Abstract
Background
Acute campylobacteriosis caused by oral infections with the enteropathogen Campylobacter jejuni represent serious threats to global human health. Since novel treatment options with safe and antibiotics-independent compounds would be highly appreciable, we here investigated the anti-bacterial and disease-alleviating effects of carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose in acute murine campylobacteriosis. To address this, secondary abiotic IL-10−/− mice were perorally infected with C. jejuni and treated with either compound alone or all four in combination via the drinking water starting two days post-infection.
Results
On day 6, the duodenal pathogen loads were lower in mice of the combination versus the vehicle treatment cohort. Importantly, mice treated with carvacrol and the combination presented with less distinct diarrheal symptoms, colonic histopathology, epithelial cell apoptosis, and immune cell responses when compared to vehicle counterparts on day 6 post-infection. Furthermore, the combination treatment did not only diminish colonic IFN-γ, TNF-α, and IL-6 secretion in C. jejuni infected mice, but also dampened extra-intestinal and even systemic pro-inflammatory cytokine concentrations to basal levels as measured in liver, kidneys, lungs, and serum samples.
Conclusions
Our preclinical placebo-controlled intervention trial provides evidence that the combined oral application of carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose alleviates acute campylobacteriosis in the vertebrate host.
Introduction
The foodborne pathogen Campylobacter jejuni causes millions of intestinal infections, which are rising worldwide and progress into a global burden with significant socioeconomic impact [1]. In 2021, campylobacteriosis was the most frequently reported zoonosis with over 127,000 confirmed cases in the European Union [2]. As member of the commensal gut microbiota in livestock, especially poultry, the Gram-negative Campylobacter bacteria can be transferred to humans through consumption of contaminated meat and other animal-derived products or surface waters [3]. Upon infection of the intestinal tract, distinct bacterial cell wall molecules such as lipo-oligosaccharide (LOS) trigger host immune responses. These LOS surface molecules interact with the mammalian Toll-like receptor-4 (TLR-4) and induce an exaggerated stimulation of the host immune system via inflammatory signaling cascades downstream of TLR-4, which additionally include the mammalian target of rapamycin (mTOR) modulated pathways [4, 5]. In this inflammatory scenario, innate immune cells including neutrophils, macrophages, and monocytes, but also adaptive T lymphocytes are recruited to the infected intestines and secrete pro-inflammatory mediators to combat the bacterial infection, but by the expenses of inducing cell damage [6, 7]. The resulting acute enterocolitis is characterized by fever, abdominal cramps, watery or bloody diarrhea, and malabsorption [8]. C. jejuni infection has been shown to directly induce gut epithelial cell damage by apoptosis and dissolution of the tight junctions [8, 9]. This cell destruction leads to the loss of water and nutrients into the intestinal lumen due to the so-called "epithelial leak flux" mechanism mounting in watery diarrhea [8–10]. C. jejuni induced symptoms usually resolve without residues after maximum two weeks post-infection (p.i.). In rare cases, post-infectious autoimmune diseases including the Guillain-Barré syndrome (GBS), reactive arthritis (RA), and intestinal diseases, such as inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) may occur after weeks to months following infection [11–14]. Importantly, the risk for the development of post-infectious collateral damages of campylobacteriosis is directly related to the severity of the initial enteritis [15]. Whereas most human C. jejuni infections require, if at all, symptomatic intervention measures such as analgetics, fluid and electrolyte replacement, antibiotic therapy may be required in severe and invasive C. jejuni induced enterocolitis affecting immune-compromised and multimorbid individuals [16, 17]. However, the effectiveness of antibiosis is significantly limited by progressive emergence of antibiotic resistance in Campylobacter strains to the commonly used quinolones and macrolides [18]. In consequence, the development of novel alternative, antibiotics-independent strategies to combat severe human campylobacteriosis has gained high importance. Natural compounds that exhibit a combination of both, anti-pathogenic and anti-inflammatory properties without inducing anti-microbial resistance constitute promising prophylactic and/or therapeutic options against enteropathogenic including Campylobacter infections [19].
Carvacrol is a phenolic monoterpenoid component of essential oils (EOs) obtained from several plants including black cumin, oregano, and thyme, for instance [20]. Previous studies provide evidence for the anti-microbial effects of carvacrol directed against food-borne pathogens such as Bacillus cereus, Escherichia coli, and Salmonella Typhimurium causing enteritis in infected humans [21]. Carvacrol possesses ATPase-inhibiting activity by reducing the cytoplasmic membrane's pH gradient, causing changes in the proton motive force and the adenosine triphosphate (ATP) pool in the Gram-positive B. cereus [22]. In Gram-negative bacteria such as Pseudomonas fragi and E. coli, for instance, carvacrol permeabilized the outer bacterial cell membrane, allowing essential molecules such as ATP to diffuse out of the cell resulting in cell death [23]. Notably, C. jejuni inhibiting effects were observed upon carvacrol application both, in vitro and in chicken in vivo, making carvacrol supplementation an attractive measure to prevent C. jejuni infection in livestock [24]. Furthermore, the reduced intestinal C. jejuni burdens and alleviated clinical signs observed in our previous preclinical intervention trial highlight the immune-modulatory properties of carvacrol in human campylobacteriosis [25].
