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 &#8220;BCRT-grant&#8221; by the German Federal Ministry of Education and Research and the Einstein Foundation. K.S.-B. and H.-D.V. are funded by FOR2165.

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 Charit&#233; Berlin, Germany, and were approved by the Landesamt f&#252;r Gesundheit und Soziales and comply with the ARRIVE guidelines.
1. Diet-induced Animal Model of Steatohepatitis
<ol>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
2. Preparation of Reagents and Solutions
<ol>
	<li>Prepare 70% ethanol, phosphate buffered saline (PBS, without calcium and magnesium) supplemented with 0.5% BSA and ACK (Ammonium-Chloride-Potassium) lysis buffer.</li>
	<li>Staining buffer
	<ol>
		<li>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.</li>
		<li>Store solution in a plastic bottle at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collagenase solution for adipose tissue digestion
	<ol>
		<li>Dissolve 2.5 g of bovine serum albumin (BSA) in 500 mL of PBS to obtain a 0.5% BSA/PBS.</li>
		<li>Dissolve 74.5 g of CaCl2 in 10 mL of 0.5% BSA/PBS to obtain a 10 mM CaCl2 solution.</li>
		<li>Add 1 mg of collagenase type II (see Table of Materials) to each mL of 0.5% BSA with 10 mM CaCl2 PBS.</li>
		<li>Prepare 3 mL of collagenase digest solution per g of adipose tissue sample. Prepare fresh collagenase solution for each isolation.</li>
	</ol>
	</li>
	<li>Collagenase solution for liver tissue digestion
	<ol>
		<li>Dissolve 2.5 g of BSA in 500 mL of Hank&#180;s Balanced Salt Solution (HBSS) to obtain 0.5% BSA HBSS.</li>
		<li>Add 10 mL of FCS in 500 mL of 0.5% BSA/HBSS to obtain 2% FCS 0.5% BSA/HBSS.</li>
		<li>Add 0.5 mg of collagenase type IV (see Table of Materials) to each mL of 2% FCS 0.5% BSA/HBSS.</li>
		<li>Add 0.02 mg of DNase per mL of 2% FCS 0.5% BSA/HBSS collagenase solution.</li>
		<li>Prepare 13 mL of collagenase digest solution per liver tissue sample.</li>
		<li>Prepare fresh collagenase solution for each isolation.</li>
	</ol>
	</li>
</ol>
3. Generation of Single cell Suspensions
<ol>
	<li>Adipose tissue digestion
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Perform the mechanical dissociation using scissors and cut adipose tissue into fine pieces in a Petri dish at 4 &#176;C. Transfer adipose tissue to 50 mL conical centrifuge tubes and rinse Petri dish with 1 mL of 0.5% BSA/PBS.</li>
		<li>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 &#176;C for 20 min under gentle shaking (200 rpm).</li>
		<li>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 &#181;m) with the aid of a plunger.</li>
		<li>Centrifuge at 500 x g for 10 min at 4 &#176;C.</li>
		<li>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.</li>
		<li>Centrifuge at 500 x g for 10 min at 4 &#176;C.</li>
		<li>Decant supernatant and resuspend cell pellet in 250 &#181;L of 2% FCS PBS.</li>
		<li>Count the number of viable cells on a hemocytometer using Trypan blue exclusion.</li>
	</ol>
	</li>
	<li>Liver tissue digestion
	<ol>
		<li>Store liver in a conical centrifuge tube filled with PBS and transport on ice.</li>
		<li>Perform the mechanical dissociation using syringe stamps in a Petri dish at 4 &#176;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.</li>
		<li>Incubate liver tissue solution at 37 &#176;C for 20 min under gentle shaking (200 rpm).</li>
		<li>Add 20 mL of HBSS. Triturate the solution numerous times with a 10 mL serological pipette and pass it through a strainer (100 &#181;m) with the aid of a plunger.</li>
		<li>Centrifuge at 500 x g for 10 min at 4 &#176;C. Decant the supernatant and resuspend the cell pellet in 20 mL of HBSS.</li>
		<li>Centrifuge at 30 x g for 1 min at room temperature to remove the hepatocyte matrix. Discard the cell pellet.</li>
		<li>Centrifuge the supernatant at 500 x g for 10 min at 4 &#176;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).</li>
		<li>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).</li>
		<li>Pass cells through a 30 &#956;m strainer in a 15 mL conical centrifuge tube and centrifuge at 500 x g for 10 min at 4 &#176;C. Decant supernatant and resuspend cell pellet in 250 &#181;L of 2% FCS PBS.</li>
		<li>Count the number of viable cells on a hemocytometer using Trypan blue exclusion.</li>
	</ol>
	</li>
</ol>
4. Surface Staining
<ol>
	<li>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 &#181;L) regarding to antibody concentrations. 
	NOTE: Lymphocytes and innate immune cells present differences in autofluorescence and should be analyzed separately.</li>
	<li>Use up to 3 x 106 cells in 100 &#181;L in a polystyrene FACS tube for surface staining. To block Fc receptors add 10 &#181;L 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.</li>
	<li>Vortex and add the appropriate volume of antibody mixture for Panel 1 and Panel 2. Add 1 &#181;L of viability dye to each sample to allow for live and dead cell discrimination. Incubate for 20 min at 4 &#176;C and protected from light.</li>
	<li>Wash two times with 2 mL of 2% FCS PBS and centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 300 &#181;L of 2% FCS PBS and store at 4 &#176;C until FACS analysis. 
	NOTE: Before starting the measurement pass cells through 30 &#181;m cell strainer into polystyrene FACS tube and vortex. Flow cytometry was performed with labeled cells stored in 2% FCS PBS at 4 &#176;C for 1-3 h. Cells should be analyzed as soon as possible for optimized results.</li>
</ol>
5. Flow Cytometry Compensation, Acquisition, and Gating
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Start the measurement, collect appropriate number of events (at least 50,000 events) and record experimental data.</li>
	<li>Export FCS data files for analysis and set the gating strategy. Gate on CD45+ leucocytes to identify subsequent cell populations.</li>
</ol>

