Correlating neighborhood-level deprivation and pediatric metabolic dysfunction-associated steatotic liver disease.

OBJECTIVES: The primary objective of this study is to further explore associations between social influencers of health and markers of disease severity at the time of presentation of patients with pediatric metabolic dysfunction-associated steatotic liver disease (MASLD) using neighborhood-level Area Deprivation Index (ADI) scores. METHODS: A retrospective cross-sectional study was conducted among 344 pediatric MASLD patients. Each patient received an ADI score based on their 9-digit zip code. Groups were defined as low (≤5) and high (6≥) ADI. Associations between ADI and symptomatology and laboratory values at presentation, as well as initial liver biopsy pathology were tested via analyses of covariance, χ2 testing, and logistic regressions. RESULTS: The mean ADI was 6.54 (standard deviation = 2.09). ADI groups did not significantly differ in age of presentation, type of presentation, or associated conditions, except for the higher ADI group having on average lower vitamin D levels (26.70 vs. 29.91, p = 0.02) and being two times more likely to also be diagnosed with low high-density lipoprotein (HDL) levels (p = 0.04, 95% CI 1.04-3.89). Mean transaminases and histopathologic nonalcoholic fatty liver disease (NAFLD) Activity Scores did not significantly differ between ADI groups. CONCLUSIONS: Pediatric patients with MASLD in this study span the entire spectrum of neighborhood deprivation. There were no differences in presentation characteristics or severity of MASLD, aside from lower HDL-cholesterol and vitamin D values in the high ADI group. Area deprivation was not predictive of more severe disease as assessed by serum transaminases and liver biopsy NAFLD activity scores.

Cell Death in Liver Disease and Liver Surgery.

Cell death is crucial for maintaining tissue balance and responding to diseases. However, under pathological conditions, the surge in dying cells results in an overwhelming presence of cell debris and the release of danger signals. In the liver, this gives rise to hepatic inflammation and hepatocellular cell death, which are key factors in various liver diseases caused by viruses, toxins, metabolic issues, or autoimmune factors. Both clinical and in vivo studies strongly affirm that hepatocyte death serves as a catalyst in the progression of liver disease. This advancement is characterized by successive stages of inflammation, fibrosis, and cirrhosis, culminating in a higher risk of tumor development. In this review, we explore pivotal forms of cell death, including apoptosis, pyroptosis, and necroptosis, examining their roles in both acute and chronic liver conditions, including liver cancer. Furthermore, we discuss the significance of cell death in liver surgery and ischemia-reperfusion injury. Our objective is to illuminate the molecular mechanisms governing cell death in liver diseases, as this understanding is crucial for identifying therapeutic opportunities aimed at modulating cell death pathways.

Extracellular Vesicles as Therapeutic and Diagnostic Tools for Chronic Liver Diseases.

Chronic liver diseases can lead to fibrotic changes that may progress to the development of cirrhosis, which poses a significant risk for morbidity and increased mortality rates. Metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), and viral hepatitis are prevalent liver diseases that may lead to cirrhosis. The advanced stages of cirrhosis can be further complicated by cancer development or end-stage liver disease and liver failure. Hence, early detection and diagnosis of liver fibrosis is crucial for preventing the progression to cirrhosis and improving patient outcomes. Traditionally, invasive liver biopsy has been considered the gold standard for diagnosing and staging liver fibrosis. In the last decade, research has focused on non-invasive methods, known as liquid biopsies, which involve the identification of disease-specific biomarkers in human fluids, such as blood. Among these alternative approaches, extracellular vesicles (EVs) have emerged as promising diagnostic and therapeutic tools for various diseases, including chronic liver diseases. EVs are released from stressed or damaged cells and can be isolated and quantified. Moreover, EVs facilitate cell-to-cell communication by transporting various cargo, and they have shown the potential to reduce the expression of profibrogenic markers, making them appealing tools for novel anti-fibrotic treatments. This review focuses on the impact of EVs in chronic liver diseases and exploring their potential applications in innovative therapeutic and diagnostic approaches.

Pyroptosis and gasdermins-Emerging insights and therapeutic opportunities in metabolic dysfunction-associated steatohepatitis.

