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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

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

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.

Ascariasis is a neglected tropical disease, widespread in the world and causing important socioeconomic impacts. The presence of various stages of worm development in the pulmonary and intestinal mucosa induces a humoral and cellular immune response. However, although there is much evidence of the protective role of mucosal immunity against various pathogens, including helminthes, there is still a gap in the knowledge about the immune response and the mechanisms of action that are involved in protection against diseases, especially in the initial phase of ascariasis. Then, the aim of this study was to evaluate the kinetic aspects of the immune parasitological parameters in intestinal and pulmonary mucosa in male mice with early ascariasis. Therefore, two mice strains showed a different susceptibility to ascariasis (BALB/c and C57BL6/j), when experimentally infected with 2,500 infective eggs of Ascaris suum from time-point 0 and the immune parasitological parameters evaluated each two days after infection, during the period of 12 days. The results were suggestive of a synergetic action of intestinal and pulmonary SIgA contributing for the protection against early ascariasis by reducing the amount of migrating larval as well as the influx of leukocytes in the lung and the consequent impair of the pulmonary capacity.

Ascariasis is a neglected tropical disease that is widespread in the world and has important socioeconomic impacts. The presence of various stages of worm development in the pulmonary and intestinal mucosae induces a humoral and cellular immune response. However, although there is much evidence of the protective role of mucosal immunity against various pathogens, including helminths, there is still a gap in the knowledge about the immune response and the mechanisms of action that are involved in protection against diseases, especially in the initial phase of ascariasis. Thus, the aim of this study was to evaluate the kinetic aspects of the immune parasitological parameters in intestinal and pulmonary mucosae in male mice with early ascariasis. Therefore, two mouse strains that showed different susceptibilities to ascariasis (BALB/c and C57BL/6J) when experimentally infected with 2,500 infective eggs of Ascaris suum from time point 0 were examined: the immune parasitological parameters were evaluated each 2 days after infection over a period of 12 days. The results were suggestive of a synergetic action of intestinal and pulmonary secretory IgA (S-IgA) contributing to protection against early ascariasis by reducing the amount of migrating larvae as well as the influx of leukocytes in the lung and the consequent impairment of pulmonary capacity.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host’s organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles., Author summary Ascariasis is the most prevalent but neglected tropical disease in the world, affecting more than 450 million people, mainly children. The parasites life cycle can be divided into the following two distinct phases after the initial infection: (i) the first stage called larval ascariasis involves migration of parasitic larval stages through several tissues (intestinal mucosa, blood circulation, liver, and lung/airways) and is marked by mechanical injuries, followed by an intense local inflammatory response, (ii) the second involves the establishment of adult worms in the lumen of the small intestine, causing the despoilment of nutrients and secretory/excretory parasitic products that downmodulate host immune response and characterize the chronic infection. Innate and adaptive immune responses are intrinsically connected and their cross-talk is very important for host homeostasis and is crucial in dealing with helminthiasis. In this study, we evaluated the importance of eosinophils mediating IgA production in mucosal sites, tissue inflammation, and remodeling, as well as controlling parasite burden during experimental larval ascariasis. This study provides a new perspective suggesting that stimulation by probiotics may prevent the lung pathology associated with ascariasis.

Aim: Characterize the course of acute Aspergillus fumigatus lung infection in immunocompetent mice, investigating the immunological, pathological and tissue functional modifications. Materials & methods: C57BL/6 mice were intranasally infected with A. fumigatus conidia and euthanized to access inflammatory parameters. Results: Mice infected with A. fumigatus showed an inoculum-dependent lethality and body weight loss. An intense proinflammatory cytokine release, neutrophil infiltrate and pulmonary dysfunction was also observed in the early phase of infection. In the late phase of infection, proresolving mediators release, apoptosis and efferocytosis increased and lung tissue architecture is restored. Conclusion: Our study characterized an immunocompetent model of acute pulmonary Aspergillus infection in mice and opened an array of possibilities for investigations on interactions of A. fumigatus with host-immune system.

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.

