Kupffer Cell Depletion Research Articles

Metabolic degradation of polysaccharides from Lentinus edodes by Kupffer cells via the Dectin-1/Syk signaling pathway

It had been shown that lentinan (LNT) was mainly distributed in the liver after intravenous administration. The study aimed to investigate the integrated metabolic processes and mechanisms of LNT in the liver, as these have not been thoroughly explored. In current work, 5-([4,6-dichlorotriazin-2-yl] amino) fluorescein and cyanine 7 were used to label LNT for tracking its metabolic behavior and mechanisms. Near-infrared imaging demonstrated that LNT was captured mainly by the liver. Kupffer cell (KC) depletion reduced LNT liver localization and degradation in BALB/c mice. Moreover, experiments with Dectin-1 siRNA and Dectin-1/Syk signaling pathway inhibitors showed that LNT was mainly taken up by KCs via the Dectin-1/Syk pathway and promoted lysosomal maturation in KCs via this same pathway, which in turn promoted LNT degradation. These empirical findings offer novel insights into the metabolism of LNT in vivo and in vitro, which will facilitate the further application of LNT and other β-glucans.

Transient Kupffer cell depletion and subsequent replacement by infiltrating monocyte-derived cells does not alter the induction or progression of hepatocellular carcinoma.

Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F+ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.

Desialylated Platelet Clearance in the Liver is a Novel Mechanism of Systemic Immunosuppression.

Platelets are small, versatile blood cells that are critical for hemostasis/thrombosis. Local platelet accumulation is a known contributor to proinflammation in various disease states. However, the anti-inflammatory/immunosuppressive potential of platelets has been poorly explored. Here, we uncovered, unexpectedly, desialylated platelets (dPLTs) down-regulated immune responses against both platelet-associated and -independent antigen challenges. Utilizing multispectral photoacoustic tomography, we tracked dPLT trafficking to gut vasculature and an exclusive Kupffer cell-mediated dPLT clearance in the liver, a process that we identified to be synergistically dependent on platelet glycoprotein Ibα and hepatic Ashwell-Morell receptor. Mechanistically, Kupffer cell clearance of dPLT potentiated a systemic immunosuppressive state with increased anti-inflammatory cytokines and circulating CD4+ regulatory T cells, abolishable by Kupffer cell depletion. Last, in a clinically relevant model of hemophilia A, presensitization with dPLT attenuated anti-factor VIII antibody production after factor VIII ( infusion. As platelet desialylation commonly occurs in daily-aged and activated platelets, these findings open new avenues toward understanding immune homeostasis and potentiate the therapeutic potential of dPLT and engineered dPLT transfusions in controlling autoimmune and alloimmune diseases.

Key Role of Kupffer Cells in IL-1 Dependent Activation of CD8+ T Cell Responses to AAV Transgene Product in Liver

Introduction: Adeno-associated virus (AAV) based viral vectors have emerged as preferred vectors for in vivo gene therapy and are being evaluated in multiple phase I/II and phase III clinical studies. However, immune responses to both the AAV capsid and its encoded transgene still remain a major hurdle. Previously, we demonstrated in a murine model that cellular responses to AAV encoded transgene in the liver are dependent on IL-1α/β mediated activation of IL-1R1-MyD88 signaling. Using flow cytometry and tissue staining we provided evidence that plasmacytoid dendritic cells (pDCs) and Kupffer cells are central players in initiation of IL-1 signaling. Further, we showed that cross-presenting XCR1+ dendritic cells (DCs) are critical for antigen presentation to transgene-specific CD8+ T cells (Blood 136 Supplement 1: 2-3, 2020). In the present study we tested the impact of Kupffer cells depletion on transgene-specific CD8+ T cell response. Since Kupffer cells have been implicated in TLR4-MyD88 mediated secretion of IL-1α in a murine model of acetaminophen induced liver injury (Cell Mol Immunol 15(11): 973, 2018). Therefore, we also explored if innate sensor TLR4 is required for IL-1 mediated CD8+ T cell response to AAV encoded transgene in liver microenvironment. Further, we tested if intrinsic IL-1R1-MyD88 signaling in DCs is essential for these cellular responses. Methods: To study the impact of Kupffer cell depletion on transgene-specific CD8+ T cells we intraperitoneally injected wild-type (WT) C57BL6 mice with 200μg of depleting α-mouse F4/80 antibody (BioXcell) one day prior and 2X/week following vector administered for up to 4 weeks. Groups of C57BL6 WT (control), C57BL6 WT (treated with α-mouse F4/80 antibody) and TLR4-/- mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8 vector encoding ovalbumin (OVA) as the transgene (Mol Ther 25(4): 880, 2017). To study the role of MyD88 intrinsic signaling in DCs, we generated mice that lacked adaptor protein MyD88 in DCs (DC-MyD88-/-) using cre-lox system (Blood 129(24): 3184, 2017). Mice were again injected with 1x109vg of AAV8-OVA via IV route. Four weeks after vector administration PBMCs from each group were analyzed for OVA-specific CD8+ T cells using MHC class I tetramer. Results: As expected, ~62% of WT mice developed OVA-specific CD8+ T cell response. Interestingly, only 12% of mice (1 out of 8) treated with α-mouse F4/80 antibody showed CD8+ T cells specific to OVA. Whereas similar to WT mice, 50% of TLR4-/- mice had OVA-specific CD8+ T cell. We conclude that Kupffer cells, which show increased surface expression of IL-1R1 within days after AAV vector administration, are essential for the IL-1R-dependent CD8+ T cells response to the hepatic transgene product but do not function through a mechanism that requires TLR4. This mechanism therefore shares some aspects of acetaminophen-induced liver injury (critical role of KCs and IL-1) but is distinct with regard to TLR involvement. IL-1R1 signaling requires the cytosolic MyD88 adapter molecule. We know from prior studies that MHC I antigen presentation of liver-derived AAV encoded transgene product in lymphoid organs is primarily mediated by cross-presenting XCR1+ DCs. Thus, we tested the requirement of intrinsic MyD88 signaling in DCs. Our results suggest that intrinsic MyD88 signaling in DCs is not required in lymphoid organs, as the CD8+ T cell response in DC-MyD88-/- mice was comparable to that in WT control mice (these mice lack MyD88 in conventional DCs in lymphoid organs but retain MyD88 in hepatic DCs and in pDCs). Conclusions: In summary, we established that Kupffer cells play a central role in elicitation of transgene product-specific CD8+ T cell responses in hepatic AAV gene transfer (through a TLR4-independent mechanisms). While direct sensing of IL-1 by DCs in the hepatic environment may very well play a role, it is not required in lymphoid organs. Following initial priming of CD8+ T cell in the liver, co-stimulation by CD4+ T helper cells may replace IL-1 signaling during the subsequent systemic response.

FRI580 - Early Kupffer cell depletion does not affect hepatocellular carcinoma progression in mice

FRI580 - Early Kupffer cell depletion does not affect hepatocellular carcinoma progression in mice

Novel Mechanisms of Thrombopoietin Generation: The Essential Role of Kupffer Cells

Thrombopoietin (TPO) is the physiological regulator of hemopoietic stem cell niche and megakaryocyte differentiation, and therefore platelet production. Prevailing theory posits that TPO is constitutively expressed by hepatocytes, and levels are fine-tuned through platelet and megakaryocyte internalization/clearance via the c-Mpl receptor. Our lab has previously shown that platelet glycoprotein (GP) Ibα is indispensable for platelet-mediated TPO generation (Blood 2018), and recent reports have demonstrated that Kupffer cells, the tissue resident macrophages of the liver, contribute to the clearance of desialylated platelets. However, whether Kupffer cells may contribute to TPO generation has never been explored.To determine the possible role of Kupffer cells in TPO production, clodronate liposome was intravenously administered to deplete Kupffer cells in wild-type mice. Wild-type, Kupffer cell depleted mice showed a TPO decrease of 43.6% (±16%) 2 days post depletion, with only a gradual insignificant increase in TPO levels to day 6. Interestingly, TPO levels could not be significantly increased in wild-type Kupffer cell depleted mice even when transfused 2x10 8 wild-type or desialylated platelets, or 50mU neuraminidase. Kupffer cell depletion in IL4Rα/GPIbα-transgenic mice, which lack platelet-mediated TPO generation, showed a TPO decrease of 22.5% (±5%) from baseline 2 days post depletion, with only a gradual increase in levels to day 6, suggesting that Kupffer cells are required for constitutive in addition to platelet-mediated TPO production. As our lab has previously shown that platelet GPIbα drives platelet-mediated TPO generation, and that Kupffer cells now required, WT and GPIbα -/- platelets were co-cultured with Kupffer cells to assess interaction. Desialylated WT platelets interacted significantly more with Kupffer cells as analyzed by flow cytometry than GPIbα -/- platelets. Interestingly, desialylation of GPIbα -/- platelets did not increase binding to Kupffer cells, consolidating that desialylated GPIbα is required for Kupffer cell interaction, and subsequent TPO generation.This data demonstrates the novel and unexpected finding that Kupffer cells are required for both platelet-mediated and baseline hepatocellular TPO generation. Elucidation of the role of Kupffer cells in this crucial mechanism will provide a better understanding of why thrombocytopenias may occur in pathological states, as well as contribute to the development of TPO mimetic therapies. DisclosuresNo relevant conflicts of interest to declare.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Platelets Stimulate Liver Regeneration in a Rat Model of Partial Liver Transplantation.

