35 Primary hepatic lymphocytes were stained with PE-Cy7-conjugated anti-CD3 (eBioscience; clone UCHT1, Catalog no. 25-0038; Hatfield, UK) and FITC-conjugated anti-CD56 (BD; Catalog no. 34058; Oxford, UK), and analyzed using Summit 4.3 software (Dako Cytomation). Formalin-fixed, paraffin-embedded liver sections from deidentified controls and subjects with biopsy-proven NASH-related

cirrhosis (n = 6/group) from the Departments of Pathology at Duke University and University Hospital Cassiano Antônio de Moraes were studied learn more in accordance with National Institutes of Health (NIH) and institutional guidelines for human subject research (see Supporting Information Materials and

Methods for immunohistochemistry protocol/antibodies). buy LDK378 The results are expressed as mean ± SEM. Statistical significance was determined using Student’s t test. Significance was accepted at the 5% level, *P < 0.05. Compared to control mice that were fed normal chow (n = 25), MCD diet-treated mice (n = 25) developed significant macrovesicular steatosis, ballooning degeneration of hepatocytes, and liver necro-inflammation (Fig. 1A), as well as fibrosis after 8 weeks. The latter was demonstrated by increased Sirius red staining (Fig. 1B,C) and hepatic hydroxyproline quantification (Fig. 1D). Collagen deposition was accompanied check details by the accumulation of alpha-smooth muscle actin (α-SMA)-immunoreactive cells (Fig. 1E,F) and induction of profibrogenic genes, including α-SMA, transforming growth factor beta (Tgf-β), collagen 1α1, mmp9, and timp1 (Supporting Information Fig. 1A-E). These fibrotic livers also demonstrated increased activity of the Hh-pathway, a morphogenic signaling system that orchestrates wound healing responses.36 Sonic hedgehog ligand (Shh) mRNA expression tripled after MCD diet treatment and mRNA levels of the Hh-regulated transcription factor, glioblastoma 2 (Gli2), increased 4-fold. This was accompanied by significant accumulation of Gli2-expressing cells, which tended to localize near portal tracts

and along fibrous septa that contained immature ductular cells and fibroblastic cells (Fig. 2A). mRNA levels of CXCL16, the Hh-inducible NKT cell chemokine, and vascular cell adhesion molecule 1 (VCAM1), a factor that promotes NKT cell adhesion, increased significantly by MCD diet treatment (Fig. 2B,C). Hh-dependent production of CXCL16 by immature ductular cells promotes NKT cell chemotaxis.37 To determine if Hh-pathway activation also promotes NKT cell adhesion, NKT cells were incubated with immature ductular cells in the presence of vehicle or Shh. Shh significantly increased adhesion of NKT cells to ductular cells; this was abrogated by adding 5E1 antibody to neutralize Shh activity (Fig. 2D).