In recent years, there has been a rapid expansion in our understanding of regulated cell death, leading to the discovery of novel mechanisms that govern diverse cell death pathways. One recently discovered type of cell death is pyroptosis, initially identified in the 1990s as a caspase-1-dependent lytic cell death. However, further investigations have redefined pyroptosis as a regulated cell death that relies on the activation of pore-forming proteins, particularly the gasdermin family. Among the key regulators of pyroptosis is the inflammasome sensor NOD-like receptor 3 (NLRP3), a critical innate immune sensor responsible for regulating the activation of caspase-1 and gasdermin D. A deeper understanding of pyroptosis and its interplay with other forms of regulated cell death is emerging, shedding light on a complex regulatory network controlling pore-forming proteins and cell fate. Cell death processes play a central role in diseases such as metabolic dysfunction-associated steatotic liver disease, metabolic dysfunction-associated steatohepatitis, autoinflammatory disorders, and cancer. Cell death often acts as a starting point in these diseases, making it an appealing target for drug development. Yet, the complete molecular mechanisms are not fully understood, and new discoveries reveal promising novel avenues for therapeutic interventions. In this review, we summarize recent evidence on pathways and proteins controlling pyroptosis and gasdermins. Furthermore, we will address the role of pyroptosis and the gasdermin family in metabolic dysfunction-associated steatotic liver disease and steatohepatitis. Additionally, we highlight new potential therapeutic targets for treating metabolic dysfunction-associated steatohepatitis and other inflammatory-associated diseases.

Antisense Oligonucleotide Therapy Decreases IL-1ß Expression and Prolongs Survival in Mutant Nlrp3 Mice.

Antisense oligonucleotides (ASOs) are a novel therapeutic strategy that targets a specific gene and suppresses its expression. The cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory diseases characterized by systemic and tissue inflammation that is caused by heterozygous gain-of-function mutations in the nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) gene. The aim of this study was to investigate the efficacy of an Nlrp3-specific ASO treatment in CAPS. An Nlrp3-specific ASO was designed and tested in murine cell lines and bone marrow-derived macrophages (BMDMs) from wild-type and CAPS mouse models. Nlrp3 knock-in mice were treated in vivo with Nlrp3-specific ASO, survival was monitored, and expression of organ-specific Nlrp3 and IL-1ß was measured. Nlrp3-specific ASO treatment of murine cell lines and BMDMs showed a significant downregulation of Nlrp3 and mature IL-1ß protein expression. Ex vivo treatment of Nlrp3 mutant mouse-derived BMDMs with Nlrp3-specific ASO demonstrated significantly reduced IL-1ß release. In vivo, Nlrp3-specific ASO treatment of Nlrp3 mutant mice prolonged survival, reduced systemic inflammation, and decreased tissue-specific expression of Nlrp3 and mature IL-1ß protein. The results of this study demonstrate that Nlrp3-specific ASO treatment downregulates Nlrp3 expression and IL-1ß release in CAPS models, suggesting ASO therapy as a potential treatment of CAPS and other NLRP3-mediated diseases.

Cell-specific Deletion of NLRP3 Inflammasome Identifies Myeloid Cells as Key Drivers of Liver Inflammation and Fibrosis in Murine Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. The NLRP3 inflammasome, a platform for caspase-1 activation and release of interleukin 1ß, is increasingly recognized in the induction of inflammation and liver fibrosis during NAFLD. However, the cell-specific contribution of NLRP3 inflammasome activation in NAFLD remains unknown. METHODS: To investigate the role of NLRP3 inflammasome activation in hepatocytes, hepatic stellate cells (HSCs) and myeloid cells, a conditional Nlrp3 knock-out mouse was generated and bred to cell-specific Cre mice. Both acute and chronic liver injury models were used: lipopolysaccharide/adenosine-triphosphate to induce in vivo NLRP3 activation, choline-deficient, L-amino acid-defined high-fat diet, and Western-type diet to induce fibrotic nonalcoholic steatohepatitis (NASH). In vitro co-culture studies were performed to dissect the crosstalk between myeloid cells and HSCs. RESULTS: Myeloid-specific deletion of Nlrp3 blunted the systemic and hepatic increase in interleukin 1ß induced by lipopolysaccharide/adenosine-triphosphate injection. In the choline-deficient, L-amino acid-defined high-fat diet model of fibrotic NASH, myeloid-specific Nlrp3 knock-out but not hepatocyte- or HSC-specific knock-out mice showed significant reduction in inflammation independent of steatosis development. Moreover, myeloid-specific Nlrp3 knock-out mice showed ameliorated liver fibrosis and decreased HSC activation. These results were validated in the Western-type diet model. In vitro co-cultured studies with human cell lines demonstrated that HSC can be activated by inflammasome stimulation in monocytes, and this effect was significantly reduced if NLRP3 was downregulated in monocytes. CONCLUSIONS: The study provides new insights in the cell-specific role of NLRP3 in liver inflammation and fibrosis. NLRP3 inflammasome activation in myeloid cells was identified as crucial for the progression of NAFLD to fibrotic NASH. These results may have implications for the development of cell-specific strategies for modulation of NLRP3 activation for treatment of fibrotic NASH.

