An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Summary

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Abstract

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.

In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.

The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

Introduction

Obesity is a multifactorial disorder and a major risk factor for developing heart disease, stroke, nonalcoholic steatohepatitis (NASH), type 2 diabetes (T2D) and some types of cancer. The prevalence of obesity is rapidly increasing globally. Today, 2.1 billion people — nearly 30% of the world’s population — are either obese or overweight¹. Obesity-associated insulin resistance can lead to T2D, when exhausted pancreatic islet beta cells fail to compensate for the increased need for insulin to maintain glucose homeostasis².

Adipose tissue is composed of various cell types ....

Protocol

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act under the supervision of our institutional Animal Care and Use Committee. Animal protocols were conducted according to institutional ethical guidelines of the Charité Berlin, Germany, and were approved by the Landesamt für Gesundheit und Soziales and comply with the ARRIVE guidelines.

1. Diet-induced Animal Model of Steatohep.......

Discussion

Steatohepatitis has a strong association with metabolic abnormalities such as obesity, insulin resistance and dyslipidemia¹⁵. Multiple studies indicate that adipose tissue inflammation can drive the pathogenesis of type 2 diabetes, including altered levels of cells of both the innate and adaptive immune system^4,5,16,17. In addition, it has been found that obesity modulat.......

Materials

Name	Company	Catalog Number	Comments
100µm cell strainers	Falcon	352340
1ml syringe	BD	309659
26G x 5/8 needles	BD	305115
35mm Petri Dishes	Falcon	353001
40µm cell strainers	Falcon	352340
ACK lysis buffer	GIBCO	A1049201
Alexa Fluor 700 anti-mouse CD45	Biolegend	103127	AB_493714 (BioLegend Cat. No. 103127)
Analysis software	FlowJo 10.0.8 software
APC anti-mouse CD11c Antibody	Biolegend	117309	AB_313778 (BioLegend Cat. No. 117309)
APC anti-mouse KLRG1 (MAFA) Antibody	Biolegend	138411	AB_10645509 (BioLegend Cat. No. 138411)
BV421 anti-mouse CD127 Antibody	Biolegend	135023	AB_10897948 (BioLegend Cat. No. 135023)
BV421 anti-mouse F4/80 Antibody	Biolegend	123131	AB_10901171 (BioLegend Cat. No. 123131)
BV605 anti-mouse CD279 (PD-1) Antibody	Biolegend	135219	AB_11125371 (BioLegend Cat. No. 135219)
BV605 anti-mouse NK-1.1 Antibody	Biolegend	108739	AB_2562273 (BioLegend Cat. No. 108739)
BV650 anti-mouse/human CD11b Antibody	Biolegend	101239	AB_11125575 (BioLegend Cat. No. 101239)
BV711 anti-mouse/human B220 Antibody	Biolegend	103255	AB_2563491 (BioLegend Cat. No. 103255)
BV785 anti-mouse CD8a Antibody	Biolegend	100749	AB_11218801 (BioLegend Cat. No. 100749)
C57Bl/6J mice, male, 5 weeks old	Forschungseinrichtungen für experimentelle Medizin (FEM)
CaCl2	Charité - Universitätsmedizin Berlin	A119.1
Collagenase NB 4G Proved Grade	SERVA	11427513
Collagenase Typ I	Worthington	LS004197
Conical centrifuge tube 15ml	Falcon	352096
Conical centrifuge tube 50ml	Falcon	352070
DNAse	Sigma-Aldrich	4716728001
Fetal bovine serum	Biochrom	S0115
Filter 30µm	Celltrics	400422316
FITC anti-mouse CD3 Antibody	Biolegend	100203	AB_312660 (BioLegend Cat. No. 100203)
Flow cytometry	BD-LSR Fortessa
Forceps	Sigma-Aldrich	F4142-1EA
HBSS	Bioanalytic GmBH	085021-0500
High-fat diet	SSNIF	E15741–34	60 kJ% from fat, 19 kJ% from proteins, and 21 kJ% from carbohydrates
micro dissecting scissors	Sigma-Aldrich	S3146	used for dissection purposes
PE anti-mouse CD25 Antibody	Biolegend	101903	AB_312846 (BioLegend Cat. No. 101903)
PE/Cy7 anti-mouse CD62L Antibody	Biolegend	104417	AB_313102 (BioLegend Cat. No. 104417)
PE/Cy7 anti-mouse I-A/I-E (MHCII) Antibody	Biolegend	107629	AB_2290801 (BioLegend Cat. No. 107629)
PE/Dazzle 594 anti-mouse CD4 Antibody	Biolegend	100565	AB_2563684 (BioLegend Cat. No. 100565)
Percoll solution	Biochrom	L6115
PerCP/Cy5.5 anti-mouse CD44 Antibody	Biolegend	103031	AB_2076206 (BioLegend Cat. No. 103031)
PerCP/Cy5.5 anti-mouse Gr-1 Antibody	Biolegend	108427	AB_893561 (BioLegend Cat. No. 108427)
Phosphate buffered saline	Gibco	12559069
Round-Bottom Tubes with cell strainer cap	STEMCELL Technologies	38030
TruStain fcX anti-mouse CD16/32	Biolegend	101301	AB_312800 (BioLegend Cat. No. 101301)
Trypan Blue	Sigma-Aldrich	T6146
Zombie NIR Fixable Viability Kit	Biolegend	423105	viablity stain