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B. Mariotti et al.
   the CD4+CD8+CD16–CD56+CD57+ (CD4 T-LGLs) immunophenotype (Online Supplementary Table S1). We have recently shown that CD8+CD16+CD56–CD57± immunophenotype identifies a subset of patients charac- terized by the presence of STAT3 mutation and/or activa- tion and neutropenia, whereas STAT3 mutations are lack- ing in CD4 T-LGLL patients, usually displaying normal neutrophil counts.5 Confirming our previous observation, high levels of STAT3-YP, detected by Western blot analy- sis (Figure 1B), characterized the CD8 T-LGLL patients, three of whom carry STAT3 mutations (Online Supplementary Table S1). Conversely, no STAT3 mutations (Online Supplementary Table S1) nor constitutive activation (Figure 1B) were detected in CD4 T-LGLs. No variation in the total STAT3 levels between CD8 and CD4 samples was detected (Figure 1B). Moreover, and consistent with our previous observation, CD8 T-LGLL patients are char- acterized by a significantly (P=0.0066) different absolute neutrophil counts (ANC) as compared to CD4 T-LGLL patients, that shows normal neutrophil counts (Figure 1C). Taken together, our data show that the global miRNome clusters with STAT3-activated/CD8 phenotype, that is, in turn, characterized by neutropenia.
miR-146b is differentially expressed in CD8 versus CD4 T-LGLs and inversely correlates with neutropenia
On the basis of the above correlation between the glob- al mature miRNA expression profile and the presence of activated STAT3 (Figure 1), and taking into account that a pathogenic link between CD8 phenotype and STAT3 activation has been demonstrated,5 we hypothesized that a STAT3 activation-dependent, CD8-specific miRNAs expression pattern is in place. To get insights into this immunophenotype-specific miRNome, miRNAs expressed in CD8 and CD4 T-LGLs were subjected to dif- ferential expression analysis. miRNAs showing threshold cycle (Ct) value <32, and Fold Change (FC) value >2 or <0.5 were considered as differentially modulated. Accordingly, twenty-four miRNA emerged as up-regulat- ed and only one miRNA, namely miR-146b, as down-reg- ulated in a statistically significant manner (P<0.05) in CD8 as compared to CD4 T-LGLs (Online Supplementary Figure S1 and Online Supplementary Table S3).
Based on our recent data indicating that high level of STAT3 activation correlates not only with CD8 T-LGLs phenotype, but also with the presence of symptomatic disease, mostly as a consequence of neutropenia,5 all the miRNAs differentially expressed in CD8 and CD4 T-LGL (Online Supplementary Table S3) were analysed for correla- tion with the ANC. Correlation analysis highlighted only two miRNAs, namely miR-630 and miR-146b, whose expression correlated with ANC (P=-0.886, P=0.033 and P=0.866, P=0.030, respectively) and simultaneously with the levels of STAT3-YP (P=1.00, P=0.003 and P=-0.866, P=0.033, respectively) (Online Supplementary Table S4). None of the remaining differentially modulated miRNA correlated with the absolute neutrophil count in a statis- tically significant manner (Online Supplementary Table S4). RT-qPCR single assay on additional T-LGLL patients con- firmed the downregulation of miR-146b expression in CD8 T-LGLs, compared to CD4 T-LGLs (P=0.018) or to T lymphocytes from healthy donors (P=0.024) (Figure 2A). Accordingly, miR-146b was found to inversely correlate simultaneously with the levels of activated STAT3 (P=- 0.846, P=0.0005) and with ANC (P=0.707, P=0.0012)
(Figure 2B). Consistently, in the additional T-LGLL patients a significant correlation between STAT3 activa- tion and neutropenia was confirmed as well (P=-0.867, P=0.0003) (Figure 2B, right panel). On the contrary, miR- 630 was confirmed as differentially expressed in CD4 T-LGLs as compared to CD8 T-LGLs, but the correlation with ANC was not validated (not shown).
To gain insights into the cause-effect relationship between the degree of STAT3 activation and the lack of miR-146b expression, CD8 T-LGLs were incubated with non-toxic doses of the STAT3 inhibitor STATTIC and the level of miR-146b expression was analyzed. Figure 2C shows that blocking STAT3 activity in CD8 T-LGLs unleashed miR-146b transcription, thus demonstrating that suppression of miR-146b expression in CD8 T-LGLs is secondary to constitutive STAT3 activation.
STAT3 is a well-known transcriptional activator for many genes,20 and it has also been reported to inhibit gene expression by promoting methylation of the target genes promoter.21-23 In normal tissues STAT3 is reported to activate miR-146b transcription.24,25 However, in several systems miR-146b promoter methylation has been shown to prevent miR-146b expression, even in the pres- ence of constitutively activated STAT3.26,27 According to the publically available methylome data (https://genome- euro.ucsc.edu/cgi-bin/hgTracks?db=hg19&last VirtModeType=default&lastVirtModeExtraState=&virtMode Type=default&virtMode=0&nonVirtPosition=&position=chr1 0%3A104196181104196428&hgsid=230688991_Xz5zjxAj b58tIT5oL9i5MkaweCLp), four cytosine located upstream (-570bp, -63bp, -56bp, -26bp) and two located down- stream (-71bp, and -273bp) the miR-146b TSS (Online Supplementary Figure S2) have been identified as differen- tially methylated in different cell lines. On these bases, we analyzed the level of miR-146b promoter methylation in CD8 and CD4 T-LGLs that are characterized by the presence or absence of activated STAT3, respectively. Our results showed a significantly higher level of 5meC in the regions -687bp/-496bp (+141.21%, P<0.01) and - 149bp/+98bp (+58.46%, P<0.05) upstream miR-146b TSS in CD8 T-LGLs compared to CD4 T-LGLs (Figure 3A). As a control, methylation of the region downstream (+44bp/+315bp) miR-146b TSS in CD8 and CD4 T-LGL was comparable (Figure 3A). Inhibition of methyl-trans- ferase activity with 5-aza-2-deoxycytidine (DAC) restored the expression of the miR-146b primary tran- script (pri-miR-146b) in CD8 T-LGLs (Figure 3B), further proving that miR-146b promoter methylation prevents miR-146b expression in CD8 T-LGLs. Remarkably, STAT3 inhibition led to a statistically significant reduc- tion of the level of DNMT1 expression, thus suggesting that constitutive STAT3 activation may lead to miR-146b promoter methylation (Figure 3B, left panel). Collectively, these data suggest a cause-effect link between the lack of miR-146b expression and constitutive STAT3-activation in CD8 T-LGLs.
FasL mRNA-stabilizing protein HuR is target of miR-146b
To gain insights into the molecular mechanisms under- lying the correlation between miR-146b expression and ANC (Figure 2B, central panel), we focused our subse- quent analysis on FasL. In fact, increased release of FasL has long been reported as one of the most relevant factor in the pathogenesis of neutropenia in LGLL patients.9,12,28
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