Nonparenchymal Cells Research Articles

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.

<i>In Vitro</i> Assessment of Drug-Induced Liver Injury Using Cell-Based Models: A Review

Drug-induced liver injury (DILI) is the reason for 15–18% of medicinal product recalls from the market. Since interspecies differences often limit the relevance of standard non-clinical tests in vivo, a promising alternative is to develop cell-based in vitro methods.The aim of the study was to review current advances in cell modelling for the in vitro identification of DILI.In vitro mechanistic studies of DILI require cells that exhibit activity specific to hepatic metabolising enzymes and transporters. This article reviews the main cell cultures (primary human hepatocytes, immortal cell lines, stem cell-derived hepatocyte-like cells, co-cultures of hepatocytes and non-parenchymal liver cells) and their configurations. The optimisation of cell systems is directed towards enhancing their viability, functionality, compositional and configurational complexity, thus bringing them closer to in vivo models. Potential DILI causes include chemically reactive metabolites, oxidative stress, mitochondrial damage, intracellular accumulation of toxic bile acids resulting from transporter inhibition, and adaptive immune system activation. Accordingly, DILI studies rely on various methods, including innovative technologies for acquisition, storage, and analysis of large datasets (e.g. high-content screening, transcriptomics, proteomics, and metabolomics). Cell models are applicable to both DILI identification and mechanistic studies. Currently, the most promising technologies are omics, complex co-culture models, and organ-on-a-chip systems.

Colorectal cancer-derived small extracellular vesicles induce TGFβ1-mediated epithelial to mesenchymal transition of hepatocytes

BackgroundMetastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called “pre-metastatic niche”. In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown.MethodssEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment.ResultsOur study shows for the first time that TGFβ1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFβ1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients.ConclusionsSince it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Hepatocyte SREBP signaling mediates clock communication within the liver.

Rhythmic intraorgan communication coordinates environmental signals and the cell-intrinsic clock to maintain organ homeostasis. Hepatocyte-specific KO of core components of the molecular clock Rev-erbα and -β (Reverb-hDKO) alters cholesterol and lipid metabolism in hepatocytes as well as rhythmic gene expression in nonparenchymal cells (NPCs) of the liver. Here, we report that in fatty liver caused by diet-induced obesity (DIO), hepatocyte SREBP cleavage-activating protein (SCAP) was required for Reverb-hDKO-induced diurnal rhythmic remodeling and epigenomic reprogramming in liver macrophages (LMs). Integrative analyses of isolated hepatocytes and LMs revealed that SCAP-dependent lipidomic changes in REV-ERB-depleted hepatocytes led to the enhancement of LM metabolic rhythms. Hepatocytic loss of REV-ERBα and β (REV-ERBs) also attenuated LM rhythms via SCAP-independent polypeptide secretion. These results shed light on the signaling mechanisms by which hepatocytes regulate diurnal rhythms in NPCs in fatty liver disease caused by DIO.

Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro.

Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.

A Scd1-mediated metabolic alteration participates in liver responses to low-dose bavachin

Hepatotoxicity induced by bioactive constituents in traditional Chinese medicines or herbs, such as bavachin (BV) in Fructus Psoraleae, has a prolonged latency to overt drug-induced liver injury in the clinic. Several studies have described BV-induced liver damage and underlying toxicity mechanisms, but little attention has been paid to the deciphering of organisms or cellular responses to BV at no-observed-adverse-effect level, and the underlying molecular mechanisms and specific indicators are also lacking during the asymptomatic phase, making it much harder for early recognition of hepatotoxicity. Here, we treated mice with BV for 7 days and did not detect any abnormalities in biochemical tests, but found subtle steatosis in BV-treated hepatocytes. We then profiled the gene expression of hepatocytes and non-parenchymal cells at single-cell resolution and discovered three types of hepatocyte subsets in the BV-treated liver. Among these, the hepa3 subtype suffered from a vast alteration in lipid metabolism, which was characterized by enhanced expression of apolipoproteins, carboxylesterases, and stearoyl-CoA desaturase 1 (Scd1). In particular, increased Scd1 promoted monounsaturated fatty acids (MUFAs) synthesis and was considered to be related to BV-induced steatosis and polyunsaturated fatty acids (PUFAs) generation, which participates in the initiation of ferroptosis. Additionally, we demonstrated that multiple intrinsic transcription factors, including Srebf1 and Hnf4a, and extrinsic signals from niche cells may regulate the above-mentioned molecular events in BV-treated hepatocytes. Collectively, our study deciphered the features of hepatocytes in response to BV insult, decoded the underlying molecular mechanisms, and suggested that Scd1 could be a hub molecule for the prediction of hepatotoxicity at an early stage.

