Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 21;10(1):898.
doi: 10.1038/s41598-020-57652-0.

Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses

Cristina I Øie #  1   2 , Deanna L Wolfson #  3 , Tanji Yasunori  4 , Gianina Dumitriu  5 , Karen K Sørensen  5 , Peter A McCourt  5 , Balpreet S Ahluwalia #  3 , Bård Smedsrød #  5
Affiliations

Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses

Cristina I Øie et al. Sci Rep. .

Abstract

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Transmission electron micrograph of T4-GFP. GFP-T4-phages diluted in PBS were placed on formvar-coated copper grids and negatively stained using uranyl acetate, prior to imaging using transmission electron microscopy. The phages were intact and found as single particles. Scale bar = 200 nm.
Figure 2
Figure 2
Time-lapse endocytosis of GFP-T4-phages by LSECs. (A) Rat LSECs preincubated with AF647-FSA were challenged with GFP-T4-phages, and endocytosis imaged in real-time by deconvolution microscopy (DV), every 5 min for about 90 min. Images represent maximum intensity projections of an 8 µm 3D z-stack. Colocalization values for FSA and phages for each time point (shown in merged images) are Pearson’s correlation coefficients calculated using the Costes threshold in Volocity Quantitation software. Only cell-containing regions of the image were used for colocalization analysis (dashed lines in t = 85 m, merged image). The results are representative of experiments performed with cells isolated from 2 animals, 3 culture dishes, and 10 fields of view including an approximate total of 150 cells. (B,C) DV maximum intensity projections of the highlighted inset at min 25 and min 85 of acquisition, respectively. (D,E) SIM image and its corresponding magnified inset of the plasma membrane of a representative live LSEC labelled with CellMask Green at the end of the acquisition time. Images represent maximum intensity projections of a 2 µm 3D SIM z-stack.
Figure 3
Figure 3
Time-lapse endocytosis of AF747-FSA in the presence or absence of monensin and LysoTracker. LSEC cultures were preincubated with LysoTracker (yellow) and challenged with AF647-FSA (magenta) in the absence or presence of monensin (10 µM). The uptake was monitored in real-time by DV overnight. Colocalization values for FSA and LysoTracker (shown in merged images) are Pearson’s correlation coefficients calculated using the Costes threshold in Volocity Quantitation software. Images represent maximum intensity projections of 4–9 µm 3D z-stacks. Right panel shows randomly selected line profiles within the cells, indicating the degree of colocalization of the two probes. The results are representative of experiments performed with cells isolated from 4 animals, and included 7 culture dishes, 49 fields of view, and approximately 350 cells; of which 2 animals, 2 culture dishes, 17 fields of view, and approximately 100 cells were in the presence of monensin).
Figure 4
Figure 4
Endocytosis of T4-phages in LSECs in vitro. (A) Time course endocytosis of 125I-GFP-T4-phages by LSECs. LSEC cultures were incubated with radiolabelled phages for various time periods. For each time point, 3 separate wells containing cells, and 1 cell-free well were used. After each time period, the supernatant from the cells and cell-free well was collected along with one 0.5 ml washing volume of PBS. Trichloroacetic acid (TCA) precipitation was then used to differentiate between free iodine = degraded phages (TCA soluble) (grey columns), and unbound, intact phages (TCA precipitable). Cell bound and internalized phages were quantified in the cell lysates, after solubilizing the cells in 1% SDS (white columns). The results were normalized by subtracting the amount of radioactivity corresponding to the non-specific binding and free 125I in cell-free wells. Each experiment was performed in triplicates, on cells isolated from four animals (Total N = 12 cell cultures for each time point). Bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 represent the statistical differences between the total endocytosis at 18 h and 24 h as compared to 4 h. (B) The specificity of uptake was studied by incubating the cells with 125I-GFP-T4-phages in the absence (Control) or presence of blocking concentrations (0.1 mg/ml) of FSA, ribonuclease B (RNaseB) and aggregated gamma globulin (AGG), inhibitors for stabilin1/2, mannose receptor and FcγRIIb2, respectively. Cell association and degradation were assessed as above. The results are presented as relative uptake compared to the control which was set to 1. Each experiment was performed in triplicates, on cells isolated from 3 animals (Total N = 9 cell cultures for each time point). Bars represent mean ± SD.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Slopek S, Weber-Dabrowska B, Dabrowski M, Kucharewicz-Krukowska A. Results of bacteriophage treatment of suppurative bacterial infections in the years 1981–1986. Arch. Immunol. Ther. Exp. (Warsz). 1987;35:569–83. - PubMed
    1. Majewska J, et al. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood. Viruses. 2015;7:4783–4799. doi: 10.3390/v7082845. - DOI - PMC - PubMed
    1. Sender R, Fuchs S, Milo R. Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans. Cell. 2016 doi: 10.1016/j.cell.2016.01.013. - DOI - PubMed
    1. Minot S, et al. Rapid evolution of the human gut virome. Proc. Natl. Acad. Sci. USA. 2013;110:12450–5. doi: 10.1073/pnas.1300833110. - DOI - PMC - PubMed
    1. Merril CR. Bacteriophage interactions with higher organisms. Transactions of the New York Academy of Sciences. 1974;36:265–72. doi: 10.1111/j.2164-0947.1974.tb01573.x. - DOI - PubMed

Publication types

Substances