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. © 2017 International Society for Advancement of Cytometry.

The pro-fibrotic effects of pregnancy in a carbon-tetrachloride-induced liver injury in mouse model.

BACKGROUND & AIMS: Immune cells interact with hepatic-stellate-cells (HSCs) in the development of liver fibrosis. Little is known about the influence of pregnancy on the development and progression of hepatic-fibrosis. In this study, we explored the influence of pregnancy on progression of hepatic fibrosis. METHODS: Female mice (C57Blc) were induced by 4 injections of peritoneal carbon-tetrachloride (CCl4) within 10 days, starting at day 10 of documented pregnancy. At end of experiment, serum samples were obtained for ALT and estradiol determination. Harvested livers were histological evaluated for liver injury and for protein αSMA expressions. Isolated intra-hepatic lymphocytes were assessed by flow cytometry. Isolated lymphocytes and serum samples were in- vitro co-cultured for 48 h with primary isolated naïve HSCs. Washed cells were analyzed for adherence (anti-αSMA+/anti-CD45 + ) and proliferations (CSFE). RESULTS: CCl4-model for liver injury was well tolerated when induced in pregnancy similar to non-pregnant state. Hepatic-fibrosis (Masson Trichrome Stain, Sirius red stain and αSMA expressions) and necro-inflammation (H&E stain and serum ALT levels) significantly increased in pregnancy. Increased liver injury was accompanied with pro-fibrotic lymphocyte profile; CD8 subsets increased and NK cells decreased. HSCs activation significantly increased when in-vitro cultured with lymphocytes from pregnant as compared to non-pregnant fibrotic ones. Pro-fibrotic profile was also explained by decreased NK activity (CD107a marker) and of their phagocytosis. Serum estradiol levels although elevated in fibrosis conditions of pregnancy was not associated with the pHSCs activations. CONCLUSION: Liver fibrosis in our murine model was severe in pregnant model; via pro-fibrotic lymphocyte and serum alterations.

Natural killer cell-dependent anti-fibrotic pathway in liver injury via Toll-like receptor-9.

The toll-like receptor-9 (TLR9) agonist cytosine phosphate guanine (CpG), activates hepatic stellate cells (HSCs) and mediates fibrosis. We investigated the TLR9 effects on lymphocyte/HSCs interactions. Liver fibrosis was induced in wild-type (WT) mice by intra-peritoneal carbon-tetrachloride (CCl4) induction for 6 weeks. Fibrotic groups were intravenously treated by a vehicle versus CpG along last 2 weeks. Compared to vehicle-treated fibrotic WT, the in-vivo CpG-treatment significantly attenuated hepatic fibrosis and inflammation, associated with decreased CD8 and increased NK liver cells. In-vitro, co-cultures with vehicle-treated fibrotic NK cells increased HSCs proliferation (P<0.001) while their CpG-treated counterparts achieved a significant decrease. To investigate the role of lymphocytes, TLR9(-/-) mice induced-hepatic fibrosis were used. Although TLR9(-/-) mice manifested lower fibrotic profile as compared to their wild-type (WT) counterparts, senescence (SA-ß-Gal activity) in the liver and ALT serum levels were significantly greater. In an adoptive transfer model; irradiated WT and TLR9(-/-) recipients were reconstituted with naïve WT or TLR9(-/-) lymphocytes. The adoptive transfer of TLR9(-/-) versus WT lymphocytes led to increased fibrosis of WT recipients. TLR9(-/-) fibrotic recipients reconstituted with TLR9(-/-) or WT lymphocytes showed no changes in hepatic fibrosis severity or ALT serum levels. TLR9 activation had inconsistent effects on lymphocytes and HSCs. The net balance of TLR9 activation in WT, displayed significant anti-fibrotic activity, accompanied by CD8 suppression and increased NK-cells, activity and adherence to HSCs. The pro-fibrotic and pro-inflammatory properties of TLR9(-/-) lymphocytes fail to activate HSCs with an early senescence in TLR9(-/-) mice.

Early fibrosis inhibits hepatocellular carcinoma mediated by free radical effects.

