Fate mapping and scRNA sequencing reveal origin and diversity of lymph node stromal precursors.

Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.

Immunosurveillance of the liver by intravascular effector CD8(+) T cells.

Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.

Nkx2-5(+)islet1(+) mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity.

Secondary lymphoid organ stromal cells comprise different subsets whose origins remain unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originate from embryonic mesenchymal progenitors of the Nkx2-5(+)Islet1(+) lineage. This lineage include embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable also of supporting ectopic lymphoid-like structures and a subset of resident spleen stromal cells that proliferate and regenerate the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.

The COP9 signalosome is a repressor of replicative stress responses and polyploidization in the regenerating liver.

UNLABELLED: Aberrant DNA replication induced by deregulated or excessive proliferative stimuli evokes a "replicative stress response" leading to cell cycle restriction and/or apoptosis. This robust fail-safe mechanism is eventually bypassed by transformed cells, due to ill-defined epistatic interactions. The COP9 signalosome (CSN) is an evolutionarily conserved regulator of cullin ring ligases (CRLs), the largest family of ubiquitin ligases in metazoans. Conditional inactivation of the CSN in several tissues leads to activation of S- or G2-phase checkpoints resulting in irreversible cell cycle arrest and cell death. Herein we ablated COPS5, the CSNs catalytic subunit, in the liver, to investigate its role in cell cycle reentry by differentiated hepatocytes. Lack of COPS5 in regenerating livers causes substantial replicative stress, which triggers a CDKN2A-dependent genetic program leading to cell cycle arrest, polyploidy, and apoptosis. These outcomes are phenocopied by acute overexpression of c-Myc in COPS5 null hepatocytes of adult mice. CONCLUSION: We propose that combined control of proto-oncogene product levels and proteins involved in DNA replication origin licensing may explain the deleterious consequences of CSN inactivation in regenerating livers and provide insight into the pathogenic role of the frequently observed overexpression of the CSN in hepatocellular carcinoma.

Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-associated HCC involves both viral and host factors. The latter include a functionally inefficient CD8(+) T-cell response that fails to clear the infection from the liver but sustains a chronic necroinflammatory process that contributes to the development of HCC. According to this scenario, amelioration of immune-mediated chronic liver injury may prevent HCC. Because platelets facilitate immune-mediated liver injury by promoting the hepatic accumulation of virus-specific CD8(+) T cells, we evaluated the long-term consequences of antiplatelet therapy in an HBV transgenic mouse model of chronic immune-mediated necroinflammatory liver disease that progresses to HCC. Treatment with aspirin and clopidogrel during the chronic phase of the disease diminished the number of intrahepatic HBV-specific CD8(+) T cells and HBV-nonspecific inflammatory cells, the severity of liver fibrosis, and the development of HCC. Antiplatelet therapy improved overall survival without causing significant side effects. In contrast, the same antiplatelet regimen had no antitumor effect when HCC was induced nonimmunologically by chronic exposure to a hepatotoxic chemical. The unprecedented observation that antiplatelet therapy inhibits or delays immune-mediated hepatocarcinogenesis suggests that platelets may be key players in the pathogenesis of HBV-associated liver cancer and supports the notion that immune-mediated necroinflammatory reactions are an important cause of hepatocellular transformation during chronic hepatitis.

Anti-platelet therapy in the prevention of hepatitis B virus-associated hepatocellular carcinoma.

Previous studies in mouse models of self-limited viral hepatitis showed that platelets contribute to acute liver damage by promoting the intrahepatic accumulation of virus-specific CD8 T cells and, secondarily, virus-non-specific inflammatory cells. Built on these observations, a recent preclinical study took advantage of a previously established hepatitis B virus (HBV) transgenic mouse model of immune-mediated chronic hepatitis that progresses to hepatocellular carcinoma (HCC), to demonstrate that clinically achievable doses of the anti-platelet drugs aspirin and clopidogrel - administered continuously after the onset of liver disease - can prevent hepatocarcinogenesis and greatly improve overall survival. These outcomes were preceded by and associated with reduced hepatic accumulation of virus-specific CD8 T cells and virus-non-specific inflammatory cells, reduced hepatocellular injury and hepatocellular proliferation, and reduced severity of liver fibrosis. The observation that anti-platelet therapy inhibits HCC development identifies platelets as key players in the pathogenesis of HBV-associated liver cancer and supports the notion that a sustained immune-mediated necroinflammatory liver disease is sufficient to trigger HCC. The results abovementioned and their clinical implications are discussed in this report.

Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.

Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology.

Variations in Biodistribution and Acute Response of Differently Shaped Titania Nanoparticles in Healthy Rodents.

Titanium dioxide nanoparticles (TiO2 NPs) are one of the main sources of the nanoparticulate matter exposure to humans. Although several studies have demonstrated their potential toxic effects, the real nature of the correlation between NP properties and their interaction with biological targets is still far from being fully elucidated. Here, engineered TiO2 NPs with various geometries (bipyramids, plates, and rods) have been prepared, characterized and intravenously administered in healthy mice. Parameters such as biodistribution, accumulation, and toxicity have been assessed in the lungs and liver. Our data show that the organ accumulation of TiO2 NPs, measured by ICP-MS, is quite low, and this is only partially and transiently affected by the NP geometries. The long-lasting permanence is exclusively restricted to the lungs. Here, bipyramids and plates show a higher accumulation, and interestingly, rod-shaped NPs are the most toxic, leading to histopathological pulmonary alterations. In addition, they are also able to induce a transient increase in serum markers related to hepatocellular injury. These results indicate that rods, more than bipyramidal and spherical geometries, lead to a stronger and more severe biological effect. Overall, small physico-chemical differences can dramatically modify both accumulation and safety.

Long-term retention of gold nanoparticles in the liver is not affected by their physicochemical characteristics.

Gold nanoparticles (GNPs) are considered promising candidates for healthcare applications, however, their toxicity after long-term exposure to the material remains uncertain. Since the liver is the main filter organ for nanomaterials, this work was aimed at evaluating hepatic accumulation, internalisation and overall safety of well-characterised and endotoxin-free GNPs in healthy mice from 15 minutes to 7 weeks after a single administration. Our data demonstrate that GNPs were rapidly segregated into lysosomes of endothelial cells (LSEC) or Kupffer cells regardless of coating or shape but with different kinetics. Despite the long-lasting accumulation in tissues, the safety of GNPs was confirmed by liver enzymatic levels, as they were rapidly eliminated from the blood circulation and accumulated in the liver without inducing hepatic toxicity. Our results demonstrate that GNPs have a safe and biocompatibile profile despite their long-term accumulation.

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

Rapamycin-sensitive signals control TCR/CD28-driven Ifng, Il4 and Foxp3 transcription and promoter region methylation.

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation.

Platelets mediate cytotoxic T lymphocyte-induced liver damage.

We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes (CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.

AAV6-mediated systemic shRNA delivery reverses disease in a mouse model of facioscapulohumeral muscular dystrophy.

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.

Arenaviral infection causes bleeding in mice due to reduced serotonin release from platelets.

Bleeding correlates with disease severity in viral hemorrhagic fevers. We found that the increase in type I interferon (IFN-I) in mice caused by infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV; an arenavirus) reduced the megakaryocytic expression of genes encoding enzymes involved in lipid biosynthesis (cyclooxygenase 1 and thromboxane A synthase 1) and a thrombopoietic transcription factor (Nf-e2). The decreased expression of these genes was associated with reduced numbers of circulating platelets and defects in the arachidonic acid synthetic pathway, thereby suppressing serotonin release from δ-granules in platelets. Bleeding resulted when severe thrombocytopenia and altered platelet function reduced the amount of platelet-derived serotonin below a critical threshold. Bleeding was facilitated by the absence of the activity of the kinase Lyn or the administration of aspirin, an inhibitor of arachidonic acid synthesis. Mouse platelets were not directly affected by IFN-I because they lack the receptor for the cytokine (IFNAR1), suggesting that transfusion of normal platelets into LCMV-infected mice could increase the amount of platelet-released serotonin and help to control hemorrhage.

