Liver-resident Immune Cells Research Articles

Abstract 1407: Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​

Abstract The incidence of liver cancer is increasing and there is an urgent need for new therapies and preventative strategies. Age is a major risk factor for liver cancer, the reasons for which are not well defined. One of the hallmarks of aging is immune dysfunction which affects liver homeostasis potentially making it prone to cancer. To understand the role of immune dysfunction in aging liver, we analyzed immune cells from young and old livers. As is reported before, in aged livers we observed an increase in CD101+PD1+CD8+ T cell population as well as an increase in an IFNγ+TNFα+ population suggesting an increase in T cell response in an aging liver. We next sought to understand the cause of T cell exhaustion in aged livers. Cell cluster analysis of the young (4-month-old) and old (22 month old) livers by scRNA seq revealed a cell population unique to the aged livers, showing increased expression of immune checkpoint ligands as well as tumor-initiating cell markers. We validated this data by flow cytometry. We next functionally probed the immune environment of aged liver, by testing the effect of cognate antigen expression in young and old hepatocytes on adoptively transferred activated P14 CD8+ T cells isolated from a young P14 mouse. Analysis of the ex vivo activated P14 CD8+ T cells from young and old antigen-expressing livers by flow cytometry and scRNA sequencing revealed increased exhaustion and cytokine-deficiency in old mice as compared to the young mice. This study explores the age-associated alterations in the immune cells in the liver, aiming to alter liver-resident immune cells and their metabolic states in a way that promotes anti-tumor immunity in an aging liver. Citation Format: Armin Aabish Gandhi, Filipe Araujo Hoffmann, Michael LaPorte II, Aaron Havas, Adarsh Rajesh, Andrew Davis, Marcos G. Teneche, Jessica Proulx, Susan Kaech, Peter D. Adams. Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1407.

Single cell RNA-sequencing analysis reveals that N-acetylcysteine partially reverses hepatic immune dysfunction in biliary atresia

Our previous study indicated that CD177+ neutrophil activation has a vital role in the pathogenesis of biliary atresia (BA), which is partially ameliorated by N-acetylcysteine (NAC) treatment. Here, we evaluated the clinical efficacy of NAC treatment and profiled liver-resident immune cells via single cell RNA-sequencing (scRNA-seq) analysis to provide a comprehensive immune landscape of NAC-derived immune regulation. A pilot clinical study was conducted to evaluate the potential effects of intravenous NAC treatment on infants with BA, and a 3-month follow-up was carried out to assess treatment efficacy. scRNA-seq analysis of liver CD45+ immune cells in the control (n= 4), BA (n= 6), and BA+ NAC (n= 6) groups was performed and the effects on innate cells, including neutrophil and monocyte-macrophage subsets, and lymphoid cells were evaluated. Intravenous NAC treatment demonstrated beneficial efficacy for infants with BA by improving bilirubin metabolism and bile acid flow. Two hepatic neutrophil subsets of innate cells were identified by scRNA-seq analysis. NAC treatment suppressed oxidative phosphorylation and reactive oxygen species production in immature neutrophils, which were transcriptionally and functionally similar to CD177+ neutrophils. We also observed the suppression of hepatic monocyte-mediated inflammation, decreased levels of oxidative phosphorylation, and M1 polarisation in Kupffer-like macrophages by NAC. In lymphoid cells, enhancement of humoral immune responses and attenuation of cellular immune responses were observed after NAC treatment. Moreover, cell-cell interaction analysis showed that innate/adaptive proinflammatory responses were downregulated by NAC. Our clinical and scRNA-seq data demonstrated that intravenous NAC treatment partially reversed liver immune dysfunction, alleviated the proinflammatory responses in BA by targeting innate cells, and exhibited beneficial clinical efficacy. BA is a serious liver disease that affects newborns and has no effective drug treatment. In this study, scRNA-seq showed that NAC treatment can partially reverse the immune dysfunction of neutrophil extracellular trap-releasing CD177+ neutrophils and Kupffer cells, and lower the inflammatory responses of other innate immune cells in BA. In consequence, intravenous NAC treatment improved the clinical outcomes of patients with BA in term of bilirubin metabolism.

