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Cathepsins are the most abundant lysosomal proteases that are mainly found in acidicendo/lysosomal compartments where they play a vital role in intracellular protein degradation,energy metabolism, and immune responses among a host of other functions. The discovery thatcathepsins are secreted and remain functionally active outside of the lysosome has caused a paradigmshift. Contemporary research has unraveled many versatile functions of cathepsins in extralysosomallocations including cytosol and extracellular space. Nevertheless, extracellular cathepsins are majorlyupregulated in pathological states and are implicated in a wide range of diseases including cancerand cardiovascular diseases. Taking advantage of the dierential expression of the cathepsinsduring pathological conditions, much research is focused on using cathepsins as diagnostic markersand therapeutic targets. A tailored therapeutic approach using selective cathepsin inhibitors isconstantly emerging to be safe and ecient. Moreover, recent development of proteomic-basedapproaches for the identification of novel physiological substrates oers a major opportunity tounderstand the mechanism of cathepsin action. In this review, we summarize the available evidenceregarding the role of cathepsins in health and disease, discuss their potential as biomarkers ofdisease progression, and shed light on the potential of extracellular cathepsin inhibitors as safetherapeutic tools.

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. Fatty liver disease is a condition characterized by the abnormal accumulation of fat in the liver. In certain cases, the fatty build-up can lead to inflammation and, in time, scarring. This advanced stage is known as MASH (short for metabolic dysfunction-associated steatohepatitis), and it can increase the risk of liver failure, cancer, and other complications. Yet the underlying mechanisms that initiate inflammation and thereby drive the disease are still poorly understood. Identifying the molecular factors contributing to this transition could aid in discovering new treatment targets. To explore this question, Peters et al. focus on microRNA-26b, a small molecule involved in many heart and metabolic diseases that helps regulate gene expression. They aimed to clarify the role of microRNA-26b in MASH using mice genetically manipulated to lack this regulatory molecule. The experiments revealed that the animals had larger amounts of fat in their livers, with the organs also showing clear signs of scarring and increased inflammation – including high levels of inflammatory signalling molecules and the presence of immune cells known as macrophages. Peters et al. then treated the animals with specially designed compounds that can act as microRNA-26b. The molecules were safely delivered to the liver within tiny fat-based spheres known as lipid nanoparticles. Following such treatment, the mice showed decreased levels of liver fat and inflammation. The anti-inflammatory effect of the microRNA-26b ‘mimics’ was also confirmed in human liver samples. Together, these results show that microRNA-26b plays a protective role in the development of MASH. Future research should focus on confirming whether these molecules could represent a viable therapeutic treatment, in particular when delivered within lipid-based nanoparticles.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases ranging from simple steatosis to non-alcoholic steatohepatitis, fibrosis, cirrhosis, and/or hepatocellular carcinoma. Due to its increasing prevalence, NAFLD is currently a major public health concern. Although a wide variety of preclinical models have contributed to better understanding the pathophysiology of NAFLD, it is not always obvious which model is best suitable for addressing a specific research question. This review provides insights into currently existing models, mainly focusing on murine models, which is of great importance to aid in the identification of novel therapeutic options for human NAFLD.

Aims/hypothesisInsulin resistance in skeletal muscle and liver plays a major role in the pathophysiology of type 2 diabetes. The hyperinsulinaemic–euglycaemic clamp is considered the gold standard for assessing peripheral and hepatic insulin sensitivity, yet it is a costly and labour-intensive procedure. Therefore, easy-to-measure, cost-effective approaches to determine insulin sensitivity are needed to enable organ-specific interventions. Recently, evidence emerged that plasma cathepsin D (CTSD) is associated with insulin sensitivity and hepatic inflammation. Here, we aimed to investigate whether plasma CTSD is associated with hepatic and/or peripheral insulin sensitivity in humans.MethodsAs part of two large clinical trials (one designed to investigate the effects of antibiotics, and the other to investigate polyphenol supplementation, on insulin sensitivity), 94 overweight and obese adults (BMI 25–35 kg/m2) previously underwent a two-step hyperinsulinaemic–euglycaemic clamp (using [6,6-2H2]glucose) to assess hepatic and peripheral insulin sensitivity (per cent suppression of endogenous glucose output during the low-insulin-infusion step, and the rate of glucose disappearance during high-insulin infusion [40 mU/(m2 × min)], respectively). In this secondary analysis, plasma CTSD levels, CTSD activity and plasma inflammatory cytokines were measured.ResultsPlasma CTSD levels were positively associated with the proinflammatory cytokines IL-8 and TNF-α (IL-8: standardised β = 0.495, p < 0.001; TNF-α: standardised β = 0.264, p = 0.012). Plasma CTSD activity was negatively associated with hepatic insulin sensitivity (standardised β = −0.206, p = 0.043), independent of age, sex, BMI and waist circumference, but it was not associated with peripheral insulin sensitivity. However, plasma IL-8 and TNF-α were not significantly correlated with hepatic insulin sensitivity.Conclusions/interpretationWe demonstrate that plasma CTSD activity, but not systemic inflammation, is inversely related to hepatic insulin sensitivity, suggesting that plasma CTSD activity may be used as a non-invasive marker for hepatic insulin sensitivity in humans.

Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory condition, hallmarked by a disturbance in the bidirectional interaction between gut and brain. In general, the gut/brain axis involves direct and/or indirect communication via the central and enteric nervous system, host innate immune system, and particularly the gut microbiota. This complex interaction implies that IBD is a complex multifactorial disease. There is increasing evidence that stress adversely affects the gut/microbiota/brain axis by altering intestinal mucosa permeability and cytokine secretion, thereby influencing the relapse risk and disease severity of IBD. Given the recurrent nature, therapeutic strategies particularly aim at achieving and maintaining remission of the disease. Alternatively, these strategies focus on preventing permanent bowel damage and concomitant long-term complications. In this review, we discuss the gut/microbiota/brain interplay with respect to chronic inflammation of the gastrointestinal tract and particularly shed light on the role of stress. Hence, we evaluated the therapeutic impact of stress management in IBD.

Nonalcoholic fatty liver disease (NAFLD) is known as the hepatic manifestation of the metabolic syndrome, and while most patients develop simple steatosis, up to one-third can develop nonalcoholic steatohepatitis (NASH) [...]

Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr(-/-) mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time. APOE2ki and Ldlr(-/-) mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control. Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr(-/-) mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr(-/-) mice. Finally, bone-marrow-derived-macrophages of Ldlr(-/-) mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice. Ldlr(-/-) mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr(-/-) mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.

Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism. Conclusion : CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

Background & AimsThe lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.MethodsLow-density lipoprotein receptor knock out ( Ldlr-/- ) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.ResultsLdlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.ConclusionWe have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs). Ldlr(-/-) mice were transplanted with wild-type (Wt), Cd36(-/-) or Msr1(-/-) bone marrow and fed a Western diet for 3 months. Cd36(-/-)- and Msr1(-/-)- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36(-/-)- and Msr1(-/-)-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation. CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.