Abstract
Incidence rates of human Campylobacter jejuni infections are progressively increasing globally. Since the risk for the development of post-infectious autoimmune diseases correlates with the severity of the preceding enteritis and campylobacteriosis treatment usually involves symptomatic measures, it is desirable to apply antibiotic-independent compounds to treat or even prevent disease. Given its health-promoting including anti-inflammatory properties carvacrol constitutes a promising candidate. This prompted us to test the disease-alleviating including immune-modulatory effects of carvacrol prophylaxis in acute murine campylobacteriosis. Therefore, human gut microbiota-associated IL-10−/− mice were orally challenged with synthetic carvacrol starting a week before C. jejuni infection and followed up until day 6 post-infection. Whereas carvacrol prophylaxis did neither affect gastrointestinal pathogen loads, nor the human commensal gut microbiota composition, it improved the clinical outcome of mice, attenuated colonic epithelial cell apoptosis, and dampened pro-inflammatory immune responses not only in the intestinal tract but also in extra-intestinal organs including the liver and the spleen. In conclusion, our preclinical placebo-controlled intervention study provides convincing evidence that oral carvacrol pretreatment constitutes a promising option to mitigate acute campylobacteriosis and in turn, to reduce the risk for post-infectious complications.
Introduction
Incidence and prevalence of reported human campylobacteriosis cases are increasing around the globe and responsible for significant morbidities and tremendous socioeconomic expenses [1–3]. The enteritis syndrome is caused by aerobic and non-spore-forming Gram-negative bacterial members of the Campylobacteraceae family. Among these, Campylobacter jejuni bacteria live commensally in the digestive tracts of warm-blooded vertebrate species, including birds, typically generating no noticeable symptoms [4, 5]. However, contaminated undercooked meat from poultry or other livestock, unpasteurized milk and its byproducts, as well as surface water may result in food-borne infections upon ingestion by humans [6]. During the acute phase of infection, the very motile enteropathogens successfully pass through the gastro-duodenum and invade the distal intestinal tissues [7, 8]. Distinct bacterial cell wall molecules, such as lipo-oligosaccharide (LOS) induce the recruitment of innate immune cells including macrophages, monocytes, and neutrophils but also of adaptive T and B lymphocytes to the inflamed sites of intestinal infection [9, 10]. The subsequently induced pro-inflammatory mediator storm damages the intestinal tissues as indicated by enhanced oxidative stress and apoptotic cell responses, ulcerations, and crypt abscesses mounting in a malabsorptive disease [9, 11–13]. After an incubation period of two to five days, the balance between enteropathogen's arsenal of virulence factors and the human host's immunological fitness determines the severity of symptoms that infected individuals display [14–16]. Patients complain about symptoms of varying severities such as general discomfort, abdominal pain, nausea, vomiting, watery or even bloody diarrhea, with or without mucous discharge, and fever, for instance, and typically recover completely within two weeks after the infection [17]. However, in rare cases, post-infectious autoimmune morbidities affecting the intestinal tract (i.e., irritable bowel syndrome, celiac disease, chronic inflammatory bowel diseases), the joints (i.e., reactive arthritis), and the central nervous system (i.e., Guillain Barré syndrome) may develop with a latency of a few weeks to months after the enteritis [2, 15, 18, 19]. Remarkably, the probability to develop post-infectious sequelae is strongly correlated with the severity of the previous enteritis episode which depends on the sialylation state of the C. jejuni-LOS [20]. Campylobacteriosis patients are typically treated with symptomatic therapies including analgesic, spasmolytic, and antipyretic medications, along with electrolyte replacement and rehydration [21]. Patients under immune-suppressive conditions developing severe C. jejuni induced disease due to bacteremia, for instance, should be treated with antibiotics such as ciprofloxacin or erythromycin [15, 21, 22]. However, the increasing prevalence of infections with multi-drug resistant C. jejuni strains can make it more difficult to sufficiently treat severe campylobacteriosis in critically ill patients [22]. Thus, it is imperative to find non-toxic, antibiotic-independent disease-alleviating therapeutic approaches to tackle C. jejuni induced morbidities.
