Polarization Of Kupffer Cells Research Articles

Mechanism of bufalin inhibition of colon cancer liver metastasis by regulating M2-type polarization of Kupffer cells induced by highly metastatic colon cancer cells.

Patients with metastatic colorectal cancer often have poor outcomes, primarily due to hepatic metastasis. Colorectal cancer (CRC) cells have the ability to secrete cytokines and other molecules that can remodel the tumor microenvironment, facilitating the spread of cancer to the liver. Kupffer cells (KCs), which are macrophages in the liver, can be polarized to M2 type, thereby promoting the expression of adhesion molecules that aid in tumor metastasis. Our research has shown that huachanshu (with bufalin as the main active monomer) can effectively inhibit CRC metastasis. However, the underlying mechanism still needs to be thoroughly investigated. We have observed that highly metastatic CRC cells have a greater ability to induce M2-type polarization of Kupffer cells, leading to enhanced metastasis. Interestingly, we have found that inhibiting the expression of IL-6, which is highly expressed in the serum, can reverse this phenomenon. Notably, bufalin has been shown to attenuate the M2-type polarization of Kupffer cells induced by highly metastatic Colorectal cancer (mCRC) cells and down-regulate IL-6 expression, ultimately inhibiting tumor metastasis. In this project, our aim is to study how high mCRC cells induce M2-type polarization and how bufalin, via the SRC-3/IL-6 pathway, can inhibit CRC metastasis. This research will provide a theoretical foundation for understanding the anti-CRC effect of bufalin.

GO-PEG Represses the Progression of Liver Inflammation via Regulating the M1/M2 Polarization of Kupffer Cells.

As a highly promising nanomaterial, exploring the impact of the liver, a vital organ, stands out as a crucial focus in the examination of its biological effects. Kupffer cells (KCs) are one of the first immune cells to contact with exotic-substances in liver. Therefore, this study investigates the immunomodulatory effects and mechanisms of polyethylene glycol-modified graphene oxide (GO-PEG) on KCs. Initial RNA-seq and KEGG pathway analyses reveal the inhibition of the TOLL-like receptor, TNF-α and NOD-like receptor pathways in continually stimulated KCs exposed to GO-PEG. Subsequent biological experiments validate that a 48-hour exposure to GO-PEG alleviates LPS-induced KCs immune activation, characterized by a shift in polarization from M1 to M2. The underlying mechanism involves the absorption of double-stranded RNA/single-stranded RNA, inhibiting the activation of TLR3 and TLR7 in KCs. Employing a Kupffer/AML12 cell co-culture model and animal studies, it is observed that GO-PEG indirectly inhibit oxidative stress, mitochondrial dysfunction, and apoptosis in AML12 cells, partially mitigating systemic inflammation and preserving liver tissue/function. This effect is attributed to the paracrine interaction between KCs and hepatocytes. These findings suggest a meaningful and effective strategy for treating liver inflammation, particularly when combined with anti-inflammatory drugs.

Mesenchymal stem cell therapy modulates macrophage dynamics in ARDS-associated liver injury in rats

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening pulmonary condition characterized by severe hypoxemia and respiratory failure. Beyond its devastating impact on the lungs, ARDS often triggers systemic responses affecting vital organs throughout the body. One such organ commonly affected is the liver, which experiences various degrees of injury during the course of ARDS. Pathophysiological changes in liver during ARDS, particularly polarization of Kupffer cells during the disease and its treatment, have drawn increasing attention. Purpose. To explore the macrophage transformation in liver injury associated with ARDS and investigate the potential of mesenchymal stem cell (MSC) therapy as a means to modulate macrophage responses and mitigate liver injury. Materials and methods. 72 mature male Wistar rats were randomly allocated to nine experimental groups as follows: the control group, groups assessed at 3 days, 7 days, and 28 days following intranasal LPS administration, groups that received 24 hours of LPS followed by 2 days of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), groups exposed to 4 days of LPS and 3 days of hUC-MSCs, groups subjected to 14 days of LPS and 14 days of hUC-MSCs, groups treated with LPS 21 days and 7 days with hUC-MSCs injection, and a control group assessed 3 days after hUC-MSCs injection. For the administration of hUC-MSCs, intraperitoneal injections were performed at a dose of 1∙106 cells/kg body weight. Immunohistochemistry was employed to analyze macrophage subpopulations in liver tissues. Animal experiments adhered to ethical guidelines. Results. Early ARDS stages showed increased M1 macrophages, indicating pro-inflammatory responses, while later stages showed M2 macrophage activation, suggestive of anti-inflammatory and tissue repair roles. MSC administration facilitated the transition from M1 to M2 macrophages, promoting an anti-inflammatory milieu. Conclusions. MSCs demonstrate the potential to modulate macrophage polarization into M2 anti-inflammatory phenotype. Such findings reflect one of the mechanisms of MSC action which holds practical significance for future ARDS therapies, aiming to mitigate excessive inflammation and enhance tissue repair.