The multifaceted role of macrophages during acute liver injury.

The liver is situated at the interface of the gut and circulation where it acts as a filter for blood-borne and gut-derived microbes and biological molecules, promoting tolerance of non-invasive antigens while driving immune responses against pathogenic ones. Liver resident immune cells such as Kupffer cells (KCs), a subset of macrophages, maintain homeostasis under physiological conditions. However, upon liver injury, these cells and others recruited from circulation participate in the response to injury and the repair of tissue damage. Such response is thus spatially and temporally regulated and implicates interconnected cells of immune and non-immune nature. This review will describe the hepatic immune environment during acute liver injury and the subsequent wound healing process. In its early stages, the wound healing immune response involves a necroinflammatory process characterized by partial depletion of resident KCs and lymphocytes and a significant infiltration of myeloid cells including monocyte-derived macrophages (MoMFs) complemented by a wave of pro-inflammatory mediators. The subsequent repair stage includes restoring KCs, initiating angiogenesis, renewing extracellular matrix and enhancing proliferation/activation of resident parenchymal and mesenchymal cells. This review will focus on the multifaceted role of hepatic macrophages, including KCs and MoMFs, and their spatial distribution and roles during acute liver injury.

Abstract B014: TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer

Abstract Metastasis remains the primary cause of mortality in cancer. This is especially true in pancreatic ductal adenocarcinoma (PDAC) - where the liver is the most common site of metastasis and the 5-year overall survival rate for these patients has remained <3% for the past 20 years. Accumulating evidence shows a critical role for a niche microenvironment in the liver, termed the pro-metastatic niche, that supports metastatic seeding and outgrowth. Our previous work demonstrated that hepatocytes orchestrate the formation of this pro-metastatic niche during early tumorigenesis through the release of Serum Amyloid A1 and 2 (SAA). However, the mechanisms by which SAA directs formation of a pro-metastatic niche in the liver, and the impact of this niche on the fate of disseminating tumor cells (DTCs) seeding the liver, remain ill-defined. To begin to identify the cellular determinants of niche formation, we first analyzed the expression of SAA receptors within the normal liver and found predominant expression of receptors on the resident F4/80+ macrophages. This is in line with current published work as SAA is well characterized to control liver biology through toll-like receptors (TLRs) expressed by liver resident immune cells and hepatic stellate cells. Consistent with this, we also found that SAA is a specific, potent agonist of TLR2 in vitro. In tumor bearing mice we then found increased expression of TLR2 transcripts as well as an accumulation of TLR2+ inflammatory monocytes within the niche. Together, these findings suggested that SAA might form a pro-metastatic niche in the liver withough a TLR2-dependent mechanism. To test this, we next examined the livers of TLR2-/- tumor bearing mice and found a decrease in the expression of surrogate markers of the pro-metastatic niche. We also sought to determine a role for TLR2 in metastatic outgrowth. Using TLR2-/- mice in a model of PDAC with direct metastasis to the liver, we found significantly decreased metastatic burden and outgrowth in the liver. Analysis of micro-metastatic lesions in TLR2-/- tumor bearing mice also revealed that DTCs demonstrated molecular markers of cellular dormancy. Altogether, our findings suggest the evolution of an inflammatory mechanism within the niche wherein SAA may continue to signal through TLR2+ inflammatory monocytes, which then act to coordinate metastatic outgrowth. Future work aims to further elucidate the role of TLR2+ resident macrophages and monocytes in the formation of a pro-metastatic niche in the liver and in regulating metastatic outgrowth. Citation Format: Jesse Lee, Meredith L. Stone, Hana Choi, Gregory L. Beatty. TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B014.

The liver-resident immune cell repertoire -A boon or a bane during machine perfusion?

