Introduction

Sarcopenia, an important component of malnutrition, is linked to poor prognosis in cirrhotic patients. Recent studies have indicated that chronic muscle inflammation plays a pivotal role in the progression of sarcopenia, although the mechanism underlying the association between cirrhosis and chronic muscle inflammation remains poorly understood. Therefore, this study aimed to elucidate the specific mechanism by which hepatic-derived extracellular vesicles (EVs) promote proinflammatory responses in muscle macrophages via circulation, leading to the advancement of muscle inflammation during cirrhosis.

Aims & Methods

Liver fibrosis was induced by carbon tetrachloride injection and bile duct ligation. EVs from cirrhotic liver were isolated and characterized. Proteomics was utilized to identify functional mitochondrial components in EVs. Lastly, we conducted a cross-sectional study on patients with cirrhosis to validate these findings.

Results

Treatment of EVs from cirrhotic liver promoted inflammation in macrophages and muscle inflammation progression via activating the STING pathway. The Sting inhibitor in cirrhotic mice abolished the role of cirrhotic-derived EVs in muscle inflammation. In addition, hepatic-specific deletion of Rab27a and the inhibitor of EV—GW4869, significantly alleviated cirrhosis-induced muscle inflammation progression in cirrhotic mice. The proteomics results showed that mitochondrial proteins and mtDNA were enriched in EVs from cirrhotic liver. Furthermore, mitochondria damage in hepatocytes caused by mitochondrial membrane potential decline led to mitochondrial DNA leakage, which caused macrophage inflammation through the STING pathway. Moreover, EVs from peripheral veins and hepatic veins who underwent transjugular intrahepatic portosystemic shunt in patients with cirrhosis and EV-mtDNA expression were associated with the severity of cirrhosis.

Conclusion

Hepatocyte-derived EVs promote inflammation in macrophages in muscle and facilitate muscle inflammation progression via the mtDNA-cGAS-STING signaling pathway . Reducing EV secretions in cirrhotic liver or targeting STING may be potential therapeutic approaches to slow the progression of cirrhosis-driven muscle inflammation.

Introduction

Dysregulated T-cell immune response-mediated inflammation is implicated in various liver diseases, yet mechanisms governing liver immune homeostasis control and therapies for limiting T-cell overactivation remain unclear. This study aims to elucidate the role of immunoregulatory myeloid cells (MCregs) in liver injury and their potential to modulate CD4⁺ T-cell hyperactivation via extracellular vesicle (EV)-mediated metabolic regulation.

Aims & Methods

Concanavalin A (ConA) mice and autoimmune hepatitis (AIH) patients were examined for alterations in purine metabolism associated with liver injury severity and therapeutic response. The expression of extracellular purine catabolism nucleases, such as CD39 and CD73, on CD11b⁺Gr-1⁺ myeloid cells (MCs) was assessed, with particular focus on liver injury conditions. The study investigated the natural expansion of the CD39⁺CD73⁺Gr-1^highCD11b⁺ regulatory MC (MCreg) subset during early liver injury phases and its effects on CD4⁺ T-cell hyperactivation and liver injury through in vitro and in vivo experiments. Mechanistic insights into MCreg-mediated immunosuppression were explored, particularly regarding the release of CD73^high EVs and their impact on extracellular AMP conversion, cAMP pathway activation, and inhibition of glycolysis and cytokine secretion in activated CD4⁺ T cells.

Results

Alterations in purine metabolism were observed in ConA mice and AIH patients, correlating with liver injury severity and therapeutic response. CD39 and CD73 expression was markedly upregulated on CD11b⁺Gr-1⁺ myeloid cells under liver injury conditions. The CD39⁺CD73⁺Gr-1^highCD11b⁺ MCreg subset expanded naturally during early-stage liver injury and demonstrated efficacy in suppressing CD4⁺ T-cell hyperactivation and liver injury both in vitro and in vivo. The release of CD73^high EVs by MCregs facilitated extracellular AMP conversion to immunosuppressive metabolites (e.g., adenosine and inosine), activating the cAMP pathway and inhibiting glycolysis and cytokine secretion in activated CD4⁺ T cells.

Conclusion

These findings shed light on the mechanisms governing immune homeostasis during early liver injury and suggest that MCreg or MCreg-EV therapy may offer a targeted approach for preventing diverse liver diseases induced by T-cell overactivation.

