Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant Staphylococcus aureus.

AIMS: The purpose was to evaluate the antimicrobial activity of highly soluble polypyrrole (Hs-PPy), alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. METHODS AND RESULTS: Ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 µg ml-1 and bactericidal action at 62.5 µg ml-1, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. CONCLUSIONS: Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Neutrophil extracellular traps (NETs) modulate inflammatory profile in obese humans and mice: adipose tissue role on NETs levels.

BACKGROUND: Neutrophil extracellular traps (NETs) are a recently discovered neutrophil defense mechanism which modulates several inflammatory conditions contributing to metabolic profile alterations. Therefore, the present study aimed to evaluate the production of NETs in obese patients and mice, verifying the possible mechanisms associated with the release of NETs by the adipose tissue. METHODS AND RESULTS: The present study investigated NETs production in human adipose tissue and also showing the neutrophils using intravital microscopy in mouse epididymal adipose tissue. Blood and white adipose tissues were obtained from eutrophic and obese individuals and from mice. Lipid, glycemic and leukocyte profiles were evaluated, as well as the levels of NETs and its markers. Bioinformatics and proteomics analyses were performed and the identified key proteins were measured. The main findings showed that the inflammatory markers interleukin-8 (IL-8), heat shock protein 90 (HSP90) and the E1 heat shock protein family (HSPE1) can be modulated by the NETs levels in obesity. Obesity has also been associated with increased cholesterol, glucose intolerance, ionic calcium and NETs. We also observed an increase in catalase and a decreased superoxide dismutase activity. Bioinformatics and proteomics analyses revealed that IL-8, HSP90 and HSPE1 were associated with obesity, inflammation and NETs release. CONCLUSIONS: In conclusion, the present study shows an increase in NETs production during obesity associated with important inflammatory markers in adipose.

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.

Lipoxin A4 impairs effective bacterial control and potentiates joint inflammation and damage caused by Staphylococcus aureus infection.

Staphylococcus aureus is the main cause of septic arthritis in humans, a disease associated with high morbidity and mortality. Inflammation triggered in response to infection is fundamental to control bacterial growth but may cause permanent tissue damage. Here, we evaluated the role of Lipoxin A4 (LXA4 ) in S aureus-induced arthritis in mice. Septic arthritis was induced by S aureus injection into tibiofemoral joints. At different time points, we evaluated cell recruitment and bacterial load in the joint, the production of pro-inflammatory molecules, and LXA4 in inflamed tissue and analyzed joint damage and dysfunction. LXA4 was investigated using genetically modified mice or by pharmacological blockade of its synthesis and receptor. CD11c+ cells were evaluated in lymph nodes by confocal microscopy and flow cytometry and dendritic cell chemotaxis using the Boyden chamber. Absence or pharmacological blockade of 5-lipoxygenase (5-LO) reduced joint inflammation and dysfunction and was associated with better control of infection at 4 to 7 days after the infection. There was an increase in LXA4 in joints of S aureus-infected mice and administration of LXA4 reversed the phenotype in 5-LO-/- mice. Blockade or absence of the LXA4 receptor FPR2 has a phenotype similar to 5-LO-/- mice. Mechanistically, LXA4 appeared to control migration and function of dendritic cells, cells shown to be crucial for adequate protective responses in the model. Thus, after the first days of infection when symptoms become evident therapies that inhibit LXA4 synthesis or action could be useful for treatment of S aureus-induced arthritis.

The Annexin A1/FPR2 pathway controls the inflammatory response and bacterial dissemination in experimental pneumococcal pneumonia.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia leading to high mortality rates. Inflammation triggered by pneumococcal infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Annexin A1 (AnxA1) mainly acts through Formyl Peptide Receptor 2 (FPR2) inducing the resolution of inflammation. Here, we have evaluated the role of AnxA1 and FPR2 during pneumococcal pneumonia in mice. For that, AnxA1, Fpr2/3 knockout (KO) mice and wild-type (WT) controls were infected intranasally with S pneumoniae. AnxA1 and Fpr2/3 KO mice were highly susceptible to infection, displaying uncontrolled inflammation, increased bacterial dissemination, and pulmonary dysfunction compared to WT animals. Mechanistically, the absence of AnxA1 resulted in the loss of lung barrier integrity and increased neutrophil activation upon S pneumoniae stimulation. Importantly, treatment of WT or AnxA1 KO-infected mice with Ac2-26 decreased inflammation, lung damage, and bacterial burden in the airways by increasing macrophage phagocytosis. Conversely, Ac2-26 peptide was ineffective to afford protection in Fpr2/3 KO mice during infection. Altogether, these findings show that AnxA1, via FPR2, controls inflammation and bacterial dissemination during pneumococcal pneumonia by promoting host defenses, suggesting AnxA1-based peptides as a novel therapeutic strategy to control pneumococcal pneumonia.

