Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury.

Background & Aims: Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury. Methods: We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver in vivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67). Results: Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, in vitro and in vivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG. Conclusion: NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery. Impact and implications: Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

Absence of CCR2 Promotes Proliferation of Alveolar Macrophages That Control Lung Inflammation in Acute Respiratory Distress Syndrome in Mice.

Acute respiratory distress syndrome (ARDS) consists of uncontrolled inflammation that causes hypoxemia and reduced lung compliance. Since it is a complex process, not all details have been elucidated yet. In a well-controlled experimental murine model of lipopolysaccharide (LPS)-induced ARDS, the activity and viability of macrophages and neutrophils dictate the beginning and end phases of lung inflammation. C-C chemokine receptor type 2 (CCR2) is a critical chemokine receptor that mediates monocyte/macrophage activation and recruitment to the tissues. Here, we used CCR2-deficient mice to explore mechanisms that control lung inflammation in LPS-induced ARDS. CCR2-/- mice presented higher total numbers of pulmonary leukocytes at the peak of inflammation as compared to CCR2+/+ mice, mainly by enhanced influx of neutrophils, whereas we observed two to six-fold lower monocyte or interstitial macrophage numbers in the CCR2-/-. Nevertheless, the time needed to control the inflammation was comparable between CCR2+/+ and CCR2-/-. Interestingly, CCR2-/- mice presented higher numbers and increased proliferative rates of alveolar macrophages from day 3, with a more pronounced M2 profile, associated with transforming growth factor (TGF)-ß and C-C chemokine ligand (CCL)22 production, decreased inducible nitric oxide synthase (Nos2), interleukin (IL)-1ß and IL-12b mRNA expression and increased mannose receptor type 1 (Mrc1) mRNA and CD206 protein expression. Depletion of alveolar macrophages significantly delayed recovery from the inflammatory insult. Thus, our work shows that the lower number of infiltrating monocytes in CCR2-/- is partially compensated by increased proliferation of resident alveolar macrophages during the inflammation control of experimental ARDS.

Corrigendum: Susceptibility to infections during acute liver injury depends on transient disruption of liver macrophage niche.

[This corrects the article DOI: 10.3389/fimmu.2022.892114.].

Susceptibility to Infections During Acute Liver Injury Depends on Transient Disruption of Liver Macrophage Niche.

Kupffer cells are the primary liver resident immune cell responsible for the liver firewall function, including clearance of bacterial infection from the circulation, as they are strategically positioned inside the liver sinusoid with intimate contact with the blood. Disruption in the tissue-resident macrophage niche, such as in Kupffer cells, can lead to a window of susceptibility to systemic infections, which represents a significant cause of mortality in patients with acetaminophen (APAP) overdose-induced acute liver injury (ALI). However, how Kupffer cell niche disruption increases susceptibility to systemic infections in ALI is not fully understood. Using a mouse model of ALI induced by APAP overdose, we found that Kupffer cells upregulated the apoptotic cell death program and were markedly reduced in the necrotic areas during the early stages of ALI, opening the niche for the infiltration of neutrophils and monocyte subsets. In addition, during the resolution phase of ALI, the remaining tissue macrophages with a Kupffer cell morphology were observed forming replicating cell clusters closer to necrotic areas devoid of Kupffer cells. Interestingly, mice with APAP-induced liver injury were still susceptible to infections despite the dual cellular input of circulating monocytes and proliferation of remaining Kupffer cells in the damaged liver. Therapy with bone marrow-derived macrophages (BMDM) was shown to be effective in occupying the niche devoid of Kupffer cells following APAP-induced ALI. The rapid BMDM migration to the liver and their positioning within necrotic areas enhanced the healing of the tissue and restored the liver firewall function after BMDM therapy. Therefore, we showed that disruption in the Kupffer cell niche and its impaired function during acute liver injury are key factors for the susceptibility to systemic bacterial infections. In addition, modulation of the liver macrophage niche was shown to be a promising therapeutic strategy for liver injuries that reduce the Kupffer cell number and compromise the organ function.

The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74-103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice.

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-1ß (IL-1ß) 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.

Genetic Background Affects the Mucosal Secretory IgA Levels, Parasite Burden, Lung Inflammation, and Mouse Susceptibility to Ascaris suum Infection.

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.

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Abstract Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases.

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.

CXCR1 and CXCR2 Inhibition by Ladarixin Improves Neutrophil-Dependent Airway Inflammation in Mice.

Rationale: Increased IL-8 levels and neutrophil accumulation in the airways are common features found in patients affected by pulmonary diseases such as Asthma, Idiopathic Pulmonary Fibrosis, Influenza-A infection and COPD. Chronic neutrophilic inflammation is usually corticosteroid insensitive and may be relevant in the progression of those diseases. Objective: To explore the role of Ladarixin, a dual CXCR1/2 antagonist, in several mouse models of airway inflammation with a significant neutrophilic component. Findings: Ladarixin was able to reduce the acute and chronic neutrophilic influx, also attenuating the Th2 eosinophil-dominated airway inflammation, tissue remodeling and airway hyperresponsiveness. Correspondingly, Ladarixin decreased bleomycin-induced neutrophilic inflammation and collagen deposition, as well as attenuated the corticosteroid resistant Th17 neutrophil-dominated airway inflammation and hyperresponsiveness, restoring corticosteroid sensitivity. Finally, Ladarixin reduced neutrophilic airway inflammation during cigarette smoke-induced corticosteroid resistant exacerbation of Influenza-A infection, improving lung function and mice survival. Conclusion: CXCR1/2 antagonist Ladarixin offers a new strategy for therapeutic treatment of acute and chronic neutrophilic airway inflammation, even in the context of corticosteroid-insensitivity.

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.

Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage.

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.

Versican and Tumor-Associated Macrophages Promotes Tumor Progression and Metastasis in Canine and Murine Models of Breast Carcinoma.

Versican and tumor-associated macrophages (TAMs) are involved in growth and metastases in several cancers. Here, we investigated the potential role of versican, a matrix proteoglycan, and its correlation with TAMs infiltrates in different stages of two different breast cancer models: spontaneous canine mammary gland carcinomas and the murine 4T1 breast cancer model. The stromal versican expression was correlated with TAMs accumulation in tumors with an advanced stage from spontaneous canine mammary carcinoma samples. Versican expression in mice, identified in late stages of tumor progression, was associated to a high number of peri-tumoral infiltrating TAMs. Indeed, TAMs were related to a pro-inflammatory and pro-angiogenic state in the primary tumor. Furthermore, TAMs accumulation was related to versican expression in the lungs and an increased number of pulmonary metastatic nodules with pulmonary mechanical dysfunction, which was due to leukocyte influx in the airways and elevated growth factor levels in the microenvironment. Thus, we suggest that versican and TAMs as attractive targets for breast cancer therapy.

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.

Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

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<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice.

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.