In the present study, we conducted transcriptome analysis of Bcell subpopulations. First, we found that expression ofLINC00487 was upregulated in B cell subsets derived frompatients with pSS. Further, LINC00487 expressionsignificantly correlated with disease activity and was induced byIFNα stimulation. Second, using WGCNA, we identified several keynetworks and hub genes in the B cells of patients with pSS.
IFN signalling is a central component of the pathogenesis of pSS[28]. To our knowledge, this is the first study to showupregulation of IFN signalling in B cells of patients with pSSaccording to developmental subsets. Type 1 IFN signalling plays acrucial role in the development of autoreactive B cells in a mousemodel of autoimmune disease [5]. Principal component analysis usinggene sets for WGCNA demonstrated that Bm1/naïve B cells of pSS wereclearly distinguished from those of HC and showed a trend ofexpression patterns closer to the more highly differentiatedsubset. These results suggest that molecular dysfunction, such asdisruption of peripheral tolerance, might start at an early stagein the maturation of B cells. Indeed, the frequencies of naïve Bcells expressing autoreactive antibodies are significantlyincreased in patients with pSS [29].
Interestingly, expression of the HLA class II gene wasupregulated. GWAS studies of pSS found associations ofHLA-DQA1 and HLA-DQB1 loci [6,7]. Further, IFNαinduces the expression of HLA-DQA1 [30]. In patients withpSS, HLA-DQA1 and HLA-DQB1alleles are associated with higher concentrations of anti-SSA andSSB antibodies [31]. These findings suggest that aberrantinteractions among IFN signalling and HLA class II genes maytrigger the breakdown of B cell tolerance, leading to thedevelopment of pSS.
LINC00487 was upregulated in all B cell subsets of pSSand its expression significantly correlated with the diseaseactivity scores of pSS and ISGs, although ESSDAI score of ourpatients skewed toward the low end. Moreover, we found that IFNαwas an upstream regulator of LINC00487 in B cells. Thesequence of LINC00487, which belongs to the class of longintergenic non-coding RNAs and resides on human chromosome 2, isatypically long (> 40,000 bases). Although LINC00487 isone of the hub genes in the normal development of human B cells[32], many of its properties, including its function, areunexplained. Further, there is no ortholog or paralog of this genein species other than humans that may provide clues to its functionin human cells. However, according to AceView, one of threetranscriptional variants derived from LINC00487 haspotential to encode a protein in silico prediction [33].
Four genomic locations are considered candidate enhancers ofLINC00487 transcription. The targets of the regulators ofLINC00487 overlap with those of other ISGs [34]. Moreover,referring to the public transcriptome database of microarrayanalyses of healthy humans, expression of LINC00487 ishigher specifically in centroblasts and centrocytes of the germinalcentre [35]. IFNα promotes the autoreactivity of B cells viagerminal centre pathways [5]. Further, LINC00487expression is upregulated in the subgroup of diffuse large B-celllymphoma with molecular characteristics of germinal centre B cells,compared with other subgroups, and is associated with the efficacyof B cell depletion therapy [36]. Therefore, our study suggeststhat upregulation of LINC00487 expression in all B cellsubsets may reflect or regulate the enrichment of a germinalcentre-like reaction by IFNα from an early stage of B celldevelopment, leading to B cell autoreactivity in patients withpSS.
Gene co-expression analysis revealed an aberrant network in Bcell subpopulations. The top significant upstream regulator of thegrey module of pSS, which was associated with clinical diseaseactivity score and enriched in the early stage of B celldevelopment in pSS, was the gene encoding T-cell acute lymphocyticleukemia protein 1 (TAL1). B cell development isstringently controlled by stage-specific transcription factors. Thetranscription factor TAL1 regulates genes such as IKAROSfamily zinc finger 3 (IKZF3), which is one of hub genes inthe grey module of pSS (Figure 5C). IKZF3 is alineage-specific transcription factor that is important in theregulation of B cell proliferation and development [37].
In the pre-GC B cells-associated module of pSS, SOX4was identified as an upstream regulator and a hub gene. In mice,SOX4 regulates the differentiation of early-stage B cellsby activating the expression of Rag1 and Rag2[38]. Further, SOX4 contributes to the formation ofectopic lymphoid-like structures via promoting CXCL13, which isligand of CXCR5 on naïve B cells and critical for migration intolight zone of germinal centre undergoing somatic hypermutation,production fromPD-1hiCXCR5–CD4+ T cells[39]. Additionally, in proteome analysis, CXCL13 positivelycorrelates with the disease activity score and serum IgG levels ofpatients with pSS [19]. Further study is needed to explore the roleof SOX4 in mature B cells.
Further, we identified miR-21 as an upstream regulator of theyellow module of pSS. Although miR-21 is upregulated in peripheralblood mononuclear cells of pSS [40], the present study is the firstto propose its involvement in pSS via B cell dysregulation.Interestingly, miR-21 regulates the immune response of memory Tcells via induction of transcription networks, such asSOX4 [41], implying that the interaction between miR-21and SOX4 may also affect function of B cells.
pSS is enormously heterogeneous disease from the molecular pointof view. In WGCNA, despite filtering out genes with high variationin each cell subpopulation, module expression showed the strongvariation among samples in the same group, in particular Bm1 subset(Figure 4B). This finding suggested that there were differentregulatory mechanisms within each subgroup, although it wasinformation that couldn’t be analyzed due to the limited samplesize.
The current study suffers from several limitations. First,patients in our cohort have low disease activity/severity. Becausethere were few patients with high disease activity beforeimmunosuppressive treatment, it was difficult to include suchpatients in this study. Therefore, correlation between expressionof LINC00487 and disease activity score is needed to bevalidated in other cohort including patients with high ESSDAIscores. Second, healthy controls were younger than the pSSpatients, and this could be a confounding variable. Third, samplesize was limited. To overcome limitation about small sample size,we validated by qPCR using another cohort, supporting resultsderived from microarray analysis. However, regarding WGCNA, wecouldn’t include subjects enough to replicate by qPCR.