Sjögren's syndrome (SS) is characterized by chronic inflammation and is manifested by impaired function of the exocrine glands, and mononuclear cell infiltrates surrounding the ducts and replacing the secretory units of the involved glands[21]. Genetically predisposed individuals might suffer from autoimmune-mediated tissue injury due to immunologically-activated or apoptotic glandular epithelial cells that expose autoantigens. It has been reported that several immune mediators are altered, such as a decrease in B-cell activating factor expression and an increase in T-helper cell activation[22]. Nine pSS-related and inflammation-associated DEGs were identified in the present study, namely LY6E, EIF2AK2, IRF7, TNFAIP6, RTP4, IL15, CXCL10, LAMP3, and CCL2 Furthermore, functional enrichment analysis revealed that the derived intersecting genes mainly involve the innate antiviral immunity process and inflammatory-related signaling pathways.
Evidence has indicated that viral infections alter the clinical manifestations of various autoimmune diseases. Viral-induced autoimmunity occurs in a variety of ways, including molecular mimicry, epitope spreading, bystander activation, and immortalization of infected B cells. On the other hand, protective effects can be achieved by suppressing autoimmune phenomena through regulatory immune responses[23]. Our functional enrichment analysis revealed the nine pSS-related and inflammation-associated DEGs involved in multiple virus infections, which were reported in previous research. Influenza viruses can induce autoimmunity by several mechanisms, such as causing T cell bystander activation and direct beta cell damage in diabetes[24, 25]. Research about pSS patients immunized with an H1N1 influenza vaccine showed pSS patients developed higher H1N1 IgG titers than healthy controls after inoculation, and off-target B cells also produced increased antibody levels against Epstein-Barr virus (EBV) and autoantibodies[26]. EBV infection is suspected of playing a central role in the pathogenesis of systemic autoimmune diseases[27]. pSS patients showed positive antibodies to EBV early antigens diffuse (anti-EA-D) more frequently and at higher levels than healthy individuals[28, 29]. This reactivity was associated with positivity for anti-Ro/SS-A and anti-La/SS-B antibodies. EBV-encoded small RNA (released from EBV-infected cells) was demonstrated to form a virus complex with La/SS-B protein or reactive environmental pollutants, consequently inducing IFN-1 production, which triggers SS[30, 31].
Toll-like receptors (TLRs) and Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) constitute distinct families of pattern-recognition receptors (PRRs) that sense nucleic acids derived from viruses and trigger antiviral innate immune responses[32, 33]. Following virus recognition, TLRs and RLRs recruit specific intracellular adaptor proteins to activate signaling pathways. This process leads to the activation of NF-kappaB, MAP kinases, and IRFs that control the transcription of genes encoding type I interferon and other inflammatory cytokines to eliminate viruses[34]. Our results found the the pSS-related and inflammation-associated DEGs involved in these two PRR-related signaling pathways, suggesting the viral defense response process may be associated with pSS. Previous studies have shown that TLRs play an essential role in the pathogenesis of pSS[35, 36]. They elevated in salivary tissue[37] and in the peripheral blood of pSS patients[38]. Emerging data indicate that damage-associated molecular patterns (DAMPs) may be significant drivers of chronic and unremitting inflammation in pSS, although the ligands activating TLRs in pSS remain unknown[39, 40]. Activating TLR signaling cascades likely reduce local and systemic inflammation, as shown in an animal study[41]. RLR-mediated signaling deregulation is also reported to be associated with disorders of the autoimmune and autoinflammatory responses[42]. Upregulated expression of DDX58/RIG-I was found in the plasmacytoid dendritic cells (pDCs) of IFN-positive pSS patients and decreased in IFN-negative pSS patients[43]. Jang et al. found that miR-1248 activates the RIG-I pathway by acting as a ligand of this sensor leading to IFN production in phSG cells cultured from pSS patients[44].
We found several pSS-related inflammation-associated DEGs functions were related to immune and inflammation signaling pathways. NOD-like receptor family, the pyrin domain containing 3 (NLRP3) is a crucial player in regulating host immune responses to infection and cell stress, its dysregulation is associated with the pathogenesis of rheumatic disease[45], and it was also found highly expressed in pSS patients than control[46]. The NLRP3 inflammasome can be triggered by the P2X7 receptor (P2X7R), leading to acute inflammatory responses. Baldini et al. proposed the P2X7R-inflammasome axis as a novel potential pathway in both pSS exocrinopathy and lymphomagenesis and seemed to involve in the pathogenesis of lymphoproliferative malignancies[47].These results suggested that NLRP3 inflammasome-mediated inflammation might be implicated in the pathogenesis of pSS. Interleukin-17 (IL-17)is a multifaceted cytokine with a well-recognized role in immune surveillance at mucosal and barrier surfaces[48]. Previous research suggests that the IL-17 axis plays a pivotal role in the pathogenesis of several autoimmune disorders, including pSS[49, 50]. Studies have demonstrated that IL-17 was overexpressed in the salivary glands (SGs)[51],serum[52], plasma[53] and tears[54] of pSS patients, and IL-17 mRNA levels in MSG biopsies seemed to be related to the degree of inflammation[53, 55]. Different IL-17 family members may play several pathogenetic roles in pSS. According to a recent study, IL-17F production in pSS patients is associated with a higher level of autoantibodies and EULAR SS disease activity index (ESSDAI) than IL-17A production in pSS patients[56].
