Volume 138, Issue 4, Pages 1546-1556.e5 (April 2010) Neutrophil Migration During Liver Injury Is Under Nucleotide-Binding Oligomerization Domain 1 Control  Sébastien Dharancy, Mathilde Body–Malapel, Alexandre Louvet, Dominique Berrebi, Emilie Gantier, Philippe Gosset, Jérôme Viala, Antoine Hollebecque, Christophe Moreno, Dana J. Philpott, Stephen E. Girardin, Philippe J. Sansonetti, Pierre Desreumaux, Philippe Mathurin, Laurent Dubuquoy  Gastroenterology  Volume 138, Issue 4, Pages 1546-1556.e5 (April 2010) DOI: 10.1053/j.gastro.2009.12.008 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Impaired PMN infiltration of Nod1−/− liver after CCl4 exposure. Nod1−/− and Nod1+/+ mice were exposed to CCl4 (50 μL/kg). They were killed at different time points (n = 6–8 at each time point) and liver was harvested. (A) Representative histology of Nod1+/+ (left) and Nod1−/− (right) mice livers, 24 hours after CCl4 injection (original magnification is indicated). The number of PMNs infiltrating the necrotic area was determined in Nod1+/+ (white bars) and Nod1−/− (black bars) mice livers, 12, 18, and 24 hours post-CCl4 exposure and is represented on a bar graph (right panel). Results are expressed as mean ± SEM. *Statistical significance. (B) Chemokine mRNA expression in the liver of Nod1+/+ (white bars) and Nod1−/− (black bars) mice 1, 2, 4, and 6 hours after CCl4 exposure (n = 6–8 at each time point). Results are expressed as mean ± SEM and statistical significances are indicated. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 NOD1 regulates the migration capacity of isolated PMNs. (A) Representative agarose gel for NOD1 mRNA expression (upper panel) in colon, small intestine (intestine), liver, skin, lung, mesenteric fat (fat), spleen, and kidney. Negative control is indicated as none. Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as a housekeeping gene (lower panel). (B) Boyden chambers were used to evaluate the migration capacity of PMNs. Neutrophils isolated from Nod1+/+ (white bars) and Nod1−/− (black bars) mice were placed in the upper compartment of a Boyden chamber and were separated from the lower compartment by a cellulose filter. The last compartment contained either HBSS (as the unstimulated condition) or a chemoattractant such as chemokines (lipopolysaccharide-induced CXC chemokine [lix], macrophage inflammatory protein–2 [mip-2], keratinocyte-derived chemokine [kc]) or fMLP. Results of 3 independent experiments are expressed as mean ± SEM, and statistical significances are indicated. (C) HBSS or NOD1 ligand FK 565 (FK) was placed in the upper well with Nod1+/+ and Nod1−/− PMNs, and HBSS, FK 565, or fMLP were placed in the lower well. Results of 3 independent experiments are expressed as mean ± SEM and statistical significances are indicated. (D) Human PMNs were isolated and allowed to migrate toward HBSS (white bar), a ligand of NOD1 (FK 565, 10−6 mol/L; gray bar), or fMLP (10−7 mol/L, black bar). Results of 3 independent experiments are expressed as mean ± SEM and statistical significances are indicated. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 NOD1 regulates CD11b expression at the PMN surface. Flow cytometry analysis of CD11b expression in mouse liver PMNs. (A) C57Bl/6 mice injected with oil (vehicle) or CCl4 also were treated with a NOD1 agonist (FK 565) or PBS (vehicle). Representative dot-plots of vehicle-treated (vehicle, left dot-plot) or FK 565–treated mice (FK 565, right dot-plot) show the effect of the NOD1 agonist on CD11b expression in GR1+ cells (upper right quadrant). This analysis was performed under basal and inflammatory (CCl4) conditions (right table). (B) Nod1+/+ (wild-type [WT]) and Nod1−/− (knock-out [KO]) mice were treated with vehicle or FK 565. Representative dot-plots show a sharp decrease in CD11b expression in GR1+ cells (upper right quadrant) in Nod1−/− (right dot-plot) compared with Nod1+/+ (left dot-plot) livers. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Effect of NOD1 deletion or activation on signaling pathways in isolated PMNs. Activation of MAPK and NF-κB pathways was determined by Western blot using specific phospho- and non–phospho-antibodies. PMNs were isolated from Nod1+/+ (Nod1+/+) or Nod1−/− (Nod1−/−) mice and remained unstimulated (Ø) or were stimulated with either FK 565 (fk) or fMLP (fmlp). (A) Left panel: representative Western blot for the active form of p38 (phosphor-p38) and total p38 (p38). Right panel: bar graph showing the median ratio between the active form of p38 and total p38 ± SEM (quantified by optical density analysis of each band of the Western blot). (B) Left panel: representative Western blot for the active form of JNK (phosphor-jnk) and total JNK (jnk). Right panel: bar graph showing the median ratio between the active form of JNK and total JNK ± SEM after evaluation of the optical density of each band of the Western blot. (C) Left panel: representative Western blot for the phosphorylated form of IκB (phospho-iκb) and total IκB (iκb). Right panel: bar graph showing the median ratio between the phosphorylated form of IκB and the total undegraded IκB ± SEM after evaluation of the optical density of each band of the Western blot. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 NOD1 regulates the phagocytic capacity of PMNs and mononuclear cells. Phagocytic capacity of immune cells from Nod1+/+ (white bars) and Nod1−/− (black bars) mice was determined using fluorescence-activated cell sorting. (A) Representative graph (open layout) of PMNs isolated from Nod1+/+ (upper) and Nod1−/− (lower) mice. The negative control is shown in gray. (B) Phagocytic capacity is determined as the mean intensity of fluorescence value. Results of 3 independent experiments are expressed as mean ± SEM of percentage of mean intensities of fluorescence compared with Nod1+/+ leukocytes. Statistical significances are indicated. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 NOD1 plays a role in liver injury after I/R in mice. After 75 minutes of ischemia and 20 hours of reperfusion, livers of Nod1+/+ mice untreated (Nod1+/+, n = 10) or treated with FK 565 (FK 565, n = 10) and livers of Nod1−/− mice (Nod1−/−, n = 10) were stained with H&E and examined blindly under a microscope. Representative images of mouse liver with original magnifications of 50× (upper panel) and 200× (lower panel). (A) As expected, Nod1+/+ mice displayed large necrotic areas delineated by dotted lines (upper). These lesions were infiltrated by PMNs, as indicated by arrows. (B) In Nod1+/+ mice treated with FK 565, necrotic areas were larger and more numerous than in untreated mice (Nod1+/+). These areas were highly infiltrated by PMNs. (C) Livers of Nod1−/− mice were characterized by limited lesions and a discrete PMN infiltrate. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 NOD1 alters necrosis and PMN infiltrate in the I/R model. Analysis of liver and plasma after I/R. (A) Number of neutrophils per necrotic areas (left panel) and semiquantification of necrosis (right panel) in liver of wild-type mice treated (WT + FK, grey bar) or not (WT, white bar) with FK565. (B) Plasma ALT and AST levels of WT and WT + FK mice after 6 hours of reperfusion, expressed as fold increase compared with basal level. (C) Number of neutrophils per necrotic areas (left panel) and semiquantification of necrosis (right panel) in liver of Nod1+/+ (white bar) and Nod1−/− mice (black bar). (D) Plasma ALT and AST levels of Nod1+/+ and Nod1−/− mice after 20 hours of reperfusion, expressed as fold increase compared with basal level. Results of 2 or 3 independent experiments are expressed as mean ± SEM. Statistical significances are indicated. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Similar macroscopic change in Nod1−/− and Nod1+/+ mice after CCl4 injection. Upper panel: kinetic of ALT plasma level in Nod1+/+ (black layout) and Nod1−/− (gray layout) mice from 1 to 48 hours after CCl4 intraperitoneal injection. Results are expressed as mean ± SEM. Lower panel: macroscopic aspect of Nod1+/+ (right panel) and Nod1−/− liver (left panel) 24 hours after CCl4 exposure (CCl4 50 μL/kg, upper panel) or vehicle administration (olive oil, lower panel). Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Decreased PMN chemoattraction after thioglycolate injection into Nod1−/− mice. (A) Percentage or (B) absolute number of PMNs counted in peritoneal liquid of Nod1+/+ (white bars) and Nod1−/− (black bars) mice 4 hours after injection of HBSS or thioglycolate 4%. Results are expressed as mean ± SEM. (C) Representative pictures of cellular content of peritoneal liquid of Nod1+/+ (upper) and Nod1−/− (lower) mice. PMNs are easily distinguishable from other cell types such as macrophages and mesothelial cells. The percentage of PMNs was determined based on 4 fields counted under a light microscope (original magnification is indicated). Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Nod1−/− mice did not display any defect in granulopoiesis or blood PMN. (A) Tukey diagram representing the number of blood leukocytes (left diagram) and the number of blood PMN (right diagram) in Nod1+/+ (white box) and Nod1−/− (black box) mice. Medians are framed; the first and the third quartile and the first and the ninth decile are indicated. Statistical significances are indicated. (B) Representative dot-plots (left panel) and bar graph (right panel) for granuloblast determination in Nod1+/+ (left dot-plot and white bar) and Nod1−/− (right dot-plot and black bar) mice. Results are indicated as a percentage of granuloblasts. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 NOD1 mRNA expression in isolated PMNs. Reverse-transcription PCR was used to determine the mouse mRNA levels of NOD1. Bar graph indicated NOD1 mRNA levels in peripheral blood mononuclear cells (pbmcs; white bars) and PMNs (black bars) from Nod1+/+ (left panel) and Nod1−/− (right panel) mice. Results of 3 independent experiments are expressed as mean ± SEM. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as a housekeeping gene. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 NOD1-regulated CD11b expression on the liver PMN surface. Diagram obtained by fluorescence-activated cell sorting (facs) representing CD11b expression on the liver PMN surface of Nod1+/+ without (grey) or with (green) stimulation with fMLP (10−6 mol/L) and Nod1−/− mice without (black) or with (red) stimulation with fMLP (10−6 mol/L). Table summarizes expression of integrins (CD11a, b, and c, and CD18) at the PMN surface obtained from Nod1+/+ and Nod1−/− mice. Results are expressed as the mean intensity of fluorescence (mif) value. Gastroenterology 2010 138, 1546-1556.e5DOI: (10.1053/j.gastro.2009.12.008) Copyright © 2010 AGA Institute Terms and Conditions