ZMIZ1 Upregulation of TET3-Mediated Hydroxymethylation Induces M2 Polarization of Kupffer Cells in Hepatocellular Carcinogenesis by Mediating Notch1/c-Myc Signaling

Hydroxymethylation of DNA, mediated by the ten-eleven translocation (TET) family of methylcytosine dioxygenases, represents a crucial epigenetic modification that manipulates gene expression in numerous biological processes. This study focuses on the effect of TET3 on the polarization of Kupffer cells (KCs) and its connection to the development of hepatocellular carcinoma (HCC). TET3 was found to be abundant in KCs, and its knockdown induced an M2–M1 phenotype shift, resulting in the suppression of viability, migration, and invasion of cocultured HCC cells. Additionally, the TET3 knockdown inhibited the tumorigenesis of HCC cells in nude mice. Downstream targets of TET3 were predicted using bioinformatics. TET3-mediated DNA hydroxymethylation of zinc finger MIZ-type containing 1 (ZMIZ1) promoter. The ZMIZ1 protein interacted with notch receptor 1 (Notch1) protein to activate the transcription of c-Myc. Silencing of ZMIZ1 in KCs similarly suppressed M2 polarization of KCs and malignant phenotype of cocultured HCC cells. However, these changes were counteracted by the overexpression of either Notch1 or c-Myc overexpression in KCs. In summary, this study demonstrates that TET3-mediated hydroxymethylation of ZMIZ1 enhances hepatocellular carcinogenesis by promoting M2 skewing of KCs through the Notch1/c-Myc axis.

HSP110 aggravates ischemia-reperfusion injury after liver transplantation by promoting NF-κB pathway

BackgroundIschemia-reperfusion injury (IRI) poses a significant challenge to liver transplantation (LT). The underlying mechanism primarily involves overactivation of the immune system. Heat shock protein 110 (HSP110) functions as a molecular chaperone that helps stabilize protein structures. MethodsAn IRI model was established by performing LT on Sprague-Dawley rats, and HSP110 was silenced using siRNA. Hematoxylin-eosin staining, TUNEL, immunohistochemistry, ELISA and liver enzyme analysis were performed to assess IRI following LT. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes. ResultsOur findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT (P < 0.05). However, when rats were injected with siRNA-HSP110, IRI subsequent to LT was notably reduced (P < 0.05). Additionally, the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced (P < 0.05). Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver (P < 0.05). ConclusionsHSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells. Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI.

Galectin-12 modulates Kupffer cell polarization to alter the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease is caused by an imbalance in lipid metabolism and immune response to pose a risk factor for liver fibrosis. Recent evidence indicates that M2 macrophages secrete transforming growth factor-β1, which contributes to liver fibrosis. Galectin-12 has been demonstrated to regulate lipid metabolism and macrophage polarization. The purpose of this study is to investigate the role of galectin-12 in the development of nonalcoholic fatty liver disease and fibrosis. Liver tissue from wild-type C57BL/6 mice fed with a high-fat diet containing cholesterol and cholic acid for 4-12weeks was used to examine galectin-12 expression and its correlation with nonalcoholic fatty liver disease. Furthermore, the effects of galectin-12 on M2 macrophages during the progression of nonalcoholic fatty liver disease were investigated by studying Kupffer cells from galectin-12 knockout mice and doxycycline-inducible Gal12-/-THP-1 cells. Ablation of galectin-12 promoted M2 polarization of Kupffer cells, as indicated by higher levels of M2 markers, such as arginase I and chitinase 3-like protein 3. Furthermore, the activation of signal transducer and activator of transcription 6 was significantly higher in Gal12-/- macrophages activated by interleukin-4, which was correlated with higher levels of transforming growth factor-β1. Moreover, Gal12-/- macrophage-conditioned medium promoted hepatic stellate cells myofibroblast differentiation, which was indicated by higher α-smooth muscle actin expression levels compared with those treated with LacZ control medium. Finally, we demonstrated that galectin-12 knockdown negatively regulated the suppressor of cytokine signaling 3 levels. These findings suggested that galectin-12 balances M1/M2 polarization of Kupffer cells to prevent nonalcoholic fatty liver disease progression.

TWEAK promotes inflammatory response in liver fibrosis.

This study aimed to investigate the role and mechanism of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in liver fibrosis. The liver Kupffer cells (KCs) and mononuclear macrophages (J774A.1) were used as the objects of study to induce M1 polarization with LPS/IFN-γ. After TWEAK intervention, the M1 cell proportion and marker cytokine levels were detected. Thereafter, CD266 expression was silenced, and NLRP3 expression was inhibited by the NLRP3 inhibitor, so as to investigate the impact of TWEAK on M1 polarization of KCs. In addition, the mouse model of liver fibrosis was constructed to observe the influence of TWEAK on mouse liver fibrosis. According to our results, TWEAK promoted M1 polarization of liver KCs and J774A.1 cells, and silencing CD266 expression or treatment with the NLRP3 inhibitor suppressed the effect of TWEAK. In the mouse experiment, it was discovered that after knocking down NLRP3 expression or using NLRP3 inhibitor to antagonize the effect of TWEAK, the mouse liver function and M1 cell level in liver tissues were improved.

PM2.5 exposure aggravates acute liver injury by creating an inflammatory microenvironment through Kupffer cell

AimThis work aimed to investigate the impact of PM2.5 exposure on acute liver injury MethodsC57BL/6 mice were used to examine the hepatic histopathological changes in PM2.5-exposed mice, as well as in CCl4-mediated acute liver injury mice after long-term exposure to PM2.5. During in vitro experiments, Kupffer cells were detected for M1 polarization level after treating with PM2.5, and the activation level of NLRP3 inflammasomes were assessed. ResultsAccording to our findings, PM2.5 can induce M1 polarization of Kupffer cells in the liver to create an inflammatory microenvironment. Long-term exposure to PM2.5 can aggravate acute liver injury in mice. Treatment with MCC950, an NLRP3 inhibitor, can inhibit the effect of PM2.5. As demonstrated by in vitro analysis, PM2.5 can promote M1 polarization of Kupffer cells. ConclusionAs suggested by our results, long-term exposure to PM2.5 can create an inflammatory microenvironment to aggravate mouse acute liver injury. The effect is related to NLRP3-mediated M1 polarization in Kupffer cells.

The role of Kupffer cell activation in immune liver damage induced by trichloroethylene associated with the IFN-γ/STAT1 signaling pathway.

Trichloroethylene (TCE) is a metal detergent commonly used in industry that can enter the human body through the respiratory tract and skin, causing occupational medicamentosa-like dermatitis due to TCE (OMDT) and multiple organ damage, including liver failure. However, the pathogenesis of liver injury remains unclear. Kupffer cells (KCs) are important tissue macrophages in the body because the polarization of KCs plays a crucial role in immune-mediated liver injury. However, the mechanism of KCs polarization in TCE-induced immune liver injury has not been thoroughly elucidated. In this study, we investigated the effect of TCE-induced KCs polarization on liver function and signal transduction pathways using the TCE sensitization model developed by our group. BALB/c mouse skin was exposed to TCE for sensitization, and an increase in the expression of M1 macrophage-specific markers (CD16/CD32, iNOS), M1 macrophage-specific cytokines IL-1β, and IFN-γ, P-JAK-1 and P-STAT1 levels were also found to be dramatically increased. When using low doses of gadolinium trichloride (GdCl3), the expression of these proteins and mRNA was significantly reduced. This phenomenon indicates that GdCl3 blocks TCE-induced polarization of KCs and suggests that the IFN-γ/STAT1 signaling pathway may be involved in the polarization process of KCs. These findings clarify the relationship between the polarization of KCs and immune liver injury and highlight the importance of further study of immune-mediated liver injury in TCE-sensitized mice.

Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

Hepatocyte-derived extracellular vesicles miR-122-5p promotes hepatic ischemia reperfusion injury by regulating Kupffer cell polarization.