Expansion of macrophage and liver sinusoidal endothelial cell subpopulations during non-alcoholic steatohepatitis progression

Liver non-parenchymal cells (NPCs) play a critical role in the progression of non-alcoholic steatohepatitis (NASH). We aimed to explore the heterogeneity of NPCs and identify NASH-specific subpopulations contributing to NASH progression. Through single-cell RNA sequencing, we uncovered a proinflammatory subpopulation of Itgadhi/Fcrl5hi macrophages with potential function of modulating macrophage accumulation and promoting NASH development. We also identified subpopulations of Egr1hi and Ly6ahi liver sinusoidal endothelial cells (LSECs), which might participate in pathological angiogenesis and inflammation regulation. The Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs, and Ly6ahi LSECs emerged in the early stage and expanded significantly along with pathological progression of liver injury during NASH. Cell-cell interactions between hepatic stellate cells (HSCs) and Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs or Ly6ahi LSECs were enhanced in NASH liver. Our results revealed that expansion of Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs or Ly6ahi LSECs was strongly associated with NASH severity, suggesting these subpopulations might be involved in NASH progression.

Responses of hepatic sinusoidal cells to liver ischemia-reperfusion injury.

The liver displays a remarkable regenerative capacity in response to acute liver injury. In addition to the proliferation of hepatocytes during liver regeneration, non-parenchymal cells, including liver macrophages, liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) play critical roles in liver repair and regeneration. Liver ischemia-reperfusion injury (IRI) is a major cause of increased liver damage during liver resection, transplantation, and trauma. Impaired liver repair increases postoperative morbidity and mortality of patients who underwent liver surgery. Successful liver repair and regeneration after liver IRI requires coordinated interplay and synergic actions between hepatic resident cells and recruited cell components. However, the underlying mechanisms of liver repair after liver IRI are not well understood. Recent technological advances have revealed the heterogeneity of each liver cell component in the steady state and diseased livers. In this review, we describe the progress in the biology of liver non-parenchymal cells obtained from novel technological advances. We address the functional role of each cell component in response to liver IRI and the interactions between diverse immune repertoires and non-hematopoietic cell populations during the course of liver repair after liver IRI. We also discuss how these findings can help in the design of novel therapeutic approaches. Growing insights into the cellular interactions during liver IRI would enhance the pathology of liver IRI understanding comprehensively and further develop the strategies for improvement of liver repair.

Preservation of Mitochondrial Health in Liver Ischemia/Reperfusion Injury.

Liver ischemia-reperfusion injury (LIRI) is a major cause of the development of complications in different clinical settings such as liver resection and liver transplantation. Damage arising from LIRI is a major risk factor for early graft rejection and is associated with higher morbidity and mortality after surgery. Although the mechanisms leading to the injury of parenchymal and non-parenchymal liver cells are not yet fully understood, mitochondrial dysfunction is recognized as a hallmark of LIRI that exacerbates cellular injury. Mitochondria play a major role in glucose metabolism, energy production, reactive oxygen species (ROS) signaling, calcium homeostasis and cell death. The diverse roles of mitochondria make it essential to preserve mitochondrial health in order to maintain cellular activity and liver integrity during liver ischemia/reperfusion (I/R). A growing body of studies suggest that protecting mitochondria by regulating mitochondrial biogenesis, fission/fusion and mitophagy during liver I/R ameliorates LIRI. Targeting mitochondria in conditions that exacerbate mitochondrial dysfunction, such as steatosis and aging, has been successful in decreasing their susceptibility to LIRI. Studying mitochondrial dysfunction will help understand the underlying mechanisms of cellular damage during LIRI which is important for the development of new therapeutic strategies aimed at improving patient outcomes. In this review, we highlight the progress made in recent years regarding the role of mitochondria in liver I/R and discuss the impact of liver conditions on LIRI.

