l -Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori

Abstract

Helicobacter pylori infection of the stomach causes an active immune response that includes stimulation of inducible nitric oxide (NO) synthase (iNOS) expression. Although NO can kill H. pylori, the bacterium persists indefinitely, suggesting that NO production is inadequate. We determined if the NO derived from iNOS in macrophages was dependent on the availability of its substrate, L-arginine (L-Arg). Production of NO by H. pylori-stimulated RAW 264.7 cells was dependent on the L-Arg concentration in the culture medium, and the 50% effective dose for L-Arg was 220 M, which is above reported plasma L-Arg levels. While iNOS mRNA induction was L-Arg independent, iNOS protein increased in an L-Arg-dependent manner that did not involve changes in iNOS protein degradation. L-Lysine, an inhibitor of L-Arg uptake, attenuated H. pylori-stimulated iNOS protein expression, translation, NO levels, and killing of H. pylori. While L-Arg starvation suppressed global protein translation, at concentrations of L-Arg at which iNOS protein was only minimally expressed in response to H. pylori, global translation was fully restored and eukaryotic translation initiation factor ␣ was dephosphorylated. H. pylori lacking the gene rocF, which codes for a bacterial arginase, induced higher levels of NO production by increasing iNOS protein levels. When murine gastric macrophages were activated with H. pylori, supraphysiologic levels of L-Arg were required to permit iNOS protein expression and NO production. These findings indicate that L-Arg is rate limiting for iNOS translation and suggest that the levels of L-Arg that occur in vivo do not permit sufficient NO generation by the host to kill H. pylori.

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

“…The glandular stomach was digested, and isolation of gastric macrophages was performed as described using CD11b-positive selection (2,39). Isolated cells were incubated with anti-F4/80 antibody conjugated with phycoerythrin (PE) (Invitrogen; 1:100 dilution).…”

“…For c-Fos and c-Jun, 1 g of mRNA was reverse-transcribed with the iScript TM cDNA synthesis kit (Bio-Rad). PCR was performed with thermal cycling conditions and methods to calculate the relative expression as described (2,8,9,35).…”

“…Infected individuals exhibit chronic active gastritis and can develop peptic ulcer disease or gastric adenocarcinoma, the second leading cause of cancer deaths worldwide (3). The infection is usually acquired in childhood and persists for the life of the host despite eliciting a vigorous innate and adaptive immune response (2). Although H. pylori has generally been considered to be a noninvasive pathogen, strong evidence has emerged that H. pylori itself and its products can invade the mucosa and have direct contact with lamina propria immune cells (4 -6).…”

“…We have reported that H. pylori induces apoptosis of macrophages by generation of polyamines from ornithine decarboxylase (ODC) In infection studies, apoptosis of macrophages has been demonstrated to play a pathogenic role in colonization of bacteria (1). Helicobacter pylori is a microaerophilic, Gram-negative bacterium that selectively colonizes the human stomach and infects half of the world population (2). Infected individuals exhibit chronic active gastritis and can develop peptic ulcer disease or gastric adenocarcinoma, the second leading cause of cancer deaths worldwide (3).…”

Pneumocystis Mediates Overexpression of Antizyme Inhibitor Resulting in Increased Polyamine Levels and Apoptosis in Alveolar Macrophages

“…H. pylori activates the transcriptional factor c-Myc, which induces ODC expression, resulting in increased intracellular polyamine pools in macrophages (44). This polyamine accumulation greatly reduces the uptake of L-arginine, which is the substrate of inducible nitric oxide synthase and is also the rate-limiting factor for inducible nitric oxide synthase translation (45). Therefore, increased polyamine levels consequently decrease the synthesis of inducible nitric oxide synthase in macrophages and result in loss of innate immune responses against H. pylori infection (46).…”