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.

Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.

Acetaminophen (APAP) is usually safe when administrated in therapeutic doses; however, APAP overdose can lead to severe liver injury. APAP can cause direct hepatocyte damage, and stimulates an inflammatory response leading to oxidative stress. Supressor of Cytokine Signaling (SOCS) 2 modulates cytokine and growth factor signaling, and plays a role in the regulation of hepatic cellular processes. Our study evaluated the role of SOCS2 in APAP liver injury. The administration of a toxic dose (600 mg/kg) of APAP caused significant liver necrosis in WT mice. In SOCS2−/− mice, there was significantly more necrosis, neutrophil recruitment, and expression of the neutrophil-active chemokine CXCL-1. Expression of proinflammatory cytokines, such as TNF-α and IL-6, was elevated, while expression of anti-inflammatory cytokines, IL-10 and TGF-β, was diminished. In vitro, SOCS2−/− hepatocytes expressed more p-NF-kB and produced more ROS than WT hepatocytes when exposed to APAP. SOCS2−/− hepatocytes were more sensitive to cell death in the presence of IL-6 and hydrogen peroxide. The administration of catalase in vitro and in vivo resulted in a pronounced reduction of cells/mice death and necrosis in the SOCS2−/− group. We have demonstrated that SOCS2 has a protective role in the liver by controlling pro-oxidative and inflammatory mechanisms induced by APAP.

Annexins are well-known Ca 2+ phospholipid-binding proteins, which have a wide variety of cellular functions. The role of annexin A1 (AnxA1) in the innate immune system has focused mainly on the anti-inflammatory and proresolving properties through its binding to the formyl-peptide receptor 2 (FPR2)/ALX receptor. However, studies suggesting an intracellular role of AnxA1 are emerging. In this study, we aimed to understand the role of AnxA1 for interleukin (IL)-1b release in response to activators of the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome. Using AnxA1 knockout mice, we observed that AnxA1 is required for IL-1b release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL-1b, NLRP3 and caspase-1, a step called NLRP3 priming. Moreover, we demonstrate that AnxA1 co-localize and directly bind to NLRP3, suggesting the role of AnxA1 in inflammasome activation is independent of its anti-inflammatory role via FPR2. Therefore, AnxA1 regulates NLRP3 inflammasome priming and activation in a FPR2-independent manner.Bone marrow-derived macrophages (BMDMs) were obtained as previously described. 31 Briefly, bone marrow cells were collected from femurs of wild-type (WT), AnxA1 À/À and FPR2/3 À/À mice and differentiated for 7 days in RPMI 1640 supplemented with 20% of fetal bovine serum (FBS) and 30% L929 conditioned medium.

The emergence of life-threatening zoonotic diseases caused by betacoronavirus, including the ongoing COVID-19 pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus MHV-3 develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired the respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic and a high virus burden could be detected in multiple organs alongside with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the pro-inflammatory cytokines IL-1β, IL-6, IL-12, IFN-γ, and TNF, thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line, TNF blockage also diminished the infection-mediated release of pro-inflammatory cytokines and virus replication of human epithelial lung cells infected with SARS-CoV-2. Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower cost, biosafety level-2 in vivo platform for evaluating the respiratory and multi-organ involvement of betacoronavirus infections. Importance Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race towards the characterization of SARS-CoV-2 infection in other animals (e.g. hamsters, cats, ferrets, bats, and monkeys) as well as the adaptation of the mouse model, by either modifying the host or the virus. In the present study, we utilized the natural pathogen of mice MHV as a prototype to model betacoronavirus-induced acute lung injure and multi—organ involvement under biosafety level 2 condition. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops a severe disease which includes acute lung damage and respiratory distress preceding systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Cocaine is a commonly abused illicit drug that causes significant morbidity and mortality. The most severe and common complications are seizures, ischemic strokes, myocardial infarction, and acute liver injury. Here, we demonstrated that acute cocaine intoxication promoted seizure along with acute liver damage in mice, with intense inflammatory infiltrate. Considering the protective role of the endocannabinoid system against cell toxicity, we hypothesized that treatment with an anandamide hydrolysis inhibitor, URB597, or with a phytocannabinoid, cannabidiol (CBD), protects against cocaine toxicity. URB597 (1.0 mg/kg) abolished cocaine-induced seizure, yet it did not protect against acute liver injury. Using confocal liver intravital microscopy, we observed that CBD (30 mg/kg) reduced acute liver inflammation and damage induced by cocaine and prevented associated seizure. Additionally, we showed that previous liver damage induced by another hepatotoxic drug (acetaminophen) increased seizure and lethality induced by cocaine intoxication, linking hepatotoxicity to seizure dynamics. These findings suggest that activation of cannabinoid system may have protective actions on both liver and brain induced by cocaine, minimizing inflammatory injury promoted by cocaine, supporting its further clinical application in the treatment of cocaine abuse.

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.

Beneficial effects of L-arginine on immune responses and bowel function have been reported. Mucositis is a side effect of chemotherapy treatment that affects approximately 40% of patients. This complication is characterized by inflammation that affects the gastrointestinal tract, increasing permeability and causing abdominal pain, nausea, vomiting, and diarrhea, which worsen the patient's nutritional status and increases morbimortality. The aim of this study was to evaluate the effect of pretreating with 2% L-arginine supplementation in water on mucositis as induced by 5-fluorouracil (5-FU; a single dose of 200 mg/kg body weight) in Swiss male mice. The effect of L-arginine on weight, intestinal permeability, morphology, and the histopathological score of the small intestine (from 0 to 12), oxidative stress, myeloperoxidase (MPO), and N-acetylglucosaminidase (NAG) activities were evaluated. Intestinal length improvement was observed, in addition to the partial recovery of the mucosal architecture. L-arginine attenuated the histopathological score and MPO activity. There was also an improvement in intestinal permeability, despite weight loss after 5-FU administration. In conclusion, L-arginine can positively impact intestinal mucositis by promoting partial mucosal recovery, reducing inflammation and improving intestinal permeability.