This study established a potential causal relationship between the expression of interferon signalling gene expression and SS, and using multi-omics data, it revealed the interactions and molecular mechanisms between the expression of IFN-associated causal genes, DNA methylation, and inflammatory cytokines. We identified 331 IFN-associated DEGs and integrated SS GWAS with eQTLs/mQTLs of DEGs to identify five IFN-associated SS-causal genes associated: LGALS9, SH2B3, CD40, GRB2, and DTX3L. Meanwhile, colocalization analyses by further integrating inflammatory cytokine GWAS data identified a common genetic effect between SH2B3, LGALS9, CD40 expression, and inflammatory cytokines. In addition, using the same approach of integrating MSG eQTLs with SS and inflammatory cytokine GWAS data, four MSG tissue IFN-associated SS-causal genes, APOBEC3G, IFI27L2, TMEM50B, and SH2B3, were identified as being involved in the interaction between SG gene expression and inflammatory cytokines. Previous research has not thoroughly examined the causal relationship between interferon signalling gene expression and SS, nor the intricate interactions between epigenetic modifications and inflammatory cytokines. Consequently, our study is crucial for identifying IFN-associated causal genes in blood and SG tissues, and for elucidating the complex interactions among interferon signalling, DNA methylation, and inflammatory cytokines in SS.
Increasingly, biomarkers derived from blood tissue are utilized to diagnose and predict SS activity, as well as to uncover the complex etiology and molecular mechanisms of the disease. SMR analysis of blood tissues revealed five potential causal relationships between IFN genes and SS, mediated by epigenomic and transcriptomic regulation. These findings underscore the significant involvement of epigenetic factors and gene expression in SS pathogenesis. Among these genes (LGALS9, SH2B3, CD40, GRB2, and DTX3L), colocalization analysis confirmed the association of LGALS9, SH2B3, and CD40 with inflammatory cytokines.
For instance, SH2B3 (also LNK, lymphocyte adaptor protein) was involved in IFN signalling activities through growth factors and cytokine receptors31,32. Several studies have found that CCL19 associated with a variety of autoimmune diseases41,64–66, is highly expressed in SS blood and salivary glands38,67. CCL19 attracts dendritic cells, and antigen-binding B cells by binding to CCR768,69, causing inflammation in the blood and salivary glands. Furthermore, it has also been shown in large cohorts to correlate with anti-Sjögren’s syndrome-related antigen A (SSA) antibody and IgG levels in SS patients38. IL-2Rβ, a subunit of IL-2R, is involved in the IL-2 signalling pathway in SS. However, elevated levels of IL-2R and impaired IL-2/IL-2R signalling lead to diminished immunosuppression of regulatory T cells (Tregs) in SS patients, correlated with the severity of SS disease46. Enhancement of regulatory T cells by low-dose IL-2 restores the Th17/Treg balance70,71, which is effective and well tolerated in SS patients72. PD-L1, a transmembrane protein, is expressed in myeloid cells and regulated by interferon signalling pathways73. In patients with SS, PD-L1 is up-regulated by IFN/JAK/STAT in SGECs. It serves a dual function, associated with epithelial cell survival and resistance to IFN-mediated apoptosis74. PD-L1 reduces the activation of T cells and IFN-γ production, while also contributing to the activation of SGECs and the persistence of the disease40. In the present study, we confirmed that hypomethylation levels upregulated SH2B3 gene expression, which potentially interacted with CCL19, IL-2Rβ, PD-L1, and TGF-α, thereby increasing the risk of SS development.
LGALS9, also known as Galectin-9, is part of the galectin family and binds beta-galactoside. It is induced by IFNα signalling, upregulated in SS, regulates B-cell activation as a feedback mechanism, and monitors SS disease activity35,75. T-cell surface glycoprotein CD5, a negative co-stimulator in TCR signalling53, shows decreased expression and functionality on peripheral blood lymphocytes of SS patients50, potentially increasing SS risk51. Galectin-9 regulates B-1a cell activation by increasing colocalization with the inhibitory co-receptor CD5, thereby modulating TLR4 signal transduction76. It is well recognized that a significant number of cells within SS lesional tissue express the CD5 cell surface molecule49. CD5 + Breg cells are crucial in restraining the effector Tfh cell response by producing IL-10 during SS development56. IL-10, a critical anti-inflammatory mediator54, is significantly elevated in the peripheral blood of SS patients55, with IL-10-producing Breg cells helping to restrain the effector Tfh cell response in SS56. Additionally, a positive correlation exists between IL-10 levels and ESSDAI, useful for monitoring SS disease activity52. Previous research indicates that the Tim-3/Galectin-9 signaling pathway may regulate the function of CD4 + T cell subsets, impacting IL-10 levels77. Importantly, our study confirms that DNA methylation positively regulates LGALS9 expression, and elevated transcript levels of LGALS9 may increase the risk of SS. Furthermore, we identified a shared genetic effect among LGALS9, CD5, IL-10, and CST5, indicating a link between DNA methylation, LGALS9 expression, these cytokines (CD5, IL-10, and CST5), and SS risk.
