Novel Clinical Campylobacter jejuni Infection Models Based on Sensitization of Mice to Lipooligosaccharide, a Major Bacterial Factor Triggering Innate Immune Responses in Human Campylobacteriosis

Human Campylobacter jejuni infections inducing campylobacteriosis including post-infectious sequelae such as Guillain-Barré syndrome and reactive arthritis are rising worldwide and progress into a global burden of high socioeconomic impact. Intestinal immunopathology underlying campylobacteriosis is a classical response of the innate immune system characterized by the accumulation of neutrophils and macrophages which cause tissue destruction, barrier defects and malabsorption leading to bloody diarrhea. Clinical studies revealed that enteritis and post-infectious morbidities of human C. jejuni infections are strongly dependent on the structure of pathogenic lipooligosaccharides (LOS) triggering the innate immune system via Toll-like-receptor (TLR)-4 signaling. Compared to humans, mice display an approximately 10,000 times weaker TLR-4 response and a pronounced colonization resistance (CR) against C. jejuni maintained by the murine gut microbiota. In consequence, investigations of campylobacteriosis have been hampered by the lack of experimental animal models. We here summarize recent progress made in the development of murine C. jejuni infection models that are based on the abolishment of CR by modulating the murine gut microbiota and by sensitization of mice to LOS. These advances support the major role of LOS driven innate immunity in pathogenesis of campylobacteriosis including post-infectious autoimmune diseases and promote the preclinical evaluation of novel pharmaceutical strategies for prophylaxis and treatment

Vitamin C alleviates acute enterocolitis in Campylobacter jejuni infected mice

Human foodborne infections with the zoonotic pathogen Campylobacter jejuni are on the rise and constitute a significant socioeconomic burden worldwide. The health-beneficial, particularly anti-inflammatory effects of vitamin C (ascorbate) are well known. In our preclinical intervention study, we assessed potential anti-pathogenic and immunomodulatory effects of ascorbate in C. jejuni-infected secondary abiotic IL-10-/- mice developing acute campylobacteriosis similar to humans. Starting 4 days prior peroral C. jejuni-infection, mice received synthetic ascorbate via the drinking water until the end of the experiment. At day 6 post-infection, ascorbate-treated mice harbored slightly lower colonic pathogen loads and suffered from less severe C. jejuni-induced enterocolitis as compared to placebo control animals. Ascorbate treatment did not only alleviate macroscopic sequelae of infection, but also dampened apoptotic and inflammatory immune cell responses in the intestines that were accompanied by less pronounced pro-inflammatory cytokine secretion. Remarkably, the anti-inflammatory effects of ascorbate pretreatment in C. jejuni-infected mice were not restricted to the intestinal tract but could also be observed in extra-intestinal compartments including liver, kidneys and lungs. In conclusion, due to the potent anti-inflammatory effects observed in the clinical murine C. jejuni-infection model, ascorbate constitutes a promising novel option for prophylaxis and treatment of acute campylobacteriosis

Preclinical Evaluation of Oral Urolithin-A for the Treatment of Acute Campylobacteriosis in Campylobacter jejuni Infected Microbiota-Depleted IL-10−/− Mice

Human campylobacteriosis represents an infectious enteritis syndrome caused by Campylobacter species, mostly Campylobacter jejuni. Given that C. jejuni infections are rising worldwide and antibiotic treatment is usually not indicated, novel treatment options for campylobacteriosis are needed. Urolithin-A constitutes a metabolite produced by the human gut microbiota from ellagitannins and ellagic acids in berries and nuts which have been known for their health-beneficial including anti-inflammatory effects since centuries. Therefore, we investigated potential pathogen-lowering and immunomodulatory effects following oral application of synthetic urolithin-A during acute campylobacteriosis applying perorally C. jejuni infected, microbiota-depleted IL-10-/- mice as preclinical inflammation model. On day 6 post infection, urolithin-A treated mice harbored slightly lower pathogen loads in their ileum, but not colon as compared to placebo counterparts. Importantly, urolithin-A treatment resulted in an improved clinical outcome and less pronounced macroscopic and microscopic inflammatory sequelae of infection that were paralleled by less pronounced intestinal pro-inflammatory immune responses which could even be observed systemically. In conclusion, this preclinical murine intervention study provides first evidence that oral urolithin-A application is a promising treatment option for acute C. jejuni infection and paves the way for future clinical studies in human campylobacteriosis

Carvacrol ameliorates acute campylobacteriosis in a clinical murine infection model

