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Targeting EFHD2 Inhibits Interferon-γ Signaling and Ameliorates Non…
Targeting EFHD2 Inhibits Interferon-γ Signaling and Ameliorates Non-Alcoholic Steatohepatitis
Introduction
Nonalcoholic steatohepatitis (NASH)
basic infromation
an advanced form of non-alcoholic fatty liver disease
can progress to cirrhosis and hepatocellular carcinoma (HCC)
The progression of NASH is currently the most rapidly increasing cause of end-stage liver disease
pathomechanisms
Not all patients progress from the simple fatty liver phase to NASH
progression remain unclear
highly noninfectious inflammatory microenvironment (compared with simple fatty liver)
Extensive liver inflammation is modulated by changes in innate immune activation
primarily associated with the diversity, dynamics, and function of hepatic macrophages and monocytes
Liver
Liver macrophages
monocyte-derived macrophages (MDM)
monocyte-derived macrophages originate from bone marrow.
Kupffer cells (KCs)
self-sustaining, non-migratory phagocytes residing in the tissue
KCs originate from precursors derived from the yolk sac during embryogenesis
widely acknowledged that NASH liver experiences significant shifts in immune composition
KC depletion/transition
the appearance of monocyte-derived macrophages (MDM) and NASH-associated macrophages (NAM)
Interferon (IFN) signaling, has emerged as a key mediator of hepatic inflammation via modulating liver macrophages
our previous finding8 and results from studies by other groups have demonstrated
IFN signaling, together with the IFN-regulated molecules such as interferon regulatory factor (IRF) and stimulator of interferon gene (STING)
control immune activation during progression of NASH
EF-hand domain family member D2 (EFHD2) (Swiprosin-1)
calcium-binding EF-hand superfamily of proteins (initially discovered in lymphocytes)
cell death
migration
neurodegenerative disorders
recent studies, have progressively expanded the roles of EFHD2 to encompass
tumorigenesis
tissue repair
atherosclerosis
sepsis
etc.....
a recent study has uncovered a potential link between EFHD2 and activation of IFNγ signaling in response to bacterial infection
the extent of EFHD2's involvement in noninfectious inflammation like NASH remains unexplored.
Hypothesis
Q1: Whether EFHD2 exhibits specific expression in hepatic macrophages/monocytes and involves the disease progression of NASH
Q2: Whether Global or myeloid cell-specific deletion of EFHD2 improves NASH and hinders NASH-HCC progression.
Q3: Whether EFHD2 regulates the replacement of resident Kupffer cells by infiltrating monocytes during NASH.
Q4: EFHD2 interacts with YWHAZ, promoting translocation of IFNγR2 to plasma membrane, and in turn, mediates IFNγ signaling
Results
A1
EFHD2 expression is triggered in patients and mice with NASH
analyzed EFHD2 expression in two distinct patient cohorts
cohort 1 (10 NASH patients (histologically diagnosed), 10 age-matched healthy ~)
Immunohistochemistry analysis of EFHD2 in liver tissues
Immunoblotting analysis of EFHD2 in liver tissues
cohort 2 (patients confirmed by biopsy ,n=23)
hepatic mRNA levels between EFHD2 and TNFα, IL6, NFKB1 or IFNγ
further investigated in two mouse models of NASH induced by
a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)
Efhd2 mRNA
immunoblotting EFHD2 protein
immunohistochemistry EFHD2 protein
a high-fat-high-cholesterol diet (HFHC)
Efhd2 mRNA
immunoblotting EFHD2 protein
immunohistochemistry EFHD2 protein
global EFHD2 knockout (western blot confirm)
CDAHFD
CDAHFD-fed KO mice & age-matched wild-type (WT) mice a CDAHFD
liver morphology
Serum ALT and AST levels
Histological analyses (H&E, Masson's trichrome, αSMA immunohistochemistry)
Quantitative PCR analysis of chemokine genes (Cxcl1, Cxcl10 and Ccl5) and pro-fibrotic genes (Col1α1 and Timp1)
Immunohistochemistry analysis of CD68
Immunoblotting analysis of pro-inflammatory factors (TNFα, IL-6, p-p65 NF-κB and MCP-1)
influence of EFHD2 deletion on ferroptosis
HFHC
serum ALT and AST levels
histological analyses (H&E, Sirius Red, and Masson's trichrome staining)
Immunoblotting analysis of pro-inflammatory factors (TNF-α, IL-6, phosphorylated p65 of NF-κB subunit and MCP-1)
EFHD2 in macrophage/monocyte promotes NASH
single-cell RNA sequencing (scRNA-seq) data from the Human Atlas Database
scRNA-seq dataset (GSE158241) in mouse liver
useing scRNA-seq in self-model myeloid cells (including macrophages/monocytes, neutrophils, and dendritic cells) & T cell
Double-immunofluorescent staining of EFHD2 in CDAHFD-fed mice liver
Double-immunofluorescent staining of EFHD2 and CD68 in liver tissue of human NASH patients.
a mouse strain with conditional EFHD2 deletion in myeloid cells ( Efhd2f/f Lysm-Cre mice )
CDAHFD
Compared with Efhd2f/f mice, Efhd2f/fLysm-Cre mice displayed
liver/body weight ratio
serum ALT/AST levels
serum TG/TC levels
Immunoblotting analysis of pro-inflammatory factors ,chemokines , pro-fibrosis genes and ferroptosis
liver weight
HFHC
A hydrocarbon-stapled peptide targeting EFHD2 inhibits NASH in mice
A2
Myeloid EFHD2 controls hepatic immune cell reprogramming during NASH
CD45+ single cells isolated from livers of Efhd2 f/f & Efhd2 f/f Lysm-Cre mice
stained with antibodies against various markers
a non-linear high-dimensional analysis approach
t-distributed stochastic neighbour embedding (t-SNE) distribution of hepatic monocyte/macrophage subsets and cluster analysis
Myeloid EFHD2 facilitates the development of NASH-associated HCC
NASH-associated HCC mouse model fed with CDAHFD for a lifetime
most of CDAHFD-fed Efhd2f/f mice died after 60 weeks of feeding due to HCC (confirmed by autopsy)
Next - Efhd2f/f and Efhd2f/fLysm-Cre mice fed a CDAHFD for 40 weeks
Serum ALT and AST levels
Gene expression levels of proinflammatory factors & chemokines & pro-fibrosis factors
Serum levels and liver mRNA expression of alpha fetoprotein (AFP)
Immunohistochemical staining for Ki67 and glypican-3 (GPC3)
The number and diameter of the HCC tumors
mRNA expressions of Tox and Cxcr6
A3
EFHD2 is vital for activating IFNγ signaling in macrophages during NASH
tissue RNA sequencing to livers of CDAHFD-fed Efhd2 f/f and Efhd2 f/f Lysm-Cre mice
KEGG & GO analysis
exogenous IFNγ signaling in the context of NASH, primary mouse bone marrow-derived macrophages (BMDMs) were isolated, cultured, primed with IFNγ
A4
EFHD2 interacts with YWHAZ/14-3-3ζ via its PxxP motif to control IFNγR2 membrane translocation