A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
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.
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.
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 ....
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.......
The protocol described allows the characterization of surface markers of innate and adaptive immune cells isolated from murine perigonadal adipose tissue and liver in a model of diet-induced NASH. In this model, NASH was induced by administration of HFD plus sucrose (6%) in drinking water for 7 to 15 weeks in C57Bl/6J mice, as previously reported13. Importantly, mice were housed in semi sterile conditions and, thus, exposed to environmental antigens throughout the experiment. HFD fed mice housed i.......
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 system4,5,16,17. In addition, it has been found that obesity modulat.......
We thank Anke Jurisch, Diana Woellner, Dr. Kathrin Witte and Cornelia Heckmann for assistance with experimental procedures and Benjamin Tiburzy from Biolegend for helpful comments on the gating strategy. J.S. was supported by the Helmholtz Grant (ICEMED). This study was supported by grants from the Clinical Research Unit of the Berlin Institute of Health (BIH), the “BCRT-grant” by the German Federal Ministry of Education and Research and the Einstein Foundation. K.S.-B. and H.-D.V. are funded by FOR2165.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved