GSE157624
Next generation sequencing of CD11b+ cells from female and male Wildtype and Cystatin C knockout mice with experimental allergic encephalomyelitis (EAE)
Rationale: Towards clarifying the role of the cysteine protease inhibitor, Cystatin C (CysC), whose expression is augmented in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental allergic encephalomyelitis (EAE - a model of MS), we discovered that CysC plays a detrimental function in MOG35-55-induced EAE, but only in female animals. Female CysC null mice displayed significantly lower clinical signs of disease compared to wildtype (WT) littermates that was associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, MHC II, LC3A/B) involved in antigen processing, presentation and co-stimulation in antigen presenting cells (APCs), specifically CD11b+ cells. To delineate if these antigen presenting markers in CD11b+ cells as well as unidentified others were expressed differentially between the two genotypes and sexes, we performed Next Generation Sequencing on total RNA from CD11b+ cells from female and male Wildtype and Cystatin C knockout mice with experimental allergic encephalomyelitis (EAE) at 7 days post-immunization. Methods: CD11b+ cells were isolated by positive selection (Miltenyi Biotec) from day 7 EAE female and male WT and CysC-/- spleens and RNA retrieved with an RNeasy Mini Kit (Qiagen). Samples with an RNA integrity number greater than 8 were subjected to Deep Sequencing and each sample is from an individual mouse. All samples were subjected to reverse transcription and Multiple Displacement Amplification with REPLI-g SensiPhi DNA Polymerase and oligo-dT primers as per the QIAGEN REPLI-g Single Cell RNA Library preparation kit and the manufacturer's protocol. Libraries were prepared using the QIAGEN GeneRead Adaptor I Set A 12-plex index adapters as per the REPLI-g single cell RNA library prep kit's protocol and subjected to on-board cluster formation and sequencing on an Illumina NextSeq 500 sequencer with a high-output v2 75 cycle sequencing kit as per the standard Illumina protocols. After sequencing, the bcl data was converted to fastq data files using the Illumina BCL2FASTQ utility. Alignment was performed using bwa 0.7.12 in ÒmemÓ alignment mode against mm10 reference genome with default parameters. Output was imported into Ingenuity Pathway Analysis software for physiological function analysis. Results: Our RNASeq data revealed that LC3B and CD80 in female EAE CysC null cells were overwhelming present in immune modulation functional pathways that were decreased (negative z-score) compared to the male cohort. Conclusions: These findings imply that CysC plays a role in promoting an appropriate molecular response needed for the maturation or antigen processing, loading and presentation by antigen presenting cells in female mice during EAE. Overall design: Spleen-derived CD11b+ cells from day 7 EAE wildtype (WT) and Cystatin C knockout female and male mice