Dual role of neutrophils in modulating liver injury and fibrosis during development and resolution of diet-induced murine steatohepatitis.

Inflammatory changes in the liver represent a key feature of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD). Innate immune activation including hepatic neutrophilic infiltration acts as an important inflammatory trigger as well as a potential mediator of inflammation resolution. In this study, we dissected the effects of neutrophil depletion via anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies administration during ongoing high fat-fructose-cholesterol (FFC) diet-induced murine NASH and during inflammation resolution by switching into a low-fat control diet. During NASH progression, protective effects were shown as HSC activation, cell infiltration and activation of pro-inflammatory macrophages were ameliorated. Furthermore, these changes were contrasted with the effects observed when neutrophil depletion was performed during the resolution phase. Impaired resolving mechanisms, such as a failure to balance the pro and anti-inflammatory cytokines ratio, deficient macrophage phenotypic switch into a pro-restorative profile, and defective repair and remodeling processes were observed when neutrophils were depleted in this scenario. This study described phase-dependent contrasting roles of neutrophils as triggers and pro-resolutive mediators of liver injury and fibrosis associated with diet-induced NASH in mice. These findings have important translational implications at the time of designing NASH therapeutic strategies.

Novel Mechanisms for Resolution of Liver Inflammation: Therapeutic Implications.

Traditional concepts have classically viewed resolution of inflammation as a passive process yet insight into the pathways by which inflammation is resolved has challenged this idea. Resolution has been revealed as a highly dynamic and active event that is essential to counteract the dysregulated inflammatory response that drives diverse disease states. Abrogation of the hepatic inflammatory response through the stimulation of proresolving mechanisms represents a new paradigm in the setting of chronic inflammatory-driven liver diseases. Elucidation of the role of different cells of the innate and adaptive immune system has highlighted the interplay between them as an important orchestrator of liver repair. A finely tuned interaction between neutrophils and macrophages has risen as revolutionary mechanism that drives the restoration of hepatic function and architecture. Specialized proresolving mediators have also been shown to act as stop signals of the inflammatory response and promote resolution as well as tissue regeneration. In this review, we discuss the discovery and understanding of the mechanisms by which inflammation is resolved and highlight novel proresolving pathways that represent promising therapeutic strategies.

Dynamic Shifts in the Composition of Resident and Recruited Macrophages Influence Tissue Remodeling in NASH.

Macrophage-mediated inflammation is critical in the pathogenesis of non-alcoholic steatohepatitis (NASH). Here, we describe that, with high-fat, high-sucrose-diet feeding, mature TIM4pos Kupffer cells (KCs) decrease in number, while monocyte-derived Tim4neg macrophages accumulate. In concert, monocyte-derived infiltrating macrophages enter the liver and consist of a transitional subset that expresses Cx3cr1/Ccr2 and a second subset characterized by expression of Trem2, Cd63, Cd9, and Gpmnb; markers ascribed to lipid-associated macrophages (LAMs). The Cx3cr1/Ccr2-expressing macrophages, referred to as C-LAMs, localize to macrophage aggregates and hepatic crown-like structures (hCLSs) in the steatotic liver. In C-motif chemokine receptor 2 (Ccr2)-deficient mice, C-LAMs fail to appear in the liver, and this prevents hCLS formation, reduces LAM numbers, and increases liver fibrosis. Taken together, our data reveal dynamic changes in liver macrophage subsets during the pathogenesis of NASH and link these shifts to pathologic tissue remodeling.

Mechanisms of nonalcoholic fatty liver disease and implications for surgery.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease in both adults and children worldwide. Understanding the pathogenic mechanisms behind NAFLD provides the basis for identifying risk factors, such as metabolic syndrome, pancreatoduodenectomy, and host genetics, that lead to the onset and progression of the disease. The progression from steatosis to more severe forms, such as steatohepatitis, fibrosis, and cirrhosis, leads to an increased number of liver and non-liver complications. PURPOSE: NAFLD-associated end-stage liver disease (ESLD) and hepatocellular carcinoma (HCC) often require surgery as the only curative treatment. In particular, the presence of NAFLD together with the coexisting metabolic comorbidities that usually occur in these patients requires careful preoperative diagnosis and peri-/postoperative management. Bariatric surgery, liver resection, and liver transplantation (LT) have shown favorable results for weight loss, HCC, and ESLD in patients with NAFLD. The LT demand and the increasing spread of NAFLD in the donor pool reinforce the already existing lack of donor organs. CONCLUSION: In this review, we will discuss the diverse mechanisms underlying NAFLD, its implications for surgery, and the challenges for patient management.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Characterization and Proteome of Circulating Extracellular Vesicles as Potential Biomarkers for NASH.