Butyrate is the salt of butyric acid and one of the short-chain fatty acids (SCFAs) that are produced by various intestinal bacteria including Faecalibacterium prausnitzii, Oscillibacter species. and Clostridium cluster XIVa [26]. Butyrate is known for its anti-inflammatory properties that help maintain the orchestrated homeostasis between the SCFAs-producing gut commensal microbiota and mucosal immunity [27]. Furthermore, previous studies revealed that the therapeutic administration of butyrate alleviated inflammatory responses and clinical symptoms in patients suffering from colonic inflammation [27]. Other health-beneficial properties of butyrate are the epithelial barrier-preserving [28] and the anti-microbial effects exerted by enhancing expression of anti-microbial peptides such as cathelicidin LL-37 in colonic epithelial cells [28]. Furthermore, due to its disease-alleviating effects in C. jejuni infection, butyrate represents a promising antibiotic-independent therapeutic strategy for combating inflammatory infections [29] and showed synergistic effects in combination with other SCFAs [30].
Ellagic acid is a polyphenolic secondary plant substance, which mainly occurs as part of the complex ellagitannin molecules. Ellagic acid can be found in a variety of fruits (e.g., pomegranates), and nuts (e.g., walnuts, almonds) [31]. In the intestines, ellagic acid is metabolized by the commensal colonic microbiota to various urolithin derivatives with health-promoting properties including anti-microbial, anti-oxidative, and anti-inflammatory activities [32, 33]. For instance, the administration of ellagic acid-enriched pomegranate extract to rats suffering from experimental colitis resulted in suppressed expression of the inducible nitric oxide synthase (iNOS) and thus lowered concentrations of distinct pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α) [34]. Besides its anti-inflammatory effects, the anti-microbial activities of ellagic acid have also been suggested given that it can inactivate bacterial enzymes, adhesins, and cell membrane transport proteins [35].
The trisaccharide 2′-fucosyl-lactose constitutes a main component of human milk oligosaccharides (HMOs) and is abundant in breast milk [36]. Various health-promoting properties are attributed to the HMOs, which help the newborn to develop a healthy and well-balanced intestinal microbiota and immune system [37]. Furthermore, 2′-fucosyl-lactose has been demonstrated to inhibit the adhesion of distinct (opportunistic) pathogens including E. coli, Pseudomonas aeruginosa, and Campylobacter to the host epithelial cells [38, 39]. These anti-adhesive properties are due to the fact that 2′-fucosyl-lactose and its derivatives are structurally related to mucosal surface glycans, which act as receptors for pathogen binding to 2′-fucosyl-lactose in the glycocalyx and on cell surfaces via their adhesins [40]. Thus, the presence of orally applied free and unbound 2′-fucosyl-lactose in excess blocks pathogen binding to the epithelial receptors which in turn, suppresses inflammatory signaling cascades and supports clearance of the pathogen by intestinal motility [41]. In support, 2′-fucosyl-lactose application attenuated the adhesion of C. jejuni to human intestinal epithelial and mucosal cells in vitro that was accompanied by less distinct intestinal colonization by C. jejuni in mice [42]. Subsequently, C. jejuni-associated diarrhea was less frequently observed in infected infants when breast milk contained high concentrations of 2′-fucosyl-lactose [43]. Furthermore, HMOs were shown to exert potent immune-modulatory activities [36].
Our previous placebo-controlled preclinical intervention studies have furnished compelling evidence for the anti-pathogenic and immune-modulatory properties of distinct natural compounds, including carvacrol [25] and butyrate [29, 30]. These studies were performed in an acute murine campylobacteriosis model, employing secondary abiotic (SAB) interleukin (IL)-10 deficient mice. The successful colonization of C. jejuni within the murine gastrointestinal tract, coupled with the pathogen-induced pro-inflammatory immune responses rising in acute enterocolitis [44], was contingent upon prior depletion of the commensal murine gut microbiota through antibiotic pre-treatment and the absence of the anti-inflammatory il10 gene. Within a timeframe of 6 days p.i., C. jejuni infected SAB IL-10−/− mice displayed characteristic features of acute human campylobacteriosis, including bloody diarrhea and wasting symptoms, colonic mucosal aggregates of innate and adaptive immune cells, and enhanced secretion of pro-inflammatory mediators in intestinal, but also in extra-intestinal and even systemic organs [44]. In this study, we used the SAB IL-10−/− mouse model to assess the therapeutic potential of individual and combined agents, namely carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose, in alleviating acute campylobacteriosis, dissecting their collective disease-mitigating properties, including the anti-pathogenic and immune-modulatory effects.
Material and methods
Gut microbiota depletion in IL-10−/− mice
IL-10−/− mice (C57BL/6j background) were bred and maintained in the Forschungsinstitute für Experimentelle Medizin, Charité – Universitätsmedizin Berlin, Germany. Mice were housed in cages including filter tops within an experimental semi-barrier under standard conditions (i.e., 22–24 °C room temperature, 55 ± 15% humidity, 12 h light/12 h dark cycle) and had free access to autoclaved water (ad libitum) and standard chow (food pellets: ssniff R/M-H, V1534-300, Sniff, Soest, Germany). To eradicate the commensal gut microbiota, 3-week-old female and male mice were transferred to sterile cages (maximum of 3–4 animals per cage) immediately after weaning and received an antibiotic treatment with ampicillin plus sulbactam (2 g L−1 plus 1 g L−1, respectively; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany) added to the drinking water (ad libitum) as reported recently [45]. Two days before the C. jejuni infection, the antibiotic solution was replaced by autoclaved tap water.
C. jejuni infection
C. jejuni strain 81-176 bacteria were thawed from frozen stocks and grown on karmali agar plates (Oxoid, Wesel, Germany) under microaerophilic conditions at 37 °C for at least 48 h as described earlier [46]. Age- and sex-matched mice (3-month-old littermates) were infected with 109 colony-forming units (CFU) of the pathogen on days 0 and 1 by oral gavage (total volume of 0.3 mL).
