Abstract
Human infections with the food-borne zoonotic enteropathogen Campylobacter jejuni are increasing globally. Since multi-drug resistant bacterial strains are further on the rise, antibiotic-independent measures are needed to fight campylobacteriosis. Given its anti-microbial and anti-inflammatory properties the polyphenolic compound resveratrol constitutes such a promising candidate molecule. In our present placebo-controlled intervention trial, synthetic resveratrol was applied perorally to human gut microbiota-associated (hma) IL-10−/− mice starting a week before oral C. jejuni infection. Our analyses revealed that the resveratrol prophylaxis did not interfere with the establishment of C. jejuni within the murine gastrointestinal tract on day 6 post-infection, but alleviated clinical signs of campylobacteriosis and resulted in less distinct colonic epithelial apoptosis. Furthermore, oral resveratrol dampened C. jejuni-induced colonic T and B cell responses as well as intestinal secretion of pro-inflammatory mediators including nitric oxide, IL-6, TNF-α, and IFN-γ to basal levels. Moreover, resveratrol application was not accompanied by significant shifts in the colonic commensal microbiota composition during campylobacteriosis in hma IL-10−/− mice. In conclusion, our placebo-controlled intervention study provides evidence that prophylactic oral application of resveratrol constitutes a promising strategy to alleviate acute campylobacteriosis and in consequence, to reduce the risk for post-infectious autoimmune sequelae.
Introduction
The incidences of human Campylobacter infections are rising all around the world and are responsible for the enteritis syndrome campylobacteriosis causing tremendous health care and economical costs [1–3]. The microaerobic and non-spore forming Gram-negative Campylobacter jejuni bacteria belong to the Campylobacteraceae family and reside as commensals in the intestinal tract of warm-blooded vertebrate species including birds usually without causing significant clinical signs [4, 5]. Humans, however, can become infected via the food chain by ingestion of contaminated undercooked meat derived from poultry or other livestock, of unpasteurized milk and derived unprocessed products, and of surface waters [6]. In the acute phase of infection, the highly motile enteropathogens invade the distal intestinal tissues after successful gastro-duodenal passage and distinct bacterial cell wall molecules including lipo-oligosaccharide (LOS) induce the recruitment of both, innate as well as adaptive immune cells such as neutrophils and T lymphocytes, respectively, to the site of infection [7, 8]. The subsequent pro-inflammatory mediator storm aims at limiting the infection, but by the expenses of causing harm to the intestinal tissues. In consequence, oxidative stress and apoptotic cell responses, ulcerations, and crypt abscesses can be observed resulting in a malabsorption syndrome [9–11]. After an incubation period of approximately 2–5 days, infected individuals present with symptoms of varying severities, depending on both, the arsenal of virulence factors expressed by the enteropathogen and the immunological fitness of the human host [12–14]. Patients may complain about nausea, vomiting, and abdominal cramps, as well as of watery or even bloody diarrhea with mucous discharge and fever, and usually fully recover within two weeks post-infection (p.i.) [12, 13]. In rare instance, however, post-infectious autoimmune morbidities affecting the central nervous system (e.g. Guillain Barré syndrome), the joints (e.g., reactive arthritis), and the intestinal tract (i.e., irritable bowel syndrome, celiac disease, chronic inflammatory bowel diseases) might develop with a latency of a few weeks to months [3, 13, 15]. Interestingly, the risk for developing post-infectious sequelae directly depends on the severity of the preceding enteritis event due to the sialylation status of the C. jejuni-LOS [16]. For the treatment of campylobacteriosis patients, usually symptomatic measures including analgetic, antipyretic, and spasmolytic drugs, as well as rehydration and electrolyte substitutions are indicated. In severely compromised patients such as immune-suppressed individuals, for instance, treatment with antibiotics including erythromycin or ciprofloxacin may be indicated [13, 17]. Successful treatment of severe campylobacteriosis cases in critically ill patients can, however, be hampered by the rising incidence of infections with multi-drug resistant C. jejuni strains [17]. Therefore, antibiotic-independent disease-alleviating treatment options with non-toxic compounds are utmost wanted.
