Biomolecules 2024, 14, 290 9 of 15

3.5. Intestinal Proinflammatory Mediator Secretion in C. jejuni Infected hma IL-10−/− Mice with
Menthol Pretreatment
Next, we determined NO concentrations in distinct intestinal organs. Whereas en-
hanced NO secretion could be detected in ex vivo biopsies derived from the colon, ileum,
and MLN of placebo control animals on day 6 p.i. (p < 0.005 vs. naive), menthol-pretreated
mice displayed basal values (n.s. vs. naive; Figure 6), indicative of diminished C. jejuni-
induced oxidative stress in respective intestinal compartments. Furthermore, C. jejuni
infection resulted in increased colonic secretion of the pro-inflammatory chemokine MCP-1
in mice from the placebo group (p < 0.05 vs. naive), but not in the menthol cohort (n.s.
vs. naive; Supplementary Figure S3). Hence, menthol pretreatment resulted in attenuated
Biomolecules 2024, 14, x FOR PEER REVIEW 1
C. jejuni-induced intestinal secretion of pro-inflammatory mediators, thereby reducing
oxidative stress in the intestinal organs.

Figure 6. Intestinal nitric oxide secretion in C. jejuni infected hma IL-10−/− mice with−/−menthol
Figure 6. Intestinal nitric oxide secretion in C. jejuni infected hma IL-10 mice with menth
pretreatment. Hma IL-10−/− mice were−/−orally pretreated with menthol (MEN, white bars) or placebo
treatment. Hma IL-10 mice were orally pretreated with menthol (MEN, white bars) or p
(PLC, grey bars)(PLC,
and infected with
grey bars) C. infected
and jejuni onwith
daysC.0 and
jejuni1 on
by days
orogastric
0 andgavage. On day gavage.
1 by orogastric 6 post- On day
infection, nitric oxide concentrations
infection, nitric oxidewere measured inwere
concentrations ex vivo biopsies
measured in sampled from thesampled
ex vivo biopsies (A) colon,
from the (A
(B) ileum, and (C) mesenteric
(B) ileum, andlymph nodes (MLN).
(C) mesenteric lymph Naive
nodes mice (non-infected
(MLN). Naive mice without pretreatment)
(non-infected without pretrea
served as negative controls.
served Box plotscontrols.
as negative (25th andBox 75th percentiles),
plots (25th andwhiskers (minimumwhiskers
75th percentiles), and maximum
(minimum and
values), mediansmum values),
(red line medians
in boxes), (red line levels
significance in boxes), significance
(p values) levels by
determined (p values) determined by the K
the Kruskal–Wallis
test with Dunn’s Wallis
post hoctest with
test, andDunn’s
numbers post hoc test, mice
of analyzed and numbers of analyzed
(in parentheses) miceexperiments
from three (in parentheses) from
experiments are given. The Grubb’s test
are given. The Grubb’s test was used to identify definite outliers. was used to identify definite outliers.

3.6. Fecal Microbiota during C. jejuni Infection −/− Mice with Menthol
3.6. Fecal Microbiota during C.ofjejuni
hma IL-10
Infection of hma IL-10−/− MicePretreatment
with Menthol Pretrea
Furthermore, we tested for we
Furthermore, fecal microbiota
tested for fecalchanges
microbiotaduring C. jejuni
changes during infection
C. jejuni in
infection in
menthol-treatedthol-treated
mice. Our quantitative culture-independent
mice. Our quantitative analyses revealed
culture-independent analysescomparable
revealed compara
fecal microbiotacalcompositions in both cohortsinon
microbiota compositions daycohorts
both 0, and hence,
on dayimmediately
0, and hence,before oral
immediately befo
C. jejuni challenge of mice
C. jejuni (n.s. menthol
challenge vs. placebo;
of mice (n.s. mentholFigure
versus 7). In theFigure
placebo; course7).ofInC.the
jejuni
course of C
infection, total infection,
eubacterial loads
total and geneloads
eubacterial numbers
and of Bacteroides/Prevotella
gene species and of species
numbers of Bacteroides/Prevotella
the Clostridiumthecoccoides group decreased irrespective of the pretreatment (p
Clostridium coccoides group decreased irrespective of the pretreatment < 0.05–0.001,(p < 0.05–
day 6 vs. day 0;day
Figure 7A,F,G). As opposed to menthol-pretreated mice,
6 versus day 0; Figure 7A,F,G). As opposed to menthol-pretreated the gene copies of the gen
mice,
< 0.01;
bifidobacteria (pies Figure 7E) and
of bifidobacteria (p of the Clostridium
< 0.01; Figure 7E)leptum group
and of (p < 0.01; Figure
the Clostridium leptum 7H)group (p
were lower in fecal
Figuresamples taken
7H) were frominplacebo
lower counterparts
fecal samples taken on dayplacebo
from 6 p.i. ifcounterparts
compared to on day 6
day 0. Hence, oral menthol
compared pretreatment
to day 0. Hence, had oral (if at all) pretreatment
menthol only minor effects
had (ifonatthe
all)human
only minor effe
fecal gut microbiota composition during C. jejuni infection of hma IL-10 −/− mice.
the human fecal gut microbiota composition during C. jejuni infection of hma IL-10−/
 Biomolecules 2024, 14, x FOR PEER REVIEW 1
Biomolecules 2024, 14, 290 10 of 15