We studied the in-vitro/in-vivo interactions between HCC/HSCs in early and advanced fibrosis-models. Hep3B-mono-cultures secreted high levels of αFetoProtein (αFP). Human-HSCs co-cultured with Hep3B-cells significantly decreased αFP and increased their apoptosis. Confocal-microscopy demonstrated Hep3B-phagocytosis inside the HSCs suggesting a direct cellular-contact mediating anti-tumor effect. Leptin-activated HSCs further suppressed Hep3B-cells with increased ROS and decreased GSH. Following intrahepatic-Hep3B-cell injections, mice with established "advanced liver-fibrosis"; had higher tumor-size and αFP serum-levels as compared to non-fibrotic livers. Mice with "early liver-fibrosis", which initiated post tumor induction had a significant decrease in tumor and high Malondialdehyde (MDA) serum levels compared to advanced-fibrosis animals. At early-fibrosis stages, activated-HSCs express direct anti-tumor effects by phagocytosis and apoptosis of tumor-cells mediated by oxidative stress.

Leptin modulates lymphocytes' adherence to hepatic stellate cells is associated with oxidative status alterations.

We investigated leptin effects on lymphocyte interactions with hepatic-stellate-cells (HSCs). Leptin showed pro-fibrotic effects on HSCs with oxidative status imbalance. In co-cultures, leptin activates HSCs and consequently adhered HCV-lymphocytes more than healthy ones. Leptin also increased healthy and HCV lymphocyte proliferations; increased their reactive-oxygen-species; decreased antioxidants (reduced-glutathione) levels while inhibited apoptosis only of HCV-lymphocytes. The leptin-treated HCV-lymphocytes activated HSCs, increase interleukin-4 while decreased their apoptosis. Leptin-receptor-deficient (db-db)-HSCs did not adhere lymphocytes. db/db-lymphocytes however showed fewer adherences to HSCs when compared to WT-counterparts. This study presents immune and oxidative modulatory effects of leptin on lymphocytes and their consequent interaction with HSCs.

Triphala (PADMA) extract alleviates bronchial hyperreactivity in a mouse model through liver and spleen immune modulation and increased anti-oxidative effects.

OBJECTIVES: Triphala (TRP), a herbal extract from Tibetan medicine, has been shown to affect lymphocytes and natural killer T (NKT) cell function. We hypothesize that TRP could ameliorate bronchial hyperreactivity through immune-cell modulations. METHODS: Asthma mouse models were generated through intraperitoneal (IP) injections of ovalbumin (OVA)/2 weeks followed by repeated intranasal OVA challenges. Mice were then treated with normal saline (OVA/NS) or Triphala (OVA/TRP). Data were compared with mice treated with inhaled budesonide. All groups were assessed for allergen-induced hyperreactivity; lymphocytes from lungs, livers and spleens were analyzed for OVA-induced proliferation and their alterations were determined by flow cytometry. Oxidative reactivity using chemiluminescence, serum anti-OVA antibodies level and lung histology were assessed. RESULTS: Both TRP and budesonide significantly ameliorated functional and histological OVA-induced bronchial hyperreactivity. TRP had no effect on serum anti-OVA antibodies as compared with decreased levels following budesonide treatment. Furthermore, a significant increase in lung and spleen CD4 counts and a decrease in the liver were noted after TRP treatments. Bronchoalveolar fluid from TRP-treated animals but not from the budesonide-treated animals showed anti-oxidative effects. CONCLUSION: TRP and budesonide caused a significant decrease in bronchial reactivity. TRP treatment altered immune-cell distributions and showed anti-oxidative properties. These findings suggest that immune-cell modulation with TRP can ameliorate lung injury.

The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor.

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Naïve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

NKp46-mediated killing of human and mouse hepatic stellate cells attenuates liver fibrosis.

BACKGROUND: Liver fibrosis, which involves activation of hepatic stellate cells (HSC), is a major health problem and is the end outcome of all chronic liver diseases. The liver is populated with lymphocytes, among which are natural killer (NK) cells, whose activity is controlled by inhibitory and activating receptors. NKp46, one of the major NK activating receptors expressed by NK cells, is also a specific NK marker that discriminates NK cells from all other lymphocyte subsets. It recognises viral haemagglutinins and unknown cellular ligands. METHODS: The anti-fibrotic activity of the NKp46 receptor was assessed in vivo and in vitro using NKp46-deficient mice (NCR1(gfp/gfp)), the carbon tetrachloride model and in vitro NK killing assays. Primary murine and human HSC were stained for the expression of the NKp46 ligand using fusion proteins composed of the extracellular portions of the murine and human NKp46 receptors fused to human IgG1. RESULTS: It was shown that murine HSC express a ligand for the murine orthologue of the NKp46 receptor, NCR1. NCR1 inhibited liver fibrosis in vivo; in vitro, murine HSC were killed in an NCR1-dependent manner. In humans it was shown that human HSC also express a ligand for the human NKp46 receptor and that the killing of human HSC is NKp46 dependent. CONCLUSIONS: In addition to NKG2D, NKp46/NCR1 play an important role in inhibition of liver fibrosis. This suggests that fibrosis can be better controlled through the manipulation of NKp46 activity.

