Positive cells in gastric mucosa of ctsz2/2 (p = 0.009) and wt mice (p = 0.001). Compared to wt animals with no further increase in F4/80 positivity, ctsz2/2 mice exhibited a prominent infiltration (p = 0.075) of F4/80-positive cells after 50 wpi (Figure 3, left). This was associated with a higher epithelial proliferation index (p = 0,029) at 50 wpi only in infected ctsz2/2, but not in wt mice as assessed by morphometric analysis of Ki-67 staining (Figure 3, right). Typically, Ki67-positive cells present as a small band within the isthmic regions of gastric glands in uninfected wt and ctsz2/2 mice. After infection, the proliferative compartment was expanded with high scores of Ki67 in the bottom of hyperplastic glands and at sites of regeneration. Spasmolytic polypeptide-expressing metaplasia (SPEM) is associated with progression to gastric cancer [1]. Interestingly, ctsz2/Cathepsin X and Premalignant Host ResponseFigure 1. Colonization efficiency of H. pylori in the corpus mucosa and the induction of Ctsz. (A) Warthin starry staining revealed a stable colonization of the SS1 strain over 50 wpi, whereas the B128 strain failed to stably colonize the mouse mucosa for more than 24 weeks. Results are shown as of H. pylori-positive tested animals for 12, 24, 36, and 50 wpi with animal numbers of 15/20, 16/20, 11/14, and 7/18 (B128/SS1), respectively. (B) Title Loaded From File Western blots of cell lysates showed induction of Ctsz in H. pylori SS1-, as well as B128-inoculated wt epithelial cells whereas ctsz2/2 cells remained unaffected. CagA was detected in all infection experiments (B) without showing delivery into the Title Loaded From File cytoplasm in fractionated cells (C). Colonization density of corpus mucosa in C57BL/6 wt mice 1315463 ( ) and ctsz2/2 ( ) challenged with H. pylori SS1 for 24, 36 or 50 weeks was semiquantitatively graded of H. pylori levels using Warthin-Starry staining with scores from minimum = 1 to maximum = 3 (D) and quantified using the DDCt method by qRT-PCR (E). doi:10.1371/journal.pone.0070242.gNmice also exhibited a significantly more severe metaplasia (p = 0.023, closed arrows) associated with oxynthic atrophy at 50 wpi compared to wt animals (Figure 3, middle). Intestinal-type acidic mucin-expressing glands were predominantly detected in ctsz2/2 mice (Figure 3, open arrow). Earlier reports have described a compensatory effect of Ctsz in ctsb2/2 mice, as well as a trend towards higher Ctsb mRNA expression in ctsz2/2 mice [17,20]. In the present study, we analyzed Ctsz and Ctsb protein expression in tissue lysates and spatial distribution in paraffin sections of proximal corpus comparing wt and ctsz2/2 mice. Ctsz expression was absent in ctsz2/2 mice, but increased significantly (p#0.002), depending on H. pylori infection after 24 wpi in wt mice (Figure 4A,B,D). Surprisingly, Ctsb was only minimally increased in non-infected ctsz2/2 mice (Figure 4A,C,F). In line with published human data, Ctsb expression was not induced by H. pylori in either wt or ctsz2/2 mice (Fig.4C,E,F). If expressed, both enzymes show a cytoplasmic staining pattern. Ctsz was predominantly expressed in macrophages, infected surface epithelium, and gastric glands, Ctsb in the surface epithelium and inflammatory cells. Ctsz seems to switch from an apical expression in surface epithelium and foveolae to a basalexpression most notably in deep gastric glands. SPEM is generally negative for Ctsz. Compared to wt animals after long term H. pylori infection, ctsz2/2 mice showed a significantly incr.Positive cells in gastric mucosa of ctsz2/2 (p = 0.009) and wt mice (p = 0.001). Compared to wt animals with no further increase in F4/80 positivity, ctsz2/2 mice exhibited a prominent infiltration (p = 0.075) of F4/80-positive cells after 50 wpi (Figure 3, left). This was associated with a higher epithelial proliferation index (p = 0,029) at 50 wpi only in infected ctsz2/2, but not in wt mice as assessed by morphometric analysis of Ki-67 staining (Figure 3, right). Typically, Ki67-positive cells present as a small band within the isthmic regions of gastric glands in uninfected wt and ctsz2/2 mice. After infection, the proliferative compartment was expanded with high scores of Ki67 in the bottom of hyperplastic glands and at sites of regeneration. Spasmolytic polypeptide-expressing metaplasia (SPEM) is associated with progression to gastric cancer [1]. Interestingly, ctsz2/Cathepsin X and Premalignant Host ResponseFigure 1. Colonization efficiency of H. pylori in the corpus mucosa and the induction of Ctsz. (A) Warthin starry staining revealed a stable colonization of the SS1 strain over 50 wpi, whereas the B128 strain failed to stably colonize the mouse mucosa for more than 24 weeks. Results are shown as of H. pylori-positive tested animals for 12, 24, 36, and 50 wpi with animal numbers of 15/20, 16/20, 11/14, and 7/18 (B128/SS1), respectively. (B) Western blots of cell lysates showed induction of Ctsz in H. pylori SS1-, as well as B128-inoculated wt epithelial cells whereas ctsz2/2 cells remained unaffected. CagA was detected in all infection experiments (B) without showing delivery into the cytoplasm in fractionated cells (C). Colonization density of corpus mucosa in C57BL/6 wt mice 1315463 ( ) and ctsz2/2 ( ) challenged with H. pylori SS1 for 24, 36 or 50 weeks was semiquantitatively graded of H. pylori levels using Warthin-Starry staining with scores from minimum = 1 to maximum = 3 (D) and quantified using the DDCt method by qRT-PCR (E). doi:10.1371/journal.pone.0070242.gNmice also exhibited a significantly more severe metaplasia (p = 0.023, closed arrows) associated with oxynthic atrophy at 50 wpi compared to wt animals (Figure 3, middle). Intestinal-type acidic mucin-expressing glands were predominantly detected in ctsz2/2 mice (Figure 3, open arrow). Earlier reports have described a compensatory effect of Ctsz in ctsb2/2 mice, as well as a trend towards higher Ctsb mRNA expression in ctsz2/2 mice [17,20]. In the present study, we analyzed Ctsz and Ctsb protein expression in tissue lysates and spatial distribution in paraffin sections of proximal corpus comparing wt and ctsz2/2 mice. Ctsz expression was absent in ctsz2/2 mice, but increased significantly (p#0.002), depending on H. pylori infection after 24 wpi in wt mice (Figure 4A,B,D). Surprisingly, Ctsb was only minimally increased in non-infected ctsz2/2 mice (Figure 4A,C,F). In line with published human data, Ctsb expression was not induced by H. pylori in either wt or ctsz2/2 mice (Fig.4C,E,F). If expressed, both enzymes show a cytoplasmic staining pattern. Ctsz was predominantly expressed in macrophages, infected surface epithelium, and gastric glands, Ctsb in the surface epithelium and inflammatory cells. Ctsz seems to switch from an apical expression in surface epithelium and foveolae to a basalexpression most notably in deep gastric glands. SPEM is generally negative for Ctsz. Compared to wt animals after long term H. pylori infection, ctsz2/2 mice showed a significantly incr.