Nonalcoholic fatty liver disease (NAFLD) is currently one of most common forms of chronic liver disease globally. NAFLD represents a wide spectrum of liver involvement from nonprogressive isolated steatosis to nonalcoholic steatohepatitis (NASH), characterized by liver necroinflammation and fibrosis and currently one of the top causes of end-stage liver disease and hepatocellular carcinoma. At present, there is a lack of effective treatments, and a central barrier to the development of therapies is the requirement for an invasive liver biopsy for diagnosis of NASH. Discovery of reliable, noninvasive biomarkers are urgently needed. In this study, we tested whether circulating extracellular vesicles (EVs), cell-derived small membrane-surrounded structures with a rich cargo of bioactive molecules, may serve as reliable noninvasive "liquid biopsies" for NASH diagnosis and assessment of disease severity. Total circulating EVs and hepatocyte-derived EVs were isolated by differential centrifugation and size-exclusion chromatography from serum samples of healthy individuals, patients with precirrhotic NASH, and patients with cirrhotic NASH. EVs were further characterized by flow cytometry, electron microscopy, western blotting, and dynamic light scattering assays before performing a proteomics analysis. Our findings suggest that levels of total and hepatocyte-derived EVs correlate with NASH clinical characteristics and disease severity. Additionally, using proteomics data, we developed understandable, powerful, and unique EV-based proteomic signatures for potential diagnosis of advanced NASH. Conclusion: Our study shows that the quantity and protein constituents of circulating EVs provide strong evidence for EV protein-based liquid biopsies for NAFLD/NASH diagnosis.

Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1ß and IL-17 upregulation in alcoholic hepatitis mice.

Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)-seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1ß and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1ß and IL-17 by HMs. KEY MESSAGES: • Circulating EVs and HC-EVs were increased in AH mice compared with control mice • AH-HC-EVs were enriched in miRNAs, organelle proteins, and mitochondrial DNA • AH-HC-EVs increased cytokine production by AH-HMs in a TLR9-dependent manner.

The NLRP3 Inflammasome in Alcoholic and Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) and alcoholic hepatitis (ASH) are advanced forms of fatty liver diseases that are associated with a high morbidity and mortality worldwide. Patients with ASH or NASH are more susceptible to the progression of fibrosis and cirrhosis up to the development of hepatocellular carcinoma. Currently, there are limited medical therapies available. Accompanied by the asymptomatic disease progression, the demand for liver transplants is high. This review provides an overview about the growing evidence for a central role of NLR family pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex that acts as a central driver of inflammation via activation of caspase 1, maturation and release of pro-inflammatory cytokines including interleukin-1ß, and trigger of inflammatory pyroptotic cell death in both NASH and ASH. We also discuss potential therapeutic approaches targeting NLRP3 inflammasome and related upstream and downstream pathways to develop prognostic biomarkers and medical treatments for both liver diseases.

MicroRNA 223 3p Negatively Regulates the NLRP3 Inflammasome in Acute and Chronic Liver Injury.

The granulocyte-specific microRNA-223 (miR-223) has recently emerged as a negative regulator of NOD-like receptor 3 (NLRP3) expression, a central key player in chronic hepatic injuries such as fibrotic nonalcoholic steatohepatitis (NASH), as well as in other liver conditions including acute hepatitis. In this study, we evaluated the therapeutic effect of the synthetic miR-223 analog miR-223 3p in a murine model of lipopolysaccharide (LPS)/D-GalN-induced endotoxin acute hepatitis (EAH) or fibrotic NASH resultant of long-term feeding with a high-fat, fructose, and cholesterol (FFC) diet. miR-223 3p ameliorated the infiltration of monocytes, neutrophils, and early activated macrophages and downregulated the transcriptional expression of the pro-inflammatory cytokines Il6 and Il12 and the chemokines Ccl2, Ccl3, Cxcl1, and Cxcl2 in EAH. In fibrotic NASH, treatment with miR-223 3p led to a remarkable mitigation of fibrosis development and activation of hepatic stellate cells (HSCs). miR-223 3p disrupted the activation of the NLRP3 inflammasome by impairing the synthesis of cleaved interleukin-1ß (IL-1ß), mature IL-1ß, and NLRP3, and the activation of caspase-1 p10 in both EAH and fibrotic NASH. Our data enlightens miR-223 3p as a post-transcriptional approach to treat acute and chronic hepatitis by silencing the activation of the NLRP3 inflammasome.

