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SummaryNatural antibodies (NAbs) are circulating polyreactive immunoglobulins that bind endogenous and exogenous antigens. Here, we investigated the role of NAbs in driving the clearance of necrotic cell debris from injury sites. Using mouse models of liver injury, we observed that IgM and IgG NAbs opsonize necrotic debrisin vivoby recognizing common self-molecules such as histones, actin, phosphoinositides and cardiolipin, but not phosphatidylserine. Importantly, mice lacking NAbs presented impaired recovery from liver injury, which was correlated to sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. Mechanistically, necrotic debris phagocytosis was dependent on NAbsin vitroandin vivo, and restitution with total immunoglobulins rescued the defective recovery from liver injury in immunodeficient mice. In summary, we showed that NAbs opsonize necrotic cell debris and act as “eat-me” signals for engulfment through FcγRs and CD11b, driving the recovery from tissue injury.HighlightsNatural antibodies opsonize exposed self-antigens upon necrotic cell death.The phagocytosis of necrotic cell debris requires natural antibodies, FcγRs and CD11b.Natural antibodies drive cellular proliferation and tissue regeneration after liver injury.Treatment with natural antibodies improves the recovery from liver injury in both immunodeficient and immunocompetent mice.Graphical abstract

Hepatic cell death occurs in response to diverse stimuli such as chemical and physical damage. The exposure of intracellular contents such as DNA during necrosis induces a severe inflammatory response that has yet to be fully explored therapeutically. Here, we sought means to neutralize the ability of extracellular DNA to induce deleterious tissue inflammation when drug‐induced liver injury had already ensued. DNA exposure and inflammation were investigated in vivo in drug‐induced liver injury using intravital microscopy. The necrotic DNA debris was studied in murine livers in vivo and in DNA debris models in vitro by using a positively charged chemokine‐derived peptide (MIG30; CXCL9[74‐103]). Acetaminophen‐induced liver necrosis was associated with massive DNA accumulation, production of CXC chemokines, and neutrophil activation inside the injured tissue. The MIG30 peptide bound the healthy liver vasculature and, to a much greater extent, to DNA‐rich necrotic tissue. Moreover, MIG30 bound extracellular DNA directly in vivo in a charge‐dependent manner and independently of glycosaminoglycans and chemokines. Post‐treatment of mice with MIG30 reduced mortality, liver damage, and inflammation significantly. These effects were not observed with a control peptide that does not bind DNA. Mechanistically, MIG30 inhibited the interaction between DNA and histones, and promoted the dissociation of histones from necrotic debris. MIG30 also inhibited the pro‐inflammatory effect of CpG DNA, as measured by a reduction in CXCL8 production, indicating that MIG30 disturbs the ability of DNA to induce hepatic inflammation. Conclusion: The use of DNA‐binding peptides reduces necrotic liver injury and inflammation, even at late timepoints., Marques et al. used a small chemokine‐derived positively‐charged peptide (MIG30) to neutralize the deleterious effects of extracellular DNA during drug‐induced liver injury. Treatment with MIG30 rescued mice from injury, inflammation and mortality by binding DNA avidly, dissociating it from histones and reducing the pro‐inflammatory activity of CpG DNA.

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. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:17 ispartof: location:Switzerland status: published

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-1β (IL-1β) 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. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:11 ispartof: location:Switzerland status: published

