Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury.

Background & Aims: Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury. Methods: We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver in vivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67). Results: Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, in vitro and in vivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG. Conclusion: NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery. Impact and implications: Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Evidence that the domesticated fungus Leucoagaricus gongylophorus recycles its cytoplasmic contents as nutritional rewards to feed its leafcutter ant farmers.

Leafcutter ants farm a fungal cultivar (Leucoagaricus gongylophorus) that converts inedible vegetation into food that sustains colonies with up to millions of workers. Analogous to edible fruits of crops domesticated by humans, L. gongylophorus has evolved specialized nutritional rewards-swollen hyphal cells called gongylidia that package metabolites and are consumed by ant farmers. Yet, little is known about how gongylidia form, and thus how fungal physiology and ant provisioning collectively govern farming performance. We explored the process of gongylidium formation using advanced microscopy to image the cultivar at scales of nanometers, and both in vitro experiments and in silico analyses to examine the mechanisms of gongylidia formation when isolated from ant farmers. We first used transmission electron, fluorescence, and confocal microscopy imaging to see inside hyphal cells. This imaging showed that the cultivar uses a process called autophagy to recycle its own cellular material (e.g. cytosol, mitochondria) and then shuttles the resulting metabolites into a vacuole whose continual expansion displaces other organelles and causes the gongylidium cell's bulging bulb-like appearance. We next used scanning electron microscopy and light microscopy to link this intracellular rearrangement to the external branching patterns of gongylidium cells as they clump together into edible bundles called staphyla. We next confirmed that autophagy plays a critical role in gongylidium formation both: (1) in vitro as gongylidium suppression occurred when isolated fungal cultures were grown on media with autophagy inhibitors, and (2) in silico as differential transcript expression (RNA-seq) analyses showed upregulation of multiple autophagy gene isoforms in gongylidia relative to undifferentiated hyphae. While autophagy is a ubiquitous and often highly derived process across the tree of life, our study reveals a new role for autophagy as a mechanism of functional integration between ant farmers and their fungal crop, and potentially as a signifier of higher-level homeostasis between uniquely life-time committed ectosymbionts.

The GAG-Binding Peptide MIG30 Protects against Liver Ischemia-Reperfusion in Mice.

Ischemia-reperfusion injury (IRI) drives graft rejection and is the main cause of mortality after liver transplantation. During IRI, an intense inflammatory response marked by chemokine production and neutrophil recruitment occurs. However, few strategies are available to restrain this excessive response. Here, we aimed to interfere with chemokine function during IRI in order to disrupt neutrophil recruitment to the injured liver. For this, we utilized a potent glycosaminoglycan (GAG)-binding peptide containing the 30 C-terminal amino acids of CXCL9 (MIG30) that is able to inhibit the binding of chemokines to GAGs in vitro. We observed that mice subjected to IRI and treated with MIG30 presented significantly lower liver injury and dysfunction as compared to vehicle-treated mice. Moreover, the levels of chemokines CXCL1, CXCL2 and CXCL6 and of proinflammatory cytokines TNF-α and IL-6 were significantly reduced in MIG30-treated mice. These events were associated with a marked inhibition of neutrophil recruitment to the liver during IRI. Lastly, we observed that MIG30 is unable to affect leukocytes directly nor to alter the stimulation by either CXCL8 or lipopolysaccharide (LPS), suggesting that its protective properties derive from its ability to inhibit chemokine activity in vivo. We conclude that MIG30 holds promise as a strategy to treat liver IRI and inflammation.

The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74-103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice.

Klebsiella pneumoniae is an important pathogen associated with hospital-acquired pneumonia (HAP). Bacterial pneumonia is characterized by a harmful inflammatory response with a massive influx of neutrophils, production of cytokines and chemokines, and consequent tissue damage and dysfunction. Targeted therapies to block neutrophil migration to avoid tissue damage while keeping the antimicrobial properties of tissue remains a challenge in the field. Here we tested the effect of the anti-inflammatory properties of the chemokine fragment CXCL9(74-103) in pneumonia induced by Klebsiella pneumoniae in mice. Mice were infected by intratracheal injection of Klebsiella pneumoniae and 6 h after infection were treated systemically with CXCL9(74-103). The recruitment of leukocytes, levels of cytokines and chemokines, colony-forming units (CFU), and lung function were evaluated. The treatment with CXCL9(74-103) decreased neutrophil migration to the airways and the production of the cytokine interleukin-1ß (IL-1ß) without affecting bacterial control. In addition, the therapeutic treatment improved lung function in infected mice. Our results indicated that the treatment with CXCL9(74-103) reduced inflammation and improved lung function in Klebsiella pneumoniae-induced pneumonia.

Method Matters: Effect of Purification Technology on Neutrophil Phenotype and Function.

Neutrophils are the most abundant leukocytes in human blood and the first cells responding to infection and injury. Due to their limited ex vivo lifespan and the impossibility to cryopreserve or expand them in vitro, neutrophils need to be purified from fresh blood for immediate use in experiments. Importantly, neutrophil purification methods may artificially modify the phenotype and functional characteristics of the isolated cells. The aim of this study was to expose the effects of 'classical' density-gradient purification versus the more expensive but faster immunomagnetic isolation on neutrophil phenotype and functionality. We found that in the absence of inflammatory stimuli, density-gradient-derived neutrophils showed increased polarization responses as well as enhanced release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and granular proteins compared to cells derived from immunomagnetic isolation, which yields mostly quiescent neutrophils. Upon exposure to pro-inflammatory mediators, immunomagnetic isolation-derived neutrophils were significantly more responsive in polarization, ROS production, phagocytosis, NETosis and degranulation assays, in comparison to density-gradient-derived cells. We found no difference in chemotactic response in Multiscreen and under-agarose migration assays, but Boyden assays showed reduced chemotaxis of immunomagnetic isolation-derived neutrophils. Finally, we confirmed that density-gradient purification induces artificial activation of neutrophils, evidenced by e.g. higher expression of CD66b, formyl peptide receptor 1 (FPR1) and CD35, and the appearance of a separate neutrophil population expressing surface molecules atypical for neutrophils (e.g. CXCR3, MHC-II and CD14). Based on these results, we recommend using immunomagnetic separation of neutrophils for studying neutrophil polarization, phagocytosis, ROS production, degranulation and NETosis, whereas for Boyden chemotaxis assays, the density-gradient purification is more suitable.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage.

Toll-like receptor 9 (TLR9) and Phosphatidylinositol-3-kinase gamma (PI3Kγ) are very important effectors of the immune response, however, the importance of such crosstalk for disease development is still a matter of discussion. Here we show that PI3Kγ is required for immune responses in which TLR9 is a relevant trigger. We demonstrate the requirement of PI3Kγ for TLR9-induced inflammation in a model of CpG-induced pleurisy. Such requirement was further observed in inflammatory models where DNA sensing via TLR9 contributes to disease, such as silicosis and drug-induced liver injury. Using adoptive transfer, we demonstrate that PI3Kγ is important not only in leukocytes but also in parenchymal cells for the progression of inflammation. We demonstrate this crosstalk between TLR9 and PI3Kγ in vitro using human PBMCs. The inhibition of PI3Kγ in CpG-stimulated PBMCs resulted in reduction of both cytokine production and phosphorylated Akt. Therefore, drugs that target PI3Kγ have the potential to treat diseases mediated by excessive TLR9 signalling.

Phagocytosis of Necrotic Debris at Sites of Injury and Inflammation.

Clearance of cellular debris is required to maintain the homeostasis of multicellular organisms. It is intrinsic to processes such as tissue growth and remodeling, regeneration and resolution of injury and inflammation. Most of the removal of effete and damaged cells is performed by macrophages and neutrophils through phagocytosis, a complex phenomenon involving ingestion and degradation of the disposable particles. The study of the clearance of cellular debris has been strongly biased toward the removal of apoptotic bodies; as a result, the mechanisms underlying the removal of necrotic cells have remained relatively unexplored. Here, we will review the incipient but growing knowledge of the phagocytosis of necrotic debris, from their recognition and engagement to their internalization and disposal. Critical insights into these events were gained recently through the development of new in vitro and in vivo models, along with advances in live-cell and intravital microscopy. This review addresses the classes of "find-me" and "eat-me" signals presented by necrotic cells and their cognate receptors in phagocytes, which in most cases differ from the extensively characterized counterparts in apoptotic cell engulfment. The roles of damage-associated molecular patterns, chemokines, lipid mediators, and complement components in recruiting and activating phagocytes are reviewed. Lastly, the physiological importance of necrotic cell removal is emphasized, highlighting the key role of impaired debris clearance in autoimmunity.

Paradoxical Role of Matrix Metalloproteinases in Liver Injury and Regeneration after Sterile Acute Hepatic Failure.

Acetaminophen (APAP) poisoning is one of the leading causes of acute hepatic failure and liver transplantation is often the only lifesaving alternative. During the course of hepatocyte necrosis, an intense accumulation of neutrophils is often observed within the liver microenvironment. Despite the classic idea that neutrophil accumulation in tissues causes collateral tissue damage, there is a growing body of evidence showing that neutrophils can also orchestrate the resolution of inflammation. In this work, drug-induced liver injury was induced by oral administration of APAP and pharmacological intervention was made 12 h after this challenge. Liver injury and repair kinetics were evaluated by a novel combination of enzyme quantifications, ELISA, specific antagonists of neutrophil enzymes and confocal intravital microscopy. We have demonstrated that neutrophil infiltration is not only involved in injury amplification, but also in liver tissue repair after APAP-induced liver injury. In fact, while neutrophil depletion led to reduced hepatic necrosis during APAP poisoning, injury recovery was also delayed in neutropenic mice. The mechanisms underlying the neutrophil reparative role involved rapid degranulation and matrix metalloproteinases (MMPs) activity. Our data highlights the crucial role of neutrophils, in particular for MMPs, in the resolution phase of APAP-induced inflammatory response.

Neutrophils: a cornerstone of liver ischemia and reperfusion injury.

Ischemia-reperfusion injury (IRI) is the main cause of morbidity and mortality due to graft rejection after liver transplantation. During IRI, an intense inflammatory process occurs in the liver. This hepatic inflammation is initiated by the ischemic period but occurs mainly during the reperfusion phase, and is characterized by a large neutrophil recruitment to the liver. Production of cytokines, chemokines, and danger signals results in activation of resident hepatocytes, leukocytes, and Kupffer cells. The role of neutrophils as the main amplifiers of liver injury in IRI has been recognized in many publications. Several studies have shown that elimination of excessive neutrophils or inhibition of their function leads to reduction of liver injury and inflammation. However, the mechanisms involved in neutrophil recruitment during liver IRI are not well known. In addition, the molecules necessary for this type of migration are poorly defined, as the liver presents an atypical sinusoidal vasculature in which the classical leukocyte migration paradigm only partially applies. This review summarizes recent advances in neutrophil-mediated liver damage, and its application to liver IRI. Basic mechanisms of activation of neutrophils and their unique mechanisms of recruitment into the liver vasculature are discussed. In particular, the role of danger signals, adhesion molecules, chemokines, glycosaminoglycans (GAGs), and metalloproteinases is explored. The precise definition of the molecular events that govern the recruitment of neutrophils and their movement into inflamed tissue may offer new therapeutic alternatives for hepatic injury by IRI and other inflammatory diseases of the liver.

