Post-transcriptional Mechanism Of Pro-inflammatory Cytokine GM-CSF In T Cells

Autoimmune diseases are disorders that the immune system mistakenly attacks and damages self-tissues.As an important proinflammatory cytokine,GM-CSF produced from CD4+ T cells has been proved to be essential to autoimmune diseases pathogenesis.Many proinflammatory cytokines,including GM-CSF,is post-transcriptionally regulated primarily through its AU rich element at 3'UTR via interaction with specific RNA-binding proteins.However,molecular mechanisms regulating GM-CSF expression at posttranscriptional level in T cells remain to be fully elucidated.In this thesis,we first constructed a fluorescence reporter for GM-CSF 3'UTR activity,and combined high-throutput sh-RNA screening to identify potential RBPs that regulate its activity in T cells.PCBP1,a KH domain containing protein recognizing poly C RNA elements,was found to be essential for GM-CSF 3'UTR activity in Jurkat T cells.PCBP1 directly bound to GM-CSF m RNA and increased its stability,as well as IL-2.Importantly,PCBP1 deficiency in autoreactive T cells abolished their capacity in inducing experimental autoimmune encephalomyelitis(EAE),an animal model for multiple sclerosis.Using RNA sequencing and CLIP(Crosslinking and immunoprecipitation)sequencing,we demonstrated that PCBP1 recognized CU-rich elements in the 3'UTRs or last exon of pro-inflammatory cytokines.Thus we identified PCBP1 as a novel post-transcriptional regulator for proinflammatory cytokines independent of AU rich element in T cells.In addition to being a RNA-binding protein,PCBP1 is known as an iron chaperon in the cytosol.Iron deposition is frequently observed in human autoinflammatory diseases,such as in the brain of patients with multiple sclerosis or in the synovial fluid of patients with rheumatoid arthritis.Yet,the functional significance of excessive iron in inflammatory conditions is largely unknown.We demonstrated that iron depletion with two structurally distinct chelators both induced rapid Caspase-mediated proteolysis of PCBP1 and inhibited the production of GM-CSF in T cells in vitro and in autoreactive T cells in vivo,a phenomenon also observed in primary human T cells.Like PCBP1,elevation of intracellular Iron also promoted GM-CSF m RNA stability and enhanced its 3'UTR activity.We also found higher iron level and PCBP1 expression in activated T cell and at inflammation sites of auto-immune diseases.More importantly,iron overload was able to enhance GM-CSF production in autoreactive T cells in vivo,which was abrogated in the Pcbp1 deficient T cells.,indicating that iron promotes GM-CSF production at least partially through enhancing PCBP1 protein stability.Our study thus demonstrates that PCBP1 is critical for the posttranscriptional regulation of proinflammatory cytokines,and indicates that RBPmediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.Furthermore,iron accumulation may precipitate autoimmune diseases by promoting proinflammatory cytokine production,suggesting a more rational design targeting iron homeostasis will likely lead to a better prognosis of these diseases.