Ischemia reperfusion injury remains a major barrier to liver transplantation, especially using grafts from donation after circulatory death, and it is also a pressing issue to be solved in clinical practice. Kupffer cell polarization toward a proinflammatory M1 phenotype is an early trigger of liver ischemia-reperfusion injury. However, the molecular mechanism regulating Kupffer cell polarization has not yet been fully elucidated. We induced liver ischemia reperfusion injury in mice and obtained samples from patients undergoing liver transplantation, serum and hepatocytes-derived extracellular vesicles were isolated by differential ultracentrifugation. Kupffer cell polarization was examined by flow cytometry and immunofluorescence histochemistry. RNA-seq was conducted to detect the differentially expressed miRNAs in extracellular vesicles. The role and mechanism of exosomal miR-122-5p in liver ischemia-reperfusion injury were determined both in vitro and in vivo. We identified ischemia reperfusion induced extracellular vesicles as a major cause of hepatic inflammation and tissue damage using adoptive transfer and release inhibition. The study also demonstrated that hepatocyte-derived exosomal miR-122-5p mediates liver ischemia reperfusion injury by polarizing Kupffer cell via PPARδ down-regulation and NF-κB pathway activation using profiling and functional analysis. Moreover, inhibiting miR-122-5p with antagomir suppressed Kupffer cell M1 polarization and attenuated liver ischemia reperfusion injury. Overall, our study demonstrated that hepatocyte-derived exosomal miR-122-5p played a critical role in promoting hepatic ischemia reperfusion injury through modulating PPARδ signaling and NF-κB pathway to introduce M1 polarization of Kupffer cell. Inhibition of miR-122-5p exhibited a protective effect against liver ischemia reperfusion injury, suggesting a potential therapeutic target for liver transplantation.

MSCs Ameliorate Hepatic IR Injury by Modulating Phenotypic Transformation of Kupffer Cells Through Drp-1 Dependent Mitochondrial Dynamics.

Hepatic ischemia and reperfusion (IR) injury, characterized by reactive oxygen species (ROS) production and immune disorders, leads to exogenous antigen-independent local inflammation and hepatocellular death. Mesenchymal stem cells (MSCs) have been shown to be immunomodulatory, antioxidative and contribute to liver regeneration in fulminant hepatic failure. We aimed to investigate the underlying mechanisms by which MSCs protect against liver IR injury in a mouse model. MSCs suspension was injected 30min prior to hepatic warm IR. Primary kupffer cells (KCs) were isolated. Hepatic injury, inflammatory responses, innate immunity, KCs phenotypic polarization and mitochondrial dynamics were evaluated with or without KCs Drp-1 overexpression RESULTS: MSCs markedly ameliorated liver injury and attenuated inflammatory responses and innate immunity after liver IR injury. MSCs significantly restrained M1 phenotypic polarization but boosted M2 polarization of KCs extracted from ischemic liver, as demonstrated by lowered transcript levels of iNOS and IL-1β but raised transcript levels of Mrc-1 and Arg-1 combined with p-STAT6 up-regulation and p-STAT1 down-regulation. Moreover, MSCs inhibited KCs mitochondrial fission, as evidenced by decreased Drp1 and Dnm2 levels. We overexpressed Drp-1 in KCs which promote mitochondrial fission during IR injury. the regulation of MSCs towards KCs M1/M2 polarization was abrogated by Drp-1 overexpression after IR injury. Ultimately, in vivo Drp-1 overexpression in KCs hampered the therapeutic effects of MSCs against hepatic IR injury CONCLUSIONS: We revealed that MSCs facilitated M1-M2 phenotypic polarization through inhibiting Drp-1 dependent mitochondrial fission and further attenuated liver IR injury. These results add a new insight into regulating mechanisms of mitochondrial dynamics during hepatic IR injury and may offer novel opportunities for developing therapeutic targets to combat hepatic IR injury.