Plant-derived molecules such as polyphenols including the 3,5,4′-trihydroxy-trans-stilbene resveratrol might be such promising candidate molecules. Resveratrol can be found, for instance, in the skin of red wine grapes and in various fruits and vegetables including berries, peanuts, and tomatoes where it acts as phytoalexin protecting against pathogenic fungi and bacteria and furthermore, in the traditional Asian remedy Reynoutria japonica [18–20]. Previous in vitro and in vivo studies provided evidence for pleiotropic health-promoting and disease-alleviating properties of synthetic resveratrol including cardio- and neuro-protective, anti-cancer, anti-dyslipidemic, anti-diabetic, anti-microbial, anti-oxidant, anti-inflammatory, and even anti-aging effects [18–20]. This prompted us to test the potential disease-alleviating properties of prophylactically applied resveratrol in experimental acute campylobacteriosis applying human gut microbiota-associated (hma) IL-10−/− mice. Therefore, secondary abiotic IL-10−/− mice generated by antibiotic gut microbial depletion were subjected to oral transplantations of a complex gut microbiota derived from human fecal donors [21, 22]. Our recent placebo-controlled intervention study where we tested the therapeutic effects of the phenolic compound carvacrol [23] further underscored that the hma IL-10−/− mouse model does not only provide a reliable experimental tool to dissect the triangular relationship between C. jejuni, vertebrate host immunity, and the human gut microbiota but also constitutes a valuable in vivo model to test novel interventive (i.e., therapeutic and/or prophylactic) measures in a preclinical setting [21]. In our present study we orally applied synthetic resveratrol to hma IL-10−/− mice in a prophylactic regimen starting a week before C. jejuni infection and assessed its impact on i.) gastrointestinal pathogen loads, ii.) clinical conditions, iii.) microscopic inflammatory changes in the colon, iv.) intestinal pro-inflammatory immune responses, and v.) shifts in the gut microbiota composition during acute campylobacteriosis.
Material and methods
Microbiota depletion in conventional IL-10−/− mice
In the Forschungsinstitute für Experimentelle Medizin, Charité – Universitätsmedizin Berlin, Germany, IL-10−/− C57BL/6j mice were bred and reared under specified pathogen-free (SPF) and standard settings. Mice were housed 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 subjected to an antibiotic regimen of ampicillin plus sulbactam (2 g/l; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany) via the drinking water for eight weeks (ad libitum) as described recently [22, 24]. Mice were handled under strict aseptic conditions to avoid contaminations and assure successful microbiota depletion. Two days before human FMT (day -16; Fig. 1), the antibiotic treatment was withdrawn and replaced by autoclaved water (ad libitum).
Experimental time line.
Conventionally colonized IL-10−/− mice were orally treated with antibiotics for 8 weeks in order to eradicate the commensal gut microbiota. Two days before human fecal microbiota transplantation (hFMT), the antibiotics were replaced by sterile drinking water (day (d)-16). The microbiota-depleted IL-10−/− mice were orally subjected to human FMT on d-14, d-13, and d-12. Synthetic resveratrol was applied to the human microbiota-associated (hma) IL-10−/− mice via the drinking water (ad libitum) starting 7 days before oral infection with Campylobacter jejuni strain 81-176 on d0 and d1 by gavage. Mice were finally dissected on d6
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Human fecal microbiota transplantation
In order to introduce a complex human intestinal microbiota into the murine host, the secondary abiotic 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. 1) as described in more detail earlier [22]. Therefore, human fecal samples that had been collected from 5 healthy individuals (all samples free of enteropathogenic bacteria, viruses, and parasites), aliquoted and stored at −80°C were thawed, resuspended in sterile phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA), and pooled before oral application to mice via gavage (0.3 mL volume). The microbiota composition of the suspensions used for the hFMT is shown in Fig. 2.