Living donor liver transplantation (LDLT) is sometimes associated with impaired regeneration and severe ischemia/reperfusion injury (IRI) in the graft, resulting in small-for-size syndrome (SFSS). Platelets were previously reported to stimulate liver regeneration in models of hepatectomy, but the evidence in partial liver transplantation (LT) is lacking. In this study, a rat model of partial LT was used, and the impact of thrombopoietin (TPO)-induced perioperative thrombocytosis on graft regeneration, IRI, and survival was investigated. In experiment 1, a 30% partial LT was performed. Under thrombocytosis, SFSS was attenuated, as shown by decreased levels of serum aminotransferases, bilirubin, and ascites. Serum hepatocyte regeneration-related cytokines, including insulin-like growth factor-1, hepatocyte growth factor, interleukin 6 (IL6), and tumor necrosis factor α (TNF-α), were elevated. In addition, the proliferative signaling pathways, Ki-67-labeling index, proliferating cell nuclear antigen (PCNA)-labeling index, mitotic index, and liver/body weight ratio were increased under thrombocytosis. The platelet-induced regeneration was independent of TPO because increases in the Ki-67-labeling and PCNA-labeling indexes were eliminated after reducing platelet counts by antiplatelet serum in rats administered with TPO. For IRI, thrombocytosis did not aggravate oxidative stress or downstream signaling pathways, necrosis, or apoptosis in the graft. After Kupffer cell (KC) depletion, the platelet-induced attenuation of serum aminotransferases, increased serum levels of IL6 and TNF-α, and proliferation-related signaling pathways were eliminated. Moreover, platelet accumulation in the graft decreased substantially. In experiment 2, a 20% partial LT was performed, and thrombocytosis improved postoperative survival. In conclusion, our results suggested that thrombocytosis stimulated graft regeneration and prolonged survival without aggregating IRI after partial LT, and KCs vitally contributed to platelet-derived regeneration. Platelet therapies to increase perioperative platelet counts may improve the outcomes after LDLT.

Kupffer cell release of platelet activating factor drives dose limiting toxicities of nucleic acid nanocarriers

This work establishes that Kupffer cell release of platelet activating factor (PAF), a lipidic molecule with pro-inflammatory and vasoactive signaling properties, dictates dose-limiting siRNA nanocarrier-associated toxicities. High-dose intravenous injection of siRNA-polymer nano-polyplexes (si-NPs) elicited acute, shock-like symptoms in mice, associated with increased plasma PAF and consequently reduced PAF acetylhydrolase (PAF-AH) activity. These symptoms were completely prevented by prophylactic PAF receptor inhibition or Kupffer cell depletion. Assessment of varied si-NP chemistries confirmed that toxicity level correlated to relative uptake of the carrier by liver Kupffer cells and that this toxicity mechanism is dependent on carrier endosome disruptive function. 4T1 tumor-bearing mice, which exhibit increased circulating leukocytes, displayed greater sensitivity to these toxicities. PAF-mediated toxicities were generalizable to commercial delivery reagent in vivo-jetPEI® and an MC3 lipid formulation matched to an FDA-approved nanomedicine. These collective results establish Kupffer cell release of PAF as a key mediator of siRNA nanocarrier toxicity and identify PAFR inhibition as an effective strategy to increase siRNA nanocarrier tolerated dose.