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 ingested by ant farmers. Yet, little is known about how gongylidia form, and thus how fungal physiology and ant provisioning interact to farming performance. We explored the mechanisms governing gongylidium formation using microscopy imaging of ant-cultivated fungus and controlled in vitro experiments with the cultivar grown in isolation from ant farmers. First, L. gongylophorus is polykaryotic (up to 17 haploid nuclei/cell) and our results suggest intracellular nucleus distributions govern gongylidium morphology with their absence in expanding edges arresting apical growth and their presence mediating complex branching patterns. Second, nanoscale imaging (SEM, TEM) shows that the cultivar recycles its own cellular material (e.g. cytosol, mitochondria) through a process called ‘autophagy’ and stores the resulting metabolites in gongylidia. This autophagic pathway is further supported by gongylidium suppression when isolated fungal cultures are grown on media with autophagy inhibitors, and differential transcript expression (RNA-seq) analyses showing upregulation of multiple autophagy genes in gongylidia. We hypothesize that autophagic nutritional reward production is the ultimate cultivar service and reflects a higher-level organismality adaptation enabled by strict symmetric lifetime commitment between ant farmers and their fungal crop.

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., In this study, we found that patients suffering from severe COVID‐19 presented with immature, activated blood neutrophils and were characterized by elevated plasma levels of G‐CSF and CXCL8 that disappeared towards ICU discharge. Elevated neutrophil‐derived proteases in plasma of ICU patients were associated with increased non‐metalloproteinase‐derived gelatinolytic activity but decreased total MMP proteolytic activity. SARS‐CoV‐2 failed to replicate inside human neutrophils, suggesting that changes observed in neutrophils from patients with COVID‐19 are indirect consequences of SARS‐CoV‐2 infection in vivo.

Several emerging lines of evidence support an anti-inflammatory role for nicotinamide and other vitamin B components. However, the mechanisms underlying their activity remain unclear. In the present study, we investigated the ability of nicotinamide to inhibit both neutrophil recruitment in IL-8-, LTB 4 - or carrageenan-induced pleurisy in mice and the rolling and adherence of neutrophils. Nicotinamide inhibited IL-8-, LTB 4 - and carrageenan-induced neutrophil migration, KC production and carrageenan-induced neutrophil rolling and adherence. We propose that the effects of nicotinamide in inhibiting neutrophil recruitment in carrageenan-induced pleurisy may be due to the ability of nicotinamide to inhibit the action of IL-8 and LTB 4 , decrease KC production, and inhibit early events that regulate leukocyte migration from blood vessels into tissue.