Treatment regimens
The treatment regimens were initiated on day 2 p.i. and persisted until day 6 p.i. Carvacrol (Sigma-Aldrich, Munich, Germany), sodium butyrate (Merck, Darmstadt, Germany), ellagic acid (Carl Roth, Karlsruhe, Germany), and 2′-fucosyl-lactose (Chr. Hansen HMO GmbH, Rheinbreitbach, Germany) were dissolved in autoclaved tap water. To enhance the aqueous solubility of carvacrol and ellagic acid, the former was dissolved in Tween® 80 (2.5 mL L−1; Sigma-Aldrich, Munich, Germany), while the latter was dissolved in 2M sodium hydroxide (400 μL L−1 NaOH, Sigma-Aldrich, Munich, Germany). Control mice were administered respective vehicles in autoclaved tap water only. In consideration of a mean murine body weight of 25 g and an approximate daily drinking volume of 5 mL, the mice were subjected to the following drinking solutions; the daily dosages, final concentrations and previously assessed minimum inhibitory concentrations (MICs) are summarized in Table 1.
Treatment regimens and concentrations of the applied substances including vehicles
| Treatment | Daily Dose (mg kg−1) | Drinking Solution (mg L−1) | Minimum Inhibitory Concentration (mg L−1) |
| Vehicle | 400 | 2.6 | 40,033 |
| Carvacrol | 100 | 500 | 150 |
| Butyrate | 4,400 | 22,000 | 4,096 |
| Ellagic acid | 0.114 | 22.8 | 8 |
| 2-Fucosyl-lactose | 480 | 2,400 | 32,768 |
| Combination | 4,980 | 24,923 | 1,558 |
Gastrointestinal pathogen loads
The numbers of viable C. jejuni bacteria were quantified in fecal samples every day p.i. and additionally, upon necropsy (day 6 p.i.) in intraluminal gastrointestinal specimens. Therefore, samples were homogenized in sterile phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) with a sterile pestle and serial dilutions plated onto karmali agar (Oxoid, Wesel, Germany) and incubated under microaerophilic conditions in a jar at 37 °C for at least 48 h (CampyGas Packs; Oxoid, Wesel, Germany) as described earlier [46]. The detection limit of viable pathogens was 100 CFU per g.
Monitoring of clinical conditions of mice
Upon initiation of respective therapeutic treatment and furthermore, immediately before and every day after infection, we conducted quantitative evaluations of the murine clinical conditions using established clinical scoring methodologies as outlined and previously described (Table 2, [45]).
Clinical scoring system (maximum 12 points)
| Clinical aspect | Scores |
| Wasting symptoms | 0: normal |
| 1: ruffled fur | |
| 2: less locomotion | |
| 3: isolation | |
| 4: severely compromised locomotion, pre-final aspect | |
| Stool consistency | 0: formed feces |
| 2: pasty feces | |
| 4: liquid feces | |
| Fecal blood | 0: no blood |
| 2: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Krefeld, Germany | |
| 4: macroscopic blood visible |
Sampling procedures
Following the sacrifice of mice by carbon dioxide asphyxiation on day 6 p.i., cardiac blood was collected for subsequent cytokine analysis, and ex vivo biopsies from liver, kidneys, lungs, mesenteric lymph nodes (MLN), and colon as well as luminal samples from stomach, duodenum, ileum, and colon were removed under strict aseptic conditions.
Histopathology
Histopathological analyses were performed in colonic ex vivo biopsies that had been immediately fixed in 5% formalin and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin (H&E), examined by light microscopy (100-times magnification), and histopathological changes in the large intestines quantitatively assessed with histopathological scores [47]: Score 0, intact epithelium, no inflammatory cell infiltrates. 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
Quantitative in situ immunohistochemical analyses were performed in colonic ex vivo biopsies following immediate fixation in 5% formalin and embedding in paraffin as reported previously [48]. In brief, to detect apoptotic epithelial cells, neutrophils, T lymphocytes, regulatory T cells, and B lymphocytes, colonic paraffin sections (5 µm) were stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA; 1:200), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), CD3 (no. N1580, Dako, Glostrup, Denmark; 1:10), 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. Positively stained cells were quantitated by a blinded independent investigator applying light microscopy. The average number of respective positively stained cells in each sample was determined within at least six high power fields (HPF, 0.287 mm2, 400-times magnification).
Pro-inflammatory cytokines
Intestinal ex vivo biopsies collected from MLN (3 nodes) as well as from the colon (longitudinally cut strips of approximately 1 cm2), the liver (approximately 1 cm3), the kidney (one half after the longitudinal cut), and one lung were washed in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA) and 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) supplemented with penicillin (100 μg mL−1) and streptomycin (100 μg mL−1; Biochrom, Berlin, Germany). After an 18-h incubation period at 37 °C, respective culture supernatants and serum samples were tested for interferon-gamma (IFN-γ), TNF-α, and interleukin-6 (IL-6) by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Heidelberg, Germany) in a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany).
Statistical analyses
Medians and significance levels were calculated using GraphPad Prism (version 8; San Diego, CA, USA). Normalization of data was assessed by the Anderson-Darling test. For multiple comparisons, the one-way ANOVA with Tukey post-correction (for normally distributed data) and the Kruskal-Wallis test with Dunn's post-correction (for not normally distributed data) were performed. Two-sided probability (p) values ≤0.05 were considered statistically significant.
Ethics statement
All animal experiments were carried out according to the European animal welfare guidelines (2010/63/EU) following approval by the commission for animal experiments (“Landesamt für Gesundheit und Soziales”, LaGeSo, Berlin; registration number G0104/19). The clinical conditions of mice were monitored daily.