Lapachol treats non-alcoholic fatty liver disease by modulating the M1 polarization of Kupffer cells via PKM2

AimThis study investigated the mechanism of action of lapachol (LAP) against non-alcoholic fatty liver disease (NAFLD). MethodsPrimary Kupffer cells (KCs) of rats were used for in-vitro experiments. The proportion of M1 cells was assayed by flow cytometry, the levels of M1 inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA) combined with real-time quantitative fluorescence PCR (RT-qPCR), the expression of p-PKM2 was detected by Western-Blotting. A SD rat model of NAFLD was established with high-fat diet. Following LAP intervention, the changes in blood glucose/lipid, insulin resistance and liver function were detected, and the hepatic histopathologic changes were examined by histological staining. ResultsThe results showed that LAP could inhibit the M1 polarization of KCs, lower the levels of inflammatory cytokines, and suppress the activation of PKM2. The effect of LAP could be counteracted after using PKM2 inhibitor PKM2-IN-1 or knocking out PKM2. Small molecule docking revealed that LAP could inhibit the phosphorylation process of PKM2 by binding to ARG-246, the phosphorylation site of PKM2. In rat experiments, LAP could ameliorate the liver function and lipid metabolism of NAFLD rats, and inhibit the hepatic histopathologic changes. ConclusionOur study found that LAP can inhibit the phosphorylation of PKM2 by binding to PKM2-ARG-246, thereby regulating the M1 polarization of KCs and inhibiting the inflammatory response of liver tissues to treat NAFLD. LAP has potential as a novel pharmaceutical for treating NAFLD.

Abstract 3808: MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs

Abstract Kupffer cells (KCs) account for 15% of the total liver cells. However, their roles in hepatocellular carcinoma (HCC) is poorly described. Clec4fCre-tdTomato knock-in mice expressing Cre recombinase and tdTomato-NLS under the KC-specific Clec4f promoter were hydrodynamically injected with oncogenic AKT and Nras (AKT/Ras) to induce HCC. A LoxP-stop-LoxP (LSL) system was used to express miR-206 in KCs of mice. AKT activation was observed in hepatocytes and KCs of HCC patients. Hydrodynamic injection (HDI) of AKT/Ras led to activation of AKT signaling in KCs and hepatocytes. AKT/Ras mice developed lethal HCC within 6-8 weeks post injection. Activation of AKT signaling led to M2 polarization of KCs, increased hepatic Tregs and exhaustion of CTLs. MicroRNA (miR) profiling revealed that AKT/Ras impaired miR-206 biogenesis in KCs by driving translation of Yin Yang 1 (YY1), a transcription repressor of miR-206. HDI of miR-206 into AKT/Ras mice fully prevented HCC, while all control mice died of HCC 6-8 weeks post injection. However, HDI of miR-206 only transfected ~ 20% of hepatocytes, indicating that it is unreasonable to conclude that the full prevention of HCC in AKT/Ras/miR-206 mice is caused by inhibited proliferation of HCC cells. We, therefore, posited that miR-206-mediated recovery of immune surveillance, at least in part, accounted for the full prevention of HCC in AKT/Ras/miR-206 mice. We next used the LSL system to overexpress miR-206 in KCs of Clec4fCretdTomato mice injected with AKT/Ras. KC-specific expression of miR-206 fully prevented HCC in AKT/Ras mice, while 100% control mice died of HCC. Mechanistically, AKT signaling in KCs drove CCL22 (C-C motif chemokine ligand 22) production by phosphorylating β-catenin that activates transcription of Ccl22. In contrast, miR-206 disrupted β-catenin signaling by targeting LEF1 (lymphoid enhancer-binding factor 1), an “architectural” transcription factor of β-catenin. CCL22 recruits Tregs. Indeed, KC-specific expression of miR-206 led to elevated CCL22, M2 to M1 transition of KCs, reduced hepatic Tregs, and elevated CTLs. Depletion of CTLs in AKT/Ras mice fully offset the ability of miR-206 to prevent HCC. Conclusions: AKT-educated KCs initiate Treg recruitment via a regulatory loop consisting of YY1, β-catenin/LEF1, miR-206, and CCL22; once this loop is activated, AKT activates β-catenin axis, which drives CCL22 production, Treg recruitment and HCC growth; while KC-specific expression of miR-206 reversed this process by blocking β-catenin axis in KCs. miR-206 represents a novel immunotherapy against HCC. Citation Format: Guisheng Song, Ningning Liu. MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3808.