Background: The prevalence of human infections with the zoonotic pathogen Campylobacter jejuni is rising worldwide. Therefore, the identification of compounds with potent anti-pathogenic and anti-inflammatory properties for future therapeutic and/or preventive application to combat campylobacteriosis is of importance for global health. Results of recent studies suggested carvacrol (4-isopropyl-2-methylphenol) as potential candidate molecule for the treatment of campylobacteriosis in humans and for the prevention of Campylobacter colonization in farm animals. Results: To address this in a clinical murine infection model of acute campylobacteriosis, secondary abiotic IL-10-/- mice were subjected to synthetic carvacrol via the drinking water starting 4 days before peroral C. jejuni challenge. Whereas at day 6 post-infection placebo treated mice suffered from acute enterocolitis, mice from the carvacrol cohort not only harbored two log orders of magnitude lower pathogen loads in their intestines, but also displayed significantly reduced disease symptoms. Alleviated campylobacteriosis following carvacrol application was accompanied by less distinct intestinal apoptosis and pro-inflammatory immune responses as well as by higher numbers of proliferating colonic epithelial cells. Remarkably, the inflammation-ameliorating effects of carvacrol treatment were not restricted to the intestinal tract, but could also be observed in extra-intestinal organs such as liver, kidneys and lungs and, strikingly, systemically as indicated by lower IFN-γ, TNF, MCP-1 and IL-6 serum concentrations in carvacrol versus placebo treated mice. Furthermore, carvacrol treatment was associated with less frequent translocation of viable C. jejuni originating from the intestines to extra-intestinal compartments. Conclusion: The lowered C. jejuni loads and alleviated symptoms observed in the here applied clinical murine model for human campylobacteriosis highlight the application of carvacrol as a promising novel option for both, the treatment of campylobacteriosis and hence, for prevention of post-infectious sequelae in humans, and for the reduction of C. jejuni colonization in the intestines of vertebrate lifestock animals

Immunopathological properties of the Campylobacter jejuni flagellins and the adhesin CadF as assessed in a clinical murine infection model

Background: Campylobacter jejuni infections constitute serious threats to human health with increasing prevalences worldwide. Our knowledge regarding the molecular mechanisms underlying host-pathogen interactions is still limited. Our group has established a clinical C. jejuni infection model based on abiotic IL-10-/- mice mimicking key features of human campylobacteriosis. In order to further validate this model for unraveling pathogen-host interactions mounting in acute disease, we here surveyed the immunopathological features of the important C. jejuni virulence factors FlaA and FlaB and the major adhesin CadF (Campylobacter adhesin to fibronectin), which play a role in bacterial motility, protein secretion and adhesion, respectively. Methods and results: Therefore, abiotic IL-10-/- mice were perorally infected with C. jejuni strain 81-176 (WT) or with its isogenic flaA/B (ΔflaA/B) or cadF (ΔcadF) deletion mutants. Cultural analyses revealed that WT and ΔcadF but not ΔflaA/B bacteria stably colonized the stomach, duodenum and ileum, whereas all three strains were present in the colon at comparably high loads on day 6 post-infection. Remarkably, despite high colonic colonization densities, murine infection with the ΔflaA/B strain did not result in overt campylobacteriosis, whereas mice infected with ΔcadF or WT were suffering from acute enterocolitis at day 6 post-infection. These symptoms coincided with pronounced pro-inflammatory immune responses, not only in the intestinal tract, but also in other organs such as the liver and kidneys and were accompanied with systemic inflammatory responses as indicated by increased serum MCP-1 concentrations following C. jejuni ΔcadF or WT, but not ΔflaA/B strain infection. Conclusion: For the first time, our observations revealed that the C. jejuni flagellins A/B, but not adhesion mediated by CadF, are essential for inducing murine campylobacteriosis. Furthermore, the secondary abiotic IL-10-/- infection model has been proven suitable not only for detailed investigations of immunological aspects of campylobacteriosis, but also for differential analyses of the roles of distinct C. jejuni virulence factors in induction and progression of disease

The Host-Specific Intestinal Microbiota Composition Impacts Campylobacter coli Infection in a Clinical Mouse Model of Campylobacteriosis

Human Campylobacter-infections are progressively rising globally. However, the molecular mechanisms underlying C. coli-host interactions are incompletely understood. In this study, we surveyed the impact of the host-specific intestinal microbiota composition during peroral C. coli infection applying an established murine campylobacteriosis model. Therefore, microbiota-depleted IL-10-/- mice were subjected to peroral fecal microbiota transplantation from murine versus human donors and infected with C. coli one week later by gavage. Irrespective of the microbiota, C. coli stably colonized the murine gastrointestinal tract until day 21 post-infection. Throughout the survey, C. coli-infected mice with a human intestinal microbiota displayed more frequently fecal blood as their murine counterparts. Intestinal inflammatory sequelae of C. coli-infection could exclusively be observed in mice with a human intestinal microbiota, as indicated by increased colonic numbers of apoptotic epithelial cells and innate as well as adaptive immune cell subsets, which were accompanied by more pronounced pro-inflammatory cytokine secretion in the colon and mesenteric lymph nodes versus mock controls. However, in extra-intestinal, including systemic compartments, pro-inflammatory responses upon pathogen challenge could be assessed in mice with either microbiota. In conclusion, the host-specific intestinal microbiota composition has a profound effect on intestinal and systemic pro-inflammatory immune responses during C. coli infection

