S. Doron

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

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. (HEPATOLOGY 2008.)

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 (NCR1gfp/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.

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-acivated 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.

Lymphocyte–hepatic stellate cell proximity suggests a direct interaction

Recent functional research studies suggest an anti‐fibrotic role for natural killer (NK) cells coupled with a profibrotic role for CD8 cells. However, the morphological cellular interplay between the different cell types is less clear. To investigate lymphocyte/hepatic stellate cell (HSC) interactions, hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) intraperitoneally (i.p.) for 4 weeks in C57Bl/6 mice. Animals were killed at 0, 1, 2, 3 and 4 weeks. Liver sections were stained for Sirius red. Confocal microscopy was used to evaluate alpha smooth‐muscle actin (αSMA) and lymphocyte subsets in liver sections. At weeks 0 and 4, liver protein extracts were assessed for αSMA by Western blotting and isolated liver lymphocytes as well as HSC were analysed by fluorescence activated cell sorter (FACS). Similar to the results obtained from classical Sirius red staining and αSMA blotting, analysis of liver sections by confocal microscopy revealed a marked and continuous accumulation of αSMA staining along sequential experimental check‐points after administering CCl4. Although the number of all liver lymphocyte subsets increased following fibrosis induction, FACS analysis revealed an increase in the distribution of liver CD8 subsets and a decrease of CD4 T cells. Confocal microscopy showed a significant early appearance of CD8 and NK cells, and to a lesser extent CD4 T cells, appearing only from week 2. Lymphocytes were seen in proximity only to HSC, mainly in the periportal area and along fibrotic septa, suggesting a direct interaction. Notably, lymphocyte subsets were undetectable in naive liver sections. Freshly isolated HCS show high expression of major histocompatibility complex (MHC) class II and CD11c. In the animal model of hepatic fibrosis, lymphocytes infiltrate into the liver parenchyma and it is thought that they attach directly to activated HSC. Because HSCs express CD11c/class II molecules, interactions involving them might reflect that HSCs have an antigen‐presenting capacity.

Amelioration of hepatic fibrosis via Padma Hepaten is associated with altered natural killer T lymphocytes

Hepatic fibrosis is the end‐stage consequence of chronic liver disease, affecting many people worldwide. Unlike the anti‐fibrotic effect of natural killer (NK) cells, CD8 and NK T subsets are considered as profibrogenic subsets. Padma Hepaten is a multi‐compound herbal preparation derived from Tibetan medicine and has proven efficacy in some clinical trials and tests at the cellular level. In this study, we evaluate the immune efficacy of Padma Hepaten administered intraperitoneally (i.p.) and/or orally in a mice model of hepatic fibrosis. Hepatic fibrosis was induced by 6 weeks of biweekly i.p. carbon tetrachloride (CCl4) injections in male C57Bl6 mice. There were four groups, including naive mice, non‐treated fibrotic mice and fibrotic mice treated by Padma Hepaten at weeks 5–6 of fibrosis induction either orally or by i.p. injections. Padma Hepaten was prepared at 10 mg/ml in saline and 250 µl (2·5 mg) were administered four times per week. After week 6, animals were killed. To isolate a Padma Hepaten‐associated effect on lymphocytes, splenocytes were harvested from either naive or Padma Hepaten‐treated non‐fibrotic donors. Isolated splenocytes were therefore reconstituted into two groups of irradiated recipients. Recipients were then administered the same CCl4 regimen. Hepatic fibrosis was determined by sirius red staining of liver sections and by assessment of alpha smooth muscle actin expression compared with β‐actin (both by mRNA as well as the protein liver extract western blotting). Hepatic fibrosis and alanine aminotransferase serum levels were decreased significantly in both Padma Hepaten‐treated groups compared with the non‐treated fibrotic group. Padma Hepaten treatment was associated with attenuation of lymphocyte subsets in both treated groups. Using a chemiluminescence technique to assess total anti‐oxidant capacities (TAC), it was found that both the plasmas and livers of mice treated by CCl4 had significantly higher TAC compared with controls. However, the levels of TAC in animals treated either by CCl4 alone or CCl4 with Padma Hepaten were similar. Adoptive transfer of Padma Hepaten‐treated lymphocytes was associated with fibrosis amelioration compared with recipients with naive lymphocytes. CCl4 generates higher levels of anti‐oxidant capacities, probably as a response to oxidative stress. Padma Hepaten administration attenuated hepatic fibrogenesis significantly, accompanied by attenuation of lymphocyte but not anti‐oxidant capacities.

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.

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

CD1d-restricted natural killer T (NKT) cells are implicated in the pathogenesis of asthma. β-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.

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.

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)

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

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.