Key Inflammatory Processes in Human NASH Are Reflected in Ldlr-/-.Leiden Mice: A Translational Gene Profiling Study

Abstract

Introduction: It is generally accepted that metabolic inflammation in the liver is an important driver of disease progression in NASH and associated matrix remodeling/fibrosis. However, the exact molecular inflammatory mechanisms are poorly defined in human studies. Investigation of key pathogenic mechanisms requires the use of pre-clinical models, for instance for time-resolved studies. Such models must reflect molecular disease processes of importance in patients. Herein we characterized inflammation in NASH patients on the molecular level by transcriptomics and investigated whether key human disease pathways can be recapitulated experimentally in Ldlr^-/-.Leiden mice, an established pre-clinical model of NASH. Methods: Human molecular inflammatory processes were defined using a publicly available NASH gene expression profiling dataset (GSE48452) allowing the comparison of biopsy-confirmed NASH patients with normal controls. Gene profiling data from high-fat diet (HFD)-fed Ldlr^-/-.Leiden mice (GSE109345) were used for assessment of the translational value of these mice. Results: In human NASH livers, we observed regulation of 65 canonical pathways of which the majority was involved in inflammation (32%), lipid metabolism (16%), and extracellular matrix/remodeling (12%). A similar distribution of pathways across these categories, inflammation (36%), lipid metabolism (24%) and extracellular matrix/remodeling (8%) was observed in HFD-fed Ldlr^-/-.Leiden mice. Detailed evaluation of these pathways revealed that a substantial proportion (11 out of 13) of human NASH inflammatory pathways was recapitulated in Ldlr^-/-.Leiden mice. Furthermore, the activation state of identified master regulators of inflammation (i.e., specific transcription factors, cytokines, and growth factors) in human NASH was largely reflected in Ldlr^-/-.Leiden mice, further substantiating its translational value. Conclusion: Human NASH is characterized by upregulation of specific inflammatory processes (e.g., "Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes," "PI3K signaling in B Lymphocytes") and master regulators (e.g., TNF, CSF2, TGFB1). The majority of these processes and regulators are modulated in the same direction in Ldlr^-/-.Leiden mice fed HFD with a human-like macronutrient composition, thus demonstrating that specific experimental conditions recapitulate human disease on the molecular level of disease pathways and upstream/master regulators.

Keywords: NASH; gene expression; human; inflammation; liver; molecular; mouse; translational.

Figure 1

Enrichment analysis of molecular pathways in NASH patients. (A) Visualization of top 20 enriched canonical pathways in human NASH patients as compared to normal controls. Values are expressed as –log(p-value). (B) Circle chart which classifies canonical pathways into more general biological processes and illustrates the proportion of pathways in each of these categories.

Figure 2

Enrichment analysis of inflammatory pathways in NASH patients. (A) Visualization of all inflammation-related canonical pathways in human NASH patients as compared to normal controls. Values are expressed as –log(p-value). (B) Visualization of the expression change in NASH patients as compared to normal controls for the canonical pathway “Leukocyte Extravasation Signaling.” Red color indicates significant upregulated genes and green color indicates significant downregulated genes.

Figure 3

Enrichment analysis of molecular pathways in HFD-fed Ldlr^−/−.Leiden mice. (A) Circle chart which classifies canonical pathways into more general biological processes and illustrates the proportion of pathways in each of these categories. (B) Visualization of all inflammation-related canonical pathways in HFD-fed Ldlr^−/−.Leiden mice. (C) Visualization of the expression change in HFD-fed Ldlr^−/−.Leiden mice as compared to chow controls for the canonical pathway “Leukocyte Extravasation Signaling.” Red color indicates significant upregulated genes and green color indicates significant downregulated genes.

Figure 4

Representation of human key molecular pathways and associated genes in HFD-fed Ldlr^−/−.Leiden mice. (A) Venn diagram to visualize the overlap in canonical pathways between human NASH biopsies (red circle) and Ldlr^−/−.Leiden mice (blue circle). (B) Network visualization of overlapping canonical pathways (gray nodes, A: Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes, B: PI3K Signaling in B Lymphocytes, C: Leukocyte Extravasation Signaling, D: IL-8 Signaling, E: Natural Killer Cell Signaling, F: Macropinocytosis Signaling, G: B Cell Receptor Signaling, H: Role of Macrophages Fibroblasts and Endothelial Cells in Rheumatoid Arthritis, I: Production of Nitric Oxide and Reactive Oxygen Species in Macrophages; J: Dendritic Cell Maturation; K: iCOS-iCOSL Signaling in T Helper Cells) and the associated significantly expressed genes in human NASH (blue and yellow nodes). The blue nodes represent genes that were also regulated in the Ldlr^−/−.Leiden mouse, the yellow nodes represent genes that were not significantly regulated in the Ldlr^−/−.Leiden mouse. (C) Heatmap visualization of genes underlying the common pathways that are regulated in both human NASH biopsies and HFD-fed Ldlr^−/−.Leiden mice relative to their respective controls. Red color indicates upregulated genes and green color indicates downregulated genes. The rightmost column shows the genes that do not share their direction of regulation between human and mouse in red.

Figure 5

Representation of human inflammation-related upstream regulators in HFD-fed Ldlr^−/−.Leiden mice. (A) Venn diagram to visualize the overlap in upstream regulators between human NASH biopsies (red circle) and Ldlr^−/−.Leiden mice (blue circle). (B) Heatmap visualization of direction of regulation (Z-score). Red color indicates activated upstream regulator and green color indicates inhibited upstream regulator.