Roles of Macrophages in Advanced Liver Fibrosis,Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells. CD11c⁺ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c⁺ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C⁺ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c⁺/Ly6C⁻ cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c⁻/Ly6C⁺ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.     

Peptide-Based Nanoarchitectonics for the Treatment of Liver Fibrosis

Liver fibrosis is a process of excessive accumulation of extracellular matrix caused by liver injury. Liver fibrosis can progress to cirrhosis or even liver cancer without proper intervention. Until now, no effective therapeutic drugs have been clinically approved for treating liver fibrosis. Hence, the development of safe and effective antifibrotic drugs is particularly important. As a representative biomaterial, peptides have been investigated as key components for constructing antifibrotic nanomaterials given their advantages of biological origination, synthetic availability, and good biocompatibility. Peptides serve as multifunctional motifs in antifibrotic nanomaterials, such as liver-targeting molecules, antifibrotic molecules, and self-assembling building blocks for the formation of the nanomaterials. In this review, we focus on peptide-based nanoarchitectonics for treating liver fibrosis, including nanomaterials modified with liver-targeting peptides, nanomaterials for the efficient delivery of antifibrotic peptides, and self-assembled peptide nanomaterials for the delivery of antifibrotic drugs. The design rules of these peptide-based nanomaterials are described. The antifibrotic mechanisms and effects of these peptide-based nanomaterials in treating liver fibrosis and related diseases are highlighted. The challenges and future perspectives of using peptide-based nanoarchitectonics for the treatment of liver fibrosis are discussed. These results are expected to accelerate the rational design and clinical translation of antifibrotic nanomaterials.     

Potential Role of Thymosin Beta 4 in Liver Fibrosis

Liver fibrosis, the main characteristic of chronic liver diseases, is strongly associated with the activation of hepatic stellate cells (HSCs), which are responsible for extracellular matrix production. As such, investigating the effective regulators controlling HSC activation provides important clues for developing therapeutics to inhibit liver fibrosis. Thymosin beta 4 (Tβ4), a major actin-sequestering protein, is known to be involved in various cellular responses. A growing body of evidence suggests that Tβ4 has a potential role in the pathogenesis of liver fibrosis and that it is especially associated with the activation of HSCs. However, it remains unclear whether Tβ4 promotes or suppresses the activation of HSCs. Herein, we review the potential role of Tβ4 in liver fibrosis by describing the effects of exogenous and endogenous Tβ4, and we discuss the possible signaling pathway regulated by Tβ4. Exogenous Tβ4 reduces liver fibrosis by inhibiting the proliferation and migration of HSCs. Tβ4 is expressed endogenously in the activated HSCs, but this endogenous Tβ4 displays opposite effects in HSC activation, either as an activator or an inhibitor. Although the role of Tβ4 has not been established, it is apparent that Tβ4 influences HSC activation, suggesting that Tβ4 is a potential therapeutic target for treating liver diseases.     

The Molecular Mechanisms of Liver Fibrosis and Its Potential Therapy in Application

Liver fibrosis results from repeated and persistent liver damage. It can start with hepatocyte injury and advance to inflammation, which recruits and activates additional liver immune cells, leading to the activation of the hepatic stellate cells (HSCs). It is the primary source of myofibroblasts (MFs), which result in collagen synthesis and extracellular matrix protein accumulation. Although there is no FDA and EMA-approved anti-fibrotic drug, antiviral therapy has made remarkable progress in preventing or even reversing the progression of liver fibrosis, but such a strategy remains elusive for patients with viral, alcoholic or nonalcoholic steatosis, genetic or autoimmune liver disease. Due to the complexity of the etiology, combination treatments affecting two or more targets are likely to be required. Here, we review the pathogenic mechanisms of liver fibrosis and signaling pathways involved, as well as various molecular targets for liver fibrosis treatment. The development of efficient drug delivery systems that target different cells in liver fibrosis therapy is also summarized. We highlight promising anti-fibrotic events in clinical trial and preclinical testing, which include small molecules and natural compounds. Last, we discuss the challenges and opportunities in developing anti-fibrotic therapies.     

The Role of Peritoneal Macrophages in Endometriosis

Endometriosis is an estrogen-dependent gynecological disorder, defined as the growth of endometrial stromal cells and glands at extrauterine sites. Endometriotic lesions are more frequently located into the abdominal cavity, although they can also be implanted in distant places. Among its etiological factors, the presence of immune dysregulation occupies a prominent place, pointing out the beneficial and harmful outcomes of macrophages in the pathogenesis of this disease. Macrophages are tissue-resident cells that connect innate and adaptive immunity, playing a key role in maintaining local homeostasis in healthy conditions and being critical in the development and sustainment of many inflammatory diseases. Macrophages accumulate in the peritoneal cavity of women with endometriosis, but their ability to clear migrated endometrial fragments seems to be inefficient. Hence, the characteristics of the peritoneal immune system in endometriosis must be further studied to facilitate the search for new diagnostic and therapeutic tools. In this review, we summarize recent relevant advances obtained in both mouse, as the main animal model used to study endometriosis, and human, focusing on peritoneal macrophages obtained from endometriotic patients and healthy donors, under the perspective of its future clinical translation to the role that these cells play on this pathology.     

Diagnosis and Therapeutic Management of Liver Fibrosis by MicroRNA

Remarkable progress has been made in the treatment and control of hepatitis B and C viral infections. However, fundamental treatments for diseases in which liver fibrosis is a key factor, such as cirrhosis, alcoholic/nonalcoholic steatohepatitis, autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis, are still under development and remain an unmet medical need. To solve this problem, it is essential to elucidate the pathogenesis of liver fibrosis in detail from a molecular and cellular perspective and to develop targeted therapeutic agents based on this information. Recently, microRNAs (miRNAs), functional RNAs of 22 nucleotides, have been shown to be involved in the pathogenesis of liver fibrosis. In addition, extracellular vesicles called “exosomes” have been attracting attention, and research is being conducted to establish noninvasive and extremely sensitive biomarkers using miRNAs in exosomes. In this review, we summarize miRNAs directly involved in liver fibrosis, miRNAs associated with diseases leading to liver fibrosis, and miRNAs related to complications of cirrhosis. We will also discuss the efficacy of each miRNA as a biomarker of liver fibrosis and pathology, and its potential application as a therapeutic agent.     

Role of Macrophages and RhoA Pathway in Atherosclerosis

The development, progression, or stabilization of the atherosclerotic plaque depends on the pro-inflammatory and anti-inflammatory macrophages. The influx of the macrophages and the regulation of macrophage phenotype, inflammatory or anti-inflammatory, are controlled by the small GTPase RhoA and its downstream effectors. Therefore, macrophages and the components of the RhoA pathway are attractive targets for anti-atherosclerotic therapies, which would inhibit macrophage influx and inflammatory phenotype, maintain an anti-inflammatory environment, and promote tissue remodeling and repair. Here, we discuss the recent findings on the role of macrophages and RhoA pathway in the atherosclerotic plaque formation and resolution and the novel therapeutic approaches.     

Liver Injury and the Macrophage Issue: Molecular and Mechanistic Facts and Their Clinical Relevance

The liver is an essential immunological organ due to its gatekeeper position to bypassing antigens from the intestinal blood flow and microbial products from the intestinal commensals. The tissue-resident liver macrophages, termed Kupffer cells, represent key phagocytes that closely interact with local parenchymal, interstitial and other immunological cells in the liver to maintain homeostasis and tolerance against harmless antigens. Upon liver injury, the pool of hepatic macrophages expands dramatically by infiltrating bone marrow-/monocyte-derived macrophages. The interplay of the injured microenvironment and altered macrophage pool skews the subsequent course of liver injuries. It may range from complete recovery to chronic inflammation, fibrosis, cirrhosis and eventually hepatocellular cancer. This review summarizes current knowledge on the classification and role of hepatic macrophages in the healthy and injured liver.     

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A⁺ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A⁺INFγ⁺ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.     

The Role of Peritoneal Alternatively Activated Macrophages in the Process of Peritoneal Fibrosis Related to Peritoneal Dialysis

