mice with colitis harbored significantly higher numbers of enterobacteria, Bacteroides/Prevotella spp. and bifidobacteria, where- as lactobacilli, Clostridium leptum group and mouse intestinal bacteroidetes rRNA were lower as compared to wildtype controls, (Fig. 2C). Thus, colonization resistance against C. jejuni is abrogated by chronic large intestinal inflammation in IL-102/2 mice accompanied by distinct changes in colonic microbiota composition. C. jejuni Infection of Infant Mice Harboring a Conventional Intestinal Microbiota In earlier studies weaned mice harboring a conventional gut microbiota were shown to be susceptible to C. jejuni infection [35,36,37,38,39]. Very recently, we could demonstrate that compared to adult animals, infant mice harbored significantly higher concentrations of E. coli, Bacteroides/Prevotella spp. and Clostridium coccoides whereas lactobacilli, bifidobacteria and Clostrid- ium leptum numbers were lower [40]. To further unravel immunopathological consequences of abrogated colonization resistance in infant mice in more detail, we infected 3-weeks-old animals right after weaning with C. jejuni strains ATCC 43431 or B2. Adult mice 3 months of age with a common commensal gut flora served as controls. Kinetic analyses of fecal pathogen burden revealed that the infant mice harbored higher C. jejuni ATCC 43431 and B2 loads until days 5 and 6 p.i., respectively, when compared to adult animals (Fig. 3A, B). Interestingly, C. jejuni strain B2, but not strain ATCC 43431, could stably infect infant mice until day 6 p.i., as indicated by 66.7% and 30.0% of culture- positive cases, respectively, with a median load of approximately 105 CFU C. jejuni B2/g feces (Fig. 3A, B). Feces samples taken from na¨ıve infant mice contained significantly higher numbers of E. coli (median 106 CFU/g), as compared to uninfected adult animals (Fig. 3C). Thus, these data provide evidence that E. coli might be causatively involved in the abrogation of murine colonization resistance against C. jejuni. The analysis of the clinical outcome revealed that acute campylobacteriosis was self-limiting in infant mice. A cumulative score addressing the clinical condition and occurrence of bloody diarrhea in infected animals revealed that either C. jejuni strain induced enterocolitis within 24 hours p.i. remaining on a comparable level until day 6 p.i. (Fig. 3D). C. jejuni infected infant mice were not severely compromised clinically but developed mild diarrhea with haemoccult-positive feces irrespec- tive of the C. jejuni strain analyzed. Furthermore, no live C. jejuni could be cultured from MLNs, spleen, liver, kidney or cardiac blood at day 6 following infection with either strain (not shown). Pro-inflammatory Immune Responses Following C. jejuni Infection in Infant Mice Since human campylobacteriosis is accompanied by an influx of immune cells to the inflamed colon [6,7,8,9], we investigated numbers of apoptotic cells and immune cells within the colon mucosa of C. jejuni infected infant mice in situ by immunohisto- chemistry. Naive infant mice served as controls. At day 6 p.i., irrespective of the C. jejuni strain used, infected infant mice displayed a two-fold increase of apoptotic cells, paralleled by a significant increase of neutrophilic granulocytes and macrophages as compared to naive controls (Fig. 4A–C). In addition, both C. jejuni strains induced multifold increases in T-lymphocytes, regulatory T-cells (Tregs) as well as B-lymphocytes in infected versus na¨ıve infant mice (Fig. 4D–F). The immune cell responses were accompanied by increased expression of pro-inflammatory cytokines and mediators in the colon. The levels of nitric oxide, E. coli Facilitates Murine Campylobacter Infection TNF-a, IL-6, and MCP-1 were significantly higher in colon biopsies derived from C. jejuni ATCC 43431 and B2 infected mice at day 6 p.i. as compared to naive controls (Fig. 5A–D). For IFN- c a significant increase was measured in C. jejuni B2 infected mice while C. jejuni ATCC 43431 infected mice displayed a trend towards elevated concentrations (Fig. 5E). Of note, following infection with either C. jejuni strain, adult mice harboring a conventional gut microbiota displayed neither clinical signs of enterocolitis nor relevant immune cell or pro-inflammatory responses in their colon (also refer to [17]). Taken together, C. jejuni infection of infant mice resulted in distinct pro-inflammatory responses and apoptotic changes in the colonic mucosa. Treatment with Live E. coli Abrogates Murine Colonization Resistance Against C. jejuni In order to dissect whether colonization resistance is abrogated by shifts in the microbiota composition or by conditions of inflammation we increased intestinal E. coli loads artificially by treating healthy adult mice with live E. coli in the drinking water (108 CFU/mL) and investigated the susceptibility to C. jejuni infection. Analysis of E. coli in feces samples revealed that the intestinal E. coli loads increased by more than 3 orders of magnitude (from median 105 CFU/g to 108 CFU/g feces) and remained stable as long as the E. coli suspension was kept (Fig. 6). Within 24 hours after withdrawl of the E. coli suspension, the intestinal E. coli loads decreased back to basal levels (Fig. 6). Oral infection of E. coli-treated mice and non-treated controls with C. jejuni strains ATCC 43431 or B2 revealed that E. coli did significantly increase the colonization capacity of either strain. (Fig. 7A and B). C. jejuni B2 could be cultured from the entire GI tract of E. coli treated animals at day 12 p.i. with highest counts in distal parts such as ileum and colon (Fig. 7C). In non-treated control animals, however, pathogen burdens were significantly lower as compared to mice with elevated intestinal E. coli load (Fig. 7). Pro-inflammatory Immune Responses in C. jejuni Infected Adult Mice with Elevated E. coli Loads To further elucidate whether C. jejuni infection of adult mice with high intestinal E. coli loads was accompanied by pro- inflammatory immune responses, we analyzed apoptosis and inflammatory cells in the colon mucosa by in situ immunohisto- chemistry. Irrespective of the C. jejuni strain analyzed, infected mice displayed significantly higher numbers of apoptotic cells, neutrophils, macrophages, T- and B-lymphocytes as well as regulatory T-cells in the colon in situ at day 12 p.i. as compared to non-infected E. coli-treated control animals (Fig. 8). Secretion of nitric oxide, IL-6, MCP-1, IFN-c and TNF-a in ex vivo colon biopsies were significantly increased at day 12 following infection with C. jejuni ATCC 43431 or B2 versus na¨ıve animals with supra-physiological intestinal E. coli loads (Fig. 9). Thus, C. jejuni infection of adult mice treated with supra- physiological E. coli loads is accompanied by pro-inflammatory immunopathology of the colon. Of note, elevation of intestinal E. coli loads alone did not affect pro-inflammatory immune responses (data not shown). This was even true in our recent study when gnotobiotic mice generated by antibiotic treatment were replen- ished with a complex human microbiota [17]. Taken together, physiological (infancy) and pathological conditions (e.g. acute and chronic intestinal inflammation in mice and men) associated with shifts of the intestinal microbiota composition such as elevated commensal E. coli loads facilitate C. jejuni infection inducing or perpetuating immunopathology. Most PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e35988