Introduction

Neutrophils could be recruited into liver in autoimmune hepatitis (AIH) and experimental AIH (Concanavalin A induced hepatitis), but the role of neutrophils in these kinds of immune related hepatitis has rare research to clarify. As previous reports, CD177 expressed exclusively on neutrophils, CD177⁺ neutrophils could alleviate inflammation in inflammatory bowel disease, and it could produce neutrophil extracellular traps that promote biliary atresia. However, there is no report about CD177⁺ neutrophils involved in AIH or experimental AIH.

Aims & Methods

In this study, we wanted to explore to role of neutrophils in AIH and wondered whether CD177⁺ neutrophils exert protective effect in it. We collected clinical samples of AIH patients and healthy control (HC), and established experimental AIH animal model by using Concanavalin A (ConA). Anti-Ly6G antibody was used as pretreatment to depleting neutrophils before ConA injection. Wild type (WT) and CD177 knock out (KO) C57BL/6J female mouse were used to construct different experimental AIH model. Flow cytometry analysis was used to detect the percentage of neutrophils in blood and liver. Detected serum biochemical indicators (ALT: alanine aminotransferase, AST: aspartate aminotransferase) and performed staining in liver tissues to evaluate liver injury.

Results

For AIH patients, CD177⁺ neutrophils in peripheral blood were significantly increased (P<0.05). And for immunohistochemistry (IHC), the positive areas of CD66b (a surface marker of neutrophils) and CD177 were more obvious in liver tissues of AIH patients (P<0.05). For animal experiment, we successfully established model of experimental AIH. In this model, liver injury was significant in ConA group than in normal control (NC) group, which showed prominent liver necrosis in hematoxylin-eosin (HE) staining and higher serum ALT and AST (P<0.05). Positive IHC areas of Ly6G and CD177 were more obvious in liver tissues of ConA group, which meant the increased infiltration of neutrophils. We found that distributions of neutrophils were different in different organ. Compared to NC group, the amount of neutrophils in ConA group was decreased in bone marrow and increased in peripheral blood and liver, and its increase in liver trended to be more obvious (P=0.096). So, we speculated that neutrophils mainly migrated to liver from marrow in ConA model. After depleting neutrophils, we through flow cytometry analysis to verify the depletion of neutrophils (CD11b⁺ Ly6G⁺ cells) in anti Ly6G-ConA group was thorough. HE staining showed liver injury appearances in ConA group and Ly6G isotype-ConA group were more severe than that in normal saline (NS) group and in anti Ly6G-ConA group. Similarly, ConA group and Ly6G isotype-ConA group had higher levels of serum ALT and AST than the other two groups. Finally, we used CD177 KO mouse to determine the role of CD177⁺ neutrophils in ConA model. HE staining showed the area of liver necrosis in CD177 KO-ConA group was largest. As for liver function, CD177 KO-ConA group also had the highest levels of serum ALT and AST (P<0.05), which improve that the liver injury in this group was the most severe.

Conclusion

Neutrophils involved in AIH and experimental AIH. In experimental AIH, depletion of neutrophils could alleviate liver injury, and CD177⁺ neutrophils could exert protective effect in it.

Introduction

The treatments for liver fibrosis are still in need so far. As the crucial initiators, activated hepatic stellate cells (aHSCs) undergo a series of epigenetic changes. The study intends to explore transcription factors (TFs) that facilitate HSCs activation and provide novel therapeutic targets for liver fibrosis.

Aims & Methods

Three distinct HSCs activation models were established in present study. Firstly, in vitro activation involved isolating primary HSCs from normal Sprague-Dawley rats and culturing them for two days (n=5) and seven days (n=5), namely natural activation model. Secondly, in vivo activation entailed isolating primary HSCs from bile duct ligated rats (n=4) and normal controls (n=4), followed by culturing until the next day. Lastly, transforming growth factor-β (TGF-β, 2.5ng/ml) (n=10) or control buffer (n=10) were added to the human HSCs cell line LX2 and cultured for 24 hours. Subsequently, assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted on the abovementioned cells, along with motif enrichment and footprint analysis. Pathway enrichment analysis was performed on differentially accessible genes (DAGs). RNA sequencing (RNA-seq) was employed to analyze gene expression profiles of primary HSCs in the natural activation model (n=4). The integration of ATAC-seq and RNA-seq further identified potential key TFs. RT-qPCR and differential expression analysis of public datasets was utilized to confirm the dysregulation of TFs in primary HSCs, fibrotic liver and fibrotic model of induced pluripotent stem cells. Finally, enrichment at enhancers and promoters of these genes was validated by H3K27Ac and H3K4me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) of human HSCs.

