Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis

In the context of MASLD and MASH, a progressive loss of KCs, where the degree of loss is correlated with the severity of the injury, has been reported.

“In the context of MASLD and MASH, a progressive loss of KCs, where the degree of loss is correlated with the severity of the injury, has been reported (36, 37, 104–106, 108).”

LAMs, SAMs and NAMs exhibit a very similar transcriptional profile, with the precise name reflecting the different studies in which these cells were identified.

“Notably, these cells exhibit a very similar transcriptional profile, with the precise name (LAM, SAM, or NAM) reflecting the different studies in which these cells were identified.”

Monocytes recruited to zones of steatosis and fibrosis appear to take on an alternative profile, which has been given multiple names, including lipid-associated macrophages (LAMs), scar-associated macrophages (SAMs) and NASH-associated macrophages (NAMs).

“On the other hand, those monocytes recruited to zones of steatosis and fibrosis appear to take on an alternative profile, which has been given multiple names, including lipid-associated macrophages (LAMs), scar-associated macrophages (SAMs) and NASH-associated macrophages (NAMs) (31, 104, 108, 109).”

The majority of murine studies suggest LAMs/LAM-like KCs play a role in tissue repair, contributing to dead cell and lipid clearance, reducing lipotoxic stress, and preventing uncontrolled inflammation.

“While the majority of murine studies to date have suggested a role for LAMs/LAM-like KCs in tissue repair, contributing to dead cell and lipid clearance, thus reducing lipotoxic stress, and preventing uncontrolled inflammation (36, 108, 122–126)”

Macrophages in the fibrotic liver called LAMs, SAMs, and NAMs in different studies largely share a conserved transcriptional profile.

“A good example of this is the population of macrophages in the fibrotic liver, which have been called LAMs, SAMs, and NAMs in different studies, despite largely sharing a conserved transcriptional profile (31, 104, 108).”

The dataset of immune cells displayed highly conserved transcriptomic signatures with data from two combined mouse MASH-related HCC studies and one human MASH study.

“Expectedly, our dataset of immune cells displayed highly conserved transcriptomic signatures with the data from two combined mouse MASH-related HCC studies [31, 46] and one human MASH study [17] (Fig. S3D,E).”

Analyses of ligand‒receptor interactions in single-cell RNA sequencing datasets have revealed that HSCs are among the most interactive cell types in the liver.

“Indeed, analyses of ligand‒receptor interactions in single-cell RNA sequencing datasets have revealed that HSCs are among the most interactive cell types in the liver [19, 223, 224].”

HSCs secrete mediators, sometimes termed "stellakines", in MASLD, including CCL2, CCL11, CXCL10, CXCL12, and CXCL16, which may also increase their recruitment and interaction with immune cells and thereby promote liver fibrosis or repair.

“HSCs secrete mediators, sometimes termed “stellakines”, in MASLD, including CCL2, CCL11, CXCL10, CXCL12, and CXCL16, which may also increase their recruitment and interaction with immune cells and thereby promote liver fibrosis or repair [223, 246].”

A TREM2+ macrophage subtype strongly linked to markers of tissue injury and fibrosis is enriched in NASH livers.

“A TREM2+ macrophage subtype that is strongly linked to markers of tissue injury and fibrosis is enriched in NASH livers (19).”

Algorithms that analyze ligand-receptor pairs to predict cell-cell signaling have demonstrated that HSCs have among the greatest number and most diverse interactions of any hepatic cell type, including with KCs, MoMFs, and to a lesser extent T, B, and NKT cells.

“Algorithms that analyze ligand-receptor pairs to predict cell-cell signaling have demonstrated that HSCs have among the greatest number and most diverse interactions of any hepatic cell type including with KCs, MoMF and to a lesser extent T, B, and NKT cells (19, 54).”

The immune-targeting stellakines upregulated in NASH include CCL2, CCL11, CXCL10, CXCL12, CXCL16, CTGF, and Gas6.

“The immune-targeting “stellakines”, which are upregulated in NASH, included CCL2, CCL11, CXCL10, CXCL12, CXCL16, CTGF, and Gas6 (19).”

Analysis of receptor ligand pairs using scRNAseq from the AMLN murine NASH model identified a set of HSC-specific secreted factors termed stellakines and established HSCs as a signaling hub that interacts extensively with LSECs, macrophages, and to a lesser extent DCs, T cells, and B cells.

“Analysis of receptor ligand pairs using scRNAseq from the AMLN murine NASH model identified a set of HSC-specific secreted factors termed “stellakines” and established HSCs as a signaling hub that interacts extensively with LSECs, macrophages, and to a lesser extent, DCs, T cells, and B cells.”

CellPhoneDB analysis has revealed numerous cell-cell interactions in mouse models of NASH and in patients with cirrhosis, including NASH-associated cirrhosis, and has revealed mesenchymal cells, macrophages and endothelial cells as having intense interactions with other cell types.

“CellPhoneDB analysis has revealed numerous cell-cell interactions in mouse models of NASH and in patients with cirrhosis, including NASH-associated cirrhosis, and has revealed mesenchymal cells, macrophages and endothelial cells as having intense interactions with other cell types.[18], [19], [20]”

Ligand-receptor analysis indicates that activated HSCs play a complex role in fibrotic signalling through the ECM secretome, with secreted proteins such as collagen serving both structural and signalling functions via receptors such as ITGA1 or DDR2, and activating receptors for vasoactive proteins such as VIPR1 and AGTR1A.

“As well as collagen production, ligand-receptor analysis indicates that activated HSCs play a complex role in fibrotic signalling through the ECM secretome itself: secreted proteins such as collagen serving both a structural and a signalling function with cognate receptors such as ITGA1 (integrin subunit α1) or DDR2. They also have activating receptors for a number of vasoactive proteins such as”

Sustained myofibroblast activation in chronic liver disease drives fibrosis, especially in MASH, where myofibroblasts are detectable by desmin and αSMA immunofluorescence and by single-cell RNA-seq gene expression.

“These functions normally contribute to tissue repair under homeostatic conditions, but sustained activation in chronic liver diseases drives fibrosis, especially in MASH (41) where they are detectable by expression of desmin and alpha smooth muscle actin (αSMA) using immunofluorescence, and by conservation of gene expression based on single-cell RNA-seq (42-46).”

Detection of both quiescent and activated HSCs in mouse liver injury models by single-cell RNA-seq supports HSC activation as a driver of fibrosis across etiologies, including MASH.

“The detection of both quiescent and activated HSCs in mouse liver injury models using single cell RNA-seq reinforces HSC activation as a driver of fibrosis common across etiologies that includes MASH (46-50).”

Additional signaling pathways directly promote or block HSC activation in MASH fibrosis, including FGF21, GLP2/GLP2R, IL11, and CCL25/CCR9.

“Additional signaling pathways also directly promote or block HSC activation in MASH fibrosis, including fibroblast growth factor 21 (FGF21) (164), glucagon-like peptide 2/ glucagon-like peptide 2 receptor (165), interleukin 11 (IL11) (46, 166), and chemokine ligand 2 (CCL25)/C-C chemokine receptor type 9 (CCR9) (167).”

scRNAseq and ligand-receptor network analysis of NASH livers shows that activated HSCs express increased CXCL12 with CXCR4 in B cells as the potential target.

“Supporting a fibrogenic function of B cells, sc‐RNAseq and ligand‐receptor network analysis of NASH livers shows that activated HSCs express increased C‐X‐C motif chemokine ligand 12 with C‐X‐C motif chemokine receptor 4 (Cxcr4) in B cells as the potential target.( 57 )”