Liver Immune Microenvironment Research Articles

Abstract LB441: An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and nearly 50% of CRC patients develop liver metastasis during the course of their disease. Treatment of colorectal cancer liver metastasis (CRLM) has been particularly challenging due to tolerogenic nature of liver immune microenvironment as well as an eventual development of therapy resistance to standard chemotherapy regimen. One promising area of cancer immunotherapy is oncolytic virotherapy that can selectively destroy tumor cells and stimulate anti-tumor immune response. In this study, we systemically administered mSJ-650, an intravenous compatible oncolytic Wyeth strain vaccinia virus that expresses human complement regulatory protein CD55 and murine GM-CSF in place of viral thymidine kinase gene and has a deletion of the viral K2L gene, and evaluated its anti-tumor efficacy in CRLM. Mouse CRLM was induced by intrasplenic injection of 5×105 MC38-OVA cancer cells. Following tumor nodules formation in the liver, low dose (1×106 pfu) or high dose (3×106 pfu) of mSJ-650 was administered multiple times via tail vein and liver was harvested for evaluation of anti-tumor efficacy. Flow cytometry analysis was performed to investigate changes in hepatic and tumor immune microenvironment upon mSJ-650 treatment. Liver and tumor tissue were stained for immunofluorescence imaging of viral replication within the tumor cells and subsequent oncolysis by the virus. Systemic administration of mSJ-650 showed potent anti-tumor efficacy and led to significant reduction in metastatic nodule formation in liver even at low dose. The anti-tumor efficacy was mediated by tumor cell-specific viral distribution and replication, followed by lytic egress of mSJ-650 from infected tumor cells. Anti-tumor efficacy of mSJ-650 also accompanied a dramatic shift in immunologically tolerant hepatic microenvironment to immune-stimulatory one by decreasing proportion of myeloid-derived suppressor cells, Kupffer cells, and monocyte-derived macrophages (MDMs) in the liver while increasing CD8+ T cell infiltration by 2~3 folds. Moreover, mSJ-650 treatment was associated with phenotypic change in hepatic immune cells, as evidenced by Kupffer cell and MDM polarization to M1 phenotype and elevated expression of activation markers and cytotoxic molecules in CD8+ T cells. Changes in hepatic immune microenvironment were also observed in tumor nodules, especially in those completely surrounded by adjacent liver tissue, suggesting that anti-tumor immune response in tumor bed is potentiated by reprogramming of tumor-adjacent hepatic immune microenvironment. Systemic administration of mSJ-650 demonstrated superior anti-tumor efficacy in CRLM which was mediated by direct tumor cell killing and tumor-adjacent hepatic immune microenvironment reprogramming by the virus. Citation Format: Eunhye Kim, Yeonsoo Yang, Solchan Won, Namhee Lee, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB441.

Abstract 3970: Impairment of tumor reactive CD8 T cells in liver cancer

Abstract Tumor reactive (TR) CD8 T cells in the liver have been studied in the setting of both immunotherapy and cellular therapy with the predominant dogma that these cells have equivalent functionality in the liver as compared to other organ sites. Herein, we detail that TR+ CD8 T cells are functionally impaired in the liver immune microenvironment in mice with liver tumors.TR+ CD8 T cells were studied in BALB/c and C57BL/6 mice injected intrahepatic, intravenous, and subcutaneously with either a colon (CT26) or a melanoma (B16F10) cancer cell line to create intrahepatic, pulmonary and subcutaneous tumors, respectively. Additionally, an ⍺CD8 antibody with IgG control was used to conduct functional studies. Samples were analyzed using flow cytometry and liver weights were measured. A high frequency of TR+ CD8 T cells were found in the liver in mice with intrahepatic tumors. Despite ~70% of CD8 T cells being tumor reactive in intrahepatic tumors, liver tumor growth kinetics were faster than subcutaneous tumors. Higher proportion of the TR+ CD8 T cells were found to be exhausted in the liver tumor by flow cytometry. Functional depletion of the CD8 T cells caused a fivefold higher tumor growth in the subcutaneous model but in contrast had no effect on intrahepatic tumors. In conclusion, despite the high frequency of “killer” tumor reactive T cells in the liver, the growth kinetics of the liver tumors, flow cytometry as well as functional studies demonstrate the impairment of TR+ CD8 T cells in the liver. We aim to dissect the mechanisms mediating intrahepatic TR CD8+ T cell exhaustion in primary liver tumors as well as metastatic models. Additionally, we aim to determine significant costimulatory molecules specific to intrahepatic exhaustion which could lead to better therapeutic models. Citation Format: Rajiv S. Trehan, Marlaine Soliman, Xin Wang, Patrick Huang, Noemi Kedei, Xiao Bin Zhu, Matthias Seifert, Mohamed-Reda Benmebarek, Amran Nur, Firouzeh Korangy, Chi Ma, Tim F. Greten. Impairment of tumor reactive CD8 T cells in liver 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 3970.

