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.
Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed ...editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all
(interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the
-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of
editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
Platelets play a known role in the maintenance of vascular homeostasis, but these cells are emerging as important cellular mediators of acute and chronic inflammatory diseases. Platelets are key ...elements in the pathogenesis of acute and chronic liver disease associated with hepatitis B virus （HBV） infection by promoting the accumulation of virus-specific CD8＋ T cells and nonspecific inflammatory cells into the liver parenchyma. This review discusses major platelet functions in immune and inflammatory responses, with an emphasis on recent pre-clinical studies that suggest that the inhibition of platelet activation pathways represent an alternative therapeutic strategy with potential use in the reduction of virus-specific T cell-mediated chronic inflammation, liver fibrosis and hepatocellular carcinoma in patients who are chronically infected with HBV.
CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified ...HMGB1 as a CpG-ODN–binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9’s redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFα secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFα, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.
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.
Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved ...inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.
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 Nkx2-5+Islet1+ mesenchymal lineage generates distinct spleen stromal cell subsets ► Spleen stromal cells derive from embryonic mesenchymal progenitors ► Nkx2-5+Islet1+ descendents include cells with lymphoid tissue organizer activity ► A subset of resident Nkx2-5+Islet1+ descendents regenerates spleen stromal network
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.
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.
Using hepatitis B virus (HBV) transgenic mice as recipients of virus‐specific cytotoxic T lymphocytes (CTLs), we recently showed that polymorphonuclear neutrophils (PMNs) and the matrix‐degrading ...metalloproteinases (MMPs) they produce are necessary for the intrahepatic recruitment of antigen nonspecific mononuclear cells that amplify the liver damage initiated by the CTLs. We now report that the high‐mobility group box 1 protein (HMGB1) is also involved in this process. Transfer of CTLs in HBV transgenic mice induces the translocation of HMGB1 from the nucleus to the cytoplasm of hepatocytes surrounding CTL‐containing necroinflammatory liver foci, without significant net synthesis of HMGB1. Treatment of CTL‐injected HBV transgenic mice with either recombinant Box‐A or glycyrrhizin, two functional inhibitors of extracellular HMGB1, significantly decreases the intrahepatic recruitment of PMNs and all other inflammatory cells, in the face of intact homing of virus‐specific CTLs into the liver. The inhibition of PMN chemoattraction explains the mode of action of glycyrrhizin, which has long been used in Japan for the treatment of hepatitis, and suggests that new and more potent inhibitors of HMGB1 may be useful for the treatment of patients chronically infected with HBV.
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