Deletion of nardilysin prevents the development of steatohepatitis and liver fibrotic changes.

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1-/- mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1-/- mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1-/- mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1-/- mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1-/- mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1-/- mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1-/- mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1-/- mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis

TNF-α was not sufficiently secreted from in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. A liver fragment was divided into two pieces. One was directly subjected to protein extraction directly and measurement of TNF-α (produced TNF-α). The other piece was cultured in serum-free medium for 12 hours, and the supernatant was subjected to measurement of TNF-α (secreted TNF-α). The relative optical density (O.D.) to that of <i>Nrd1^+/+</i> fed the CSAA diet was determined by ELISA for TNF-α. Production of TNF-α from liver specimens were not significantly different between <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet for 20 weeks (“produced TNF-α”). In contrast, TNF-α secreted from liver specimens was significantly increased in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those fed the CSAA diet; however, the elevation was not observed in <i>Nrd1^−/−</i> mice under the same condition (“secreted TNF-α”). *<i>P</i><0.05. B. Production of IL6 and IL1-β proteins were significantly increased only in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those in <i>Nrd1^−/−</i> mice fed the CSAA diet for 20 weeks. *<i>P</i><0.05. C. mRNA (upper, ‘produced’) and protein (lower, ‘secreted’) production of IL6 and IL1-β were significantly increased after LPS treatment in <i>Nrd1^+/+</i> mouse peritoneal macrophages. However, administration of anti-TNF-α neutralizing antibodies significantly suppressed the production of IL6 and IL1-β in the presence of LPS. *<i>P</i><0.05.</p

Liver fibrotic area was not observed in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Liver fibrosis was determined by Sirius red staining (red) in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice at 4 (upper), 12 (middle), and 20 (lower) weeks in the livers of <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA or CDAA diet. Fibrotic changes were not observed in <i>Nrd1^+/+</i> or <i>Nrd1^−/−</i> mice fed the CSAA diet (left). Fibrotic changes were prominent in <i>Nrd1^+/+</i> mice, but not in <i>Nrd1^−/−</i> mice fed the CDAA diet (right). Bars indicate 100 µm. B. Quantification of fibrotic areas. Fibrotic areas was observed and increased in a time-dependent manner only in the livers of <i>Nrd1^+/+</i> mice fed the CDAA diet. n = 5–8, each. *<i>P</i><0.05.</p

TNF-α was expressed in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Immunohistochemistry showed that TNF-α protein (red, arrowheads) was expressed in F4/80-positive Kupffer cells or macrophages (green, arrowheads) in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mouse fed the CDAA diet for 20 weeks (right), but not in mice fed the CSAA diet for 20 weeks (left). A blue color indicates DAPI-positive nuclei. Bars indicate 50 µm. B. The number of F4/80-positive cells/×100 high-power field (HPF) in livers slightly increased (approximately 1.2 times) only in <i>Nrd1^+/+</i> mice fed the CDAA diet (right). C. Relative expression of mRNA are shown as relative values compared to those at 0 w. The mRNA expression level of CCR2 was increased in <i>Nrd1^+/+</i> mice fed the CDAA diet, and the levels were significantly higher than respective values in <i>Nrd1^−/−</i> mice fed the CDAA diet. *<i>P</i><0.05.</p

CDAA diet did not cause steatohepatitis in <i>Nrd1^−/−</i> mice.

<p>A. Serum ALT was not elevated in both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA diet (left). Serum ALT levels were significantly elevated in <i>Nrd1^+/+</i> mice, but were not elevated in <i>Nrd1^−/−</i> mice upon administration of the CDAA diet (right). Each value depicts the mean ± standard errors (n = 5–15, each). *<i>P</i><0.05. B. Relative expression of mRNA are shown as relative values compared to those at 0 w. mRNA of TNF-α was increased in both <i>Nrd1^+/+</i> (significantly) and <i>Nrd1^−/−</i> mice fed the CDAA diet compared with those fed the CSAA diet, with no significant difference between the two groups. In contrast, the mRNA expression levels of IL6 and IL1-β were increased only in <i>Nrd1^+/+</i> mice, and these levels were significantly higher than respective values in <i>Nrd1^−/−</i> mice fed the CDAA diet. (n = 5–16, each) *<i>P</i><0.05.</p