OBJECTIVE:In the present study, we investigated the role of the gut-liver crosstalk in the pathogenesis of steatotic liver disease (SLD) induced by the compounding and deleterious effects of alcohol and metabolic risk factors, and explored the potential translational aspects of microbiome-based interventions. DESIGN:The effects of combined exposure to alcohol and a high-fat, high-cholesterol diet (HFHC) Western diet (WD) were tested in a dietary mouse DUAL model and compared to mice fed only with WD. Liver and gut phenotypes were evaluated via histochemistry, flow cytometry, gene expression, proteomic, and metabolomic analyses. The effects on the gut microbiota were studied in both DUAL mice and MASLD patients with a history of alcohol consumption. Antibiotic-induced microbiota depletion (AIMD) and microbiota modulation therapies (probiotics and fecal microbiota transplant (FMT)) were performed in mice. Primary human hepatocytes and HepG2 cells were used to study the underlying mechanisms. Zebrafish larvae exposed to alcohol and a HFHC diet were used as a validation model. RESULTS:Alcohol in combination with WD synergistically exacerbated SLD. DUAL-diet-induced disruption of the intestinal barrier led to LPS leakage into the bloodstream and subsequent TLR4-mediated hepatic inflammation. This, together with enhanced intestinal fat absorption, and impaired intrahepatic lipid oxidation - particularly due to insufficient CPT-1 activity - contributed to prominent steatohepatitis. The DUAL-induced changes in the gut microbiota showed similarities to human dysbiosis in MASLD patients who consumed alcohol, including an increase in Bacteroides and Alistipes. AIMD improved pathology, indicating a causal role of the microbiota in the pathophysiology of DUAL steatohepatitis, whilst early microbiome modulation via FMT induced mild improvements in liver and gut physiology. CONCLUSIONS:These results indicated that the microbiota‒gut‒liver axis plays a crucial role in the progression of SLD intensified by alcohol and concurrent metabolic risk factors, thus providing a promising translational target for potential therapeutic interventions.

Introduction 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. Objectives 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. Methods 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. Results 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. Conclusion 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.

The prevalence of metabolic dysfunction-associated steatohepatitis (MASH) is increasing, urging more research into the underlying mechanisms. MicroRNA-26b ( Mir26b ) might play a role in several MASH-related pathways. Therefore, we aimed to determine the role of Mir26b in MASH and its therapeutic potential using Mir26b mimic-loaded lipid nanoparticles (LNPs). Apoe -/- Mir26b -/- , Apoe -/- Lyz2 cre Mir26b fl/fl mice, and respective controls were fed a Western-type diet to induce MASH. Plasma and liver samples were characterized regarding lipid metabolism, hepatic inflammation, and fibrosis. Additionally, Mir26b mimic-loaded LNPs were injected in Apoe -/- Mir26b -/- mice to rescue the phenotype and key results were validated in human precision-cut liver slices. Finally, kinase profiling was used to elucidate underlying mechanisms. Apoe -/- Mir26b -/- mice showed increased hepatic lipid levels, coinciding with increased expression of scavenger receptor a and platelet glycoprotein 4. Similar effects were found in mice lacking myeloid-specific Mir26b . Additionally, hepatic TNF and IL-6 levels and amount of infiltrated macrophages were increased in Apoe -/- Mir26b -/- mice. Moreover, Tgfb expression was increased by the Mir26b deficiency, leading to more hepatic fibrosis. A murine treatment model with Mir26b mimic-loaded LNPs reduced hepatic lipids, rescuing the observed phenotype. Kinase profiling identified increased inflammatory signaling upon Mir26b deficiency, which was rescued by LNP treatment. Finally, Mir26b mimic-loaded LNPs also reduced inflammation in human precision-cut liver slices. Overall, our study demonstrates that the detrimental effects of Mir26b deficiency in MASH can be rescued by LNP treatment. This novel discovery leads to more insight into MASH development, opening doors to potential new treatment options using LNP technology.

