| BackgroundB cells perform several immunological functions but have been considered mainly as positive regulators of immune responses and central contributors to the pathogenesis of immunerelated diseases because of their ability to produce antibodies. However in the past decades, investigators gradually realized that B cells may embrace unique immune suppressive abilities, tremendous works had been conducted to ascribe the features of regulatory B cells, a functionally named B cell subset which were proposed to negatively regulate immune responses and keep the fine equilibrium required for immune homeostasis.As a result of lacking specific markers, Bregs are functionally defined, currently identified mainly by their ability to secrete IL-10. Though various mechanisms had been reported to be applicable for Breg to exert their immune suppressive function, IL-10-depending way is still the most important one. Bregs with IL-10 defect failure to exert their immune regularly functions in vitro and in vivo. And the absence of IL-10-producing Bregs is implicated in the etiology of numerous immune related diseases. Thus, according to the central role of IL-10 in knowing how IL-10 production is regulated in B cells will be of great scientific significance.IL-10 is a key orchestrator of the immune system that has been shown to be anti-inflammatory in many model systems. It mediates a plethora of immune regulatory events and dysregulation of IL-10 leads to various immunological diseases..As clinical application of extrinsic IL-10 is handled for its short half-life and insufficient mucosal penetration, making use of endogenous IL-10 seemed to be a alternative way. Though IL-10 could be derived from various cell types,it has been reported that B cells seem to be the main source of IL-10.Therefore, knowing how IL-10 expression is regulated in B cells would be of great practical significance.Previous researches had demonstrated that IL-10 expression is mainly regulated on the transcriptional and posttranscriptional levels.Although NF-kB and Blimp1 have been found to regulate IL-10 transcription in B cells, it has been shown that they also influence other biological process of B cells, however the availability of its protein is significantly determined by posttranscriptional mechanisms. It has been demonstrated that via the binding on AU-rich element (ARE) locus in 3’-untranslated region (3’UTR), IL-10 mRNA could be regulated by mico-RNA and RNA binding proteins in different cells. But how IL-10 is posttranscriptionally regulated in B cells is unknown.By searching differentially expressed gene between IL-10-producing cells and non-IL-10-producing cells with Affymetrix microarrays analysis, we focus on genes participate in posttranscription regulation. RBM47 is one of our candidates.as unregulated gene in IL-10 producing B cells encoding a protein contains three classical RNA recognition motifs involved in mRNA sticking. Though it has been demonstrated that Rbm47 could exert regulatory function on mRNA molecules via various way and participate in several biological progress, its influence on immune system is still speculative.Manipulated by different means, gene modified immune cells, including T cell, DC cell, NK cell and mesenchymal stem cells, had been applied to treat various kinds of diseases. Now that Bregs have elicited the interest of a broad spectrum of immunologists and clinicians, peoples tried to modify B cells to improve their immune suppressive effect. According to the crucial role of IL-10 for B cells’ regulatory effect, and considering IL-10 is unstable both on protein and mRNA level, we wonder if it is a feasible way to facilitate the regulatory function of B cells by manipulating transcriptional regulators who can influence IL-10 production in B cells.Object:This study is designed not only to figure out the influence of Rbm47 on IL-10 producing in B cells and the underlying mechanism, but also to determine if charging the expression of posttranscription regulator, as Rbm47, could be a feasible way to improve the regulatory function of B cells.Methods and results:1. We sorted IL-10-producing and IL-10-nonproducing B cells by IL-10 antibody labeled micro-beads with B10 separating kit, and take a Affymetrix microarrays analysis to explore the molecular that might be relative to IL-10 expression in B cells. And we focus on RBM47, a gene encoding a RNA-binding protein, for its high expression level in IL-10-producing B cells in compare with IL-10-noproducing B cells.2. We employed IL-10-GFP-reporter mice and LPS induction to further confirm Rbm47 expression profile in B cells. The result showed that Rbm47 expression increased during IL-10 producing induction, and the Rbm47 overexpression arises in IL-10 producing B cells specifically.3. We performed infection of Rbm47-specific shRNA-expressing lentivirus in SP2/0 cells followed by puromycin selection and detected IL-10 expression change by qPCR and ELISA analysis. The results showed that IL-10 expression significantly declined with Rbm47 knockdown, both on mRNA and protein level.4. By performing luciferase reporter assay and and RNA-binding protein immunoprecipitation assay, we demonstrate that Rbm47 may directly interact with IL-10 mRNA but not binds to promoter of IL-10.5. To evaluate the influence of Rbm47on the steady of IL-10 mRNA in B cell, we employed actinomycin D to stop transcription Total RNA was harvested after 0,15, 30 and 45 minutes and was subjected to q-PCR. As the result revealed that knockdown of Rbm47 accelerate the decay of IL-10 mRNA, meanwhile the overexpression of Rbm47 delayed the decay of IL-10 mRNA.6. To further demonstrate the physiological significance of Rbm47 in regulating IL-10 production in B cells, we overexpressed Rbm47 in primary B cells by employing Rbm47-expressing lentivirus. The qPCR and FACS analysis showed that Rbm47 overexpression promote IL-10 production in B cells.7. To evaluate the influence of Rbm47 on the regulatory function of B cells in maintenance of CD4+Foxp3+T cells, B cell infected with empty-GFP-expressing or Rbm47-GFP-expressing retrovirus were purified by sorting and then co-cultured with CD4+T cells. The result showed that Rbm47 overexpressing B cells facilitate Treg induction in vitro.8. By employing IL-10-GFP-reporter mice in our preliminary experiment, we found deduced abundances of IL-10 secreting B cells in the mLN in mice fed with 3% DSS.Meanwhile we found that mLN B cells in mice suffered DSS-induced colitis possess an remarkable decrease of Rbm47 and IL-10 expression.9. We further examined the effect of Rbm47 on the regulatory function of B cells in vivo by using DSS-induced colitis model. C57BL/6 mice were injected with PBS, B-Rbm47, or B-Ctrl via tail veil on the day 1 and day3 since from of the ingestion of 3% DSS. The body weights were recorded daily for 7 days, and the mice were subsequently sacrificed to assess the resulting colonic damage. The result showed that Rbm47-overexpressing B cells adoption release severity of urgent colitis.Conclution:This suggest that Rbm47 could positively regulate IL-10 production on posttranscriptional level, and the immune suppressive function of B cells could also be promoted by ruling the expression of this posttranscriptional regulator. |
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