Helicobacter hepaticuscytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice

Abstract

Summary Multiple pathogenic Gram-negative bacteria produce the cytolethal distending toxin (CDT) with activity of DNase I; CDT can induce DNA double strand breaks (DSBs), G2/M cell cycle arrest and apoptosis in cultured mammalian cells. However, the link of CDT to in vivo tumorigenesis is not fully understood. In this study, 129/SvEv Rag2−/− mice were gavaged with wild-type Helicobacter hepatics 3B1(Hh) and its isogenic cdtB mutant HhcdtBm7, followed by infection for 10 and 20 weeks (WPI). HhCDT-deficiency did not affect cecal colonization levels of HhcdtBm7, but attenuated severity of cecal pathology in HhcdtBm7-infected mice. Of importance, preneoplasic dysplasia was progressed to cancer from 10 WPI to 20 WPI in the Hh-infected mice but not in the HhcdtBm7-infected mice. In addition, the loss of HhCDT significantly dampened transcriptional upregulation of cecal Tnfα and Il-6, but elevated Il-10 mRNA levels when compared to Hh at 10 WPI. Furthermore, the presence of HhCDT increased numbers of lower bowel intestinal γH2AX-positive epithelial cells (a marker of DSBs) at both 10 WPI and 20 WPI, and augmented phospho-Stat3 foci+ intestinal crypts (activation of Stat3) at 20 WPI. Our findings suggest that CDT promoted Hh carcinogenesis by enhancing DSBs and activation of the Tnfα /Il-6-Stat3 signaling pathway.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2−/‐Il10−/−gpt delta mice is influenced by sex

“…This difference was unlikely attributable solely to H. hepaticus CDT activity, since H. hepaticus colonization levels were comparable between RagIl10gpt males and females. Given that infection with the cdtB ‐defective H. hepaticus mutant induced less epithelial DSBs in concert with a decreased severity of dysplastic lesions compared to CDT‐producing H. hepaticus in the lower bowel of Rag2‐deficient mice . It is plausible to speculate that deficiency in CDT activity could attenuate the ability of H. hepaticus to promote lower bowel MFs in Rag2Il10gpt males.…”

“…females at 21 WPI suggests a longer duration (>21 WPI) of H. hepaticus infection in this mouse strain is needed to differentiate the differences in the lower bowel pathology between males and females. This premise is supported by our previous study showing that wild‐type (WT) H. hepaticus infection in Rag2‐deficient mice induced higher levels of cecal Tnfα and γH2AX, but similar severity of intestinal pathology compared to CDT‐deficient H. hepaticus mutant infection at 10 WPI; more severe preneoplastic lesions in the lower bowel were noted in the WT H. hepaticus ‐infected mice compared to the H. hepaticus mutant‐infected mice at 20 WPI …”

“…Our previous studies indicated that H. hepaticus infection in Rag2 −/− mice induced progression of intestinal pathology from inflammation, dysplasia to carcinoma during the duration of infection, and this process is mediated in part by elevated production of NO . The increased production of NO as evidenced by elevation of urinary nitrate excretion was correlated with H. hepaticus ‐induced overexpression of Tnfα and iNos primarily in macrophages and lower bowel epithelial cells in concert with neutrophils . Consistent with these previous findings in H. hepaticus ‐infected Rag2 −/− mice, there were significantly higher levels of epithelial iNos expression in the cecum and colon as well as the upregulation of Tnfα and iNos , Il‐17A , Il‐22 and Ifnγ in H. hepaticus ‐infected RagIl10gpt mice of both sexes compared to their uninfected controls.…”

“…qPCR assays for quantifying H. hepaticus and pks+ E. coli were performed in the 7,500 Fast Real‐Time PCR system (Life Technologies, Carlsbad, CA). Genome copy numbers of H. hepaticus or pks + E. coli were expressed per microgram of murine chromosomal DNA, which was measured by qPCR using a mammalian 18S rRNA gene‐based primer and probe mixture (Thermo Fisher Scientific, Waltham, MA) as described previously . Additional information is described in the Supporting Information.…”

“…H. hepaticus infection causes IBD and colon cancer in susceptible mouse strains; these infectious models have been widely used to delineate etiopathogenesis of IBD and intestinal carcinogenesis . Inactivation of H. hepaticus CDT attenuated H. hepaticus ‐induced colitis accompanied by a reduction of double‐strand DNA breaks in 129/SvEv Rag2 −/− mice . The mechanisms underlying the role of H. hepaticus in promoting intestinal tumorigenesis have been extensively characterized .…”