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Congestion enriches intra-hepatic macrophages through reverse zonation of CXCL9 in liver sinusoidal endothelial cells

ABSTRACT: Background & Aims Congestion alters the microenvironment of the liver sinusoid along the portal-central axis. We studied spatial changes in immune cells in the sinusoid which contribute to congestive fibrosis and portal hypertension (PHTN). Methods To visualize the distribution of immune cells in congestive hepatopathy (CH), we performed imaging mass cytometry (IMC) on liver tissue from patients with CH, Fontan-associated liver disease (FALD), and controls. We performed partial ligation of the inferior vena cava (pIVCL) to simulate CH in mice and isolated primary liver cells for single cell RNA-sequencing (scRNA-seq) to study zonation of liver sinusoidal endothelial cells (LSECs). After pIVCL, mice were treated with intraperitoneal injections of AMG487, an inhibitor of the CXCL9 receptor. Results Peri-central macrophages are enriched in CH and FALD. Given the role of CXCL9 in macrophage patterning in the liver, we performed RNA in situ hybridization (RNAish) in CH and determined that CXCL9 was highly expressed in LSECs in FALD, suggesting that LSECs recruit macrophages in CH. After pIVCL, treatment with AMG487 attenuated portal pressures, fibrosis, and intra-hepatic macrophages. To study changes in LSECs which promote macrophage chemotaxis, we performed scRNA-seq after pIVCL and sham procedures. Analysis revealed 3 LSEC subpopulations according to sinusoidal location. RNANish identified peri-central LSECs as the predominant source of CXCL9 in FALD. In vitro analyses revealed that -catenin and hypoxia inducible factor-1α regulate CXCL9 transcription in peri-central LSECs. Conclusions CXCL9 derived from peri-central LSECs enriches intra-hepatic macrophages in CH and FALD, contributing to congestive fibrosis and PHTN. Strategies to target LSEC-derived CXCL9 may prevent the progression of CH and FALD.

ORGANISM(S): Mus musculus

PROVIDER: GSE253915 | GEO | 2024/01/27

REPOSITORIES: GEO

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Project description:The liver is located at the interface between the intestinal portal vasculature and the general circulation, employing a complex set of innate and adaptive immune cells to protect from the continuous systemic threat of gut-translocating pathogens. In contrast to lymphoid tissues in which asymmetric, polarized spatial positioning of distinct cell types plays an essential role in optimal host defense, the relationship between liver phagocytes and lymphoid element distribution and their immune functions is currently unknown. This lack of organization stands in marked contrast to the extensively documented zonation of hepatocyte metabolic function controlled by Wnt signaling during development. Here we used quantitative multi-parameter confocal imaging to re-examine resident immune cells distribution in the liver, finding that both myeloid (Kupffer cell -KC) and lymphoid (NKT cell) components are substantially enriched in the peri-portal regions of the liver lobule, the entry point for portal venous blood rich in gut-derived pathogens and molecules. Peri-portal immune enrichment of KCs was lost in germ-free (GF) animals and coincided temporally with weaning and microbial colonization, suggesting that immune cell positioning was not a developmentally programmed process but rather dependent on extrinsic factors. Conditional ablation of MyD88-dependent bacterial product sensing by liver sinusoidal endothelial cells (LSECs) disrupted KC spatial organization animals, consistent with immune cell positioning within the liver architecture arising from sustained, active responses of LSECs to microbial stimuli. Bacterial challenge studies revealed that this asymmetric peri-portal-biased immune cell localization protects the liver and the host against bacterial dissemination. Together, these data reveal that LSECs act as microbiome sensors, actively promoting concentration of protective immune components in the sub-regions of the liver most susceptible to infection to provide optimal host defense against systemic pathogen spread.

Project description:Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo cell phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1 and the anti-inflammatory drug dexamethasone. Methods: LSECs from male Sprague Dawley rats were cultured on type I collagen in a 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology were used to examine proteins in cells and supernatants. Validation was done with ELISA and multiplex immunobead assays, cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. Results: LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1, displaying upregulation of cellular responses to cytokines and interferon-, cell-cell adhesion, and glycolysis, and downregulation of membrane/endocytosis receptors (MCAM, STAB1, STAB2, LYVE1, CLEC4G) and proteins involved in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone improved LSEC viability in culture and repressed the culture-induced stimulation of glycolysis, inflammatory and immune regulatory pathways, and secretion of pro-inflammatory cytokines while increasing IL-10 secretion compared to time-matched control. The number of sieve plates in LSECs was reduced at 24 h both in the presence and absence of dexamethasone but the dexamethasone-treated cells showed a more quiescent phenotype. Conclusion: Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation and improves cell viability.

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Congestion enriches intra-hepatic macrophages through reverse zonation of CXCL9 in liver sinusoidal endothelial cells

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