Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease lacking effective therapies. Mesenchymal stem cells (MSCs) show therapeutic potential; however, their efficacy is often limited. Aggregates of human umbilical cord-derived MSCs (hUC-MSCs) were generated using AggreWell™ 400 plates. The paracrine and mechanotransduction profiles of three-dimensional (3D)-MSCs were assessed by reverse transcription quantitative PCR and western blot. MASH was induced in C57BL/6J mice via a 32-week high-fat, high-fructose, and high-cholesterol (HFFC) diet, followed by tail vein injection of PBS, two-dimensional (2D)-MSCs, or 3D-MSCs. Therapeutic efficacy was evaluated via histological staining, immunohistochemistry, and serum biochemistry. RNA sequencing was performed to elucidate underlying molecular mechanisms. Under 3D culture conditions, hUC-MSCs formed relatively uniform aggregates with preserved MSC phenotypic characteristics after recovery. 3D aggregate culture enhanced the paracrine, anti-inflammatory, mechanosensitive phenotype, pro-survival, and matrix-remodeling properties of hUC-MSCs, evidenced by increased expression of hepatocyte growth factor, tumor necrosis factor-stimulated gene 6, cyclooxygenase-2, prostaglandin E synthase, B-cell lymphoma-2, and mechanotransduction-related genes, accompanied by elevated matrix metalloproteinase-2 (MMP2) and MMP9 expression in vitro. In vivo fluorescence imaging showed no significant difference in early hepatic retention between intravenously infused single-cell hUC-MSCs and 3D aggregates. In the MASH model, 3D-MSCs more effectively reduced lipid accumulation, collagen deposition, α-smooth muscle actin, hepatomegaly, and serum total cholesterol levels compared with 2D-MSCs. Moreover, 3D-MSCs enhanced the expression of anti-inflammatory interleukin-10, while suppressing inducible nitric oxide synthase. 3D-MSC therapy induced broader transcriptional remodeling than conventional MSCs, with enrichment in extracellular matrix organization, focal adhesion, and the phosphatidylinositol 3-kinase-protein kinase B signaling pathway. Consistently, western blot analysis showed that 3D-MSCs more effectively reduced hepatic p-PI3K and p-AKT levels, indicating stronger inhibition of PI3K/AKT pathway activation. 3D aggregate culture enhances the therapeutic efficacy of hUC-MSCs against MASH, supporting its translational potential for MSC-based liver therapy.

Background Mesenchymal stem cells (MSCs) have become a promising treatment of liver fibrosis which is a key process in liver diseases. Recent studies have shown that transplanted MSCs undergo rapid apoptosis and the apoptotic extracellular vesicles (ApoEVs) derived from MSCs exhibited stronger immunosuppressive capability. However, the effect and the mechanisms of ApoEVs in liver fibrosis remain unclear. The functional differences between ApoEVs and extracellular vesicles (EVs) have yet to be elucidated. This study aims to compare their therapeutic effects on liver fibrosis in order to optimize existing treatment strategies. Methods ApoEVs and EVs were isolated by density gradient centrifugation and illustrated by TEM and NTA. A CCl4-induced liver fibrosis mouse model was treated with equal doses of ApoEVs and EVs. Histopathological analysis was performed on liver sections, serological indicators, fibrosis-related gene expression, macrophage polarization, and the activation status of hepatic stellate cells (HSCs) were analyzed. Subsequently, miRNA-sequencing and untargeted metabolomics analysis were conducted to identify potential pathway. Results Our results demonstrated that ApoEVs had fourfold higher protein yield than EVs, and ApoEVs exhibited a significant superior ability to improve liver fibrosis. In vitro, ApoEVs enhanced macrophage polarization and suppressed HSC activation more effectively, thereby reducing the degree of fibrosis. The underlying molecular mechanism likely due to the enrichment of more miRNAs targeting the PI3K-AKT pathway in ApoEVs and more metabolite molecules that mediate inflammatory metabolic processes. Conclusion These findings showed that ApoEVs exhibit better effects than EVs in alleviating liver fibrosis. Besides, the findings highlighted their therapeutic potential which suggested that ApoEVs could be a promising approach for the treatment of liver diseases and further lay a research foundation for clarifying the therapeutic mechanism of MSCs.

Background: Unhealthy diet and microbiota dysbiosis are known risk factors of type 2 diabetes (T2D), but the value of microbial metabolites as indicators of diet quality and T2D risk has rarely been explored. Objectives: In this prospective study, we examined the correlations of dietary intake and circulating microbial metabolism-associated metabolites with T2D parameters in adults enrolled in the Coronary Artery Risk Development in Young Adults study. Methods: A cohort of 2296 nondiabetic participants was examined on their diet quality, plasma metabolome, fasting glucose, and insulin in year 7 of the coronary artery risk development in young adults study, and the occurrence of incident T2D afterward. Dietary intake was assessed by an interviewer-administered diet history. Diet quality was characterized by the Healthy Eating Index 2020 score. Spearman correlation analysis assessed the associations of plasma metabolites with healthy eating index, fasting glucose, insulin, and homeostatic model assessment indexes. Subsequent propensity matching of 131 incident T2D cases with controls yielded a paired dataset for logistic and multivariate regression analyses, resulting in the predictive markers that were further validated by Cox proportional hazard models on 3 random cohorts selected from the full cohort. Results: Among 611 circulating plasma metabolites, 41 were classified as microbial metabolites or their dietary precursors. Cinnamoylglycine, a metabolite produced jointly by microbial phenylalanine fermentation and hepatic glycine conjugation, was positively correlated with diet quality and inversely associated with incident T2D risk [odds ratio (OR): 0.66; 95% confidence interval (CI): 0.49, 0.87]. Isoleucine was inversely correlated with diet quality and positively associated with T2D risk (OR: 1.98; 95% CI: 1.36, 2.87). This contrast between cinnamoylglycine and isoleucine provided a cinnamoylglycine/isoleucine ratio as a predictive indicator of diet quality and incident T2D risk (OR: 0.61; 95% CI: 0.46, 0.82), which was validated from the 3 randomly selected samples (hazard ratio: 0.75; 95% CI: 0.59, 0.96; P = 0.02). Conclusions: The cinnamoylglycine/isoleucine ratio may be an effective indicator linking diet quality, microbial metabolism, and T2D risk.

BACKGROUND/AIMS:The molecular mechanisms driving nonalcoholic steatohepatitis (NASH) progression are poorly understood. This research examines the involvement of chaperone-mediated autophagy (CMA) in NASH progression. METHODS:Hepatic CMA activity was analysed in NASH mice and patients. Lysosome-associated membrane protein 2A (LAMP2A) was knocked down or overexpressed to assess the effects of hepatocyte-specific CMA on NASH progression. Mice received a high-fat diet or a methionine and choline-deficient diet to induce NASH. Palmitic acid was employed to mimic lipotoxicity-induced hepatocyte damage in vitro. The promoter activity of FOXM1 was evaluated via ChIP and dual-luciferase reporter assays. RESULTS:Hepatic CMA activity was substantially low in NASH mice and patients. LAMP2A knockdown resulted in hepatocyte-specific CMA deficiency, which promoted fibrosis and hepatic inflammation in NASH mice. Both in vitro and in vivo, CMA deficiency also exacerbated hepatocyte damage and endoplasmic reticulum (ER) stress. Mechanistically, CMA deficiency in hepatocytes increased cholesterol accumulation by blocking the degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCR), a key cholesterol synthesis-related enzyme, and the accumulated cholesterol subsequently induced ER stress and hepatocyte damage. The restoration of hepatocyte-specific CMA activity effectively ameliorated diet-induced NASH and ER stress in vivo and in vitro. FOXM1 directly bound to LAMP2A promoter and negatively regulated its transcription. The upregulation of FOXM1 expression impaired CMA and enhanced ER stress, which in turn increased FOXM1 expression, resulting in a vicious cycle and promoting NASH development. CONCLUSIONS:This study highlights the significance of the FOXM1/CMA/ER stress axis in NASH progression and proposes novel therapeutic targets for NASH. KEY POINTS:Chaperone-mediated autophagy (CMA) deficiency in hepatocytes promotes hepatic inflammation and fibrosis in mice with nonalcoholic steatohepatitis (NASH) by inducing cholesterol accumulation and endoplasmic reticulum (ER) stress. Upregulated FOXM1 impairs CMA by suppressing the transcription of lysosome-associated membrane protein 2A (LAMP2A), a rate-limiting component of CMA. ER stress increases FOXM1 expression and cholesterol accumulation. FOXM1/CMA/ER stress axis forms a vicious circle and promotes the development of NASH.

Adult stem cell therapy holds great promise for treating decompensated liver cirrhosis on the basis of animal studies, despite uncertainty about its clinical therapeutic efficacy and unclear underlying mechanisms. Here, we investigated the role of follistatin-like 1 (FSTL1), a profibrotic and proinflammatory matricellular protein, in inflammation-related heterogeneity in stem cell therapy. Our results showed that a high level of circulating FSTL1 is significantly correlated with therapeutic response in patients with cirrhosis. FSTL1 facilitated MSC-mediated early recruitment of Ly6C+ inflammatory macrophages within 24 h postinfusion, which was essential for the empowerment of MSCs and subsequent Ly6C−CX3CR1+ macrophage remodelling at 48 h postinfusion. Fstl1 deficiency abrogated early macrophage recruitment and effective Ly6C−CX3CR1+ macrophage accumulation, resulting in the poor antifibrotic effect of MSCs in mice. Whereas, recombinant FSTL1 protein restored the therapeutic efficacy of MSCs in CCl4-injured Fstl1+/− mice. Mechanistically, host FSTL1 enhanced rapid recycling of CCR2 to the membrane via activation of the CD14/TLR4/NF-κB/ATP6V1G2 axis, leading to early recruitment of Ly6C+ monocytes /macrophages. Taken together, our findings revealed that FSTL1 is a critical regulator of the fibrotic immune microenvironment and facilitates subsequent stem cell therapy. These data suggest that FSTL1 could serve as a predictive biomarker of stem cell therapy response in patients with liver cirrhosis.

Primary biliary cholangitis is characterized by breaking of immune tolerance and disorders of bile acid metabolism. Our previous study found that abnormal expression of Lamp2 was detected in PBC patients. However, the specific role of Lamp2a in disease progression is still unclear. In this study, we showed that hepatic-specific Lamp2a deficiency could aggravate the inflammatory phenotype of murine autoimmune cholangitis. Mechanistically, the loss of Lamp2a in hepatocytes contributed to the abnormal accumulation of Acot8, thus altered the bile acid components, thereby enhancing the lymphocyte activities, and ultimately promoting the inflammatory phenotype of model mice. Moreover, we also found that Acot8 knockdown could alleviate the liver inflammation caused by Lamp2a deficiency. Altogether, our findings explored the effect of Lamp2a deficiency on the murine autoimmune cholangitis by the perspective of bile acid metabolism, and marked the possibility of Acot8 as a new target for the treatment of PBC disease.

