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

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

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

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

Prevalence of human papillomavirus (HPV) in the semen of patients submitted to assisted reproductive technology treatment in a private clinic in Brazil.

OBJECTIVE: The aim of our study was to identify the prevalence of HPV in the semen of men submitted to ART treatment and look into the possible impacts of the virus on sperm parameters. METHODS: Thirty-five patients treated for infertility from March to August 2016 were invited to join the study. Samples with a minimum concentration of 40x106 spermatozoa per milliliter were included in the study. After the evaluation of semen parameters, DNA extraction and PCR were performed to verify the presence of HPV by electrophoresis in 8% polyacrylamide gel. RESULTS: Patient age ranged from 27 to 68 years (mean 39.2 years). Semen analysis showed a mean volume of 2.5mL; mean concentration of 58.9x106; and mean motility of 51.8%. HPV DNA was identified in seven semen samples from 25 patients (28%). Ten samples with DNA concentrations below 10ng/µL were excluded from the study due to poor amplification quality. There was no statistical difference in sperm concentration when HPV-negative and HPV-positive samples were compared (65.9x106 vs. 62.3x106; p=0.70). However, sperm motility was significantly higher in HPV-positive semen (65% vs. 46.6%; p=0.02). CONCLUSIONS: HPV prevalence was 28% in the semen of patients submitted to ART treatment. HPV-positive samples had statistically increased motility compared to negative samples (65% vs. 46.6%; p=0.02).

Paradoxical Role of Matrix Metalloproteinases in Liver Injury and Regeneration after Sterile Acute Hepatic Failure.

Acetaminophen (APAP) poisoning is one of the leading causes of acute hepatic failure and liver transplantation is often the only lifesaving alternative. During the course of hepatocyte necrosis, an intense accumulation of neutrophils is often observed within the liver microenvironment. Despite the classic idea that neutrophil accumulation in tissues causes collateral tissue damage, there is a growing body of evidence showing that neutrophils can also orchestrate the resolution of inflammation. In this work, drug-induced liver injury was induced by oral administration of APAP and pharmacological intervention was made 12 h after this challenge. Liver injury and repair kinetics were evaluated by a novel combination of enzyme quantifications, ELISA, specific antagonists of neutrophil enzymes and confocal intravital microscopy. We have demonstrated that neutrophil infiltration is not only involved in injury amplification, but also in liver tissue repair after APAP-induced liver injury. In fact, while neutrophil depletion led to reduced hepatic necrosis during APAP poisoning, injury recovery was also delayed in neutropenic mice. The mechanisms underlying the neutrophil reparative role involved rapid degranulation and matrix metalloproteinases (MMPs) activity. Our data highlights the crucial role of neutrophils, in particular for MMPs, in the resolution phase of APAP-induced inflammatory response.

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

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

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

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

Neutrophil biology within hepatic environment.

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

IL-33 signalling in liver immune cells enhances drug-induced liver injury and inflammation.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the contribution of IL-33/ST2 axis in the onset and progression of acute liver injury using a mice model of drug-induced liver injury (DILI). MATERIAL AND TREATMENTS: DILI was induced by overdose administration of acetaminophen (APAP) by oral gavage in wild-type BALB/c, ST2-deficient mice and in different bone marrow chimeras. Neutrophils were depleted by anti-Ly6G and macrophages with clodronate liposomes (CLL). METHODS: Blood and liver were collected for biochemical, immunologic and genetic analyses. Mice were imaged by confocal intravital microscopy and liver non-parenchymal cells and hepatocytes were isolated for flow cytometry, genetic and immunofluorescence studies. RESULTS: Acetaminophen overdose caused a massive necrosis and accumulation of immune cells within the liver, concomitantly with IL-33 and chemokine release. Liver non-parenchymal cells were the major sensors for IL-33, and amongst them, neutrophils were the major players in amplification of the inflammatory response triggered by IL-33/ST2 signalling pathway. CONCLUSION: Blockage of IL-33/ST2 axis reduces APAP-mediated organ injury by dampening liver chemokine release and activation of resident and infiltrating liver non-parenchymal cells.

Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice.

BACKGROUND & AIMS: Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be replaced by bone marrow precursors during extensive injuries, irradiation, and infections. We investigated the liver populations of myeloid immune cells and their location, as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original phagocytes by bone marrow-derived surrogates were also examined. METHODS: We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt, and CX3CR1-DTR-EYFP. Liver nonparenchymal cells were immunophenotyped using mass cytometry and gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. RESULTS: Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory cytokines, and dexamethasone accelerated the repopulation of liver phagocytes. CONCLUSIONS: In studies of hepatic phagocyte depletion in mice, we found that myeloid precursors can differentiate into liver macrophages and dendritic cells, which each localize to distinct tissue compartments. During replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.

Dietary supplementation with omega-3-PUFA-rich fish oil reduces signs of food allergy in ovalbumin-sensitized mice.

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.

Experimental food allergy leads to adipose tissue inflammation, systemic metabolic alterations and weight loss in mice.

To investigate the consequences of food allergy in adipose tissue and metabolism, we used a murine model in which mice have been sensitized subcutaneously with ovalbumin and further received antigen-containing diet. Allergic mice presented a significant weight loss 7 days after oral challenge with a concomitant decrease in epididymal adipose tissue mass. This decrease was associated with increased lipolysis and local inflammation. In adipose tissue of allergic mice there were increased leukocyte rolling and adhesion in the microvasculature, increased number of leukocytes in the tissue, especially macrophages (F4/80(+) cells) and increased pro-inflammatory cytokines levels, including TNF-α, IL-6 and CCL2. In addition, we observed low serum concentrations of triglyceride, glucose, total cholesterol and free fatty acids in the allergic mice. Our results suggest that the induction of food allergy in mice leads to adipose tissue inflammation and systemic metabolic alterations that contribute to the weight loss observed.