Achieving remote control of biological processes remains a significant challenge in genetics. Although ultrasound has been employed to remotely regulate biological functions by targeting mechanosensitive ion channels, existing systems are constrained by the limited responsiveness of specific channels to specific ultrasound frequencies and their applicability to only a few cell types. Sonogenetics has shown promise for promoter control, thereby regulating gene transcription in eukaryotes. Here, we introduce a new modular toolkit for regulating gene expression using ultrasound-responsive RNA carriers capable of releasing small molecule modulators in response to a broad spectrum of ultrasound frequencies. The cells contain engineered mRNA structures encoding riboswitches or aptazymes, which respond specifically to these small molecule modulators finally controlling downstream protein expression by biocompatible ultrasound. This toolkit is versatile, functioning across various eukaryotic systems-from yeast to mammalian cells-and offers control over gene expression by regulating mRNA translation. We demonstrated that this sonogenetic toolkit robustly modulates gene expression, achieving up to a six-fold downregulation of protein levels in response to ultrasound stimulation. By expanding the application of sonogenetics across eukaryotes, this RNA-based toolkit might provide a promising platform for remotely controlling protein function in specific tissues through on-demand ultrasound activation in the future.

Background and Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is associated with c-Jun N-terminal kinases (JNK) activation across various cell types, but its hepatocyte-specific function in steatotic liver disease remains unclear. Our study investigates the role of JNK1/JNK2 during MASH progression and dissect its hepatocyte-specific function. Approach and Results: We showed that UK biobank patients with a predicted-loss-of-function (pLOF) variant of JNK1 presented an increased prevalence of MASLD and liver damage. Analysis of a pathology cohort of patients with steatotic liver disease revealed increased oxidative stress response and apoptosis. After subjecting mice deficient for Jnk1 and Jnk2 in hepatocytes ( Jnk1/2 Δhepa ) to two different MASH models, we observed enhanced liver injury, fibrosis and oxidative stress. RNA sequencing revealed highly up-regulated pathways in Jnk1/2 Δhepa livers, including inflammatory signals and apoptotic pathways. Additional blocking of Caspase 8 signaling improved HFD-induced liver damage, fibrogenesis and oxidative stress. Ultimately, a therapeutic approach using lipid nanoparticles containing small interfering RNA (siRNA) targeting Caspase 8 during MASH progression attenuated liver injury and cell death in mice. Conclusions: Our findings define a protective role of JNK1/JNK2 in hepatocytes during the oxidative stress response driving progression of MASH. This process is mainly mediated by Caspase 8-dependent apoptosis, thereby discovering that Caspase 8 is a downstream target of JNK1/2. Caspase 8 directed therapy in hepatocytes might be a promising treatment for patients with an increased oxidative stress response and MASH.

BACKGROUND:Atherosclerosis is a leading cause of cardiovascular diseases, and microRNA-26b (miR-26b) has emerged as a significant regulator in its development. This study investigates the role of nonhematopoietic miR-26b in atherosclerosis.METHODS:To study the specific role of nonhematopoietic cell miR-26b in atherosclerosis development, we used a reverse bone marrow transplantation model combined with 12-week Western-type diet feeding.RESULTS:Nonhematopoietic-specific miR-26b deficiency exacerbated atherosclerosis, characterized by larger plaques with increased collagen and necrotic core content. Enhanced VCAM-1 (vascular cell adhesion molecule 1) expression correlated with elevated leukocyte adhesion in ex vivo perfusion studies. Restoration of miR-26b levels in human coronary artery endothelial cells reduced inflammatory responses and leukocyte adhesion.CONCLUSIONS:Our findings highlight that nonhematopoietic miR-26b plays a protective role in atherosclerosis by modulating endothelial cell function, suggesting potential therapeutic applications for miR-26b mimics in cardiovascular disease management.

