Gastroenterology

Gastroenterology

Volume 138, Issue 4, April 2010, Pages 1546-1556.e5
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Neutrophil Migration During Liver Injury Is Under Nucleotide-Binding Oligomerization Domain 1 Control

https://doi.org/10.1053/j.gastro.2009.12.008Get rights and content

Background & Aims

A more complete understanding of the mechanisms involved in pathogen-associated molecular pattern signaling is crucial in the setting of liver injury. In intestinal diseases, nucleotide-binding oligomerization domain 1 (NOD1), a receptor for bacteria, appears to regulate cross-talk between innate and adaptive immunity, involving polymorphonuclear neutrophils (PMNs). Our aim was to explore the role of NOD1 in PMN-induced liver injury.

Methods

Nod1+/+ and Nod1−/− mice were challenged with carbon tetrachloride (CCl4). Migration and phagocytosis of Nod1+/+ and Nod1−/− PMN were studied in vivo and ex vivo. We evaluated main inflammatory pathways in PMNs by Western blot and CD11b expression using fluorescence-activated cell sorting. Mice were submitted to liver ischemia/reperfusion.

Results

After CCl4 exposure, livers of Nod1−/− mice had more than 50% less PMN infiltration within necrotic areas than those of Nod1+/+. PMNs isolated from Nod1−/− mice displayed a 90% decrease in migration capacity compared with Nod1+/+ PMNs, whereas FK 565, a potent NOD1 ligand, increased PMN migration. Upon FK 565 stimulation, mitogen-activated protein kinase and nuclear factor κB were activated in Nod1+/+ PMNs, but less so in Nod1−/− PMNs. Expression of CD11b on the Nod1−/− PMN was decreased compared with Nod1+/+. The phagocytic capacity of Nod1−/− PMNs was decreased by more than 50% compared with Nod1+/+. In an ischemia/reperfusion model of PMN-induced liver injury, FK 565 increased lesions, whereas Nod1−/− mice were protected.

Conclusions

The identification of NOD1 as a modulator of PMN function and migration in the liver suggests that this receptor may represent a new therapeutic target in PMN-dependent liver diseases.

Section snippets

Migration Assays

Random and directed migration of PMNs was studied using the Boyden chamber technique as previously described.20 N-formyl-methionyl-leucyl-phenylalanine (fMLP) was purchased from Sigma (Sigma-Aldrich, Saint-Quentin Fallavier, France). Forty-five microliters of a suspension of 1 × 106 PMNs in Hank's balanced salt solution (HBSS) containing 0.5% bovine serum albumin were incubated in the upper compartment of a chamber separated from the lower compartment by a cellulose filter (3-μm–diameter pores;

Nod1−/− Mice Displayed Impaired Hepatic PMN Recruitment

Challenge with CCl4 induced acute hepatitis with an increase in mice plasma ALT levels that peaked, as expected, at 18 hours after injection (Supplementary Figure 1). Nod1−/− and Nod1+/+ mice displayed similar kinetics of plasma ALT. Likewise, there was no difference in terms of macroscopic appearance of excised livers between the 2 groups of mice (Supplementary Figure 1). As expected, microscopic analysis of excised liver showed extensive hepatocyte necrosis in pericentrilobular areas (Figure 1

Discussion

Progress in our understanding of the mechanisms of neutrophil function is necessary to design new therapeutic approaches for patients with PMN-mediated liver injury. In the present study, we showed that NOD1 is an intrinsic central regulator of PMN migration and phagocytic capacity. In such cells, NOD1 acted, at least in part, through activation of MAPKs and NF-κB, and expression of β2 integrins at the PMN surface.

It has been shown that NOD1 indirectly influenced PMN recruitment in intestinal

Acknowledgments

S.D. and M.B.-M. contributed equally to this work.

The authors thank Nathalie Jouy for her help in flow cytometry analysis and the “Service Commun de Cytométrie et de Tri Cellulaire” set-up by Institut Fédératif de Recherche 114 (Institut de Medecine Prédictive et de Recherche Thérapeutique). The authors are grateful to Candice Duburque for valuable technical help and Aurore Saudemont for hematologic analysis. The authors thank Professor Daniel Poulain for critical reading of the manuscript. For

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by French grants from Arcir (from the “Conseil Regional of Nord Pas-de-Calais”), Institut de Recherches Scientifiques sur les Boissons, Paris, France, INSERM “ATC Alcool,” the “Institut Universitaire de France,”“Agence Nationale de la Recherche”ANR-06-PHYSIO-022, and Fonds européen de développement régional (FEDER).

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