PTEN Expression, Regulation And Dysfunction In The Pathogenesis Of Systemic B Cell Abnormalities

ObjectivePTEN is involved in maintaining normal B cell function. Since B cell overactivity is characteristic of systemic lupus erythematosus (SLE), we sought to determine whether abnormalities in PTEN might contribute to increase B cell responsiveness in this disease.MethodsThis study recuited26newly-diagnosed SLE patients and24sex and age matched health volunteers as health controls(HC). Peripheral mononuclear cells were isolated from collected anti-coagulated blood. Puried B cells were sorting by micro beads.Flow cytometry and real time quantitative PCR were used for detecting the level of PTEN from each subsets of B cells of HC and SLE patients.The relationship between PTEN levels and the disease activity and severity of SLE was analysised.Purified B cells were incubated with human rIL-2, rIL-4, rIL-6, rIL-21, rIL-10, rCD40L or anti-IgM (10μg/ml; eBioscience) alone or in combination for72hr and after that the PTEN level was detected by flow cytometry.The proportion and surface activated marker of B cells were determined by flow cytometry.Results1. PTEN was expressed by all B cell populations in HC and the density of PTEN expression was the greatest in CD19+IgD+CD38int/hi B cells, a circulating immature B cell subset, and IgD-CD38hi plasma cells. 2. Compared to HC, the expression of PTEN was diminished in each SLE B cell population except IgD-CD38low/-memory B cells. In addition, PTEN mRNA was also decreased in SLE B cells.3. The level of PTEN in CD19B cells was inversely correlated with disease activity as determined by SLEDAI score (r=-0.5, p=0.03), and also directly correlated with the serum C3level, but not with the titer of anti-dsDNA autoantibodies. Notably, PTEN reduction in CD19B cells was significantly decreased in SLE patients with nephritis manifested by proteinuria (p<0.05).4. The capacity of IL-21to induce PTEN expression in B cells of HC was found by flow cytometry and confirmed by western blotting. Importantly, neither IL-21alone nor CD40L plus anti-IgM, nor the three in combination stimulated PTEN protein up-regulation in IgD+B cells in SLE patients. In contrast, PTEN mRNA level was up-regulated by IL-21stimulation in both SLE and HC B cells., suggesting that the defective IL-21-mediated PTEN induction in SLE occurred at the post-transcriptional stage.5. SLE patients had significantly higher proportions of CD19lgD CD38int/hi immature B cells than HC (p=0.01). These immature B cells in SLE patients had a greater expression of CD86and CD95but less PTEN compared to HC, suggesting that down-regulation of PTEN was associated with an increasing proportion of immature B cells with a more activated phenotype in SLE patients.ConclusionSLE B cell showed decreased levels of PTEN and the decrease was associated with increases in disease activity and severity, but not anti-dsDNA autoantibody titers in SLE patients. The capacity of IL-21to induce PTEN was defect in SLE while IL-21stimulation of upregulation of PTEN mRNA was intact. Together, these data suggesting that the defective IL-21-mediated PTEN induction in SLE occurred at the post-transcriptional stage.Defective PTEN expression, regulation and function contribute to B cell hyper-responsiveness in systemic lupus erythematosus. ObjectiveSLE is an autoimmune disease characterized by abnormally B cells activation and proliferation. We found unique abnormalities in PTEN expression in SLE B cells. The nature of the abnormal regulation of PTEN suggested a defect in post-transcriptional regulation of this molecule. Abnormal microRNA expression may contribute to this defective regulation of PTEN. Our lab previously found that miR-7expressed abnormally high in SLE patients and identified PTEN as one of the targets of mir-7, which was proved by dual luciferase report gene assay. In this study, we sought to determine whether abnormalities in miRNAs might contribute to decrease PTEN expression of B cell in this disease.MethodsThis study recuited20newly-diagnosed SLE patients and15sex and age matched health volunteers as health controls(HC). Peripheral mononuclear cells were isolated from collected anti-coagulated blood. Puried B cells were sorting by micro beads.Taqman real time quantitative PCR were used for detecting the level of miR-7、 miR-21and miR-22from B cells(ex vivo) of HC and SLE patients.Purified B cells were incubated with human rIL-21for48hr and after that the level of miR-7、 miR-21and miR-22were detected by Taqman real time quantitative PCR.B cells were transfected with pre-miR-7、 anti-miR-7or control microRNA electroporately. The expression levels of miR-7was detected by Taqman RT-PCR. Transfected cells were cultured for48hr. The level of PTEN mRNA was detected by SYBR Green RT-PCR and PTEN level was detected by flow cytometry.B cells of SLE patients were transfected with miR-7antagomir or negative control. Transfected cells were cultured for48hr. The expression levels of PTEN was detected by flow cytometry.Results1. The ex vivo expression of miR-7,21and22in SLE B cells was significantly higher than that of HC B cells (p=0.03,0.0001, and0.0001, respectively).2. Moreover, IL-21significantly up-regulated the expression of miR-7and22in SLE B cells as well as in B cells from HC3. Peripheral B cells were electroporated with pre-miR-7or an miR-7antagomir that resulted in overexpression miR-7or inhibition of the function of mir-7.4. Inhibition of miR-7function by electroporated with anti-miR-7increased PTEN mRNA levels in SLE B cells (p<0.05)5. Overexpression of miR-7reduced PTEN expression in HC B cells and inhibition of miR-7function increased PTEN levels in SLE B cells (p<0.05)。