Macrophage Arginase Restricts Host Defense to Helicobacter pylori by Inhibiting Inducible NO Synthase

Nuruddeen D. Lewis1, Rupesh Chaturvedi2,3, Kshipra Singh2,3, Mohammad Asim2,3, Daniel P. Barry2, Thibaut Saltet de Sablet2, Jean-Luc Boucher4, and Keith T.

Wilson1,2,3

1 Cancer Biology Department, Vanderbilt University School of Medicine2 Division of Gastroenterology, Vanderbilt University School of Medicine3 Veterans Affairs Tennessee Valley Health Care System, Nashville, TN4 Universite Rene Descartes, Paris, France

Gram-negative, microaerophilic bacterium that effectively colonizes the stomach

Infects more than half of the world’s population

Main cause of peptic ulceration, distal gastric adenocarcinoma, and gastric lymphoma

Considered non-invasive, but bacterial antigens have been demonstrated in the gastric lamina propria

Despite a vigorous immune response, the bacterium generally persists for the life of the host.

H. pylori is an Important Human Pathogen

Macrophages in HP Infection

Kaparakis et al. Infect Immun (2008).

Nitric Oxide Produced in copious amounts

by iNOS

Many diverse functions, including antimicrobial properties

iNOS–/– mice have increased susceptibility to various infections

Can react with proteins to form S-nitrosothiols, which can be bactericidal

NO kills HP in vitro

Description • Increasing strength and

stamina • Signaling muscle

growth and speeding recovery

• Promotion of an extended pump

Nitric Oxide is the REAL DEAL

Gobert et al. PNAS, 2001.

H. pylori Arginase Inhibits Host NO Production by Competition for L-Arg

Log 1

0 (CF

U/m

l)

2

876543

1

9

0 24 hrs 0 24 hrs

WT rocF::aphA3

medium medium

macrophages

macrophages

Bacterial Arginase Modulates NO-derivedH. pylori Killing

**p < 0.01 vs control

Gobert et al. PNAS 2001.

Macrophage-derived Killing of H. pylori rocF::aphA3 is NO-dependent

5

6

7

8

Log 1

0 (CF

U/m

l)

Wild-type iNOS–/– **p < 0.01 vs control

Gobert et al. PNAS, 2001.

H. pylori Arginase Inhibits iNOS Protein Expression

Chaturvedi et al. Infect. Immun., 2007.

Arginine Metabolism in H. pylori Pathogenesis

L-arginine

L-ornithineODC

Putrescine

H2O2

NO

Spermine

SpermidineApoptosis

Arginase II

SMO

H. pylori

iNOS

Hypothesis

In macrophages stimulated by H. pylori, L-arginine metabolism by arginase II reduces L-arginine availability. This limits iNOS protein expression and NO production.

Aims

Arginase activity restricts NO production and iNOS protein expression.

These effects can be attributed to arginase II. These effects occur in vivo.

To determine in H. pylori-activated macrophages if:

Design and Methods

C57BL/6 Mice Arginase II–/–Mice RAW 264.7 Macrophage

Cell Line

Stimulate with SS1-Treat w/ BEC-Treat w/ Arg2-siRNA Stimulate w/

HP for 24h

Real Time-PCRiNOS, ArgII

Western BlotiNOS

NO LevelsGriess Assay

Isolate Gastric Macrophages

after 48h

-Infect w/ SS1-Give BEC in drinking water

IsolatePMacs

4 month infectionHP Load, Gastritis

Flow Cyt.iNOS

Arginase Inhibition Increases NO Production in H. pylori-activated Macrophages

*p < 0.05 vs. no BEC**p < 0.01 vs. no BEC

***p < 0.001 vs. no BEC

RAW 264.7 cells

Arginase Inhibition Has No Effect on iNOS or Arginase II mRNA

RAW 264.7 cells

Arginase Inhibition Increases H. pylori-stimulated iNOS Protein Levels

RAW 264.7 cells

Knockdown of Arginase II Increases NO Production in H. pylori-activated Macrophages

**p < 0.01 vs. no BEC RAW 264.7 cells

Knockdown of Arginase II Increases iNOS Protein Without Affecting mRNA Levels

RAW 264.7 cells

Arginase II –/– Peritoneal Macrophages Have Increased NO Production and iNOS Protein

ArgII–/– + HPWT + HP

iNOS

Inhibition of Arginase in vivo Increases NO Production in Gastric Macrophages from H. pylori-infected Mice

**p < 0.01 vs. controlIsolated Gastric Macrophages

In vivo BEC Administration Increases iNOS Protein Levels in Gastric Macrophages

* p < 0.05, ***p < 0.001 vs. Ctrl # p < 0.05, ## p < 0.01, ### p < 0.001 vs. HP

Isolated Gastric Macrophages

Chronic Infection of Wild Type and Arginase II –/– Mice

Bacterial Colonization and Gastritis Scores from Arginase II–/– Mice

Gastritis Score and Bacterial Load Correlate in Arginase II–/– but not Wild Type Mice

Summary When arginase was inhibited, macrophages were

able to produce more NO in response to H. pylori, whether activated in vitro or in vivo.

Arginase II knockdown by siRNA and arginase-deficient peritoneal macrophages mimicked the effect seen with BEC.

Arginase inhibition or knockdown did not increase iNOS mRNA, but substantially increased iNOS protein levels.

Gastritis scores correlate with bacterial load in Arg2–/– mice, but not in WT mice

Conclusions

Arginase limits the availability of substrate for iNOS and this competition, along with its downstream products results in decreased iNOS protein synthesis.

Arginase activity in macrophages may contribute to the failure of the host innate immune response to H. pylori, and its inhibition could result in a more successful defense against H. pylori.

Future Directions

Determine if the mechanism of arginase II inhibition of iNOS is due to competition for L-Arg or the downstream effectors.

Determine if the phenotype of Arg2–/– mice is iNOS-dependent.

Determine the presence/role of myeloid-derived suppressor cells (MDSC) in H. pylori infection.