Ricin toxin is a plant-derived, ribosome-inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)—with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a “lactose-sensitive” pathway mediated by ricin's galactose/N-acetylgalactosamine-specific lectin subunit (RTB), and a “mannose-sensitive” pathway mediated by the mannose receptor (MR; CD206) or other C-type lectins capable of recognizing the mannose-side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin-specific mouse MAb and camelid single-domain (VHH) antibodies to protect KCs and LSECs from ricin-induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VHHs afforded little (<40%) or even no protection to LSECs against ricin-induced death. Complete protection of LSECs was only achieved with MAb or VHH cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECsmore »
Liver homeostasis requires the presence of both parenchymal and non-parenchymal cells (NPCs). However, systems biology studies of the liver have primarily focused on hepatocytes. Using an organotypic three-dimensional (3D) hepatic culture, we report the first transcriptomic study of liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) cultured with hepatocytes. Through computational pathway and interaction network analyses, we demonstrate that hepatocytes, LSECs and KCs have distinct expression profiles and functional characteristics. Our results show that LSECs in the presence of KCs exhibit decreased expression of focal adhesion kinase (FAK) signaling, a pathway linked to LSEC dedifferentiation. We report the novel result that peroxisome proliferator-activated receptor alpha (PPARα) is transcribed in LSECs. The expression of downstream processes corroborates active PPARα signaling in LSECs. We uncover transcriptional evidence in LSECs for a feedback mechanism between PPARα and farnesoid X-activated receptor (FXR) that maintains bile acid homeostasis; previously, this feedback was known occur only in HepG2 cells. We demonstrate that KCs in 3D liver models display expression patterns consistent with an anti-inflammatory phenotype when compared to monocultures. These results highlight the distinct roles of LSECs and KCs in maintaining liver function and emphasize the need for additional mechanistic studies of NPCs in more »
- Publication Date:
- NSF-PAR ID:
- 10154095
- Journal Name:
- Scientific Reports
- Volume:
- 8
- Issue:
- 1
- ISSN:
- 2045-2322
- Publisher:
- Nature Publishing Group
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract -
INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP)more »
-
Abstract Background Emerging RNA viruses that target the central nervous system (CNS) lead to cognitive sequelae in survivors. Studies in humans and mice infected with West Nile virus (WNV), a re-emerging RNA virus associated with learning and memory deficits, revealed microglial-mediated synapse elimination within the hippocampus. Moreover, CNS-resident memory T (TRM) cells activate microglia, limiting synapse recovery and inducing spatial learning defects in WNV-recovered mice. The signals involved in T cell-microglia interactions are unknown.
Methods Here, we examined immune cells within the murine WNV-recovered forebrain using single-cell RNA sequencing to identify putative ligand-receptor pairs involved in intercellular communication between T cells and microglia. Clustering and differential gene analyses were followed by protein validation and genetic and antibody-based approaches utilizing an established murine model of WNV recovery in which microglia and complement promote ongoing hippocampal synaptic loss.
Results Profiling of host transcriptome immune cells at 25 days post-infection in mice revealed a shift in forebrain homeostatic microglia to activated subpopulations with transcriptional signatures that have previously been observed in studies of neurodegenerative diseases. Importantly, CXCL16/CXCR6, a chemokine signaling pathway involved in TRM cell biology, was identified as critically regulating CXCR6 expressing CD8+TRM cell numbers within the WNV-recovered forebrain. We demonstrate that CXCL16 is highlymore »
Conclusions We provide a comprehensive assessment of the role of CXCL16/CXCR6 as an interaction link between microglia and CD8+T cells that maintains forebrain TRM cells, microglial and astrocyte activation, and ongoing synapse elimination in virally recovered animals. We also show that therapeutic targeting of CXCL16 in mice during recovery may reduce CNS CD8+TRM cells.
-
Abstract Despite considerable efforts in modeling liver disease in vitro, it remains difficult to recapitulate the pathogenesis of the advanced phases of non‐alcoholic fatty liver disease (NAFLD) with inflammation and fibrosis. Here, a liver‐on‐a‐chip platform with bioengineered multicellular liver microtissues is developed, composed of four major types of liver cells (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) to implement a human hepatic fibrosis model driven by NAFLD: i) lipid accumulation in hepatocytes (steatosis), ii) neovascularization by endothelial cells, iii) inflammation by activated Kupffer cells (steatohepatitis), and iv) extracellular matrix deposition by activated stellate cells (fibrosis). In this model, the presence of stellate cells in the liver‐on‐a‐chip model with fat supplementation showed elevated inflammatory responses and fibrosis marker up‐regulation. Compared to transforming growth factor‐beta‐induced hepatic fibrosis models, this model includes the native pathological and chronological steps of NAFLD which shows i) higher fibrotic phenotypes, ii) increased expression of fibrosis markers, and iii) efficient drug transport and metabolism. Taken together, the proposed platform will enable a better understanding of the mechanisms underlying fibrosis progression in NAFLD as well as the identification of new drugs for the different stages of NAFLD.
-
Abstract Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroidsmore »
