Name: an advanced murine model for nonalcoholic steatohepatitis in association with type 2 diabetes
Uploaded: 2019-04-26T14:30:00
Description: Watch this Scientific Journal Video about An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes at JoVE.com

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 Steatohep...

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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.

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			<td height="21" style="height:21px;width:351px;">100&#181;m cell strainers&#160;</td>
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			<td height="21" style="height:21px;width:351px;">1ml syringe&#160;</td>
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			<td style="width:119px;">352340</td>
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			<td height="21" style="height:21px;width:351px;">ACK lysis buffer&#160;</td>
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			<td>A1049201</td>
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			<td style="width:119px;">103127</td>
			<td>AB_493714 (BioLegend Cat. No. 103127)</td>
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			<td height="21" style="height:21px;width:351px;">Analysis software&#160;</td>
			<td>FlowJo 10.0.8 software</td>
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			<td height="21" style="height:21px;width:351px;">APC anti-mouse CD11c Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">117309</td>
			<td>AB_313778 (BioLegend Cat. No. 117309)</td>
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			<td height="21" style="height:21px;width:351px;">APC anti-mouse KLRG1 (MAFA) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">138411</td>
			<td>AB_10645509 (BioLegend Cat. No. 138411)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV421 anti-mouse CD127 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">135023</td>
			<td>AB_10897948 (BioLegend Cat. No. 135023)</td>
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			<td height="21" style="height:21px;width:351px;">BV421 anti-mouse F4/80 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">123131</td>
			<td>AB_10901171 (BioLegend Cat. No. 123131)</td>
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			<td height="21" style="height:21px;width:351px;">BV605 anti-mouse CD279 (PD-1) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">135219</td>
			<td>AB_11125371 (BioLegend Cat. No. 135219)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV605 anti-mouse NK-1.1 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">108739</td>
			<td>AB_2562273 (BioLegend Cat. No. 108739)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV650 anti-mouse/human CD11b Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101239</td>
			<td>AB_11125575 (BioLegend Cat. No. 101239)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV711 anti-mouse/human B220 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103255</td>
			<td>AB_2563491 (BioLegend Cat. No. 103255)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV785 anti-mouse CD8a Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100749</td>
			<td>AB_11218801 (BioLegend Cat. No. 100749)</td>
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			<td height="38" style="height:39px;width:351px;">C57Bl/6J mice, male, 5 weeks old&#160;</td>
			<td style="width:169px;">Forschungseinrichtungen f&#252;r experimentelle Medizin (FEM)</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">CaCl2&#160;</td>
			<td>Charit&#233; - Universit&#228;tsmedizin Berlin</td>
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			<td style="width:169px;">SERVA&#160;</td>
			<td style="width:119px;">11427513</td>
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			<td height="36" style="height:36px;width:351px;">Collagenase Typ I&#160;</td>
			<td style="width:169px;">Worthington&#160;</td>
			<td style="width:119px;">LS004197</td>
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			<td height="21" style="height:21px;width:351px;">Conical centrifuge tube 15ml&#160;</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352096</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Conical centrifuge tube 50ml&#160;</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352070</td>
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			<td height="21" style="height:21px;width:351px;">DNAse&#160;&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">4716728001</td>
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			<td height="21" style="height:21px;width:351px;">Fetal bovine serum&#160;</td>
			<td style="width:169px;">Biochrom</td>
			<td style="width:119px;">S0115</td>
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			<td height="21" style="height:21px;width:351px;">Filter 30&#181;m&#160;</td>
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			<td style="width:119px;">400422316</td>
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			<td height="21" style="height:21px;width:351px;">FITC anti-mouse CD3 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100203</td>
			<td>AB_312660 (BioLegend Cat. No. 100203)</td>
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			<td height="21" style="height:21px;width:351px;">Flow cytometry&#160;</td>
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			<td height="21" style="height:21px;width:351px;">Forceps&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td>F4142-1EA</td>
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			<td height="21" style="height:21px;width:351px;">HBSS&#160;</td>
			<td style="width:169px;">Bioanalytic GmBH&#160;</td>
			<td style="width:119px;">085021-0500&#160;</td>
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			<td height="21" style="height:21px;width:351px;">High-fat diet&#160;</td>
			<td style="width:169px;">SSNIF</td>
			<td>E15741&#8211;34&#160;</td>
			<td>60 kJ% from fat, 19&#8201;kJ% from proteins, and 21&#8201;kJ% from carbohydrates</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">micro dissecting scissors&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">S3146</td>
			<td>used for dissection purposes&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE anti-mouse CD25 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101903</td>
			<td>AB_312846 (BioLegend Cat. No. 101903)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Cy7 anti-mouse CD62L Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">104417</td>
			<td>AB_313102 (BioLegend Cat. No. 104417)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Cy7 anti-mouse I-A/I-E (MHCII) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">107629</td>
			<td>AB_2290801 (BioLegend Cat. No. 107629)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Dazzle 594 anti-mouse CD4 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100565</td>
			<td>AB_2563684 (BioLegend Cat. No. 100565)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Percoll solution&#160;</td>
			<td style="width:169px;">Biochrom</td>
			<td style="width:119px;">L6115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse CD44 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103031</td>
			<td>AB_2076206 (BioLegend Cat. No. 103031)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse Gr-1 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">108427</td>
			<td>AB_893561 (BioLegend Cat. No. 108427)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Phosphate buffered saline&#160;</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:119px;">12559069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Round-Bottom Tubes with cell strainer cap</td>
			<td style="width:169px;">STEMCELL Technologies&#160;</td>
			<td style="width:119px;">38030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TruStain fcX anti-mouse CD16/32</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101301</td>
			<td>AB_312800 (BioLegend Cat. No. 101301)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Trypan Blue&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T6146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Zombie NIR Fixable Viability Kit</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">423105</td>
			<td>viablity stain&#160;</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, ...