The role of neutrophils in neuro-immune modulation.

Neutrophils are peripheral immune cells that represent the first recruited innate immune defense against infections and tissue injury. However, these cells can also induce overzealous responses and cause tissue damage. Although the role of neutrophils activating the immune system is well established, only recently their critical implications in neuro-immune interactions are becoming more relevant. Here, we review several aspects of neutrophils in the bidirectional regulation between the nervous and immune systems. First, the role of neutrophils as a diffuse source of acetylcholine and catecholamines is controversial as well as the effects of these neurotransmitters in neutrophil's functions. Second, neutrophils contribute for the activation and sensitization of sensory neurons, and thereby, in events of nociception and pain. In addition, nociceptor activation promotes an axon reflex triggering a local release of neural mediators and provoking neutrophil activation. Third, the recruitment of neutrophils in inflammatory responses in the nervous system suggests these immune cells as innovative targets in the treatment of central infectious, neurological and neurodegenerative disorders. Multidisciplinary studies involving immunologists and neuroscientists are required to define the role of the neurons-neutrophils communication in the pathophysiology of infectious, inflammatory, and neurological disorders.

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.

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.

Tissue macrophages as mediators of a healthy relationship with gut commensal microbiota.

Mammals and microorganisms have evolved a complex and tightly controlled mutual relationship. This interaction grants protection and energy source for the microorganisms, and on the other hand, provides several immunologic, metabolic and physiological advantages for the host. The gastrointestinal tract (GI) harbors the largest bacteria diversity within the body and complex mechanisms control microbiota community under homeostasis. However, once disrupted, microbiota imbalance can lead to overt growth of resident and invasive populations, with potential risk for lethal diseases. In these cases, bacteria might also escape from the intestines and reach different organs through the blood and lymphatic circulation. To control these unwanted conditions, all body tissues are populated with resident macrophages that have the ability to capture and eliminate pathogens, avoiding their dissemination. Here we discuss the different routes for bacterial translocation from the intestinal tract, and how macrophages act in the removal of these microorganisms to prevent systemic infections and restore the homeostasis.

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.

Modulation of experimental arthritis by vagal sensory and central brain stimulation.

Articular inflammation is a major clinical burden in multiple inflammatory diseases, especially in rheumatoid arthritis. Biological anti-rheumatic drug therapies are expensive and increase the risk of systemic immunosuppression, infections, and malignancies. Here, we report that vagus nerve stimulation controls arthritic joint inflammation by inducing local regulation of innate immune response. Most of the previous studies of neuromodulation focused on vagal regulation of inflammation via the efferent peripheral pathway toward the viscera. Here, we report that vagal stimulation modulates arthritic joint inflammation through a novel "afferent" pathway mediated by the locus coeruleus (LC) of the central nervous system. Afferent vagal stimulation activates two sympatho-excitatory brain areas: the paraventricular hypothalamic nucleus (PVN) and the LC. The integrity of the LC, but not that of the PVN, is critical for vagal control of arthritic joint inflammation. Afferent vagal stimulation suppresses articular inflammation in the ipsilateral, but not in the contralateral knee to the hemispheric LC lesion. Central stimulation is followed by subsequent activation of joint sympathetic nerve terminals inducing articular norepinephrine release. Selective adrenergic beta-blockers prevent the effects of articular norepinephrine and thereby abrogate vagal control of arthritic joint inflammation. These results reveals a novel neuro-immune brain map with afferent vagal signals controlling side-specific articular inflammation through specific inflammatory-processing brain centers and joint sympathetic innervations.

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.

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.

Cannabidiol rescues acute hepatic toxicity and seizure induced by cocaine.

Cocaine is a commonly abused illicit drug that causes significant morbidity and mortality. The most severe and common complications are seizures, ischemic strokes, myocardial infarction, and acute liver injury. Here, we demonstrated that acute cocaine intoxication promoted seizure along with acute liver damage in mice, with intense inflammatory infiltrate. Considering the protective role of the endocannabinoid system against cell toxicity, we hypothesized that treatment with an anandamide hydrolysis inhibitor, URB597, or with a phytocannabinoid, cannabidiol (CBD), protects against cocaine toxicity. URB597 (1.0 mg/kg) abolished cocaine-induced seizure, yet it did not protect against acute liver injury. Using confocal liver intravital microscopy, we observed that CBD (30 mg/kg) reduced acute liver inflammation and damage induced by cocaine and prevented associated seizure. Additionally, we showed that previous liver damage induced by another hepatotoxic drug (acetaminophen) increased seizure and lethality induced by cocaine intoxication, linking hepatotoxicity to seizure dynamics. These findings suggest that activation of cannabinoid system may have protective actions on both liver and brain induced by cocaine, minimizing inflammatory injury promoted by cocaine, supporting its further clinical application in the treatment of cocaine abuse.

Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.

OBJECTIVE: Interleukin-4 (IL-4) is a multifunctional cytokine involved in many diseases such as autoimmune hepatitis and idiosyncratic drug reactions. However, its role in acetaminophen (APAP)-induced liver injury remains unclear. Our objective was to evaluate the contribution of IL-4 to the pathogenesis of APAP-induced liver injury. METHODS: Balb/C (WT) and IL-4 knockout (IL-4(-/-)) mice were orally overdosed with APAP. After 24 h, survival percentage, biochemical and morphological markers of liver injury, and tissue inflammation were assessed. RESULTS: IL-4(-/-) mice were protected from APAP toxicity. Intravital confocal microscopy, tissue histology and serum ALT levels showed significantly less liver injury and inflammation than in the WT group, which may explain the increased survival rate of IL-4(-/-) mice. In addition, IL-4(-/-) mice had decreased production of tumor necrosis factor α, CXCL1 and interleukin-1ß in the liver, but not in a remote site such as the lungs. Hepatic macrophage activation was markedly reduced in IL-4-deficient mice. In addition, glutathione depletion-a primary cause of APAP-mediated injury-was significantly attenuated in IL-4(-/-) mice. CONCLUSIONS: Taken together, our data demonstrate that IL-4(-/-) mice are protected from APAP-induced liver injury due to reduced depletion of glutathione, which prevented liver damage and tissue inflammation.

The neonatal liver hosts a spontaneously occurring neutrophil population, exhibiting distinct spatial and functional characteristics from adults.

The elusive nature of the liver immune system in newborns remains an important challenge, casting a shadow over our understanding of how to effectively treat and prevent diseases in children. Therefore, deeper exploration into the intricacies of neonatal immunology might be crucial for improved pediatric healthcare. Using liver intravital microscopy, we unveiled a significant population of granulocytes in the hepatic parenchyma of fetuses and newborns. Utilizing high-dimensional immunophenotyping, we showed dynamic alterations predominantly in granulocytes during neonatal development. Liver intravital microscopy from birth through adulthood captures real-time dynamics, showing a substantial presence of Ly6G + cells that persisted significantly up to 2 weeks of age. Using CyTOF, we characterized neonatal Ly6G + cells as neutrophils, confirmed by morphology and immunohistochemistry. Surprisingly, the embryonic liver hosts a distinct population of neutrophils established as early as the second gestational week, challenging conventional notions about their origin. Additionally, we observed that embryonic neutrophils occupy preferentially the extravascular space, indicating their early establishment within the liver. Hepatic neutrophils in embryos and neonates form unique cell clusters, persisting during the initial days of life, while reduced migratory capabilities in neonates are observed, potentially compensating with increased reactive oxygen species (ROS) release in response to stimuli. Finally, in vivo imaging of acute neutrophil behavior in a newborn mouse, subjected to focal liver necrosis, unveils that neonatal neutrophils exhibit a reduced migratory response. The study provides unprecedented insights into the intricate interplay of neutrophils within the liver, shedding light on their functional and dynamic characteristics during development.

Prolonged ingestion of ovalbumin diet by Ova sensitized mice suppresses mBSA-induced arthritis.

Concomitant chronic diseases are a common finding in clinics and may consist in a major issue in therapeutics. Here, we investigated whether prolonged ingestion of ovalbumin (Ova) by sensitized mice would reduce the severity of an associated concurrent immunomediated condition such as antigen-induced arthritis (AIA). AIA was induced by administration of methylated bovine albumin (mBSA) into the knee joints of previously immunized mice, and evaluated by articular leukocyte trafficking and levels of cytokines (TNF-α, IL-1ß) and chemokine (CXCL-1) in the periarticular tissue. Continuous Ova feeding by Ova sensitized mice decreased serum levels of anti-Ova IgE, and led to a significant suppression of leukocyte adhesion and infiltration into synovial tissue and cavity. Also, a marked cytokine reduction was observed, suggesting that prolonged ingestion of ovalbumin by sensitized mice suppresses specific IgE production with concomitant reduction in peripheral T cells, which may impact in the pathogenesis of AIA, a non-related condition.