Associating Appendicitis with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): A Novel Insight into an Unexpected Connection.

The gut microbiome modulates the liver immune microenvironment and is deeply integrated into the pathophysiology of metabolic dysfunction-associated steatotic liver disease (MASLD). Appendectomies, which are performed in almost all patients diagnosed with appendicitis, cause long-term alterations to the gut microbiome, providing a potential link with the development of MASLD. We therefore investigated a potential link between appendicitis and the presence of MASLD in a large cohort of outpatients in Germany. The present study included 26,717 individuals with and 26,717 without appendicitis. Univariable Cox-regression analyses were conducted to assess the association between appendicitis and MASLD. During the long-term follow-up, 4.8% of patients with appendicitis and 3.4% of those in the non-appendicitis group were diagnosed with MASLD (p < 0.001), corresponding to an incidence of 5.4 (appendicitis cohort) versus 3.5 (non-appendicitis cohort) cases per 1000 patient years. These findings were confirmed in regression analysis, revealing a strong and statistically significant association between appendicitis and the development of MASLD (HR: 1.57; 95% CI: 1.39-1.78). This link was observed for all age groups and was independent of patients' sex. We provide evidence from a large cohort of outpatients in Germany suggesting a link between appendicitis and MASLD. This might help to better stratify patients according to their individual risk for the development of chronic liver diseases.

Retreatment with HBV siRNA Results in Additional Reduction in HBV Antigenemia and Immune Stimulation in the AAV-HBV Mouse Model.

Treatment with siRNAs that target HBV has demonstrated robust declines in HBV antigens. This effect is also observed in the AAV-HBV mouse model, which was used to investigate if two cycles of GalNAc-HBV-siRNA treatment could induce deeper declines in HBsAg levels or prevent rebound, and to provide insights into the liver immune microenvironment. C57Bl/6 mice were transduced with one of two different titers of AAV-HBV for 28 days, resulting in stable levels of HBsAg of about 103 or 105 IU/mL. Mice were treated for 12 weeks (four doses q3wk) per cycle with 3 mg/kg of siRNA-targeting HBV or an irrelevant sequence either once (single treatment) or twice (retreatment) with an 8-week treatment pause in between. Blood was collected to evaluate viral parameters. Nine weeks after the last treatment, liver samples were collected to perform phenotyping, bulk RNA-sequencing, and immunohistochemistry. Independent of HBsAg baseline levels, treatment with HBV-siRNA induced a rapid decline in HBsAg levels, which then plateaued before gradually rebounding 12 weeks after treatment stopped. A second cycle of HBV-siRNA treatment induced a further decline in HBsAg levels in serum and the liver, reaching undetectable levels and preventing rebound when baseline levels were 103 IU/mL. This was accompanied with a significant increase in inflammatory macrophages in the liver and significant upregulation of regulatory T-cells and T-cells expressing immune checkpoint receptors. Retreatment induced an additional decline in HBsAg levels, reaching undetectable levels when baseline HBsAg levels were 3log10 or less. This correlated with T-cell activation and upregulation of Trem2.