MiR-27a-5p inhibits acute rejection of liver transplantation in rats by inducing M2 polarization of Kupffer cells through the PI3K/Akt pathway

Liver transplantation (LT), a major therapy for end-stage liver disease, is often associated with acute rejection (AR). MicroRNAs (miRNAs) have been implicated in AR-related gene regulation. In this experiment, the mechanism of miR-27a-5p in AR of LT was studied. Allotransplantation model (LEW-BN) and syngeneic transplantation model (LEW-LEW) of rat orthotopic liver transplantation (OLT) were established. miR-27a-5p was overexpressed in recipient rats 28 days before LT to detect its effects on LT pathology, liver function, and survival time. Kupffer cells (KCs) were isolated and treated with lipopolysaccharide (LPS) and miR-27a-5p overexpression. miR-27a-5p overexpression reduced lymphocyte numbers around portal areas and central veins after LT and mitigated degeneration of epithelial cells of the bile duct. Expression levels of IL-10 and TGF-β1 were increased while IL-12 was decreased. Liver function damage was alleviated and the survival time of rats with LT was prolonged. miR-27a-5p induced M2 polarization of rats with AR after LT and LPS-treated KCs in vitro and promoted activation of the PI3K/Akt pathway in KCs. Inhibition of the PI3K/Akt pathway averted induction of miR-27a-5p on M2 polarization of KCs. Taken together, miR-27a-5p inhibited AR after LT in rats by inducing M2 polarization of KCs through the PI3K/Akt pathway.

Extracellular vesicles from bone marrow mesenchymal stem cells alleviate acute rejection injury after liver transplantation by carrying miR-22-3p and inducing M2 polarization of Kupffer cells.

Acute rejection (AR) is a major problem following liver transplantation. Extracellular vesicles (EVs) are involved in various pathological processes including liver disease. The present study investigated the effect of EVs derived from bone marrow mesenchymal stem cells (BMSCs) on AR injury after orthotopic liver transplantation (OLT) in mice. BMSCs and EVs were isolated and identified. The OLT mouse model was established using Kamada's two-cuff method and injection with EVs, followed by liver function detection and measurement of inflammatory cytokines (interleukin-10, interferon-γ, and tumor necrosis factor-α), M1 and M2 markers (tumor necrosis factor-α, inducible nitric oxide synthase, resistin like alpha, and Arg1) were detected. Kupffer cells (KCs) were cultured and treated with lipopolysaccharides. miR-22-3p expression was detected. The effect of EVs-shuttled miR-22-3p on Kupffer cell polarization was studied. Binding relation of miR-22-3p and interferon regulatory factor 8 (IRF8) was verified. The effect of IRF8 on KC polarization was verified. BMSC-EV treatment enhanced liver function of OLT mice and alleviated AR and apoptosis, which were annulled after removing KCs. EVs induced KC M2 polarization. Mechanically, EVs carried miR-22-3p into KCs, upregulated miR-22-3p in KCs, and inhibited IRF8 expression. Upregulation of IRF8 in KCs inhibited EV-induced KC M2 polarization. BMSCs-EVs carry miR-22-3p into KCs and upregulate miR-22-3p, inhibit IRF8 expression, induce KC M2 polarization, and attenuate AR injury after liver transplantation.

The effect of miR-155-5p on M1 polarization of Kupffer cells and immune response during liver transplantation through regulating the expression of KDM5D.

To explore the effect and its specific mechanism of miR-155-5p on M1 polarization of Kupffer cells (KCs) and immune response in liver transplantation (LT) through KDM5D. Primary KCs were isolated from Wistar rats and identified by cell culture, ink-swallowing test and flow cytometry. The cells identified as KCs were induced into LT acute rejection (AR) model cells by LPS/IFN-γ, flow cytometry was used for cell sorting and apoptosis detection. Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the levels of inflammatory factors, macrophages and liver function markers. RT-qPCR detected the expression of miR-155-5p and KDM5D mRNA. The protein expression of KDM5D was detected by Western blot. Dual luciferase reporter gene experiment verified the targeting relationship between miR-155-5p and KDM5D. The separated KCs adhered after being cultured for 24h, had pseudopodia and phagocytosis, and the proportion of F4/80 positive cells was more than 90%. The expression of miR-155-5p was increased in LPS/IFN-γ-induced KCs. And knockdown of miR-155-5p inhibited H3K4me3 and H3K27me3 of TNF-α promoter, M1 polarization of KCs and the immune response of AR model cells by upregulating KDM5D. In animal experiments, knockdown of miR-155-5p was found to inhibit liver damage and immune response in rats with allogeneic orthotopic LT. These results confirmed that miR-155-5p inhibited M1 polarization of KCs induced by LPS/IFN-γ, thereby alleviating AR and liver function impairment after LT by upregulating KDM5D.

CoQ10 alleviates hepatic ischemia reperfusion injury via inhibiting NLRP3 activity and promoting Tregs infiltration.

