TLR7 Joint With BCR Tiggers Activation Induced B Cell Death

The Physiological state of B cells in active SLE patients has been changed extensively. We investigated the variations of physiological and biochemical pathways in the peripheral blood B cells of SLE patients, based on the differentially expressed genes in the microarray. Among them, the up-regulated expression of TLR7, and the changes in apotosis and necrosis related molecules catches our attention. We stimulated mouse spleen B cells with ligands of TLR7 and BCR to mimic the pathological state of B cells in SLE patients and found that combination of the two stimulant can induce intensive activation and subsequent aggravated death of B cells. Futher more, we discovered that the aggravated death of B cells showed both elevation in apotosis and necrosis. Thus, we can reach a conclusion that:the increased apoptosis and necrosis of B cells is part of the mechanism that TLR7 promote the development of SLE.1. Analysis of the microarry of peripheral blood B cells in SLE patientsSLE is a systemic autoimmune disease, the etiology of which is considered to be interaction between genetic and inducible factors and the immune system. The antibodies produced by autoreactive B cells in SLE patients form immune complexes with the autoantigen and deposite in the mesangium and capillary wall,causing glomerulonephritis, vasculitis and multiple organ inflammation. B cell is supposed to play a pivotal role in the pathogenesis of SLE, due to the frequent correlation between the clinical manifestation of SLE and immune complexes. Moreover, researches for B cells in SLE focused on the immune function.In order to fully understand the physiological and pathological state of SLE B cells, the whole genome transcriptome microarray of B cells from six active SLE patients and six healthy human peripheral blood was performed in our prophase experiment. We chose 476 down-regulated and 955 up-regulated genes from the microarrays. GO annotation and functional enrichment of SLE B cell differential expressed genes showed that, changed genes mainly enriched in type I interferon pathway, apoptotic pathway, and antiviral pathway. The microarray analysis indicates that, these differential expression genes are not only related to the immune function, but also related to the cell survival. The apoptosis-related differential expressed genes are mostly related to the mitochondrial apoptotic pathway.2. TLR7 joint BCR stimulation promotes apoptosis of activated B cellsIn the first chapter, we noticed that in the B cell gene chips:The expression level of many genes was changed in the PBMC of SLE B cells, including those pro-apoptotic and anti-apoptotic genes. And in SLE patients the TLR7 pathway and BCR were activated simultaneously. To simulate the lupus patients’ in vivo environment in vitro, we used TLR7 ligand R848, BCR activators anti-IgM and anti-CD40, respectively, or their combination to stimulate the spleen B cells of mice. And after in vitro treatment with above stimulators, we resolved RT-qPCR method to verify part of the genes differently expressed in the B cell chip. And results from the flow cytometry showed significant late apoptosis of B cells. Thus, we further hypothesized that there may exist accelerated B cell apoptosis in patients with lupus.3. TLR7 joint BCR stimulation promotes necrosis of B cellsIn the second chapter, we found that TLR7 joint BCR stimulation promotes B cells to produce large amounts of Annexin V/PI double-positive death cells. And in the next analysis we found that the Transmission Electron Microscopy photographs of the death cells showed B cells in necrosis. And this is accompanied by the disappearance of the mitochondrial membrane potential, increased intracellular Ca ions, and decreased ATP levels in B cells after TLR7 joint BCR stimulation. Because under certain conditions, programmed cell apoptosis and programmed cell necrosis can be converted to each other. Whether the increased necrotic cell with joint stimulation is dependent on the molecular pathways of apoptosis needs further investigation.Meanwhile, in the SLE B cells chip, genes has been reported to associate with programmed cell necrosis such as PARP family have significantly increased.we will further investigate whether the joint BCR and TLR7 activation on B cells would promote their necrosis. And a step further, the possibility of necrosis is associated with PARP1 or RIP3, namely whether it is programmed necrosis.