Identification of actin network proteins, talin-1 and filamin-A, in circulating extracellular vesicles as blood biomarkers for human myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, debilitating disorder with a wide spectrum of symptoms, including pain, depression, and neurocognitive deterioration. Over 17 million people around the world have ME/CFS, predominantly women with peak onset at 30-50 years. Given the wide spectrum of symptoms and unclear etiology, specific biomarkers for diagnosis and stratification of ME/CFS are lacking. Here we show that actin network proteins in circulating extracellular vesicles (EVs) offer specific non-invasive biomarkers for ME/CFS. We found that circulating EVs were significantly increased in ME/CFS patients correlating to C-reactive protein, as well as biological antioxidant potential. Area under the receiver operating characteristic curve for circulating EVs was 0.80, allowing correct diagnosis in 90-94% of ME/CFS cases. From two independent proteomic analyses using circulating EVs from ME/CFS, healthy controls, idiopathic chronic fatigue, and depression, proteins identified from ME/CFS patients are involved in focal adhesion, actin skeletal regulation, PI3K-Akt signaling pathway, and Epstein-Barr virus infection. In particular, talin-1, filamin-A, and 14-3-3 family proteins were the most abundant proteins, representing highly specific ME/CFS biomarkers. Our results identified circulating EV number and EV-specific proteins as novel biomarkers for diagnosing ME/CFS, providing important information on the pathogenic mechanisms of ME/CFS.

Neutrophils contribute to spontaneous resolution of liver inflammation and fibrosis via microRNA-223.

Persistent, unresolved inflammation in the liver represents a key trigger for hepatic injury and fibrosis in various liver diseases and is controlled by classically activated pro-inflammatory macrophages, while restorative macrophages of the liver are capable of reversing inflammation once the injury trigger ceases. Here we have identified a novel role for neutrophils as key contributors to resolving the inflammatory response in the liver. Using two models of liver inflammatory resolution, we found that mice undergoing neutrophil depletion during the resolution phase exhibited unresolved hepatic inflammation, activation of the fibrogenic machinery and early fibrosis. These findings were associated with an impairment of the phenotypic switch of pro-inflammatory macrophages into a restorative stage after removal of the cause of injury and an increased NLRP3 / miR-223 ratio. Mice with a deletion of the granulocyte specific miR-223 gene showed a similarly impaired resolution profile that could be reversed by restoring miR-223 levels using a miR-223 3p mimic or infusing neutrophils from wildtype animals. Collectively, our findings reveal a novel role for neutrophils in the liver as resolving effector cells that induce pro-inflammatory macrophages into a restorative phenotype, potentially via miR-223.

NAFLD in children: new genes, new diagnostic modalities and new drugs.

Nonalcoholic fatty liver disease (NAFLD) has rapidly become the most common form of chronic liver disease in children and adolescents. Over the past 5 years, developments have revolutionized our understanding of the genetic factors, natural history, diagnostic modalities and therapeutic targets for this disease. New polymorphisms, such as those in PNPLA3, TM6SF2, MBOAT7 and GCKR, have been identified and used to predict the development and severity of NAFLD in both adults and children, and their interaction with environmental factors has been elucidated. Studies have demonstrated the true burden of paediatric NAFLD and its progression to end-stage liver disease in adulthood. In particular, nonalcoholic steatohepatitis can progress to advanced fibrosis and cirrhosis, emphasizing the importance of early diagnosis. Non-invasive imaging tests, such as transient elastography, will probably replace liver biopsy for the diagnosis of nonalcoholic steatohepatitis and the assessment of fibrosis severity in the near future. The therapeutic landscape is also expanding rapidly with the development of drugs that can modify liver steatosis, inflammation and fibrosis, indicating that pharmacotherapy for NAFLD will become available in the future. In this Review, we summarize current knowledge and new advances related to the pathogenesis and management of paediatric NAFLD.

NLR Family Pyrin Domain-Containing 3 Inflammasome Activation in Hepatic Stellate Cells Induces Liver Fibrosis in Mice.

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.