Pretreatment using celecoxib, a cyclooxygenase (COX) 2 inhibitor, or indomethacin, a nonselective COX inhibitor, reduced lypopolyssaccharide (LPS)-induced leukocyte migration to the rat peritoneal cavity. The effect of celecoxib (12 mg/kg) or indomethacin (2 mg/kg) on neutrophil chemotaxis induced by formyl-methionyl-leucyl-phenylalanine (FMLP) in an in vitro chemotactic assay (Boyden chamber) was investigated. Celecoxib and indomethacin inhibited chemotaxis induced by FMLP (Control=26.6+/-1.45, Celecoxib=12.8+/-3.04, Indomethacin=6.26+/-2.19 cells/field). When observed under intravital microscopy, a mouse cremaster preparation was used to assess the microvasculature to further investigate which step of cell recruitment was affected by these drugs. Celecoxib and indomethacin inhibited leukocyte migration induced by 0.05 microg/kg LPS injected into the cremaster muscle. However, the effect of celecoxib was associated with reduced cell rolling and adhesion, whereas indomethacin was only effective at inhibiting cell adhesion. Furthermore, SC560 pretreatment (a COX-1 selective inhibitor) of normal or LPS-challenged tissues did not alter leukocyte migration or cell adhesion, but it did enhance leukocyte rolling activity in both cases. Taken together, these results indicate that: 1) COX-1 activity is mainly related to leukocyte traffic under physiological conditions, and 2) COX-2 activity is mainly related to cell traffic under inflammatory conditions in vascular beds, suggesting a possible effect of selective COX-2 inhibitors on the expression of adhesion molecules.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A lesão hepática por medicamentos (DILI) é um problema crescente de saúde pública. Ela pode evoluir para falência hepática aguda (FHA), um transtorno associado a alta mortalidade e com poucas opções de tratamento. Durante a DILI, a morte necrótica de hepatócitos leva à liberação de moléculas intracelulares capazes de causar um intenso processo inflamatório (DAMPs), podendo amplificar a lesão tecidual causada inicialmente pelo medicamento. Dentre estas moléculas, o potencial citotóxico do DNA já está associado a várias doenças inflamatórias e autoimunes, entretanto, a dinâmica de sua liberação e acúmulo em tecidos lesados, e os mecanismos de seu reconhecimento por leucócitos são pouco conhecidos. Neste trabalho, nós demonstramos que durante a intoxicação por diferentes agentes hepatotóxicos, paracetamol (APAP) e tioacetamida (TAA), ocorre acúmulo de DNA aberrante no citoplasma de hepatócitos, que culmina na liberação de DNA para o meio e extracelular morte destas células. Ao avaliarmos a lesão hepática por estes agentes in vivo, encontramos um enorme acúmulo de DNA no fígado, principalmente em zonas de necrose e na forma de depósitos intravasculares. Em paralelo ao DNA depositado, observamos um grande infiltrado de neutrófilos no fígado, que se direcionavam especificamente para o interior das zonas de necrose e exerciam um patrulhamento intenso destas áreas. Enquanto neutrófilos se movimentavam ativamente dentro da necrose, macrófagos se mantinham imóveis e restritos à área viável do fígado. Observamos que neutrófilos aumentam a expressão de TLR9 durante a lesão hepática, e usam este receptor para reconhecer e responder à deposição de DNA por meio do aumento de expressão de NF-κB e CXCR2. A eliminação dos depósitos de DNA no fígado pelo pós-tratamento com DNASE1 ou bloqueio do reconhecimento por TLR9 reduziram significativamente a lesão hepática, o infiltrado de neutrófilos e a inflamação tecidual. A expressão de TLR9 em neutrófilos é essencial para seu papel danoso, pois a transferência de neutrófilos selvagens para camundongos deficientes em TLR9 reverte completamente o fenótipo protegido da lesão por APAP. Em conjunto, nossos dados demonstram um novo fenômeno envolvendo a deposição de DNA no fígado durante a lesão hepática, e sugerem que o controle do reconhecimento de DNA pelo sistema imune é uma estratégia promissora para o tratamento de DILI e FHA. Drug-induced liver injury (DILI) is an important cause of acute liver failure (FHA), but with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Amongst these molecules, DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, the mechanisms involved in DNA release from damaged hepatocytes, whether it accumulates into the necrotic liver, and the impact of its recognition by the immune system remains elusive. Here we showed that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release to the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within the liver necrotic areas, together with a widespread intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to hepatotoxic overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibited an active patrolling behavior. DNA removal by intravenous DNASE1 injection or blockage of TLR9-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils upregulated TLR9 expression in the plasma membrane during liver necrosis, and these cells sensed and reacted to extracellular DNA by up-regulating NF-κB and CXCR2 expression. Interestingly, adoptive transfer of wild-type neutrophils to TLR9-/- mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. We concluded that hepatic DNA accumulation is a novel feature of DILI pathogenesis and blockage of DNA recognition by the innate immune system may consist in a promising therapeutic venue for DILI and FHA.

We investigated the effect of dietary supplementation with n-3 PUFA (fish oil source) in an experimental model of food allergy. Mice were sensitized (allergic group) or not (nonallergic group) with OVA and were fed with OVA diet to induce allergy signals. Mice were fed with regular diet in which 7% of lipid content was provided by soybean (5% of n-3 PUFA) or fish (25% of n-3 PUFA) oil. Allergic group mice had increased serum levels of antiovalbumin IgE and IgG1 and changes in small intestine, characterized by an increased edema, number of rolling leukocytes in microcirculation, eosinophil infiltration, mucus production, and Paneth cell degranulation, in comparison to non-allergic group. All these inflammatory parameters were reduced in mice fed high-n-3-PUFA diet. Our data together suggest that diet supplementation with n-3 PUFA from fish oil may consist of a valid adjuvant in food allergy treatment. ispartof: CLINICAL & DEVELOPMENTAL IMMUNOLOGY vol:2012 ispartof: location:Egypt status: published