The liver has been proposed as an important “immune organ” of the body, as it is critically involved in a variety of specific and unique immune tasks. It contains a huge resident immune cell repertoire, which determines the balance between tolerance and inflammation in the hepatic microenvironment. Liver-resident immune cells, populating the sinusoids and the space of Disse, include professional antigen-presenting cells, myeloid cells, as well as innate and adaptive lymphoid cell populations. Machine perfusion (MP) has emerged as an innovative technology to preserve organs ex vivo while testing for organ quality and function prior to transplantation. As for the liver, hypothermic and normothermic MP techniques have successfully been implemented in clinically routine, especially for the use of marginal donor livers. Although there is evidence that ischemia reperfusion injury-associated inflammation is reduced in machine-perfused livers, little is known whether MP impacts the quantity, activation state and function of the hepatic immune-cell repertoire, and how this affects the inflammatory milieu during MP. At this point, it remains even speculative if liver-resident immune cells primarily exert a pro-inflammatory and hence destructive effect on machine-perfused organs, or in part may be essential to induce liver regeneration and counteract liver damage. This review discusses the role of hepatic immune cell subtypes during inflammatory conditions and ischemia reperfusion injury in the context of liver transplantation. We further highlight the possible impact of MP on the modification of the immune cell repertoire and its potential for future applications and immune modulation of the liver.

TREM-2 plays a protective role in cholestasis by acting as a negative regulator of inflammation.

Inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis. TREM-2 expression was analyzed in the livers of patients with primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Primary cultured Kupffer cells were incubated with lipopolysaccharide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed. TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/- mice. This was characterized by enhanced necroptotic cell death, inflammatory responses and biliary expansion. Antibiotic treatment partially abrogated the effects observed in Trem-2-/- mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and dampened inflammatory gene transcription via a TREM-2-dependent mechanism. TREM-2 acts as a negative regulator of inflammation during cholestasis, representing a novel potential therapeutic target. Cholestasis (the reduction or cessation of bileflow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors (specifically Toll-like receptors or TLRs) on liver-resident immune cells, promoting inflammation. Herein, we show that the anti-inflammatory receptor TREM-2 dampens TLR-mediated signaling and hence protects against cholestasis-induced liver injury. Thus, TREM-2 could be a potential therapeutic target in cholestasis.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

IntroductionNormothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells.MethodsAdult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions.ResultsThere were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1β), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group.ConclusionsLiver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