Living on the edge: how stromal cells restrict liver cancer invasion

The global incidence of primary liver cancer is increasing and currently ranks amongst the top-five causes of premature death from cancer annually. Like in many solid tumours, liver cancer cells are closely associated with stromal cells, known as non-parenchymal cells (NPCs), in the liver, including fibroblasts and endothelial cells, which normally support liver function and structure. Although single-cell RNA sequencing has begun to unpick the interactions between tumour cells and this non-epithelial microenvironment, these -omic approaches cannot mechanistically resolve how the non-parenchymal stroma interacts with tumour cells, nor can they resolve whether a fibroblast exerts differential outcomes on mutant versus healthy epithelial cells.In this issue of Disease Models & Mechanisms, Liqin Zhu and colleagues describe a novel in vitro microwell culture system to co-culture NPCs with either healthy hepatocytes or tumour cells of intrahepatic cholangiocarcinoma (iCCA), an aggressive type of primary liver cancer. When the authors co-cultured NPCs with primary hepatocytes, the NPCs supported hepatocyte spheroid growth, maintaining the phenotype of the healthy hepatocytes in culture. Interestingly, when cultured with mutant iCCA cells, NPCs supressed the growth of cancer spheroids, suggesting a divergent role of NPCs in tumour growth.To understand the role of these stromal cells in tumour growth further, the authors then co-cultured healthy hepatocytes and tumour cells in their microwell system and found that tumour cells invade and kill healthy hepatocytes. The addition of NPCs to this system significantly limited this process, and in vitro cancer cells were constrained, no longer killing healthy hepatocytes. Following on from this, the authors used an orthotopic transplantation mouse model of liver cancer and liver tumour samples from patients to show that activated myofibroblasts localised at the interface between tumour and normal tissue. Together, the data suggest that, in liver cancer, NPCs, in particular myofibroblasts, form a physical barrier that limits the ability of cancer cells to invade and damage normal tissue.Targeting the tumour microenvironment to limit disease progression is an attractive therapeutic prospect that could be agnostic to the underlying genetic and molecular heterogeneity seen in cancer. However, as NPCs have previously been shown to provide pro-proliferative signals in cancer and modify the intratumoral immune landscape, more research is required to understand the nuanced NPC-epithelial cell relationship in cancer. We should carefully consider the diverse and potentially conflicting roles of NPCs in tumour progression, and advanced in vitro systems, such as those developed by Zhu and colleagues, will help us decipher this.

Time-resolved single-cell transcriptomics reveals the landscape and dynamics of hepatic cells in sepsis-induced acute liver dysfunction

Sepsis-induced acute liver dysfunction often occurs early in sepsis and can exacerbate the pathology by triggering multiple organ dysfunction and increasing lethality. Nevertheless, our understanding of the cellular heterogeneity and dynamic regulation of major nonparenchymal cell lineages remains unclear. Here, single-cell RNA sequencing was used to profile multiple nonparenchymal cell subsets and dissect their crosstalk during sepsis-induced acute liver dysfunction in a clinically relevant polymicrobial sepsis model. The transcriptomes of major liver nonparenchymal cells from control and sepsis mice were analysed. The alterations in the endothelial cell and neutrophil subsets that were closely associated with acute liver dysfunction were validated using multiplex immunofluorescence staining. In addition, the therapeutic efficacy of inhibiting activating transcription factor 4 (ATF4) in sepsis and sepsis-induced acute liver dysfunction was explored. Our results present the dynamic transcriptomic landscape of major nonparenchymal cells at single-cell resolution. We observed significant alterations and heterogeneity in major hepatic nonparenchymal cell subsets during sepsis. Importantly, we identified endothelial cell (CD31+Sele+Glut1+) and neutrophil (Ly6G+Lta4h+Sort1+) subsets that were closely associated with acute liver dysfunction during sepsis progression. Furthermore, we found that ATF4 inhibition alleviated sepsis-induced acute liver dysfunction, prolonging the survival of septic mice. These results elucidate the potential mechanisms and subsequent therapeutic targets for the prevention and treatment of sepsis-induced acute liver dysfunction and other liver-related diseases. Sepsis-induced acute liver dysfunction often occurs early in sepsis and can lead to the death of the patient. Nevertheless, the pathogenesis of sepsis-induced acute liver dysfunction is not yet clear. We identified the major cell types associated with acute liver dysfunction and explored their interactions during sepsis. In addition, we also found that ATF-4 inhibition could be invoked as a potential therapeutic for sepsis-induced acute liver dysfunction.

A hepatoprotective role of peritumoral non-parenchymal cells in early liver tumorigenesis.

Various 3-dimensional (3D) models of hepatocytes (HCs) have been established to assess liver functions in vitro. Contribution of the hepatic non-parenchymal cells (NPCs), however, is largely neglected in these models. Here, we report a comparative study of hepatic spheroids generated from freshly isolated mouse whole liver cells (WLCs) and HCs (referred to as SphWLC and SphHC, respectively). We found that HC differentiation was preserved better in SphWLC than in SphHC, and when cocultured with liver tumor spheroids (SphT), SphWLC showed a more potent suppression on SphT growth compared to SphHC. Histological characterization revealed a marked activation and accumulation of hepatic stellate cells (HSCs) at the SphWLC:SphT interface. We found that mixing HSCs in both 3D and 2D HC:T cocultures provided a potent protection to HCs over tumor-induced cell death. Activation of HSCs at the tumor border was similarly found in liver tumors from both mice and patients. Overall, our study suggests a hepatoprotective role of peritumoral HSCs in liver tumorigenesis, and the potential application of SphWLC as a useful 3D model for dissecting liver's response to tumorigenesis in vitro.

