E. coli Facilitates Murine Campylobacter Infection Figure 9. Colonic pro-inflammatory cytokine responses of C. jejuni infected adult mice with increased E. coli loads. In conventionally colonized 3-months-old adult mice intestinal E. coli loads were raised up to 108 bacteria per gram feces by adding live E. coli into the drinking water (+ E. coli) as described in Methods. Then, mice were orally infected with C. jejuni strain ATCC 43431 (crossed circles) or B2 (solid circles). Concentrations (pg per mg colon) of (A) nitric oxide, (B) IL-6, (C) MCP-1, (D) IFN-c, and (E) TNF-a were determined in supernatants of ex vivo colon cultures at day 12 p.i. by Griess reaction or CBA-ELISA, respectively. Uninfected mice (Na¨ıve, open circles) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (P-values) as compared to the uninfected (Na¨ıve) group determined by Student’s t-test are indicated. Data shown were pooled from at least three independent experiments. doi:10.1371/journal.pone.0035988.g009 Materials and Methods Ethics Statement Animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval of the commission for animal experiments headed by the ‘‘Landesamt fu¨ r Gesundheit und Soziales’’ (LaGeSo, Berlin, Germany; Registra- tion numbers: G0173/07 and G135/10). Animal welfare was monitored twice daily by assessment of clinical conditions. Mice Mice were bred and maintained in the facilities of the ‘‘Forschungsinstitut f u¨r Experimentelle Medizin’’ (FEM, Charite´ - Universita¨tsmedizin, Berlin, Germany), under specific pathogen- PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e35988