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The authors have nothing to disclose.

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...

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 &#8212;&#160;nearly 30% of the world&#8217;s population &#8212; 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 tissue3,4. 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 cells5,6. 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 resistance7,8,9.
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 approaches12,13,14.
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.

<table><tbody><tr><td>100 &amp;micro;m cell strainers&amp;nbsp;</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>1 mL syringe&amp;nbsp;</td><td>BD&amp;nbsp;&amp;nbsp;</td><td>309659</td><td></td></tr><tr><td>26 G x 5/8 needles&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>305115</td><td></td></tr><tr><td>35 mm Petri Dishes&amp;nbsp;</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainers&amp;nbsp;</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>ACK lysis buffer&amp;nbsp;</td><td>GIBCO</td><td>A1049201</td><td></td></tr><tr><td>Alexa Fluor 700 anti-mouse CD45</td><td>Biolegend&amp;nbsp;</td><td>103127</td><td>AB_493714 (BioLegend Cat. No. 103127)</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>FlowJo 10.0.8 software</td><td></td><td></td></tr><tr><td>APC anti-mouse CD11c Antibody</td><td>Biolegend&amp;nbsp;</td><td>117309</td><td>AB_313778 (BioLegend Cat. No. 117309)</td></tr><tr><td>APC anti-mouse KLRG1 (MAFA) Antibody</td><td>Biolegend&amp;nbsp;</td><td>138411</td><td>AB_10645509 (BioLegend Cat. No. 138411)</td></tr><tr><td>BV421 anti-mouse CD127 Antibody</td><td>Biolegend&amp;nbsp;</td><td>135023</td><td>AB_10897948 (BioLegend Cat. No. 135023)</td></tr><tr><td>BV421 anti-mouse F4/80 Antibody</td><td>Biolegend&amp;nbsp;</td><td>123131</td><td>AB_10901171 (BioLegend Cat. No. 123131)</td></tr><tr><td>BV605 anti-mouse CD279 (PD-1) Antibody</td><td>Biolegend&amp;nbsp;</td><td>135219</td><td>AB_11125371 (BioLegend Cat. No. 135219)</td></tr><tr><td>BV605 anti-mouse NK-1.1 Antibody</td><td>Biolegend&amp;nbsp;</td><td>108739</td><td>AB_2562273 (BioLegend Cat. No. 108739)</td></tr><tr><td>BV650 anti-mouse/human CD11b Antibody</td><td>Biolegend&amp;nbsp;</td><td>101239</td><td>AB_11125575 (BioLegend Cat. No. 101239)</td></tr><tr><td>BV711 anti-mouse/human B220 Antibody</td><td>Biolegend&amp;nbsp;</td><td>103255</td><td>AB_2563491 (BioLegend Cat. No. 103255)</td></tr><tr><td>BV785 anti-mouse CD8a Antibody</td><td>Biolegend&amp;nbsp;</td><td>100749</td><td>AB_11218801 (BioLegend Cat. No. 100749)</td></tr><tr><td>C57Bl/6J mice, male, 5 weeks old&amp;nbsp;</td><td>Forschungseinrichtungen f&amp;uuml;r experimentelle Medizin (FEM)</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Charit&amp;eacute; - Universit&amp;auml;tsmedizin Berlin</td><td>A119.1&amp;nbsp;</td><td></td></tr><tr><td>Collagenase NB 4G Proved Grade&amp;nbsp;</td><td>SERVA&amp;nbsp;</td><td>11427513</td><td></td></tr><tr><td>Collagenase Typ I&amp;nbsp;</td><td>Worthington&amp;nbsp;</td><td>LS004197</td><td></td></tr><tr><td>Conical centrifuge tube 15mL&amp;nbsp;</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>Conical centrifuge tube 50 mL&amp;nbsp;</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>DNAse&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>4716728001</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S0115</td><td></td></tr><tr><td>Filter 