Epigenetic repression of hepatocyte FoxO1 disrupts local immune homeostasis and promotes liver inflammation.

Novel Model of Antigen-Specific Induction of Bile Duct Injury

NKT-cell subsets: Promoters and protectors in inflammatory liver disease

Depletion of the CD1d-Restricted NKT Cells Suppresses In Vitro Alloreactivity: A Possible Means To Prevent aGVHD.

Hepatitis B is a noncytopathic virus, which exclusively replicates within the liver and affects 350 million people worldwide [1]. Chronic infection can lead to variable disease manifestations such as cirrhosis, decompensated liver disease and hepatocellular carcinoma causing 1 million deaths per year [2]. These important clinical outcomes are a consequence of the host immune response to HBV, which constitutes a double-edged sword responsible for both viral clearance and hepatocellular damage. The precipitants for natural history milestones such as HBV-related hepatic flares (HF) and hepatitis B e Antigen (HBeAg) seroconversion remain unknown. Virus-specific and non-specific cytotoxic T lymphocytes (CTLs), T regulatory (Treg) cells, Natural Killer (NK), Natural Killer T (NKT) cells and dendritic cells (DCs) have been postulated to play a role [3]. The contribution of these immune cells and the nature of their interaction in the immune pathogenesis of HBV-related liver disease require further characterization. The logistical restraints of longitudinal, peripheral and intrahepatic sampling of the human host as well as inadequate small animal and cell culture models have hampered investigation of these immune mechanisms. As a caveat to human based studies in HBV, the circulating immunological cells may not reflect the phenotype and function of equivalent cells sequestered within the human liver. Current knowledge about the immune response to HBV is extrapolated from transgenic mouse models, many of which are models of viral replication rather than liver injury. Natural Killer (NK) and Natural Killer T (NKT) cells are cytotoxic lymphocytes that constitute a key effector arm of the innate immune system. Efforts to characterise the immunological determinants of Hepatitis B virus (HBV) infection have focused on the adaptive immune system whilst overlooking the potential interaction between virus, hepatocyte and NK or NKT cells, which play an important role in host defense against viral pathogens through direct cytotoxicity and the production of proinflammatory and immune regulatory cytokines. There is debate as to whether NK or NKT cells are effectors of antiviral activity or mediators of hepatic injury and fibrogenesis in chronic hepatitis B infection. NK and NKT cells have been implicated in the pathogenesis of liver disease due to other hepatotropic viruses such as hepatitis C and E as well as autoimmune liver disease, as shown in animal models of liver injury [4]. Human intrahepatic lymphocytes consist of 30%-50% NK and 5-10% NKT cells. Peripheral blood lymphocytes contain 13% NK cells and 2% NKT cells [5]. The hepatic enrichment of NK and NKT cells reflects their role as regulators at the interface between the innate and adaptive immune response to liver disease. NK and NKT cells in the peripheral and intrahepatic compartments share effector functions such as direct killing of viral-infected cells and cytokine production. The latter is considered the more important effector function in CHB [4]. NK and NKT cells demonstrate reciprocal interactions (“crosstalk”) with hepatic macrophages, Kupffer cells (KC), DCs and T cells as part of an amalgamated immune response to HBV [4]. The role of NK and NKT cells in the initiation and orchestration of a dynamic host immune response against HBV-related liver disease is investigated in this thesis. This hepatotrophic virus has evolved direct and indirect strategies to evade or inhibit the large hepatic reservoir of NK and NKT cells. In this thesis, I will focus on the dynamic phenotype and function of NK and NKT cells throughout the different phases of HBV infection, which so far have been poorly characterized. I will also examine the effect of activated NK and NKT cells on liver injury, fibrosis, and their attenuation following HBV treatment, which remains controversial. Understanding the role of NK and NKT cells in the pathogenesis of CHB may help to develop new biomarkers for disease and treatment activity and design novel immunotherapies.

BACKGROUND & AIMS Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T-cell receptors (NKT cells) in APAP-induced liver injury. METHODS C57BL/6 mice were administered a toxic dose of APAP intraperitoneally to cause liver injury with or without depletion of NK and NKT cells by anti-NK1.1 monoclonal antibody (MAb). Serum alanine transaminase (ALT) levels, liver histology, hepatic leukocyte accumulation, and cytokine/chemokine expression were assessed. RESULTS Compared with APAP-treated control mice, depletion of both NK and NKT cells by anti-NK1.1 significantly protected mice from APAP-induced liver injury, as evidenced by decreased serum ALT level, improved survival of mice, decreased hepatic necrosis, inhibition of messenger RNA (mRNA) expression for interferon-gamma (IFN-gamma), Fas ligand (FasL), and chemokines including KC (Keratinocyte-derived chemokine); MIP-1 alpha (macrophage inflammatory protein-1 alpha); MCP-1 (monocyte chemoattractant protein-1); IP-10 (interferon-inducible protein); Mig (monokine induced by IFN-gamma) and decreased neutrophil accumulation in the liver. Hepatic NK and NKT cells were identified as the major source of IFN-gamma by intracellular cytokine staining. APAP induced much less liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice compared with that in wild-type mice. CONCLUSIONS NK and NKT cells play a critical role in the progression of APAP-induced liver injury by secreting IFN-gamma, modulating chemokine production and accumulation of neutrophils, and up-regulating FasL expression in the liver, all of which may promote the inflammatory response of liver innate immune system, thus contributing to the severity and progression of liver injury downstream of the metabolism of APAP and depletion of reduced glutathione (GSH) in hepatocytes.

Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity

Heterogeneity of Human Invariant Natural Killer T Cells, Including Novel Naïve-like CD8 + and Temra-like CD4 -CD8 - Populations, Revealed through Single Cell RNA-Sequencing

1084 CYCLIN El AND Dl CAN DRIVE LIVER REGENERATION IN MICE WITHOUT CDK2

To clarify the roles of innate immune cells in liver regeneration, here, we investigated the alteration in regenerative responses after partial hepatectomy (PH) under selective depletion of natural killer (NK) and/or NKT cells. Male, wild-type (WT; C57Bl/6), and CD1d-knockout (KO) mice were injected with anti-NK1.1 or anti-asialo ganglio-N-tetraosylceramide (GM1) antibody and then underwent the 70% PH. Regenerative responses after PH were evaluated, and hepatic expression levels of cytokines and growth factors were measured by real-time RT-PCR and ELISA. Phosphorylation of STAT3 was detected by Western blotting. Depletion of both NK and NKT cells with an anti-NK1.1 antibody in WT mice caused drastic decreases in bromodeoxyuridine uptake, expression of proliferating cell nuclear antigen, and cyclin D1, 48 h after PH. In mice given NK1.1 antibody, increases in hepatic TNF-α, IL-6/phospho-STAT3, and hepatocyte growth factor (HGF) levels following PH were also blunted significantly, whereas IFN-γ mRNA levels were not different. CD1d-KO mice per se showed normal liver regeneration; however, pretreatment with an antiasialo GM1 antibody to CD1d-KO mice, resulting in depletion of both NK and NKT cells, also blunted regenerative responses. Collectively, these observations clearly indicated that depletion of both NK and NKT cells by two different ways results in impaired liver regeneration. NK and NKT cells most likely upregulate TNF-α, IL-6/STAT3, and HGF in a coordinate fashion, thus promoting normal regenerative responses in the liver.

LYMPHOCYTE PROFILE ON PATIENTS WITH CHRONIC AND ACUTE ALCOHOL CONSUMPTION

Invariant NKT (iNKT) cells are a group of innate-like T cells that plays important roles in immune homeostasis and activation. We found that iNKT cells, compared with CD4+ T cells, have significantly higher levels of lipid peroxidation in both mice and humans. Proteomic analysis also demonstrated that iNKT cells express higher levels of phospholipid hydroperoxidase glutathione peroxidase 4 (Gpx4), a major antioxidant enzyme that reduces lipid peroxidation and prevents ferroptosis. T cell-specific deletion of Gpx4 reduces iNKT cell population, most prominently the IFN-γ-producing NKT1 subset. RNA-sequencing analysis revealed that IFN-γ signaling, cell cycle regulation, and mitochondrial function are perturbed by Gpx4 deletion in iNKT cells. Consistently, we detected impaired cytokine production, elevated cell proliferation and cell death, and accumulation of lipid peroxides and mitochondrial reactive oxygen species in Gpx4 knockout iNKT cells. Ferroptosis inhibitors, iron chelators, vitamin E, and vitamin K2 can prevent ferroptosis induced by Gpx4 deficiency in iNKT cells and ameliorate the impaired function of iNKT cells due to Gpx4 inhibition. Last, vitamin E rescues iNKT cell population in Gpx4 knockout mice. Altogether, our findings reveal the critical role of Gpx4 in regulating iNKT cell homeostasis and function, through controlling lipid peroxidation and ferroptosis.

Interleukin-33 (IL-33) is thought to be released during cellular death as an alarming cytokine during the acute phase of disease, but its regulation in vivo is poorly understood. We investigated the expression of IL-33 in two mouse models of acute hepatitis by administering either carbon tetrachloride (CCl(4) ) or concanavalin A (ConA). IL-33 was overexpressed in both models but with a stronger induction in ConA-induced hepatitis. IL-33 was weakly expressed in vascular and sinusoidal endothelial cells from normal liver and was clearly induced in CCl(4) -treated mice. Surprisingly, we found that hepatocytes strongly expressed IL-33 exclusively in the ConA model. CD1d knock-out mice, which are deficient in NKT cells and resistant to ConA-induced hepatitis, no longer expressed IL-33 in hepatocytes following ConA administration. Interestingly, invariant NKT (iNKT) cells adoptively transferred into ConA-treated CD1d KO mouse restored IL-33 expression in hepatocytes. This strongly suggests that NKT cells are responsible for the induction of IL-33 in hepatocytes.

CD1d-dependent type I NKT cells, which are activated by lipid antigen, are known to play important roles in innate and adaptive immunity, as are a portion of type II NKT cells. However, the heterogeneity of NKT cells, especially NKT-like cells, remains largely unknown. Here, we report the profiling of NKT (NK1.1+CD3e+) cells in livers from wild type (WT), Jα18-deficient and CD1d-deficient mice by single-cell RNA sequencing. Unbiased transcriptional clustering revealed distinct cell subsets. The transcriptomic profiles identified the well-known CD1d-dependent NKT cells and defined two CD1d-independent NKT cell subsets. In addition, validation of marker genes revealed the differential organ distribution and landscape of NKT cell subsets during liver tumor progression. More importantly, we found that CD1d-independent Sca-1−CD62L+ NKT cells showed a strong ability to secrete IFN-γ after costimulation with IL-2, IL-12 and IL-18 in vitro. Collectively, our findings provide a comprehensive characterization of NKT cell heterogeneity and unveil a previously undefined functional NKT cell subset.

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.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells invivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets invivo and uncovered a hidden immunoregulatory mechanism.