Terpenoids are known for their broad anti-microbial potential [23]. Carvacrol (4-isopropyl-2-methylphenol) is a phenolic monoterpenoid and a predominant constituent of essential oils derived from oregano, thyme, and other medicinal plants known for numerous health-promoting effects [24, 25]. For instance, carvacrol can modulate different enzymatic pathways resulting in anxiolytic, spasmolytic, cell regenerative, and anti-cancer activities [24, 25]. Due to its naturally occurring anti-bacterial effects against various food-borne pathogens, including Campylobacter species, carvacrol is also being studied in the field of infection research. In vitro studies demonstrated that carvacrol can alter the fatty acid composition of bacterial cell walls at bacteriostatic doses [26, 27], whereas at bactericidal concentrations, the monoterpenoid even permeabilizes the outer membrane of Gram-negative bacteria [28]. Furthermore, carvacrol has been shown to have ATPase-inhibiting activities [29, 30]. It is also thought to function as a proton exchanger, lowering the pH gradient across the cytoplasmic membrane, and altering the ATP pool and proton motive force, ultimately resulting in cell death [29, 31]. Notably, carvacrol application was shown to effectively reduce the C. jejuni loads in gut specimens taken from poultry including chicken [32–35]. Moreover, carvacrol could successfully down-regulate distinct virulence genes expressed in C. jejuni and inhibit the pathogenic invasion of chicken cells [33, 36]. Particularly, the discovery that the application of carvacrol inhibited the motility and invasion characteristics of C. jejuni in vitro suggests that carvacrol might be a potentially effective candidate molecule in the fight against human campylobacteriosis [37].
This prompted us to test prophylactic oral carvacrol in acute experimental campylobacteriosis employing human gut microbiota-associated (hma) IL-10−/− mice. The reasons for the application of this particular C. jejuni infection and inflammation model are the following. Firstly, conventional as opposed to microbiota-depleted and hma mice are protected from C. jejuni infection due to the complex murine microbiota providing a potent physiological colonization resistance [38, 39]. Furthermore, even upon successful C. jejuni colonization in microbiota-depleted or hma wildtype mice, the pathogen does not induce overt disease including enteritis since mice are approximately 10,000 times more resistant to Toll-like recptor-4 (TLR-4) ligands such as lipo-polysaccharide (LPS) and LOS if compared to chicken and humans. This experimental limitation can, however, be overcome by il10 gene deletion [40]. Therefore, oral transplants of a diverse gut microbiota obtained from healthy human fecal donors were administered to microbiota-depleted IL-10−/− mice created by antibiotic pretreatment [41, 42]. Overall, the hma IL-10−/− mouse model constitutes a reliable experimental tool to dissect the triangular relationship between C. jejuni, vertebrate host immunity, and the human gut microbiota and mimics key features of severe campylobacteriosis in humans [42]. In addition, it enables us to test novel interventive measures, such as therapeutic and/or prophylactic regimens with defined molecules alone or in combination within a preclinical setting [41] as validated by our recent placebo-controlled intervention studies [43–46].
In our actual study, we subjected hma IL-10−/− mice to oral carvacrol beginning seven days before C. jejuni infection. We evaluated the effects of this prophylactic regimen on i.) gastrointestinal C. jejuni burdens; ii.) clinical outcome; iii.) macroscopic and iv.) microscopic inflammatory complications in the colon; v.) intestinal, vi.) extraintestinal, vii.) and systemic pro-inflammatory immune responses; and viii.) finally, human gut microbiota shifts during acute campylobacteriosis.
Material and methods
Mice, gut microbiota depletion
IL-10−/− C57BL/6j mice were bred, reared, and maintained under specified pathogen-free (SPF) and standard settings in the Forschungsinstitute für Experimentelle Medizin, Charité – Universitätsmedizin Berlin, Germany. Mice were kept in autoclaved cages with filter tops within an experimental semi-barrier and had free access to standard chow diet (food pellets: ssniff R/M-H, V1534-300, Sniff, Soest, Germany) and autoclaved tap water (ad libitum). In order to deplete the commensal gut microbiota, 3-week-old female and male mice were treated with the antibiotics ampicillin plus sulbactam (2 g L−1 and 1 g L−1, respectively; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany) via the drinking water for eight weeks (ad libitum) as described recently [42, 47]. All mice were handled under strict aseptic conditions to avoid contaminations and assure successful microbiota depletion. Two days before human FMT (day −16; Fig. 1A), the antibiotics were withdrawn and replaced by autoclaved water (ad libitum).
Timeline of experimental procedures and microbiota composition of human fecal transplants
(A) In order to remove the commensal gut microbiota, conventional IL-10−/− mice were treated with the antibiotics ampicillin plus sulbactam for 8 weeks. On day (d)-16, the antibiosis was withdrawn and replaced by autoclaved tap water. On d-14, d-13, and d-12, microbiota-depleted mice were subjected to oral human fecal microbiota transplantation (hFMT). Starting on d-7, synthetic carvacrol was applied via the drinking water (until necropsy on d6), and human gut microbiota-associated mice were perorally infected with Campylobacter jejuni strain 81-176 by gavage on d0 and d1. (B) The microbiota compositions of the human fecal donor suspensions used for hFMT (n = 3 per experiment) were quantitatively assessed by culture-independent molecular analyses. Median (red bars) of the total eubacterial loads (TEL), enterobacteria (EB), enterococci (EC), lactobacilli (LB), bifidobacteria (BB), Bacteroides/Prevotella species (BP), Clostridium coccoides (CC) and Clostridium leptum (CL) groups, and Mouse Intestinal Bacteroides (MIB) are given (expressed as gene copies per ng DNA). Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Human fecal microbiota transplantation
The microbiota-depleted IL-10−/− mice were subjected to human fecal microbiota transplantation (hFMT) starting two weeks prior C. jejuni infection on three consecutive days (i.e., on d−14, d−13, and d−12; Fig. 1A) in order to introduce a complex human intestinal microbiota into the murine host as stated recently [42]. In brief, human fecal samples were collected from 5 healthy individuals (all samples free of enteropathogenic bacteria, viruses and parasites), aliquoted, and stored at −80 °C. Immediately before use, the fecal samples were thawed, resuspended in sterile phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA), and pooled before oral application to the mice via gavage (0.3 mL volume). The microbiota compositions of the suspensions used for the hFMT are shown in Fig. 1B.