Immune microenvironment in hepatocellular carcinoma: current concepts and the role of blockade of immune checkpoints

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. The specificity of the immune microenvironment of an organ limits the possibilities of traditional therapeutic and surgical approaches to treatment, therefore one of the most important tasks of modern medicine is the search for new therapeutic targets targeting the tumor microenvironment. The introduction of checkpoint inhibitors into clinical practice expands immunotherapeutic options in the fight against liver cancer. The purpose of our review is to summarize the available data on the liver immune microenvironment in hepatocellular carcinoma and to present advances in cancer immunotherapy using immune checkpoint blockade. Material and methods. An analysis of the main foreign and domestic sources was carried out using the PubMed/Medline, ClinicalTrials.gov databases over the past 5 years. Results. In terms of morbidity and mortality, hepatocellular carcinoma is included in the list of the most common malignant neoplasms in the world and the forecasts for the coming decades are disappointing. Modern approaches to immunotherapy, taking into account the tumor microenvironment, are associated with better survival rates and safety profiles than standard therapy. Conclusion. The effectiveness of checkpoint inhibitors as monotherapy and combination strategies offers hope for improving the prognosis and quality of life of patients with unresectable HCC.

Neuregulin 4 mediates the metabolic benefits of mild cold exposure by promoting beige fat thermogenesis.

Interorgan crosstalk via secreted hormones and metabolites is a fundamental aspect of mammalian metabolic physiology. Beyond the highly specialized endocrine cells, peripheral tissues are emerging as an important source of metabolic hormones that influence energy and nutrient metabolism and contribute to disease pathogenesis. Neuregulin 4 (Nrg4) is a fat-derived hormone that protects mice from nonalcoholic steatohepatitis (NASH) and NASH-associated liver cancer by shaping hepatic lipid metabolism and the liver immune microenvironment. Despite its enriched expression in brown fat, whether NRG4 plays a role in thermogenic response and mediates the metabolic benefits of cold exposure are areas that remain unexplored. Here we show that Nrg4 expression in inguinal white adipose tissue (iWAT) is highly responsive to chronic cold exposure. Nrg4 deficiency impairs beige fat induction and renders mice more susceptible to diet-induced metabolic disorders under mild cold conditions. Using mice with adipocyte and hepatocyte-specific Nrg4 deletion, we reveal that adipose tissue-derived NRG4, but not hepatic NRG4, is essential for beige fat induction following cold acclimation. Furthermore, treatment with recombinant NRG4-Fc fusion protein promotes beige fat induction in iWAT and improves metabolic health in mice with diet-induced obesity. These findings highlight a critical role of NRG4 in mediating beige fat induction and preserving metabolic health under mild cold conditions.

Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

Single-cell landscape of immune cells during the progression from HBV infection to HBV cirrhosis and HBV-associated hepatocellular carcinoma.

Immune cells play crucial roles in the development of chronic hepatitis B virus (HBV) infection, leading to cirrhosis and hepatocellular carcinoma (HCC). However, their functions at different disease stages are not fully understood. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize the human liver immune microenvironment at different disease stages. We analyzed scRNA-seq data from 118,455 immune cells obtained from livers of six healthy individuals, four patients with HBV infection, five patients with HBV cirrhosis, and three patients with HBV-associated HCC. Our results showed an accumulation of scar-associated macrophages during disease progression, and we identified two relevant immune subsets, Macrophage-CD9/IL18 and macrophage-CD9/IFI6. Macrophage-CD9/IL18 expanded from HBV infection to cirrhosis, while macrophage-CD9/IFI6 expanded from cirrhosis to HCC. We verified the existence of Macrophage-CD9/IFI6 using multiplex immunofluorescence staining. We also found an increase in cytotoxic NK Cell-GNLY during progression from cirrhosis to HCC. Additionally, the proportion of CD4 T cell-TNFAIP3, CD8 T cell-TNF (effector CD8 T cells), and CD8 T cell-CD53 increased, while the proportion of Treg cells decreased from HBV infection to cirrhosis. The proportion of Treg and CD8 T cell-LAG3 (Exhausted CD8 T cell) enhanced, while the proportion of CD8 T cell-TNF (effector CD8 T cells) decreased from cirrhosis to HCC. Furthermore, GSEA enrichment analyses revealed that MAPK, ERBB, and P53 signaling pathways in myeloid cells were gradually inhibited from HBV infection to cirrhosis and HCC. Our study provides important insights into changes in the hepatic immune environment during the progression of HBV-related liver disease, which may help improve the management of HBV-infected liver diseases.