Microbiota composition of human fecal donor transplants.
The microbiota composition of the human fecal donor suspensions (n = 3 for each experiment) was quantitatively surveyed by culture-independent molecular analyses. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes) 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 are indicated (expressed as gene copies per ng DNA. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Prophylactic regimen
Prophylactic application of synthetic resveratrol (Sigma-Alderich, München, Germany) was initiated seven days before C. jejuni infection (i.e., d-7; Fig. 1). Therefore, resveratrol was dissolved in 2% carboxy-methyl-cellulose (to a final concentration of 0.05%), and administered to mice via the autoclaved tap water (ad libitum). The final concentrations of the resveratrol solution was 300 mg L−1, resulting in daily treatment dosages of 60 mg per kg body weight. Placebo counterparts received vehicle only.
C. jejuni infection
Viable C. jejuni strain 81-176 bacteria stored at −80 °C were thawed, streaked out, and incubated on selective karmali agar plates (purchased from Oxoid, Wesel, Germany) at 37 °C for 48 h under microaerophilic conditions. In order to generate an inoculum of 109 bacterial cells, bacteria were harvested in sterile PBS. 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 on two consecutive days (i.e., on d0 and d1; Fig. 1) by gavage as state in detail elsewhere [25].
Gastrointestinal C. jejuni loads
For determination of gastrointestinal pathogen loads, the numbers of live C. jejuni bacteria after oral infection by gavage were monitored in fecal samples daily, and upon necropsy in intraluminal gastrointestinal samples taken from the stomach, duodenum, ileum, and colon lumen that were subsequently homogenized in PBS. C. jejuni was quantified by counting of CFU after growth of serial dilutions of intestinal samples on karmali agar for at least 48 h at 37°C under microaerophilic conditions as described in detail previously [25]. The detection limit of viable pathogens was 100 CFU per g fecal sample.
Gut microbiota composition
The microbiota composition of human fecal donor suspensions used for human FMT and of murine fecal samples of hma mice was analyzed immediately before (i.e, day 0) and 6 days after C. jejuni infection as described in detail previously [26–28]. 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 respective samples and the main bacterial groups that are abundant in the human gut microbiota were determined by quantitative real-time polymerase chain reaction applying species-, genera- or group-specific 16S rRNA primers (Tib MolBiol, Berlin, Germany) and expressed as gene copies per ng DNA [26–28].
Clinical conditions of mice
Immediately before and every day after C. jejuni infection the clinical outcome of mice was monitored quantitatively by using a cumulative clinical score (maximum 12 points), addressing the clinical aspect of animals (i.e., 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 stated elsewhere [29].
Sampling procedures
At day 6 p.i., mice were sacrificed by carbon dioxide asphyxiation (Fig. 1). 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 colon 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 by applying an established scoring scheme [30]: 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 colonic ex vivo biopsies that had been fixed in 5% formalin and embedded in paraffin as reported earlier [31]. In brief, in order to detect apoptotic epithelial cells, neutrophils, T lymphocytes, 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, 1:5), and B220 (no. 14-0452-81, eBioscience; 1:200), respectively. The mean number of positively stained cells was 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), 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−1; Biochrom, Berlin, Germany) and streptomycin (100 μg mL−1; Biochrom, Berlin, Germany). After an 18-h incubation period at 37°C, culture supernatants were tested for interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) by the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Germany) in a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide (NO) was determined by the Griess reaction [26].
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-correction (for normally distributed data) and Kruskal-Wallis test with Dunn's post-correction (for not normally distributed data). Two-sided probability (p) values ≤0.05 were considered significant. Definite outliers were identified by the Grubb's test (α = 0.001).