Food-Grade Titanium Dioxide Induces Toxicity in the Nematode Caenorhabditis elegans and Acute Hepatic and Pulmonary Responses in Mice.

Food-grade titanium dioxide (E171) contains variable percentages of titanium dioxide (TiO2) nanoparticles (NPs), posing concerns for its potential effects on human and animal health. Despite many studies, the actual relationship between the physicochemical properties of E171 NPs and their interaction with biological targets is still far from clear. We evaluated the impact of acute E171 administration on invertebrate and vertebrate animals. In the nematode, Caenorhabditis elegans, the administration of up to 1.0 mg/mL of E171 did not affect the worm's viability and lifespan, but significantly impaired its pharyngeal function, reproduction, and development. We also investigated whether the intravenous administration of E171 in mice (at the dose of 6 mg/kg/body weight) could result in an acute over-absorption of filter organs. A significant increase of hepatic titanium concentration and the formation of microgranulomas were observed. Interstitial inflammation and parenchymal modification were found in the lungs, coupled with titanium accumulation. This was probably due to the propensity of TiO2 NPs to agglomerate, as demonstrated by transmission electron microscopy experiments showing that the incubation of E171 with serum promoted the formation of compact clusters. Overall, these data emphasize the actual risk for human and animal exposure to E171.

Continuous sensing of IFNα by hepatic endothelial cells shapes a vascular antimetastatic barrier.

Hepatic metastases are a poor prognostic factor of colorectal carcinoma (CRC) and new strategies to reduce the risk of liver CRC colonization are highly needed. Herein, we used mouse models of hepatic metastatization to demonstrate that the continuous infusion of therapeutic doses of interferon-alpha (IFNα) controls CRC invasion by acting on hepatic endothelial cells (HECs). Mechanistically, IFNα promoted the development of a vascular antimetastatic niche characterized by liver sinusoidal endothelial cells (LSECs) defenestration extracellular matrix and glycocalyx deposition, thus strengthening the liver vascular barrier impairing CRC trans-sinusoidal migration, without requiring a direct action on tumor cells, hepatic stellate cells, hepatocytes, or liver dendritic cells (DCs), Kupffer cells (KCs) and liver capsular macrophages (LCMs). Moreover, IFNα endowed LSECs with efficient cross-priming potential that, along with the early intravascular tumor burden reduction, supported the generation of antitumor CD8+ T cells and ultimately led to the establishment of a protective long-term memory T cell response. These findings provide a rationale for the use of continuous IFNα therapy in perioperative settings to reduce CRC metastatic spreading to the liver.

Colorectal cancer remains one of the most widespread and deadly cancers worldwide. Poor health outcomes are usually linked to diseased cells spreading from the intestine to create new tumors in the liver or other parts of the body. Treatment involves surgically removing the initial tumors in the bowel, but patient survival could be improved if, in parallel, their immune system was 'boosted' to destroy cancer cells before they can form other tumors. Interferon alpha is a small protein which helps to coordinate how the immune system recognizes and deactivates foreign agents and cancerous cells. It has recently been trialed as a colorectal cancer treatment to prevent tumors from spreading to the liver, but only with limited success. This partly because interferon-alpha is usually administered in high and pulsed doses, which cause severe side effects through the body. Instead, Tran, Ferreira, Alvarez-Moya et al. aimed to investigate whether continuously delivering lower amounts of the drug could be a better approach. This strategy was tested on mice in which colorectal cancer cells had been implanted into the wall of the large intestine. Continuous administration minimized the risk of the implanted cancer cells spreading to the liver while also creating fewer side effects. The team was able to identify an optimum delivery strategy by varying how much interferon-alpha the animals received and when. Further experiments also revealed a new mechanism by which interferon-alpha prevented the spread of colorectal cancer. Upon receiving continuous doses of the drug, a group of liver cells started to generate a physical barrier which stopped cancer cells from being able to invade the organ. The treatment also promoted long-term immune responses that targeted diseased cells while being safe for healthy tissues. If confirmed in clinical trials, these results suggest that colorectal patients undergoing tumor removal surgery may benefit from also receiving interferon-alpha through continuous delivery.

