Data in support of dyslipidemia-associated alterations in B cell subpopulations frequency and phenotype during experimental atherosclerosis

Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE−/−) mice fed or not with high-fat diet (HFD), by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis (DOI: 10.1016/j.atherosclerosis.2015.12.022, article reference: ATH14410) [1]. The data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia.

data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia. & 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Immunology. More specific subject area Immunology of Atherosclerosis. Data accessibility Data is within this article.

Value of the data
Similar alterations in frequency and phenotype of B cell subsets could be presented in murine models of other chronic inflammatory diseases.
The data can be used for further investigations concerning B cells biology in other lipid associated and metabolic diseases.
The data and procedures can be used by other scientists investigating the effects of dyslipidemia on murine B cells.
Characterization strategy for B cell subsets presented herein, can be used in other murine models.

Data
Here we describe detailed materials and methods, the analysis strategy for discriminating B cells from aortas, lymph nodes and spleens and some aspects of the phenotype of B cell subsets in the atherosclerosis mouse model apoE -/and control mice (Figs. 1-14). A similar approach for the analysis of B cells in apoE -/mice were previously published in [1].

Mice
C57BL/6 WT and apoE -/mice were purchased from Charles River (MI) and Jackson Laboratory (ME), respectively. Mice were housed and kept under a specific pathogen-free environment, at the animal facility, Sede de Investigación Universitaria, Universidad de Antioquia (Medellín, Colombia). The institutional animal ethical committee at Universidad de Antioquia approved all the animal protocols and experiments. Water and a standard laboratory diet (SLD, Labdiet Company, IN) or high fat diet (HFD; 1.5% cholesterol, 42% anhydrous milk fat) (Harlan Teklad, WI) were given ad libitum to the animals.

Sample collection
Mice were euthanatized with CO 2 exposition and heart, spleen, lymph nodes and aorta were harvested by dissection. Hearts were embedded in paraformaldehyde solution (4%, JT Baker, NJ) at 4°C during 48 h, and then in sucrose solution (30%, Sigma-Aldrich, MO) for 24 h. Finally, heart tissues were stored at À 20°C in Shandon cryomatrix (Thermo Scientific, PE) until their use. Spleens, aortas and lymph nodes were collected in phosphate buffered saline (PBS, Gibco, NY) at 4°C and immediately processed. Peripheral blood obtained by cardiac puncture was centrifuged at 10.000 Â g during 10 min at 4°C to obtain serum. After centrifugation, serum was stored at À 20°C until used.
Heart tissues were serially sectioned in a CM-1850 Cryostat (Leica Microsystems, Germany) for 6-7 mm thickness on slides with positive charge (Thermo scientific), and stained with Hematoxylin- Eosin (Sigma-Aldrich). Microphotografies were taken from aortic root and ascendant aorta at 4 Â with a digital camera Nikon DS-Fi1 coupled to a Nikon E2000 microscope (Nikon, Japan). Images were analyzed with NIS Element Software (Nikon) to determinate the size of atherosclerotic lesions.

Isolation of murine splenocytes
Spleens were perfused and macerated with syringe plunger (Rymco, Colombia) and cold media compound of RPMI 1640 Glutamax (Gibco) with 2% inactivated Fetal Bovine Serum (FBS, Gibco) and 1% Penicillin/Streptomycin (Gibco) on ice. Cells were pelleted at 800 Â g, for 10 min at 4°C. Erythrocytes were lysed with RBC Lysis Buffer (eBiosciences, CA) for 5 min with constant movement. Splenocytes were washed with PBS, resuspended in cold media and immediately used after obtention. Number of cells was determined in Neubauer camera. Viability (around 90%, data not shown) was evaluated by trypan blue exclusion and Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).

Isolation of aortic cells
Aortas were processed as previously reported [2]. Each aorta was washed in cold PBS before the treatment and were cut with sterilized scissors in 1 mL of enzymatic cocktail, containing 450 U/ml Collagenase I (Sigma-Aldrich), 125 U/ml Collagenase XI (Sigma-Aldrich), 60 U/ml DNAse I (Sigma-Aldrich) and 60 U/ml Hyaluronidase I (Sigma-Aldrich) for 1 h at 37°C. Cell suspensions were passed through a 74 mm mesh, centrifuged at 400 Â g for 5 min at 4°C and suspended in cold media. Cells were processed immediately after obtention and their number and viability was determined with trypan blue exclusion and Live/Dead Fixable Aqua Dead Cell Stain Kit (475%, data not shown). Blood contamination was determined with TER-119 (o 18%, data not shown).

Multiparametric flow cytometry
Cell suspensions (1 Â 10 6 splenocytes and 1-2 Â 10 5 aortic cells) were washed in 1 ml of FACS buffer (0.01% sodium azide (Sigma-Aldrich) and 3% FBS in PBS, pH ¼ 7.35) and centrifuged at 600 Â g for 5 min, 4°C. Cell pellets were incubated with blocking buffer (10% FBS, 0.1% bovine serum albumin (Sigma-Aldrich) and 0.01% sodium azide in PBS, pH ¼7.35) for 15 min at 4°C. Specific monoclonal antibodies were added in 100 ml FACS buffer and incubated for 20 min at 4°C in dark. Cells marked with biotinylated antibodies were washed twice with FACS buffer and incubated with streptavidin for another 20 min at 4°C in dark. Cells were washed two times with FACS buffer and immediately acquired in the flow cytometer FACS Canto-II (BD Biosciences). Data were analyzed using FlowJo software (Tree star, CA) and reported as percentages, absolute numbers and median fluorescence intensity (MFI).

Determination of cholesterol levels
Total cholesterol serum levels were determined by conventional wet chemistry method using an A-15 automated biochemical analyzer (Biosystems, Spain) in the School of Veterinary, Universidad de Antioquia.

Immunoglobulin levels
Serum IgM and IgG1 levels were measured by Enzyme-linked Immunosorbent Assay (ELISA) Ready-Set-Go! Kits (eBiosciences), following manufacturer's instructions and using an ELx800 Absorbance Microplate Reader (Biotek instruments, VT) at 450 nm emission.

Cell culture
Culture media containing 10 mM L-glutamine (Gibco), 0.05 mM 2-mercaptoethanol (Gibco), 1% Penicillin/Streptomycin in RPMI 1640 Glutamax. Two types of serum were used: 20% lipemic serum from apoE À / À HFD mice 20 weeks old, and 20% non-lipemic serum from WT-SLD mice 20 weeks old. Total splenocytes (2 Â 10 6 ) were cultured in 1 ml of media for 5 and 12 hours at 37°C and 5% CO 2 . Cells were washed with PBS and the immunofluoresce with specific antibodies was performed as previously described. Cells were fixed with a commercial solution (fixation/permeabilization kit, Foxp3/Transcription Factor, eBioscience) during 1 h and washed twice with PBS. Finally, cells were stained with Nile Red (Sigma-Aldrich) for 15 min at 4°C in dark. Cells were washed twice with PBS and immediately acquired in the flow cytometer.

Statistical analysis
Differences between groups were examined using two ways ANOVA with Šidák´s multiple comparisons post-test. Differences in MFI of CD95 and Nile Red between groups were examined using non-parametric Mann-Whitney test. Spearman rank correlation coefficient test was performed to assess the association between two variables. Data was analyzed in Prism 6.0 software (GraphPad, CA). P-values o0.05 were considered statistically significant. The classical principal component analysis was performed with the Statgraphics Centurion XVI software (StatPoint Technologies, VA).