Results
C. jejuni counts in the gastrointestinal tract following oral treatment of infected SAB IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination
First, we investigated whether oral treatment of C. jejuni infected SAB mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or all in combination would interfere with the intestinal colonization capabilities of the enteropathogen. Therefore, we quantitated fecal C. jejuni numbers by culture. 24 h after the latest of two oral pathogen applications and hence, immediately before initiation of the treatment regimens on day 2 p.i., C. jejuni were detectable at high median numbers of approximately 109 live bacteria per gram feces in all groups versus vehicle (not significant (n.s.); Fig. 1). Furthermore, our cultural analyses over time revealed that irrespective of the four compounds applied alone or in combination, fecal C. jejuni numbers were comparable to those obtained from vehicle control mice on days 3, 4, and 5 p.i. (n.s.; Fig. 1). Upon sacrifice on day 6 p.i., we surveyed the C. jejuni loads in distinct gastrointestinal parts by culture (Fig. 2) and found similar luminal pathogen numbers in the stomach, the ileum, and the colon, irrespective of the treatment regimen (n.s. versus vehicle; Fig. 2A, C and D). In the duodenum of combination treated mice, however, median C. jejuni counts were ca. 1.5 to 2.5 log lower if compared to mice from the ellagic acid, the 2′-fucosyl-lactose, and the vehicle treatment groups (P < 0.05–0.01; Fig. 2B). Of note, the vehicle solution alone did not exert anti-C. jejuni directed effects as shown in vitro (Table 1). Hence, the combined oral treatment of C. jejuni infected SAB IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose exclusively lowered the pathogen loads in the duodenum.
Fecal C. jejuni counts over time following oral treatment of infected secondary abiotic IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. Secondary abiotic IL-10−/− mice were orally infected with C. jejuni 81-176 strain on day (d) 0 and d1 and treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on d2. C. jejuni numbers were enumerated in cultured fecal samples taken on (A) d2, (B) d3, (C) d4, and (D) d5 and indicated as colony-forming units per gram (CFU/g). Medians (black bars) and mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
C. jejuni counts in the gastrointestinal tract following oral treatment of infected secondary abiotic IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. Secondary abiotic IL-10−/− mice were orally infected with C. jejuni 81-176 strain on day (d) 0 and d1 and treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on d2. Placebo (PLC) control mice received vehicle only. Following sacrifice on d6, C. jejuni numbers were enumerated in cultured samples taken from the (A) stomach, (B) duodenum, (C) ileum, and (D) colon and indicated as colony-forming units per gram (CFU/g). Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test and Dunn's post-correction, and numbers of culture-positive mice out of the total cohort (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Clinical signs of campylobacteriosis following oral treatment of infected mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Next, we analyzed whether the oral treatment regimens would improve the clinical outcome in infected mice despite comparable fecal C. jejuni burdens. Therefore, we quantitated the acute campylobacteriosis symptoms with a total clinical score assessing wasting, stool consistency, and abundance of fecal blood over time p.i. (Fig. 3). Twenty-four hours after the latest C. jejuni challenge, higher total clinical scores were obtained in all infected groups when compared to uninfected control mice (P < 0.01–0.001; Fig. 3A). On day 5 p.i., however, butyrate and combination treated mice suffered less distinctly from C. jejuni infection as compared to vehicle mice as indicated by lower total clinical scores in the former versus the latter (P < 0.05–0.01; Fig. 3D). At the end of the experiment, carvacrol treated mice presented with lower total clinical scores if compared to vehicle counterparts (P < 0.05; Fig. 4A). In the case of butyrate and combination treated mice, there was at least a trend towards lower total clinical scores versus vehicle counterparts that did not reach statistical significance in the multivariate analyses due to high standard deviations (n.s.; Fig. 4A). Notably, lower scores for diarrheal symptoms were obtained in the carvacrol and combination groups when compared to vehicle treated mice (P < 0.01; Fig. 4C), which was also the case in the combination cohort versus the ellagic acid and 2′-fucosyl-lactose groups (P < 0.01–0.001; Fig. 4C). Of note, in the carvacrol, butyrate, and combination treatment cohorts, the diarrheal scores did not differ from uninfected mice (n.s. versus naive; Fig. 4C). In the case of wasting symptoms and abundance of fecal blood, the scores were all elevated in comparison to naive controls (P < 0.01–0.001; Fig. 4B and D), but did not differ between the treatment cohorts on day 6 p.i. (n.s.; Fig. 4B and D). Hence, application of carvacrol and the combination to infected mice alleviated diarrheal symptoms in acute campylobacteriosis.