Macrophage STING signaling promotes NK cell to suppress colorectal cancer liver metastasis via 4-1BBL/4-1BB co-stimulation

Background and aimsMacrophage innate immune response plays an important role in tumorigenesis. However, the role and mechanism of macrophage STING signaling in modulating tumor microenvironment to suppress tumor growth at...

Monocyte-derived cells invade brain parenchyma and amyloid plaques in human Alzheimer’s disease hippocampus

Microglia are brain-resident myeloid cells and play a major role in the innate immune responses of the CNS and the pathogenesis of Alzheimer's disease (AD). However, the contribution of nonparenchymal or brain-infiltrated myeloid cells to disease progression remains to be demonstrated. Here, we show that monocyte-derived cells (MDC) invade brain parenchyma in advanced stages of AD continuum using transcriptional analysis and immunohistochemical characterization in post-mortem human hippocampus. Our findings demonstrated that a high proportion (60%) of demented Braak V–VI individuals was associated with up-regulation of genes rarely expressed by microglial cells and abundant in monocytes, among which stands the membrane-bound scavenger receptor for haptoglobin/hemoglobin complexes or Cd163. These Cd163-positive MDC invaded the hippocampal parenchyma, acquired a microglial-like morphology, and were located in close proximity to blood vessels. Moreover, and most interesting, these invading monocytes infiltrated the nearby amyloid plaques contributing to plaque-associated myeloid cell heterogeneity. However, in aged-matched control individuals with hippocampal amyloid pathology, no signs of MDC brain infiltration or plaque invasion were found. The previously reported microglial degeneration/dysfunction in AD hippocampus could be a key pathological factor inducing MDC recruitment. Our data suggest a clear association between MDC infiltration and endothelial activation which in turn may contribute to damage of the blood brain barrier integrity. The recruitment of monocytes could be a consequence rather than the cause of the severity of the disease. Whether monocyte infiltration is beneficial or detrimental to AD pathology remains to be fully elucidated. These findings open the opportunity to design targeted therapies, not only for microglia but also for the peripheral immune cell population to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying the progression of AD.

Antisense oligodeoxynucleotides: the certain but limited efficacy and the uncovering mechanisms for the cure of chronic hepatitis B

Recently, several phase I and phase II clinical trials of antisense oligodeoxynucleotides (ASOs) targeting to the commonly shared conserved sequences of HBV transcripts brought us some promising results. Particularly in the report of phase IIb clinical trial of Bepirovirsen (GSK3228836), approximately 9-10% patients with low baseline serum HBsAg (> 100 IU/ml & < 3 000 IU/ml) achieved functional cure after 24 weeks' of Bepirovirsen treatment. After reviewing the results of other clinical trials, one would be impressed to know that ALG-020572 (Aligos), RO7062931 (Roche) and GSK3389404 (GSK) all failed to sufficiently suppress serum HBsAg expression though the hepatocyte-targeted delivery of these ASOs were enhanced via N-acetyl galactosamine conjugation. Bepirovirsen enabled some patients to achieve sustained disappearance of serum HBsAg. The analysis of its distribution in different tissues of patients after drug administration showed that only a few fractions of ASOs entered liver tissues and far fewer eventually entered hepatocytes. Taking into consideration that only a few hepatocytes could be expected positive for HBsAg staining among these participants with low serum HBsAg level. We suspect that the mechanistic contribution of ASOs declining the serum HBsAg is not only via directly acting on the HBV transcripts in hepatocytes, but also via entering non-parenchymal cells such as Kupffer cells and resulting in stimulation and activation of innate immunity. Eventually the serum HBsAg declines in most participants and even disappears in a small fraction of patients with low baseline HBsAg level, via attack the infected hepatocytes evidenced by the aberrant elevation of ALT. Nevertheless, the functional cure of CHB remains a challenging issue and more efforts are needed.

Non-Parenchymal Cells and the Extracellular Matrix in Hepatocellular Carcinoma in Non-Alcoholic Fatty Liver Disease.