Role of the immune system in liver transplantation and its implications for therapeutic interventions

AbstractLiver transplantation (LT) stands as the gold standard for treating end‐stage liver disease and hepatocellular carcinoma, yet postoperative complications continue to impact survival rates. The liver's unique immune system, governed by a microenvironment of diverse immune cells, is disrupted during processes like ischemia–reperfusion injury posttransplantation, leading to immune imbalance, inflammation, and subsequent complications. In the posttransplantation period, immune cells within the liver collaboratively foster a tolerant environment, crucial for immune tolerance and liver regeneration. While clinical trials exploring cell therapy for LT complications exist, a comprehensive summary is lacking. This review provides an insight into the intricacies of the liver's immune microenvironment, with a specific focus on macrophages and T cells as primary immune players. Delving into the immunological dynamics at different stages of LT, we explore the disruptions after LT and subsequent immune responses. Focusing on immune cell targeting for treating liver transplant complications, we provide a comprehensive summary of ongoing clinical trials in this domain, especially cell therapies. Furthermore, we offer innovative treatment strategies that leverage the opportunities and prospects identified in the therapeutic landscape. This review seeks to advance our understanding of LT immunology and steer the development of precise therapies for postoperative complications.

Radiation and Immune Checkpoint Inhibitors: Combination Therapy for Treatment of Hepatocellular Carcinoma.

The liver tumor immune microenvironment has been thought to possess a critical role in the development and progression of hepatocellular carcinoma (HCC). Despite the approval of immune checkpoint inhibitors (ICIs), such as programmed cell death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitors, for several types of cancers, including HCC, liver metastases have shown evidence of resistance or poor response to immunotherapies. Radiation therapy (RT) has displayed evidence of immunosuppressive effects through the upregulation of immune checkpoint molecules post-treatment. However, it was revealed that the limitations of ICIs can be overcome through the use of RT, as it can reshape the liver immune microenvironment. Moreover, ICIs are able to overcome the RT-induced inhibitory signals, effectively restoring anti-tumor activity. Owing to the synergetic effect believed to arise from the combination of ICIs with RT, several clinical trials are currently ongoing to assess the efficacy and safety of this treatment for patients with HCC.

Single-cell transcriptome analysis of liver immune microenvironment changes induced by microplastics in mice with non-alcoholic fatty liver

Recent studies have discovered that tiny particles of microplastics (MPs) at the nano-scale level can enter the body of organisms from the environment, potentially causing metabolic ailments. However, further investigation is required to understand the alterations in the immune microenvironment associated with non-alcoholic fatty liver disease (NAFLD) occurrence following exposure to MPs.Experiments were performed using mice, which were given a normal chow or high-fat diet (NCD or HFD, respectively) plus free drinking of sterile water with or without MPs, respectively. Employing an impartial technique known as unbiased single-cell RNA-sequencing (scRNA-seq), the cellular (single-cell) pathology landscape of NAFLD and related changes in the identified immune cell populations induced following MPs plus HFD treatment were assessed. The results showed that mice in the HFD groups had remarkably greater NAFLD activity scores than those from the NCD groups. Moreover, administration of MPs plus HFD further worsened the histopathological changes in the mice's liver, leading to hepatic steatosis, inflammatory cell infiltrations and ballooning degeneration. Following the construction of a sing-cell resolution transcriptomic atlas of 43,480 cells in the mice's livers of the indicated groups, clear cellular heterogeneity and potential cell-to-cell cross-talk could be observed. Specifically, we observed that MPs exacerbated the pro-inflammatory response and influenced the stemness of hepatocytes during HFD feeding. Importantly, treatment with MPs significantly increase the infiltration of the infiltrating liver-protecting Vsig4+ macrophages in the liver of the NAFLD mouse model while remarkably decreasing the angiogenic S100A6+ macrophage subpopulation. Furthermore, mice treated with MPs plus HFD exhibited significantly increased recruitment of CD4+ cells and heightened exhaustion of CD8+ T cells than those from the control group, characteristics typically associated with the dysregulation of immune homeostasis and severe inflammatory damage. Overall, this study offers valuable perspectives into comprehending the potential underlying cellular mechanisms and regulatory aspects of the microenvironment regarding MPs in the development of NAFLD.

