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Liver inflammation is greater in nonalcoholic steatohepatitis (NASH) than steatosis, suggesting that immune responses contribute to nonalcoholic fatty liver disease (NAFLD) progression. Livers normally contain many natural killer T (NKT) cells that produce factors that modulate inflammatory and fibrogenic responses. Such cells are relatively depleted in steatosis, but their status in more advanced NAFLD is uncertain. We hypothesized that NKT cells accumulate and promote fibrosis progression in NASH. We aimed to determine if livers become enriched with NKT cells during NASH-related fibrosis; identify responsible mechanisms; and assess if NKT cells stimulate fibrogenesis. NKT cells were analyzed in wildtype mice and Patched-deficient (Ptc(+/-)) mice with an overly active Hedgehog (Hh) pathway, before and after feeding methionine choline-deficient (MCD) diets to induce NASH-related fibrosis. Effects of NKT cell-derived factors on hepatic stellate cells (HSC) were examined and fibrogenesis was evaluated in CD1d-deficient mice that lack NKT cells. NKT cells were quantified in human cirrhotic and nondiseased livers. During NASH-related fibrogenesis in wildtype mice, Hh pathway activation occurred, leading to induction of factors that promoted NKT cell recruitment, retention, and viability, plus liver enrichment with NKT cells. Ptc(+/-) mice accumulated more NKT cells and developed worse liver fibrosis; CD1d-deficient mice that lack NKT cells were protected from fibrosis. NKT cell-conditioned medium stimulated HSC to become myofibroblastic. Liver explants were 2-fold enriched with NKT cells in patients with non-NASH cirrhosis, and 4-fold enriched in patients with NASH cirrhosis.Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH.

Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of NKT cells, which comprise a substantial sub-population of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of pro-fibrogenic iNKT cell populations during certain types of fibrosing liver damage.

We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; however, female mice transiently increased adipocyte area within regenerated muscle in a sex hormone-dependent manner. We extended these observations to investigate the effect of aging and sex on sarcopenia and muscle regeneration. Cardiotoxin injury to the tibialis anterior muscle of young, middle, and old-aged C57Bl/6J male and female mice was used to measure regenerated myofiber cross-sectional area (CSA), adipocyte area, residual necrosis, and inflammatory cell recruitment. Baseline (uninjured) myofiber CSA was decreased in old mice of both sexes compared to young and middle-aged mice. Regenerated CSA was similar in male mice in all age groups until baseline CSA was attained but decreased in middle and old age female mice compared to young females. Furthermore, adipocyte area within regenerated muscle was transiently increased in young females compared to young males and these sex-dependent increases persisted in middle and old age female mice and were associated with increased Pparg Young female mice had more pro-inflammatory monocytes/macrophages in regenerating muscle than young male mice and increased Sca-1(+)CD45(-)cells. In conclusion, sex and age influence pro-inflammatory cell recruitment, muscle regeneration, and adipocyte area following skeletal muscle injury.

Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of natural killer T (NKT) cells, which comprise a substantial subpopulation of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to Sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly-active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of profibrogenic iNKT cell populations during certain types of fibrosing liver damage.

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results Platelet-derived growth factor–treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Background & Aims Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors. Methods Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc +/− ) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD. Results In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc +/− mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD. Conclusions Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.