Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein.

Infection with SARS-CoV-2, the causative agent of COVID-19, causes mild to moderate disease in most patients but carries a risk of morbidity and mortality. Seriously affected individuals manifest disorders of hemostasis and a cytokine storm, but it is not understood how these manifestations of severe COVID-19 are linked. Here, we showed that the SARS-CoV-2 spike protein engaged the CD42b receptor to activate platelets via 2 distinct signaling pathways and promoted platelet-monocyte communication through the engagement of P selectin/PGSL-1 and CD40L/CD40, which led to proinflammatory cytokine production by monocytes. These results explain why hypercoagulation, monocyte activation, and a cytokine storm are correlated in patients severely affected by COVID-19 and suggest a potential target for therapeutic intervention.

Early-Derived Murine Macrophages Temporarily Renounce Tissue Identity during Acute Systemic Inflammation.

In mice, a subset of cardiac macrophages and Kupffer cells derive from fetal precursors, seed the developing tissues, self-renew locally, and persist into adulthood. In this study we investigated how these cells survive acute systemic inflammation. In both tissues, early-derived subsets rapidly responded to acute systemic inflammation by assuming a temporary nonclassical activation state featuring upregulation of both proinflammatory (Il1b, Tnf, Nfkb1), and anti-inflammatory (Il10, Il4ra, Nfkbiz) genes. During this process, transcription factor genes associated with myeloid identity (Spi1, Zeb2) were upregulated, whereas those associated with tissue specificity (Nr1h3 for Kupffer cells and Nfatc2 and Irf4 for cardiac macrophages) were downregulated, suggesting that the cells reasserted their myeloid identity but renounced their tissue identity. Most of these changes in gene expression reverted to steady-state levels postresolution. We conclude that these early-derived macrophage subsets are resilient in the face of acute stress by temporary loss of adaptation to local tissue-specific niches while reasserting their generic myeloid identity.

Fetal origin confers radioresistance on liver macrophages via p21cip1/WAF1.

BACKGROUND & AIMS: Cells of hematopoietic origin, including macrophages, are generally radiation sensitive, but a subset of Kupffer cells (KCs) is relatively radioresistant. Here, we focused on the identity of the radioresistant KCs in unmanipulated mice and the mechanism of radioresistance. METHODS: We employed Emr1- and inducible CX3Cr1-based fate-mapping strategies combined with the RiboTag reporter to identify the total KCs and the embryo-derived KCs, respectively. The KC compartment was reconstituted with adult bone-marrow-derived KCs (bm-KCs) using clodronate depletion. Mice were lethally irradiated and transplanted with donor bone marrow, and the radioresistance of bone-marrow- or embryo-derived KCs was studied. Gene expression was analyzed using in situ mRNA isolation via RiboTag reporter mice, and the translatomes were compared among subsets. RESULTS: Here, we identified the radioresistant KCs as the long-lived subset that is derived from CX3CR1-expressing progenitor cells in fetal life, while adult bm-KCs do not resist irradiation. While both subsets upregulated the Cdkn1a gene, encoding p21-cip1/WAF1 protein, radioresistant embryo-derived KCs showed a greater increase in response to irradiation. In the absence of this molecule, the radioresistance of KCs was compromised. Replacement KCs, derived from adult hematopoietic stem cells, differed from radioresistant KCs in their expression of genes related to immunity and phagocytosis. CONCLUSIONS: Here, we show that, in the murine liver, a subset of KCs of embryonic origin resists lethal irradiation through Cdkn1a upregulation and is maintained for a long period, while bm-KCs do not survive lethal irradiation. LAY SUMMARY: Kupffer cells (KCs) are the tissue-resident macrophages of the liver. KCs can be originated from fetal precursors and from monocytes during the fetal stage and post-birth, respectively. Most immune cells in mice are sensitive to lethal-irradiation-induced death, while a subset of KCs resists radiation-induced death. These radioresistant KCs continue to live in the irradiated mice. We discovered that this relatively radioresistant KC subset are the fetal-derived KCs, and they achieve this through cell-cycle arrest. Understanding the radiobiology of KCs will provide valuable insights into the mechanisms that elicit radiation-induced liver disease.

Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.

Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured, despite many recent successes with immunotherapy for other malignancies. As the human disease is heavily infiltrated by effector T cells, we postulated that accurately modeling the PDA immune microenvironment would allow us to study mechanisms of immunosuppression that could be overcome for therapeutic benefit. Using viable precision-cut slices from fresh PDA, we developed an organotypic culture system for this purpose. We confirmed that cultured slices maintain their baseline morphology, surface area, and microenvironment after at least 6 d in culture, and demonstrated slice survival by MTT assay and by immunohistochemistry staining with Ki-67 and cleaved-Caspase-3 antibodies. Immune cells, including T cells (CD3+, CD8+, and FOXP3+) and macrophages (CD68+, CD163+ and HLA-DR+), as well as stromal myofibroblasts (αSMA+) were present throughout the culture period. Global profiling of the PDA proteome before and after 6 d slice culture indicated that the majority of the immunological proteins identified remain stable during the culture process. Cytotoxic effects of drug treatment (staurosporine, STS and cycloheximide, CHX) on PDA slices culture confirmed that this system can be used to assess functional response and cell survival following drug treatment in both a treatment time- and dose-dependent manner. Using multicolor immunofluorescence, we stained live slices for both cancer cells (EpCAM+) and immune cells (CD11b+ and CD8+). Finally, we confirmed that autologous CFSE-labeled splenocytes readily migrate into co-cultured tumor slices. Thus, our present study demonstrates the potential to use tumor slice cultures to study the immune microenvironment of PDA.

Infiltrating monocytes in liver injury and repair.

Noninfectious liver injury causes many acute and chronic liver diseases around the globe, and particularly in developed nations. Bone marrow-derived monocytes infiltrate the damaged liver tissue and are a critical component of the innate immune response that may drive injury resolution or host death in the short term or chronic inflammation, fibrosis and hepatocellular carcinoma in the long term. Monocytes often play dual roles in liver injury-both perpetuating inflammation and promoting resolution of inflammation and fibrosis. Thus, we will address the role that monocytes play in different experimental forms of noninfectious liver injury; considering in particular the importance of the transition from inflammatory Ly6Chi monocytes to pro-resolution Ly6Clo monocyte-derived macrophages and the consequences of this transition for disease progression and resolution.

TLR4-Dependent Secretion by Hepatic Stellate Cells of the Neutrophil-Chemoattractant CXCL1 Mediates Liver Response to Gut Microbiota.

BACKGROUND & AIMS: The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. METHODS: Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. RESULTS: TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. CONCLUSIONS: Showing the specific activation of TLR4 and the secretion of one major functional chemokine-CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state.

Hepatocytes as Immunological Agents.

Hepatocytes are targeted for infection by a number of major human pathogens, including hepatitis B virus, hepatitis C virus, and malaria. However, hepatocytes are also immunological agents in their own right. In systemic immunity, they are central in the acute-phase response, which floods the circulation with defensive proteins during diverse stresses, including ischemia, physical trauma, and sepsis. Hepatocytes express a variety of innate immune receptors and, when challenged with pathogen- or damage-associated molecular patterns, can deliver cell-autonomous innate immune responses that may result in host defense or in immunopathology. Important human pathogens have evolved mechanisms to subvert these responses. Finally, hepatocytes talk directly to T cells, resulting in a bias toward immune tolerance.

CD40 is required for protective immunity against liver stage Plasmodium infection.

The costimulatory molecule CD40 enhances immunity through several distinct roles in T cell activation and T cell interaction with other immune cells. In a mouse model of immunity to liver stage Plasmodium infection, CD40 was critical for the full maturation of liver dendritic cells, accumulation of CD8(+) T cells in the liver, and protective immunity induced by immunization with the Plasmodium yoelii fabb/f(-) genetically attenuated parasite. Using mixed adoptive transfers of polyclonal wild-type and CD40-deficient CD8(+) T cells into wild-type and CD40-deficient hosts, we evaluated the contributions to CD8(+) T cell immunity of CD40 expressed on host tissues including APC, compared with CD40 expressed on the CD8(+) T cells themselves. Most of the effects of CD40 could be accounted for by expression in the T cells' environment, including the accumulation of large numbers of CD8(+) T cells in the livers of immunized mice. Thus, protective immunity generated during immunization with fabb/f(-) was largely dependent on effective APC licensing via CD40 signaling.

Ineffective CD8(+) T-cell immunity to adeno-associated virus can result in prolonged liver injury and fibrogenesis.

Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. This results in a sustained immune response accompanied by tissue injury and fibrogenesis. We have created a mouse model that reproduces these effects, based on the response of CD8(+) T cells to hepatocellular antigen delivered by an adeno-associated virus (AAV) vector. Ten thousand antigen-specific CD8(+) T cells undergo slow expansion in the liver and can precipitate a subacute inflammatory hepatitis with stellate cell activation and fibrosis. Over time, antigen-specific CD8(+) T cells show signs of exhaustion, including high expression of PD-1, and eventually both inflammation and fibrosis resolve. This model allows the investigation of both chronic liver immunopathology and its resolution.

Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.

UNLABELLED: Both hepatitis B and C viruses frequently establish chronic infection, raising the question whether T cells are poorly primed in the liver. To determine the role of different cell types in the activation of CD8+ T cells against hepatocellular antigens, we used an Adeno-associated virus to deliver ovalbumin to hepatocytes. In contrast to CD8+ T cells, CD4+ T cells were not activated. The CD8+ T cells were activated even in the absence of endogenous CD4+ T cells; however, in the liver, these cells were high in the programmed death-1 protein and low in CD127. Chimera experiments revealed that these CD8+ T cells were activated on a solid tissue cell. CONCLUSION: Priming of CD8+ T cells directly on nonhematopoietic cells, in the absence of CD4+ T cell help, results in suboptimal T cell activation. This could explain the impaired function of CD8+ T cells seen in chronic liver infection.

Polyketal copolymers: a new acid-sensitive delivery vehicle for treating acute inflammatory diseases.

Acute inflammatory diseases are a major cause of death in the world, and effective treatments are greatly needed. Macrophages play a central role in causing acute inflammatory diseases, and there is currently great interest in developing drug delivery vehicles that can target therapeutics to macrophages. Microparticles formulated from aliphatic polyketals have great potential to enhance the treatment of acute inflammatory diseases, due to their ability to passively target therapeutics to macrophages, their acid sensitivity, and their biocompatible degradation products. However, existing aliphatic polyketals are unsuitable for treating acute inflammatory diseases because they require weeks to hydrolyze, and strategies for accelerating their hydrolysis kinetics are greatly needed. In this report, we demonstrate that the hydrolysis kinetics of aliphatic polyketals can be accelerated by increasing their hydrophilic/hydrophobic balance. Aliphatic polyketals of varying hydrophobicity were synthesized, via the acetal exchange reaction, and their hydrolysis kinetics were investigated at the pH values of 4.5 and 7.4. A polyketal termed PK3 was developed, which had the hydrolysis kinetics suitable for treating acute inflammatory diseases. PK3 has a hydrolysis half-life of 2 days at pH 4.5, but requires several weeks to hydrolyze at pH 7.4. Microparticles were formulated with PK3, which encapsulated the anti-inflammatory drug, imatinib. In vivo experiments demonstrated that PK3 microparticles were able to significantly improve the efficacy of imatinib in treating acute liver failure. We anticipate that aliphatic polyketals will have numerous applications for the treatment of acute inflammatory diseases, given their pH sensitivity, tunable hydrolysis kinetics, and biocompatible degradation products.

E2F4 modulates differentiation and gene expression in hematopoietic progenitor cells during commitment to the lymphoid lineage.

The E2F4 protein is involved in gene repression and cell cycle exit, and also has poorly understood effects in differentiation. We analyzed the impact of E2F4 deficiency on early steps in mouse hematopoietic development, and found defects in early hematopoietic progenitor cells that were propagated through common lymphoid precursors to the B and T lineages. In contrast, the defects in erythromyeloid precursor cells were self-correcting over time. This suggests that E2F4 is important in early stages of commitment to the lymphoid lineage. The E2F4-deficient progenitor cells showed reduced expression of several key lymphoid-lineage genes, and overexpression of two erythromyeloid lineage genes. However, we did not detect effects on cell proliferation. These findings emphasize the significance of E2F4 in controlling gene expression and cell fate.

Early intrahepatic accumulation of CD8+ T cells provides a source of effectors for nonhepatic immune responses.

Interactions between the liver and CD8+ T cells can lead to tolerance, due in part to CD8+ T cell death. To test whether this was the case in an extrahepatic infection, we investigated the fate and effector capacity of intrahepatic CD8+ T cells during lung-restricted influenza infection in mice. Virus-specific T cells accumulated in livers without detectable intrahepatic presentation of viral Ags, and this accumulation was not restricted to the contraction phase, but was apparent as early as day 5. Intrahepatic influenza-specific cells were functionally similar to those recovered from the bronchioalveolar lavage, based on ex vivo cytokine production and specific target lysis. Both adoptive transfer of liver lymphocytes and orthotopic liver transplant of organs containing accumulated effector T cells revealed that activated CD8s from the liver were viable, expanded during reinfection, and generated a memory population that trafficked to lymphoid organs. Thus, intrahepatic CD8+ T cells re-enter circulation and generate functional memory, indicating that the liver does not uniformly incapacitate activated CD8+ T cells. Instead, it constitutes a substantial reservoir of usable Ag-specific effector CD8+ T cells involved in both acute and recall immune responses.