Clinical campylobacteriosis signs over time following oral treatment of C. jejuni infected secondary abiotic IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. Secondary abiotic IL-10−/− mice were orally infected with C. jejuni 81-176 strain on day (d) 0 and d1 and treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on d2 or received placebo (PLC; vehicle only). Naive mice (N) served as untreated and uninfected controls. Clinical signs of campylobacteriosis such as wasting and bloody diarrhea were quantitated with defined clinical scores (see methods) on (A) d2, (B) d3, (C) d4, and (D) d5. Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test and Dunn's post-correction and mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Clinical signs of campylobacteriosis following oral treatment of C. jejuni infected secondary abiotic IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. Following oral C. jejuni infection on day (d) 0 and d1, secondary abiotic IL-10−/− mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on d2. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. Before sacrifice on d6, clinical signs of campylobacteriosis were quantitated with a (A) total clinical score assessing (B) wasting, (C) diarrhea, and (D) fecal blood. Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test and Dunn's post-correction, and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Macroscopic and microscopic inflammatory signs following oral treatment of C. jejuni infected mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Since intestinal inflammation leads to a shrinkage of the affected part of the intestinal tract [44], we determined the colonic lengths of mice at the end of the experiment. Whereas on day 6 p.i. lower values were obtained in all infected groups when compared to uninfected counterparts (P < 0.001 versus naive; Fig. 5A), the colonic lengths in the treatment cohorts were comparable to those measured in the vehicle controls (n.s.; Fig. 5A). Interestingly, the colons were longer in the combination group when compared to those from butyrate, ellagic acid, and 2′-fucosyl-lactose treated mice indicative for less macroscopic inflammatory signs of C. jejuni infection following combination treatment (P < 0.05–0.001; Fig. 5A). Furthermore, we quantitated the C. jejuni induced microscopic tissue destruction with histopathological scores and found lower values in the carvacrol and combination groups when compared to vehicle controls on day 6 p.i. (P < 0.001; Fig. 5B). Moreover, the histopathological scores in the combination cohort were also lower when compared to those obtained from butyrate, ellagic acid, and 2′-fucosyl-lactose treated mice (P < 0.05–0.001; Fig. 5B). Furthermore, we counted apoptotic colonic epithelial cells given that apoptosis constitutes a reliable parameter used for the grading of intestinal inflammatory diseases including campylobacteriosis [46]. On day 6 p.i., C. jejuni induced increases in apoptotic colonic epithelial cell counts were less pronounced in mice from the carvacrol, the butyrate, and the combination treatment groups as compared to vehicle controls (P < 0.05–0.001; Fig. 5C). Remarkably, apoptotic cell numbers did not differ in colonic epithelia of infected mice from the combination cohort and naive control mice (n.s.; Fig. 5C). Hence, treatment with carvacrol, butyrate, and the combination of the four compounds alleviated microscopic C. jejuni induced inflammatory changes in the colon during murine campylobacteriosis.
Macroscopic and microscopic inflammatory signs following oral treatment of C. jejuni infected IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. C. jejuni infected secondary abiotic IL-10−/− mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on day 2 post-infection. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. On day 6, (A) the colonic lengths were measured (in cm) and (B) the microscopic inflammatory changes of the colon assessed with a histopathological score (see methods). Furthermore, (C) the apoptotic colonic epithelial cell numbers were quantitated in paraffin sections of colonic explants stained with cleaved caspase 3 (Casp3) and indicated as average numbers of six representative high-power fields (HPF, 400-times magnification). Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test with Dunn's post-correction (A, B) and the one-way ANOVA with Tukey's post-correction (C), and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Colonic immune cell populations in C. jejuni infected mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Next, we surveyed the effect of the treatment regimens on the C. jejuni induced immune cell responses. Therefore, we applied in situ immunohistochemical stainings of colonic paraffin sections with antibodies directed against distinct innate and adaptive immune cell populations (Fig. 6). On day 6 p.i., mice from the carvacrol and the combination treatment groups exhibited lower MPO7+ neutrophil numbers in their colonic mucosa and lamina propria when compared to vehicle counterparts (P < 0.001; Fig. 6A), which also held true for CD3+ T lymphocytes (P < 0.001; Fig. 6B). Remarkably, the colonic neutrophil and T cell counts in carvacrol and combination treated mice were comparable to those obtained from uninfected control animals (n.s. versus naive; Fig. 6A and B). Furthermore, the combination treatment resulted in less pronounced C. jejuni induced increases in colonic neutrophil and T lymphocyte numbers if compared to butyrate and 2′-fucosyl-lactose application (P < 0.05–0.01; Fig. 6A and B). Irrespective of the treatment regimen, however, B220+ B lymphocyte counts were elevated in the colon taken on day 6 p.i. (P < 0.01–0.001 versus naive; Fig. 6D). This was also the case when determining FOXP3+ regulatory T cells that were increased in the colonic mucosa and lamina propria of all single treatment groups including vehicle controls (P < 0.05–0.001; Fig. 6C), but not the combination treatment cohort (n.s. versus naive; Fig. 6C). Hence, the combination of all four compounds resulted in dampened C. jejuni induced innate and adaptive immune cell responses.
Colonic immune cell populations in C. jejuni infected secondary abiotic IL-10−/− mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. C. jejuni infected mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on day 2 post-infection. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. On day 6, (A) neutrophils (MPO7+), (B) T lymphocytes (CD3+), (C) regulatory T cells (FOXP3+), and (D) B lymphocytes (B220+) were counted in paraffin sections of large intestinal explants stained with respective antibodies and indicated as average numbers out of six representative high-power fields (HPF, 400-times magnification). Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test with Dunn's post-correction, and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Colonic pro-inflammatory cytokine secretion in infected mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
In addition, we assessed pro-inflammatory cytokine secretion in colonic ex vivo biopsies following treatment of infected mice. As for colonic neutrophil and T lymphocyte numbers, C. jejuni infection enhanced the secretion of IFN-γ, TNF-α, and IL-6 secretion in the colon samples taken from mice of the vehicle, the butyrate, the ellagic acid, and the 2′-fucosyl-lactose groups (P < 0.05–0.001 versus naive; Fig. 7). In the carvacrol and the combination treatment cohorts, however, basal cytokine values were detected on day 6 p.i. (n.s. versus naive; Fig. 7). When compared to vehicle, lower colonic IFN-γ concentrations were obtained from carvacrol and combination treated mice (P < 0.05 and P < 0.001, respectively; Fig. 7A), which was also the case for IL-6 in the colon taken from the latter on day 6 p.i. (P < 0.05 versus vehicle; Fig. 7C). Furthermore, IFN-γ, TNF-α, and IL-6 concentrations were even lower in colonic explants taken from mice subjected to the combination treatment when compared to butyrate, ellagic acid, and 2′-fucosyl-lactose single applications (P < 0.05–0.001; Fig. 7). Hence, the combination treatment resulted in diminished C. jejuni induced pro-inflammatory cytokine concentrations in the colon.