AIMS:Increasing evidence has shown that microRNAs (miRs) play a fundamental role in atherosclerosis, but the exact role of various miRs remains elusive. Preliminary data showed that, with a five-fold increase, miR-26b was highly expressed in human atherosclerotic plaques compared to healthy vessels. Therefore, we aimed to determine its cell-specific effects on atherosclerosis development and its therapeutic potential. METHODS AND RESULTS:We examined the role of miR-26b in atherosclerosis by using whole-body Apoe-/-Mir26b-/- and myeloid cell-specific miR-26b-deficient (LysM-Cre) mice on a Western-type diet (WTD). Atherosclerotic plaque size and phenotype, as well as the phenotype and function of bone marrow-derived macrophages (BMDMs) from Apoe-/-Mir26b-/- mice, were investigated. Lipid nanoparticles (LNPs) served as vehicles for miR-26b mimics to restore miR-26b levels in miR-26b-deficient BMDMs in vitro and in mice in vivo. Apoe-/-Mir26b-/- mice have a striking 2.8-fold increase in atherosclerotic lesion size in the aortic arch after 12-week WTD, compared to control Apoe-/-, while lesions in the aortic root were unaffected. Consistent with a more advanced plaque phenotype, collagen, smooth muscle cell, and necrotic core content were all significantly increased in plaques from Apoe-/-Mir26b-/- mice, whilst the relative macrophage content was significantly reduced. This phenotype could also be observed in Apoe-/-Mir26b-/- mice after 4-week WTD. Intriguingly, relative plaque size in the arches of Apoe-/-LysmCre+Mir26bfl/fl mice was increased by 2.5-fold, suggesting a role for myeloid-specific miR-26b in atherosclerosis development. Further highlighting its myeloid-specific effects, Apoe-/-Mir26b-/- BMDMs showed an increase in pro-inflammatory cytokine secretion, which could be rescued by LNPs containing miR-26b mimics. MiR-26b pull-down analysis revealed AnnexinA2 as one of the novel targets playing a key role in these effects, which could be validated in BMDMs in vitro. Furthermore, in vivo treatment of Apoe-/-Mir26b-/- mice as well as ex vivo treatment of human plaques with miR-26b-mimic-loaded LNPs demonstrated their therapeutic potential and human relevance, respectively. CONCLUSION:Overall, our results clearly demonstrate an atheroprotective role of miR-26b by attenuating lesion formation, mainly by suppressing inflammation and stimulating collagen breakdown. Furthermore, the therapeutic potential of miR-26b mimic-loaded LNPs could be proven, opening up new avenues for miRNA-based treatment options in the future.

Background: The N6-methyladenosine (m6A) modification of eukaryotic mRNA is the most prevalent of such epigenetic modifications and has recently been identified as a potential player in the pathogenesis and progression of hepatocellular carcinoma (HCC). With the increasing emergence of immunotherapy in the treatment of HCC, we have evaluated the potential of m6A-related genes in predicting overall survival and the therapeutic efficacy of immunotherapy in HCC patients. Methods: We employed transcriptomic data from TCGA-LIHC and GSE76427, comprising a total of 485 HCC patients, as the training set. Based on 23 recognized m6A regulators, we performed clustering analysis on HCC patients. The intersecting differentially expressed genes (DEGs) among subtypes were used in least absolute shrinkage and selection operator (LASSO) Cox and multivariate Cox regression analyses to construct the risk model. For the quantification of a risk model of HCC patients, a risk score was developed and correlated with clinical and immunological parameters. Furthermore, a single-cell transcriptomic atlas was used to analyze the relationship between model genes and immune cell subpopulations. Mechanistic studies included in vitro assays to validate the association between the m6A-related gene ANLN and the progression of HCC. Results: Internal (TCGA and GEO) and external validation (ICGC) suggested that an 8-gene risk score provides an accurate and stable prognostic assessment for HCC. Furthermore, the high-risk score, characterized by elevated TP53 mutation frequency, tumor mutation burden (TMB), and tumor stem cell characteristics indicated a poor prognosis. The prognostic signature was associated with immune cell infiltration in HCC. Those patients with a high-risk score had lower immune tolerance with a better prediction of the efficacy of immunotherapy. The risk model helps to assess and predict the response and prognosis of HCC patients to immune checkpoint inhibitors (ICIs). Additionally, single-cell RNA sequencing data revealed that the high-risk group had a higher proportion of T cells and fewer immunosuppressive T cells, potentially correlating with a better response to immunotherapy. Finally, in vitro experiments showed that ANLN, an m6A-related gene, promoted the proliferation and migration of HCC cells. Conclusions: In this study, we identified and validated an m6A gene signature consisting of eight genes that can be used to predict prognosis and immunotherapy efficacy in HCC patients.

