In order to expand the sample size, we merged the three datasets and finally obtained the gene expression data of 351 cases and 91 healthy controls, which provided a reliable basis for this study. Further analysis for differential gene expression, protein function, enrichment pathways and immune infiltration identified some key factors for pSS. We identified a total of 89 up-regulated genes and 11 down-regulated genes in the dataset. These DEGs were enriched in NOD-like and RIG-I-like receptor signaling pathways, which were significantly associated with the expression of immune cells such as neutrophils, activated dendritic cells, resting NK cells and memory B cells, respectively.
Pattern recognition receptors (PRRs), which include NOD-like receptors (NLRs) and RIG-I like receptors (RLRs), are important sensors for sensing invading viruses (17–19). The primary function of NLRs is to regulate a series of signaling cascades as well as participate in the body's acquired and intrinsic immune responses (20). Members of the NLRs family regulate the development and progression of a variety of diseases, mainly by participating in the inflammatory response or interacting with key proteins of the immune signaling pathway. Important members of the NLRs family include NOD-like receptor pyrin domain-containing protein 1 (NLRP1), NLRP3, NLR family CARD domain-containing protein 4 (NLRC4), neuronal apoptosis inhibitor protein (NAIP), etc. Working as inflammasomes sensors, they induce the secretion of interleukin-1 (IL-1) and IL-18 through binding to their cognate ligands and participate in the immune regulation process of the body. They can create tissue damage and lead to a status of chronic inflammation in some tissues and organs (21–23). In the study by Aigli et al (24), a comparative analysis of gene and protein expression between patients and controls showed that NLRP3 was activated in the infiltrated immune cells of peripheral blood monocytes and saliary glands of patients with pSS. The expression level of NLRP3 is further increased during this process and may affect the severity of the disease. Activation of NLRP3 inflammatory vesicles has been shown to lead to the release of mature IL-1β and IL-18. IL-18 is also significantly elevated in serum and saliva of pSS patients. It is thought to exert a wide range of pro-inflammatory activities and perform a key role in the further expansion of infiltrating damaged tissues of pSS patients (25–27).
The findings of our study revealed that in addition to the NOD-like and RIG-I-like receptor pathways, DEGs were predominantly enriched in infectious diseases, including hepatitis C, measles, influenza A and EBV infections. As an important environmental factor, infection acts as a key player in the initiation and development of pSS (28–30). Innate immunity is the first barrier for the host to block viral infection, and RLRs play a critical role in this process. There are three members of RLRs including retinoic-acid inducible gene I (RIG-I), laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) (31). RLRs are cytosolic sensors for virus RNA and the RIG-I-like receptor signaling triggers the secretion of type I interferon and proinflammatory cytokines, which further activate the immune responses (32). Preliminary experiments have been performed to identify the expression patterns of RIG-I and MDA5 in pDCs and monocytes from pSS patients (33). Therefore, the effect of RLRs on pSS is also worth further attention.
Activation of RIG-I and MDA5 triggers the activation of mitochondrial antiviral signaling protein (MAVS), an important junction molecule on downstream mitochondria or peroxisomes, which in turn activates downstream pathways leading to phosphorylation and activation of nuclear factor-κB (NF-κB) and interferon regulating factor 3 (IRF3), eventually resulting in the secretion of type I interferon and proinflammatory factors (34). Interestingly, there is a specific association between NLRs and RLRs in antiviral signaling, actually, NLRs are also involved in the regulation of the type I interferon pathway. Sabbah et al (35) demonstrated that nucleotide-binding oligomerization domain 2 (NOD2) produces an immune response to viral infection and mediates the activation of IRF3, and that this action mediates the direct interaction between NOD2 and MAVS. This reaction occurs when RSV-derived ssRNA is recognized by NOD2 and activates the production of IRF3 and IFN. NLRs have also been demonstrated to negatively regulate the RLR signaling pathway. NLR family member NLRX1 localizes to the outer mitochondrial membrane and interacts with MAVS to inhibit the activation of IRF3 and NF-κB. NLRP5 can also inhibit NF-κB and IFN immune response pathways, possibly by regulating inhibitor of nuclear factor kappa-B kinase (IKK) phosphorylation or by directly binding RIG-I and MDA5 (36–38). In conclusion, NLRs both positively and negatively regulate the RLR signaling pathway.
In addition to immune pathways, immune cell is also the major point of research in pSS. We found that DCs and neutrophils play a more prominent role in pSS. Our findings also suggest a significant association of both DCs and neutrophils with NOD-like and RIG-l-like signaling pathways. DCs not only participate in the body's intrinsic immune response by recognizing and ingesting antigens, but also participate in the adaptive immune response by processing and presenting antigens, as well as regulating the function of other immune cells by secreting chemokines and cytokines. DCs act as important players in the immune process against viral infections mainly through the expression of multiple PRRs, including the NOD-like receptors and RIG-I-like receptors (39). Moreover, it was found that NLRC3 not only reduced the antigen presentation function of DCs, but also decreased the activation and differentiation of CD4+ T cells, resulting in a reduction of Th1 and Th17 subsets, further leading to an inflammatory response(40). With the expression of NOD-like receptors and RIG-like receptors, neutrophils are involved in not only recognizing pathogens but also sensing tissue damage (41). Neutrophils activate NLRP3 inflammasome by releasing danger signals, which can block IL-1β production (42). In addition, neutrophils can also express both RIG-I and MDA5 receptors and release cytokines as well as alter the expression of genes (43, 44).
In this study, bioinformatic methods were used to identify some possible novel associations between pSS and two receptor signaling pathways, NOD-like and RIG-I-like, thus providing some inspiration for researchers. However, in the absence of experiments, the results of this study have yet to be validated.