Prophylactic regimen
Prophylactic application of synthetic carvacrol (Sigma-Aldrich, München, Germany) was started seven days prior C. jejuni infection (i.e., d−7; Fig. 1A). To improve its aqueous solubility, carvacrol was dissolved in Tween® 80 (2.5 mL L−1; Sigma-Aldrich, München, Germany) and then diluted in autoclaved tap water. The final concentration of the carvacrol drinking solution was 500 mg L−1 (ad libitum) resulting in daily treatment doses of 100 mg per kg body weight when considering a mean mouse body weight of 25 g and an average drinking volume of approximately 5 mL per day. Placebo counterparts received vehicle instead (ad libitum).
C. jejuni infection
C. jejuni strain 81-176 bacterial stocks were stored at −80 °C. After thawing, the bacteria were streaked out and incubated on selective karmali agar plates (purchased from Oxoid, Wesel, Germany) at 37 °C for 48 h in a jar under microaerophilic conditions (CampyGas Packs; Oxoid, Wesel, Germany). Grown C. jejuni bacteria were harvested in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA) immediately before infection. Age- and sex-matched human intestinal microbiota-associated (hma) IL-10−/− mice (3-month-old littermates) were then infected perorally with 109 colony forming units (CFU) of the pathogen (in 0.3 mL) on d0 and d1 (Fig. 1A) by gavage as reported earlier [48].
C. jejuni loads in the gastrointestinal tract
For the determination of gastrointestinal pathogen loads, the numbers of live C. jejuni bacteria were monitored in fecal samples daily post-infection (p.i.), and upon necropsy in intraluminal gastrointestinal samples taken from the stomach, duodenum, ileum, and colon lumen that were subsequently homogenized in PBS (Thermo Fisher Scientific, Waltham, MA, USA). The C. jejuni bacteria were quantified by counting of CFU after growth of serial dilutions of intestinal samples on karmali agar plates placed in a jar for at least 48 h at 37 °C under microaerophilic conditions (CampyGas Packs; Oxoid, Wesel, Germany) as described earlier [48]. The detection limit of viable pathogens was 100 CFU per g fecal sample.
Fecal microbiota composition
The microbiota composition of human fecal donor suspensions used for hFMT and of murine fecal samples derived from hma mice was analyzed immediately before (i.e, day 0) and 6 days after C. jejuni infection as described in detail previously [49–51]. In brief, culture-independent 16S rRNA based methods were applied to quantitatively assess even fastidious and non-cultivable bacteria. Therefore, the total genomic DNA was extracted from the samples and the main bacterial groups that are abundant in the human gut microbiota were determined by quantitative real-time polymerase chain reaction (qRT-PCR) applying species-, genera- or group-specific 16S rRNA primers (Tib MolBiol, Berlin, Germany) and expressed as gene copies per ng DNA [49–51].
Clinical conditions of mice
Immediately before and every day after C. jejuni infection the clinical conditions of mice were monitored quantitatively by using a cumulative clinical score (maximum 12 points), addressing wasting symptoms (0: normal; 1: ruffled fur; 2: less locomotion; 3: isolation; 4: severely compromised locomotion, pre-final aspect), the occurrence of fecal blood (0: no blood; 2: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Germany; 4: macroscopic blood visible), and the stool consistency (0: formed feces; 2: pasty feces; 4: liquid feces), as described earlier [52].
Sampling procedures
Mice were sacrificed by carbon dioxide asphyxiation on day 6 p.i. (Fig. 1A). Immediately thereafter, ex vivo biopsies were taken from the colon and ileum as well as luminal samples derived from the stomach, duodenum, ileum, and colon under aseptic conditions for microbiological and immunopathological analyses.
