Transcriptomic analysis of pathways associated with alpha(v) integrin-dependent autophagy in human B cells

Autophagy proteins have been linked with development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to complex roles of these proteins in multiple immune cell types. We have previously shown that a form of non-canonical autophagy induced by av-integrins regulates B cell activation by viral and self-antigens, in mice. Here we investigated the involvement of this pathway in B cells from human tissue. Our data revealed that autophagy is specifically induced in germinal-center and memory B cell sub-populations from human tonsil and spleen. Transcriptomic analysis showed that induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/av integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells we found that ?????-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B cell-mediated immune dysregulation in diseases such as lupus. Overall design: Sorted tonsil B cell subsets with and without stimulation subjected to RNAseq, cell lines with CRISPR of autophagy genes or ITGAV gene subjected to RNA seq

自噬（autophagy）蛋白已被发现与包括狼疮在内的免疫介导疾病的发生发展密切相关，但由于这类蛋白在多种免疫细胞类型中作用复杂，其具体关联机制仍未阐明。我们此前的研究表明，在小鼠体内，αv整合素（αv integrins）诱导的非经典自噬可调控B细胞（B cell）对病毒抗原与自身抗原的活化过程。本研究针对人体组织中的B细胞，探究了该通路的参与作用。我们的实验数据显示，自噬特异性诱导于人扁桃体与脾脏的生发中心（germinal center）B细胞及记忆B细胞（memory B cell）亚群。转录组分析（transcriptomic analysis）表明，自噬的诱导与活化B细胞的独特生物学特征相关，例如线粒体代谢过程。为阐明ITGAV/αv整合素依赖型自噬在人B细胞中的功能，我们采用CRISPR介导的基因敲低（CRISPR-mediated knockdown）技术靶向自噬相关基因。整合原代B细胞（primary B cells）与基因敲除细胞的相关数据后，我们发现，αv整合素依赖型自噬可限制与B细胞应答相关的特定通路的活化，同时促进另一部分通路的激活。上述研究结果为自噬与狼疮等疾病中B细胞介导的免疫失调提供了新的机制关联。实验整体设计：对分选获得的经刺激与未刺激的扁桃体B细胞亚群进行RNA测序（RNA-seq）；对经CRISPR编辑靶向自噬相关基因或ITGAV基因的细胞系进行RNA测序。