Leukotriene B4 receptor 1 (BLT1) does not mediate disease progression in a mouse model of liver fibrosis.

MASH is a prevalent liver disease that can progress to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death, but there are no approved therapies. Leukotriene B4 (LTB4) is a potent pro-inflammatory chemoattractant that drives macrophage and neutrophil chemotaxis, and genetic loss or inhibition of its high affinity receptor, leukotriene B4 receptor 1 (BLT1), results in improved insulin sensitivity and decreased hepatic steatosis. To validate the therapeutic efficacy of BLT1 inhibition in an inflammatory and pro-fibrotic mouse model of MASH and fibrosis, mice were challenged with a choline-deficient, L-amino acid defined high fat diet and treated with a BLT1 antagonist at 30 or 90 mg/kg for 8 weeks. Liver function, histology, and gene expression were evaluated at the end of the study. Treatment with the BLT1 antagonist significantly reduced plasma lipids and liver steatosis but had no impact on liver injury biomarkers or histological endpoints such as inflammation, ballooning, or fibrosis compared to control. Artificial intelligence-powered digital pathology analysis revealed a significant reduction in steatosis co-localized fibrosis in livers treated with the BLT1 antagonist. Liver RNA-seq and pathway analyses revealed significant changes in fatty acid, arachidonic acid, and eicosanoid metabolic pathways with BLT1 antagonist treatment, however, these changes were not sufficient to impact inflammation and fibrosis endpoints. Targeting this LTB4-BLT1 axis with a small molecule inhibitor in animal models of chronic liver disease should be considered with caution, and additional studies are warranted to understand the mechanistic nuances of BLT1 inhibition in the context of MASH and liver fibrosis.

Loss of low-molecular-weight protein tyrosine phosphatase shows limited improvement in glucose tolerance but causes mild cardiac hypertrophy in mice.

Insulin resistance is a major public health burden that often results in other comorbidities including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease. An insulin sensitizer has the potential to become a disease-modifying therapy. It remains an unmet medical need to identify therapeutics that target the insulin signaling pathway to treat insulin resistance. Low-molecular-weight protein tyrosine phosphatase (LMPTP) negatively regulates insulin signaling and has emerged as a potential therapeutic target for insulin sensitization. Genetic studies have demonstrated that LMPTP is positively associated with obesity in humans and promotes insulin resistance in rodents. A recent study showed that pharmacological inhibition or genetic deletion of LMPTP protects mice from high-fat diet-induced insulin resistance and diabetes. Here, we show that loss of LMPTP by genetic deletion has no significant effects on improving glucose tolerance in lean or diet-induced obese mice. Furthermore, our data demonstrate that LMPTP deficiency potentiates cardiac hypertrophy that leads to mild cardiac dysfunction. Our findings suggest that the development of LMPTP inhibitors for the treatment of insulin resistance and type 2 diabetes should be reevaluated, and further studies are needed to characterize the molecular and pathophysiological role of LMPTP.NEW & NOTEWORTHY Inhibition of LMPTP with a small-molecule inhibitor, Cmpd23, improves glucose tolerance in mice as reported earlier. However, genetic deficiency of the LMPTP-encoding gene, Acp1, has limited effects on glucose metabolism but leads to mild cardiac hypertrophy in mice. The findings suggest the potential off-target effects of Cmpd23 and call for reevaluation of LMPTP as a therapeutic target for the treatment of insulin resistance and type 2 diabetes.

The deubiquitinating enzyme USP19 modulates adipogenesis and potentiates high-fat-diet-induced obesity and glucose intolerance in mice.