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
Oral resveratrol prophylaxis and gastrointestinal C. jejuni colonization in human microbiota-associated (hma) IL-10−/− mice following infection
First we tested whether prophylactic oral application of synthetic resveratrol to hma IL-10−/− mice that had been initiated a week before C. jejuni infection (Fig. 1) would affect the colonization efficiencies of the enteropathogen within the murine gut. To address this, we performed daily cultural analyses of fecal samples, but found comparable C. jejuni numbers in the feces of mice from the resveratrol and placebo cohorts between days 2 and 6 p.i. (not significant (n.s.); Fig. 3). Furthermore, the C. jejuni colonization efficiency alongside the gastrointestinal tract was independent of the prophylactic regimen as indicated by similar pathogen counts in luminal samples taken from the stomach, duodenum, terminal ileum, and colon upon necropsy of resveratrol and placebo challenged mice (n.s.; Fig. 4). Hence, oral resveratrol prophylaxis did not interfere with the establishment of C. jejuni within the gastrointestinal tract of hma IL-10−/− mice upon infection.
Fecal Campylobacter jejuni numbers in human microbiota-associated (hma) IL-10−/− mice over time following resveratrol prophylaxis and C. jejuni infection.
On day (d) 0 and d1, hma IL-10−/− mice that had undergone oral resveratrol (RESV, white bars) or placebo (PLC, grey bars) prophylaxis were infected with C. jejuni strain 81-176 by gavage. Fecal pathogen numbers were determined daily post-infection until necropsy on d6 by culture and expressed as colony-forming units per gram (CFU/g). Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), and numbers of included mice (in parentheses) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Gastrointestinal C. jejuni loads in infected hma IL-10−/− mice following resveratrol prophylaxis.
Six days following C. jejuni infection, the enteropathogenic loads were determined in distinct compartments of the gastrointestinal tract (as indicated) in hma IL-10−/− mice that had undergone oral resveratrol (RESV, white bars) or placebo (PLC, grey bars) prophylaxis by culture and expressed as colony-forming units per gram (CFU/g). Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), and the numbers of culture-positive mice out of the total numbers of included animals (in parentheses) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Clinical outcome of resveratrol prophylaxis in C. jejuni infected IL-10−/− mice harboring a human gut microbiota
Further, we surveyed the clinical outcome of campylobacteriosis in hma mice with a preceding oral resveratrol prophylaxis by assessing wasting symptoms and bloody diarrhea (Figs 5–8). When focussing on individual parameters contributing to the cumulative campylobacteriosis score, mice from the placebo group suffered more distinctly from wasting symptoms if compared to mice with preceding resveratrol prophylaxis (P < 0.01–0.001), whereas the latter exhibited basal values (Fig. 5). In case of the abundance of fecal blood, only infected placebo controls showed elevated scores between days 3 and 5 p.i. (P < 0.05–0.001 versus naive; Fig. 6A–C), whereas on day 6 p.i., a trend towards lower scores was detected in resveratrol as compared to placebo prophylactically treated mice (n.s.; Fig. 6D). With regard to stool consistencies, placebo, but not resveratrol challenged mice exhibited increased diarrheal scores on day 6 p.i. (P < 0.05 versus naive; Fig. 7D). When assessing the overall clinical conditions of mice by the cumulative campylobacteriosis score we found increased values in mice from the placebo control group (P < 0.01–0.001 versus naive) as opposed to resveratrol challenged mice between days 3 and 5 p.i. (Fig. 8A–C). At the end of the experiment (i.e., on day 6 p.i.), however, the C. jejuni-induced cumulative clinical scores were increased in mice from both cohorts (P < 0.05–0.001 versus naive), but with a trend towards lower values in resveratrol as compared to placebo challenged mice (n.s.; Fig. 8D). Hence, oral resveratrol prophylaxis could alleviate clinical signs of campylobacteriosis in infected hma IL-10−/− mice.
Wasting symptoms in hma IL-10−/− mice over time following resveratrol prophylaxis and C. jejuni infection.
