The liver revisited: CLEM reveals the Kupffer cell niche

Session

LST.5 - Correlative Microscopy across the scales

Authors

Anna Kremer (1, 2), Evelien Van Hamme (1, 2), Peter Borghgraef (1, 2), Johnny Bonnardel (2), Djoere Gaublomme (2), Wouter T'Jonck (2), Martin Guilliams (2), Chris Guerin (1, 2), Saskia Lippens (1, 2)

Affiliations

1. VIB BioImaging Core Ghent, VIB
2. VIB Center of Inflammation Research

Keywords

3D-CLEM

Kupffer cell

near-infrared branding

Abstract text

Macrophages are strongly adapted to their tissue of residence by performing day-to-day functions that are essential for tissue homeostasis. Most of tissue-resident macrophages are embryonically-derived and locally self-maintain without any input of monocytes. However, monocytes contribution to macrophage pools has been shown after genetic depletion or in infection models. During this process, they acquire the exact same genetic and functional profile as their embryonic counterpart. This is perfectly demonstrated for Kupffer cells, the main tissue-resident macrophage population of the liver. It is thought that the environment itself instructs the monocytes to become a certain type of macrophage. Kupffer cells are believed to be strictly located in the liver sinusoids, lining endothelial cells, but the exact mechanisms and cellular interactions that determine their identity are not fully understood. 

In order to study monocyte to Kupffer cell transition we used a 3D CLEM pipeline that we previously reported for brain tissue [1]. It is based on near infra-red branding which creates marks that are visible in both LM and EM, defining the region of interest (ROI) thus enabling identification of the ROI between LM and EM imaging and also serving as reference points for making overlays between the LM and EM datasets. We also made use of an anti-CD31 marker to generate a blood-vessel map, to further facilitate the correlative process. A mouse model was generated in which both Kupffer cells and monocytes were genetically labelled by different endogenous fluorescent proteins and in which Kupffer cells could be specifically depleted. Using this model the monocytes could be identified and imaged at high resolution in LM by AiryScan detection and the ultrastructure of these particular monocytes could be subsequently imaged with SBF-SEM. 

LM and intra-vital imaging revealed that upon Kupffer cell depletion, circulating monocytes slow down, settle in a specific spot, and change properties while they gradually transition into Kupffer cells. SBF-SEM showed that the recruited monocytes don’t reside entirely in the blood vessels, but protrude into the endothelium and reach into the perisinusoidal space where they closely interact with the Stellate cells. Similarly, Kupffer cells from wild type mice also have large parts of the cell body in the space of Disse and they clearly intertwine with Stellate cells and put them in close contact with hepatocytes.

We conclude that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity [2].

References

[1] Urwyler O, et al. Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy. Development. 2015 Jan 15;142(2):394-405.

 

[2] Bonnardel J, et al. Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche. Immunity. 2019 Oct 15;51(4):638-654.