Tocilizumab As a Novel Immunomodulatory Regimen for Hemophilia Gene Therapy

Hemophilia is an X-linked bleeding disorder typically resulting in deficiency of factor VIII (FVIII) or factor IX (FIX) due to mutations in the F8 or F9 genes, respectively. Data from clinical trials have shown that the investigational delivery of functional F8 or F9 gene by recombinant adeno-associated virus (AAV) to hepatocytes can substantially eliminate the need for infusions of clotting factor in hemophilia patients. Despite major advances, the durability and redosing of these investigational gene therapies have been limited by the host immune response against the AAV capsid (Verdera et al. Mol Ther 2020). Currently, an oral corticosteroid, prednisone, is commonly used to prevent cytotoxic T cells from killing AAV-transduced hepatocytes and to sustain the production of transgenic clotting factor. However, in some instances, transgene expression was lost despite prednisone administration and prolonged use of prednisone can be associated with adverse effects. Previously, we demonstrated that the anti-capsid humoral response depends on interleukin 6 (IL-6) secretion from human monocyte-related dendritic cells (Kuranda et al. JCI 2018). IL-6 signaling in response to AAV was also observed in human non-parenchymal liver cells in vitro, animal gene transfer models and AAV-based gene therapy trial for hemophilia B (Konkle et al. Blood 2021).Here, we investigated the effects of the IL-6 signaling blockade as a possible targeted approach to modulate AAV vector immunogenicity in hemophilia gene therapy using a non-human primate (NHP) model. Spk100 AAV capsid was used to deliver the human F9 gene. To prevent IL-6 signaling, we used a monoclonal antibody, tocilizumab (TCZ), which blocks the IL-6 receptor. TCZ is currently approved for use in several forms of arthritis and cytokine release syndrome. Ten male cynomolgus monkeys received an intravenous injection of Spk100-hFIX vector (4x10 12 vg/kg) and 5 of those animals received a single dose of TCZ (8 mg/kg) the day prior to vector administration and were monitored for 13 weeks.As assessed by an array of clinical and anatomic pathology parameters, the investigational use of gene therapy combined with prophylactic TCZ administration was safe and well-tolerated. TCZ did not interfere with vector biodistribution, liver transduction or the transgenic FIX production. Spk100-hFIX alone modestly increased IL-6 secretion from NHP peripheral blood mononuclear cells (PBMC) in vitro but, following the vector infusion in animals, plasma IL-6 levels did not change significantly. Overall, cytokine secretion in response to Spk100 capsid and hFIX protein was lower in PBMCs isolated from TCZ-treated animals compared to cells obtained from control animals. As expected, animals administered Spk100-hFIX alone developed anti-capsid antibodies. The administration of TCZ was associated with a lower level of anti-Spk100 IgM, IgG and neutralizing antibodies (NAb). While the blockade of IL-6 signaling was effective for about for 14 days post-vector infusion, the lower level of anti-Spk100 antibodies persisted for the entire study duration. In the TCZ group, 4 out of 5 animals had NAb titers equal to or below 1:10, theoretically compatible with vector readministration. In contrast, 4 out of 5 animals in the control group developed high titer NAbs following vector infusion. Importantly, TCZ reduced the detection of the capsid-specific TNFα-positive T cells that were observed in animals after vector infusion. Finally, livers from sacrificed animals were used to prepare liver non-parenchymal cells for ex vivo phenotyping. Compared to the control animals, liver-resident immune cells from the TCZ-treated group had increased basal IL-10 secretion, while liver sinusoidal endothelial cells (albumin-CD45-CD31+CD146+MHCII+) had lower surface levels of MHC class II molecules, suggesting an anti-inflammatory milieu in the TCZ-treated livers.Our results show that short-term prophylactic blockade of IL-6 signaling was safely used in NHPs and has the potential to reduce immune responses commonly observed post AAV vector infusion. These results support the continued investigation of tocilizumab as a targeted immunomodulatory regimen in liver gene therapy for hemophilia. DisclosuresKuranda: Spark Therapeutics: Current Employment. Weisshaar: Spark Therapeutics: Current Employment. Chen: Spark Therapeutics: Ended employment in the past 24 months. Smith: Spark Therapeutics: Current Employment. Beck: Spark Therapeutics: Current Employment. Kahle: Spark Therapeutics: Current Employment. Mingozzi: Spark Therapeutics: Current Employment.

Gut-Liver Immune Traffic: Deciphering Immune-Pathogenesis to Underpin Translational Therapy.

The tight relationship between the gut and liver on embryological, anatomical and physiological levels inspired the concept of a gut-liver axis as a central element in the pathogenesis of gut-liver axis diseases. This axis refers to the reciprocal regulation between these two organs causing an integrated system of immune homeostasis or tolerance breakdown guided by the microbiota, the diet, genetic background, and environmental factors. Continuous exposure of gut microbiome, various hormones, drugs and toxins, or metabolites from the diet through the portal vein adapt the liver to maintain its tolerogenic state. This is orchestrated by the combined effort of immune cells network: behaving as a sinusoidal and biliary firewall, along with a regulatory network of immune cells including, regulatory T cells and tolerogenic dendritic cells (DC). In addition, downregulation of costimulatory molecules on hepatic sinusoids, hepatocytes and biliary epithelial cells as well as regulating the bile acids chain also play a part in hepatic immune homeostasis. Recent evidence also demonstrated the link between changes in the gut microbiome and liver resident immune cells in the progression of cirrhosis and the tight correlation among primary sclerosing cholangitis (PSC) and also checkpoint induced liver and gut injury. In this review, we will summarize the most recent evidence of the bidirectional relationship among the gut and the liver and how it contributes to liver disease, focusing mainly on PSC and checkpoint induced hepatitis and colitis. We will also focus on completed therapeutic options and on potential targets for future treatment linking with immunology and describe the future direction of this research, taking advantage of modern technologies.

Interleukin-10 and Transforming Growth Factor-β Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver.

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor β (TGF-β) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4hours at 37°C with or without the addition of 20ng/mL of each IL10 and TGF-β (n=7). Naïve and cold storage (4hours at 4°C) livers served as controls (n=4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-β treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-β treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-β.

