As a service to our customers we are providing this early version of the manuscript. 2004; Mundy et al., 2005). We previously reported that CRAMP, an epithelial cell antimicrobial protein belonging to the cathelicidin family, is important in determining early colonization of the host with (Iimura et al., 2005), whereas CD4+ T cells, B cells and IgG antibodies to are important in controlling infection in the later periods and are required for ultimate pathogen clearance(Bry and Brenner, 2004; Maaser et al., 2004; Simmons et al., 2003). Furthermore, several cytokine knockout mice (e.g. interferon-, tumor necrosis factor-, IL-6, and either p19 or IL-12p40) have delayed clearance of infection (Dann et al., 2008; Goncalves et al., 2001; Mangan et al., 2006; Simmons et al., 2002). We used the A/E pathogen, acts in a nonredundant manner to enhance host protection to an A/E pathogen through mechanisms that involve DC and epithelial cells. Results Colonic GM-CSF induction after infection To probe the functions of GM-CSF in mucosal defense, WT B6 mice were infected with the A/E pathogen, for 2 weeks was immunostained with anti-GM-CSF antibody. Infected mice manifest marked colonic crypt hyperplasia. Original magnification, x 100. (B) GM-CSF in colon tissue was determined by ELISA at the indicated times after infection. Data are mean SEM (n = 5 mice at each time point). ND = none detected. *; < 0.05 vs. uninfected control. (C). Top row. Colon from B6 mice infected with for 2 weeks was immunostained for GM-CSF (left panel, red) and CD11c (middle panel, green). Right panel is a merged image of the left and middle panels and also includes nuclear staining. Bottom row. Colon was stained for GM-CSF (left panel, red) and F4/80 (middle panel, green). Right panel is a merged image including nuclear staining. Merged images were created using Adobe Photoshop software. Arrows indicate GM-CSF producing cells. Original magnification, x 400. Increased susceptibility of GM-CSF-/- mice to infection To test the importance of GM-CSF during infection, we employed gene-targeted mice deficient in GM-CSF. Bacterial colonization was comparable early NS11394 after infection (4 days), indicating that GM-CSF-/- mice had no apparent defect in innate antibacterial defense. Consistent with this conclusion, NS11394 GM-CSF deficiency did not impact expression of the epithelial cell-produced antimicrobial peptide mCRAMP (data not shown), which is critical in early defense against (Iimura et al., 2005). However, at one week after infection, GM-CSF-/- mice had significantly increased mucosal colonization with compared to WT B6 controls (Fig. 2A), significantly greater fecal counts of (Fig. 2B), and significantly greater systemic infection in spleen and MLN KIAA1235 (Fig. 2C). In parallel, GM-CSF-/- mice had lost significantly more body weight after 2 weeks than WT B6 mice (97.8 1.5% vs. 103.2 NS11394 1.1% of pre-infection weight, respectively; p<0.05), underlining the overall clinical impact of GM-CSF deficiency on the course of the infection. B6 mice had cleared infection by 3 weeks, whereas clearance did not occur until 4 weeks after infection in GM-CSF-/- mice (Fig. 2B). Open in a separate window Figure 2 infection in NS11394 B6 and GM-CSF-/- mice(A) One week after infection, colon sections from B6 and GM-CSF-/- mice were co-stained for (red) and F-actin (green). Co-localization of and F-actin is shown in yellow. Original magnification, x 100. (B) Fecal counts (CFU) of were determined at the indicated times after oral gavage NS11394 of 5 108 to B6 and GM-CSF-/-.