Asymmetric control of food intake by left and right vagal sensory neurons.

We investigated the lateralization of gut-innervating vagal sensory neurons and their roles in feeding behavior. Using genetic, anatomical, and behavioral analyses, we discovered a subset of highly lateralized vagal sensory neurons with distinct sensory responses to intestinal stimuli. Our results demonstrated that left vagal sensory neurons (LNG) are crucial for distension-induced satiety, while right vagal sensory neurons (RNG) mediate preference for nutritive foods. Furthermore, these lateralized neurons engage different central circuits, with LNG neurons recruiting brain regions associated with energy balance and RNG neurons activating areas related to salience, memory, and reward. Altogether, our findings unveil the diverse roles of asymmetrical gut-vagal-brain circuits in feeding behavior, offering new insights for potential therapeutic interventions targeting vagal nerve stimulation in metabolic and neuropsychiatric diseases.

Central Administration of Hydrogen Sulfide Donor NaHS Reduces Iba1-Positive Cells in the PVN and Attenuates Rodent Angiotensin II Hypertension.

Hydrogen sulfide (H2S) is a gaseous signaling molecule with neuromodulatory, anti-inflammatory, and anti-hypertensive effects. Here, we investigate whether chronic intracerebroventricular (ICV) infusion of sodium hydrosulfide (NaHS), an H2S donor, can alleviate angiotensin II (Ang II)-induced hypertension (HTN), improve autonomic function, and impact microglia in the paraventricular nucleus (PVN) of the hypothalamus, a brain region associated with autonomic control of blood pressure (BP) and neuroinflammation in HTN. Chronic delivery of Ang II (200 ng/kg/min, subcutaneous) for 4 weeks produced a typical increase in BP and sympathetic drive and elevated the number of ionized calcium binding adaptor molecule 1-positive (Iba1+) cells in the PVN of male, Sprague-Dawley rats. ICV co-infusion of NaHS (at 30 and/or 60 nmol/h) significantly attenuated these effects of Ang II. Ang II also increased the abundance of cecal Deltaproteobacteria and Desulfovibrionales, among others, which was prevented by ICV NaHS co-infusion at 30 and 60 nmol/h. We observed no differences in circulating H2S between the groups. Our results suggest that central H2S may alleviate rodent HTN independently from circulating H2S via effects on autonomic nervous system and PVN microglia.

Demystifying functional role of cocaine- and amphetamine-related transcript (CART) peptide in control of energy homeostasis: A twenty-five year expedition.

Cocaine- and amphetamine-related transcript (CART) is a neuropeptide first discovered in the striatum of the rat brain. Later, the genetic sequence and function of CART peptide (CARTp) was found to be conserved among multiple mammalian species. Over the 25 years, since its discovery, CART mRNA (Cartpt) expression has been reported widely throughout the central and peripheral nervous systems underscoring its role in diverse physiological functions. Here, we review the localization and function of CARTp as it relates to energy homeostasis. We summarize the expression changes of central and peripheral Cartpt in response to metabolic states and make use of available large data sets to gain additional insights into the anatomy of the Cartpt expressing vagal neurons and their expression patterns in the gut. Furthermore, we provide an overview of the role of CARTp as an anorexigenic signal and its effect on energy expenditure and body weight control with insights from both pharmacological and transgenic animal studies. Subsequently, we discuss the role of CARTp in the pathophysiology of obesity and review important new developments towards identifying a candidate receptor for CARTp signalling. Altogether, the field of CARTp research has made rapid and substantial progress recently, and we review the case for considering CARTp as a potential therapeutic target for stemming the obesity epidemic.

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD.

BACKGROUND & AIMS: The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. METHODS: Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2-3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. RESULTS: We observed major immunologic changes in patients with NAS 2-3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. CONCLUSION: The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. LAY SUMMARY: Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease.

Prolonged neutrophil survival at necrotic sites is a fundamental feature for tissue recovery and resolution of hepatic inflammation.

Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of "resolutive neutrophils" harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.

Vagus nerve regulates the phagocytic and secretory activity of resident macrophages in the liver.

The gastrointestinal (GI) tract harbors commensal microorganisms as well as invasive bacteria, toxins and other pathogens and, therefore, plays a pivotal barrier and immunological role against pathogenic agents. The vagus nerve is an important regulator of the GI tract-associated immune system, having profound effects on inflammatory responses. Among GI tract organs, the liver is a key site of immune surveillance, as it has a large population of resident macrophages and receives the blood drained from the guts through the hepatic portal circulation. Although it is widely accepted that the hepatic tissue is a major target for vagus nerve fibers, the role of this neural circuit in liver immune functions is still poorly understood. Herein we used in vivo imaging techniques, including confocal microscopy and scintigraphy, to show that vagus nerve stimulation increases the phagocytosis activity by resident macrophages in the liver, even on the absence of an immune challenge. The activation of this neural circuit in a non-lethal model of sepsis optimized the removal of bacteria in the liver and resulted in the production of anti-inflammatory and pro-regenerative cytokines. Our findings provide new insights into the neural regulation of the immune system in the liver.

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.

Immune and metabolic shifts during neonatal development reprogram liver identity and function.

BACKGROUND & AIMS: The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. METHODS: We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. RESULTS: Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. CONCLUSION: In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. LAY SUMMARY: Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.

Liver Immune Cells Release Type 1 Interferon Due to DNA Sensing and Amplify Liver Injury from Acetaminophen Overdose.