Colonic pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10−/− mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. C. jejuni infected mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on day 2 post-infection. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. (A) IFN-γ, (B) TNF-α, and (C) IL-6 concentrations were measured in colonic explants taken on day 6. Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test with Dunn's post-correction, and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Extra-intestinal pro-inflammatory cytokines in infected mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Further, we addressed whether the treatment regimens affected C. jejuni induced cytokine secretion in extra-intestinal organs such as the liver, the kidneys, and the lungs (Fig. 8). On day 6 p.i., hepatic IFN-γ and TNF-α concentrations were lower upon combination as compared to vehicle treatment (P < 0.001 and P < 0.05, respectively; Fig. 8A and B), which also was the case when measuring renal IFN-γ and pulmonal TNF-α concentrations (P < 0.001 and P < 0.05, respectively; Fig. 8C and F). Furthermore, mice from the carvacrol group exhibited less pronounced IFN-γ secretion in the kidneys as compared to vehicle counterparts on day 6 p.i. (P < 0.05; Fig. 8C). Compared to single butyrate, ellagic acid, and 2′-fucosyl-lactose applications, lower hepatic and renal IFN-γ as well as renal TNF-α concentrations were determined following the combination treatment of mice (P < 0.05–0.001; Fig. 8A, C and D). Notably, mice from the combination treatment group presented with basal IFN-γ concentrations measured in the liver and kidneys on day 6 p.i. (n.s. versus naive; Fig. 8A and C), which also held true for hepatic and pulmonal TNF-α concentrations (n.s. versus naive; Fig. 8B and F). Hence, the combination treatment also lowered extra-intestinal cytokine concentrations in C. jejuni infected mice.
Extra-intestinal pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10−/− mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. C. jejuni infected mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on day 2 post-infection. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. IFN-γ (A,C,E) and TNF-α (B,D,F) concentrations were measured in liver (A,B), kidney (C,D), and lung (E,F) explants taken on day 6. Medians (black bars), significance levels (P values) determined by the Kruskal Wallis test with Dunn's post-correction, and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Systemic pro-inflammatory cytokines in C. jejuni infected mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Then, we assessed the impact of the treatment regimens on the systemic cytokine secretion. On day 6 p.i., IFN-γ, TNF-α, and IL-6 concentrations were lower in serum samples derived from mice of the combination treatment cohort versus the vehicle counterparts (P < 0.05–0.01; Fig. 9), which was also the case when measuring serum IL-6 concentrations in carvacrol treated mice (P < 0.05; Fig. 9C). Remarkably, both, the carvacrol and the combination treatment could dampen C. jejuni induced systemic IFN-γ, TNF-α, and IL-6 secretions to basal levels (n.s. versus naive; Fig. 9).
Systemic pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10−/− mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. C. jejuni infected mice were treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on day 2 post-infection. Placebo (PLC) control mice received vehicle only. Naive mice served as untreated and uninfected controls. (A) IFN-γ, (B) TNF-α, and (C) IL-6 concentrations were measured in serum samples taken on day 6. Medians (black bars), significance levels (p values) determined by the Kruskal Wallis test with Dunn's post-correction, and the mouse numbers (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Bacterial translocation in C. jejuni infected mice following oral treatment with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose
Finally, we asked whether the treatment regimens would impact bacterial translocation from the infected intestines to extra-intestinal organs. Our cultural analyses of ex vivo biopsies taken from the liver, the kidneys, and the lungs on day 6 p.i. revealed C. jejuni growth in single cases only, with no differences in pathogen counts when comparing the infected cohorts with each other (n.s.; Fig. 10). Furthermore, all cardiac blood samples were free of viable C. jejuni bacteria (data not shown). The results of our placebo-controlled intervention trial are summarized in Table 3.