Results

For ATAC-seq, primary HSCs exhibited higher chromatin accessibility after activation compared with the controls. After peak annotation, the up-regulated peaks were mainly located in the promoter region in all comparison groups, uncovering the dynamic transcriptional regulation during HSC activation. In the motif enrichment analysis, 150 aHSCs related TFs and 54 quiescent HSCs (qHSCs) related TFs were identified in the in vitro activation model. The three models shared 12 aHSCs related TFs, namely Runx-aml, Runx1, Runx2, Runx, Ets:Runx, Atf2, Atf4, Atf7, c-Jun-Cre, Creb5, Smad3 and CArG; and 19 qHSCs related TFs, including Foxf1, Foxo3, Foxk1, Foxp1, Hoxa1, Hoxa2, Stat3, Hnf4a, Hnf6, Elk1, Elk4, Cux1, Crx, Duxbl, Vdr, Tr4, Ebf2, Gata2, Gata6. Footprint analysis validated the alteration of accessibility in Atf2, Atf4, Atf7, Creb5, Hnf6 and Gata2. Actin filament organization, Ras protein signal transduction and intrinsic apoptotic signal pathways were enriched in DAGs. Integration of ATAC-seq and RNA-seq showed that gene expression was significantly correlated with chromatin accessibility, among which Runx2, Creb5, and Smad3 were significantly upregulated in aHSCs at transcription level. The dysregulation of these three genes was verified in primary HSCs, fibrotic liver and fibrotic model of human induced pluripotent stem cells. Finally, enrichment at active enhancers and promoters of these TFs was validated by H3K27Ac and H3K4me3 ChIP-seq of human HSCs, revealing the crucial role of genes in the transcription.

Conclusion

We generated chromatin accessibility profiles of different HSCs activation models, highlighting the significance of Runx2, Creb5, and Smad3 as key TFs mediating HSCs activation. These findings proposed these TFs as promising targets for future liver fibrosis treatment.

Disclosure

The author declare no competing interests and financial relationships with commercial interests.

Introduction

Sarcopenia defined as having low muscle mass, muscle strength and if combined with low performance indicate severe sarcopenia¹. It is associated with adverse outcomes in cirrhosis, including hepatic encephalopathy, ascites, infection, and increased hospitalisation. Prevalence of sarcopenia has been reported between 40 to 70% and are more common with alcoholic liver disease².

Aims & Methods

We aim to assess relationship between nutritional status/assessment, muscle quality and sarcopenia. Cirrhotic patients were recruited from a tertiary-hepatology-clinic for this prospective cross-sectional study. BIA was performed as the standard (SECA mBCA-525) for nutritional status and sarcopenia assessment. Appendicular skeletal muscle mass (ASM) calculated through sum of limb muscle masses in kg. sarcopenia was diagnosed with ASM/height²<7.0 kg/m² and <5.5 kg/m² for male and female respectively. Muscle quality was measured by mean handgrip strength in kg divided by ASM (HGS/ASM). Validated functional muscle metrics (handgrip and sit-to-stand), liver-frailty-index (LFI)³, Chronic Liver Disease Questionnaire (CLD-Q)⁴ and food-frequency-questionnaire (FFQ) were also analysed.

Results

49 patients, 57% (28) alcohol-liver-disease, 18% (9) non-alcoholic steatohepatitis, 5% (4) viral, 4% (3) autoimmune and 7% (5) mixed aetiologies, were included. 44 healthy controls (HC) were recruited as well. Most patients were male (n=28, 57%), with mean age 56yrs (SD=9), mean Child-Pugh score 6 (IQR= 5-7) and MELD 10 (IQR= 7-12). 35% (n=17) were actively drinking alcohol. 9 (18%) of patients were frail and 34 (69%) were pre-frail. 25 (89%) cirrhotic male and 14 (66%) cirrhotic female had sarcopenia diagnosed via BIA. 24/42 (57%) had lower total daily energy intake than is recommended by EASL guidelines⁵. Of those, 11 (44%) male and 6 (42%) had diagnosis of sarcopenia.
There was positive correlation between nutritional status measured with BIA as Phase angle analysis (PhA) with total skeletal muscle mass adjusted for height, SMM/H², (r=0.51, p<0.0001) and ASM/H² (r=0.04,p=0.7). There was strong correlation between PhA and HGS (r=0.71, p=<0.001). Higher PhA score predicts lower MELD score therefore longer survivals (β = -0.092, p=0.004).
Healthy control has significantly better muscle quality (HGS/ASM) compared to cirrhotic patient (mean 2.52 vs 1.85 kg, SD 0.6 and 0.4 respectively, p<0.001).
Using functional testing, sit-to-stand time was lower in HCs (13.35 vs 8.94 secs, p=0.0001) and hand-grip strength higher in HC (p< 0.001). QoL was negatively correlate with sarcopenia (r=-0.06,p=0.6), LFI (r=-0.2,p=0.1), MELD (r=-0.15,p=0.3) and Child-Pugh score (r= -0.08, p=0.6) although they were not statically significant.