Hepatocytes may rupture after a drug overdose, and their intracellular contents act as damage-associated molecular patterns (DAMPs) that lead to additional leukocyte infiltration, amplifying the original injury. Necrosis-derived DNA can be recognized as a DAMP, activating liver non-parenchymal cells (NPCs). We hypothesized that NPCs react to DNA by releasing interferon (IFN)-1, which amplifies acetaminophen (APAP)-triggered liver necrosis. We orally overdosed different knockout mouse strains to investigate the pathways involved in DNA-mediated amplification of APAP-induced necrosis. Mice were imaged under intravital confocal microscopy to estimate injury progression, and hepatocytes and liver NPCs were differentially isolated for gene expression assays. Flow cytometry (FACS) using a fluorescent reporter mouse estimated the interferon-beta production by liver leukocytes under different injury conditions. We also treated mice with DNase to investigate the role of necrosis DNA signaling in IFN-1 production. Hepatocytes released a large amount of DNA after APAP overdose, which was not primarily sensed by these cells. However, liver NPCs promptly sensed such environmental disturbances and activated several DNA sensing pathways. Liver NPCs synthesized and released IFN-1, which was associated with concomitant hepatocyte necrosis. Ablation of IFN-1 recognition in interferon α/ß receptor (IFNAR-/-) mice delayed APAP-mediated liver necrosis and dampened IFN-1 sensing pathways. We demonstrated a novel loop involving DNA recognition by hepatic NPCs and additional IFN-1 mediated hepatocyte death.

Neutrophil biology within hepatic environment.

Neutrophils are the most abundant leukocyte in the human circulation. These short-lived cells are constantly produced from hematopoietic stem cells (HSC) within the bone marrow from which they daily reach the blood and perform major roles in innate immunity. Neutrophils are the first cells to reach inflamed tissues and are armed with a plethora of enzymes that help both with their trafficking within tissues and the killing of pathogens. Damaged or infected organs are rapidly invaded by neutrophils. Their erroneous activation within parenchyma or the vasculature is involved in the pathogenesis of several inflammatory diseases including arthritis, colitis, sepsis, acute lung injury and liver failure. Despite the proposal of a canonical pathway that governs neutrophil migration into tissues, the liver has been extensively described as a unique environment for leukocyte recruitment. Since the control of inflammatory responses is considered one of the most promising avenues for novel therapeutics, the expansion of our understanding of the mechanisms behind neutrophil accumulation within injured liver might add to the development of specific and more efficacious treatments. In this review, we discuss the basic concepts of neutrophil ontogeny and biology, with a focus on the particularities and the molecular steps involved in neutrophil recruitment to 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.

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.

Detecção de Espécies Reativas de Oxigênio no sangue periférico de pacientes com leptospirose grave / Detection of Reactive Oxygen Species in peripheral blood patients with severe leptospirosis

Leptospirose é uma zoonose que pode levar a graves complicações, como a síndrome de Weil e a síndrome pulmonar hemorrágica, porém os mecanismos patogênicos que levam ao desenvolvimento das formas graves da doença ainda são desconhecidos. Após a penetração no indivíduo, as leptospiras invadem a corrente sanguínea e se disseminam para os órgãos. Dessa forma, a leptospirose apresenta características semelhantes as da sepse, doença que tem o estresse oxidativo como um dos principais responsáveis pelo seu agravamento. Entretanto, pouco se sabe sobre o envolvimento do estresse oxidativo na leptospirose. O presente estudo teve como objetivo avaliar se a produção de espécies reativas de oxigênio (ROS) e os níveis do antioxidante glutationa (GSH) estão relacionados com as manifestações clínicas mais graves de pacientes hospitalizados com leptospirose. A produção de ROS e os níveis de GSH foram avaliados nas amostras de sangue de doze pacientes e nove indivíduos saudáveis através dos ensaios de quimioluminescência e de absorbância, respectivamente. Nós observamos que os níveis de ROS estavam aumentados (p=0.0012) e os de GSH diminuídos (p=0.0002) nos pacientes quando comparados com os indivíduos saudáveis. Dentre os pacientes, a diminuição de GSH estava correlacionada com a trombocitopenia (r=0.63) e com elevados níveis de creatinina (r= -0.64), enquanto que a produção de ROS estava fortemente correlacionada com os níveis elevados de potássio sérico (r=0.8). A compreensão da importância biológica de ROS e do GSH na leptospirose faz-se necessária, pois uma investigação mais detalhada pode levar ao desenvolvimento de terapias adjuvantes focadas no estresse oxidativo.

Leptospirosis is a zoonotic disease that causes severe manifestations such as Weil’s disease and pulmonary hemorrhage syndrome, however the underlying mechanisms that lead to the development of severe forms are not clear. Leptospires penetrate through skin, reach the bloodstream and disseminate to the organs. Thus, leptospirosis and sepsis have similar characteristics. Although there is vast literature demonstrating that oxidative stress play an important role in the severity of sepsis, none is known about it in leptospirosis. The aim of this study was to evaluate whether reactive oxygen species (ROS) production and antioxidant reduced glutathione (GSH) levels are related to complications in patients hospitalized with leptospirosis. ROS production and GSH levels were measured in blood samples of twelve patients and nine healthy controls using chemiluminescence and absorbance assays. We found that ROS production was higher (p=0.0012) and GSH levels were lower (p=0.0002) in leptospirosis patients compared with healthy individuals. Among patients, GSH depletion was correlated with thrombocytopenia (r=0.63) and elevated serum creatinine (r= -0.64), while a strong positive correlation was observed between ROS production and elevated serum potassium (r=0.8). Additional investigation of the biological significance of ROS production and GSH levels is warranted as they may guide the development of novel adjuvant therapies for leptospirosis targeting oxidative stress.