Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models.

BACKGROUND: Obesity is a growing problem worldwide and a major risk factor for many chronic diseases. The accumulation of adipose tissue leads to the release of significant amounts of pro-inflammatory cytokines and adipokines, resulting in a low-grade systemic inflammation. However, the mechanisms behind the development of obesity-related diseases are not fully understood. Therefore, our study aimed to investigate the pathological changes and inflammatory processes at systemic level and in individual organs in two different diet-induced mouse obesity models. METHODS: Male C57BL6/J mice were fed by high-fat diet (HFD), high-fat/high-fructose diet (HFD + FR) or normal chow for 21 weeks starting at 3 months of age (n = 15 animals/group). Insulin resistance was tested by oral glucose tolerance test. Pathological changes were investigated on hematoxylin-eosin-stained liver and brown adipose tissue sections. The gene expression levels of adipokines and cytokines were analyzed by qPCR in adipose tissues, whereas serum protein concentrations were determined by multiplex immunoassays. Immunophenotyping of isolated blood, bone marrow and spleen cells was performed by single-cell mass cytometry. RESULTS: Weight gain, glucose intolerance and hepatic steatosis were more severe in the HFD + FR group than in the control and HFD groups. This was accompanied by a higher level of systemic inflammation, as indicated by increased expression of pro-inflammatory genes in visceral white adipose tissue and by a higher serum TNFα level. In addition, immunophenotyping revealed the increase of the surface expressions of CD44 and CD69 on various cell types, such as CD8+ and CD4 + T-cells, B-cells and macrophages, in animals with obesity. CONCLUSIONS: The combination of HFD with fructose supplementation promotes more properly the symptoms of metabolic syndrome. Therefore, the combined high-fat/high-fructose nutrition can be a more suitable model of the Western diet. However, despite these differences, both models showed immunophenotypic changes that may be associated with increased risk of obesity-related cancer.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury.

BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Male and Female Animals Respond Differently to High-Fat Diet and Regular Exercise Training in a Mouse Model of Hyperlipidemia.

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.

Exercise training worsens cardiac performance in males but does not change ejection fraction and improves hypertrophy in females in a mouse model of metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) refers to a cluster of co-existing cardio-metabolic risk factors, including visceral obesity, dyslipidemia, hyperglycemia with insulin resistance, and hypertension. As there is a close link between MetS and cardiovascular diseases, we aimed to investigate the sex-based differences in MetS-associated heart failure (HF) and cardiovascular response to regular exercise training (ET). METHODS: High-fat diet-fed male and female APOB-100 transgenic (HFD/APOB-100, 3 months) mice were used as MetS models, and age- and sex-matched C57BL/6 wild-type mice on standard diet served as healthy controls (SD/WT). Both the SD/WT and HFD/APOB-100 mice were divided into sedentary and ET groups, the latter running on a treadmill (0.9 km/h) for 45 min 5 times per week for 7 months. At month 9, transthoracic echocardiography was performed to monitor cardiac function and morphology. At the termination of the experiment at month 10, blood was collected for serum low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol measurements and homeostatic assessment model for insulin resistance (HOMA-IR) calculation. Cardiomyocyte hypertrophy and fibrosis were assessed by histology. Left ventricular expressions of selected genes associated with metabolism, inflammation, and stress response were investigated by qPCR. RESULTS: Both HFD/APOB-100 males and females developed obesity and hypercholesterolemia; however, only males showed insulin resistance. ET did not change these metabolic parameters. HFD/APOB-100 males showed echocardiographic signs of mild HF with dilated ventricles and thinner walls, whereas females presented the beginning of left ventricular hypertrophy. In response to ET, SD/WT males developed increased left ventricular volumes, whereas females responded with physiologic hypertrophy. Exercise-trained HFD/APOB-100 males presented worsening HF with reduced ejection fraction; however, ET did not change the ejection fraction and reversed the echocardiographic signs of left ventricular hypertrophy in HFD/APOB-100 females. The left ventricular expression of the leptin receptor was higher in females than males in the SD/WT groups. Left ventricular expression levels of stress response-related genes were higher in the exercise-trained HFD/APOB-100 males and exercise-trained SD/WT females than exercise-trained SD/WT males. CONCLUSIONS: HFD/APOB-100 mice showed sex-specific cardiovascular responses to MetS and ET; however, left ventricular gene expressions were similar between the groups except for leptin receptor and several stress response-related genes.