The utilization of mesenchymal stem cells (MSCs) serves as an encouraging strategy for treating liver fibrosis. However, precise mechanisms are not completely understood. Recently, small extracellular vesicles (sEVs) have emerged as major paracrine effectors mediating the anti-fibrotic effects of MSCs. This study seeks to examine the healing properties of MSCs-sEVs on liver fibrosis and decipher the associated signaling pathways. Herein, MSCs substantially ameliorated carbon tetrachloride (CCL4)-induced liver inflammation and fibrosis in mice, with this effect predominantly attributed to their derived sEVs. Both in vivo and in vitro experiments verified that MSCs-sEVs skewed the phenotype of liver macrophages into an anti-fibrotic phenotype. Mass spectrometry analysis showed that ubiquitin-specific peptidase 10 (USP10) was significantly enriched in MSCs-sEVs, which was critical for protection against liver fibrosis. USP10 stabilizes Krüppel-like factor 4 (KLF4) via deubiquitination, participating in the modulation of macrophage phenotypes. Mechanistically, KLF4 reprograms macrophages to enhance their anti-inflammatory and repairing functions by modulating NF-κB/STAT6 signaling and regulating the transcription of MMP12. Finally, the exogenous incorporation of USP10 into MSCs-sEVs via genetic engineering further potentiated their antifibrotic effects. These findings deepen the knowledge regarding the cellular pathways through which MSCs ameliorate liver fibrosis, offering a theoretical basis for sEV-based therapeutic strategies.

Background Metabolic dysfunction-associated steatohepatitis (MASH), a more severe subtype of Metabolic dysfunction-associated steatotic liver disease (MASLD), can lead to cirrhosis and hepatocellular carcinoma. Monocyte-derived macrophages (MDMs) play a central role in NASH. Single-cell and spatial transcriptomic technologies have revealed that MDMs react to niche-specific and inflammatory signals to differentiate into Monocyte-derived Kupffer cells (MoKCs) or hepatic lipid-associated macrophages (LAMs)/CCR2+ lipid-associated macrophages (C-LAMs). However, we still lack further descriptions of specific subsets of hepatic macrophages in different MASH models. Methods Two MASH models were established by either giving a methionine-choline-deficient (MCD) diet for 4 weeks or a high-fat‒fructose‒cholesterol (HFFC) diet for 16 weeks. Liver tissues were collected for pathological analyses with hematoxylin and eosin, Oil Red O and F4/80 staining. The expression of lipid metabolism enzymes and inflammatory cytokines were detected using quantitative reverse transcription-polymerase chain reaction (RT‒qPCR). Flow cytometry was utilized to analyze the composition of isolated hepatic macrophages. Results Our study revealed that after a HFFC diet or MCD diet feeding, two MASH models presented opposite changes in the FFA synthesis pathway. The MCD and HFFC diets induce the same alternation in the composition of hepatic macrophages characterized by a decrease in Embryo-derived Kupffer cells (EmKCs) and a concomitant increase in MDMs. However, the composition of the KC pool differed between MCD- and HFFC-fed mice. The MCD diet induced a greater loss of EmKCs, accompanied by more recruited monocytes. HFFC-fed mice contain more MoKCs than MCD-fed mice, whereas MCD-fed mice have more C-LAMs and LAMs than HFFC-fed mice. Conclusions MCD- and HFFC-fed mice have a different composition of KC pool

Background and AimPrimary biliary cholangitis (PBC) is an autoimmune liver disease characterized by destructive lymphocytic cholangitis and specific anti-mitochondrial antibodies. Innate lymphoid cells (ILCs) have been reported to play a role in liver homeostasis and autoimmunity. MethodsWe evaluated the features of peripheral ILC1s and ILC3 in patients with PBC and hepatic ILC1 and ILC3 in two different PBC mouse models (dominant-negative transforming growth factor-beta receptor II [dnTGF beta RII] and 2-octynoic acid-bovine serum albumin [2OA-BSA]). ResultsA total of 115 patients and 18 healthy controls were enrolled in the study. Decreased circulating ILC1/3s were observed in early-stage PBC patients, and the numbers of ILC1/3s were negatively correlated with specific parameters and the proportion of T-helper (Th) 1 and Th17 cells. Reduced numbers of ILC1s were observed in PBC mouse models with different etiologies. ILC1-deficient mice had more severe hepatic inflammation after inducing the 2OA-BSA model. Continuous low-dose injections of lipopolysaccharide (LPS) reduced ILC1 levels in mice, consistent with the lower level of ILC1s in PBC patients with high LPS (> 50 ng/mL), and aggravated hepatic lymphocyte infiltration. ConclusionPatients with PBC had decreased ILC1s, which were negatively correlated with CD4+ T cells. Deficient ILC1 populations led to disease exacerbations in mice. Our results indicated that ILC1s may participate in the pathogenesis of PBC.