Immune modulation of ovalbumin-induced lung injury in mice using ß-glucosylceramide and a potential role of the liver.

CD1d-restricted natural killer T (NKT) cells are implicated in the pathogenesis of asthma. ß-Glucosylceramide (GC), a naturally occurring lipid, was previously shown to alter NKT cell distribution in the liver. We hypothesized that GC can affect lung and liver NKT cell distribution and ameliorate asthma. Mice were sensitized by intra-peritoneal injection of ovalbumin (OVA) for 2 weeks followed by repeated intranasal OVA challenges to induce lung injury mimicking asthma. OVA induced asthma groups were either treated by intranasal instillation of normal saline, intranasal instillation of GC or inhaled budesonide. To investigate the role of the liver, hepatic fibrosis was induced using carbon tetrachloride prior to asthma induction. Allergen induced bronchoconstriction was measured prior to sacrifice. Isolated lymphocytes from lungs, livers and spleens were analyzed for OVA induced proliferation and flow cytometry. Liver and lung histology, serum aminotransferase and anti-OVA antibodies level were assessed. Treatment with GC significantly reduced OVA induced airway responsiveness (p<0.001) similar to inhaled budesonide. GC significantly reduced the peri-bronchial and peri-vascular inflammatory infiltration mainly through an effect on T cells, as suggested by decreased T cell proliferation (p=0.009). Liver CD4 and NKT cells significantly increased after GC treatment suggesting liver involvement. Inducing hepatic fibrosis blunted the propagation of asthma in spite of sufficient increase of serum anti-OVA titers. GC has an immunomodulatory effect on a murine model of experimental asthma. We also suggest that the liver acts as an immunomodulatory organ and might have a regulatory effect on pulmonary diseases.

Amelioration of hepatic fibrosis by NK cell activation.

BACKGROUND AND AIMS: Interactions between hepatic stellate cells (HSCs) and immune cell subsets have emerged as important determinants of liver fibrosis progression and regression. Natural killer (NK) cells have an antifibrotic activity through killing of activated HSCs. In liver injury NK cell expression of activating/inhibitory killer immunoglobulin-related receptors (aKIR/iKIR) and their ratio are significantly increased, while class I major histocompatibilty (MHC) expression by activated HSCs is decreased. The aim of this study was to amplify the antifibrotic activity of NK cells and ameliorate hepatic fibrosis by iKIR silencing. METHODS: Human lymphocytes from patients with hepatitis C virus (HCV) infection were transfected with specific iKIR small interfering RNAs (siRNAs) or non-silencing control siRNAs, then co-cultured with a human HSC line and assessed for fibrogenic activity. To induce hepatic fibrosis, carbon tetrachloride was administrated to BALBc SCID-Beige male mice (lacking B/T/NK cells) for 4 weeks. Splenocytes from naive SCID donors (lacking B/T cells but with preserved NK cells) were transfected in vitro with either iKIR siRNA or non-silencing control siRNA, and then were transferred to the fibrotic SCID-Beige recipients. RESULTS: Transfection with iKIR or positive control siRNAs (mice and human) decreased mRNA expression of iKIR and mitogen-activated protein kinase 1 (MAPK1). Consequently, total NK cells and NK cell degranulation were increased (p=0.01), consistent with NK cell stimulation. Compared with healthy lymphocytes, when HCV lymphocytes were transfected with non-silencing control siRNA and co-cultured with HSCs there was increased α-smooth muscle actin (αSMA) expression, reflecting HSC activation. Expression of αSMA in co-cultures was attenuated when HCV lymphocytes were transfected with iKIR siRNA. In SCID-Beige recipients, hepatic fibrosis and serum alanine aminotransferase (ALT) levels were significantly attenuated as a result of receiving iKIR siRNA. CONCLUSIONS: iKIR knockdown stimulates NK cells and promotes their antifibrogenic activity in mice and human co-cultures. These findings have implications for possible immune therapeutic strategies in patients with advanced liver disease.

