(1) Background: With new potential drug targets emerging, combination therapies appear attractive to treat non-alcoholic steatohepatitis (NASH) and fibrosis. Chemokine receptor CCR2/5 antagonists can improve fibrosis by reducing monocyte infiltration and altering hepatic macrophage subsets. Fibroblast growth factor 21 (FGF21) may improve NASH by modulating lipid and glucose metabolism. We compared effects of single drug to combination treatment as therapeutic strategies against NASH. (2) Methods: We analyzed serum samples and liver biopsies from 85 nonalcoholic fatty liver disease (NAFLD) patients. A CCR2/5 inhibitor (BMS-687681-02-020) and a pegylated FGF21 agonist (BMS-986171) were tested in male C57BL/6J mice subjected to dietary models of NASH and fibrosis (choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) up to 12 weeks; short- (2w) or long-term (6w) treatment). (3) Results: In NAFLD patients, chemokine CCL2 and FGF21 serum levels correlated with inflammatory serum markers, only CCL2 was significantly associated with advanced liver fibrosis. In rodent NASH, CCR2/5 inhibition significantly reduced circulating Ly6C+ monocytes and hepatic monocyte-derived macrophages, alongside reduced hepatic inflammation and fibrosis. FGF21 agonism decreased body weight, liver triglycerides and histological NASH activity. Combination treatment reflected aspects of both compounds upon short- and long-term application, thereby amplifying beneficial effects on all aspects of steatohepatitis and fibrosis. (4) Conclusions: CCR2/5 inhibition blocks hepatic infiltration of inflammatory monocytes, FGF21 agonism improves obesity-related metabolic disorders. Combined therapy ameliorates steatohepatitis and fibrosis more potently than single drug treatment in rodent NASH, corroborating the therapeutic potential of combining these two approaches in NASH patients.

Hepatocellular carcinoma (HCC) is one of the most severe malignancies with increasing incidence and limited treatment options. Typically, HCC develops during a multistep process involving chronic liver inflammation and liver fibrosis. The latter is characterized by the accumulation of extracellular matrix produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs in an ordered sequence that is highly regulated by cyclins and associated cyclin-dependent kinases (CDKs) such as the Cyclin E1 (CCNE1)/CDK2 kinase complex. In the present study, we examined the role of Cyclin E1 (Ccne1) and Cdk2 genes in HSCs for liver fibrogenesis and hepatocarcinogenesis. To this end, we generated conditional knockout mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1^∆HSC or Cdk2^∆HSC). Ccne1^∆HSC mice showed significantly reduced liver fibrosis formation and attenuated HSC activation in the carbon tetrachloride (CCl₄) model. In a combined model of fibrosis-driven hepatocarcinogenesis, Ccne1^∆HSC mice revealed decreased HSC activation even after long-term observation and substantially reduced tumor load in the liver when compared to wild-type controls. Importantly, the deletion of Cdk2 in HSCs also resulted in attenuated liver fibrosis after chronic CCl₄ treatment. Single-cell RNA sequencing revealed that only a small fraction of HSCs expressed Ccne1/Cdk2 at a distinct time point after CCl₄ treatment. In summary, we provide evidence that Ccne1 expression in a small population of HSCs is sufficient to trigger extensive liver fibrosis and hepatocarcinogenesis in a Cdk2-dependent manner. Thus, HSC-specific targeting of Ccne1 or Cdk2 in patients with liver fibrosis and high risk for HCC development could be therapeutically beneficial.

Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.

Chimeric antigen receptor (CAR) technology represents a groundbreaking advancement in gene therapy. While CAR-T cells are approved for specific types of leukemia, the therapeutic potential of CAR-macrophages remains largely unexplored. Additionally, potentially synergistic effects between CAR-T cells and CAR-macrophages in cancer cell clearance are still elusive. This study aimed to investigate the efficiency and mechanisms of human CAR-T cells and CAR-macrophages for clearing cancer cells using co-culture experiments. We encapsulated a second generation CD19 CAR mRNA or enhanced green fluorescent protein (Egfp) encoding control mRNA into lipid nanoparticles (LNP) using different methods. We transfected human primary T cells or macrophages with these LNP and studied their CAR expression using flow cytometry or confocal microscopy. Killing and phagocytosis assays were conducted to evaluate the impact of CAR-T cells and CAR-macrophages on CD19⁺ NALM6 leukemia and CD19^- control cells. Motility analyses of macrophages, T cells, and cancer cells were done using live-cell imaging. Activated T cells upregulated the low-density lipoprotein receptor (LDLR), resulting in higher CAR surface expression post-transfection than in non-activated T cells. CAR-T cells efficiently induced death of lymphoma cells. CAR-macrophages cleared cancer cells mainly via phagocytosis and CAR mRNA altered the expression of both M1 and M2-associated genes. Interestingly, CAR macrophages also upregulated the Signal regulatory protein alpha (SIRPA), a receptor of the CD47 ligand expressed by cancer cells, and knockdown of SIRPA by siRNA efficiently counteracted this phenomenon. We noted a decrease in the motility of macrophages, T cells, and cancer cells in co-cultures containing CAR-transfected effector cells. Co-culturing CAR-T cells with CAR-macrophages synergistically enhanced tumor cell eradication. Our findings underscore the superior cancer cell-killing capability of CAR-T cells. Additionally, CAR-macrophages play a crucial role in clearing cancer cells through phagocytosis, thereby synergistically supporting the activity of CAR-T cells.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1 ^−/− mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1 -specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1 ^−/− mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4 ⁺ and CD8 ⁺ lymphoid cells. Following concanavalin A administration, Ccne1 ^−/− mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8 ⁺ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1 ^−/− macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-κB1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-κB-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.