This method can help answer questions in the immunometabolism field about the contribution of immune cells to obesity-induced inflammation in adipose and steatohepatitis in hepatic tissue. These techniques have been optimized for the isolation of adipose and hepatic immune cells in a diet-induced model of nonalcoholic steatohepatitis achieved through a non-specific pathogen-free rodent housing. These methods can help to deepen our understanding of the immunological mechanisms involved in the maintenance of metabolic homeostasis.Generally, individuals new to this technique will struggle with maximizing the yield of functionally viable dissociated cells and setting up the proper gating in flow cytometry. To generate an adipose tissue single-cell suspension, spray the chest of a euthanized mouse with 70%ethanol, and carefully make a five to six-centimeter central incision through the integument and abdominal wall along the entire length of the rib cage to expose the pleural cavity and heart. Using a 26-gauge needle, inject at least 10 milliliters of 0.9%saline solution into the apex of the left ventricle, and use scissors to open the peritoneal cavity.Then, dissect the epigonadal adipose tissue, and weigh it. Mechanically dissociate the adipose tissue into fine pieces in a Petri dish at four degrees Celsius before transferring the tissue fragments into a 50-milliliter conical tube. Rinse the Petri dish with one milliliter of 0.5%bovine serum albumin, or BSA, in PBS, and add three milliliters of freshly prepared adipose tissue digest solution per gram of adipose tissue to the tube.After 20 minutes at 37 degrees Celsius and 200 rotations per minute, add five milliliters of 0.5%BSA in PBS, and place the adipose digest on ice. Triturate the solution multiple times with a 10-milliliter serological pipette, and use a plunger to pass the solution through a 100-micrometer strainer. Separate the tissue fractions by centrifugation, and use a pipette to discard the floating adipocyte fraction.Resuspend the stromal vascular fraction cell pellet in one milliliter of ammonium-chloride-potassium, or ACK, lysis buffer, stopping the lysis reaction with 10 milliliters of 2%fetal calf serum, or FCS, in PBS after a few seconds. Then, collect the isolated white blood cells by centrifugation, and resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. Harvest the liver into a conical tube of PBS on ice before using syringe stamps to mechanically dissociate the tissue in a Petri dish at four degrees Celsius.Transfer the dissected liver tissue fragments into a 50-milliliter tube containing 10 milliliters of warm liver digest solution, and rinse the Petri dish with three milliliters of fresh liver digest solution. After 20 minutes at 37 degrees Celsius and 200 rotations per minute, stop the digestion with 20 milliliters of HBSS, and triturate the tissue slurry multiple times with a 10-milliliter serological pipette. Use a plunger to pass the tissue solution through a 100-micrometer strainer, and collect the liver cells by centrifugation.Resuspend the pellet in 20 milliliters of fresh HBSS, and remove the hepatocyte matrix by centrifugation. Collect the supernatant for centrifugation, resuspending the pellet in 10 milliliters of 33%low viscosity density gradient medium solution in HBSS. Separate the liver immune cells by density gradient centrifugation, and resuspend the leukocyte pellet in one milliliter of ACK lysis