It has been confirmed that alternatively activated macrophages (M2) participate in tissue remodeling and fibrosis occurrence, but the effect of M2 on peritoneal fibrosis related to peritoneal dialysis (PD) hasn’t been elucidated. This study was therefore conducted to assess the association between M2 and peritoneal fibrosis related to PD. In this study, peritoneal fibrosis was induced by intraperitoneal (i.p.) injection of Lactate-4.25% dialysate (100 mL/kg) to C57BL/6J mice for 28 days, and liposome-encapsulated clodronate (LC, the specific scavenger of macrophages) was used to treat the peritoneal fibrosis mice model by i.p. injection at day 18 and day 21. All animals were sacrificed at day 29. Parietal peritonea were stained with Masson’s trichrome, and the expression of type I collagen (Col-I), fibronectin, mannose receptor (CD206), transforming growth factor beta (TGF-β), chemokine receptor 7 (CCR7), chitinase 3-like 3 (Ym-1) and arginase-1 (Arg-1) was determined by Western blotting, immunofluorescence and quantitative real-time PCR. Our results revealed that peritoneal thickness, Col-I, fibronectin, CD206, TGF-β, Ym-1 and Arg-1 were upregulated in the peritoneal fibrosis mice model, and all of these indexes were downregulated in those treated with LC. Additionally, there was no difference in the level of CCR7 between the model and treatment group. Our study indicated that peritoneal M2 played an important role in the process of peritoneal fibrosis related to PD and might be a potential target for intervention therapy of peritoneal fibrosis.     

MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

Chronic liver disease mediated by the activation of hepatic stellate cells (HSCs) leads to liver fibrosis. The signal adaptor MyD88 of Toll-like receptor (TLR) signaling is involved during the progression of liver fibrosis. However, the specific role of MyD88 in myeloid cells in liver fibrosis has not been thoroughly investigated. In this study, we used a carbon tetrachloride (CCl₄)-induced mouse fibrosis model in which MyD88 was selectively depleted in myeloid cells. MyD88 deficiency in myeloid cells attenuated liver fibrosis in mice and decreased inflammatory cell infiltration. Furthermore, deficiency of MyD88 in macrophages inhibits the secretion of CXC motif chemokine 2 (CXCL2), which restrains the activation of HSCs characterized by NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. Moreover, targeting CXCL2 by CXCR2 inhibitors attenuated the activation of HSCs and reduced liver fibrosis. Thus, MyD88 may represent a potential candidate target for the prevention and treatment of liver fibrosis.     

There Is Strength in Numbers: Quantitation of Fc Gamma Receptors on Murine Tissue-Resident Macrophages

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.     

Ivermectin Attenuates CCl4-Induced Liver Fibrosis in Mice by Suppressing Hepatic Stellate Cell Activation

Liver fibrosis, a common liver dysfunction with high morbidity and mortality rates, is the leading cause of cirrhosis and hepatocellular carcinoma, for which there are no effective therapies. Ivermectin is an antiparasitic drug that also has been showing therapeutic actions in many other diseases, including antiviral and anticancer actions, as well as treating metabolic diseases. Herein, we evaluated the function of ivermectin in regulating liver fibrosis. Firstly, carbon tetrachloride (CCl₄)-injected Balb/c mice were used to assess the antifibrosis effects of ivermectin in vivo. Further, CFSC, a rat hepatic stellate cell (HSC) line, was used to explore the function of ivermectin in HSC activation in vitro. The in vivo data showed that ivermectin administration alleviated histopathological changes, improved liver function, reduced collagen deposition, and downregulated the expression of profibrotic genes. Mechanistically, the ivermectin treatment inhibited intrahepatic macrophage accumulation and suppressed the production of proinflammatory factors. Importantly, the ivermectin administration significantly decreased the protein levels of α-smooth muscle actin (α-SMA) both in vivo and in vitro, suggesting that the antifibrotic effects of ivermectin are mainly due to the promotion of HSC deactivation. The present study demonstrates that ivermectin may be a potential therapeutic agent for the prevention of hepatic fibrosis.     

Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.     

The Role of the Gut Microbiome in Liver Cirrhosis Treatment

Liver cirrhosis is one of the most prevalent chronic liver diseases worldwide. In addition to viral hepatitis, diseases such as steatohepatitis, autoimmune hepatitis, sclerosing cholangitis and Wilson’s disease can also lead to cirrhosis. Moreover, alcohol can cause cirrhosis on its own and exacerbate chronic liver disease of other causes. The treatment of cirrhosis can be divided into addressing the cause of cirrhosis and reversing liver fibrosis. To this date, there is still no clear consensus on the treatment of cirrhosis. Recently, there has been a lot of interest in potential treatments that modulate the gut microbiota and gut-liver axis for the treatment of cirrhosis. According to recent studies, modulation of the gut microbiome by probiotics ameliorates the progression of liver disease. The precise mechanism for relieving cirrhosis via gut microbial modulation has not been identified. This paper summarizes the role and effects of the gut microbiome in cirrhosis based on experimental and clinical studies on absorbable antibiotics, probiotics, prebiotics, and synbiotics. Moreover, it provides evidence of a relationship between the gut microbiome and liver fibrosis.     