Toll-like receptor 9 (TLR9) and Phosphatidylinositol-3-kinase gamma (PI3Kγ) are very important effectors of the immune response, however, the importance of such crosstalk for disease development is still a matter of discussion. Here we show that PI3Kγ is required for immune responses in which TLR9 is a relevant trigger. We demonstrate the requirement of PI3Kγ for TLR9-induced inflammation in a model of CpG-induced pleurisy. Such requirement was further observed in inflammatory models where DNA sensing via TLR9 contributes to disease, such as silicosis and drug-induced liver injury. Using adoptive transfer, we demonstrate that PI3Kγ is important not only in leukocytes but also in parenchymal cells for the progression of inflammation. We demonstrate this crosstalk between TLR9 and PI3Kγ in vitro using human PBMCs. The inhibition of PI3Kγ in CpG-stimulated PBMCs resulted in reduction of both cytokine production and phosphorylated Akt. Therefore, drugs that target PI3Kγ have the potential to treat diseases mediated by excessive TLR9 signalling. ispartof: SCIENTIFIC REPORTS vol:9 issue:1 ispartof: location:England status: published

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.

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 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.

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 &alpha, /&beta, receptor (IFNAR&minus, /&minus, ) 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.

PI3K activation plays a central role in the development of pulmonary inflammation and tissue remodeling. PI3K inhibitors may thus offer an improved therapeutic opportunity to treat non-resolving lung inflammation but their action is limited by unwanted on-target systemic toxicity. Here we present CL27c, a prodrug pan-PI3K inhibitor designed for local therapy, and investigate whether inhaled CL27c is effective in asthma and pulmonary fibrosis. Mice inhaling CL27c show reduced insulin-evoked Akt phosphorylation in lungs, but no change in other tissues and no increase in blood glycaemia, in line with a local action. In murine models of acute or glucocorticoid-resistant neutrophilic asthma, inhaled CL27c reduces inflammation and improves lung function. Finally, inhaled CL27c administered in a therapeutic setting protects from bleomycin-induced lung fibrosis, ultimately leading to significantly improved survival. Therefore, local delivery of a pan-PI3K inhibitor prodrug reduces systemic on-target side effects but effectively treats asthma and irreversible pulmonary fibrosis., Activation of PI3K plays a role in pulmonary inflammation. Here, the authors develop a drug inhibitor of PI3K, and show that it inhibits lung inflammation and damage in mouse models of asthma and lung fibrosis.

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.

Purpose – This paper aims to investigate the electroporation phenomenon in a single cell exposed to ultra short (μs) and high voltage (kV) electric pulses. Design/methodology/approach – The problem is addressed by two complementary approaches. First, numerical simulations based on an asymptotic approximation derived from the Smoluchowski theory are used to calculate the pore generation, growth and size evolution at the membrane of a spherical cell model, immersed in a suspension medium and consisting of cytoplasm and membrane. The numerical calculations are solved using the finite difference method. Second, an in vitro experiment with LLC-MK2 cells is carried out in which electroporation was monitored with molecules of propidium iodide. This part also comprehended the design and manufacturing of a portable electric pulse generator capable of providing rectangular pulses with amplitude of 1,000 V and duration in the range of 1-μs to 100-μs. The pulse generator is composed of three modules: a high voltage D...

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P

Ascaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host’s homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris., Author Summary Human ascariasis caused by the helminths Ascaris lumbricoides and Ascaris suum, is the most prevalent neglected tropical disease in the world, affecting more than 800 million people and mainly school-aged children. The parasite life cycle may be divided in two distinct phases after the initial infection: (i) migration of parasitic larval stages through several tissues (intestinal mucosa, blood circulation, liver and lung/airways), namely larval ascariasis; and (ii) establishment of adult worms in the lumen of the small intestine, causing the despoilment of nutrients and secretion/excretion of parasitic products, which down modulate the immune response of the host and characterizing the chronic infection. Over the past decades special focus has been dedicated to chronic Ascaris infection with the assessment of immunopathological aspects of the disease in chronically infected individuals from endemic areas. However, knowledge about larval ascariasis still remains scarcedue the limitations of diagnostic techniques and the need for an experimental model that would mimic natural infection. Indeed, many aspects of the immunobiology of the early Ascaris infection are still poorly understood. In this context, single and multiple infections in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of larval ascariasis.