Reiners-Koch, P.-S. | Search bioRxiv

bioRxiv2024

A perfusion-independent high-throughput method to isolate liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) critically regulate homeostatic liver function and liver pathogenesis. However, the isolation of LSECs remains a major technological bottleneck in studying molecular mechanisms governing LSEC functions. Current techniques to isolate LSECs, relying on perfusion-dependent liver digestion, are cumbersome with limited throughput. We here describe a perfusion-independent high-throughput procedure to isolate LSECs with high purity. Indifferently from previous perfusion-independent approaches, chopped liver tissue was incubated in the digestion mix for 30 minutes with intermittent mixing with a serological pipette. This led to the safeguarding of LSEC integrity and yielded 10 {+/-} 1.0 million LSECs per adult mouse liver, which is far higher than previous perfusion-independent protocols and comparable yield to established perfusion-dependent protocols for isolating LSECs. Combining magnetic and fluorescence-activated cell sorting (FACS), LSECs from different zones of the hepatic sinusoid can now be isolated in high numbers in less than two hours for downstream applications including proteomics. Our protocol enabled the isolation of LSECs from fibrotic liver tissues from mice and healthy liver tissues from higher vertebrate species (pigs), where traditional perfusion-based digestion protocols have very limited application. In conclusion, these technical advancements reduce post-mortem changes in the LSEC state and aid in reliable investigation of LSEC functions.

physiology↗

bioRxiv2022

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury

In both steady-state and during endotoxicosis, liver sinusoidal endothelial cells (LSECs) can rapidly clear lipopolysaccharide (LPS) from the bloodstream. They are located along blood sinusoids of the liver, and establish intimate contact with circulating and tissue-resident immune cells. However, their role in regulating immune responses during LPS-induced endotoxicosis remains poorly understood. Here, we show that LSECs play a dual role in regulating inflammatory responses, acting as modulators of NK cell pro-inflammatory output and as major producers of immune cell-attracting chemokines. We demonstrate that LSECs switch their chemokine expression pattern driven by LPS and IFN-{gamma}, resulting in the production of the myeloid-attracting chemokine CCL2 and the lymphoid-attracting chemokine CXCL10, which accumulate in the serum of LPS-challenged animals. In livers of LPS-injected mice, monocytes and Kupffer cells expressed highest amounts of the pro-inflammatory cytokine Il12a and Il18 transcripts, while NK cells expressed the highest amounts of Ifng. NK cell exposure to LSECs in vitro led to global transcriptomic changes, and primed NK cells to produce higher amounts of IFN-{gamma} in response to IL-12 and IL-18. LSECs required exposure to IFN-{gamma} for Cxcl10 expression, and Cxcl10 gene-deletion in endothelial cells abrogated NK cell accumulation in the liver after LPS treatment. Thus, our data indicate that LSECs occupy a unique temporal and spatial position acting as central regulators that respond to both LPS and immune-derived inflammatory signals, and fuel a positive feedback loop of immune cell attraction and activation in the inflamed liver tissue. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/500206v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@653c53org.highwire.dtl.DTLVardef@5ba4bdorg.highwire.dtl.DTLVardef@9caad8org.highwire.dtl.DTLVardef@1945eb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