Obesity Intensifies Sex-specific Interferon (IFN) Signaling to Selectively Worsen Central Nervous System Autoimmunity in Females
Description
Human Blood Collection and Processing For OLINK analysis, serum samples were collected in BD Vacutainer Serum tubes. Blood was allowed to clot for 60 minutes and then centrifuged at 1300 x g for 10 minutes. Serum was then aliquoted into tubes for storage at -80 °C. For naïve CD4+ T cell studies, peripheral blood was collected in BD Vacutainer tubes with sodium heparin. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll-Paque density gradient centrifugation (GE Healthcare). Cells were washed in 1 x MACS buffer and were counted. Proteomic Analysis on Human Serum Proteins were measured from serum samples using Olink Explore 1536 from Olink Proteomics which combines the proximity extension assay (PEA)78 with next-generation sequencing (NGS) technology79. The complete library contains antibodies targeting 1472 proteins, of which 1463 are unique proteins. Patient samples were randomized and incubated overnight with antibodies conjugated to oligonucleotide PEA probes at 4°C. After antibody binding, oligonucleotide annealing, and extension steps, the pre-amplification mix was added to the samples at room temperature. PCR amplification was performed, and PCR amplicons were then pooled and subjected to another PCR amplification step after addition of individual sample index sequences. After pooling of the samples, bead purification and quality control of the generated libraries were performed. Sequencing was carried out using Illumina’s NovaSeq 6000 instrument. Quality control and normalization processes were then performed to translate barcode sequence counts into normalized protein expression (NPX) units. Assays on the samples were performed blinded from the clinical data. Significant differences in individual protein levels (NPX units) between obese and non-obese was determined using T tests (two groups were compared) and adjusted for multiple comparisons by the Bonferroni and Hochberg method (R v4.2.3). Statistical comparisons of individual proteins between three or more groups were determined using one-way ANOVA (GraphPad Prism v9.5.0). For bioinformatic analysis, differentially abundant serum proteins were mapped to biological pathways using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.)80.
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Proteins were measured from serum samples using Olink Explore 1536 from Olink Proteomics which combines the proximity extension assay (PEA)78 with next-generation sequencing (NGS) technology79. The complete library contains antibodies targeting 1472 proteins, of which 1463 are unique proteins. Patient samples were randomized and incubated overnight with antibodies conjugated to oligonucleotide PEA probes at 4°C. After antibody binding, oligonucleotide annealing, and extension steps, the pre-amplification mix was added to the samples at room temperature. PCR amplification was performed, and PCR amplicons were then pooled and subjected to another PCR amplification step after addition of individual sample index sequences. After pooling of the samples, bead purification and quality control of the generated libraries were performed. Sequencing was carried out using Illumina’s NovaSeq 6000 instrument. Quality control and normalization processes were then performed to translate barcode sequence counts into normalized protein expression (NPX) units. Assays on the samples were performed blinded from the clinical data. Significant differences in individual protein levels (NPX units) between obese and non-obese was determined using T tests (two groups were compared) and adjusted for multiple comparisons by the Bonferroni and Hochberg method (R v4.2.3). Statistical comparisons of individual proteins between three or more groups were determined using one-way ANOVA (GraphPad Prism v9.5.0). For bioinformatic analysis, differentially abundant serum proteins were mapped to biological pathways using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.)80.