Conclusion

Cirrhotic patients had lower muscle quality and functional status compared to HC. FFQ and PhA are good assessment tool to establish nutritional status. PhA correlated much better with SMM/H2 than ASM/H2 and is a good predictor for disease severity. QoL is multifactorial and couldn’t be explained with frailty scores or clinical severity scores.

References

1. Alfonso J Cruz-Jentoft et al. Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 2019 Jan 1;48(1):16-31
2. Xinxing Tantai et al. Effect of sarcopenia on survival in patients with cirrhosis: A meta-analysis Journal of Hepatology 2022 vol. 76 j 588–599
3. Jennifer C Lai et al. Development of a novel frailty index to predict mortality in patients with end-stage liver disease. Hepatology 2017 Aug;66(2):564-574
4. Z M Younossi et al. Development of a disease specific questionnaire to measure health related quality of life in patients with chronic liver disease. Gut 1999;45:295–300
5. EASL Clinical Practice Guidelines on nutrition in chronic liver disease. J Hepatol. 2019 Jan;70(1):172-193.

Introduction

Portal hypertension (PH), a key driver of liver-related morbidity, arises as a consequence of static and dynamic changes in the liver parenchyma. While the static PH component is determined by the degree of fibrosis, the analysis of dynamic PH components remains challenging.

Aims & Methods

We aimed to investigate the influence of inflammation on the dynamic component of PH in the setting of liver disease regression in (i) well-standardised mouse models and (ii) abstinent patients with alcohol-related liver cirrhosis (ALD). In male C57BL/6JRj mice, disease regression was modelled by administering carbon tetrachloride or thioacetamide for 12 weeks to induce cirrhosis before discontinuing the toxic stimulus and thus enabling regression for either one (R1; n=15) or two (R2; n=15) weeks. In our patient cohort, we included 128 ALD patients with prolonged abstinence (>1 month) undergoing same-day hepatic venous pressure gradient (HVPG) and liver stiffness measurement (LSM). The static component of PH during regression (histological collagen proportionate area [CPA; %] in mice; LSM in patients) was used to predict portal pressure (PP) based on a linear model. Subsequently, we explored parameters affecting the dynamic PH component (i.e., difference between the predicted PP based on LSM/CPA and the true/measured PP) by analysing the linear model’s residuals.

Results

When compared to mice at peak disease severity, withdrawing the toxic stimulus led to a significant decrease in PP, CPA and intrahepatic expression of inflammatory genes (see Table). Despite improvements in hepatic inflammation during regression, the linear model demonstrated a significant association between an increased expression of inflammatory genes and higher-than-expected PP levels in R1 and R2 mice, as shown by the positive correlation between Tnfα (Spearman’s r:0.46; p=0.012), Il6 (r:0.48; p=0.008), Il1β (r:0.39; p=0.036), Cxcl1 (r:0.33; p=0.075) and Mcp1 (r:0.26; p=0.159) with the model’s residuals. Similarly, a longer duration of abstinence in ALD patients was associated with a significantly lower HVPG (r:-0.21; p=0.017), LSM (r:-0.37; p<0.001), IL-6 (r:-0.24; p=0.006) and CRP (r:-0.28; p=0.001). Nevertheless, patients with higher levels of systemic inflammation also demonstrated a higher HVPG than predicted by LSM in the linear model. Specifically, IL-6 (r:0.18; p=0.043) and CRP (r:0.21; p=0.016) were positively correlated with the model’s residuals. Furthermore, levels of complement factor C3c (r:-0.22, p=0.049) and C4 (r:-0.15, p=0.203) were markedly decreased in patients with higher-than-expected HVPG. Interestingly, while the impact of systemic inflammation was primarily found in long-term abstinence (>6 months), the negative correlation with C3c was only present in short-term abstinence (≤6 months).

