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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.

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 Malária continua sendo uma grande ameaça à saúde pública mundial, e crianças menores de 5 anos são as principais vítimas fatais da doença. O fígado possui papel essencial no curso da infecção, em que as características particulares do endotélio hepático, juntamente com diferentes populações de células imunes, inclusive de macrófagos residentes do fígado, estão diretamente envolvidas em complicações grave, como a anemia da malária grave, responsável por grande parte dos óbitos infantis. Estudos prévios realizados por nosso grupo apontam que o fígado passa por uma transição hematopoiética, caracterizada pela mudança de um perfil mieloide para linfoide, ao longo do desenvolvimento pós-natal, e que o baço e medula óssea de neonatos também apresentam perfis celulares diferentes de adultos. Entretanto, faltam entendimentos da relação do desenvolvimento hematopoiético com a gravidade da malária. Além disso, há ausência de modelos experimentais que reproduzam a suscetibilidade infantil à malária. Objetivo: Propor um modelo experimental em camundongos que possa ser útil no entendimento da maior susceptibilidade de crianças à malária, caracterizando a dinâmica e os mecanismos envolvidos na eritropoiese em resposta à infecção por Plasmodium chabaudi. Resultados e conclusão: Padronizamos a infecção em animais de diferentes idades ao longo da vida e observamos que a parasitemia é maior em camundongos infantes do que em neonatos e adultos. Além disso, camundongos infantes infectados por P. Chabaudi apresentam anemia no pico da infecção e não sobrevivem. Por citometria de fluxo, vimos que os nichos hematopoiéticos de infantes (medula óssea, baço e fígado) apresentam aumento dos precursores eritroides durante a infecção por P. chabaudi, enquanto que os adultos suprimem a eritropoiese. Também mostramos que em resposta à infecção, há maior quantidade de precursores de eritrócitos no sangue periférico de animais infantes do que em adultos, e que estes precursores também podem ser infectados por P. Chabaudi, e contribuir para a hiperparasitemia do grupo e consequente letalidade. Malaria remains a major threat to public health worldwide, and children under 5 years old are the main fatal victims of this disease. The liver plays an essential role in the course of infection. The singular characteristics of the hepatic endothelium, together with different immune cell populations, including tissue macrophages, are directly involved in serious complications, such as severe malarial anemia, responsible for a large number of infant deaths. Studies developed by our group show that the liver undergoes a hematopoietic transition throughout postnatal development, and the spleen and bone marrow of neonates have different cell profiles when compared to adults. However, studies on the relationship between hematopoietic development and the severity of malaria and experimental models that reproduce childhood susceptibility are lacking. Aim: To propose an experimental model in mice that can be useful in understanding the mortality of children from malaria, characterizing the dynamics and mechanisms involved in erythropoiesis in response to Plasmodium chabaudi infection. Results and conclusion: We standardized malaria infection in mice in different ages throughout life, and a higher parasitemia in infant mice than in neonates and adults was observed. In addition, infant mice infected with P. chabaudi presented anemia at the peak of infection and did not survive. Using flow cytometry, we saw that the hematopoietic niches of infants - bone marrow, spleen and liver - showed increased erythroid precursors during malarial infection, whereas adults suppress erythropoiesis. We also showed that there is a greater number of erythrocyte precursors in the peripheral blood of infant animals than in adults, and these precursors can be infected by P. chabaudi, contributing to the group's hyperparasitemia and consequent lethality.

Objectives To investigate putative different outcomes on non-alcoholic fatty liver disease development in mice using fat options regularly used in human nutrition. Methods Male C57BL/6 mice were fed control diet, and four different high fat diets (HFD - 40% calories from fat, Research Diets, USA) for 16 and 30 weeks. High fat diets had different common fat sources, including trans fat, non-trans fat palm oil (Primex-Z®), palm oil alone and corn oil alone. Mice were euthanized and samples were collected for analysis. Results Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that HFD induced a major increase in hepatic lipid droplet deposition in comparison to controls, being significantly higher in Primex-Z® fed mice. All HFDs had similar or less weight gain compared with control mice; however, Primex-Z® ingestion led to a higher increasing in adiposity index (∼90% increase) in comparison to other fat sources. Gene expression of isolated liver immune cells revealed large changes expression of several inflammatory pathways, which were also more elevated in Primex-Z fed mice, including Tnf (∼20-fold), Il1b (∼60-fold) and Tgfb (2.5-fold). Immunophenotyping and in vivo analysis showed that the frequency of hepatic immune cells was also disturbed during different HFD contents, rendering not only Kupffer cell depletion, but also reduced bacterial arresting ability. Conclusion Different fat dietary sources imprint different immune and metabolic impacts in the liver during high fat diet consumption. Our data highlight that Primex-Z® – a novel non-trans fat – is not only able to damage hepatocytes, but also impair liver ability to clear blood-borne infections.

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., Graphical abstract, Highlights • Hepatic immune response is already altered in liver biopsies from patients with mild NAFLD. • We designed a novel mouse model to mimic mild NAFLD, enabling the chronological mapping of liver changes. • This revealed an increased mortality rate upon secondary liver damage and a window of increased susceptibility to infection. • NAFLD diagnosis may be significantly improved by a more profound investigation of changes in hepatic immunology. • These data could guide customized nutritional and therapeutic interventions at different stages of NAFLD.

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. ispartof: CELLS vol:7 issue:12 ispartof: location:Switzerland status: published

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.