Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells

Abstract

Infection with the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and oxidative DNA damage in gastric epithelial cells that can lead to gastric cancer (GC). However, the underlying pathogenic mechanism is largely unclear. Here, we report that the suppression of Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase that specifically removes oxidized bases, is one mechanism through which H. pylori infection may fuel the accumulation of DNA damage leading to GC. Using cultured cell lines, gastric biopsies, primary cells, and human enteroid-derived monolayers from healthy human stomach, we show that H. pylori infection greatly reduces NEIL2 expression. The H. pylori infection–induced down-regulation of NEIL2 was specific, as Campylobacter jejuni had no such effect. Using gastric organoids isolated from the murine stomach in co-culture experiments with live bacteria mimicking the infected stomach lining, we found that H. pylori infection is associated with the production of various inflammatory cytokines. This response was more pronounced in Neil2-knockout (KO) mouse cells than in WT cells, suggesting that NEIL2 suppresses inflammation under physiological conditions. Notably, the H. pylori–infected Neil2-KO murine stomach exhibited more DNA damage than WT. Furthermore, H. pylori–infected Neil2-KO mice had greater inflammation and more epithelial cell damage. Computational analysis of gene expression profiles of DNA glycosylases in gastric specimens linked the reduced Neil2 level to GC progression. Our results suggest that NEIL2 down-regulation is a plausible mechanism by which H. pylori infection impairs DNA damage repair, amplifies the inflammatory response, and initiates GC.

Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19

“…The plate was inverted and incubated at 37°C for 10 min to allow complete polymerization of the Matrigel before the addition of 1 mL Lung Expansion Medium per well. Lung expansion media was prepared by modifying the GI-organoid media (50% conditioned media, prepared from L-WRN cells with Wnt3a, R-spondin, and Noggin, ATCC-CRL-3276 TM ) (Ghosh et al, 2020;Sayed et al, 2020a;Sayed et al, 2020b;Sayed et al, 2020c) with a proprietary cocktail from the HUMANOID CoRE containing B27, TGF-β receptor inhibitor, antioxidants, p38 MAPK inhibitor, FGF 7, FGF 10 and ROCK inhibitor.…”

“…Lung-organoid-derived monolayers were prepared using a modified protocol of GI-organoid-derived monolayers (Ghosh et al, 2020;Sayed et al, 2020a;Sayed et al, 2020b;Sayed et al, 2020c). Briefly, transwell inserts (6.5 mm diameter, 0.4 um pore size, Corning) were coated in Matrigel diluted in cold PBS at a 1:40 ratio and incubated for 1hr at room temperature.…”

The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells

“…Similarly, ELISA analyses also revealed that Neil2 KO EDMs have a significantly higher KC expression compared to the WT EDMs following Fn infection ( Figure 3 C). As we have shown that NEIL2 prevents DNA damage accumulation following infection with H. pylori [ 44 ], we therefore examined the level of oxidized DNA bases in WT vs. Neil2 KO EDMs. Fn infection indeed led to the accumulation of oxidized DNA base lesions, and Fn -infected Neil2 KO EDMs accumulated a relatively higher level of oxidized DNA base lesions compared to Fn WT EDMs ( Figure 3 D).…”

“…Genomic DNA (gDNA) was extracted from WT, NEIL2- KO, and APC Min/+ EDMs, either uninfected or infected with Fn , using a Gentra Puregene Cell Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Accumulation of DNA strand-break was assessed in both transcribed genes ( polß and globin ) following the protocol described previously [ 31 , 44 ]. Briefly, the extracted gDNA was quantified by Pico Green (Molecular Probes, ThermoFischer, Waltham, MA, USA).…”

“…BER is the major pathway for the repair of such DNA lesions, and thereby a robust DNA damage response through BER can reduce the risk of colon carcinogenesis [ 43 ]. Recently, we have shown that Helicobacter pylori infection leads to degradation of NEIL2 and subsequent accumulation of oxidative DNA damage, a plausible cause of gastric cancer [ 44 ]. This result is in agreement with our earlier studies showing that the Neil2 -null mice are highly sensitive to inflammatory stress and also accumulate oxidized DNA bases in their genome [ 31 ].…”