A. Salhab

Insulin signaling as a potential natural killer cell checkpoint in fatty liver disease

Insulin resistance is a key risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) and may lead to liver fibrosis. Natural killer (NK) cells are thought to exert an antifibrotic effect through their killing of activated hepatic stellate cells (HSCs). Here, we investigated how the interplay between NK cells and HSCs are modified by insulin resistance in NAFLD. Fresh peripheral blood NK cells (clusters of differentiation [CD]56dim, CD16+) were collected from 22 healthy adults and 72 patients with NAFLD not currently taking any medications and without signs of metabolic syndrome. NK cells were assessed for insulin receptor expressions and cytotoxic activity when cultured in medium with HSCs. Fibrosis severities in patients with NAFLD were correlated linearly with elevated serum proinflammatory cytokine expression and insulin resistance severity. At the same time, fibrosis severities inversely correlated with insulin receptor expressions on NK cells as well as with their cytotoxic activities determined by CD107a by flow cytometry. NK cells from donors exhibiting severe fibrosis and insulin resistance exhibited significant mammalian target of rapamycin and extracellular signal‐regulated kinase depletion (through NK cell western blot quantitation), increased apoptosis, and failure to attenuate HSC activation in vitro. While exposure to insulin stimulated the cytotoxic activity of healthy NK cells, rapamycin prevented this effect and reduced NK insulin receptor expressions. Conclusion: Elevated insulin levels in F1 and F2 fibrosis enhances NK cell cytotoxic activity toward HSCs and prevents fibrosis progression by insulin receptors and downstream mammalian target of rapamycin and extracellular signal‐regulated kinase pathways. At more advanced stages of insulin resistance (F3 and F4 fibrosis), impaired NK cell activity rooted in low insulin receptor expression and or low serum insulin levels could further deteriorate fibrosis and may likely lead to cirrhosis development. (Hepatology Communications 2018;2:285‐298)

A novel flow cytometry tool for fibrosis scoring through hepatic stellate cell differentiation

Hepatic stellate cells (HSCs) are a central fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. Peripheral blood lymphocytes from HCV patients are phagocytized by HSCs and induce their differentiation. This study aimed to characterize HSCs differentiation using a flow cytometry tool for fibrosis scoring. NK cells from healthy donors and from patients with chronic HCV with various severities of fibrosis were co‐cultured with a human HSC line (LX2). LX2 phagocytosis of NK cells were stained for NK cells (CD45/CD56/CD3) and NK activation marker (CD107a) as well as INF‐γ, apoptosis (Annexin‐V) and α‐smooth‐muscle‐actin (αSMA, as a marker of LX2 activation). In addition, reactive oxygen species (ROS) and the senescence marker P15 were analyzed prior to flow cytometry analysis. LX2 mono‐cultures demonstrated a homogenous cell‐population according to size (forward‐scattered; FSC), granularity and αSMA expressions. However, on their co‐culture with NK cells, the HSCs formed four subpopulations, which were stratified by αSMA intensities and cell size. NK cells isolated from heathy donors did not activate LX2‐cells. In contrast, HCV exposed to NK cells from both F1 and F4 fibrosis grade patients, showed elevated CD107a and INF‐γ levels and increased αSMA intensities in two of the four cell populations, with fibrosis scoring showing a linear correlation with αSMA intensities and NK phagocytosis. The αSMAintermediate/SizeLow HSCs sub‐population showed higher proliferation following F4‐NK cells with higher phagocytosis ability, suggesting an active/regulatory population. The αSMAhigh/Sizehigh subpopulations showed low proliferation and phagocytosis capacity, and were correlated with higher apoptosis, increased ROS and P15 intensities, suggesting senescing cells. Taken together, NK cells lead to heterogeneous differentiation of HSCs. Flow‐cytometry may provide a novel means of characterizing HSCs in relation to the severity of liver fibrosis.

The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis

Background A high prevalence of thyroid papillary cancer was reported in hepatitis-C-virus (HCV) positive patients. However, the mechanistic role of hepatic-fibrosis in thyroid malignancy progressions is still unclear. Aim We aimed to study the immune-modulatory interactions between thyroid papillary carcinoma and hepatic-fibrosis. Methods Hepatic-fibrosis was induced in nude-nu-male mice by intra-peritoneal administration of carbon-tetrachloride. To induce thyroid-tumor, a thyroid papillary carcinoma cell line (NPA) was injected subcutaneously in the backs. Fibrotic profile was estimated by α-smooth-muscle-actin (αSMA) expression in liver tissue extracts using western-blots and RT-PCR. Intra-hepatic NK cells were isolated and stained for NK activity (CD107a) by flow cytometry. Liver histopathology (H&E staining), thyroid tumor mass and serum alanine aminotransferase (ALT), serum vascular endothelial growth factor (VEGF) and free-T4 levels were also assessed. Results Ex-vivo: NPA cells were co-cultured with intra-hepatic NK cells isolated from fibrotic mice with/without the tumor were analyzed for CFSE-proliferations. Both tumor groups (with/without hepatic-fibrosis) excreted higher serum free T4 levels. Hepatic-fibrosis increased tumor weight and size and serum free-T4 levels. In addition, tumor induction increased liver injury (both hepatic-fibrosis, necro-inflammation and serum ALT levels). In addition, tumor-bearing animals with hepatic-fibrosis had increased NK activity. NPA tumor-bearing animals increased fibrosis in spite of increased NK activity; probably due to a direct effect through increased serum free-T4 excretions. Serum VEGF levels were significantly increased in the fibrotic- bearing tumor groups compared to the non-fibrotic groups. In-vitro, NK cells from fibrotic tumor-bearing animals reduced proliferation of NPA cells. This decrease is attributed to increase NK cells activity in the fibrotic animals with the NPA tumors. Conclusions Our results propose that NK cells although were stimulated in advanced fibrosis with tumor, they lost their anti-tumor and anti-fibrotic activity probably due to secretions of T4 and VEFG and may explain increased risk of thyroid tumors in chronic HCV patients.