On day (d) 0 and d1, hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis were infected with C. jejuni strain 81-176 by gavage. Severity of clinical signs such as wasting symptoms were quantitatively assessed daily post-infection until necropsy on d6 with a defined scoring system (see methods). Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), numbers of included mice (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Fecal blood in hma IL-10−/− mice over time following resveratrol prophylaxis and C. jejuni infection.
On day (d) 0 and d1, hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis were infected with C. jejuni strain 81-176 by gavage. The abundance of fecal blood was quantitatively assessed daily post-infection until necropsy on d6 with a defined scoring system (see methods). Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), numbers of included mice (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Diarrheal symtoms in hma IL-10−/− mice over time following resveratrol prophylaxis and C. jejuni infection.
On day (d) 0 and d1, hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis were infected with C. jejuni strain 81-176 by gavage. The stool consistencies were quantitatively assessed daily post-infection until necropsy on d6 with a defined scoring system (see methods). Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), numbers of included mice (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Overall clinical conditions in hma IL-10−/− mice over time following resveratrol prophylaxis and C. jejuni infection.
On day (d) 0 and d1, hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis were infected with C. jejuni strain 81-176 by gavage. The overall clinical conditions were quantitatively assessed daily post-infection until necropsy on d6 with a defined scoring system assessing wasting symptoms, fecal blood, and stool consistency (see methods). Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), numbers of included mice (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Microscopic inflammatory changes in the colon of hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection
Next, we tested for potential microscopic inflammation-alleviating effects of resveratrol prophylaxis in the large intestines of infected mice. Mice from both, the resveratrol and placebo groups displayed increased histopathological changes in the colon on day 6 p.i. with a trend towards lower scores in the former versus the latter (n.s.; Fig. 9A). When assessing apoptotic cell responses in the large intestines by quantitative in situ immunohistochemistry, we found C. jejuni-induced increases in cleaved caspase-3 positive colonic epithelial cells (P < 0.05–0.001), but with much lower apoptotic cell numbers in resveratrol as compared to placebo pretreated mice on day 6 p.i. (P < 0.05; Fig. 9B). Hence, resveratrol prophylaxis resulted in less pronounced C. jejuni-induced microscopic inflammatory including apoptotic cell responses in the colon.
Microscopic inflammatory changes in the colon of hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection.
Six days following C. jejuni infection of hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis, (A) the histopathological changes were quantitated in hematoxylin and eosin-stained colonic paraffin sections applying a standardized histopathological scoring system (see methods). Furthermore, (B) apoptotic colonic epithelial cells were counted in large intestinal paraffin sections stained against cleaved caspase-3 (Casp3), and the average numbers of positively stained cells out of six representative high-power filed (HPF, 400-times magnification, light microscopy) were determined. Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), the numbers of included animals (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments. Definite outliers were identified by the Grubb's test
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Immune cell responses in the colon of hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection
In order to assess the effects of resveratrol prophylaxis on the C. jejuni-induced immune cell responses in the large intestines, we performed quantitative in situ immunohistochemical analyses of colonic paraffin sections that had been stained with antibodies directed against distinct innate and adaptive immune cell subsets. On day 6 p.i., increased numbers of neutrophils could be observed in the colonic mucosa and lamina propria irrespective of the prophylactic regimen (P < 0.01–0.001 versus naive; Fig. 10A). Interestingly, the medians of colonic neutrophils were more than 50% lower in the resveratrol as compared to placebo challenged mice, but did not reach statistical significance in the multi-variate analysis due to high standard deviations (n.s.; Fig. 10A). In case of T and B lymphocytes, however, mice from the placebo, but not the resveratrol cohorts displayed increased adaptive immune cells counts in the colonic mucosa and lamina propria on day 6 p.i. (P < 0.001 versus naive; P < 0.01 versus resveratrol; Fig. 10B and C). Hence, resveratrol prophylaxis dampened C. jejuni-induced colonic T and B cell responses.
Immune cell responses in the colon of hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection.
