To assess changes in the amount of inflammation-induced leucocytes, 5 × 106 washed spleen cells were stained with the following fluorescence-coupled monoclonal antibodies anti-CD11b-phycoerythrin (PE) or -allophycocyanin (APC), granulocyte-differentiation antigen-1 (Gr-1)-PE, B220-fluorescein isothiocyanate (FITC), anti-CD4-PE, anti-CD25-FITC

and biotinylated anti-CD3ε followed by incubation with streptavidin-PE-Cy5 (PharMingen Canada for conjugated monoclonal antibodies, and Cedarlane, Hornby, Ontario, Canada for streptavidin) for flow cytometry according to published procedures. The remaining splenic lymphocytes were placed into the wells of 96-well plates at a concentration of 2 × 105 cells per well. Cultures were stimulated with either sterile sonicates

prepared from pure strains of selected endogenous bacteria, as detailed in Sydora et al.[8], or with sterile lysates prepared from faecal material PLX-4720 cost using glass beads as described in Sydora et al.[9]. Bacterial sonicates and faecal lysates were added at a protein concentration of 50 µg/ml, which was found to be optimal for cytokine production. Control stimuli included plate-bound anti-CD3ε clone 145-2C11 (PharMingen Canada) and medium alone. After 48 h of incubation at 37°C in a humidified www.selleckchem.com/products/VX-809.html incubator at 5% CO2, the plates were centrifuged, and the amounts of the indicated cytokines in the supernatants were quantified using standard ELISA techniques, as described above. Data are expressed as means ± standard error of the mean (s.e.m.) or means ± standard deviation (s.d.) dependent upon whether data were combined from both experiments of the same mouse strain or whether they were derived from only one experimental group, respectively. Differences between mean values were evaluated using analysis of variance or paired t-tests, where appropriate (SigmaStat; Jandel Corporation, San Rafael, CA, USA). In axenic mice, spontaneous release

of cytokines from colonic and caecal mucosal tissue was low (Fig. 1, day 0), similar to cytokine release in wild-type mice raised under conventional, non-pathogenic conditions in the presence of commensal intestinal bacteria [8]. However, inoculation of the axenic mice with faecal bacteria slurry resulted in a significant colonic and caecal immune response of proinflammatory cytokines, IFN-γ, TNF-α and IL-17 that peaked at Vitamin B12 3–7 days after faecal slurry exposure (Fig. 1 and data not shown). Similarly, there was a significant increase in G-CSF 3 days post-faecal slurry feeding. In contrast, colonic and caecal immune response of anti-inflammatory cytokines, IL-4 and IL-10, followed that of the proinflammatory cytokines and peaked at day 7 (Fig. 1). While small increases in production of IL-6 were noted on days 3 and 7, these increases were not significant (data not shown). By day 14 following faecal slurry exposure, production of all cytokines was diminished and reached background levels by 28 days (Fig. 1 and data not shown).