6. The expression of PTEN in miR-7antagomir-transfected SLE B cells was significantly up-regulated, as compare to that in negative control transfected SLE B cells (p=0.03).ConclusionIncreased expression of several miRs, including miR-21,22and7can result in defective expression of PTEN in SLE. The decrease in PTEN expression in SLE B cells and their heightened responsiveness can be reversed by an antagomir of miR-7, pointing to this miR as playing a dominant role in the hyperactivity of lupus B cell. Therefore, decreased expression of PTEN regulated by miR-7and perhaps miR-21and22, contributes to B cell hyper-responsiveness and disturbed B cell homeostasis in SLE ObjectiveB cell development, activation and immune tolerance is an interrelated process under the control of the signal transmitted by the B cell receptor (B cell receptor, BCR). The activation of the PI3K signaling is an essential component of the BCR-induced signal. The BCR signal regulates the transmembrane calcium (Ca2+) and plays an important role in B cell proliferation, differentiation and apoptosis. Although the initial Ca2+signal is directly induced by a phospholipase C-γ dependent inositol-1,4,5-trisphosphate-mediated mechanism, PI(3,4,5)P3plays a critical amplifying role. PI3K pathway in maintain the B cell homeostasis and survival, as well as the differentiation of B cells. Interleukin-21, produced mainly by activated CD4+T cells, is an essential cytokine involved in the differenciation of B cell. The upregulation of AID, blimp1promotes B-cell differentiation. In this part of study, we will focus on whether PTEN plays a role in the dysregulation of calcium influx and the relationship of IL-21and B cells proliferation and differentiation. Novel pathways of PTEN regulation by microRNAs and the regulation of these microRNAs by IL-21would be identified, and their role in altering B cell responsiveness in SLE would be delineated..MethodsThis study recuited21newly-diagnosed SLE patients and15sex and age matched health volunteers as health controls(HC). Peripheral mononuclear cells were isolated from collected anti-coagulated blood. Puried B cells were sorting by micro beads.Flow cytometry were used for detecting the expression of phospho-STAT3 and phospho-Akt after fixation, permeabilization, and labeling with specific mAbs (BD PhosflowTM) in f B cells of HC and SLE patients.Freshly isolated B cells or B cells that had been transfected with human hsa-miR-7-5p (MIMAT0000252) antagomir(or its negative control) were incubated with Fluo-4AM analyzed by flow cytometry to record the intracellular Ca2+.Real time quantitative PCR were used for detecting the level of FOXO1, BLIMP1and AID from B cells(cultured with medium, rIL-21, rCD40L+algM and rCD40L+algM+rIL-21) of HC and SLE patients.Freshly isolated B cells or B cells that had been transfected with human hsa-miR-7-5p (MIMAT0000252) antagomir were cultured with IL-21for5days. The proportion of plasma cells were determined by flow cytometry by labeling CD19, CD27, CD38and CD138. Flow cytometry were used for detecting the level of PTEN from each groups of B cells as well.Results1. IL-21induced Akt phosphorylation in the absence of CD40L plus anti-IgM stimulation in SLE B cells(p=0.008) but not HC(ns). IL-21reduced Akt phosphorylation stimulated by CD40L plus anti-IgM in HC B cells(p<0.05). As expected from the lack of induction of PTEN by IL-21in SLE B cells, IL-21failed to suppress Akt phosphorylation induced by CD40L plus anti-IgM in lupus B cells (p>0.05).2、 IL-21induced STAT3phosphorylation in the presence or absence of CD40L plus anti-IgM stimulation in both HC and SLE B cells.3、 The baseline [Ca2+]i was also measured and there was a significant (p=0.02) difference in the resting [Ca2+]i of SLE and HC B cells. In addition, anti-IgM-induced higher [Ca2+]i responses(p=0.001) in B cells from SLE patients than B cells from HC.4、Importantly, disruption of mir-7activity with its antagomir in SLE B cells resulted in significant (p=0.0004) reduction of the calcium flux upon BCR stimulation to the level noted in HC B cells.5、 The mRNA levels of FOXO1, BLIMP1and AID from the four cultured groups(medium, rIL-21, rCD40L+aIgM and rCD40L+aIgM+rIL-21) of SLE B cells were abnormal in SLE B cells while compared to that of HC.6、 Furthermore, IL-21significantly (p=0.02) increased the proportion of plasmablasts (CD19+CD27+CD38hi) and plasma cells (CD138+CD38hi) from SLE B cells in the absence of BCR signaling and T cell help (Fig.7B). This effect of IL-21in SLE could be reversed by the miR-7antagomir, which upregulated IL-21-induced PTEN in SLE B cells.7、 MiR-7antagomir had no effect on IL-21-induced Stat3phosphorylation.ConclusionIncreased expression of several miRs, including miR-21,22and7can result in defective expression of PTEN and enhanced BCR signaling. Notably,2of the miRs, miR-7and22are regulated by IL-21, and appear to provide a negative feedback loop in normal B cells that can limit BCR signaling. The decrease in PTEN expression in SLE B cells and their heightened responsiveness can be reversed by an antagomir of miR-7, pointing to this miR as playing a dominant role in the hyperactivity of lupus B cell. Therefore, decreased expression of PTEN regulated by miR-7and perhaps miR-21and22, contributes to B cell hyper-responsiveness and disturbed B cell homeostasis in SLE.