Six days following C. jejuni infection of hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis, the numbers of distinct immune cell populations such as (A) neutrophils (MPO7+), (B) T lymphocytes (CD3+), and (C) B lymphocytes (B220+) were counted in the mucosa and lamina propria of immunohistochemically stained colonic paraffin sections, and the average numbers of positively stained cells out of six representative high-power filed (HPF, 400-times magnification, light microscopy) were determined. Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), the numbers of included animals (in parentheses), and significance levels (P values, determined by the one-way ANOVA test with Tukey post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Intestinal pro-inflammatory mediator secretion in hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection
Next, we assessed the effect of prophylactic resveratrol application on intestinal pro-inflammatory mediator secretion during campylobacteriosis. Our measurements revealed that C. jejuni infection was accompanied by increased colonic concentrations of NO, IL-6, TNF-α, and IFN-γ in placebo, but not resveratrol challenged mice (P < 0.05–0.01 versus naive; Fig. 11), which also held true for enhanced NO secretion in the terminal ileum of mice from the placebo as opposed to the resveratrol cohorts (P < 0.05; Fig. 12). Hence, oral resveratrol prophylaxis could dampen C. jejuni-induced intestinal pro-inflammatory mediator secretion to basal levels.
Colonic pro-inflammatory mediator responses in hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection.
Six days following C. jejuni infection of hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis, (A) nitric oxide (NO), (B) IL-6, (C) TNF-α, and (D) IFN-γ concentrations were measured in colonic ex vivo biopsies. Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), the numbers of included animals (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Ileal nitric oxide secretion in hma IL-10−/− mice following resveratrol prophylaxis and C. jejuni infection.
Six days following C. jejuni infection of hma IL-10−/− mice that had undergone oral resveratrol (RESV) or placebo (PLC) prophylaxis, nitric oxide (NO) concentrations were measured in ileal ex vivo biopsies. Naive mice served as untreated and non-infected controls. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), the numbers of included animals (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Fecal microbiota changes during C. jejuni infection of hma IL-10−/− mice following resveratrol prophylaxis
Finally, we surveyed the potential impact of resveratrol prophylaxis on the commensal human microbiota composition in the large intestines during acute campylobacteriosis. Our quantitative culture-independent molecular measurements revealed that immediately before C. jejuni infection (i.e., on day 0), the colonic total eubacterial loads and the gene numbers of aerobic as well as obligate anaerobic bacterial phyla under investigation did not differ between mice from the resveratrol and placebo prophylaxis cohorts (n.s.; Fig. 13). In the course of C. jejuni infection, lower total eubacterial copies (P < 0.05–0.001; Fig. 13A) as well as lower gene numbers of obligate anaerobic bacteria such as bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides and Clostridium leptum groups as well as Mouse Intestinal Bacteroides could be measured on day 6 p.i. as compared to day 0 (P < 0.05–0.001; Fig. 13E–I), irrespective of the prophylaxis regimen. Notably, fecal loads of enterobacteria increased during infection of placebo control animals (P < 0.05 versus d0), but not in mice from the resveratrol cohort (n.s.; Fig. 13B). Hence, oral resveratrol prophylaxis was not associated with significant shifts in the colonic commensal microbiota composition during campylobacteriosis of hma IL-10−/− mice.
Fecal microbiota changes during C. jejuni infection of hma IL-10−/− mice with resveratrol prophylaxis.