Research Highlights

Longevity and Replenishment of Human Liver-resident Memory T Cells and Mononuclear Phagocytes Pallett LJ, Burton AR, Amin OE, et al. J Exp Med. 2020;217(9):e20200050. There is an increasing interest in tissue resident leukocytes and their role in mediating rejection and tissue regeneration after transplantation.1 However, not a great deal is known about graft-resident populations in the liver after transplantation.2 Pallett et al3 investigated the composition of liver-resident immune cells in HLA-mismatched donor–recipient pairs after transplantation. They found that the majority of cells were recipient-derived cells recruited from the periphery but that a donor-derived population persisted in the graft, even up to 10 years after transplantation. Interestingly, this appeared to be unique to the liver, as no such chimerism was detected in the peripheral blood. Additionally, certain populations including polymorphonuclear cells, NK, T, and NKT cells persisted more effectively than other cell types, such as dendritic cells. The infiltrating cells acquired tissue-resident and liver-resident features and even replicated the ratios of cell types normally found in the liver, such as the higher CD8:CD4 ratio that is normally found in the periphery. This suggests that the liver microenvironment can recruit cells and alter their phenotype to fit liver-resident requirements, regardless of HLA provenance. The infiltrating cells acquired phenotypic markers including the expression of Kupffer/macrophage cell marker CD68 but still were distinguishable from embryonically seeded donor cells in the expression of a few markers, such as HMOX1, CD206, and CD163, indicating that the phenotype induced is not identical to native tissue-resident cells. This phenotypic malleability has implications in cellular therapy, if the graft is capable of controlling which cell types it recruits and is capable of then morphing their phenotype according to its homeostatic composition. The presence of a long-lived donor-derived resident population is also of interest for localized chimerism within the graft, which may help facilitate tolerance.4 This is particularly relevant as donor T cells persisted, but dendritic cells did not. Whether this long-lived chimerism is restricted to the liver or can be achieved in other organs is a matter of great relevance for adoptive cellular therapies. Sympathetic Nervous Tone Limits the Development of Myeloid-derived Suppressor Cells Nevin JT, Moussa M, Corwin WL, et al. Sci Immunol. 2020;5(51):eaay9368. The immune system can respond to a huge variety of physiological cues. Sympathetic nervous system (SNS) release of norepinephrine directly signals to various immune cells via adrenergic receptors (ARs) expressed on their surface, known to modify their function.1 In the study from Nevin et al,2 the authors investigated the impact of ablation of SNS signaling on myeloid cells in mice. Sympathetic signaling was targeted using neurotoxin 6-hydroxydopamine, which is selectively taken up by sympathetic terminals and results in their degradation. Sympathectomized mice had increased polymorphonuclear (PMN) and monocytic cell populations with phenotypes consistent with polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) and mononuclear MDSCs (M-MDSCs). They found that the CD11b+ compartment from sympathectomized mice was more suppressive than that of control mice, specifically the CD11b+Ly6CintLy6G+ PMN-MDSC subpopulation. Single-cell RNA sequencing revealed a shift in the PMN population in SNS-ablated mice, with an enrichment of immature cells and a decrease in cells expressing the mature neutrophil marker CD101, as well as an increase in proliferation. Sympathectomized mice also had an increase in regulatory T cell (Treg) populations in a Gr1-dependent manner. The mechanism behind this was further elucidated with the use of α-AR and ß-AR agonists and antagonists, which revealed that ß-AR agonism and α-AR antagonism both reproduced the effects of SNS ablation and induced MDSC proliferation and impaired maturity. As α-ARs have higher affinity for norepinephrine than ß-ARs, the researchers suggest that tonal SNS signaling preferentially affects α-ARs and that SNS ablation therefore reduces this to induce MDSC development. Additionally, they suggest that s100a8-s100a9 heterodimers secreted by MDSCs may be responsible for the observed Treg induction, as this heterodimer has been shown to signal via CD69 to induce STAT5 phosphorylation. Indeed, iTreg induced in the presence of s100a8-s100a9 heterodimers showed increased Foxp3 expression and proliferation even in the absence of IL-2, suggesting that this dimer can compensate via alternative STAT5 phosphorylation. Naturally suppressive cell populations such as Tregs and MDSCs are of significant interest to the field of transplantation for their ability to therapeutically control alloresponses.3,4 Understanding the various physiological changes that modify leukocyte behavior after transplantation is critical. This study sheds some light on the physiological mechanisms that control these leukocyte populations and has implications for transplantation where transplanted organs are denervated or the settings of organ failure and surgery in which there is autonomic system activation.