Impact of chemotherapeutic agents on liver microenvironment: oxaliplatin create a pro-metastatic landscape

BackgroundChemotherapeutic agents are used to control tumor proliferation. However, their influence in the pre-metastatic niche of target organs has not been well studied. Oxaliplatin (OXA) is a drug applied in standard treatments of colorectal cancer (CRC), while the direct effect of which on the pre-metastatic microenvironment of the liver remains unclear.MethodsModels of liver metastases were established with luciferase expressing CT26 cells in BALB/c and BALB/c-nude mice. Single-cell RNA Sequencing was performed to examine the immune microenvironment in the liver elicited by OXA. Immunofluorescence and flowcytometry were utilized to confirm the changes in the number of immune cells. LDH, CellTrace CFSE Cell Proliferation and apoptosis assays were conducted to explore the impact of OXA on T cells ex vivo. The correlation between chemotherapy-related lymphopenia and metastases was assessed by meta-analysis.ResultsHerein we discovered that administration of OXA prior to the occurrence of liver metastasis actually accelerated tumor development and colonization in the liver. Single-cell RNA sequencing revealed that the landscape of the liver immune microenvironment had been changed to immunosuppressive phenotype. Macrophages after the treatment of OXA exhibited a high ability to inhibit the activation of T cells. Further investigation revealed a significant decrease in the number of T cells in the liver, particularly CD8+ T cells with reduced capacity of proliferation, activation, and killing. When mice were treated with T cell supplementation, the OXA-induced metastasis was notably abolished, indicating that the OXA-primed liver microenvironment could be reversed by the infusion of T cells. Consistent with our findings in mice, a meta-analysis was performed to verify that chemotherapy-related lymphopenia was associated with an inferior prognosis related with high incidence of metastasis, suggesting the pivotal role of chemotherapy in pre-metastatic niche formation. Furthermore, a notable reduction in the count of both macrophages and T cells was observed in the liver of colorectal cancer (CRC) patient undergoing OXA-based chemotherapy.ConclusionsOur findings proposed that immunosuppressive microenvironment in liver induced by OXA enhanced liver metastasis of colorectal cancer, which highlighted a new consideration to balance the pro metastases and anti-cancer possibility of OXA treatment.

Corilagin alleviates liver fibrosis in zebrafish and mice by repressing IDO1-mediated M2 macrophage repolarization

BackgroundLiver fibrosis caused by chronic liver injury, eventually develops into liver cirrhosis and hepatocellular carcinoma. Currently, there are no effective drugs to relieve liver fibrosis due to the lack of molecular pathogenesis characteristics. Former research demonstrates that the hepatic immune microenvironment plays a key role in the pathogenesis of liver fibrosis, thus macrophages are important immune cells in the liver. Our previous study has found that IDO1 plays an important role in the liver immune microenvironment. CRG is a gallic acid tannin found in medicinal plants of many ethnicities that protects against inflammation, tumors and chronic liver disease. However, the mechanism of by which CRG mediates the interaction of IDO1 with macrophages during hepatic immune maturation is not clear. PurposeTo investigate the regulatory mechanism of CRG in liver fibrosis and the intrinsic relationship between IDO1 and macrophage differentiation. MethodsZebrafish, RAW264.7 cells and mice were used in the study. IDO1 overexpression and knockdown cell lines were constructed using lentiviral techniques. ResultsWe discovered that CRG remarkably reduced the AST and ALT serum levels. Histological examination revealed that CRG ameliorates CCL4-induced liver fibrosis and depressed the expression of α-SMA, Lamimin, Collagen-Ι and fibronectin. Besides, we found that CRG promoted increased MerTK expression on partly macrophages. Interestingly, in vitro, we found that CRG suppressed IDO1 expression and regulated macrophage differentiation by upregulating CD86, CD80 and iNOS, while downregulating CD206, CD163, IL-4 and IL-10 expression. Additionally, we found that CRG could inhibit hepatic stellate cell activation by direct or indirect action. ConclusionOur findings suggest that CRG alleviates liver fibrosis by mediating IDO1-mediated M2 macrophage repolarization.

