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 including adipocytes, endothelial cells, fibroblasts and immune cells. During progression of obesity, changes in the number and activity of immune cells can lead to low-grade inflammation of hypertrophic adipose tissue^3,4. Specifically, it has been found that excessive energy intake, accompanied by chronically elevated levels of blood glucose, triglycerides and free fatty acids, leads to adipocyte hypoxia, endoplasmic reticulum stress, impaired mitochondrial function and enhanced cytokine secretion, resulting in the activation of pro-inflammatory adipose immune cells^5,6. Past research has mainly focused on innate immunity, but more recently adaptive immune cells (T and B cells) have emerged as important regulators of glucose homeostasis. They possess inflammatory (including CD8⁺ T cells, Th1, and B cells) or primarily regulatory functions (including regulatory T (Treg) cells, Th2 cells) and can both exacerbate or protect against insulin resistance^7,8,9.

Furthermore, several mechanisms were proposed to explain how obesity increases steatohepatitis, including increased production of cytokines by adipose tissue¹⁰. NASH, the progressive form of nonalcoholic fatty liver disease and a major health burden in developed countries, is histologically characterized by ballooned hepatocytes, lipid accumulation, fibrosis and pericellular inflammation and may progress to cirrhosis, end stage liver disease or hepatocellular carcinoma. Several regimen (for instance the methionine and choline deficient diet¹¹) are known to induce NASH-like liver pathology in non-human animal models, but most of these approaches do not recapitulate human conditions of NASH and its metabolic consequences as they either require specific gene knockout, non-physiological dietary manipulations or lack insulin resistance typical of human NASH. Moreover, our understanding of the underlying mechanisms of metabolic diseases is currently based on experiments carried out with laboratory mice housed under standard specific pathogen free (SPF) conditions. Those barrier facilities are abnormally hygienic and do not consider the microbial diversity humans have to encounter, which may account for difficulties in the translation process of animal studies to clinical approaches^12,13,14.

To investigate the different immune cell subsets in adipose tissue and liver during the development of insulin resistance and NASH in an advanced mouse model reproducing human immunological conditions, mice were housed in individual cages in semi sterile conditions without a barrier. Mice housed under antigen exposed conditions developed NASH-like liver pathology already after 15 weeks of high-fat diet (HFD) feeding¹³. Compared to age-matched SPF mice they developed macrovesicular steatosis, hepatic infiltration and activation of immune cells.

This manuscript describes a robust flow cytometry analysis to define and count immune cell subsets from mouse adipose tissue and liver in a model of NASH. Flow cytometry analysis allows the detection of multiple parameters of individual cells simultaneously in contrast to RT-PCR or immunohistochemistry approaches.

In summary, our study offers a mouse model of short-term HFD for investigating the development of insulin resistance and NASH and the underlying mechanisms that also exhibits fidelity to the human condition.

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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 Steatohepatitis

1. Transfer mice (C57Bl/6J, male) to non-SPF housing at the age of 4 weeks and expose them continuously to a broad range of environmental pathogens/antigens. Guarantee the exposure by daily handling of the laboratory animals as antigens are distributed by air passage in this way.
2. Maintain mice (C57Bl/6J, male, 12 weeks old) in open caging systems with conventional filters on a 12 h:12 h light/dark cycle at a temperature of 22 °C. Do not irradiate or autoclave experimental diet and bedding. Access animal housing facility without mask or hairnet. Open doors between animal rooms. Do not use an airshower.
3. Guarantee daily handling of the laboratory animals and switch rooms on a regular basis so that antigens are distributed by air passage. Complement dirty housing with daily non-specific microbial exposure to antigens from bedding of mammalian laboratory animals housed in rooms next to the laboratory mice.
4. Measure proportions of CD44^-CD62L^- effector memory CD4⁺ and CD8⁺ T cells in blood and spleen using flow cytometry (as described in refefence¹³).
   NOTE: In this regard we defined an increase of 20% of effector memory CD8⁺ T cells from CD8⁺ T cells as evidence of sufficient microbial exposure.
5. Start HFD (60 kJ% from fat, 19 kJ% from proteins and 21 kJ% from carbohydrates and ad libitum consumption of water with 6% sucrose content with five-week old C57Bl/6J male mice for 7-15 weeks. The HFD should contain 60% kJ from fat (as described above) in order to induce the development of insulin resistance throughout the course of the experiment.
   NOTE: Hematoxylin staining should be performed and histological characteristics as hepatocyte ballooning, Mallory Denk bodies, immune cell infiltration and macrovesicular steatosis must be found to demonstrate NASH-like liver pathology (as shown in reference¹³).