AIMS/HYPOTHESIS: Elucidating the molecular mechanisms of fat accumulation and its metabolic consequences is crucial to understanding and treating obesity, an epidemic disease. We have previously observed that Usp19 deubiquitinating enzyme-null mice (Usp19-/-) have significantly lower fat mass than wild-type (WT) mice. Thus, this study aimed to provide further understanding of the role of ubiquitin-specific peptidase 19 (USP19) in fat development, obesity and diabetes. METHODS: In this study, the metabolic phenotypes of WT and Usp19-/- mice were compared. The stromal vascular fractions (SVFs) of inguinal fat pads from WT and Usp19-/- mice were isolated and cells were differentiated into adipocytes in culture to assess their adipogenic capacity. Mice were fed a high-fat diet (HFD) for 18 weeks. Body composition, glucose metabolism and metabolic variables were assessed. In addition, following insulin injection, signalling activity was analysed in the muscle, liver and adipose tissue. Finally, the correlation between the expression of Usp19 mRNA and adipocyte function genes in human adipose tissue was analysed. RESULT: Upon adipogenic differentiation, SVF cells from Usp19-/- failed to accumulate lipid and upregulate adipogenic genes, unlike cells from WT mice. Usp19-/- mice were also found to have smaller fat pads throughout the lifespan and a higher percentage of lean mass, compared with WT mice. When fed an HFD, Usp19-/- mice were more glucose tolerant, pyruvate tolerant and insulin sensitive than WT mice. Moreover, HFD-fed Usp19-/- mice had enhanced insulin signalling in the muscle and the liver, but not in adipose tissue. Finally, USP19 mRNA expression in human adipose tissue was positively correlated with the expression of important adipocyte genes in abdominal fat depots, but not subcutaneous fat depots. CONCLUSIONS/INTERPRETATION: USP19 is an important regulator of fat development. Its inactivation in mice exerts effects on multiple tissues, which may protect against the negative metabolic effects of high-fat feeding. These findings suggest that inhibition of USP19 could have therapeutic potential to protect from the deleterious consequences of obesity and diabetes.

A Standardized Clinical Pathway to Decrease Hospital Admissions Among Febrile Children With Sickle Cell Disease.

BACKGROUND AND OBJECTIVE: Recurrent hospital admissions for patients with sickle cell disease (SCD) are costly and contribute to a low quality of life for patients. We implemented a clinical pathway to safely discharge SCD patients with fever who are evaluated in the emergency department (ED) of a large tertiary care center. METHODS: An interdisciplinary team of ED and hematology physicians, nurses, and an improvement advisor developed a clinical pathway that identified febrile SCD patients at low risk of serious bacterial infection based on historical, clinical, and laboratory criteria who could be discharged from the ED. Phone follow-up was planned through the use of an automated electronic notification that was sent to an established hematology follow-up pool at the time of ED discharge. We conducted two "fake front end" trials in the ED to receive feedback on our process before full implementation. A postpathway implementation quality improvement team monitored discharge rates, phone follow-up rates and adverse events. RESULTS: In the first 9 weeks postpathway implementation, 100 SCD patients were evaluated for fever; 84 (24%) met low-risk criteria and were discharged home. This reduction in admission rate has been maintained throughout the 3 years postimplementation. Successful phone follow-up was achieved in all discharged patients within 24 hours and no adverse events were identified. CONCLUSIONS: Low-risk febrile patients with SCD can be safely discharged from the ED. An automated notification system within the electronic medical record system can facilitate patient follow-up after ED discharge. Future quality improvement efforts aimed to further reduce admissions in this population should target patients with modifiable risk factors for serious bacterial infection.

The ubiquitin proteasome system in atrophying skeletal muscle: roles and regulation.

Muscle atrophy complicates many diseases as well as aging, and its presence predicts both decreased quality of life and survival. Much work has been conducted to define the molecular mechanisms involved in maintaining protein homeostasis in muscle. To date, the ubiquitin proteasome system (UPS) has been shown to play an important role in mediating muscle wasting. In this review, we have collated the enzymes in the UPS whose roles in muscle wasting have been confirmed through loss-of-function studies. We have integrated information on their mechanisms of action to create a model of how they work together to produce muscle atrophy. These enzymes are involved in promoting myofibrillar disassembly and degradation, activation of autophagy, inhibition of myogenesis as well as in modulating the signaling pathways that control these processes. Many anabolic and catabolic signaling pathways are involved in regulating these UPS genes, but none appear to coordinately regulate a large number of these genes. A number of catabolic signaling pathways appear to instead function by inhibition of the insulin/IGF-I/protein kinase B anabolic pathway. This pathway is a critical determinant of muscle mass, since it can suppress key ubiquitin ligases and autophagy, activate protein synthesis, and promote myogenesis through its downstream mediators such as forkhead box O, mammalian target of rapamycin, and GSK3ß, respectively. Although much progress has been made, a more complete inventory of the UPS genes involved in mediating muscle atrophy, their mechanisms of action, and their regulation will be useful for identifying novel therapeutic approaches to this important clinical problem.

Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

Hedgehog signaling regulates MyoD expression and activity.

The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

CyTOF protocol for immune monitoring of solid tumors from mouse models.

Techniques for robust immune profiling of mouse tumor and blood are key to understanding immunological responses in mouse models of cancer. Here, we describe mass cytometry (cytometry by time-of-flight) procedures to facilitate high-parameter profiling of low-volume survival blood samples and end-of-study tumor samples. We employ live-cell barcoding systems to mark all cells from each tumor and blood to improve cost-effectiveness and minimize batch effects. For complete details on the use and execution of this protocol, please refer to Charmsaz et al. (2021).1.

Preventing NK cell activation in the damaged liver induced by cabozantinib/PD-1 blockade increases survival in hepatocellular carcinoma models.

The addition of anti-VEGF antibody treatment to immune checkpoint blockade (ICB) has increased the efficacy of immunotherapy in advanced hepatocellular carcinoma (HCC). Despite an initial promise, adding multitargeted kinase inhibitors of VEGFR with ICB has failed to increase survival in HCC. To reveal the mechanisms underlying treatment failure, we studied the effects of cabozantinib/ICB using orthotopic murine HCC models with or without liver damage. We monitored tumor growth and liver function, recorded survival outcomes, and performed immune profiling studies for intra-tumoral and surrounding liver. Cabozantinib/ICB treatment led to tumor regression and significantly improved survival in mice with normal livers. However, consistent with the clinical findings, combination therapy failed to show survival benefits despite similar tumor control when tested in the same models but in mice with liver fibrosis. Moreover, preclinical and clinical data converged, showing that activating immune responses by cabozantinib/ICB treatment induced hepatoxicity. Immune profiling revealed that combination therapy effectively reprogrammed the tumor immune microenvironment and increased NK cell infiltration and activation in the damaged liver tissue. Surprisingly, systemic depletion of NK reduced hepatotoxicity elicited by the combination therapy without compromising its anti-cancer effect, and significantly enhanced the survival benefit even in mice with HCC and underlying liver fibrosis. These findings demonstrate that preventing NK activation allowed for maintaining a favorable therapeutic ratio when combining ICB with cabozantinib in advanced HCC models.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.

Implementation of a learning healthcare system for sickle cell disease.

OBJECTIVE: Using sickle cell disease (SCD) as a model, the objective of this study was to create a comprehensive learning healthcare system to support disease management and research. A multidisciplinary team developed a SCD clinical data dictionary to standardize bedside data entry and inform a scalable environment capable of converting complex electronic healthcare records (EHRs) into knowledge accessible in real time. MATERIALS AND METHODS: Clinicians expert in SCD care developed a data dictionary to describe important SCD-associated health maintenance and adverse events. The SCD data dictionary was deployed in the EHR using EPIC SmartForms, an efficient bedside data entry tool. Additional data elements were extracted from the EHR database (Clarity) using Pentaho Data Integration and stored in a data analytics database (SQL). A custom application, the Sickle Cell Knowledgebase, was developed to improve data analysis and visualization. Utilization, accuracy, and completeness of data entry were assessed. RESULTS: The SCD Knowledgebase facilitates generation of patient-level and aggregate data visualization, driving the translation of data into knowledge that can impact care. A single patient can be selected to monitor health maintenance, comorbidities, adverse event frequency and severity, and medication dosing/adherence. CONCLUSIONS: Disease-specific data dictionaries used at the bedside will ultimately increase the meaningful use of EHR datasets to drive consistent clinical data entry, improve data accuracy, and support analytics that will facilitate quality improvement and research.

