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.

A conserved pathway of transdifferentiation in murine Kupffer cells.

Abundant long-lived liver-resident macrophages, termed Kupffer cells, are activated during chronic liver injury. They secrete both pro-inflammatory and pro-fibrotic cytokines, which act on hepatic stellate cells causing their transdifferentiation into myofibroblasts that deposit collagen. In other tissues, wound-associated macrophages go further, and transdifferentiate into fibrocytes, secreting collagen themselves. We tested Kupffer cells for this property in two experimental models: mixed non-parenchymal cell culture, and precision-cut liver slice culture. Using the Emr1-Cre transgene as a driver and the RiboTag transgene as a reporter, we found that Kupffer cells undergo transdifferentiation under these circumstances. Over time, they lose the expression of both Kupffer cell-specific and macrophage-specific genes and the transcription factors that control their expression, and they begin to express multiple genes and proteins characteristic of either myofibroblasts or tissue fibroblasts. These effects were strongly conserved between non-parenchymal cell culture and liver tissue slice culture, arguing that such transdifferentiation is a conserved function of Kupffer cells. We conclude that in addition to supporting fibrosis through an action on stellate cells, Kupffer cells also participate in liver fibrosis through transdifferentiation into fibrocytes.

Functional responses of resident human T cells in intact liver tissue.

The liver contains a rich mix of T cells, including activated T cells, tissue-resident memory T cells and cells undergoing apoptosis. When antigens are presented in this milieu the default result is functional tolerance. T cell tolerance in the liver could be constitutive, or it could be adaptive, in which case liver cells would become unresponsive after encountering antigen in the liver context. To test this model, we evaluated the potential of human liver T cells to respond to T cell receptor ligation in liver tissue slice cultures. These T cells contained an actively motile subset of CD4+ T cells marked by CCR7 and CD62L, and fully functional subsets of CD4+ and CD8+ T cells that synthesized effector cytokines but subsequently assumed an exhausted phenotype. These data favor the model that human liver T cells are not constitutively tolerant but undergo adaptive tolerance after activation.

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.

Central role of the TIR-domain-containing adaptor-inducing interferon-ß (TRIF) adaptor protein in murine sterile liver injury.

Multiple pathways drive the sterile injury response in the liver; however, it is unclear how the type of cells injured or the mechanism of injury activates these pathways. Here, we use a model of selective hepatocyte death to investigate sterile liver injury. In this model, the TIR-domain-containing adaptor-inducing interferon-ß (TRIF) was a central mediator of the resulting intrahepatic inflammatory response that was independent of both upstream Toll-like receptor (TLR) 4 signaling and downstream type I interferon (IFN) signaling. TRIF was required for induction of interleukin (IL)-10, IL-6, and IL-1ß cytokines. Conversely, although induction of C-C motif chemokine ligand (CCL) 2 and C-X-C motif chemokine ligand (CXCL) 1 chemokines and up-regulation of chemokine (Ccl2, Ccl7, Cxcl1, Cxcl2, and Cxcl10) and cell-adhesion (intracellular adhesion molecule 1 and vascular cell adhesion molecule 1) genes involved in myeloid cell recruitment was reduced in a majority of TRIF-/- mice, a subset of TRIF-/- mice showed breakthrough inflammation and the ability to induce these genes and proteins, indicating that redundant pathways exist to respond to hepatocyte death. Furthermore, we found that hepatocytes themselves were the main responders to hepatocyte death, increasing transcription of genes involved in myeloid cell recruitment more than either liver sinusoidal endothelial cells or Kupffer cells. CONCLUSION: Our studies define a TRIF-dependent, TLR4- and type I IFN-independent pathway of sterile liver injury in which hepatocytes are both the targets of damage and the principal responding cell type. (Hepatology 2017;65:1336-1351).

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.

Immune tolerance in liver disease.

UNLABELLED: Liver tolerance is manifest as a bias toward immune unresponsiveness, both in the context of a major histocompatibility complex-mismatched liver transplant and in the context of liver infection. Two broad classes of mechanisms account for liver tolerance. The presentation of antigens by different liver cell types results in incomplete activation of CD8(+) T cells, usually leading to initial proliferation followed by either clonal exhaustion or premature death of the T cell. Many liver infections result in relatively poor CD4(+) T-cell activation, which may be because liver antigen-presenting cells express a variety of inhibitory cytokines and coinhibitor ligands. Poor CD4(+) T-cell activation by liver antigens likely contributes to abortive activation, exhaustion, and early death of CD8(+) T cells. In addition, a network of active immunosuppressive pathways in the liver is mediated mostly by myeloid cells. Kupffer cells, myeloid-derived suppressor cells, and liver dendritic cells both promote activation of regulatory T cells and suppress CD8(+) and CD4(+) effector T cells. This suppressive network responds to diverse inputs, including signals from hepatocytes, sinusoidal endothelial cells, and hepatic stellate cells. CONCLUSION: Though liver tolerance may be exploited by pathogens, it serves a valuable purpose. Hepatitis A and B infections occasionally elicit a powerful immune response sufficient to cause fatal massive liver necrosis. More commonly, the mechanisms of liver tolerance limit the magnitude of intrahepatic immune responses, allowing the liver to recover. The cost of this adaptive mechanism may be incomplete pathogen eradication, leading to chronic infection.

