Because immune cells were found to have detectable levels of ASL even during basal conditions, no dietary intervention was necessary to reveal differential ASL expression between Vav1: Aslmice and controls; thus, mice were given a regular diet ( Figure S1 D). Following colitis induction by a standard dose of DSS (1.5%–2.5%), the degree of colonic inflammation was significantly milder in Vav1: Aslmice compared with control littermates ( Figures 3 A–3E). In addition, although control mice had decreased hemoglobin levels following colitis, Vav1: Aslmice displayed normal hemoglobin levels, likely reflecting decreased blood loss to the stool ( Figure 3 F). Hence, modulation of ASL levels in a cell type-specific manner differentially affects colitis severity; although ASL produced by enterocytes appears to have a protective role, ASL expression in immune cells exacerbates colonic injury.

(F) Anemia developed only in the control group after colitis induction, as shown by decreased red blood cells (RBCs), hemoglobin (HgB), and hematocrit (HCT) levels. No differences between groups were detected in the white blood cell (WBC) count (n = 3 in each group).

(C) A T2 map of representative colon sections, demonstrating increased relaxation time in the control group compared with Vav1 Cre : Asl f/f mice. Right: quantification of the images using Image-pro Pulse software. The experiment was performed on a total of n = 5 mice in each group.

(B) A representative experiment showing a significant increase in colon length in Vav1 Cre : Asl f/f mice compared with the control group. The experiment was performed three times with a total of n ≥ 19 mice in each group.

(A) A representative graph of endoscopic evaluation scores on day 12 after colitis induction, showing a significant improvement in the colitis score of the Vav1 Cre : Asl f/f group compared with the control group. The experiment was performed 3 times with a total of n ≥ 20 mice in each group with 3 repetitions. Right: a representative image of the colon taken by colonoscopy.

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Figure 4 Decreased NO Levels in ASL CKO Immune Cells Show full caption (A) Nitrite (NO 2 −) levels were measured by dedicated high-performance liquid chromatography (HPLC) in blood samples before and after colitis induction. Results show a significant elevation in NO levels after colitis induction in RBCs of the control group but not in Vav1Cre: Aslf/f mice (n ≥ 3 in each group). (B) A significant elevation in NO 2 − levels is seen in peritoneal macrophages growth medium 18 hr following LPS administration in the control group compared with the Vav1Cre: Aslf/f group (n = 6 in each group). (C and D) A significant reduction in glucose levels (C) and elevation in lactate levels (D) in response to LPS administration is demonstrated in control but not in Vav1Cre: Aslf/f macrophages. Measurements were performed using a NOVA measuring instrument, (n ≥ 4 in each group). (E) Immunohistochemistry staining for the total number of macrophages (using the F4-80 marker, top) as well as for activated macrophages (using the MAC2 marker of activation, bottom), in distal colon sections after colitis induction. The ratio between F4-80- and MAC2-positive cells shows a significant reduction in the number of activated macrophages in Vav1Cre: Aslf/f mice compared with controls. Cells were counted from at least 8 microscopic fields taken from 3 different animals. Right: quantification analysis. (F and G) Colitis was induced by DSS in CD11cCre:Aslf/f and in control Aslf/f mice. (F) Right: a representative graph showing that CD11cCre:Aslf/f mice have decreased colitis severity compared with control mice, as demonstrated by endoscopic evaluation on day 12 after colitis induction. The experiment was performed on a total of n ≥ 13 mice in each group. Left: a representative image of the colon taken by colonoscopy. (G) A representative graph showing a significant increase in colon length in CD11cCre:Aslf/f mice compared with the control group. The experiment was performed on a total of n ≥ 10 mice in each group. (H) ELISA measurement of colon tissues extracted from mice on day12 following colitis induction, showing significant elevation of pro-inflammatory cytokine levels in the control group compared with CD11cCre:Aslf/f mice. (I) Human data taken from a GEO dataset analysis (GEO: GSE57945 ; ID:200057945), showing significant upregulation of ASL and iNOS RNA expression in patients suffering from Crohn’s disease (CD) and ulcerative colitis (UC) compared with controls, with no significant change in ornithine decarboxylase (ODC) expression. Error bars represent SEM.

Increased NO production by immune cells is a well-known phenomenon associated with inflammation (). In addition, it was recently shown that red blood cells (RBCs) control systemic NO bioavailability by synthesizing, transporting, and releasing NO metabolic products (). Indeed, following colitis induction, we found a robust increase in nitrite production by RBCs only in control mice and not in Vav1: Aslanimals ( Figure 4 A). To confirm that the effect on the inflammatory cascade is caused specifically by decreased NO production by the immune cells of the mutant mice, we conducted ex vivo studies with peritoneal macrophages isolated from the Vav1: Aslmice, the major immune cell type producing NO (), and measured their response to lipopolysaccharide (LPS). Similar to the finding at the whole-organism level, Vav1:Aslmacrophages produced less nitrite in response to LPS ( Figure 4 B). Lately, several studies have shown that, in addition to the paracrine effects of macrophage-derived NO on other cells, NO has an autocrine effect in polarizing macrophages toward a pro-inflammatory (or “M1”) state. The metabolic mechanism behind this phenomenon was proposed to be a prominent induction of glycolysis (). We hence measured lactate and glucose levels in the medium of cultured peritoneal macrophages as a readout for macrophage activation (). Although macrophages of control mice showed decreased glucose levels and increased lactate production, consistent with increased glycolysis, no such changes were observed in Vav1: Aslmacrophages, suggesting their decreased activation ( Figures 4 C and 4D). Furthermore, colon histology after induction of colitis revealed decreased recruitment of activated monocytes/macrophages to the intestine in immune Asl CKO mice, as demonstrated by a decreased ratio between the general number of macrophages (marked with F-4/80) and the number of activated macrophages (marked with MAC-2) ( Figure 4 E). To specifically investigate intestinal macrophages, we analyzed the levels of iNOS and NO in gut hematopoietic cells (CD45+) and enterocytes (CD326+) (). As expected, we found ASL levels to be significantly lower in intestinal CD45+ cells of Vav1:Aslmice, correlating with decreased iNOS expression and decreased NO production; in contrast, there were no significant differences in these parameters in enterocytes of the same animals ( Figures S4 A–S4F). To corroborate these results, we generated CD11c:Aslmice and induced DSS colitis in these animals. Challenged CD11c:Aslmice displayed a decrease in weight loss, colitis scores, and pro-inflammatory cytokines and an increase in colon length, all supporting ameliorated colitis ( Figures 4 F–4H). Collectively, our data suggest that loss of ASL in enterocytes has a significant effect on increasing the severity of inflammation, whereas loss of ASL in immune cells, and in particular CD11cmononuclear phagocytes, alleviates colitis severity, likely because of alterations in NO levels. Interestingly, analysis of the GEO database revealed that ASL expression is increased in chronic IBD patients with Crohn’s disease or ulcerative colitis compared with controls. Moreover, in contrast to the expression of the rate-limiting enzyme in polyamine synthesis (i.e., ornithine decarboxylase [ODC]), ASL expression correlates with iNOS expression ( Figure 4 I; Figure S4 J). These data provide further support for our assumption of the importance of ASL re-expression in enterocytes during colitis for NO synthesis and for the potential relevance of our findings to human IBD.