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A doença hepática gordurosa não alcoólica (NAFLD) é uma das doenças crônicas mais frequentes no mundo. As limitações na compreensão de sua patogênese, especialmente nos estágios iniciais, impedem o desenvolvimento de ferramentas de diagnóstico mais eficazes e de novos tratamentos. No presente trabalho, mapeamos as alterações imunológicas no fígado durante os estágios iniciais da NAFLD em camundongos e em seres humanos, e propusemos um novo modelo animal de esteatose hepática inicial, que pode servir como uma plataforma para impulsionar os avanços no diagnóstico rápido e precoce auxiliando também nos métodos terapêuticos. Para tal, biópsias hepáticas de pacientes diagnosticados com NAFLD branda, foram coletadas para desenvolvimento de um perfil de expressão gênica, e camundongos foram alimentados com dieta high fat por curtos períodos para mimetizar a esteatose inicial. A resposta imune do fígado em camundongos foi investigada detalhadamente usando uma combinação de imagem intravital confocal, expressão gênica, isolamento celular, citometria de fluxo e ensaios de transplante de medula óssea. Sendo assim, foi possível observar grandes alterações imunológicas em humanos e camundongos nas fases iniciais da esteatose hepática. Em camundongos, essas alterações aumentaram significativamente a taxa de mortalidade por lesão hepática aguda induzida por drogas e também predispuseram os animais a infecções bacterianas. Além disso, a deleção de TLR4 tanto nas células imunológicas do fígado quanto nos hepatócitos reduziu a tolerância hepática, especialmente nas células de Kupffer, nos momentos iniciais de ingestão da dieta high fat. Dessa forma, concluímos que o sistema imunológico do fígado é capaz de sentir de maneira precoce o acúmulo de gordura hepático, gerando uma resposta bifásica ao consumo de dieta rica em gordura. Observamos ainda que as células do fígado parecem estar sendo primadas por ligantes de TLR4, o que confere ao fígado uma tolerância imunológica nas fases iniciais mas que a manutenção do insulto dietético pode levar a uma hiper-responsividade a lesões hepáticas agudas e a uma inabilidade em controlar infecções bacterianas. Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent chronic disease in the world, and limitations in our understanding of its pathogenesis, especially in initial stages, are precluding the development of diagnosis tools and new treatments. Here we mapped the immunological changes in liver during the early stages of fatty liver disease in mice and humans, and proposed a novel mouse model of initial liver steatosis, which may serve as a platform to drive advances in rapid diagnosis and therapeutic fields. For that, liver biopsies from patients in early stages of NAFLD were collected to further profiling of gene expression, and mice received high fat diet for short periods to mimic initial steatosis. Liver immune response in mice was detailed investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. We observed major immunologic changes in both humans and mice in the early phases of liver steatosis. In mice, these changes significantly enhanced mortality rate upon drug-induced liver injury and also predisposed mice to bacterial infections. Also, deletion of TLR4 in liver cells dampened liver tolerogenesis, especially in Kupffer cells, in the initial time points of dietary insult. In conclusion, liver immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon high fat diet insult. Priming of liver immune cells by gut-derived TLR4 ligands may add to liver tolerogenesis in initial phases, but continuous insults may drive hyper-responsiveness to damage and reduced ability to control infections.