IL-33 signalling in liver immune cells enhances drug-induced liver injury and inflammation.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the contribution of IL-33/ST2 axis in the onset and progression of acute liver injury using a mice model of drug-induced liver injury (DILI). MATERIAL AND TREATMENTS: DILI was induced by overdose administration of acetaminophen (APAP) by oral gavage in wild-type BALB/c, ST2-deficient mice and in different bone marrow chimeras. Neutrophils were depleted by anti-Ly6G and macrophages with clodronate liposomes (CLL). METHODS: Blood and liver were collected for biochemical, immunologic and genetic analyses. Mice were imaged by confocal intravital microscopy and liver non-parenchymal cells and hepatocytes were isolated for flow cytometry, genetic and immunofluorescence studies. RESULTS: Acetaminophen overdose caused a massive necrosis and accumulation of immune cells within the liver, concomitantly with IL-33 and chemokine release. Liver non-parenchymal cells were the major sensors for IL-33, and amongst them, neutrophils were the major players in amplification of the inflammatory response triggered by IL-33/ST2 signalling pathway. CONCLUSION: Blockage of IL-33/ST2 axis reduces APAP-mediated organ injury by dampening liver chemokine release and activation of resident and infiltrating liver non-parenchymal cells.

Intravital Microscopic Evaluation of the Effects of a CXCR2 Antagonist in a Model of Liver Ischemia Reperfusion Injury in Mice.

BACKGROUND: Ischemia-reperfusion (IR) is a major contributor to graft rejection after liver transplantation. During IR injury, an intense inflammatory process occurs in the liver. Neutrophils are considered central players in the events that lead to liver injury. CXC chemokines mediate hepatic inflammation following reperfusion. However, few studies have demonstrated in real-time the behavior of recruited neutrophils. We used confocal intravital microscopy (IVM) to image neutrophil migration in the liver and to analyze in real-time parameters of neutrophil recruitment in the inflamed tissue in animals treated or not with reparixin, an allosteric antagonist of CXCR1/2 receptors. MATERIALS AND METHODS: WT and LysM-eGFP mice treated with reparixin or saline were subjected to 60 min of ischemia followed by different times of reperfusion. Mice received Sytox orange intravenously to show necrotic DNA in IVM. The effect of reparixin on parameters of local and systemic reperfusion-induced injury was also investigated. RESULTS: IR induced liver injury and inflammation, as evidenced by high levels of alanine aminotransferase and myeloperoxidase activity, chemokine and cytokine production, and histological outcome. Treatment with reparixin significantly decreased neutrophil influx. Moreover, reparixin effectively suppressed the increase in serum concentrations of TNF-α, IL-6, and CCL3, and the reperfusion-associated tissue damage. The number of neutrophils in the liver increased between 6 and 24 h of reperfusion, whereas the distance traveled, velocity, neutrophil size and shape, and cluster formation reached a maximum 6 h after reperfusion and then decreased gradually. In vivo imaging revealed that reparixin significantly decreased neutrophil infiltration and movement and displacement of recruited cells. Moreover, neutrophils had a smaller size and less elongated shape in treated mice. CONCLUSION: Imaging of the liver by confocal IVM was successfully implemented to describe neutrophil behavior in vivo during liver injury by IR. Treatment with reparixin decreased not only the recruitment of neutrophils in tissues but also their activation state and capacity to migrate within the liver. CXCR1/2 antagonists may be a promising therapy for patients undergoing liver transplantation.

Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice.

BACKGROUND & AIMS: Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be replaced by bone marrow precursors during extensive injuries, irradiation, and infections. We investigated the liver populations of myeloid immune cells and their location, as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original phagocytes by bone marrow-derived surrogates were also examined. METHODS: We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt, and CX3CR1-DTR-EYFP. Liver nonparenchymal cells were immunophenotyped using mass cytometry and gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. RESULTS: Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory cytokines, and dexamethasone accelerated the repopulation of liver phagocytes. CONCLUSIONS: In studies of hepatic phagocyte depletion in mice, we found that myeloid precursors can differentiate into liver macrophages and dendritic cells, which each localize to distinct tissue compartments. During replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.

Exploring the Homeostatic and Sensory Roles of the Immune System.

Immunology developed under the notion of the immune system exists to fight pathogens. Recently, the discovery of interactions with commensal microbiota that are essential to human health initiated a change in this old paradigm. Here, we argue that the immune system has major physiological roles extending far beyond defending the host. Immune and inflammatory responses share the core property of sensing, defining the immune system also as a sensory system. The inference with the immune system collects, interprets, and stores information, while creating an identity of self, places it in close relationship to the nervous system, which suggests that these systems may have a profound evolutionary connection.

Type I Interferon Transcriptional Signature in Neutrophils and Low-Density Granulocytes Are Associated with Tissue Damage in Malaria.

Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1ß and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis.

Understanding liver immunology using intravital microscopy.

The liver has come a long way since it was considered only a metabolic organ attached to the gastrointestinal tract. The simultaneous ascension of immunology and intravital microscopy evidenced the liver as a central axis in the immune system, controlling immune responses to local and systemic agents as well as disease tolerance. The multiple hepatic cell populations are organized in a vascular environment that promotes intimate cellular interactions, including initiation of innate and adaptive immune responses, rapid leukocyte recruitment, pathogen clearance and production of a variety of immune mediators. In this review, we focus on the advances in liver immunology supported by intravital microscopy in diseases such as isquemia/reperfusion, acute liver injury and infections.

Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.

UNLABELLED: Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. CONCLUSION: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue.

Murine model to study brain, behavior and immunity during hepatic encephalopathy.

AIM: To propose an alternative model of hepatic encephalopathy (HE) in mice, resembling the human features of the disease. METHODS: Mice received two consecutive intraperitoneal injections of thioacetamide (TAA) at low dosage (300 mg/kg). Liver injury was assessed by serum transaminase levels (ALT) and liver histology (hematoxylin and eosin). Neutrophil infiltration was estimated by confocal liver intravital microscopy. Coagulopathy was evaluated using prolonged prothrombin and partial thromboplastin time. Hemodynamic parameters were measured through tail cuff. Ammonia levels were quantified in serum and brain samples. Electroencephalography (EEG) and psychomotor activity score were performed to show brain function. Brain edema was evaluated using magnetic resonance imaging. RESULTS: Mice submitted to the TAA regime developed massive liver injury, as shown by elevation of serum ALT levels and a high degree of liver necrosis. An intense hepatic neutrophil accumulation occurred in response to TAA-induced liver injury. This led to mice mortality and weight loss, which was associated with severe coagulopathy. Furthermore, TAA-treated mice presented with increased serum and cerebral levels of ammonia, in parallel with alterations in EEG spectrum and discrete brain edema, as shown by magnetic resonance imaging. In agreement with this, neuropsychomotor abnormalities ensued 36 h after TAA, fulfilling several HE features observed in humans. In this context of liver injury and neurological dysfunction, we observed lung inflammation and alterations in blood pressure and heart rate that were indicative of multiple organ dysfunction syndrome. CONCLUSION: In summary, we describe a new murine model of hepatic encephalopathy comprising multiple features of the disease in humans, which may provide new insights for treatment.

Acute intake of a high-fructose diet alters the balance of adipokine concentrations and induces neutrophil influx in the liver.

The postprandial state is a period of metabolic fluxes, biosynthesis and oxidative metabolism. A considerable amount is known about the inflammatory response to the chronic consumption of fructose, but little is known about its effects in the postprandial state. The aim of the present study was to investigate the inflammatory effects of a single meal containing fructose on healthy mice. Male BALB/c and LysM-eGFP mice at 12-14 weeks were divided into three groups: fasted, control (mice fed with a sucrose-containing diet) and fructose (mice fed with a fructose-containing diet). One, 2 or 4 h postprandial, the BALB/c mice were killed, and samples were collected. LysM-eGFP mice were submitted to intravital microscopy. The fed mice showed a low-grade inflammatory response apart from dietary composition, which was characterized by increased numbers of leukocytes and high serum concentrations of pentraxin 3, leptin and resistin. TNF-α and CCL2 concentrations rose in the liver after the meal. IL-6 concentration increased and IL-10 decreased in the adipose tissue of the fed mice. Mice fed with the fructose-containing diet showed an intensification of the inflammatory response. Furthermore, the adiponectin concentration dropped, and the liver influx of neutrophils increased after fructose intake. Overall, this study showed a rapid increase in the systemic and tissue-specific immune response after a balanced meal. The study also showed an increased neutrophil influx in liver associated with an imbalance of adipokine concentrations and an increase of cytokine in the liver and adipose tissue following a fructose-containing meal.