Polymer mechanochemistry utilizes mechanical force to activate latent functionalities in macromolecules and widely relies on ultrasonication techniques. Although ultrasound is a clinically established modality, fundamental constraints of frequency and power intensity have prohibited the application of the polymer mechanochemistry principles in a biomedical context up to now. Here, a universal polynucleotide framework is presented that allows the binding and release of therapeutic oligonucleotides, both DNA- and RNA-based, as cargo by biocompatible imaging ultrasound. It is shown that the high molar mass, colloidal assembly, and a distinct mechanochemical mechanism enable the force-induced release of cargo and subsequent activation of biological function in vitro and in vivo. Thereby, an avenue for the exploration of biological questions and therapeutics development steered by mechanical force is uncovered.

Most gene therapies exert their actions via manipulation of hepatocytes (parenchymal cells) and the reasons behind the suboptimal performance of synthetic mRNA in non-parenchymal cells (NPC) such as Kupffer cells (KC), and liver macrophages, remain unclear. Here, the spatio-temporal distribution of mRNA encoding enhanced green fluorescent protein (Egfp), siRNA, or both co-encapsulated into lipid nanoparticles (LNP) in the liver in vivo using real-time intravital imaging is investigated. Although both KC and hepatocytes demonstrate comparable high and rapid uptake of mRNA-LNP and siRNA-LNP in vivo, the translation of Egfp mRNA occurs exclusively in hepatocytes during intravital imaging. Despite attempts such as inhibiting intracellular ribonuclease, substituting uridine bases in mRNA with pseudouridine, and using a different ionizable lipid in the LNP mixture, no substantial increase in Egfp translation by NPC is possible. The investigation reveals that hepatocytes, which are distinct from other liver cells due to their polyploidy, exhibit significantly elevated levels of total RNA and protein, along with a higher proportion of ribosomal protein per individual cell. Consequently, fundamental cellular differences account for the low mRNA translation observed in NPC. The findings therefore suggest that cellular biology imposes a natural limitation on synthetic mRNA translation that is strongly influenced by cellular ploidy.

Polymer mechanochemistry utilizes mechanical force to activate latent functionalities in macromolecules and widely relies on ultrasonication techniques. Fundamental constraints of frequency and power intensity have prohibited the application of the polymer mechanochemistry principles in a biomedical context up to now, although medical ultrasound is a clinically established modality. Here, a universal polynucleotide framework is presented that allows the binding and release of therapeutic oligonucleotides, both DNA- and RNA-based, as cargo by biocompatible medical imaging ultrasound. It is shown that the high molar mass, colloidal assembly, and a distinct mechanochemical mechanism enable the force-induced release of cargo and subsequent activation of biological function in vitro and in vivo. Thereby, this work introduces a platform for the exploration of biological questions and therapeutics development steered by mechanical force.

Injectable poly-L-lactic acid (PLLA-SCA) is used for the correction of shallow to deep nasolabial fold contour deficiencies, cheek wrinkles, and other facial wrinkles. In contrast to hyaluronan (HA) fillers, PLLA-SCA has a biostimulatory effect by activating resident fibroblasts to produce collagen, but the mechanisms are not known in detail at the molecular level. Therefore, our aim was to investigate the molecular effects of PLLA-SCA in a comprehensive in vitro study. Since PLLA-SCA-dependent collagen production in fibroblasts depends on the interaction with macrophages, we generated novel macrophage-containing 3D skin models. According to the clinical application, PLLA-SCA was injected once into the dermal equivalent of the 3D skin model. Histological analysis showed a significant increase in epidermal thickness in these models after 5 and 14 days. Gene expression profiling revealed an upregulation of integrins and laminins (e.g., LAMA3, ITGA6), which are essential components of the dermal-epidermal junction. In addition, we found an upregulation of cytokines and chemokines (TGFB2, CXCL6, IL1B) at day 14 after PLLA-SCA injection. Interestingly, immunohistochemical analyses exhibited a significantly stimulated collagen I production in our models. These effects might be attributed, at least in part, to the upregulation of IL1B and subsequently CXCL6, which stimulates collagen I synthesis in human dermal fibroblasts as we could demonstrate. Taken together, our data provide for the first time molecular insights into the biostimulatory effects of PLLA-SCA on collagen I production in novel human 3D skin models comprising macrophages.