The fecal microbiota compositions were surveyed in hma IL-10−/− mice with preceding resveratrol (RESV) or placebo (PLC) prophylaxis immediately before (i.e., day (d)0) and on d6 after C. jejuni infection by culture-independent, molecular methods. The (A) total eubacterial loads, (B) enterobacteria, (C) enterococci, (D) lactobacilli, (E) bifidobacetria, (F) Bacteroides/Prevotella species, (G) Clostridium coccoides and (H) Clostridium leptum groups, and (I) Mouse Intestinal Bacteroides are expressed as copies per ng DNA. Box plots (25th and 75th percentiles), whiskers (minimum and maximum values), medians (black bar in boxes), the numbers of mice with bacteria-positive detection out of the total number of analyzed animals (in parentheses), and significance levels (P values, determined by the Kruskal-Wallis test with Dunn's post-correction (A-F, H) and the one-way ANOVA test with Tukey post-correction (G, I)) are indicated. Data were pooled from three independent experiments
Citation: European Journal of Microbiology and Immunology 13, 4; 10.1556/1886.2023.00042
Discussion
In our present preclinical placebo-controlled intervention trial we tested the disease-alleviating effects of resveratrol prophylaxis in acute murine campylobacteriosis. The oral application of synthetic resveratrol to hma IL-10−/− mice starting a week before C. jejuni infection did not affect the establishment of the enteropathogen in the gut as shown by comparable luminal bacterial loads in defined compartments of the gastrointestinal tract. This result might not be surprising given that the concentration of the resveratrol drinking solution (i.e., 300 mg/L) was below the minimum inhibitory concentration (MIC) of 456.5 mg/L as measured against the here applied C. jejuni 81-176 strain before [32]. Nevertheless, a previous study revealed that resveratrol even when applied in concentrations below its MIC, could antagonize biofilm formation by Aliarcobacter as well as Campylobacter species [33]. One need to take into consideration, however, that resveratrol is metabolized rapidly upon ingestion and its oral bioavailability is regarded as rather low even though the absorption rate may reach 70% [18, 34]. Despite comparable gastrointestinal C. jejuni loads, resveratrol prophylaxis could alleviate clinical signs of campylobacteriosis, and in particular wasting symptoms, which also held true for dampened C. jejuni-induced microscopic inflammatory including apoptotic cell responses in the colon. Pronounced disease-alleviating as well as anti-apoptotic effects were also observed in our previous study where we tested the therapeutic effects of an oral 4-day course of resveratrol in the same concentration, but after C. jejuni infection of secondary abiotic IL-10−/− mice [32]. These results are supported by data from an acute pharyngitis in vivo model showing that exogenous resveratrol down-regulated the expression of caspase-3 and of pro-inflammatory mediators including IL-6 and TNF-α [35]. In both, our actual prophylactic and recent therapeutic [32] treatment studies exogenous resveratrol dampened C. jejuni-induced adaptive immune cell responses in the colon as shown for both, T and B lymphocytes. These results are well in line with our previous study providing evidence for diminished T cell responses preventing mice from acute Toxoplasma gondii-induced ileitis [36]. Furthermore, resveratrol could inhibit B cell proliferation in a murine model of systemic lupus erythemotodes [37].
Remarkably, our actual study revealed that oral resveratrol prophylaxis dampened C. jejuni induced intestinal secretion of pro-inflammatory mediators including IL-6, TNF-α, and IFN-γ to basal levels. Furthermore, NO concentrations measured in both, the colon and the terminal ileum of C. jejuni infected mice with resveratrol prophylaxis did not differ from those obtained from uninfected and untreated control mice. These results are well in line with our previous study showing that even a short-term resveratrol treatment of infected secondary abiotic IL-10−/− mice suffering from acute enterocolitis reduced NO secretion in the colon, thereby diminishing oxidative stress [32] and attenuating intestinal epithelial barrier dysfunction [38]. In support, oral resveratrol could alleviate acute dextran sulfate sodium- (DSS-)induced colitis in mice as evidenced by improved clinical conditions, by down-regulated colonic expression of IL-6 and TNF-α, and conversely, by up-regulated expression of colonic tight junction molecules thereby enhancing intestinal repair mechanisms [39]. Remarkably, a randomized double-blind and placebo-controlled pilot study revealed that a 6-week resveratrol treatment of patients suffering from mild to moderate ulcerative colitis could decrease distinct pro-inflammatory disease markers including systemic TNF-α concentrations [40].
