GSE175424
CRAC channel controls the differentiation of pathogenic B cells in lupus nephritis
Systemic lupus erythematous (SLE) is a prototype of autoimmune disease. Lupus nephritis (LN) is one of the most serious complications of SLE. The development of autoreactive B cells and the production of autoantibodies have been critical for the initiation and progression of LN.   Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for immune response . SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels which are comprised of stromal interaction molecule (STIM) and calcium release-activated calcium modulators (ORAI) . Mutations in genes encoding the CRAC channel abolish SOCE in cells of the immune system and cause severe combined immunodeficiency . Calcium signaling via ORAI1 has been involved in the pathogenesis of autoimmune diseases by driving Th17 differentiation . STIM1 deficiency significantly reduced Th1/Th17 responses and resulted in complete protection from experimental autoimmune encephalomyelitis . Compared to T cells, the roles of CRAC channel in B cells is far less clear.   Ca²?/calmodulin (CaM)-dependent protein kinase2 (CaMK2) is a serine/threonine-specific protein kinase that is regulated by the Ca²?/CaM complex. CaMK2 has been involved in many signaling pathways and is necessary for Ca²? homeostasis, T cell development and activation. CaMK4, another member of the CaMK family, has been shown to compromises podocyte function and promote renal diseases in LN.   In the current study, we found that CRAC channel mediated calcium signaling is enhanced in B cells from patients with LN. CRAC channel inhibition by YM-58483 and knocked down of CRAC channel by ORAI1 or STIM2 siRNA led to suppression of CaMK2 signaling and decreased B cell differentiation. Lupus mice treated with CRAC channel inhibitor showed reduced anti-double stranded DNA antibodies (anti-dsDNA), decreased immune deposition in the glomeruli and improved renal function. CRAC channel mediates the development and progression of LN by promoting the differentiation of B cells into plasma cells. Overall design: Naïve human B cells were isolated from 3 donors and stimulated with anti-human IgM (5µg/ml) and anti-human CD40 (1µg/ml) antibodies in the presence of YM-58483 or vehicle for 72h. RNA purification was performed using Qiagen RNeasy Kit (Valencia, CA) and DNA was removed by DNase digestion. RNA quality was defined by RIN and OD260/280. The RNA-seq libraries were made with the Ovation® Ultralow Library Systems and sequenced on an Illumina HiSeq at POHC@IGB. Human reference genome (hg38) was used for alignment using HISAT2. Aligned reads were mapped to known genes. The samples were compared in terms of their total reads mapped to genes; distribution of reads across genes; and global correlation with each other.  50 pg of total RNA was used as input for the SMART-seq v3 cDNA synthesis kit (Takara) using 10 cycles of PCR amplification. 1 ng of cDNA was used as input for the NexteraXT kit (Illumina) using 9 cycles of PCR amplification. Final libraries were quantitated by qPCR and sized distribution determined by Bioanalyzer prior to pooling and sequencing on a HiSeq2500 using 50bp PE chemistry.