MTOR INHIBITORS ENHANCE ANTITUMOR ACTIVITY OF LIVER NATURAL KILLER CELLS IN MICE

Background: Liver transplantation (LT) is the best available option for patients with end stage liver disease and HCC. However, recipients are at high risk of cancer recurrence and infection, due to immunosuppressive (IS) therapy. IS used in LT commonly consist of calcineurin inhibitor (CNI) or a mechanistic target of rapamycin (mTOR), anti-metabolite and corticosteroids. Liver is an important immunological organ with a unique microenvironment. Innate and adaptive immune responses shapes the balance between immune tolerance and activation. It is well-known that IS drugs targeting the adaptive component of immunity, while innate immune cells become the only self-defense mechanism against neoplastic cells and infectious agents. However, the effect of IS drugs on liver resident immune cells are not well known. In this study, we analyzed the effect of two main IS drugs, calcineurin inhibitors and mTOR inhibitors on adaptive and innate immune components of the liver in mice Method: C57/BL6 mice were treated by intraperitoneal injection of mTORi (rapamycin and everolimus) and CNI (cyclosporine) for 7 days. Twenty-four hours after the last injection, liver mononuclear cells (LMNCs), splenocytes were collected. The proportion of T cells, NKT cells and NK cells and various functional molecules were analyzed by flow cytometry. Result: The proportion of TCR+NK1.1- T cells, TCR+NK1.1+ NKT cells in LMNC did not differ in both CNI mTORi treated groups. TCR-NK1.1+NK fraction in LMNCs was significantly increased in mTORi group (9.5 in mTORi groups versus 7.8 in control group), CNI treatment had no effect on NK cells frequency. mTORi treatment significantly upregulated the TRAIL positive NK cells (38.5%±4.0 in mTORi group vs 21.0%±8.3 in untreated, p =0.003). CNI treatment oppositely significantly decreased the TRAIL positive NK cell population (16.4%±4.2 in CNI group vs 21.0%±8.3 in untreated, p <0.05). CD 69 and Nkp46 expression on liver NK cells were not changed significantly in both mTORi and CNI treated groups in comparison to control group. Moreover, liver NK cells from mTORi treated mice showed significantly higher cytotoxicity against TRAIL-sensitive Hepa1-6 cells (30.1% ± 13.0 vs 13.5 %± 6.0, p=0.009). Cytotoxicity against TRAIL resistant YAC-1 mouse lymphoma was not significantly enhanced after mTORi treatment (53.6 % ± 4.4 vs 39.9% ± 1.2). Conclusion: mTOR inhibitors has ability to enhance liver resident NK cell activity. This result suggests that mTOR inhibitors might be useful to maintain anti-microbe and anti-tumor immunity even under immunosuppressive condition after transplantation.

Single-cell RNA sequencing reveals the heterogeneity of liver-resident immune cells in human

The liver plays a critical role in both immune defense and tolerance in the body. The liver-resident immune cells (LrICs) determine the immune properties, but the unique composition and heterogeneity of these cells are incompletely understood. Here, we dissect the diversity of LrICs by a comprehensive transcriptomic profiling using the unbiased single-cell RNA-sequencing (scRNA-seq). A total of 70, 706 of CD45+ immune cells from the paired liver perfusion, spleen and peripheral blood as references were profiled. We identified more than 30 discrete cell populations comprising 13 of T and NK cell, 7 of B cell, 4 of plasma cell, and 8 of myeloid cell subsets in human liver and donor-paired spleen and blood, and characterized their tissue distribution, gene expression and functional modules. Especially, four of CXCR6+ T and NK cell subsets were found to be present preferentially in the liver, where they manifested heterogeneity, distinct function and prominent homeostatic proliferation. We propose a universal category system of T and NK cells based on distinct chemokine receptors, confirmed subsequently by phenotype, transcriptional factors and functionality. We also identified adaptive changes by the spleen and liver-derived monocyte and macrophage populations. Finally, we give a global glimpse on B cell and plasma cell subsets in human spleen and liver. We, therefore, reveal the heterogeneity and functional diversity of LrICs in human. This study presents comprehensively the landscape of LrICs and will enable further study on their roles in various human diseases.