Figure 7. Fecal microbiota during C. jejuni infection of hma IL-10−/− mice with menthol pretreatment.
Figure 7. Fecal microbiota during C. jejuni infection of hma IL-10−/− mice with menthol pretrea
Immediately before (i.e., day (d)0) and on d6 post C. jejuni infection, the fecal microbiota compositions
Immediately before (i.e., day (d)0) and on d6 post C. jejuni infection, the fecal microbiota co
were assessed intions IL-10−
hma were /− mice with menthol (MEN; white bars) or placebo (PLC; grey bars)
assessed in hma IL-10−/− mice with menthol (MEN; white bars) or placebo (PLC
prophylaxis by culture-independent methods. The (A) total
bars) prophylaxis by culture-independent eubacterial
methods. loads,
The (A) total(B) enterobacteria,
eubacterial loads, (B) entero
(C) enterococci, (D) lactobacilli,
ria, (C) (E) bifidobacteria,
enterococci, (E)Bacteroides/Prevotella
(D) lactobacilli,(F) species, (G) Clostridium
bifidobacteria, (F) Bacteroides/Prevotella species, (G) C
coccoides and (H)ium
Clostridium leptum
coccoides and (H)groups, and leptum
Clostridium (I) Mouse Intestinal
groups, Bacteroides
and (I) are expressed
Mouse Intestinal as are exp
Bacteroides
as copies
copies per ng DNA. per(25th
Box plots ng DNA. Boxpercentiles),
and 75th plots (25th whiskers
and 75th (minimum
percentiles), whiskers
and maximum (minimum
values), and max
medians (red linevalues), medians
in boxes), (red line
significance in (p
levels boxes),
values)significance
determinedlevels
by the(pone-way
values) ANOVA
determinedtestby the on
ANOVA
with Tukey post hoc test
test (A) with
and theTukey post hoc test
Kruskal–Wallis test(A)
withand the Kruskal–Wallis
Dunn’s test with
post hoc test (B–I), Dunn’s post hoc t
and numbers
I), and numbers of analyzed mice (in parentheses) from three experiments are given.
of analyzed mice (in parentheses) from three experiments are given.

4. Discussion 4. Discussion
In our present
In our present preclinical preclinical placebo-controlled
placebo-controlled intervention study, intervention
we tested thestudy, we tested th
disease-
ease-mitigating,
mitigating, anti-pathogenic, anti-pathogenic, anti-oxidant
anti-inflammatory, anti-inflammatory, anti-oxidanteffects
and anti-apoptotic and anti-apopto
of
oral menthol in fects
acuteof experimental
oral menthol in acute experimental
campylobacteriosis hma IL-10−employing
campylobacteriosis
employing /− mice. hma I

At first, it was mice.