Toll-Like Receptor-4 Is Involved in Mediating Intestinal and Extra-Intestinal Inflammation in Campylobacter coli-Infected Secondary Abiotic IL-10−/− Mice

Human Campylobacter infections are emerging worldwide and constitute significant health burdens. We recently showed that the immunopathological sequelae in Campylobacter jejuni-infected mice were due to Toll-like receptor (TLR)-4 dependent immune responses induced by bacterial lipooligosaccharide (LOS). Information regarding the molecular mechanisms underlying Campylobacter coli-host interactions are scarce, however. Therefore, we analyzed C. coli-induced campylobacteriosis in secondary abiotic IL-10-/- mice with and without TLR4. Mice were infected perorally with a human C. coli isolate or with a murine commensal Escherichia coli as apathogenic, non-invasive control. Independent from TLR4, C. coli and E. coli stably colonized the gastrointestinal tract, but only C. coli induced clinical signs of campylobacteriosis. TLR4-/- IL-10-/- mice, however, displayed less frequently fecal blood and less distinct histopathological and apoptotic sequelae in the colon versus IL-10-/- counterparts on day 28 following C. coli infection. Furthermore, C. coli-induced colonic immune cell responses were less pronounced in TLR4-/- IL-10-/- as compared to IL-10-/- mice and accompanied by lower pro-inflammatory mediator concentrations in the intestines and the liver of the former versus the latter. In conclusion, our study provides evidence that TLR4 is involved in mediating C. coli-LOS-induced immune responses in intestinal and extra-intestinal compartments during murine campylobacteriosis

Inflammatory Immune Responses and Gut Microbiota Changes Following Campylobacter coli Infection of IL-10 -/- Mice with Chronic Colitis

Human infections with the food-borne enteropathogens Campylobacter are progressively rising. Recent evidence revealed that pre-existing intestinal inflammation facilitates enteropathogenic infection subsequently exacerbating the underlying disease. Given that only little is known about C. coli-host interactions and particularly during intestinal inflammation, the aim of the present study was to survey gastrointestinal colonization properties, gut microbiota changes and pro-inflammatory sequelae upon peroral C. coli-infection of IL-10-/- mice with chronic colitis. C. coli colonized the gastrointestinal tract of mice with varying efficiencies until day 28 post-infection and induced macroscopic and microscopic inflammatory changes as indicated by shorter colonic lengths, more distinct histopathological changes in the colonic mucosa and higher numbers of apoptotic colonic epithelial cells when compared to mock-infected controls. Furthermore, not only colonic innate and adaptive immune cell responses, but also enhanced systemic TNF-α secretion could be observed following C. coli as opposed to mock challenge. Notably, C. coli induced intestinal inflammatory sequelae were accompanied with gut microbiota shifts towards higher commensal enterobacterial loads in the infected gut lumen. Moreover, the pathogen translocated from the intestinal tract to extra-intestinal tissue sites in some cases, but never to systemic compartments. Hence, C. coli accelerates inflammatory immune responses in IL-10-/- mice with chronic colitis

Metabolomic signatures of intestinal colonization resistance against Campylobacter jejuni in mice

Introduction: Campylobacter jejuni stands out as one of the leading causes of bacterial enteritis. In contrast to humans, specific pathogen-free (SPF) laboratory mice display strict intestinal colonization resistance (CR) against C. jejuni, orchestrated by the specific murine intestinal microbiota, as shown by fecal microbiota transplantation (FMT) earlier. Methods: Murine infection models, comprising SPF, SAB, hma, and mma mice were employed. FMT and microbiota depletion were confirmed by culture and culture-independent analyses. Targeted metabolome analyses of fecal samples provided insights into the associated metabolomic signatures. Results: In comparison to hma mice, the murine intestinal microbiota of mma and SPF mice (with CR against C. jejuni) contained significantly elevated numbers of lactobacilli, and Mouse Intestinal Bacteroides, whereas numbers of enterobacteria, enterococci, and Clostridium coccoides group were reduced. Targeted metabolome analysis revealed that fecal samples from mice with CR contained increased levels of secondary bile acids and fatty acids with known antimicrobial activities, but reduced concentrations of amino acids essential for C. jejuni growth as compared to control animals without CR. Discussion: The findings highlight the role of microbiota-mediated nutrient competition and antibacterial activities of intestinal metabolites in driving murine CR against C. jejuni. The study underscores the complex dynamics of host-microbiota-pathogen interactions and sets the stage for further investigations into the mechanisms driving CR against enteric infections