2b). The colons, in addition, had significantly higher levels of the cytokines Csf2, Csf3, Il9 and Tnfa. The observed chemokine and inflammatory gene expression pattern was clearly reflected in the composition of the inflammatory infiltrates in the caeca and colons of the C. difficile-infected mice. Both organs contained significantly higher numbers of neutrophils after the infection (Fig. 3a), a finding consistent with the significant up-regulation of Cxcl1, Cxcl2 and Il17f. In addition, there was a substantial increase in CD11b expression on the recruited neutrophils
(Fig. 3b). Flow cytometric analysis showed a significant increase in the number of dendritic cells and cells of the monocyte/macrophage lineage in the caeca of the C. difficile-infected mice (Fig. 4a,b; compare with Supplementary material, Figure S3A and B); which was consistent with the increased expression levels of Ccl2. The infected colons showed a similar GSI-IX molecular weight trend. A substantial fraction of the monocyte/macrophage lineage cells in the caeca and colons of the infected mice
expressed low levels of MHC II (Fig. 4c), which was consistent with their recent recruitment. The significant increase in the number BAY 80-6946 mouse of lymphocytes (B cells, CD4 T cells and CD8 T cells) in the caeca and colons of the C. difficile-infected mice (Fig. 5a; compare with Supplementary material, Figure S4A) also correlated with the heightened expression of chemokines and pro-inflammatory genes. Nonetheless, the recruited CD4 T cells expressed levels of CD69 that were comparable with that found in their untreated counterparts (Fig. 5b; compare with Figure S4B) and had low levels of CD25 expression (assessed on CD4 T cells with gating to exclude the FoxP3+ subset) (Fig. 5c; compare with Figure S4C). These observations were in full biological concordance with the lack of any significant change in Tbx21, Gata3 or Rorc expression levels or in that of cytokines secreted by polarized T cells (data not PRKACG shown). There was a significant up-regulation of Il22 expression and
a number of anti-microbial peptides in the caeca and colons of the infected mice. These included Defa1, Defa28, Defb1 and Slpi in the colon and Reg3g in the caecum (Fig. 2c). There was also an increase in Reg3g levels in the colons of infected mice; however, in these experiments, the increase did not reach statistical significance. To assess the activation of the UPR in C. difficile infection in mice, caecal and colonic samples from untreated and C. difficile-infected mice were examined for their expression of numerous UPR markers. Immunoblotting showed a significant increase in the level of eIF2α phosphorylation, the most rapid aspect of the UPR, in the caeca and colons of the infected mice (Fig. 6a). This coincided with the significant up-regulation of Gadd34 and Wars mRNA expression levels, both downstream of eIF2α phosphorylation, in the infected samples (Fig. 6b).