It is well known that the Campylobacter-LOS serves as an endotoxin and leads to an immune hyperactivation upon enteropathogenic infection that is dependent on Toll-like receptor-4 (TLR-4) leading to the acute infectious and chronic post-infectious collateral damages of campylobacteriosis [8, 41]. Interestingly, several bioactive molecules from plant-derived products are known to interact with TLR-4-mediated signalling pathways in an inhibitory fashion as reviewed previously [42]. Among these, also resveratrol has been shown to interact with TLR-4 [43] and was able to ameliorate inflammation-induced vascular endothelial cell damage by inhibiting the TLR-4-dependent nuclear factor (NF)-kB signalling pathway [44]. It is therefore tempting to speculate, that the observed immune-modulatory and in consequence, disease-alleviating effects of resveratrol in acute campylobacteriosis might have been, at least in part, due to its potential TLR-4-antagonistic properties.
Given the importance of the commensal gut in the initiation and perpetuation of intestinal inflammatory morbidities in mice and men [25, 26, 45–48], we performed a comprehensive survey of the gut microbiota compositions in hma IL-10−/− mice during acute campylobacteriosis. Immediately before infection (i.e. at day 0), our culture-independent analyses revealed comparable gene copy numbers of distinct bacterial phyla from the human fecal transplants that had engrafted in the mice from the resveratrol and the placebo cohorts. This points towards an if at all, negliable impact of the preceding resveratrol prophylaxis on the transplanted gut microbiota and hence, comparable gut microbial conditions upon induction of campylobacteriosis. Until day 6 p.i., only the fecal loads of obligate anaerobic bacterial species such as bifidobacteria, clostridia, Bacteroides/Prevotella species, and Mouse Intestinal Bacteroides decreased during campylobacteriosis, irrespective of the prophalaxis regimen. Interestingly, in placebo, but not resveratrol treated mice, commensal enterobacteria such as Escherichia coli increased between day 0 and day 6 p.i. In support, Larrosa et al. found that a 25-day course of oral low-dose resveratrol in rats starting 20 days before induction of acute DSS colitis did not only reduce colonic inflammatory signs, but also resulted in decreased abundance of enterobacteria including E. coli in the large intestines of diseased mice until day 5 post-induction [49]. Interestingly, our previous studies revealed that irrespective of their etiology, acute as well as chronic inflammatory morbidities of the intestinal tract were accompanied by distinct gut microbiota shifts towards enterobacterial overgrowth of the inflamed gut lumen further perpetuating the inflammatory process in a TLR-4-dependent fashion [36, 41, 45–47, 50–54]. Hence, the lacking increase in fecal enterobacterial gene numbers in C. jejuni-infected mice from the resveratrol prophylaxis cohort might be considered as a surrogate marker for the less severe gut inflammatory process during campylobacteriosis.
Conclusion
Our placebo-controlled intervention study provides evidence that prophylactic oral application of resveratrol constitutes a promising strategy to alleviate acute campylobacteriosis and in consequence, to reduce the risk for post-infectious autoimmune sequelae. Resveratrol might be considered as safe given that even long-term clinical trials proved that daily doses of up to 5 g were well tolerated [55, 56]. Furthermore, the here applied hma IL-10−/− mice have been further proven as a suitable acute campylobacteriosis in vivo model to dissect the interplay between enteropathogens, human gut microbiota, and host immunity and to test the impact of prophylactic as well as therapeutic interventive strategies in a preclinical setting.
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.
NS: Performed experiments, analyzed data.
LQL: Performed experiments.
NWS: Performed experiments.
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 interests
MMH and SB are Editorial Board members. Therefore, the submission was handled by a different member of the editorial board, and they did not take part in the review process in any capacity.
Acknowledgements
We thank Alexandra Bittroff-Leben, Ines Puschendorf, Ulrike Fiebiger, Sumaya Abdul-Rahman, Gernot Reifenberger, 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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