Extra-intestinal C. jejuni counts following oral treatment of infected secondary abiotic IL-10−/− mice with carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose either alone or in combination. Secondary abiotic IL-10−/− mice were orally infected with C. jejuni 81-176 strain on day (d) 0 and d1 and treated with carvacrol (CAR), butyrate (BUT), ellagic acid (EGA), and 2′-fucosyl-lactose (2-FL) either alone or all in combination (COMBI) via the drinking water (ad libitum) starting on d2. Placebo (PLC) control mice received vehicle only. Following sacrifice on d6, C. jejuni numbers were enumerated in cultured samples taken from the (A) liver, (B) kidney, and (C) lung and indicated as colony-forming units per gram (CFU/g). Medians (black bars) and numbers of culture-positive mice out of the total cohort (in parentheses) are shown from four independent experiments
Citation: European Journal of Microbiology and Immunology 13, 3; 10.1556/1886.2023.00037
Summary of the results
| Treatment | Results (versus placebo) |
| Carvacrol |
|
| Butyrate |
|
| Ellagic acid | No significant effect |
| 2′-Fucosyl-lactose | No significant effect |
| Combination |
|
Discussion
Since the prevalence of Campylobacter infections is rising all around the globe [1], developing antibiotic-independent intervention strategies for the prevention and treatment of campylobacteriosis and finding alternative measures to limit antibiotic resistance in both, livestock and humans are necessary. Most importantly, novel treatment options alleviating the severity of the initial inflammatory enteritis would reduce the risk for the onset of secondary autoimmune sequelae of C. jejuni infection such as GBS, RA, IBD, and IBS [11–14]. Therefore, we assessed potential disease-alleviating effects of therapeutic oral application of carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose alone or all four in combination in acute murine campylobacteriosis. The applied compounds are listed as “generally recognized as safe” by the U.S. Food and Drug Administration [49]. Despite reported anti-microbial effects for the applied substances against various bacteria such as group B streptococci, Helicobacter pylori, E. coli, Salmonella Typhimurium, and C. jejuni in vitro [24, 38, 50–52], the daily quantification of intestinal C. jejuni loads revealed comparably high fecal bacterial numbers in all groups, which also held true for similar luminal pathogen counts in the stomach, the ileum, and the colon assessed upon sacrifice of mice. Although carvacrol did not lower gastrointestinal C. jejuni numbers when administered therapeutically (i.e, starting 2 days following infection), our previous study showed that the prophylactic application of carvacrol to SAB IL-10−/− mice at the same concentration like in the present study significantly lowered pathogen numbers in the ileal and colonic lumen at day 6 p. i. [25]. This provides evidence that oral carvacrol may prevent stable C. jejuni colonization when administered before oral infection, but did not exhibit sufficient anti-pathogenic effects when the C. jejuni bacteria had already occupied the ecological niches in the gastrointestinal tract. In support, also oral butyrate did not lower intestinal C. jejuni loads when starting treatment 2 days p.i. [29], which in addition, might have been due to its short half-life [53]. Although it was demonstrated by others that 2′-fucosyl-lactose treatment in antibiotic-pretreated C. jejuni infected mice reduced pathogen colonization and induced inflammation [52], we did not observe anti-Campylobacter effects of 2′-fucosyl-lactose in the applied SAB IL-10−/− campylobacteriosis model. Different aspects could explain the earlier observations: i) the presence of the anti-inflammatory cytokine IL-10; ii) the higher applied daily dose of 2′-fucosyl-lactose (5.0 g L−1 versus 2.4 g L−1 in our study); and iii) the application in parallel to the infection. One cannot exclude that the applied compounds would have exerted significant pathogen-lowering effects upon a longer-term treatment period following infection. This could, however, not be tested for ethical reasons given the severity of acute enterocolitis reaching humane endpoints by day 6 p.i.
Moreover, in our study, we applied carvacrol, butyrate, and ellagic acid at concentrations that were higher than the measured MICs, whereas the 2′-fucosyl-lactose concentration was far below its MIC (Table 1). It is, however, highly likely that the intraluminal intestinal and hence, biologically active concentrations of the substances even when applied in concentrations exceeding the in vitro measured MICs by far, were much lower in the infected intestinal lumen due to mixing and diluting with the secretory intestinal fluids resulting in a rather subtle to absent antibacterial effect. Interestingly, the combination of carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose exclusively lowered the pathogen numbers in the duodenum. It is tempting to speculate that this phenomenon might be explained by the interactions of the compounds within the specific luminal milieu of the duodenum characterized by an alkaline pH and the abundance of pancreatic enzymes and bile acids, for instance, which in concert may have contributed to an eradication of the pathogen in more than 50% of the infected mice. Furthermore, it can be assumed that due to the absence of the commensal gut microbiota in the SAB mouse model used, ellagic acid could not exhibit any antibacterial effect, since it neither could be metabolized to its active forms nor have acted in a prebiotic fashion on the intestinal microbiota with consequences for the pathogen-host interactions.
With respect to the reported anti-inflammatory effects of the substances used here, we quantitated macroscopic inflammatory signs of infection including diarrhea in line with microscopic inflammatory parameters such as histopathological changes and apoptotic cell responses in the colonic mucosa. Despite the absence of a significant pathogen-lowering effect in the lower intestines of C. jejuni infected mice, oral application of carvacrol and of all four compounds in combination did not only alleviate diarrheal symptoms, but also prevented from any diarrhea development in approximately 80% of infected mice. This could be confirmed by our previous study showing potent anti-diarrheal effects of prophylactic oral carvacrol in C. jejuni infected SAB IL10−/− mice [25]. When further dissecting the disease-alleviating effects of the applied compounds on the microscopic level, we found that mice treated with carvacrol alone and its combination with butyrate, ellagic acid, and 2′-fucosyl-lactose displayed less severe histopathological changes in the infected colon when compared to vehicle. Furthermore, application of carvacrol, butyrate, and the combination of the four compounds dampened C. jejuni induced apoptotic changes in the colonic epithelia. Remarkably, the numbers of apoptotic colonic epithelial cells in mice from the combination cohort did not differ from those in uninfected controls. Our results are supported by previous studies demonstrating potent gut epithelial barrier-preserving properties for carvacrol and butyrate due to anti-apoptotic and anti-inflammatory actions as indicated by less C. jejuni-induced apoptosis and secretion of pro-inflammatory mediators such as IFN-γ and TNF-α in colonic ex vivo biopsies [25, 29, 54]. This would in turn alleviate the leaky gut syndrome observed in acute intestinal morbidities including campylobacteriosis characterized by watery diarrhea and malabsorption leading to the loss of water, electrolytes, and nutrients into the intestinal lumen [8–10].
The improved clinical outcome and alleviated microscopic inflammatory including apoptotic sequelae of C. jejuni infection in carvacrol and combination treated mice were paralleled by dampened pro-inflammatory immune responses in the colon on day 6 p.i., as indicated by less accumulation of neutrophils and T lymphocytes in the large intestinal mucosa and lamina propria and by diminished colonic secretion of pro-inflammatory cytokines such as IFN-γ, TNF-α, and IL-6. Remarkably, pro-inflammatory immune cell numbers as well as cytokine concentrations in the colon of carvacrol and combination treated mice did not differ from basal values. In support, not only the prophylactic oral application of carvacrol to SAB IL-10−/− mice [25], but also the therapeutic treatment of C. jejuni infected IL-10−/− mice harboring a human gut microbiota [54] underscored the disease-alleviating and anti-inflammatory properties of the phenolic compound. Further in vivo studies confirmed the inhibitory effects of carvacrol on the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 in the intestinal tract during both, lipo-polysaccharide- (LPS-) and acetic acid-induced inflammation [55–57].