Histopathology
For histopathological analyses colonic ex vivo biopsies were immediately fixed in 5% formalin, embedded in paraffin and 5-µm-sections were stained with hematoxylin and eosin (H&E). In order to evaluate the severity of histopathological changes of the colonic mucosa respective biopsies were assessed by light microscopy (100-times magnification) and quantitated according to an established scoring scheme [53]: Score 1, minimal inflammatory cell infiltrates in the mucosa with intact epithelium. Score 2, mild inflammatory cell infiltrates in the mucosa and submucosa with mild hyperplasia and mild goblet cell loss. Score 3, moderate inflammatory cell infiltrates in the mucosa and submucosa with moderate goblet cell loss. Score 4, marked inflammatory cell infiltration into the mucosa and submucosa with marked goblet cell loss, multiple crypt abscesses, and crypt loss.
In situ immunohistochemistry
In situ immunohistochemical analyses were performed in ex vivo biopsies taken from the distal colon and the liver that had been fixed in 5% formalin and embedded in paraffin as reported earlier [54]. In brief, in order to detect apoptotic epithelial cells, macrophages/monocytes, neutrophils, T lymphocytes, regulatory T cells, and B lymphocytes paraffin sections (5 µm) were stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), CD3 (no. N1580, Dako, Glostrup, Denmark; 1:5), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, San Diego, CA, USA; 1:100), and B220 (no. 14-0452-81, eBioscience, San Diego, CA, USA;1:200), respectively. The mean numbers of positively stained cells were determined within at least six high power fields (HPF, 0.287 mm2, 400-times magnification) by an independent blinded investigator.
Pro-inflammatory mediators
Ex vivo biopsies from the terminal ileum and colon (approximately 1 cm2 each) were cut longitudinally and washed 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) and the spleen (one third) were transferred to 24-flat-bottom well culture plates (Thermo Fisher Scientific, 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). After an 18-h incubation period at 37 °C, culture supernatants were tested for monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), and interleukin-6 (IL-6) by the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Heidelberg, Germany) using a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide concentrations were determined by the Griess reaction [55].
Statistical analyses
After pooling of data from three independent experiments, medians and significance levels were calculated using GraphPad Prism (version 9; San Diego, CA, USA). The normalization of data sets was assessed by the Anderson-Darling test. The Student's t-test and Mann-Whitney test were used for pairwise comparisons of normally and not normally distributed data, respectively. Multiple comparisons were performed using the one-way ANOVA with Tukey post hoc test (for normally distributed data) and Kruskal-Wallis test with Dunn's post hoc test (for not normally distributed data). Two-sided probability (p) values ≤0.05 were considered significant.
Ethics statement
All animal experiments were carried out in accordance to the European animal welfare guidelines (2010/63/EU) upon approval by the commission for animal experiments (“Landesamt für Gesundheit und Soziales”, LAGeSo, Berlin; registration number G0104/19). The clinical conditions of mice were surveyed daily.
Results
Gastrointestinal pathogens loads in C. jejuni infected hma IL-10−/− mice following oral carvacrol prophylaxis
First, we addressed whether prophylactic oral carvacrol application to hma IL-10−/− mice had an impact on fecal pathogen loads following C. jejuni infection. Our cultural analyses revealed that within 24 h after the latest infectious challenge, the enteropathogen could be isolated from fecal samples at median loads of more than 108 CFU ger gram in both, carvacrol and placebo pretreated mice (not significant (n.s.)) and remained at comparable levels until day 5 p.i. (n.s.; Fig. 2). On day 6 p.i., we assessed the C. jejuni numbers in luminal samples taken from the stomach, duodenum, terminal ileum, and colon and again, found pathogen loads that did not differ between both cohorts (n.s.; Fig. 3). Hence, oral carvacrol prophylaxis did not affect gastrointestinal C. jejuni loads in infected hma IL-10−/− mice.
Kinetic analysis of fecal Campylobacter jejuni loads in human microbiota-associated (hma) IL-10−/− mice following prophylactic carvacrol application and C. jejuni infection
On day (d) 0 and d1, hma IL-10−/− mice with oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis were infected with C. jejuni strain 81-176 by gavage. Fecal enteropathogen loads were determined over time post-infection (as indicated) by culture and expressed as colony-forming units per gram (CFU/g). Medians (red bars) and numbers of analyzed mice (in parentheses) are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Gastrointestinal pathogens loads in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis.
On day 6 following infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, the gastrointestinal C. jejuni loads were determined by culture and expressed as colony-forming units per gram (CFU/g). Medians (red bars) and the numbers of analyzed mice (in parentheses) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Kinetic analysis of campylobacteriosis symptoms in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
Furthermore, we surveyed the severities of developing clinical signs of campylobacteriosis over time p.i. (Fig. 4). Our clinical scoring revealed that mice from the carvacrol cohort presented with a better clinical outcome given lower scores for the overall clinical conditions in the verum versus placebo pretreated mice on day 6 p.i. (P < 0.05; Fig. 4A). When focusing on individual parameter contributing to the campylobacteriosis syndrome, carvacrol pretreated mice exhibited lower scores for diarrhea on days 4, 5, and 6 p.i. if compared to placebo counterparts (P < 0.05–0.01; Fig. 4C), whereas the scores for wasting symptoms and fecal blood were not significantly different between the cohorts over time p.i. (n.s.; Fig. 4B and D). Hence, carvacrol pretreatment improved the clinical outcome, particularly diarrheal symptoms upon C. jejuni infection.