30&amp;micro;m&amp;nbsp;</td><td>Celltrics&amp;nbsp;</td><td>400422316</td><td></td></tr><tr><td>FITC anti-mouse CD3 Antibody</td><td>Biolegend&amp;nbsp;</td><td>100203</td><td>AB_312660 (BioLegend Cat. No. 100203)</td></tr><tr><td>Flow cytometry&amp;nbsp;</td><td>BD-LSR Fortessa&amp;nbsp;</td><td></td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F4142-1EA</td><td></td></tr><tr><td>HBSS&amp;nbsp;</td><td>Bioanalytic GmBH&amp;nbsp;</td><td>085021-0500&amp;nbsp;</td><td></td></tr><tr><td>High-fat diet&amp;nbsp;</td><td>SSNIF</td><td>E15741&amp;ndash;34&amp;nbsp;</td><td>60 kJ% from fat, 19&amp;thinsp;kJ% from proteins, and 21&amp;thinsp;kJ% from carbohydrates</td></tr><tr><td>micro dissecting scissors&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S3146</td><td>used for dissection purposes&amp;nbsp;</td></tr><tr><td>PE anti-mouse CD25 Antibody</td><td>Biolegend&amp;nbsp;</td><td>101903</td><td>AB_312846 (BioLegend Cat. No. 101903)</td></tr><tr><td>PE/Cy7 anti-mouse CD62L Antibody</td><td>Biolegend&amp;nbsp;</td><td>104417</td><td>AB_313102 (BioLegend Cat. No. 104417)</td></tr><tr><td>PE/Cy7 anti-mouse I-A/I-E (MHCII) Antibody</td><td>Biolegend&amp;nbsp;</td><td>107629</td><td>AB_2290801 (BioLegend Cat. No. 107629)</td></tr><tr><td>PE/Dazzle 594 anti-mouse CD4 Antibody</td><td>Biolegend&amp;nbsp;</td><td>100565</td><td>AB_2563684 (BioLegend Cat. No. 100565)</td></tr><tr><td>Percoll solution&amp;nbsp;</td><td>Biochrom</td><td>L6115</td><td></td></tr><tr><td>PerCP/Cy5.5 anti-mouse CD44 Antibody</td><td>Biolegend&amp;nbsp;</td><td>103031</td><td>AB_2076206 (BioLegend Cat. No. 103031)</td></tr><tr><td>PerCP/Cy5.5 anti-mouse Gr-1 Antibody</td><td>Biolegend&amp;nbsp;</td><td>108427</td><td>AB_893561 (BioLegend Cat. No. 108427)</td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Gibco</td><td>12559069</td><td></td></tr><tr><td>Round-Bottom Tubes with cell strainer cap</td><td>STEMCELL Technologies&amp;nbsp;</td><td>38030</td><td></td></tr><tr><td>TruStain fcX anti-mouse CD16/32</td><td>Biolegend&amp;nbsp;</td><td>101301</td><td>AB_312800 (BioLegend Cat. No. 101301)</td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T6146</td><td></td></tr><tr><td>Zombie NIR Fixable Viability Kit</td><td>Biolegend&amp;nbsp;</td><td>423105</td><td>viablity stain&amp;nbsp;</td></tr></tbody></table>

an advanced murine model for nonalcoholic steatohepatitis in association with type 2 diabetes

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.

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

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, ...

an-advanced-murine-model-for-nonalcoholic-steatohepatitis-association

Research

JoVE Journal

Immunology and Infection

7.0K Views.  Humboldt-Universität zu Berlin. Deze methode kan helpen bij het beantwoorden van vragen op het gebied van immunometabolisme over de bijdrage van immuuncellen aan obesitas-geïnduceerde ontsteking in vet en steatohepatitis in leverweefsel. Deze technieken zijn geoptimaliseerd voor de isolatie van vet- en lever immune cellen in een door voeding geïnduceerd model van niet-alcoholische steatohepatitis bereikt door een niet-specifieke pathogene-vrije knaagdierbehuizing. Deze methoden kunnen helpen om ons begrip van de immunolog...

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

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicine

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Biology

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

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

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