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.

Cross-presentation of antigen by diverse subsets of murine liver cells.

UNLABELLED: Antigen cross-presentation is a principal function of specialized antigen-presenting cells of bone marrow origin such as dendritic cells. Although these cells are sometimes known as "professional" antigen-presenting cells, nonbone marrow-derived cells may also act as antigen-presenting cells. Here, using four-way liver cell isolation and parallel comparison of candidate antigen-presenting cells, we show that, depending on the abundance of antigen-donor cells, different subsets of liver cells could cross-present a hepatocyte-associated antigen. This function was observed in both liver sinusoidal endothelial cells and Kupffer cells even at very low antigen concentration, as well as when using soluble protein. Antigen cross-presentation by liver cells induced efficient CD8+ T-cell proliferation in a similar manner to classical dendritic cells from spleen. However, proliferated cells expressed a lower level of T-cell activation markers and intracellular interferon-gamma levels. In contrast to classical spleen dendritic cells, cross-presentation by liver antigen-presenting cells was predominantly dependent on intercellular adhesion molecule-1. CONCLUSION: Hepatic sinusoids are an environment rich in antigen cross-presenting activity. However, the liver's resident antigen-presenting cells cause partial T-cell activation. These results clarify how the liver can act as a primary site of CD8+ T-cell activation, and why immunity against hepatocyte pathogens is sometimes ineffective.

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.

Response of Human Liver Tissue to Innate Immune Stimuli.

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

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.

Altered vitamin A metabolism in human liver slices corresponds to fibrogenesis.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, has antifibrogenic properties in vitro and in animal models. Liver vitamin A homeostasis is maintained by cell-specific enzymatic activities including storage in hepatic stellate cells (HSCs), secretion into circulation from hepatocytes, and formation and clearance of atRA. During chronic liver injury, HSC activation is associated with a decrease in liver retinyl esters and retinol concentrations. atRA is synthesized through two enzymatic steps from retinol, but it is unknown if the loss of retinoid stores is associated with changes in atRA formation and which cell types contribute to the metabolic changes. The aim of this study was to determine if the vitamin A metabolic flux is perturbed in acute liver injury, and if changes in atRA concentrations are associated with HSC activation and collagen expression. At basal levels, HSC and Kupffer cells expressed key genes involved in vitamin A metabolism, whereas after acute liver injury, complex changes to the metabolic flux were observed in liver slices. These changes include a reproducible spike in atRA tissue concentrations, decreased retinyl ester and atRA formation rate, and time-dependent changes to the expression of metabolizing enzymes. Kinetic simulations suggested that oxidoreductases are important in determining retinoid metabolic flux after liver injury. These early changes precede HSC activation and upregulation of profibrogenic gene expression, which were inversely correlated with atRA tissue concentrations, suggesting that HSC and Kupffer cells are key cells involved in changes to vitamin A metabolic flux and signaling after liver injury. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Vitamin A is metabolized in the liver for storage as retinyl esters in hepatic stellate cell (HSCs) or to all-trans-retinoic acid (atRA), an active metabolite with antifibrogenic properties. Following chronic liver injury, vitamin A metabolic flux is perturbed, and HSC activation leads to diminished retinoid stores. WHAT QUESTION DID THIS STUDY ADDRESS? Do changes in the expression of vitamin A metabolizing enzymes explain changes in atRA concentrations and the regulation of fibrosis following acute liver injury? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? In healthy liver, both HSC and Kupffer cells may mediate vitamin A homeostasis. Following acute liver injury, complex changes in metabolizing enzyme expression/activity alter the metabolic flux of retinoids, resulting in a transient peak in atRA concentrations. The atRA concentrations are inversely correlated with profibrogenic gene expression, HSC activation, and collagen deposition. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Improved understanding of altered vitamin A metabolic flux in acute liver injury may provide insight into cell-specific contributions to vitamin A loss and lead to novel interventions in liver fibrosis.

Human Liver Macrophage Subsets Defined by CD32.

Human liver myeloid cells are imperfectly defined, but it is broadly agreed that cells of stellate appearance in situ, expressing the markers CD11b and CD68, are the liver's resident macrophages, classically termed Kupffer cells. Recent investigations using single cell RNA sequencing and unsupervised clustering algorithms suggest there are two populations of cells with the characteristics of tissue macrophages in human liver. We therefore analyzed dissociated human liver tissue using the markers CD11b and CD68 to define macrophage-like cells and found within this population two subsets that differ in their expression of multiple surface markers. These subsets were FACS-sorted based on CD32 expression, and gene expression analysis identified them with human liver myeloid cell subsets that were previously defined by two independent single cell RNA sequencing studies. Using qRT-PCR we found that the two subsets differed in the expression of genes associated with T cell activation and immunosuppression, suggesting distinct roles in T cell tolerance. In addition, one subset expressed two markers, CD1C and CD11c, more often seen on classical dendritic cells. Criteria used to distinguish macrophages from dendritic cells in other tissues may need to be revised in the human liver.

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.

Precision-cut human liver slice cultures as an immunological platform.

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. It is not easy to address those differences experimentally. Therefore, to improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Therefore, precision-cut liver slices emerge as a valuable tool to study human innate immunity.