Sepsis is characterized by a dysregulated immune response and is very difficult to treat. In the cecal ligation and puncture (CLP) mouse model, we show that nanomedicines can effectively alleviate systemic and local septic events by targeting neutrophils. Specifically, by decorating the surface of clinical-stage dexamethasone liposomes with cyclic arginine-glycine-aspartic acid (cRGD) peptides, we promote their engagement with neutrophils in the systemic circulation, leading to their prominent accumulation at primary and secondary sepsis sites. cRGD-targeted dexamethasone liposomes potently reduce immature circulating neutrophils and neutrophil extracellular traps in intestinal sepsis induction sites and the liver. Additionally, they mitigate inflammatory cytokines systemically and locally while preserving systemic IL-10 levels, contributing to lower IFN-γ/IL-10 ratios as compared to control liposomes and free dexamethasone. Our strategy addresses sepsis at the cellular level, illustrating the use of neutrophils both as a therapeutic target and as a chariot for drug delivery.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IZKF Increasing evidence has shown that microRNAs (miRs) are fundamental players in cardio-metabolic diseases. MiR-26b has been associated with the development of metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis, although causality has not been demonstrated yet. Therefore, we aimed to determine the causal role of miR-26b in MASH and atherosclerosis. Apoe-/-Mir-26b-/- mice were fed a 4- or 12-week western type diet (WTD). Plasma and liver samples were subjected to various assays to investigate the effects on lipid metabolism, inflammation and fibrosis. Additionally, miR-26b mimic-loaded lipid nanoparticles (LNPs) were used to rescue the phenotype. A 1.5-fold increase in hepatic lipid levels was observed in Apoe-/-Mir26b-/- mice, while plasma cholesterol/triglyceride levels remained unchanged. These results were further confirmed by an increase in steatosis. Additionally, the proinflammatory cytokines TNF-α, IL-6 and CXCL1 were significantly elevated, which coincided with increased macrophage infiltration into the liver. Moreover, loss of miR-26b resulted in more hepatic fibrosis. Finally, a treatment with miR-26b mimic-loaded LNPs reduced hepatic lipid levels, rescuing the observed phenotype. Additionally, Apoe-/-Mir26b-/- mice demonstrated a striking 10-fold increase in atherosclerotic lesion size. Consistent with a more advanced plaque phenotype, collagen content and relative necrotic core area were significantly increased in plaques from mice lacking miR-26b. Intriguingly, this increased atherosclerotic lesion size could also be rescued by repetitive injections of miR-26b mimic-loaded LNPs. Overall, our results demonstrate a protective role of miR-26b in cardio-metabolic diseases, thereby opening doors to future research and potential new treatment options using LNP technology.

Sepsis, marked by organ dysfunction, necessitates reliable biomarkers. Ribonuclease inhibitor 1 (RNH1), a ribonuclease (RNase) inhibitor, emerged as a potential biomarker for acute kidney injury and mortality in thoracoabdominal aortic aneurysm patients. Our study investigates RNH1 dynamics in sepsis, its links to mortality and organ dysfunction, and the interplay with RNase 1 and RNase 5. Furthermore, we explore RNH1 as a therapeutic target in sepsis-related processes like inflammation, non-canonical inflammasome activation, and iron homeostasis. We showed that RNH1 levels are significantly higher in deceased patients compared to sepsis survivors and correlate with creatine kinase, aspartate and alanine transaminase, bilirubin, serum creatinine and RNase 5, but not RNase 1. RNH1 mitigated LPS-induced TNFα and RNase 5 secretion, and relative mRNA expression of ferroptosis-associated genes HMOX1, FTH1 and HAMP in PBMCs. Monocytes were identified as the predominant type of LPS-positive PBMCs. Exogenous RNH1 attenuated LPS-induced CASP5 expression, while increasing IL-1β secretion in PBMCs and THP-1 macrophages. As RNH1 has contradictory effects on inflammation and non-canonical inflammasome activation, its use as a therapeutic agent is limited. However, RNH1 levels may play a central role in iron homeostasis during sepsis, supporting our clinical observations. Hence, RNH1 shows promise as biomarkers for renal and hepatic dysfunction and hepatocyte injury, and may be useful in predicting the outcome of septic 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.