Periodic Reporting for period 1 - Liver ID3ntity (Unravelling the molecular basis of the Kupffer cell-hepatocyte crosstalk and its role in the functional specialization of liver Kupffer cells.)
Okres sprawozdawczy: 2021-05-01 do 2023-04-30
Each organ is thought to primarily comprise of 4 main cell types that form a minimal tissue module: parenchymal cells, endothelial cells, fibroblasts and macrophages. The liver is mainly constituted of hepatocytes (~60%), liver sinusoidal endothelial cells (LSECs) (~15%), liver-resident fibroblasts called stellate cells (~15%) and liver-resident macrophages called Kupffer cells (KCs) (~15%).Macrophages (Macs) are found in all tissues and perform unique functions that are essential to maintain homeostasis in their respective organ, such as synaptic pruning in the brain, recycling of surfactant in the lung or electrical conduction in the heart. Transcriptomic profiling has revealed that each tissue-resident Mac expresses a relatively unique gene expression profile controlled by a restricted set of transcription factors. Little is known, however, about the precise cell-cell circuits that underlie the tissue-specific imprinting of Macs. In the case of KCs, previous studies indicate that this identity and functionality is imprinted by the other cells that constitute the liver module (hepatocytes, LSECs and stellate cells) that together form the Kupffer cell niche.
The host lab has previously shown that the transcription factor LXRa controls 30% of liver-specific KC identity and is essential for KC development and survival. ID3, is a transcription factor that is highly expressed in KCs and conserved across species (human, mouse, pig, zebrafish,etc). We hypothesize that the cell-cell circuits within the sinusoidal liver module not only form the blueprint of liver homeostasis, but that perturbations in these cell-cell interactions will lead to the development of liver diseases. The Liver ID3ntity project sought to identify the molecular cues driving ID3 expression, a key transcription factor in KCs, and identify whether mice lacking these signals would display aberrant liver responses.
The overall objectives of this project were to: Design an in vivo CRISPR pipeline to screen and identify key genes driving ID3 expression, identify the cell-cell interactions and define the pathophysiological implications of these cell-cell interactions.
The effect of KCs on the steady-state identity of the other module cells remains almost completely unknown. Deciphering the reciprocal cell-cell interactions by which these cells imprint the liver sinusoidal identity on one another in vivo is not only key to understand liver biology, it also paves the way to the development of in vitro liver organoids that will more closely resemble the in vivo liver.
The host lab has previously shown that the transcription factor LXRa controls 30% of liver-specific KC identity and is essential for KC development and survival. ID3, is a transcription factor that is highly expressed in KCs and conserved across species (human, mouse, pig, zebrafish,etc). We hypothesize that the cell-cell circuits within the sinusoidal liver module not only form the blueprint of liver homeostasis, but that perturbations in these cell-cell interactions will lead to the development of liver diseases. The Liver ID3ntity project sought to identify the molecular cues driving ID3 expression, a key transcription factor in KCs, and identify whether mice lacking these signals would display aberrant liver responses.
The overall objectives of this project were to: Design an in vivo CRISPR pipeline to screen and identify key genes driving ID3 expression, identify the cell-cell interactions and define the pathophysiological implications of these cell-cell interactions.
The effect of KCs on the steady-state identity of the other module cells remains almost completely unknown. Deciphering the reciprocal cell-cell interactions by which these cells imprint the liver sinusoidal identity on one another in vivo is not only key to understand liver biology, it also paves the way to the development of in vitro liver organoids that will more closely resemble the in vivo liver.
The first stages of the project were dedicated to developing a CRISPR pipeline that would allow us to perform an in vivo CRISPR screen. We started the optimization steps in vitro, to first optimize the use of lentiviral particles (MOIs, transduction methods) as well as the culturing of stem cells isolated from Cas9 mice. In addition, we identified potential upstream signals that induce KC genes such as Id3 in vitro by co-culturing bone-marrow derived monocytes with DLL4-OP9 cells and BMP9.
The next significant step of the project was to optimize the in vivo CRISPR screen by transplanting CRISPR-edited stem cells into irradiated mice. By depletion of the host KCs, we were able to generate livers containing solely CRISPR-edited KCs. This was a major step forward in the project.
From our findings in vitro and from the newly optimized CRISPR set-up, we were able to perform an in vivo CRISPR screen to identify KC receptors upstream of Id3 expression. BMP9 was previously predicted to be important in driving Id3 expression in KCs, and indeed the CRISPR screen revealed that the knock-out of ALK1 (the receptor for Bmp9), resulted in the decrease in Id3 expression and the inability of the circulating monocytes to fully differentiate into KCs.
To validate these findings, we generated CD64-Cre x ALK1 floxed mice, to knock-out ALK1 specifically in the macrophages. We found that mice lacking ALK1 on macrophages have a blocked Kupffer cell development and contain livers without any Kupffer cells. We found that these mice display a disrupted modular architecture with malformed portal triads and aberrant collagen deposition pointing towards an essential role of KCs as architects in the liver.
The next significant step of the project was to optimize the in vivo CRISPR screen by transplanting CRISPR-edited stem cells into irradiated mice. By depletion of the host KCs, we were able to generate livers containing solely CRISPR-edited KCs. This was a major step forward in the project.
From our findings in vitro and from the newly optimized CRISPR set-up, we were able to perform an in vivo CRISPR screen to identify KC receptors upstream of Id3 expression. BMP9 was previously predicted to be important in driving Id3 expression in KCs, and indeed the CRISPR screen revealed that the knock-out of ALK1 (the receptor for Bmp9), resulted in the decrease in Id3 expression and the inability of the circulating monocytes to fully differentiate into KCs.
To validate these findings, we generated CD64-Cre x ALK1 floxed mice, to knock-out ALK1 specifically in the macrophages. We found that mice lacking ALK1 on macrophages have a blocked Kupffer cell development and contain livers without any Kupffer cells. We found that these mice display a disrupted modular architecture with malformed portal triads and aberrant collagen deposition pointing towards an essential role of KCs as architects in the liver.
This MSCA has allowed us to develop a cutting edge in vivo CRISPR pipeline that can be applied in many disease settings. In this project, we applied the CRISPR pipeline in steady-state to validate a predicted upstream driver of Id3 expression, a key transcription factor in KCs. In future, however, this pipeline could be used to screen up to 30-40 receptors in parallel in one mouse. This means, without generating 30-40 Cre/Lox mice, we could now screen multiple knock-out KCs in one mouse and select for the most promising target genes. These screens could also be applied in disease settings such as fibrosis or metastasis to identity key drivers in these diseases and identify potential pharmaceutical targets. All-in-all, the design of the CRISPR pipeline will significantly advance the field of KC biology as it will increase the speed at which we can screen and validate targets in both homeostasis and disease settings. Finally, the identification of BMP9-ALK1 signaling axis between stellate cells and KCs further advances the field in understanding the importance of KCs and their relationship with the surrounding cells within the liver niche (stellate cells, LSECs and hepatocytes).