Dual roles of immune cells and cytokines in hepatic ischemia reperfusion injury

Ischemia-reperfusion injury is one of the main causes of complications related to liver transplantation, hepatectomy, trauma and hemorrhagic shock. The cells of ischemia and hypoxic injury release of injury-related molecular patterns, lead to the activation of immune cells and cytokine, which further aggravates the inflammatory response and enlarges the injury. It’s indicated that injury-related molecular patterns, liver resident immune cells and cytokines play a key role in promoting inflammation and liver ischemia-reperfusion injury. However, recent studies suggested that the ischemia cells and cytokines played acomplex role in this process. Relevant progresses were reviewed in this article. Key words: Reperfusion injury; Liver; Cytokines; High mobility group protein B1; Dendritic cells

MSC-triggered metabolomic alterations in liver-resident immune cells isolated from CCl4-induced mouse ALI model

Clinical trials testing mesenchymal stem cell (MSC) as a cellular remedy for acute liver injury (ALI) are underway, but its underlying mechanism has not been thoroughly scrutinized. We highlight that the metabolomic profile of the liver-resident immune cells is significantly altered after MSC administration; its potential correlation with ALI remission is discussed in this study. C57BL/6 mice are randomly divided into three groups: the sham group, MSC-treated ALI group and PBS-treated ALI group; acute liver injury is induced by intraperitoneal injection of carbon tetrachloride. A high-performance chemical isotope labeling liquid chromatography–mass spectrometry (CIL LC–MS) is exploited to profile amine, phenol and carbonyl submetabolome of the liver-resident immune cells in different treatments. 4295 peak pairs are quantified and 2461 peak pairs are further identified in zero-reaction and one-reaction libraries. Clear separation of the three groups is observed in the global PCA and OPLS-DA analyses. We identified 256 metabolites to be candidate biomarkers for ALI-activated immunity and 114 metabolites to be candidate biomarkers for MSC-modulated immunity. Ariginine, aspartate and glutamate metabolism are most affected in both cases, with eight significantly regulated metabolites as joints (glutamic-gamma-semialdehyde, aspartate acid, glutamate acid, gamma-Aminobutyric acidorinithine, 2-keto-glutaramic acid, N-acetylornithine, citrulline and ornithine). These findings shed new light on the therapeutic benefit of immune modulation during ALI rescue. It needs to be further investigated whether exogenous supply of certain metabolites will exert a profound impact on the metabolic network, crosstalking with immune responses and modulating ALI prognosis.

The Role of CD1d and MR1 Restricted T Cells in the Liver

The liver is one of the most important immunological organs that remains tolerogenic in homeostasis yet promotes rapid responses to pathogens in the presence of a systemic infection. The composition of leucocytes in the liver is highly distinct from that of the blood and other lymphoid organs, particularly with respect to enrichment of innate T cells, i.e., invariant NKT cells (iNKT cells) and Mucosal-Associated Invariant T cells (MAIT cells). In recent years, studies have revealed insights into their biology and potential roles in maintaining the immune-environment in the liver. As the primary liver-resident immune cells, they are emerging as significant players in the human immune system and are associated with an increasing number of clinical diseases. As such, innate T cells are promising targets for modifying host defense and inflammation of various liver diseases, including viral, autoimmune, and those of tumor origin. In this review, we emphasize and discuss some of the recent discoveries and advances in the biology of innate T cells, their recruitment and diversity in the liver, and their role in various liver diseases, postulating on their potential application in immunotherapy.

