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.

Maternal chronic caffeine intake impairs fertility, placental vascularization and fetal development in mice.

Caffeine is commonly consumed by pregnant women to avoid fatigue or as a habit. However, it is not clearly determined its side effects to the conceptuses. This study evaluated placental morphofunctional alterations after maternal chronic caffeine intake and the effects on fetal growth. Female Swiss mice received, via gavage, caffeine doses (either 60, 120 or 240 mg/kg/day) seven days before mating until gestational days-(GD) 11.5 or 17.5. Fetal biometrical parameters were assessed, and placentae were either submitted to histomorphometrical or molecular evaluation of angiogenesis (placental growth factor-1[PlGF-1]), apoptosis (Caspase-3) and proliferation (Ki-67) markers (evaluated in Swiss dams) and to intravital microscopy (evaluated in C57BL/6 dams). Caffeine exposed fetuses exhibited intrauterine growth restriction in a sex-dependent manner, with greater commitment of female fetuses (P < 0.05). In addition, placentae from dams that received 120 mg/kg/day showed less irrigation by maternal blood and greater development of fetal vasculature, characterized by higher number of larger vessels (P < 0.05). Although no effects on apoptosis (Caspase-3) and angiogenesis (PlGF-1) were observed, dams treated with 60 mg/kg/day showed greater placental cell proliferation (Ki-67 staining) at GD 11.5 (P < 0.05). The group treated with 240 mg/kg/day exhibited only one pregnant dam for each gestational age, suggesting that this high caffeine consumption may compromise fertility. Taken together, even in the doses currently ingested by many pregnant women, caffeine has detrimental effects on placental vasculature and fetal development in mice. Therefore, our results strongly suggest that caffeine consumption in human pregnancies greater than the recommended doses should be avoided.

Chronic ingestion of Primex-Z, compared with other common fat sources, drives worse liver injury and enhanced susceptibility to bacterial infections.

OBJECTIVES: The aim of this study was to investigate putative different outcomes on the development of non-alcoholic fatty liver disease in mice using fat options regularly used in human nutrition. METHODS: Male C57BL/6 mice were fed a control diet, and four different high-fat diets (HFD: 40% calories from fat; Research Diet, Inc., New Brunswick, New Jersey, USA) for 16 and 30 wk. HFDs had different common fat sources, including trans-fat, non-trans-fat palm oil (Primex-Z), palm oil alone, and corn oil alone. Mice were sacrificed and samples were collected for analysis. RESULTS: Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that a HFD induced a major increase in hepatic lipid droplet deposition compared with control mice, being significantly higher in Primex-Z-fed mice. All HFD mice had similar or less weight gain as control mice; however, Primex-Z ingestion led to a higher increase in adiposity index (~90% increase) compared with other fat sources. Gene expression of isolated liver immune cells revealed large changes in 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 effects in the liver during consumption of an HFD. The present data highlighted that Primex-Z-a novel non-trans-fat-is not only able to damage hepatocytes, but also to impair liver ability to clear blood-borne infections.

Photobiomodulation drives pericyte mobilization towards skin regeneration.

Photobiomodulation is being widely applied for improving dermal or mucosal wound healing. However, the underlying cellular and molecular processes that directly contribute to its effects remain poorly understood. Pericytes are relevant cells involved in the wound microenvironment and could be one of the main targets of photobiomodulation due to their plasticity and perivascular localization. Herein, we investigate tissue repair under the photobiomodulation stimulus using a pericyte labeled (or reporter) transgenic mice. Using a model of two contralateral back wounds, one the control and the other photoactivated daily (660 nm, 20 mW, 0.71 W/cm2, 5 J/cm2, 7 s, 0.14 J), we showed an overall influx of immune and undifferentiated cells and higher mobilization of a potent pericyte subpopulation (Type-2 pericytes) in the photoactivated wounds in comparison to the controls. Doppler analysis showed a significant increase in the blood flow in the photoactivated wounds, while marked vascular supply was observed histologically. Histochemical analysis has indicated more advanced stages of tissue repair after photoactivation. These data suggest that photobiomodulation significantly accelerates tissue repair through its vascular effects with direct recruitment of pericytes to the injury site.

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.

Gelatin-based membrane containing usnic acid-loaded liposomes: A new treatment strategy for corneal healing.

PURPOSE: To evaluate the safety and potential healing efficacy of the topical ocular administration of a gelatin membrane containing usnic acid/liposomes (UALs) for corneal cicatrization. UALs have shown healing activity in animal models of dermal burn lesions. We evaluated the safety of topical ocular administration of UAL and its potential healing efficacy as an ophthalmic treatment on chemical lesions in rabbit eyes. METHOD: The Draize test was used to check for ocular toxicity and the score was zero at each observation, indicating the ocular safety of a gelatin membrane containing usnic acid/liposome. Its potential healing efficacy as an ophthalmic treatment on chemical lesions in rabbit eyes was also assessed. RESULTS: After epithelial removal and treatment with UAL, there was a 49.4 % reduction in injury under in vivo conditions compared with a 36.6 % reduction in the control, a gelatin membrane containing liposome without usnic acid. Histological analysis of ocular surface chemical injury-tissue sections after treatment with UAL supported these observations. The corneal expression of VEGF and TGF-ß1increased by 70 % and 50 % respectively following treatment with UAL gelatin membrane. CONCLUSION: These results indicate the potential therapeutic application of UAL gelatin membranes as an ophthalmic treatment that may be used for corneal cicatrization.

The liver as a nursery for leukocytes.

Leukocytes are a large population of cells spread within most tissues in the body. These cells may be either sessile (called as resident cells) or circulating leukocytes, which travel long journeys inside the vessels during their lifespan. Although production and maturation of these leukocytes in adults primarily occur in the bone marrow, it is well known that this process-called hematopoiesis-started in the embryonic life in different sites, including the yolk sac, placenta, and the liver. In this review, we will discuss how the liver acts as a pivotal site for leukocyte maturation during the embryo phase, and also how the most frequent liver-resident immune cell populations-namely Kupffer cells, dendritic cells, and lymphocytes-play a vital role in both tolerance and inflammatory responses to antigens from food, microbiota, and pathogens.

Generation of a triple-fluorescent mouse strain allows a dynamic and spatial visualization of different liver phagocytes in vivo.

Resident and circulating immune cells have been extensively studied due to their almost ubiquitous role in cell biology. Despite their classification under the "immune cell department", it is becoming increasingly clear that these cells are involved in many different non-immune related phenomena, including fetus development, vascular formation, memory, social behavior and many other phenotypes. There is a huge potential in combining high-throughput assays - including flow cytometry and gene analysis - with in vivo imaging. This can improve our knowledge in both basic and clinical cell biology, and accessing the expression of markers that are relevant in the context of both homeostasis and disease conditions might be instrumental. Here we describe how we generated a novel mouse strain that spontaneously express three different fluorescence markers under control of well-studied receptors (CX3CR1, CCR2 and CD11c) that are involved in a plethora of stages of cell ontogenesis, maturation, migration and behavior. Also, we assess the percentage of the expression and co-expression of each marker under homeostasis conditions, and how these cells behave when a local inflammation is induced in the liver applying a cutting-edge technology to image cells by confocal intravital microscopy.

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.