Kinetic analysis of campylobacteriosis symptoms in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day (d) 0 and d1, hma IL-10−/− mice with oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis were infected with C. jejuni strain 81-176 by gavage. (A) The overall clinical conditions of campylobacteriosis further specified by (B) wasting, (C) diarrhea, and (D) fecal blood were quantitatively assessed immediately before and after infection over time until necropsy on d6 applying defined scoring schemes (see methods). Medians (red bars), numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the Mann-Whitney test are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Macroscopic and microscopic inflammatory changes in the colon of C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
Next, we tested the impact of exogenous carvacrol on overt macroscopic and microscopic disease induced by C. jejuni infection. Since intestinal inflammation is known to cause significant shrinkage of the inflamed gut tissue [49, 56, 57], we measured the colonic lengths upon necropsy. In fact, shorter colons were determined in the placebo control mice (P < 0.01 versus naive; Fig. 5A) as opposed to the carvacrol cohort on day 6 p.i. (n.s. versus naive; P < 0.05 versus placebo) (Fig. 5A) indicative for attenuated gross disease in the latter. When performing quantitative histopathological grading of the colonic mucosa and lamina propria we found enhanced C. jejuni induced microscopic disease in both infected groups upon necropsy (P < 0.001 versus naive; Fig. 5B), but observed a trend towards lower histopathological scores in the carvacrol versus placebo treated mice that did not reach statistical difference due to relatively high standard deviations particularly in the latter group (n.s.; Fig. 5B). When counting the apoptotic colonic epithelial cells, however, the C. jejuni induced increases in cleaved caspase-3 positive cells were far less pronounced in the large intestines of the carvacrol as compared to placebo treated mice (P < 0.001; Fig. 5C). Notably, the median apoptotic colonic epithelial cell numbers were more that 50% lower in the former versus the latter (Fig. 5C). Hence, carvacrol pretreatment resulted in attenuated overt macroscopic disease and epithelial cell apoptosis in the colon following C. jejuni infection.
Macroscopic and microscopic inflammatory changes in the colon of C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day 6 following C. jejuni infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, (A) the colonic lengths were measured with a ruler (in cm) and (B) the histopathological changes quantitatively assessed in hematoxylin and eosin-stained colonic paraffin sections by using a standardized histopathological scoring scheme (see methods). In addition, (C) apoptotic colonic epithelial cell numbers were determined in large intestinal paraffin sections stained with antibodies against cleaved caspase-3 (Casp3), and the mean numbers of positively stained cells out of six representative high-power fields (HPF, 400-times magnification, light microscopy) indicated. Naive mice were used as non-infected controls without prophylaxis (white triangles). Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the one-way ANOVA test with Tukey post-correction (A,C) or the Kruskal-Wallis test with Dunn's post hoc test (B) are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Immune cell responses in the colon of C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
Then, we surveyed the immune-modulatory effects of exogenous carvacrol and determined the colonic immune cell responses by quantitative in situ immunohistochemistry. C. jejuni infection led to increases in numbers of innate immune cells such as macrophages, monocytes, and neutrophils (P < 0.01–0.001 versus naive; Fig. 6A and B). In case of macrophages and monocytes, the numbers determined in the colonic mucosa and lamina propria on day 6 p.i. were lower in the carvacrol as compared to placebo pretreated mice (P < 0.001; Fig. 6A), whereas a trend towards lower neutrophil counts was detected in the colon of the former versus the latter (n.s.; Fig. 6B). Following staining of the colonic paraffin section with antibodies directed against defined adaptive immune cell subsets (Fig. 6C–E), we found increased numbers of T lymphocytes in infected mice from the placebo control (P < 0.01 versus naive; Fig. 6C), but not the carvacrol cohort (n.s. versus naive; Fig. 6C). Moreover, C. jejuni infected mice exhibited increases in both, regulatory T cells and B lymphocytes (P < 0.05–0.001; Fig. 6D and E). Whereas fewer regulatory T cells were detected in the colonic samples taken from the carvacrol as compared to the placebo pretreated mice on day 6 p.i. (P < 0.05; Fig. 6D), a trend towards lower large intestinal B cell numbers became evident with approximately 50% lower median values in the former if compared to the latter (n.s.; Fig. 6E). Hence, carvacrol prophylaxis dampened both, innate and adaptive immune responses upon C. jejuni infection.