The anti-inflammatory effects of carvacrol particularly when given in combination with butyrate, ellagic acid, and 2′-fucosyl-lactose were not limited to the intestines. In fact, also in extra-intestinal organs including the liver, the kidneys, and the lungs attenuated concentrations of pro-inflammatory cytokines such as IFN-γ and TNF-α were measured on day 6 p.i. that were similar to basal concentrations. Strikingly, this was also the case systemically given basal IFN-γ, TNF-α, and IL-6 serum concentrations determined in carvacrol and combination treated C. jejuni infected mice. In support, carvacrol showed anti-inflammatory effects in LPS-induced lung damage and endotoxemia, presumably via the inhibition of NF-κB signaling [55]. In support, our previous study revealed an attenuated secretion of IFN-γ and TNF-α in the liver, kidneys, lungs, and serum, as well as of IL-6 in serum, when C. jejuni infected IL-10−/− mice were pre-treated with carvacrol [25]. In summary of the assessed parameters (Table 3), the most prominent effects following quadruple treatment of diseased mice could be attributed to carvacrol given that in the multi-variate analyses no significant differences were obtained between the carvacrol and the combination cohorts. When comparing with the vehicle controls, however, significantly lower duodenal C. jejuni numbers, lower colonic and renal IFN-γ as well as serum IL-6 concentrations were detected upon the combination treatment, but not following carvacrol application alone. Which compound within the mixture was responsible for the additional anti-inflammatory effects remains unanswered, however. The absence of the intestinal microbiota may explain the lack of anti-inflammatory effects of ellagic acid and 2′-fucosyl-lactose. Whereas ellagic acid is metabolized by the colonic commensal microbes into the bioactive urolithins [58, 59], 2′-fucosyl-lactose acts as a prebiotic and is used by bacterial groups such as Bifidobacterium and Lactobacillus species as nutrients, supporting intestinal colonization by a “healthy” gut microbiota [60–62]. This hypothesis can be supported by other studies. For instance, ellagic acid treatment did not only eradicate H. pylori from murine stomach tissue (in the presence of lactobacilli), but also enhanced the repair of pathogen-induced gastric mucosal damage in H. pylori infected mice colonized with a commensal gastrointestinal microbiota [50]. Furthermore, when conventionally colonized IL-10−/− mice were treated with 2′-fucosyl-lactose, not only a shift in the gut microbiota composition, but also a decreased severity of spontaneous chronic microbiota-induced enterocolitis were observed, characterized by reduced inflammatory responses, histopathological, and diarrhea scores [63]. Therefore, the disease-alleviating effects of ellagic acid and 2′-fucosyl-lactose should be further addressed in mice harboring a commensal murine or even a human gut microbiota.
Conclusions
Our actual preclinical placebo-controlled intervention trial provides evidence that the oral treatment of mice suffering from acute campylobacteriosis with a combination of carvacrol, butyrate, ellagic acid, and 2′-fucosyl-lactose alleviates diarrheal symptoms that are accompanied by enhanced anti-inflammatory immune responses in intestinal, extra-intestinal, and systemic organs. In consequence, these novel intervention options might provide great benefit to lower the risk for the development of secondary sequelae of campylobacteriosis including GBS, IBD, IBS, and RA. Future studies should address further antibiotics-independent approaches for the treatment and/or prophylaxis of food-borne diseases caused by Campylobacter species and other enteropathogens.
Declarations
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
SM: Performed experiments, analyzed data, critically discussed results, co-wrote the paper.
LVB: Performed experiments, analyzed data.
RB: Performed experiments, analyzed data.
NWS: Performed experiments.
SB: Provided advice in experimental design, critically discussed results.
MMH: Designed and performed experiments, analyzed data, critically discussed results, wrote the paper.
Conflict of interests
SB and MMH are members of the Editorial Board of the journal. 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, Ulrike Fiebiger, Sumaya Abdul-Rahman, Gernot Reifenberger, and the staff of the animal research facility at the FEM of Charité – Universitätsmedizin Berlin for excellent technical assistance and animal breeding.
List of abbreviations
| ATP | adenosine triphosphate |
| CBA | cytometric bead assay |
| CFU | colony-forming units |
| EO | essential oil |
| GBS | Guillain-Barré syndrome |
| HMOs | human milk oligosaccharides |
| H&E | hematoxylin and eosin |
| IBD | inflammatory bowel diseases |
| IBS | irritable bowel syndrome |
| IFN-γ | interferon-gamma |
| IL | interleukin |
| iNOS | inducible nitric oxide synthase |
| LOS | lipo-oligosaccharide |
| LPS | lipo-polysaccharide |
| MIC | minimum inhibitory concentration |
| MLN | mesenteric lymph nodes |
| mTOR | mammalian target of rapamycin |
| NaOH | sodium hydroxide |
| NF-κB | nuclear factor kappa-light-chain-enhancer of activated B cells |
| n.s. | not significant |
| PBS | phosphate-buffered saline |
| p.i. | post infection |
| RA | reactive arthritis |
| SAB | secondary abiotic |
| SCFA | short-chain fatty acid |
| TLR-4 | Toll-like receptor-4 |
| TNF-α | tumor necrosis factor-alpha |
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