Hepatocellular carcinoma (HCC) in the setting of non-alcoholic fatty liver disease (NAFLD)-related cirrhosis and even in the pre-cirrhotic state is increasing in incidence. NAFLD-related HCC has a poor clinical outcome as it is often advanced at diagnosis due to late diagnosis and systemic treatment response is poor due to reduced immune surveillance. Much of the focus of molecular research has been on the pathological changes in hepatocytes; however, immune cells, hepatic stellate cells, liver sinusoidal endothelial cells and the extracellular matrix may play important roles in the pathogenesis of NAFLD-related HCC as well. Here, we review the role of non-parenchymal cells in the liver in the pathogenesis of HCC in the context of NAFLD-NASH, with a particular focus on the innate and the adaptive immune system, fibrogenesis and angiogenesis. We review the key roles of macrophages, hepatic stellate cells (HSCs), T cells, natural killer (NK) cells, NKT cells and liver sinusoidal endothelial cells (LSECs) and the role of the extracellular matrix in hepatocarcinogenesis within the steatotic milieu.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/β-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.

Dysregulation of innate cell types in the hepatic immune microenvironment of alcoholic liver cirrhosis.

The risk of alcoholic cirrhosis increases in a dose- and time-dependent manner with alcohol consumption and ethanol metabolism in the liver. Currently, no effective antifibrotic therapies are available. We aimed to obtain a better understanding of the cellular and molecular mechanisms involved in the pathogenesis of liver cirrhosis. We performed single-cell RNA-sequencing to analyze immune cells from the liver tissue and peripheral blood form patients with alcoholic cirrhosis and healthy controls to profile the transcriptomes of more than 100,000 single human cells and yield molecular definitions for non-parenchymal cell types. In addition, we performed single-cell RNA-sequencing analysis to reveal the immune microenvironment related to alcoholic liver cirrhosis. Hematoxylin and eosin, Immunofluorescence staining and Flow cytometric analysis were employed to study the difference between tissues and cells with or without alcoholic cirrhosis. We identified a fibrosis-associated M1 subpopulation of macrophages that expands in liver fibrosis, differentiates from circulating monocytes, and is pro-fibrogenic. We also define mucosal-associated invariant T (MAIT) cells that expand in alcoholic cirrhosis and are topographically restricted to the fibrotic niche. Multilineage modeling of ligand and receptor interactions between the fibrosis-associated macrophages, MAIT, and NK cells revealed the intra-fibrotic activity of several pro-fibrogenic pathways, including responses to cytokines and antigen processing and presentation, natural killer cell-mediated cytotoxicity, cell adhesion molecules, Th1/Th2/Th17 cell differentiation, IL-17 signaling pathway, and Toll-like receptor signaling pathway. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ alcoholic fibrosis at the single-cell level and provides a conceptual framework for the discovery of rational therapeutic targets in liver alcoholic cirrhosis.

A Pathogenic Role of Non-Parenchymal Liver Cells in Alcohol-Associated Liver Disease of Infectious and Non-Infectious Origin.

Now, much is known regarding the impact of chronic and heavy alcohol consumption on the disruption of physiological liver functions and the induction of structural distortions in the hepatic tissues in alcohol-associated liver disease (ALD). This review deliberates the effects of alcohol on the activity and properties of liver non-parenchymal cells (NPCs), which are either residential or infiltrated into the liver from the general circulation. NPCs play a pivotal role in the regulation of organ inflammation and fibrosis, both in the context of hepatotropic infections and in non-infectious settings. Here, we overview how NPC functions in ALD are regulated by second hits, such as gender and the exposure to bacterial or viral infections. As an example of the virus-mediated trigger of liver injury, we focused on HIV infections potentiated by alcohol exposure, since this combination was only limitedly studied in relation to the role of hepatic stellate cells (HSCs) in the development of liver fibrosis. The review specifically focusses on liver macrophages, HSC, and T-lymphocytes and their regulation of ALD pathogenesis and outcomes. It also illustrates the activation of NPCs by the engulfment of apoptotic bodies, a frequent event observed when hepatocytes are exposed to ethanol metabolites and infections. As an example of such a double-hit-induced apoptotic hepatocyte death, we deliberate on the hepatotoxic accumulation of HIV proteins, which in combination with ethanol metabolites, causes intensive hepatic cell death and pro-fibrotic activation of HSCs engulfing these HIV- and malondialdehyde-expressing apoptotic hepatocytes.

Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH.

The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear. Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4+ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining. Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163+ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4+-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4+-KCs than in those cocultured with lipotoxic KCs. A subgroup of Vsig4+ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.