Preparation and Culture of Human Liver Resident Immune Cells

Co-cultivation of tumor cells and liver resident immune cells or other non-parenchymal cells (NPCs) from the same donor is important for the study of cancer metastasis. So far, little is known about the mechanism of tumor cell or pathogen clearance, leukocyte infiltration, and immune cell recruitment in the human liver. To investigate these processes in vitro, the use of primary human hepatocytes and non-parenchymal cell, especially immune cell, co-culture systems play essential roles in the establishment of cell-cell and cell-extracellular matrix communications similar to native liver tissues. Hepatic non-parenchymal cells mainly comprise liver sinusoid endothelial cells (LSECs), microvascular endothelial cells, hepatic stellate cells, Kupffer cells (KCs), natural killer T (iNKT) cells, and dendritic cells (DCs). Here we describe procedures for preparation, isolation, and culture of human liver resident immune cells and other non-parenchymal cells. © 2018 by John Wiley & Sons, Inc.

Hepatic immune response to environmental carcinogens

Aim: Environmental carcinogenic substances contribute to increasing incidence of hepatocellular carcinoma (HCC). We employed a sensitive method for the detection of DNA damage combined with analysis of the immune response to gain better knowledge how environmental carcinogens mediate pathology. Materials and Methods: Rat hepatocytes were isolated and stimulated with carcinogenic substances for the assessment of DNA damage. The mycotoxin aflatoxin B1(AFB1), two heterocyclic amines from the cooking of meat amino-3-methylimidazo[4,5-f] quinoline (IQ) and 3-amino-1-methyl-5H-pyrido-(4,3-b)-indole (TRP-P-2), and protein extract from the fungus Lactarius necator were assayed. Unscheduled DNA synthesis in hepatocytes was measured by the incorporation of radioactive thymidine during DNA repair. Stimulation of hepatocyte/immune cell preparation with the substances and measurement of IFNγ release at different time points determined their ability to induce an inflammatory response. Results: DNA repair in the hepatocytes was induced in response to 10−7 M AFB1 and 10−9 M IQ. TRP-P-2 did not induce DNA repair; however, at 10−4 M, the fungus extract did this. Furthermore, liver-resident immune cells responded with differential production of IFNγ over time in response to stimulation by all the carcinogens, with AFB1 being the most potent. TRP-P-2 showed the most significant reduction in IFNγ response over time. Conclusion: DNA damage in hepatocytes induced by environmental substances was detected at low molecular concentrations. The system did provide novel evidence for hepatic carcinogenicity by the fungus L. necator. Analysis of the response by liver-resident immune cells to the substances suggested that highly mutagenic substances induce prolonged inflammatory response. Abbreviations used: HCC: Hepatocellular carcinoma, AFB1: Aflatoxin B1, IQ: Amino-3-methylimidazo[4,5-f] quinoline, TRP-P-2: 3-amino-1-methyl-5H-pyrido-(4,3-b)-indole, UDS: Unscheduled DNA synthesis, EGTA: Ethylene glycol-bis-(2-aminoethyl ether) N-N-N'-N'-tetraacetic acid, WME: Williams' medium E, 2-AAF: 2-Acetylaminofluorene, HBSS: Hanks' balanced salt solution, CYPs: Cytochrome p450 enzymes.

Kupffer cells facilitate the acute effects of leptin on hepatic lipid metabolism.

Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120 min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on phosphoinositol-3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes. Notably, liver TG were elevated in both lean and obese myeloid cell ObR, but the degree of obesity and insulin resistance induced by a high-fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the coculture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually [granulocyte macrophage colony-stimulating factor, IL-1α, IL-1β, IL-6, IL-10, and IL-18] or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin.

The Liver at the Nexus of Host-Microbial Interactions.

The liver receives blood from the intestine, from the spleen, and directly from the heart and holds a vital position in vertebrate physiology. It plays a role in intermediary metabolism, bile secretion, maintaining blood sterility, serum homeostasis, xenobiotic detoxification, and immunological activity. This article provides our perspective on the liver as a nexus in establishing and maintaining host microbial mutualism. We discuss the role of the liver not only in sanitizing the blood stream from penetrant live microbes, but also in metabolizing xenobiotics that are synthesized or modified by intestinal microbes, and how microbiota modify the signaling potential of bile acids. The combination of bile acids as hormones and the metabolic control from pervasive effects of other absorbed microbial molecules powerfully shape hepatic metabolism. In addition, intestinal microbial metabolites can be sensed by liver-resident immune cells, which may disturb liver homeostasis, leading to fibrosis and liver cancer.