ratherAtdisappointing that menthol
first, it was rather pretreatment
disappointing did not
that menthol significantly
pretreatment didim-
not signifi
prove overt C. jejuni-induced
improve overtclinical signs such as
C. jejuni-induced wasting
clinical symptoms
signs and bloody
such as wasting diarrheaand bloo
symptoms
(Figure 2). Onearrhea
needs to take into
(Figure consideration,
2). One however,
needs to take that the severity
into consideration, of the observed
however, that the severity
symptoms ranged observed symptoms ranged from almost no observable clinical signs at in
from almost no observable clinical signs at all to full-blown disease all to full-
both cohorts. In our previous
disease in vivo study
in both cohorts. In ouremploying
previous ina vivo
different
studymurine campylobacte-
employing a different murine
riosis model, we found that oral
pylobacteriosis menthol
model, of the same
we found concentration
that oral menthol oftherapeutically given
the same concentration thera
to secondary abiotic IL-10−
cally given
/− mice from day 2 until−/−
to secondary abiotic IL-10 daymice6 p.i.
fromresulted
day 2 in an day
until improved
6 p.i. resulted
clinical outcome [45].
improved clinical outcome [45].
In our actual trial, oral
In our actualmenthol
trial,pretreatment
oral mentholcould prevent inflammation-induced
pretreatment could prevent inflammation-in
colonic shrinkage, whereas placebo-treated mice displayed
colonic shrinkage, whereas placebo-treated mice shorter
displayedcolons C. je- upon C.
uponcolons
shorter
juni infection ifinfection
compared to untreated and non-infected controls (Figure
if compared to untreated and non-infected controls (Figure 4A). The anti-4A). The
Biomolecules 2024, 14, 290 11 of 15

inflammatory effects of exogenous menthol were also evident on the microscopic level,
given that treated animals displayed diminished pathogen-induced colonic epithelial apop-
tosis (Figure 4C). In support, previous in vivo studies reported potent anti-apoptotic effects
upon menthol treatment, given decreases in pro-apoptotic molecules such as cleaved
caspase-3 and Bax protein and/or increases in anti-apoptotic molecules including Bcl-2
and HSP-70 [32,33,46,47]. The dampened apoptotic responses upon C. jejuni infection were
accompanied by lower numbers of macrophages and monocytes in the colonic mucosa
in menthol-pretreated mice on day 6 p.i. (Figure 5A). In support, menthol application
could decrease infiltration of infected brain tissues with respective innate immune cell
subsets during brain ischemia [48]. Furthermore, C. jejuni-induced increases in colonic
T lymphocytes could be reversed to basal values in menthol-treated mice (Figure 5C). In
support, a recent in vitro study revealed that menthol inhibited lymphocytic proliferation in
a dose-dependent manner and reduced the numbers of T cells expressing pro-inflammatory
cytokines [49]. Moreover, in our study, oral menthol could effectively reduce pathogen-
induced oxidative stress in different intestinal compartments, as shown by NO concentra-
tions measured in the colon, ileum, and MLN of treated mice on day 6 p.i. that did not differ
from those obtained from naive mice (Figure 6). In parallel, enhanced colonic secretion of
the pro-inflammatory chemokine MCP-1 secretion could be assessed in the infected placebo
as opposed to the verum cohort upon necropsy (Supplementary Figure S3).
The potent anti-oxidant and anti-inflammatory properties of exogenous menthol were
shown in vitro to inhibit NO and pro-inflammatory mediator secretion in LPS-stimulated
monocytes [50,51]. Furthermore, in vivo studies revealed that menthol mitigated acute
intestinal morbidities. Intraperitoneal injection of synthetic menthol before colitis induction,
for instance, could exert potent anti-colitogenic effects in rats [52], which also held true for
an improved clinical outcome and attenuated macroscopic and microscopic disease due to
diminished oxidative stress and inflammation in rats suffering from acute colitis [53].
When addressing the anti-pathogenic effects of the compounds, we found less than one
order of magnitude lower C. jejuni numbers in the colon lumen of menthol as compared
to placebo treated mice on day 6 p.i. (Figure 3). It is, however, questionable whether
the (statistically significant) slightly lower pathogen burden might explain the observed
biological effects. In our previous study, we did not observe significant differences in
colonic C. jejuni counts in menthol- vs. placebo-treated mice with acute campylobacteriosis.
In that study, however, we applied a therapeutic regimen to secondary abiotic IL-10−/−
mice from day 2 until day 6 p.i. only [45]. Furthermore, the menthol concentration in
the drinking solutions given in our previous and actual studies (i.e., 500 mg/L) was far
below the minimum inhibitory concentration (MIC) of the compound measured against
the applied C. jejuni 81–176 strain (16,000 mg/L). Anyhow, it is much more likely that
the mitigated enterocolitis following menthol pretreatment of C. jejuni-infected mice was
due to immune-modulatory, including anti-inflammatory, anti-oxidant, and anti-apoptotic
effects, as, in support, had been reported in a murine gastritis model earlier [32].
Furthermore, we surveyed the impact of menthol pretreatment on influencing the
(human) commensal gut microbiota composition following human FMT but found com-
parable fecal microbiota conditions on day 0 (i.e., immediately before C. jejuni infection)
(Figure 7). This result points towards comparably effective engraftments of fecal human
transplants and speaks against the significant microbiota-shifting effects of exogenous
menthol that had already been applied for one week by that time point. In addition, loads
of human bacterial communities were comparable in fecal samples derived at the end of
the experiment on day 6 p.i. (Figure 7). When focusing on microbiota shifts during C. jejuni
infection within respective treatment cohorts (i.e., changes between day 0 and day 6 p.i.),
we found statistically significant decreases in fecal gene numbers of potentially probiotic
bifidobacteria and Clostridium leptum in the placebo, but only a trend towards lower counts
of respective phyla in the menthol cohort (n.s.; Figure 7). Given these rather marginal
differences, one should not overemphasize these observations.
Biomolecules 2024, 14, 290 12 of 15