Bone marrow as an alternative site for islet transplantation.

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

Platelets prevent IFN-alpha/beta-induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus.

We found that mice infected with different isolates of lymphocytic choriomeningitis virus (LCMV) develop a mild hemorrhagic anemia, which becomes severe and eventually lethal in animals depleted of platelets or lacking integrin beta3. Lethal hemorrhagic anemia is mediated by virus-induced IFN-alpha/beta that causes platelet dysfunction, mucocutaneous blood loss and suppression of erythropoiesis. In addition to the life-threatening hemorrhagic anemia, platelet-depleted mice fail to mount an efficient cytotoxic T lymphocyte (CTL) response and cannot clear LCMV. Transfusion of functional platelets into these animals reduces hemorrhage, prevents death and restores CTL-induced viral clearance in a manner partially dependent on CD40 ligand (CD40L). These results indicate that, upon activation, platelets expressing integrin beta3 and CD40L are required for protecting the host against the induction of an IFN-alpha/beta-dependent lethal hemorrhagic diathesis and for clearing LCMV infection through CTLs.

Advances in Bio-Based Polymers for Colorectal CancerTreatment: Hydrogels and Nanoplatforms.

Adenocarcinoma of the colon is the most common malignant neoplasia of the gastrointestinal tract and is a major contributor to mortality worldwide. Invasiveness and metastatic behavior are typical of malignant tumors and, because of its portal drainage, the liver is the closest capillary bed available in this case, hence the common site of metastatic dissemination. Current therapies forecast total resection of primary tumor when possible and partial liver resection at advanced stages, along with systemic intravenous therapies consisting of chemotherapeutic agents such as 5-fluorouracil. These cures are definitely not exempt from drawbacks and heavy side effects. Biocompatible polymeric networks, both in colloids and bulk forms, able to absorb large quantities of water and load a variety of molecules-belong to the class of innovative drug delivery systems, thus suitable for the purpose and tunable on each patient can represent a promising alternative. Indeed, the implantation of polymeric scaffolds easy to synthesize can substitute chemotherapy and combination therapies scheduling, shortening side effects. Moreover, they do not require a surgical removal thanks to spontaneous degradation and guarantees an extended and regional cargo release, maintaining high drug concentrations. In this review, we focus our attention on the key role of polymeric networks as drug delivery systems potentially able to counteract this dramatic disease.

Organosilica Cages Target Hepatic Sinusoidal Endothelial Cells Avoiding Macrophage Filtering.

Over the last years, advancements in the use of nanoparticles for biomedical applications have clearly showcased their potential for the preparation of improved imaging and drug-delivery systems. However, compared to the vast number of currently studied nanoparticles for such applications, only a few successfully translate into clinical practice. A common "barrier" that prevents nanoparticles from efficiently delivering their payload to the target site after administration is related to liver filtering, mainly due to nanoparticle uptake by macrophages. This work reports the physicochemical and biological investigation of disulfide-bridged organosilica nanoparticles with cage-like morphology, OSCs, assessing in detail their bioaccumulation in vivo. The fate of intravenously injected 20 nm OSCs was investigated in both healthy and tumor-bearing mice. Interestingly, OSCs exclusively colocalize with hepatic sinusoidal endothelial cells (LSECs) while avoiding Kupffer-cell uptake (less than 6%) under both physiological and pathological conditions. Our findings suggest that organosilica nanocages hold the potential to be used as nanotools for LSECs modulation, potentially impacting key biological processes such as tumor cell extravasation and hepatic immunity to invading metastatic cells or a tolerogenic state in intrahepatic immune cells in autoimmune diseases.