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl₄)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl₄-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (^bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl₄-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl₄-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.     

Phenotype Diversity of Macrophages in Osteoarthritis: Implications for Development of Macrophage Modulating Therapies

Chronic inflammation is implicated in numerous human pathologies. In particular, low-grade inflammation is currently recognized as an important mechanism of osteoarthritis (OA), at least in some patients. Among the signs of the inflammatory process are elevated macrophage numbers detected in the OA synovium compared to healthy controls. High macrophage counts also correlate with clinical symptoms of the disease. Macrophages are central players in the development of chronic inflammation, pain, cartilage destruction, and bone remodeling. However, macrophages are also involved in tissue repair and remodeling, including cartilage. Therefore, reduction of macrophage content in the joints correlates with deleterious effects in OA models. Macrophage population is heterogeneous and dynamic, with phenotype transitions being induced by a variety of stimuli. In order to effectively use the macrophage inflammatory circuit for treatment of OA, it is important to understand macrophage heterogeneity and interactions with surrounding cells and tissues in the joint. In this review, we discuss functional phenotypes of macrophages and specific targeting approaches relevant for OA treatment development.     

Role of Hepatic Progenitor Cells in Nonalcoholic Fatty Liver Disease Development: Cellular Cross-Talks and Molecular Networks

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of diseases ranging from simple fatty liver to nonalcoholic steatohepatitis, (NASH) which may progress to cirrhosis and hepatocellular carcinoma. NASH has been independently correlated with atherosclerosis progression and cardiovascular risk. NASH development is characterized by intricate interactions between resident and recruited cells that enable liver damage progression. The increasing general agreement is that the cross-talk between hepatocytes, hepatic stellate cells (HSCs) and macrophages in NAFLD has a main role in the derangement of lipid homeostasis, insulin resistance, danger recognition, immune tolerance response and fibrogenesis. Moreover, several evidences have suggested that hepatic stem/progenitor cell (HPCs) activation is a component of the adaptive response of the liver to oxidative stress in NAFLD. HPC activation determines the appearance of a ductular reaction. In NASH, ductular reaction is independently correlated with progressive portal fibrosis raising the possibility of a periportal fibrogenetic pathway for fibrogenesis that is parallel to the deposition of subsinusoidal collagen in zone 3 by HSCs. Recent evidences indicated that adipokines, a class of circulating factors, have a key role in the cross-talk among HSCs, HPCs and liver macrophages. This review will be focused on cellular cross-talk and the relative molecular networks which are at the base of NASH progression and fibrosis.     

Ficolin-2 Plasma Level Assesses Liver Fibrosis in Non-Alcoholic Fatty Liver Disease

Fibrosis is the strongest predictor for disease-specific mortality in non-alcoholic fatty liver diseases (NAFLD), but the need for liver biopsy limits its diagnosis. We assessed the performance of plasma ficolin-2 (FCN-2) as a biomarker of fibrosis identified by an in silico discovery strategy. Two hundred and thirty-five morbidly obese (MO) subjects with biopsy-proven NAFLD stratified by fibrosis stage (F0, n = 44; F1, n = 134; F2, n = 46; F3/F4, n = 11) and 40 cirrhotic patients were enrolled. The cohort was subdivided into discovery (n = 76) and validation groups (n = 159). The plasma level of FCN-2 and other candidate markers was determined. FCN-2 was inversely correlated with the stage of liver fibrosis (ρ = −0.49, p < 0.001) independently of steatosis (p = 0.90), inflammation (p = 0.57), and ballooning (p = 0.59). In the global cohort, FCN-2 level decreased significantly in a stepwise fashion from F0/F1 (median 4753 ng/mL) to F2–F3–F4 (2760 ng/mL) and in cirrhotic subjects (1418 ng/mL). The diagnostic performance of FCN-2 in detecting F ≥ 2 was higher than other indexes (APRI, FIB-4) (AUROC 0.82, 0.68, and 0.6, respectively). The accuracy improved when combined with APRI score and HDL values (FCNscore, AUROC 0.85). Overall, the FCN-2 plasma level can accurately discriminate liver fibrosis status (minimal vs. moderate/advanced) significantly improving the fibrosis diagnostic algorithms.