PPARγ-dependent hepatic macrophage switching acts as a central hub for hUCMSC-mediated alleviation of decompensated liver cirrhosis in rats

BackgroundDecompensated liver cirrhosis (DLC), a terminal-stage complication of liver disease, is a major cause of morbidity and mortality in patients with hepatopathies. Human umbilical cord mesenchymal stem cell (hUCMSC) therapy has emerged as a novel treatment alternative for the treatment of DLC. However, optimized therapy protocols and the associated mechanisms are not entirely understood.MethodsWe constructed a DLC rat model consistent with the typical clinical characteristics combined use of PB and CCL4. Performing dynamic detection of liver morphology and function in rats for 11 weeks, various disease characteristics of DLC and the therapeutic effect of hUCMSCs on DLC in experimental rats were thoroughly investigated, according to ascites examination, histopathological, and related blood biochemical analyses. Flow cytometry analysis of rat liver, immunofluorescence, and RT-qPCR was performed to examine the changes in the liver immune microenvironment after hucMSCs treatment. We performed RNA-seq analysis of liver and primary macrophages and hUCMSCs co-culture system in vitro to explore possible signaling pathways. PPARγ antagonist, GW9662, and clodronate liposomes were used to inhibit PPAR activation and pre-exhaustion of macrophages in DLC rats’ livers, respectively.ResultsWe found that changing the two key issues, the frequency and initial phase of hUCMSCs infusion, can affect the efficacy of hUCMSCs, and the optimal hUCMSCs treatment schedule is once every week for three weeks at the early stage of DLC progression, providing the best therapeutic effect in reducing mortality and ascites, and improving liver function in DLC rats. hUCMSCs treatment skewed the macrophage phenotype from M1-type to M2-type by activating the PPARγ signaling pathway in the liver, which was approved by primary macrophages and hUCMSCs co-culture system in vitro. Both inhibition of PPARγ activation with GW9662 and pre-exhaustion of macrophages in DLC rats’ liver abolished the regulation of hUCMSCs on macrophage polarization, thus attenuating the beneficial effect of hUCMSCs treatment in DLC rats.ConclusionsThese data demonstrated that the optimal hUCMSCs treatment effectively inhibits the ascites formation, prolongs survival and significantly improves liver structure and function in DLC rats through the activation of the PPARγ signaling pathway within liver macrophages. Our study compared the efficacy of different hUCMSCs infusion regimens for DLC, providing new insights on cell-based therapies for regenerative medicine.

A Treg-specific long noncoding RNA maintains immune-metabolic homeostasis in aging liver.

Regulatory T (Treg) cells modulate several aging-related liver diseases. However, the molecular mechanisms regulating Treg function in this context are unknown. Here we identified a long noncoding RNA, Altre (aging liver Treg-expressed non-protein-coding RNA), which was specifically expressed in the nucleus of Treg cells and increased with aging. Treg-specific deletion of Altre did not affect Treg homeostasis and function in young mice but caused Treg metabolic dysfunction, inflammatory liver microenvironment, liver fibrosis and liver cancer in aged mice. Depletion of Altre reduced Treg mitochondrial integrity and respiratory capacity, and induced reactive oxygen species accumulation, thus increasing intrahepatic Treg apoptosis in aged mice. Moreover, lipidomic analysis identified a specific lipid species driving Treg aging and apoptosis in the aging liver microenvironment. Mechanistically, Altre interacts with Yin Yang 1 to orchestrate its occupation on chromatin, thereby regulating the expression of a group of mitochondrial genes, and maintaining optimal mitochondrial function and Treg fitness in the liver of aged mice. In conclusion, the Treg-specific nuclear long noncoding RNA Altre maintains the immune-metabolic homeostasis of the aged liver through Yin Yang 1-regulated optimal mitochondrial function and the Treg-sustained liver immune microenvironment. Thus, Altre is a potential therapeutic target for the treatment of liver diseases affecting older adults.