BACKGROUND & AIMS:Nonalcoholic steatohepatitis (NASH), a more severe subtype of nonalcoholic fatty liver disease, can cause cirrhosis and hepatocellular carcinoma. Macrophages play critical roles in initiating and maintaining NASH-induced liver inflammation and fibrosis. However, the underlying molecular mechanism of macrophage chaperone-mediated autophagy (CMA) in NASH remains unclear. We aimed to investigate the effects of macrophage-specific CMA on liver inflammation and identify a potential therapeutic target for NASH treatment.METHODS:The CMA function of liver macrophages was detected using Western blot, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and flow cytometry. By constructing myeloid-specific CMA deficiency mice, we evaluated the effects of deficient CMA of macrophages on monocyte recruitment, liver injury, steatosis and fibrosis in NASH mice. A label-free mass spectrometry was utilized to screen the substrates of CMA in macrophages and their mutual interactions. The association between CMA and its substrate was further examined by immunoprecipitation, Western blot and RT-qPCR.RESULTS:A typical hallmark in murine NASH models was impaired CMA function in hepatic macrophages. Monocyte-derived macrophages (MDM) were the dominant macrophage population in NASH, and CMA function was impaired in MDM. CMA dysfunction aggravated liver-targeted recruitment of monocyte and promoted steatosis and fibrosis. Mechanistically, Nup85 functions as a substrate for CMA and its degradation was inhibited in CMA-deficient macrophages. Inhibition of Nup85 attenuated the steatosis and monocyte recruitment caused by CMA deficiency in NASH mice.CONCLUSIONS:We proposed that the impaired CMA-induced Nup85 degradation aggravated monocyte recruitment, promoting liver inflammation and disease progression of NASH.

Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

Background Despite emerging evidence on the therapeutic potential of mesenchymal stem cells (MSCs) for liver fibrosis, the underlying mechanisms remain unclear. At present, MSC-derived exosomes (MSC-EXOs) are widely accepted as crucial messengers for intercellular communication. This study aimed to explore the therapeutic effects of MSC-EXOs on liver fibrosis and identify the mechanisms underlying the action of MSC-EXOs. Methods Carbon tetrachloride was used to induce a liver fibrosis model, which was intravenously administered with MSCs or MSC-EXOs to assess treatment efficacy. The resulting histopathology, fibrosis degree, inflammation and macrophage polarization were analyzed. RAW264.7 and BMDM cells were used to explore the regulatory effects of MSC-EXOs on macrophage polarization. Then, the critical miRNA mediating the therapeutic effects of MSC-EXOs was screened via RNA sequencing and validated experimentally. Furthermore, the target mRNA and downstream signaling pathways were elucidated by luciferase reporter assay, bioinformatics analysis and western blot. Results MSCs alleviated liver fibrosis largely depended on their secreted exosomes, which were visualized to circulate into liver after transplantation. In addition, MSC-EXOs were found to modulate macrophage phenotype to regulate inflammatory microenvironment in liver and repair the injury. Mechanically, RNA-sequencing illustrates that miR-148a, enriched in the MSC-EXOs, targets Kruppel-like factor 6 (KLF6) to suppress pro-inflammatory macrophages and promote anti-inflammatory macrophages by inhibiting the STAT3 pathway. Administration of miR-148a-enriched MSC-EXOs or miR-148a agomir shows potent ameliorative effects on liver fibrosis. Conclusions These findings suggest that MSC-EXOs protect against liver fibrosis via delivering miR-148a that regulates intrahepatic macrophage functions through KLF6/STAT3 signaling and provide a potential therapeutic target for liver fibrosis.