CD40, belonging to the tumor necrosis factor (TNF) gene superfamily, encodes proteins serving as receptors on antigen-presenting cells of the immune system78 and are essential in SS for mediating a variety of immune and inflammatory responses79,80. For example, CD40-CD154 mediated T-B cell interactions result in aberrant lymphocyte activation, SG inflammation, and subsequent tissue damage81,82. CD40 gene expression correlates with CCL4, which has been demonstrated in previous studies83. CCL4 levels were significantly elevated in the MSG, submandibular gland, and lacrimal gland, suggesting that it is closely involved in the pathogenesis of SS84–87. In addition, IL-13 release from B cells requires CD40 cross-linking and cytokine signalling88. IL-13 regulates the function of glandular tissue and the recruitment of mast cells to the gland, causing the progression of SS immunopathology89. The present study confirms that the interaction of CD40 expression with inflammatory cytokines (CCL4, IL-10) is involved in the pathogenesis of SS.
SS is a chronic inflammatory autoimmune disease characterized by lymphocytic infiltration of exocrine glands, with a predominance of salivary glands. Using MSG eQTLs (the most relevant tissue type) to study their genetic influence on IFN gene expression may be more relevant than studies in blood. We identified APOBEC3G, IFI27L2, TMEM50B, and SH2B3 as potential IFN-associated SS causal genes in MSG tissues based on SMR analyses, where SH2B3 was also a relevant gene in blood. Nonetheless, the relationship between SH2B3 expression and SS in blood was different from that in MSG, indicating a tissue-specific influence on the pathogenesis of SS. In addition, colocalization analyses of gene expression with inflammatory cytokines confirmed the presence of common genetic effects of these gene expressions (APOBEC3G, IFI27L2, TMEM50B, and SH2B3) with inflammatory cytokines.
APOBEC3G (Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3G), one of the APOBEC3 genes, is a member of the large apolipoprotein B mRNA editing enzyme catalytic polypeptide-like family which plays a critical role in innate immunity90. The APOBEC3 family is highly expressed in macrophages, lymphoid cells, and dendritic cells, and dysregulated APOBEC3 activity has been implicated in genome mutagenesis in cancer90,91. APOBEC3G was notably upregulated in MSG tissues obtained from SS lymphoma patients92, which exhibited a more widespread distribution across myeloid, B-lymphocyte, and T-lymphocyte clusters93. Furthermore, our research colocalized the genetic influences on APOBEC3G expression with those of inflammatory cytokines. Both Flt3L and DNER exhibited shared genetic effects with APOBEC3G expression, indicating possible interactions among this gene, Flt3L and DNER. Interestingly, serum levels of Flt3L are elevated in patients with SS and are associated with high disease activity scores, aberrant B cell distribution, and increased risk of developing lymphoma94,95. Flt3L serves as a predictor of SS lymphoma60, and may prove valuable as a predictive marker for lymphoproliferative disorders in SS61. Elevated levels of Flt3L preferentially enhance the proliferation of type-I interferon-producing plasmacytoid dendritic cells96. Increased levels of Flt3L may trigger autoimmune diseases97. For example, patients with rheumatoid arthritis exhibit increased Flt3L levels in both serum and the synovial fluid of inflamed joints98. Moreover, DNER is associated with inflammation of articular cartilage99. This suggests that the genetic variant regulating APOBEC3G might simultaneously regulate its gene expression as well as the levels of Flt3L and DNER, thereby participating in the pathogenesis of SS.
Our study found that IFI27L2 expression was positively associated with the risk of developing SS and shared genetic effects with NGF-β. IFI27L2 (Interferon Alpha Inducible Protein 27 Like 2, also ISG12B), a member of the interferon-stimulated gene(ISG)12 family, has an important role in the apoptotic properties induced by type 1 interferon100. Notably, IFI27 was selected as a prospective biomarker for SS101. NGF-β has also been reported to have increased expression in SS salivary gland epithelial cells (SGECs), correlating with MSG inflammatory grade62. Thus, we hypothesize that genetic variation regulates IFI27L2 expression and interacts with NGF-β via the interferon signalling pathway, thereby promoting SS pathogenesis. Additionally, this study highlighted a potential interaction between TMEM50B (Transmembrane Protein 50B) gene expression, negatively correlated with SS risk, and IL-1α in SS pathogenesis. IL-1α, one of the Interleukin (IL)-1 family members, is a widely distributed and pivotal pro-inflammatory cytokine63. Studies suggest that inhibiting IL-1 may be a therapeutic strategy worth considering in SS36.
This study has certain limitations. Firstly, although it utilizes GWAS data from FinnGen and UK Biobank, it may not fully capture the genetic and epigenetic diversity of the general population. This limitation could restrict the generalizability of the findings to all SS patients, especially those from varied ethnic backgrounds. Secondly, the cross-sectional nature of the genetic and epigenetic data may not adequately reflect the dynamic changes in gene expression and methylation status, which are crucial for understanding the chronic progression of SS.