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis.

UNLABELLED: Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. CONCLUSION: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis.

Beneficial effect of glatiramer acetate (Copaxone) on immune modulation of experimental hepatic fibrosis.

While CD8 subsets activate hepatic fibrosis, natural killer (NK) cells exhibit antifibrotic activity. Glatiramer acetate (GA) is an immune modulator for multiple sclerosis. We assessed the potential impact of GA on mouse hepatic fibrogenesis. Hepatic fibrosis was induced in C57BL/6 mice by intraperitoneal administration of carbon tetrachloride (CCl(4)) for 6 wk. During the last 2 wk, animals were also treated with either GA (200 mu/day ip) or medium and compared with naive and fibrotic mice (8 animals/group). GA markedly attenuated fibrosis without altering reactive oxygen species production. By morphometric measurement of Sirius red-stained tissue sections, the relative fibrosis area decreased from 5.28 +/- 0.32% (mean +/- SE) in the untreated CCl(4) group to 2.01 +/- 0.28% in CCl(4)+GA-treated animals, compared with 0.38 +/- 0.07% in naive mice. alpha-Smooth muscle actin immunoblotting and mRNA expression revealed a similar pattern. Serum aminotransferase and Ishak-Knodell necroinflammatory score were markedly elevated, to the same extent, in both CCl(4)-treated groups. Fibrosis induction was associated with significant increase in CD8 subsets and decrease in CD4 T cells. After GA treatment, however, NK content, CD4(+)CD25(+)FoxP3(+) cells, hepatic expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and apoptosis of hepatic stellate cells were all increased. Serum interleukin (IL)-10 levels markedly rose, whereas IL-4 fell. In vitro activation of human hepatic stellate cells cocultured with hepatitis C virus-derived peripheral blood lymphocytes decreased when lymphocytes were preincubated with GA before coculture. In an animal model of hepatic fibrosis, GA has an antifibrotic effect associated with decreased CD8 cells and reduced serum IL-4 levels and increased NK cells, CD4(+)CD25(+)FoxP3(+) cells, TRAIL, and elevated serum IL-10 levels.

Amelioration of experimental colitis by Copaxone is associated with class-II-restricted CD4 immune blocking.

UNLABELLED: Copaxone modifies TH1 immune response in multiple sclerosis. As Crohn's disease shares TH1 predominance, this study came to investigate the anti-inflammatory response of Copaxone in animal model of colitis. METHODS: Colitis was induced by intra-rectal instillation of TNBS in 2 animal groups; one of them was daily treated intraperitoneally by 300 mug Copaxone starting 48 h post-colitis induction. Both colitis groups were compared to naive group. Eight male C57Bl6 mice were used in each group. At day 12, distal colon was excised for standard scoring, splenocytes were isolated for FACS and serum cytokines were assessed. Splenocytes were in-vitro-stimulated with colitis protein extracts in the presence or absence of Copaxone. Lymphocytes were blocked by either MHC anti-class I or anti-class II antibodies prior to Copaxone administration. RESULTS: Copaxone markedly alleviated macro/microscopic colitis scoring as they decreased from 2.9 +/- 1.1/2.6 +/- 0.8 in colitis group to 1.7 +/- 1/1.5 +/- 0.5 in Copaxone-treated mice (P = 0.03/P = 0.008, respectively) compared to 0 +/- 0/1 +/- 0 in naives (P < 0.001/P < 0.01, respectively). CD4 subsets significantly decreased following Copaxone administration as compared to naive mice (P = 0.05). Although Copaxone-treated mice manifested a block of both serum TH1/TH2 responses, only interferon gamma secreting CD4 cells significantly decreased. NK cells tend to increase following colitis induction (P = 0.08), however, they significantly decreased in Copaxone-treated animals (P = 0.006). NK-T followed NK pattern. Using in vitro studies, Copaxone showed amelioration of T-cell proliferation that was significantly blocked when cells were pre-incubated with anti-MHC class II but not class I antibodies. CONCLUSIONS: Copaxone had class-II-restricted anti-inflammatory effect in our animal colitis model associated with CD4/NK/NKT/TH1/TH2 suppression.