buffer.After four minutes at room temperature, stop the lysis with 10 milliliters of HBSS, and filter the cells through a 30-micrometer pore strainer into a 15-milliliter tube for centrifugation. Then, resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. For flow cytometric analysis of the isolated leukocyte populations, add up to three times 10 to the six cells in 100 microliters of 2%FCS in PBS from the tissue of interest into individual polystyrene fluorescence-activated cell sorting, or FACS, tubes.Add 10 microliters of anti-CD16/CD32 antibody to each tube to block any non-specific binding for 10 minutes on ice, followed by staining with the appropriate volume of antibody mixture and one microliter of viability dye per sample to allow live and dead cell discrimination. After 20 minutes at four degrees Celsius protected from light, wash the samples two times with two milliliters of 2%FCS plus PBS per wash, and centrifuge at 300 times g for five minutes at four degrees Celsius. Then, resuspend the pellet in fresh PBS plus FCS.To analyze the cells by flow cytometry, use an unstained negative control sample to set the forward and side scatter, and adjust the voltages of the flow cytometer to detect the viable leukocyte population and to exclude the debris. Next, run single stained control samples for multicolor compensation before running the experimental samples, collecting at least five times 10 to the four events per tube. Then, export the FCS data files for analysis, and set the gating strategy for CD45-positive leukocytes to allow the identification of the subsequent immune cell populations of interest.Here, a representative gating strategy for T cell subpopulations including doublet discrimination and viability staining in murine perigonadal fat is shown. CD45-positive leukocytes are first gated for CD4 and CD8 and subsequently for CD44 and CD62 ligand to discriminate between naive, central memory, effector memory, and effector T cells. CD44-positive cells are then further characterized with CD127, killer cell lectin-like receptor G1, and programmed death-1.Here, the gating strategy for analyzing B cells, granulocytes, natural killer cells, macrophages, and dendritic cells is shown. A higher percentage of effector memory CD4-positive and CD8-positive cells can be detected in high-fat diet mice housed in non-SPF conditions, whereas intrahepatic naive CD4-positive and CD8-positive T cells are found to be considerably lower in these animals compared to SPF high-fat diet mice at week seven. Severe steatosis, including an increase of large lipid droplets resulting in macrovesicular steatosis, lobular inflammation, hepatocellular ballooning, and destroyed lobular structure is found in high-fat diet exposed mice while only some SPF mice display a mild fat accumulation in the liver.In addition, the percentage of natural killer cells is higher in the perigonadal adipose tissue of high-fat diet exposed mice, whereas high-fat diet SPF mice demonstrated higher percentages of dendritic cells. When attempting this procedure, it is important to keep the cells on ice and in dark unless otherwise noted. You should now have a good understanding of how to properly prepare single-cell suspensions and how to isolate murine adipose and hepatic tissue for flow cytometry experiments.

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

Research

JoVE Journal

Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...