Immune cell responses in the colon of C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day 6 following C. jejuni infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, the numbers of defined immune cell populations including (A) macrophages and monocytes (F4/80+), (B) neutrophils (MPO7+), (C) T lymphocytes (CD3+), (D) regulatory T cells (FOXP3+), and (E) B lymphocytes (B220+) were enumerated in the mucosa and lamina propria of immunohistochemically stained colonic paraffin sections and the mean numbers of positively stained cells out of six representative high-power filed (HPF, 400-times magnification, light microscopy) indicated. Naive mice were used as non-infected controls without prophylaxis (white triagnles). Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the one-way ANOVA test with Tukey post hoc test (A) or the Kruskal-Wallis test with Dunn's post hoc test (B–E) are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Intestinal pro-inflammatory mediator secretion in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
Next, we tested the effect of prophylactic carvacrol application on C. jejuni induced pro-inflammatory mediator secretion in the intestinal tract and found elevated MCP-1 concentrations in the colon of placebo (P < 0.05 versus naive; Fig. 7A), but not carvacrol pretreated mice (n.s. versus naive; Fig. 7A). This also held true for enhanced IFN-γ secretion assessed in the terminal ileum of placebo counterparts (P < 0.01 versus naive; Fig. 7B), whereas mice from the carvacrol cohort presented basal values on day 6 p.i. (n.s. versus naive; Fig. 7B). Hence, carvacrol pretreatment could mitigate C. jejuni induced pro-inflammatory mediator secretion to basal levels.
Intestinal pro-inflammatory mediator secretion in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day 6 following C. jejuni infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, (A) colonic MCP-1 and (B) ileal IFN-γ concentrations were determined. Naive mice were used as non-infected controls without prophylaxis (white triangles). Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the Kruskal-Wallis test with Dunn's post hoc test are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Extra-intestinal pro-inflammatory inflammatory responses in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
Furthermore, we addressed whether the inflammation-dampening effects of carvacrol pretreatment was also effective beyond the intestinal tract. In fact, C. jejuni infected mice displayed increased numbers of apoptotic cells as well as of neutrophils in liver ex vivo biopsies taken from placebo (P < 0.5 and P < 0.001 versus naive, respectively; Fig. 8) as opposed to carvacrol pretreated mice on day 6 p.i. (n.s. versus naive; P < 0.05 and 0.01 versus placebo, respectively; Fig. 8). When measuring pro-inflammatory mediators in splenic samples on day 6 p.i., we detected decreased nitric oxide and IL-6 concentrations in the spleens taken from mice of the placebo (P < 0.05 versus naive; Fig. 9) as opposed to the verum cohort (n.s. versus naive; P < 0.01 and P < 0.05 versus placebo, respectively; Fig. 9), indicative for enhanced recruitment of pro-inflammatory immune cells from the spleen to infected intestinal compartments. Hence, the inflammation-dampening effects of carvacrol prophylaxis in acute campylobacteriosis became also evident in extra-intestinal compartments such as the liver and the spleen.
Hepatic inflammatory changes in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day 6 following C. jejuni infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, (A) apoptotic cells and (B) neutrophils were quantitated in liver paraffin sections stained with antibodies against cleaved caspase-3 (Casp3) and MPO7, respectively, and the mean numbers of positively stained cells out of six representative high-power fields (HPF, 400-times magnification, light microscopy) were indicated. Naive mice served as non-infected controls without prophylaxis (white triangles). Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the Kruskal-Wallis test with Dunn's post hoc test are indicated. Data were pooled from at least two independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Splenic pro-inflammatory mediator secretion in C. jejuni infected hma IL-10−/− mice following carvacrol prophylaxis
On day 6 following C. jejuni infection of hma IL-10−/− mice that had been subjected to oral carvacrol (CAR, white circles) or placebo (PLC, black circles) prophylaxis, (A) nitric oxide and (B) IL-6 concentrations were measured in splenic ex vivo biopsies. Naive mice were used as non-infected controls without prophylaxis (white triangles). Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the one-way ANOVA test with Tukey post hoc test (A) or the Kruskal-Wallis test with Dunn's post hoc test (B) are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Fecal microbiota changes during C. jejuni infection of hma IL-10−/− mice following carvacrol prophylaxis
In addition, we surveyed the changes in the fecal microbiota composition of carvacrol pretreated mice during campylobacteriosis. Our comprehensive culture-independent survey of fecal pellets assessing also fastidious and uncultivable bacterial phyla revealed that immediately before infection (i.e., on day 0) mice from both cohorts displayed comparable fecal microbial conditions that were independent from the carvacrol pretreatment (n.s.; Fig. 10). This also held true at the end of the observation period given that the fecal gene numbers of the analyzed intestinal commensals did not differ between both infected groups on day 6 p.i. (Fig. 10). When assessing changes within each cohort during campylobacteriosis development by comparing respective bacterial phyla on days 0 and 6 p.i., we found decreases in fecal gene numbers of Bacteroides/Prevotella spp. (P < 0.01–0.001; Fig. 10F) and Clostridium coccoides (P < 0.05–0.001; Fig. 10G) that were independent of the pretreatment regimen. Slight decreases in total eubacterial loads were, however, obtained in placebo, as opposed to carvacrol treated mice (P < 0.01; Fig. 10A), which was also the case when measuring bifidobacteria (P < 0.01; Fig. 10E) and Clostridium leptum group members (P < 0.01; Fig. 10H). Whereas fecal enterobacterial loads increased in carvacrol pretreated mice from day 0 until day 6 p.i. (P < 0.001; Fig. 10B), a trend towards higher numbers were detected placebo counterparts (n.s.; Fig. 10B). Hence, carvacrol pretreatment did neither affect the fecal gut microbiota composition immediately before nor 6 days after C. jejuni infection.
Fecal microbiota changes during C. jejuni infection of hma IL-10−/− mice following carvacrol prophylaxis
Immediately before (i.e., day (d)0) and on d6 after C. jejuni infection, the fecal microbiota compositions were assessed in hma IL-10−/− mice with carvacrol (CAR, white circles) or placebo (PLC, black circles) 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. Medians (red bars), the numbers of analyzed mice (in parentheses), and significance levels (P values) determined by the one-way ANOVA test with Tukey post hoc test (A) or the Kruskal-Wallis test with Dunn's post hoc test (B–I) are indicated. Data were pooled from three independent experiments.
Citation: European Journal of Microbiology and Immunology 14, 2; 10.1556/1886.2024.00009
Discussion
Our actual preclinical placebo-controlled intervention study revealed that oral carvacrol application to hma IL-10−/− mice starting a week before C. jejuni infection did i.) neither affect gastrointestinal pathogen loads, ii.) nor the human commensal gut microbiota composition; iii.) improved the clinical outcome of mice; iv.) attenuated colonic epithelial cell apoptosis; and v.) dampened pro-inflammatory immune responses not only in the intestinal tract but also in extra-intestinal organs including the liver and the spleen.
Whereas in our study the oral carvacrol application did not interfere with the pathogen burdens in the gastrointestinal tract (Figs 2 and 3), slightly lower C. jejuni number were detected in the large and/or small intestines of infected microbiota-depleted [58] and hma IL-10−/− mice [43] following oral carvacrol application with the same concentration (i.e., 500 mg L−1) starting five days before [58] or two days after oral pathogen challenge [43]. Since the carvacrol concentration of the applied drinking solution was more than 3-times exceeding the minimum inhibitory concentration (MIC) of 150 mg L−1 as measured previously [58], the comparable gastrointestinal C. jejuni numbers in the mice from the verum and placebo cohorts appear somewhat surprising. One might hypothesize that the secreted gastrointestinal fluids may have diluted the applied phenolic compound. Despite comparable intestinal pathogen loads, the mice from the carvacrol cohort were in much better clinical conditions at the end of the experiment (Fig. 4A) and suffered less distinctly from diarrheal symptoms if compared to placebo counterparts (Fig. 4C). In support, previous in vitro studies highlighted that carvacrol can down-regulate the expression of distinct virulence factors responsible for motility, cell adhesion, and invasion [33, 34, 36, 37, 59], which might explain the ameliorated campylobacteriosis symptoms in the verum cohort. In line with the improved clinical outcome, carvacrol as opposed to placebo pretreated mice did not exhibit colonic shrinkage (Fig. 5A) indicative for mitigated C. jejuni induced macroscopic disease. Furthermore, the disease-alleviating effect of the phenolic compound became also evident on the microscopic levels given that carvacrol pretreatment resulted in attenuated apoptotic cell responses in the colon (Fig. 5C). The anti-apoptotic effects of exogenous carvacrol could also be observed in our previous interventive studies where carvacrol application before [58], but also after C. jejuni infection [43] resulted in less severe colonic cell apoptosis in acute murine campylobacteriosis. In support, a previous in vitro study revealed that carvacrol down-regulated the expression of the apoptosis-inducing factors (AIF) leading to suppressed cleavage of caspase-3 [60]. In addition, the authors reported a down-regulated expression of mammalian target of rapamycin (mTOR) upon carvacrol treatment in vitro [60]. This result is of particular interest, since the immune-suppressive agent rapamycin (sirolimus) for which mTOR is a cellular target has been shown to alleviate acute enterocolitis in C. jejuni infected IL-10−/− mice [61].