Knockout of USP19 Deubiquitinating Enzyme Prevents Muscle Wasting by Modulating Insulin and Glucocorticoid Signaling.

Muscle atrophy arises because of many chronic illnesses, as well as from prolonged glucocorticoid treatment and nutrient deprivation. We previously demonstrated that the USP19 deubiquitinating enzyme plays an important role in chronic glucocorticoid- and denervation-induced muscle wasting. However, the mechanisms by which USP19 exerts its effects remain unknown. To explore this further, we fasted mice for 48 hours to try to identify early differences in the response of wild-type and USP19 knockout (KO) mice that could yield insights into the mechanisms of USP19 action. USP19 KO mice manifested less myofiber atrophy in response to fasting due to increased rates of protein synthesis. Insulin signaling was enhanced in the KO mice, as revealed by lower circulating insulin levels, increased insulin-stimulated glucose disposal and phosphorylation of Akt and S6K in muscle, and improved overall glucose tolerance. Glucocorticoid signaling, which is essential in many conditions of atrophy, was decreased in KO muscle, as revealed by decreased expression of glucocorticoid receptor (GR) target genes upon both fasting and glucocorticoid treatment. This decreased GR signaling was associated with lower GR protein levels in the USP19 KO muscle. Restoring the GR levels in USP19-deficient muscle was sufficient to abolish the protection from myofiber atrophy. Expression of GR target genes also correlated with that of USP19 in human muscle samples. Thus, USP19 modulates GR levels and in so doing may modulate both insulin and glucocorticoid signaling, two critical pathways that control protein turnover in muscle and overall glucose homeostasis.

The business of deubiquitination - location, location, location.

A majority of proteins in the cell can be modified by ubiquitination, thereby altering their function or stability. This ubiquitination is controlled by both ubiquitinating and deubiquitinating enzymes (DUBs). The number of ubiquitin ligases exceeds that of DUBs by about eightfold, indicating that DUBs may have much broader substrate specificity. Despite this, DUBs have been shown to have quite specific physiological functions. This functional specificity is likely due to very precise regulation of activity arising from the sophisticated use of all mechanisms of enzyme regulation. In this commentary, we briefly review key features of DUBs with more emphasis on regulation. In particular, we focus on localization of the enzymes as a critical regulatory mechanism which when integrated with control of expression, substrate activation, allosteric regulation, and post-translational modifications results in precise spatial and temporal deubiquitination of proteins and therefore specific physiological functions. Identification of compounds that target the structural elements in DUBs that dictate localization may be a more promising approach to development of drugs with specificity of action than targeting the enzymatic activity, which for most DUBs is dependent on a thiol group that can react non-specifically with many compounds in large-scale screening.

USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling.

The USP19 deubiquitinating enzyme modulates the expression of myogenin and myofibrillar proteins in L6 muscle cells. This raised the possibility that USP19 might regulate muscle cell differentiation. We therefore tested the effects of adenoviral-mediated overexpression or small interfering RNA (siRNA)-mediated silencing of either the cytoplasmic or endoplasmic reticulum (ER)-localized isoforms of USP19. Only the ER-localized isoform of USP19 (USP19-ER) modulated myoblast fusion as well as the expression of myogenin and myofibrillar proteins, and these effects were also dependent on USP19 catalytic activity. USP19-ER inhibited muscle cell differentiation and the induction of CHOP, a transcription factor in the unfolded-protein response (UPR) that is activated during differentiation. Inducing the UPR by creating mild ER stress with thapsigargin was able to reverse the defect in myoblast fusion caused by the overexpression of USP19-ER, suggesting strongly that USP19 exerts its effects on fusion through its effects on UPR signaling. USP19 also functions similarly in vivo, as USP19(-/-) mice display improved muscle regeneration concomitant with enhanced expression of CHOP. Collectively these results implicate a deubiquitinating enzyme as a regulator of the UPR. They also suggest that inhibition of USP19 may be a therapeutic approach for the enhancement of muscle growth following injury.