2. Preparation of Reagents and Solutions

1. Prepare 70% ethanol, phosphate buffered saline (PBS, without calcium and magnesium) supplemented with 0.5% BSA and ACK (Ammonium-Chloride-Potassium) lysis buffer.
2. Staining buffer
   1. Dissolve 10 mL of fetal calf serum (FCS) in 500 mL of PBS to obtain 2% FCS PBS. Place staining buffer on ice before use.
   2. Store solution in a plastic bottle at 4 °C.
3. Collagenase solution for adipose tissue digestion
   1. Dissolve 2.5 g of bovine serum albumin (BSA) in 500 mL of PBS to obtain a 0.5% BSA/PBS.
   2. Dissolve 74.5 g of CaCl₂ in 10 mL of 0.5% BSA/PBS to obtain a 10 mM CaCl₂ solution.
   3. Add 1 mg of collagenase type II (see Table of Materials) to each mL of 0.5% BSA with 10 mM CaCl₂ PBS.
   4. Prepare 3 mL of collagenase digest solution per g of adipose tissue sample. Prepare fresh collagenase solution for each isolation.
4. Collagenase solution for liver tissue digestion
   1. Dissolve 2.5 g of BSA in 500 mL of Hank´s Balanced Salt Solution (HBSS) to obtain 0.5% BSA HBSS.
   2. Add 10 mL of FCS in 500 mL of 0.5% BSA/HBSS to obtain 2% FCS 0.5% BSA/HBSS.
   3. Add 0.5 mg of collagenase type IV (see Table of Materials) to each mL of 2% FCS 0.5% BSA/HBSS.
   4. Add 0.02 mg of DNase per mL of 2% FCS 0.5% BSA/HBSS collagenase solution.
   5. Prepare 13 mL of collagenase digest solution per liver tissue sample.
   6. Prepare fresh collagenase solution for each isolation.

3. Generation of Single cell Suspensions

1. Adipose tissue digestion
   1. Euthanize mice via isoflurane anesthesia followed by cervical dislocation. Spray the chest with 70% ethanol. Carefully make a 5-6 cm central incision through the integument and abdominal wall along the entire length of the rib cage to expose the pleural cavity and heart.
      NOTE: Do not damage underlying organs and keep scissors tips up.
   2. Inject at least 10 mL of 0.9% saline solution in the apex of the left ventricle using a 26 G needle.
      NOTE: Successful perfusion is noted by blanching of the liver.
   3. Open the peritoneal cavity with scissors and cut out the perigonadal fat pads on each side with fine curved scissors. Dissect gonadal tissues with fine curved scissors and weigh adipose tissue.
   4. Perform the mechanical dissociation using scissors and cut adipose tissue into fine pieces in a Petri dish at 4 °C. Transfer adipose tissue to 50 mL conical centrifuge tubes and rinse Petri dish with 1 mL of 0.5% BSA/PBS.
   5. Add 3 mL of adipose tissue digest solution (as prepared in step 2.3) per gram of adipose tissue. Incubate adipose tissue solution at 37 °C for 20 min under gentle shaking (200 rpm).
   6. Add 5 mL of 0.5% BSA/PBS per gram of adipose tissue and place on ice. Triturate the solution numerous times with a 10 mL serological pipette and pass it through a strainer (100 µm) with the aid of a plunger.
   7. Centrifuge at 500 x g for 10 min at 4 °C.
   8. Remove the floating adipocyte fraction by pipetting. Resuspend the cell pellet (stromal vascular fraction) in 1 mL of ACK lysis buffer (see Table of Materials). Add 10 mL of 2% FCS PBS.
   9. Centrifuge at 500 x g for 10 min at 4 °C.
   10. Decant supernatant and resuspend cell pellet in 250 µL of 2% FCS PBS.
   11. Count the number of viable cells on a hemocytometer using Trypan blue exclusion.
2. Liver tissue digestion
   1. Store liver in a conical centrifuge tube filled with PBS and transport on ice.
   2. Perform the mechanical dissociation using syringe stamps in a Petri dish at 4 °C. Put dissected liver tissue in a 50 mL conical centrifuge tube containing 10 mL of warm liver digest solution. Rinse the Petri dish with 3 mL of liver digest solution.
   3. Incubate liver tissue solution at 37 °C for 20 min under gentle shaking (200 rpm).
   4. Add 20 mL of HBSS. Triturate the solution numerous times with a 10 mL serological pipette and pass it through a strainer (100 µm) with the aid of a plunger.
   5. Centrifuge at 500 x g for 10 min at 4 °C. Decant the supernatant and resuspend the cell pellet in 20 mL of HBSS.
   6. Centrifuge at 30 x g for 1 min at room temperature to remove the hepatocyte matrix. Discard the cell pellet.
   7. Centrifuge the supernatant at 500 x g for 10 min at 4 °C. Resuspend the pellet in 10 mL of 33% low viscosity density gradient medium solution (see Table of Materials) in HBSS followed by centrifugation (800 x g; 30 min; room temperature; no brake).
   8. Resuspend the pellet in 1 mL of ACK lysis buffer and incubate for 4 min at room temperature. Then add 10 mL of HBSS.
      NOTE: To discard the supernatant aspirate the supernatant with interphase (hepatocytes) very carefully with a transfer pipette (as much as possible without taking the pellet with immune cells and erythrocytes).
   9. Pass cells through a 30 μm strainer in a 15 mL conical centrifuge tube and centrifuge at 500 x g for 10 min at 4 °C. Decant supernatant and resuspend cell pellet in 250 µL of 2% FCS PBS.
   10. Count the number of viable cells on a hemocytometer using Trypan blue exclusion.