Our quantitative in situ immunohistochemical analyses further underscored significant immune-modulatory effects of preventive carvacrol application given that lower colonic numbers of neutrophils and T lymphocytes were assessed in carvacrol as compared to placebo treated mice on day 6 p.i. (Fig. 6). The dampened innate as well as adaptive immune cell responses upon C. jejuni infection could also be observed in our previous therapeutic carvacrol intervention study applying hma IL-10−/− mice [43]. In addition, oral carvacrol pretreatment resulted in diminished intestinal secretion of pro-inflammatory mediators (Fig. 7), which was also the case when carvacrol treatment was initiated after C. jejuni infection [43]. In support, carvacrol application could decrease pro-inflammatory mediator secretion from stimulated macrophages and T lymphocytes in vitro [62, 63], whereas the expression of pro-inflammatory cytokines was down-regulated in LPS-challenged broiler chicken following oral carvacrol challenge [64]. Since the TLR-4 dependent sensing of the C. jejuni-derived endotoxin LOS is a cornerstone in the immunopathogenesis of acute campylobacteriosis [10, 19], anti-TLR-4-directed treatment strategies appear of particular importance in the combat of C. jejuni induced disease. In fact, several in vitro studies showed that carvacrol could act in a TLR-4-antagonistic manner [65–68].
Our actual study further revealed that the anti-inflammatory effects of oral carvacrol prophylaxis were not restricted to the intestines, but also effective in extra-intestinal organs given that C. jejuni induced increases in apoptotic cells and neutrophils in the liver were observed in the placebo as opposed to the carvacrol cohort (Fig. 8). Our previous study supported the anti-apoptotic effects upon prophylactic application of the phenolic compound in microbiota-depleted IL-10−/− mice suffering from acute campylobacteriosis [58], whereas carvacrol application to hma IL-10−/− mice in a therapeutic regimen starting post-infection could diminish hepatic pro-inflammatory cytokine secretion to basal levels [43]. Our results are further supported by a study showing that carvacrol reduced inflammatory including apoptotic cell responses in cadmium induced liver toxicity in rats [69]. Importantly, when carvacrol was applied perorally to diabetic mice, the induced liver damage could be prevented by down-regulating the TLR-4 and mTOR signaling pathways [68]. Remarkably, the anti-inflammatory effects of carvacrol became evident even systemically given decreased nitric oxide and IL-6 concentrations in the spleens of pretreated mice from the placebo opposed to the verum cohort (Fig. 9), pointing towards an enhanced recruitment of pro-inflammatory immune cells from the spleen to the infected intestines. The systemic anti-inflammatory properties of carvacrol were also shown in our previous intervention studies given diminished C. jejuni induced IL-6 serum concentrations [43, 58]. In line, carvacrol improved survival in mice with LPS induced endotoxinemia further presenting with diminished neutrophilic IL-6 secretion [70].
Notably, our molecular analyses of fecal pellets collected on day 0 and day 6 post-infection confirmed that mice from the verum and the placebo cohorts were equipped with a comparable human gut microbiota immediately before C. jejuni infection, which was also the case at the end of the observation period (Fig. 10). This points towards a comparably effective engraftment of the human fecal transplants upon human FMT and furthermore, speaks against significant effects of the compound on the human fecal microbiota composition.
Conclusion
Our preclinical placebo-controlled intervention study provides convincing evidence that oral carvacrol pretreatment constitutes an antibiotic-independent option to mitigate acute campylobacteriosis and in turn, to reduce the risk for post-infectious complications. The application of synthetic carvacrol has been considered as safe by the Federal Drug Administration (FDA) and is being used as preservative by the food industry [24]. Furthermore, carvacrol has been approved as an animal food supplement to reduce Campylobacter burdens in livestock by the European Union [71].
Funding
This work was supported by grants from the German Federal Ministries of Education and Research (BMBF) in frame of the zoonoses research consortium PAC-Campylobacter to MMH and SB (IP7/01KI2007D) and from the Federal Ministry for Economic Affairs and Energy following a resolution of the German National Parliament, Deutscher Bundestag to MMH and SB (ZIM, ZF4117908 AJ8). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Authors' contributions
MMH: Designed and performed experiments, analyzed data, wrote the paper.
LQL: Performed experiments, analyzed data, edited the paper.
NS: Performed experiments, analyzed data, edited the paper.
SM: Performed experiments, analyzed data, critically discussed results, edited the paper.
SB: Provided advice in experimental design, critically discussed results, edited the paper.
Conflict of interest
MMH and SB are members of the editorial board. Therefore, they did not take part in the review process in any capacity and the submission was handled by a different member of the editorial board.
Acknowledgements
We thank Alexandra Bittroff-Leben, Ines Puschendorf, Sumaya Abdul-Rahman, Gernot Reifenberger, Nizar W. Shayya, and the staff of the animal research facility at FEM of Charité - University Medicine Berlin for excellent technical assistance and animal breeding.
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