Therapeutic modulation of the liver immune microenvironment.

Inflammation is a hallmark of progressive liver diseases such as chronic viral or immune-mediated hepatitis, alcohol-associated liver disease, and NAFLD. Preclinical and clinical studies have provided robust evidence that cytokines and related cellular stress sensors in innate and adaptive immunity orchestrate hepatic disease processes. Unresolved inflammation and liver injury result in hepatic scarring, fibrosis, and cirrhosis, which may culminate in HCC. Liver diseases are accompanied by gut dysbiosis and a bloom of pathobionts, fueling hepatic inflammation. Anti-inflammatory strategies are extensively used to treat human immune-mediated conditions beyond the liver, while evidence for immunomodulatory therapies and cell therapy-based strategies in liver diseases is only emerging. The development and establishment of novel immunomodulatory therapies for chronic liver diseases has been dampened by several clinical challenges, such as invasive monitoring of therapeutic efficacy with liver biopsy in clinical trials and risk of DILI in several studies. Such aspects prevented advancements of novel medical therapies for chronic inflammatory liver diseases. New concepts modulating the liver immune environment are studied and eagerly awaited to improve the management of chronic liver diseases in the future.

Single-cell RNA sequencing reveals a novel inhibitory effect of ApoA4 on NAFL mediated by liver-specific subsets of myeloid cells.

The liver immune microenvironment is a key element in the development of hepatic inflammation in NAFLD. ApoA4 deficiency increases the hepatic lipid burden, insulin resistance, and metabolic inflammation. However, the effect of ApoA4 on liver immune cells and the precise immune cell subsets that exacerbate fatty liver remain elusive. The aim of this study was to profile the hepatic immune cells affected by ApoA4 in NAFL. We performed scRNA-seq on liver immune cells from WT and ApoA4-deficient mice administered a high-fat diet. Immunostaining and qRT-PCR analysis were used to validate the results of scRNA-seq. We identified 10 discrete immune cell populations comprising macrophages, DCs, granulocytes, B, T and NK&NKT cells and characterized their subsets, gene expression profiles, and functional modules. ApoA4 deficiency led to significant increases in the abundance of specific subsets, including inflammatory macrophages (2-Mφ-Cxcl9 and 4-Mφ-Cxcl2) and activated granulocytes (0-Gran-Wfdc17). Moreover, ApoA4 deficiency resulted in higher Lgals3, Ctss, Fcgr2b, Spp1, Cxcl2, and Elane levels and lower Nr4a1 levels in hepatic immune cells. These genes were consistent with human NAFLD-associated marker genes linked to disease severity. The expression of NE and IL-1β in granulocytes and macrophages as key ApoA4 targets were validate in the presence or absence of ApoA4 by immunostaining. The scRNA-seq data analyses revealed reprogramming of liver immune cells resulted from ApoA4 deficiency. We uncovered that the emergence of ApoA4-associated immune subsets (namely Cxcl9+ macrophage, Cxcl2+ macrophage and Wfdc17+ granulocyte), pathways, and NAFLD-related marker genes may promote the development of NAFL. These findings may provide novel therapeutic targets for NAFL and the foundations for further studying the effects of ApoA4 on immune cells in various diseases.

The role of gut microbiota in liver regeneration.

The liver has unique regeneration potential, which ensures the continuous dependence of the human body on hepatic functions. As the composition and function of gut microbiota has been gradually elucidated, the vital role of gut microbiota in liver regeneration through gut-liver axis has recently been accepted. In the process of liver regeneration, gut microbiota composition is changed. Moreover, gut microbiota can contribute to the regulation of the liver immune microenvironment, thereby modulating the release of inflammatory factors including IL-6, TNF-α, HGF, IFN-γ and TGF-β, which involve in different phases of liver regeneration. And previous research have demonstrated that through enterohepatic circulation, bile acids (BAs), lipopolysaccharide, short-chain fatty acids and other metabolites of gut microbiota associate with liver and may promote liver regeneration through various pathways. In this perspective, by summarizing gut microbiota-derived signaling pathways that promote liver regeneration, we unveil the role of gut microbiota in liver regeneration and provide feasible strategies to promote liver regeneration by altering gut microbiota composition.