Menthol is generally considered a safe compound by the United States Food and Drug
Administration and the Flavoring Extract Manufacturer’s Association, and only very rarely
have intoxications resulting in dizziness, agitation, hallucination, convulsion, and coma
been reported [54,55]. A medical integrative international organization (Natural Standards)
has indicated a dose of up to 1 g per kg of body weight as fatal [54], whereas in our trial,
mice received one tenth.
In summary, our actual preclinical trial provides the first evidence that oral pretreat-
ment of C. jejuni-infected IL-10−/− mice harboring a commensal human gut microbiota
mitigates acute enterocolitis due to its immune-modulatory, including anti-inflammatory,
anti-oxidant, and anti-apoptotic modes of action.

5. Conclusions
Menthol constitutes a promising antibiotic-independent option to fight acute campy-
lobacteriosis, thereby decreasing the risk for the development of post-infectious autoim-
mune morbidities given that the severity of the enteritis is associated with the risk of
complications including Guillain–Barré syndrome, reactive arthritis, and chronic intestinal
inflammatory morbidities later on. Furthermore, menthol might be a useful measure to
prevent C. jejuni infection in individuals with an increased risk of exposure to the en-
teropathogen. Future research should focus on developing antibiotic-independent methods
for the prevention and/or treatment of food-borne illnesses brought on by enteropathogens
besides Campylobacter species.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/biom14030290/s1, Table S1: Clinical scores; Table S2: Histopatho-
logical scores; Table S3: Primary antibodies for in situ immunohistochemical analyses; Figure S1:
Clinical signs of campylobacteriosis over time following C. jejuni infection of hma IL-10−/− mice
with menthol pre-treatment. Figure S2: Pathogen numbers in fecal samples taken following C. jejuni
infection of hma IL-10−/− mice with menthol pre-treatment. Figure S3: Colonic MCP-1 secretion in
C. jejuni infected hma IL-10−/− mice with menthol pretreatment.
Author Contributions: Conceptualization, supervision, and methodology: M.M.H. and S.B.; In-
vestigation, M.M.H., L.Q.L., N.S., N.W.S. and S.M.; Visualization: L.Q.L. and S.M.; Evaluation and
interpretation of data: M.M.H., L.Q.L., S.M. and S.B.; Writing—original draft preparation: M.M.H.;
Writing—review and editing: N.W.S., S.M. and S.B. All authors have read and agreed to the published
version of the manuscript.
Funding: Grants were provided from the German Federal Ministries of Education and Research
(BMBF) in the framework of the PAC-Campylobacter zoonoses research consortium to M.M.H. and
S.B. (IP7/01KI2007D) and from the Federal Ministry for Economic Affairs and Energy in response to
a resolution of the Deutscher Bundestag, the German National Parliament, to M.M.H. and S.B. (ZIM,
ZF4117908 AJ8) supporting this work. The funders were not involved in the design of the study, the
gathering and analysis of data, the publishing decision, or the writing of the manuscript.
Institutional Review Board Statement: Mouse experiments were approved by the commission
for animal experiments headed by the “Landesamt für Gesundheit und Soziales” (LaGeSo, Berlin;
registration numbers G0104/19, approved on 15 July 2019) and carried out in accordance with the
European Guidelines for animal welfare (2010/63/EU) and the ARRIVE guidelines.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We acknowledge the great technical help and animal breeding provided by
Alexandra Bittroff-Leben, Ines Puschendorf, Sumaya Abdul-Rahman, Gernot Reifenberger, and the
personnel of the animal research facility at FEM of Charité-University Medicine Berlin, respectively.
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Biomolecules 2024, 14, 290 13 of 15

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