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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

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.......

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 system4,5,16,17. In addition, it has been found that obesity modulat.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
100µm cell strainers Falcon352340
1ml syringe BD  309659
26G x 5/8 needles BD 305115
35mm Petri Dishes Falcon353001
40µm cell strainers Falcon352340
ACK lysis buffer GIBCOA1049201
Alexa Fluor 700 anti-mouse CD45Biolegend 103127AB_493714 (BioLegend Cat. No. 103127)
Analysis software FlowJo 10.0.8 software
APC anti-mouse CD11c AntibodyBiolegend 117309AB_313778 (BioLegend Cat. No. 117309)
APC anti-mouse KLRG1 (MAFA) AntibodyBiolegend 138411AB_10645509 (BioLegend Cat. No. 138411)
BV421 anti-mouse CD127 AntibodyBiolegend 135023AB_10897948 (BioLegend Cat. No. 135023)
BV421 anti-mouse F4/80 AntibodyBiolegend 123131AB_10901171 (BioLegend Cat. No. 123131)
BV605 anti-mouse CD279 (PD-1) AntibodyBiolegend 135219AB_11125371 (BioLegend Cat. No. 135219)
BV605 anti-mouse NK-1.1 AntibodyBiolegend 108739AB_2562273 (BioLegend Cat. No. 108739)
BV650 anti-mouse/human CD11b AntibodyBiolegend 101239AB_11125575 (BioLegend Cat. No. 101239)
BV711 anti-mouse/human B220 AntibodyBiolegend 103255AB_2563491 (BioLegend Cat. No. 103255)
BV785 anti-mouse CD8a AntibodyBiolegend 100749AB_11218801 (BioLegend Cat. No. 100749)
C57Bl/6J mice, male, 5 weeks old Forschungseinrichtungen für experimentelle Medizin (FEM)
CaCl2 Charité - Universitätsmedizin BerlinA119.1 
Collagenase NB 4G Proved Grade SERVA 11427513
Collagenase Typ I Worthington LS004197
Conical centrifuge tube 15ml Falcon352096
Conical centrifuge tube 50ml Falcon352070
DNAse  Sigma-Aldrich 4716728001
Fetal bovine serum BiochromS0115
Filter 30µm Celltrics 400422316
FITC anti-mouse CD3 AntibodyBiolegend 100203AB_312660 (BioLegend Cat. No. 100203)
Flow cytometry BD-LSR Fortessa 
Forceps Sigma-Aldrich F4142-1EA
HBSS Bioanalytic GmBH 085021-0500 
High-fat diet SSNIFE15741–34 60 kJ% from fat, 19 kJ% from proteins, and 21 kJ% from carbohydrates
micro dissecting scissors Sigma-Aldrich S3146used for dissection purposes 
PE anti-mouse CD25 AntibodyBiolegend 101903AB_312846 (BioLegend Cat. No. 101903)
PE/Cy7 anti-mouse CD62L AntibodyBiolegend 104417AB_313102 (BioLegend Cat. No. 104417)
PE/Cy7 anti-mouse I-A/I-E (MHCII) AntibodyBiolegend 107629AB_2290801 (BioLegend Cat. No. 107629)
PE/Dazzle 594 anti-mouse CD4 AntibodyBiolegend 100565AB_2563684 (BioLegend Cat. No. 100565)
Percoll solution BiochromL6115
PerCP/Cy5.5 anti-mouse CD44 AntibodyBiolegend 103031AB_2076206 (BioLegend Cat. No. 103031)
PerCP/Cy5.5 anti-mouse Gr-1 AntibodyBiolegend 108427AB_893561 (BioLegend Cat. No. 108427)
Phosphate buffered saline Gibco12559069
Round-Bottom Tubes with cell strainer capSTEMCELL Technologies 38030
TruStain fcX anti-mouse CD16/32Biolegend 101301AB_312800 (BioLegend Cat. No. 101301)
Trypan Blue Sigma-Aldrich T6146
Zombie NIR Fixable Viability KitBiolegend 423105viablity stain 

References

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