The innate immune molecule, NOD1, regulates direct killing ofHelicobacter pyloriby antimicrobial peptides

Abstract

SummaryThe cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI + H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human b-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI + bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1 'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAIdependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI + H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human b-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI + H. pylori infection.

Nucleotide Oligomerization Domain 1 Enhances IFN-γ Signaling in Gastric Epithelial Cells during Helicobacter pylori Infection and Exacerbates Disease Severity

“…Secretion of the potent chemoattractants, IL-8 and IP-10, were measured to assess NF-kB and IFN-g proinflammatory signaling, respectively, and as a means of investigating the ability of epithelial cells to recruit and direct adaptive immune responses. Consistent with previous data (1,34,40), AGS cells treated with siRNA to NOD1 secreted significantly less IL-8 than AGS control cells treated with scrambled siRNA (Fig. 1A), in response to stimulation with wild-type (WT) H. pylori bacteria.…”

“…5B; C. Allison and R. Ferrero, unpublished observations). As we have previously found NOD1 to be a crucial mediator of cagPAI-dependent epithelial signaling (1, 40), we next investigated the role of this pathogen recognition molecule in H. pyloriinduced IRF1 expression using AGS cells stably expressing siRNA to NOD1 or an irrelevant gene: AGS NOD1 KD and control cells, respectively (34). In agreement with the responses in MKN28 cells (Fig.…”

“…In addition, we used AGS cells stably expressing small interfering RNA (siRNA) to either the caspase activation and recruitment domain of NOD1 or an irrelevant gene, enhanced GFP (referred to henceforth as AGS NOD1 KD or AGS control cells, respectively) (34). All cell lines were cultured as described previously (34).…”

“…In addition, we used AGS cells stably expressing small interfering RNA (siRNA) to either the caspase activation and recruitment domain of NOD1 or an irrelevant gene, enhanced GFP (referred to henceforth as AGS NOD1 KD or AGS control cells, respectively) (34). All cell lines were cultured as described previously (34). For STAT1 overexpression, cells were transfected with STAT1 plasmid (35) (100 ng/well), standardized to 1150 ng/well with pcDNA3 (1) in polyethyleneimine (Polysciences, Warrington, PA) 16 h prior to stimulation.…”

Activation of type I IFN signaling by NOD1 mediates mucosal host defense againstHelicobacter pyloriinfection

“…Thus, Grubman et al has shown that H. pylori-induced activation of NOD1 in human gastric ECs results in enhanced β-defensin secretion (hBDs) in a cag-PAI-dependent manner. 25 In addition, it has been shown that gastric expression of murine β-defensin 4 is decreased in NOD1-deficient mice as compared with NOD1-intact mice. 29 These data suggest that NOD1-induced hBDs also exert anti-microbial activity in the mucosal environment.…”