Choline-deficient Diet-induced NAFLD Animal Model Recaptures Core Human Pathophysiology With Similar Gene Co-expression Networks.

BACKGROUND/AIM: Nonalcoholic fatty liver disease (NAFLD) is a wide spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recently, the prevalence of NAFLD has dramatically increased, and treatment is urgently needed. Animal models are often used to understand the molecular mechanisms of disease development and progression, but their relevance to human diseases has not been fully understood. This study aimed to establish the usefulness of the animal model for preclinical research, we evaluated its relevance to human disease by gene expression analysis. MATERIALS AND METHODS: We performed weighted gene co-expression network analysis of liver tissues from a choline-deficient L-amino acid-defined (CDAA) diet-induced NAFLD animal model. In addition, module preservation analysis was conducted to evaluate similarity across species. RESULTS: Several modules were identified to be associated with disease severity, and their gene co-expression network was found to be preserved in the human NAFLD datasets. Of note, module brown (immune cell clusters involved in inflammatory responses) was positively associated with disease severity, and its gene co-expression network was highly preserved in the human datasets. Tyrobp, Laptm5 and Lgals3 were identified as hub genes in the brown module, and their increased expression was confirmed in the human datasets. CONCLUSION: CDAA diet-induced NAFLD animal model recaptured key aspects of human pathophysiology (especially immune cell functions) and is thought to be a powerful tool for understanding the molecular mechanisms of NAFLD development and progression.

Peptides of major basic protein and eosinophil cationic protein activate human mast cells.

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca2+ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99-110 (MBP (99-110)) and ECP residues 29-45 (ECP (29-45)), respectively, induced degranulation of HCMCs and intracellular Ca2+ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99-110) or ECP (29-45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99-110) and ECP (29-45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.

Novel MRGPRX2 antagonists inhibit IgE-independent activation of human umbilical cord blood-derived mast cells.

Human MCs are primary effectors implicated in immune surveillance and defense by secreting histamine and various inflammatory mediators, a mechanism termed as degranulation. MCs can be activated by two pathways: IgE-dependent classical pathway and the IgE-independent pathway that utilizes various cationic molecules including substance P (SP) and pituitary adenylate cyclase-activating polypeptides, which are host defense peptides collectively known as basic secretagogues. Our pharmacological study investigated whether or not IgE-independent MC activation is mediated via MRGPRX2. We identified two novel MRGPRX2 antagonists, which completely inhibited the degranulation of human cord blood-derived MCs (hCMCs) induced by basic secretagogues and pseudoallergic drug, icatibant, but IgE- or A23187-challenged hCMCs were resistant to MRGPRX2 antagonists. The MRGPRX2 antagonists markedly inhibited the de novo synthesis of SP-induced prostaglandin D2 in hCMCs. Moreover, the antagonists were able to inhibit p42/44 mitogen-activated protein kinase signal in hCMCs activated by SP. This study strongly suggests that MRGPRX2 antagonists may be a promising drug to prevent the IgE-independent allergic reactions, and thus, MRGPRX2 antagonist development may lead to a promising therapeutic medication for the IgE-independent allergic reactions.

Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors.

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through two main pathways, immunoglobulin E-dependent and -independent activation. In the latter, mast cells are activated by a diverse range of basic molecules, including peptides and amines such as substance P, neuropeptide Y, and compound 48/80. These secretagogues are thought to activate the G proteins in mast cells through a receptor-independent mechanism. Here, we report that the basic molecules activate G proteins through the Mas-related gene (Mrg) receptors on mast cells, leading to mast cell degranulation. We suggest that one of the Mrg receptors, MrgX2, has an important role in regulating inflammatory responses to non-immunological activation of human mast cells.

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.