O fígado possui papel vital desempenhando funções biosintéticas, metabólicas e detoxificantes. Apesar de sua conhecida capacidade de regeneração, em algumas condições como excesso de medicamentos, ele pode ser danificado, comprometendo suas funções e a vida do indivíduo. A lesão hepática induzida por droga (DILI) tem sido mais frequente em todo o mundo devido à maior facilidade de acesso a fármacos pela população. Dentre as diversas drogas comercializadas, o paracetamol (APAP) está sendo usado de forma indiscriminada, sendo a sobredose de APAP a maior causa de falência hepática aguda (ALF). A lesão estéril causada por APAP leva a um processo inflamatório com recrutamento de vários leucócitos ao fígado, mas em casos de inflamação aguda como na necrose hepática, neutrófilos são os primeiros a serem recrutados e migram em grande quantidade. Essas células amplificam a lesão hepática e pouco se sabe sobre o mecanismo efetor dessa amplificação. Considerando isso, tivemos como objetivo identificar os componentes granulares presentes nos neutrófilos responsáveis pela amplificação da lesão hepática após sobredose de APAP. Iniciamos o trabalho utilizando inibidores de degranulação e observamos uma diminuição da lesão hepática com o uso da doxiciclina e do cromoglicato nas doses de 50 e 100 mg/kg, e um aumento da função hepática com a maior dose de cromoglicato. Dentre os componentes presentes nos grânulos, inibimos primeiro as proteases com um coquetel inespecífico e observamos diminuição da lesão hepática e redução do número de neutrófilos com a dose de 1,4 mg/animal. Após isso buscamos identificar quais proteases estavam sendo responsáveis pelo resultado encontrado. Para isso, inibimos cada protease separadamente e observamos que a inibição de MMPs com 2 mg/kg do inibidor de MMP1 foi capaz de melhorar a lesão e a função hepática, além de reduzir em 40% a atividade de MPO. Para entendermos se a redução da lesão era decorrente da redução dos neutrófilos, inibimos o seu recrutamento, com o inibidor DF 2156A, e observamos que mesmo com a diminuição no número de neutrófilos a lesão não foi alterada. Inibimos também a produção de ROS por diversas vias e não houve alteração na lesão e na função hepática. Concluímos assim, que as MMPs são responsáveis por aumentar a morte de hepatócitos e diminuir a funcionalidade hepática após sobredose de APAP. The liver plays pivotal role in biosynthetic, metabolic and detoxifying functions. Despite its known capacity for regeneration, in some conditions like excess medication, it can be damaged, compromising their function and the individual healthy. Drug-induced liver injury (DILI) has been more frequent in the world due to the easy access to drugs by the population. Among the several marketed drugs, acetaminophen (APAP) is being used indiscriminately, and the APAP overdose is the leading cause of acute liver failure (ALF). The sterile injury caused by APAP leads to inflammatory process with recruitment of many leukocytes to the liver, but in cases of acute inflammation, such as the liver necrosis, neutrophils are the first to be recruited and migrate in large numbers. These cells eventually amplify the liver damage and little is known about the effector mechanism of this amplification. Considering this, our objective was to identify the granular components present in neutrophils responsible for amplification of liver injury after APAP overdose. We started working using degranulation inhibitors and a reduction of the liver damage with the use of doxycycline and cromoglycate at doses of 50 and 100 mg/kg, and an increase in liver function with the highest dose of cromoglycate was observed. Among the components present in the granules, we first inhibit unspecific proteases with a cocktail and observed decreased liver damage and decreased number of neutrophils at the dose of 1.4 mg/mouse. After that, we tried to identify which proteases were being responsible for the results found. To this end, we inhibited each protease separately and found that the inhibition of MMPs with 2 mg/kg of MMP1 inhibitor was able to reduce the damage and improve liver function in addition to 40% reduction in MPO activity. To understand whether the reduction of the injury was due to the reduction of neutrophils, we inhibited neutrophils recruitment, with DF2156A inhibitor, and noted that even with the decrease in the number of neutrophils injury has not changed. We also inhibited ROS production in many pathways and there was no change in the injury and liver function. This way, we concluded that MMPs are responsible for increase hepatocyte death and decrease the hepatic functionality after APAP overdose.