Hepatic ischemia-reperfusion injury (IRI) has been concerned as a main complication of liver surgery and transplantation. Previous studies show that reactive oxygen species (ROS) associated inflammation response and contribute to the liver damage during IRI. Coenzyme Q10 (CoQ10) has shown many beneficial effects on abrogating ROS production and ameliorating liver injury. This study found lower CoQ10 level in the process of liver IRI in a mouse model of hepatic IRI. Meanwhile, our results showed that CoQ10 administration significantly attenuate hepatic IRI proved by HE staining, serum ALT/AST. The NOD-like receptor protein 3 (NLRP3) inflammasome is activated by ROS which triggers the activation of inflammatory caspases. In this study, NLRP3 was significantly suppressed by CoQ10 while Foxp3 exhibited increased expression in liver. Furthermore, Kupffer cells (KCs) pretreated with CoQ10 under the condition of hypoxia and reoxygenation contributed to improved CD4+CD25+Foxp3+ regulatory T cells (Tregs) ratio in co-culture system. Furthermore, NLRP3 inflammasome activator treatment in vivo resulted in higher expression of caspase-1 and NLRP3 and reduction of Tregs in liver, which reversed the protection of CoQ10 in the liver injury. Taken together, our study discovered that CoQ10 can suppress NLRP3 activity in KCs and improves Foxp3+ Tregs differentiation depending on M2 macrophage polarization of KCs to ameliorate hepatic IRI.

Exosomal RBP4 potentiated hepatic lipid accumulation and inflammation in high-fat-diet-fed mice by promoting M1 polarization of Kupffer cells.

Exosomes containing various biological cargoes have potential to be novel diagnostic biomarkers for metabolic diseases. In this study, retinol-binding protein 4 (RBP4) was found to be enriched in serum exosomes, and its increased levels could be considered as an independent risk factor for the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Exosomal RBP4 (exo-RBP4), primarily derived from hepatocytes, significantly enhanced the M1-like polarization of Kupffer cells (KCs) via promoting the activation of NOX2 and NF-κB and reactive oxygen species (ROS) accumulation, resulting in the over-production of inflammatory cytokines including TNF-α. Subsequently, those excess cytokines remarkably increased the levels of intracellular free fatty acid uptake and lipogenesis-related genes (FAS and SREBP-1c) but decreased fatty acid degradation-related genes (CPT-1 and PPARα) in palmitic acid-treated LO2 cells. More notably, TNF-α significantly elevated RBP4 transcription by activating STAT3 in hepatocytes, playing a positive role in NAFLD development. Intravenous injection with RBP4 (50μg/kg) potentiated hepatic lipid accumulation, M1-type KC proportion, and serum pro-inflammatory cytokine levels in the hepatic tissues of high-fat-diet-fed mice. Collectively, these data indicated that exo-RBP4 converted KCs to M1 subtype by mediating the NOX2/ROS/NF-κB pathway, subsequently promoting de novo lipogenesis in hepatocytes by TNF-α secretion to activate the JAK2/STAT3 signaling pathway. Therefore, this study uncovered a novel intercellular communication between the inflammatory microenvironment and lipid metabolism for fostering NAFLD progression and found the potential of exo-RBP4 as a novel diagnostic biomarker and therapeutic target for NAFLD.

MicroRNA-124 expression in Kupffer cells modulates liver injury by targeting IL-6/STAT3 signaling

MicroRNA-124 (miR-124) is related to liver injury due to chronic hepatitis B (CHB) and hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). However, the mechanism whereby miR-124 regulates liver inflammation remains unknown. In this study, we show that serum miR-124 serves as a compensatory predictive factor for organ failure and the 28-day prognosis of patients with HBV-ACLF. Moreover, within a mouse model of concanavalin A-induced acute liver injury, miR-124 is highly expressed in Kupffer cells. Overexpression of miR-124 significantly decreases interleukin-6 (IL-6) secretion, and relieves pathological liver necrosis to a great extent. Mechanistically, miR-124 directly targets the 3′-untranslated region of signal transducer and activator of transcription 3 (STAT3) and inhibits IL-6/STAT3 signaling, which reduces pro-inflammatory Kupffer cell polarization. Collectively, our findings suggest that miR-124 can potentially serve as a predictive biomarker for HBV-ACLF prognosis and may represent a promising therapeutic target for relieving severe liver injury resulting from cytokine storms.