Multiparametric Flow Cytometry-Based Immunophenotyping of Mouse Liver Immune Cells.

The liver is a complex organ that governs many types of metabolisms, including energy metabolism and other cellular processes. The liver also plays a crucial role in important functions in immunity, and the activity of liver tissue-associated immunity affects the outcome of many liver pathologies. A thorough characterization of the liver immune microenvironment may contribute to a better understanding of immune signaling, the mechanisms of specific immune responses, and even to improved predictions about therapy outcomes. In this paper, we present an optimized, simple, and rapid protocol to characterize the liver-associated immune cell milieu. We believe that the most suitable technique for obtaining a complex immune cell suspension and for removing contaminating blood cells is to perform mouse liver perfusion, using only phosphate buffer saline. Combining an enzymatic digestion and a mechanical dissociation of liver tissue, followed by cell purification, improves downstream applications. This combination is an essential prerequisite for immune cell determination and characterization. We then demonstrate a flow cytometry-based multiparametric immunophenotyping along with a gating strategy to detect and quantify liver endothelial cells, T cells (helper and cytotoxic), B cells, NK cells, NKT cells, neutrophils, monocytes (subsets included), dendritic cells (subsets included), macrophages and Kupffer cells.

Abstract PO026: Bile acid signaling remodels the iCCA immune microenvironment

Abstract Intrahepatic cholangiocarcinoma (iCCA) primarily arises from the transformation of cholangiocytes (epithelial cells of the bile ducts), is diagnosed at late stage and has a dismal prognosis (average 5-year survival after resection is 32%). An alarming increase in its incidence over the past decades and the scarce treatment options available urges for the development of novel therapeutic strategies to tackle this malignancy. Immunotherapy has had limited success in iCCA. Possible explanations are that iCCA immune microenvironment is poor in CD8+ T cells and rich in immunosuppressive innate immune cells. The immune landscape of iCCA is determined by the aetiology of the liver insult (e.g. viral, cholestatic) and these different iCCA immune landscapes have not yet been characterized. Therefore, in depth studies on the immunobiology of iCCA are essential to develop new immunotherapy strategies where innate immune cells play a prominent role. We hypothesized that bile acids (BA) are a potential, valid therapeutic target for iCCA, as they strongly influence liver immune microenvironment and proliferation. This idea is supported by the fact that BA have an immunomodulatory role through inhibition of NF-KB and reduction of macrophage production of pro-inflammatory cytokines, suppressing cholangiocyte transformation in iCCA. Since BA and derivatives have recently been approved for the treatment of human chronic cholestatic diseases and primary sclerosis cholangitis, but have not yet been tested to treat iCCA, the proposed project might directly offer evidence for a second use of these drugs in patients with iCCA. Using novel preclinical mouse models of iCCA and human iCCA samples, our objective is to develop new therapeutic strategies for iCCA treatment based on BA signalling, that would impair tumour growth and foster the immune response against the tumour. In order to investigate the effects that BA can exert in tumour initiation and development, we adopted a iCCA mouse model based on the hydrodynamic tail-vein delivery of genetic elements specifically in hepatocytes, which differentiate into cholangiocytes and ultimately leads to the development of iCCA, while conserving a competent immune system. We found that by feeding the animals with an endogenous BA-enriched diet (BA diet), we reduced the overall tumor burden and strikingly increased overall survival. The positive response to the BA diet was associated to an inhibition of tumor proliferation and a robust increase in the abundance and activation of inflammatory monocytes and T cells. Our preliminary results indicate that mimicking the effect of BA could represent a novel immunotherapeutic strategy for iCCA. We will present these data as well as ongoing studies including fluorescence-activated cell sorting and single-cell RNA seq sequencing, focused on understanding the cooperation between different immune cell types that foster response against the tumor. Citation Format: María García-Beccaria, Mirian Fernández-Vaquero, Maria Reich, Dominik Pfister, Diran Herebian, Benjamin Goeppert, Darjus Felix Tschaharganeh, Verena Keitel, Mathias Heikenwälder. Bile acid signaling remodels the iCCA immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO026.