Induction of Premalignant Host Responses by Cathepsin X/Z-Deficiency in <i>Helicobacter Pylori-</i>Infected Mice

<div><p><i>Helicobacter pylori</i> are responsible for the induction of chronic gastric inflammation progressing to atrophy, metaplasia, and gastric cancer. The overexpression of Cathepsin X/Z (Ctsz) in <i>H. pylori-</i>infected mucosa and gastric cancer is mediated predominantly by an augmented migration of ctsz^−/−positive macrophages and the up-regulation of Ctsz in tumor epithelium. To explore the Ctsz-function in the context of chronic inflammation and the development of preneoplastic lesions, we used <i>Ctsz</i>-deficient mice in a <i>H. pylori</i> gastritis model. <i>Ctsz</i>^−/− and wild-type (wt) mice were infected with <i>H. pylori</i> strain SS1. The mice were sacrificed at 24, 36, and 50 weeks post infection (wpi). The stomach was removed, and gastric strips were snap-frozen or embedded and stained with H&E. Tissue sections were scored for epithelial lesions and inflammation. Ki-67 and F4/80 immunostaining were used to measure epithelial cell proliferation and macrophage infiltration, respectively. The upregulation of compensating cathepsins and cytokines were confirmed by Western blotting and quantitative RT-PCR. SS1-infected wt and <i>ctsz</i>^−/− mice showed strong inflammation, foveolar hyperplasia, atrophy, and cystically-dilated glands. However, at 50 wpi, <i>ctsz</i>^−/− mice developed significantly more severe spasmolytic polypeptide-expressing metaplasia (SPEM), showed enhanced epithelial proliferation, and higher levels of infiltrating macrophages. Induction of cytokines was higher and significantly prolonged in <i>ctsz</i>^−/− mice compared to wt. Ctsz deficiency supports <i>H. pylori</i>-dependent development of chronic gastritis up to metaplasia, indicating a protective, but not proteolytic, function of Ctsz in inflammatory gastric disease.</p></div

Histochemical (PAS/Alcian blue) and immunohistochemical (F4/80, Ki-67) stainings in gastric mucosa.

<p>Uninfected and <i>H. pylori</i> SS1-infected mice at 24 and 50 wpi were analyzed for proliferative activity, macrophage infiltration, and SPEM development. Expression of F4/80, indicating infiltrating macrophages, was much higher (<i>p</i> = 0.075) in infected <i>ctsz^−/−</i> mice compared to wt at 50 wpi. This was accompanied by a higher proliferation rate as shown by nuclear Ki-67 immunoreactivity (p = 0.029) and significantly stronger SPEM formation (p = 0.023) in <i>ctsz^−/−</i> mice (closed arrows) with intestinal-type acidic mucin-expressing glands (open arrows). Macrophages and proliferating cells were evaluated for their quantity per visual field. SPEM was quantified as outlined by Rogers et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070242#pone.0070242-Rogers1" target="_blank">[23]</a>. Results from data sets (n = 5–11) are presented in the box plots (IRS, immunoreactive score). All box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). Solid dots are outliers above 95%. The line in the box represents the median.</p

Histological evaluation of inflammation, hyperplasia, and glandular ectasia.

<p>Blinded H&E-stained gastric sections from n = 5–11 wt and <i>ctsz^−/−</i> mice infected or non-infected with <i>H. pylori</i> SS1 for 24, 36, or 50 weeks were assessed. Sections were graded from 0–5 based on the criteria of Rogers <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070242#pone.0070242-Rogers1" target="_blank">[23]</a>. Compared to sham-inoculated mice, gastric mucosa of infected mice exhibited marked inflammation (<i>p</i> = 0.001) with abscesses (Ab) and lymph follicles (Lf), as well as mucosal thickening (<i>p</i> = 0.001), glandular ectasia (<i>p</i> = 0.001), and loss of parietal cells with development of mucus metaplasia (closed arrows). There were no statistically significant differences between wt and <i>ctsz^−/−</i> mice for all three criteria. All box plots show 25^th to 75^th percentiles (box) and 5^th to 95^th percentiles (whiskers). Solid dots are outliers above 95%. The line in the box represents the median.</p

Colonization efficiency of <i>H.</i><i>pylori</i> in the corpus mucosa and the induction of Ctsz.

<p>(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 <i>H. pylori</i>-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 <i>H. pylori</i> SS1-, as well as B128-inoculated wt epithelial cells whereas <i>ctsz^−/−</i> 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 (•) and <i>ctsz^−/−</i> (□) challenged with <i>H. pylori</i> SS1 for 24, 36 or 50 weeks was semiquantitatively graded of <i>H. pylori</i> levels using Warthin-Starry staining with scores from minimum = 1 to maximum = 3 (D) and quantified using the ΔΔCt method by qRT-PCR (E).</p

Differential expression of Ctsz and Ctsb.

<p>(A) Western blot analysis revealed <i>H. pylori</i>-dependent induction of Ctsz expression in wt mice after 36 wpi, whereas Ctsb remains unaffected in both <i>ctsz^−/−</i> and wt mice. (B and C) Immunohistochemical (IHC) grading and (D–E) staining of Ctsz and Ctsb of corpus mucosa from wt (D,E) and <i>ctsz^−/−</i> mice infected and uninfected (inserts) with <i>H. pylori</i> SS1. <i>H. pylori</i> infection significantly increased Ctsz expression in macrophages and deep glands, but only slightly in surface epithelium (<i>p</i> = 0.002–0.008). Ctsb was predominantly expressed in surface epithelium and some macrophages with no significant changes after <i>H. pylori</i> infection. All box plots show 25^th to 75^th percentiles. The line in the box represents the median.</p