Besides, four genes (LY6E, EIF2AK2, IL15, and CXCL10) were authenticated as inflammation-associated pSS biomarkers, and the reliability of them in discriminating pSS samples from normal samples was also demonstrated, suggesting a potential clinical diagnostic value. Functional enrichment results and immune infiltration analysis pointed to the involvement of the four genes in the immune process and inflammation-related pathways in pSS. The Lymphocyte antigen 6E (LY6E) protein belongs to the Ly6/uPAR family of plasminogen activator receptors and is known as one of the IFN type I response genes. Recent studies have reported its essential role in immunological regulation, T cell physiology, oncogenesis, and viral infection [57]. Our study found higher LY6E levels in the peripheral blood of pSS patients, which has been proven by previous clinical studies [58–60]. These findings may reveal the importance of the peripheral blood LY6E levels and the monocyte IFN type I signature in pSS patients. The eukaryotic translation initiation factor 2-α kinase 2 (EIF2AK2) gene locates on chromosome 2 and encodes modifying protein kinase R (PKR, interferon-induced, double-stranded RNA-activated protein kinase)[61]. Marc et al. performed a series of studies on the activation of B-cell-activating factor (BAFF) in salivary gland epithelial cells (SGECs, the resident targeted cells of autoimmunity in pSS), and found PKR may be an essential target for inhibiting BAFF following the induction of innate immunity in pSS patients[37, 62, 63]. Interleukin-15 (IL-15) is a crucial regulatory inflammatory cytokine that is up-regulated in autoimmunity disorders[64, 65]. Previous studies revealed a higher IL-15 expression level in the peripheral blood of pSS patientss[66], which is consistent with our results. Moreover, a similar action was observed in salivary gland (SG) tissues[67, 68]. Sisto et al. demonstrated the number of IL-15(+) cultured SGECs was significantly higher in cells derived from pSS patients, which suggested an intensified epithelial expression of IL-15 in salivary glands[69]. Their following research suggested that TLR2 activation is involved in the induction of IL-15 production by pSS SGECs and promotes inflammation through NF-κB activation[70]. C-X-C motif chemokine ligand 10 (CXCL10) protein is categorized functionally as a Th1-chemokine, and its secretion is regulated by interferon (IFN)-γ[71]. The serum and/or tissue expressions of CXCL10 increased in various autoimmune diseases[71–74]. A study that assessed CXCL10 plasma levels in pSS patients showed that the ratio of full-length (active) CXCL10 to truncated DPP4-truncated (inactive) CXCL10 was significantly increased and provided the highest correlation with disease activity[75]. Elevated CXCL10 levels were also found in the salivary gland of pSS patients, which were associated with decreased circulating CXCR3 + helper cells, suggesting facilitating their concerted migration[76].
The identified miRNAs in the present study exhibited consistency with other research on autoimmune or immune-mediated related diseases. The miR-26 expression level was downregulated in multiple sclerosis (MS) patients[77]. The neuroregulatory miRNA miR-9-5p was significantly upregulated in the peripheral blood samples of HLA-B27(+) radiographic axial spondyloarthropathy (rad-AxSpA) patients[78]. Immuno-miRNAs miR-21-5p and let-7f-5p were significantly elevated in the serum of patients with acetylcholine receptor myasthenia gravis (AChR+-MG) [79–82], and miR-21-5p was also upregulated in type 1 autoimmune pancreatitis (AIP) [83] and psoriatic arthritis (PsA)[84]. Kim et al. found significantly downregulated expression of miR-30d-5p in the tear samples of pSS patients[85]. These miRNAs may be involved in disease pathogenesis via immune-related processes.
Furthermore, drug-gene interaction pairs were predicted, identifying IL15 and CXCL10 as the potential gene targets of these drugs. The predicted drugs included glucocorticoids and immunosuppressive drugs, which have been effectively applied for the treatment of autoimmune diseases. Cevimeline has already been proved to be effective as a topical therapy for pSS patients. It was associated with improvement in xerostomia and xerophthalmia[86]. Reina et al. found atorvastatin inhibited the production of IL-1β, PGE (2), and MMP-3 in rats’ submandibular glands treated with anti-M (3) peptide IgG purified from SS patients, suggesting that atorvastatin may exhibit an anti-inflammatory effect in SS[87]. Sirolimus is an effective treatment option for pSS, as it restores the PD-1 + ICOS + Tfh:CD45RA-Foxp3 high activated Tfr cell balance[88]. Methylprednisolone is reported to apply in pSS combined with other clinical symptoms and comorbid diseases[89–91]. Zidovudine was effective and well tolerated in pSS patients with a significant improvement in all subjective manifestations[92]. The effects of the other screened drugs were verified for many other diseases such as Parkinson's disease [93], inflammatory bowel diseases[94, 95], and colorectal cancer[96]. Nevertheless, additional studies and clinical trials are required to identify and explore effective drugs for treating pSS in the future. This study utilized multiple databases to provide new insights into pSS pathogenesis and treatment and identified conventional drugs that may have new indications in the future.
In conclusion, 4 hub genes (LY6E, EIF2AK2, IL15, CXCL10) that might be potential diagnostic biomarkers of pSS in peripheral blood were identified by bioinformatics analysis, and their expression were validated by RT-qPCR. Furthermore, the molecular mechanisms of these genes were preliminarily explored by generating a lncRNA-miRNA-mRNA regulatory network and investigating their targeted drugs. The results provided a basis for understanding the pathogenesis and improving clinical diagnosis and treatment for pSS. However, there are still several limitations: first, clinical data was needed to confirm clinical inflammation in those proposed inflammatory biomarkers; second, all results were predictions and should be verified based on a larger cohort of patients, additional follow-up RNA-seq and animal studies. Overall, a better understanding of the mechanisms of pSS progression would promote novel therapeutic strategies. With advances in genomics, the mechanistic details will certainly be further elucidated. Further research will provide potential new approaches in treating pSS.