4. Surface Staining

1. Prepare antibody mix for T-cell-subsets (Panel 1) and innate immune cells (Panel 2) as described in Table 1 and Table 2. Volumes are optimized for one sample (100 µL) regarding to antibody concentrations.
   NOTE: Lymphocytes and innate immune cells present differences in autofluorescence and should be analyzed separately.
2. Use up to 3 x 10⁶ cells in 100 µL in a polystyrene FACS tube for surface staining. To block Fc receptors add 10 µL of anti-CD16/CD32 antibody (diluted 1:100) and incubate for 10 min on ice. Use an unstained negative control sample to adjust side scatter (SSC) and forward scatter (FSC) to determine the location of the negative cell population.
3. Vortex and add the appropriate volume of antibody mixture for Panel 1 and Panel 2. Add 1 µL of viability dye to each sample to allow for live and dead cell discrimination. Incubate for 20 min at 4 °C and protected from light.
4. Wash two times with 2 mL of 2% FCS PBS and centrifuge at 300 x g for 5 min at 4 °C.
5. Resuspend the cell pellet in 300 µL of 2% FCS PBS and store at 4 °C until FACS analysis.
   NOTE: Before starting the measurement pass cells through 30 µm cell strainer into polystyrene FACS tube and vortex. Flow cytometry was performed with labeled cells stored in 2% FCS PBS at 4 °C for 1-3 h. Cells should be analyzed as soon as possible for optimized results.

5. Flow Cytometry Compensation, Acquisition, and Gating

1. Run unstained negative control sample to set the FSC and SSC and adjust voltages of the flow cytometer to detect leucocyte populations and to distinguish between debris and viable cells. Exclude debris and dead cells.
   NOTE: Viability of the cell suspensions from the liver might be lower than the viability from the cell suspensions from perigonadal adipose tissue due to an additional density separation step.
2. Run single stained control samples for multi-color compensation.
   NOTE: Antibody capture beads could also be used to adjust for spectral overlap if the number of cells of a cell population of interest is too low to compensate using cells. To detect possible autofluorescence signals of the cells fluorescence minus one (FMO) controls for every antibody are recommended but were not applied in this protocol.
3. Start the measurement, collect appropriate number of events (at least 50,000 events) and record experimental data.
4. Export FCS data files for analysis and set the gating strategy. Gate on CD45⁺ leucocytes to identify subsequent cell populations.

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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...

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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...

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Materials

Name	Company	Catalog Number	Comments
100 µm cell strainers	Falcon	352340
1 mL syringe	BD	309659
26 G x 5/8 needles	BD	305115
35 mm Petri Dishes	Falcon	353001
40 µm 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 50 mL	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