Kupffer cell depletion attenuates IL-6/STAT3 mediates hepatocyte apoptosis in immunological liver injury of trichloroethylene sensitized mice

Trichloroethylene (TCE) induced TCE hypersensitivity syndrome which makes immune injuries in multi-system. The multiple organ damage included skin, liver, kidney and so on. The main manifestations of liver injuries were apoptosis and edema of hepatocytes. In our previous research, we found the activation of Kupffer cells (KCs) which increased IL-6 can aggravate liver cell apoptosis in TCE sensitized mice. However, the mechanism of IL-6 in liver damages induced by TCE was not clear. This study explored the function of IL-6/STAT3 signal pathway on the TCE induced apoptosis of liver cell. We established a TCE sensitized BALB/c mouse model with a KCs inhibitor GdCl3, we found that the expressions of ALT and AST in TCE sensitization positive mice were higher than other mice, and the expressions of apoptosis-related proteins were up-regulated in TCE sensitization positive mice, GdCl3 could alleviate this process. Meanwhile, GdCl3 could significantly decrease the expressions of IL-6/STAT3 proteins. All in all, the activation of KCs can increase the expression of IL-6, IL-6R and phosphorylate STAT3, induces hepatocyte apoptosis, and participates in immunity damage of liver which induced by TCE.

Chronic ingestion of Primex-Z, compared with other common fat sources, drives worse liver injury and enhanced susceptibility to bacterial infections.

The aim of this study was to investigate putative different outcomes on the development of non-alcoholic fatty liver disease in mice using fat options regularly used in human nutrition. Male C57BL/6 mice were fed a control diet, and four different high-fat diets (HFD: 40% calories from fat; Research Diet, Inc., New Brunswick, New Jersey, USA) for 16 and 30 wk. HFDs had different common fat sources, including trans-fat, non-trans-fat palm oil (Primex-Z), palm oil alone, and corn oil alone. Mice were sacrificed and samples were collected for analysis. Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that a HFD induced a major increase in hepatic lipid droplet deposition compared with control mice, being significantly higher in Primex-Z-fed mice. All HFD mice had similar or less weight gain as control mice; however, Primex-Z ingestion led to a higher increase in adiposity index (~90% increase) compared with other fat sources. Gene expression of isolated liver immune cells revealed large changes in expression of several inflammatory pathways, which were also more elevated in Primex-Z-fed mice, including Tnf (~20-fold), Il1b (~60-fold), and Tgfb (2.5-fold). Immunophenotyping and in vivo analysis showed that the frequency of hepatic immune cells was also disturbed during different HFD contents, rendering not only Kupffer cell depletion, but also reduced bacterial arresting ability. Different fat dietary sources imprint different immune and metabolic effects in the liver during consumption of an HFD. The present data highlighted that Primex-Z-a novel non-trans-fat-is not only able to damage hepatocytes, but also to impair liver ability to clear blood-borne infections.

Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets.

Every day, megakaryocytes produce billions of platelets that circulate for several days and eventually are cleared by the liver. The exact removal mechanism, however, remains unclear. Loss of sialic acid residues is thought to feature in the aging and clearance of platelets. Using state-of-the-art spinning disk intravital microscopy to delineate the different compartments and cells of the mouse liver, we observed rapid accumulation of desialylated platelets predominantly on Kupffer cells, with only a few on endothelial cells and none on hepatocytes. Kupffer cell depletion prevented the removal of aged platelets from circulation. Ashwell-Morell receptor (AMR) deficiency alone had little effect on platelet uptake. Macrophage galactose lectin (MGL) together with AMR mediated clearance of desialylated or cold-stored platelets by Kupffer cells. Effective clearance is critical, as mice with an aged platelet population displayed a bleeding phenotype. Our data provide evidence that the MGL of Kupffer cells plays a significant role in the removal of desialylated platelets through a collaboration with the AMR, thereby maintaining a healthy and functional platelet compartment.

Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Abstract 3899: Nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake

Abstract Background: One of the major concerns in cancer treatment and clinical translation of many anti-cancer compounds has been their potential toxicities, especially hepatotoxicity. Nanoparticle (NP) holds great potential to solve the problem, as many clinical studies have not shown increased hepatotoxicity for nanotherapeutics with higher accumulation in the liver. However, there are few studies investigating how nanoparticle assists in reducing hepatotoxicity. Herein, we demonstrated that nanoformulation of known hepatotoxic anti-cancer compounds showed lower hepatotoxicity than their small molecule counterparts. Importantly, we demonstrated that slower drug release is associated with reduced hepatotoxicity. We also showed that that Kupffer cell uptake in the liver reduce nanotherapeutics’ hepatotoxicity. Methods: Two different antineoplastic drugs with high hepatotoxicity, SN-38 and wortmannin (Wtmn), were encapsulated into lipid shell-PLGA core nanoparticles separately. NP release kinetics were controlled by adjusting lipids/polymer ratio for fast (Fast-NPs), medium (Medium-NPs) and slow drug release profile (Slow-NPs), respectively. Hepatotoxicity of NPs or free drugs was analyzed by assessing serum ALT and AST levels post intravenous injection at ½ MTD in CD-1 mice. IHC staining of HO-1 and Mn-SOD was also used to show liver damage. In vitro hepatotoxicity of primary hepatocytes after Kupffer cell uptake was assessed by MTS assay and LDH assay. In vivo macrophage depletion was achieved using clodrosome. Results: Nanoformulations of SN-38 and Wtmn showed lower ALT and AST levels than their small molecule counterparts 12 h and 24 h post treatment. The liver damage was further confirmed with IHC staining of Mn-SOD and HO-1. To address the effect of drug release kinetics on hepatotoxicity, we showed that over 90% of drugs were released within 24 h in Fast-NP. While the release rate of Slow-NPs was 66.8% for SN-38 and 67.5% for Wtmn. We showed that Slow-NPs of Wtmn resulted in minimal increase of ALT and AST levels. Compared to the baseline in untreated mice, the ALT level was 2.3-fold for Slow-NPs, 4.4-fold for Medium-NPs and 6-fold for fast-NPs; meanwhile, the AST level was 1.6-fold, 2.5-fold and 3.6-fold, respectively. To assess the effect of Kupffer cell uptake in hepatotoxicity, we found that the toxicity of the nanotherapeutics for primary hepatocytes significantly reduced after Kupffer cell uptake in vitro. We further confirmed it in vivo by depleting Kupffer cells in CD-1 mice. We demonstrated that ALT and AST levels of nanotherapeutics significantly increased to the levels comparable to free drugs after Kupffer cell depletion. Conclusions: We demonstrate that nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake. Our work bridges an important knowledge in nanoparticle drug delivery and clinical translation of nanomedicine. Citation Format: Yu Mi, Feifei Yang, Andrew Z. Wang. Nanoparticle reduces hepatotoxicity of cancer treatment by controlled release and Kupffer cell uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3899.

Deciphering liver environmental signaling pathways for Kupffer cell identity

Abstract Functional specialization of tissue resident macrophages occurs through environmental signals controlling activity and/or expression of transcription factors. Kupffer cells are resident macrophages in the hepatic sinusoids and have critical roles in the innate immune response and iron metabolism. Here, we characterize transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. Nr1h3 encoding LXRα is rapidly and highly induced in repopulating liver macrophages, suggesting its induction plays a crucial role in Kupffer cell differentiation. Restricted deletion of Nr1h3 in Kupffer cells reveal that it is required for shaping the Kupffer cell-specific enhancer landscape. Further, we obtain evidence that combinatorial interactions of DLL4 and TGF-β/BMP produced by sinusoidal endothelial cells and endogenous LXR ligands are required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of RBPJ through Notch signaling plays a key role in activating poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogram the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Using molecules which mimic these liver environment signals, we show that it is possible to induce Kupffer cell-specific genes in mouse bone marrow progenitor cells and human monocytes in vitro. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

The gut microbiota metabolite indole alleviates liver inflammation in mice.

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p-cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision-cut liver slices prepared from control mice, Kupffer cell (KC)-depleted mice, and obese mice (ob/ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose-dependently reduced the LPS-induced up-regulation of proinflammatory mediators at both mRNA and protein levels in precision-cut liver slices prepared from control or ob/ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF-κB pathway and downstream proinflammatory gene up-regulation. Indole also prevented LPS-induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4β-hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.—Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.-B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice.

Kupffer cell depletion by gadolinium chloride aggravates liver injury after brain death in rats.

Brain death (BD) impairs liver function in potential donors, and is associated with hormonal and metabolic changes or molecular effects with pro-inflammatory activation. Resident macrophages in the liver named Kupffer cells (KCs) undergo pro- or anti-inflammatory pathway activation, which affects liver function. However, the role of the KCs in liver dysfunction following BD has not been fully elucidated. The aim of the present study was to investigate the role of KCs in liver dysfunction in the context of BD and the effects of their inhibition by gadolinium chloride (GdCl3). Rats were randomly divided into the following groups: Control, BD with GdCl3 pretreatment and BD with normal saline pretreatment. Liver function, hepatic pathological histology and cytokine levels in the liver were assessed. Apoptosis and apoptosis-related proteins [cleaved caspase-3, caspase-3 and apoptosis regulator Bcl-2 (Bcl-2)] were evaluated. GdCl3 significantly aggravated liver injury by elevating alanine aminotransferase and aspartate aminotransferase levels (P<0.05) by inhibiting KCs. Interleukin (IL)-1β and tumor necrosis factor α levels in the GdCl3 group were significantly increased compared with those in the control and saline groups (P<0.01). However, IL-10 levels in the GdCl3 group were significantly reduced compared with those in the saline group (P<0.05). Caspase-3 and cleaved caspase-3 activation, and apoptosis induction in the context of BD were also significantly aggravated by the depletion of KCs, whereas Bcl-2 was significantly suppressed by the administration of GdCl3. The present study indicated that GdCl3 efficiently inhibits the activity of KCs, and is involved in the onset of liver injury through its effects on pro-inflammatory and anti-inflammatory activation. KCs are protective in the liver in the context of BD. This protection appears to be due to KCs secretion of the potent anti-inflammatory cytokine IL-10, suggesting that KCs are an attractive target for the prevention and treatment of liver injury in the context of BD in rats.

Contribution of Kupffer cells to liposome accumulation in the liver

The liver is a major barrier for site-specific delivery of systemically injected nanoparticles, as up to 90% of the dose is usually captured by this organ. Kupffer cells are thought to be the main cellular component responsible for nanoparticle accumulation in the liver. These resident macrophages form part of the mononuclear phagocyte system, which recognizes and engulfs foreign bodies in the circulatory system. In this study, we have compared two strategies for reducing nanoparticle accumulation in the liver, in order to investigate the specific contribution of Kupffer cells. Specifically, we have performed a comparison of the capability of pegylation and Kupffer cell depletion to reduce liposome accumulation in the liver. Pegylation reduces nanoparticle interactions with all types of cells and can serve as a control for elucidating the role of specific cell populations in liver accumulation. The results indicate that liposome pegylation is a more effective strategy for avoiding liver uptake compared to depletion of Kupffer cells, suggesting that nanoparticle interactions with other cells in the liver may also play a contributing role. This study highlights the need for a more complete understanding of factors that mediate nanoparticle accumulation in the liver and for the exploration of microenvironmental modulation strategies for reducing nanoparticle-cell interactions in this organ.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Autophagy-deficient Kupffer cells promote tumorigenesis by enhancing mtROS-NF-κB-IL1α/β-dependent inflammation and fibrosis during the preneoplastic stage of hepatocarcinogenesis

As a cellular degradation mechanism, autophagy exerts crucial and complicated effects on HCC development. Liver non-parenchymal cells, including hepatic resident macrophage Kupffer cells, also play important roles in this process. However, most associated studies have focused on the influence of the autophagy level in hepatic cells and HCC cells, but not liver non-parenchymal cells. Based on our previous study, we confirmed that Atg5 silence in the liver during the preneoplastic stage facilitated liver fibrosis, inflammation and, ultimately, tumorigenesis. We further found that autophagy deficiency promotes the production of inflammatory and fibrogenic factors in macrophages. Moreover, Kupffer cell depletion rescued the tumor-promoting effect of autophagy deficiency during the preneoplastic stage. In autophagy-deficient macrophages, mitochondrial ROS mediated inflammation- and fibrosis-promoting effects by increasing IL1α/β production via enhancing NF-κB-associated pathways. Both blocking of mitochondrial ROS and blocking the IL1 receptor stopped the promotion of fibrosis, inflammation and tumorigenesis resulting from Atg5 knockdown during the preneoplastic stage. In conclusion, autophagy-deficient Kupffer cells promote liver fibrosis, inflammation and, finally, hepatocarcinogenesis during the preneoplastic stage by enhancing mitochondrial ROS- NF-κB-IL1α/β pathways.