Conclusion

Despite significant improvements during disease regression, intrahepatic inflammation in mice and systemic inflammation in ALD patients remain critical determinants of the dynamic component of PH.

Disclosure

Benedikt Simbrunner: Travel support from AbbVie and Gilead. Georg Semmler: Travel Support from Gilead. Michael Trauner: Grant support from Albireo, Alnylam, Cymabay, Falk, Gilead, Intercept, MSD, Takeda and UltraGenyx; Honoraria for consulting from Albireo, Boehringer Ingelheim, BiomX, Falk, Genfit, Gilead, Intercept, MSD, Novartis, Phenex, Pliant, Regulus and Shire; Speaker fees from Bristol-Myers Squibb, Falk, Gilead, Intercept and MSD; Travel support from AbbVie, Falk, Gilead and Intercept. Mattias Mandorfer: Speaker and/or consultant and/or advisory board member for AbbVie, Collective Acumen, Gilead, and W. L. Gore & Associates; Travel support from AbbVie and Gilead. Philipp Schwabl: Consulting fees from PharmaIN; Travel support from Falk and Phenex Pharmaceuticals. Thomas Reiberger: Grant support from Abbvie, Boehringer-Ingelheim, Gilead, Gore, Intercept, MSD, Myr Pharmaceuticals, Philips Healthcare, Pliant, and Siemens; Speaking honoraria from Abbvie, Gilead, Gore, Intercept, Roche, MSD; Consulting/advisory board fee from Abbvie, Bayer, Boehringer-Ingelheim, Gilead, Intercept, MSD, Siemens; Travel support from Abbvie, Boehringer-Ingelheim, Gilead and Roche.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) presents as a complex multifactorial disease, in which dysregulation of metabolic homeostasis in the liver, as well as in distant organs, plays a substantial role. Recent evidence places skeletal muscle and the muscle-liver axis at the center of the MASLD pathogenic cascade. In fact, low muscle mass and muscle fat infiltration associates with increased risk of advanced MASLD, namely metabolic-associated steatohepatitis (MASH). In parallel, we showed that microRNA-21 (miR-21) is increased in the liver and skeletal muscle of MASH patients, with its abrogation ameliorating disease progression in vivo.

Aims & Methods

Here, we investigated the effects of miR-21 on muscle function in the context of experimental MASLD.Mir21 whole body knockout (KO); Mir21Alb-Cre KO (hepatocyte-specific KO) and wild-type (WT) mice were fed ad libitum with a high-fat/calorie diet (HFD) with high fructose/glucose corn syrup in drinking water for 20 weeks, or with a standard diet (SD), to recapitulate key, complementary metabolic features of human MASLD. Muscle phenotype and function were evaluated through grip strength and wheel running assays one week before sacrifice. In parallel, glucose tolerance test was also performed. At sacrifice, liver/muscle biopsies and total serum were collected for subsequent analyses.

Results

Both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice displayed significant lower weight increments throughout time, compared to WT HFD-fed mice. Moreover, all HFD-fed mice presented reduced voluntary wheel-running distance, except for Mir21KO HFD-fed mice, which showed SD-like wheel-running distance. Mir21 KO mice also displayed a tendency to have grip strength levels higher than WT mice. Furthermore, both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice had AUC of glucose significantly lower than WT HFD-fed mice, and that was similar to WT SD-fed mice. In agreement, Mir21 KO HFD-fed mice showed increased mRNA levels of MyoD and Pax7, two transcription factors responsible for the development and regeneration of skeletal muscle through the differentiation of myoblasts into myotubes, thereby counteracting the decrease observed in WT HFD-fed mice. Moreover, both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice had significantly decreased mRNA expression levels of Mafbx (atrofin-1), a ubiquitin ligase responsible for marking muscle cell proteins for degradation, when compared with WT mice.

Conclusion

Our results show that Mir21 KO mice on a high-fat diet exhibit higher skeletal muscle function compared to their WT counterparts, further suggesting that the ablation of miR-21 contributes to muscle regeneration while inhibiting muscle degradation. These findings highlight the relevance of the muscle-liver axis in MASLD pathogenesis and, possibly, therapeutic targeting. (2022.08837.PTDC, FCT).