SjS is a complex rheumatic disease characterized by the infiltration of immune cells into exocrine glands such as salivary glands, and effective targeted therapies for SjS are still lacking (1, 2). Current studies suggest that some factors such as disease susceptibility genes, immune abnormalities, and viral infections are synergistically involved in its pathogenesis of SjS (3–5). Among those factors, abnormal immune factors such as B cell hyperactivity have been considered as key players in SjS pathogenesis and potential targets for SjS treatment (6, 7). Nevertheless, the immune mechanisms involved in SjS pathogenesis and progression have not been fully clarified. To reveal potential targets of immunotherapy, it is necessary to further study the immune cell subsets that play a critical pathogenic role in SjS.

Mononuclear phagocytes (MNPs) are the most common innate immune cells with key roles in both immunity and autoimmunity (8–11). MNPs in blood are mainly composed of monocytes and dendritic cells (DCs), both of which have heterogenerous subsets with distinct phenotypes (12, 13). Recent studies using scRNA-seq have demonstrated the existence of distinct monocyte subsets and they have crucial roles in the development of some rheumatic diseases such as SLE (14, 15). For instance, monocytes can participate in the pathogenesis of SLE by immune mechanisms such as secreting pro-inflammatory cytokines and assisting in the activation of B cells and T cells (16–19). In recent years, the roles of monocytes in the pathogenesis of SjS have also gained increased attentions, and some studies have proved possible key roles of monocytes in the development and progression of SjS (20–24). However, the mechanisms of monocytes in SjS have not been fully clarified, and further research is required. At present, there is a lack of relevant research exploring the changes of monocyte subsets in SjS patients via scRNA-seq. This study aimed to analyze the abnormal changes of monocyte subsets in peripheral blood of SjS patients by scRNA-seq data, and further explore the key transcriptomic changes in monocytes of SjS patients.

The roles and underlying mechanisms of monocytes in SjS development have not been fully clarified. This study analyzed the abnormal changes of monocyte subsets in SjS patients by scRNA-seq data, and further explored the key transcriptomic changes of monocytes in SjS patients. We found a new monocyte subset characterized by higher expressions of VNN2 (GPI-80) and S100A12 (Monocyte cluster 3), which was increased in SjS patients. Moreover, virus infection-associated pathways, IFN-related pathways and neutrophil activation-associated pathways were the major up-regulated pathways in the monocytes of SjS patients. Finally, compared with controls, almost all monocyte subsets from SjS patients had increased expression of TNFSF10 (TRAIL). Therefore, this study uncovered the abnormal changes in monocyte subsets and their transcriptomic changes in SjS patients, and identified TNFSF10 ^high/+ monocytes as a potential key player in SjS pathogenesis and a promising target for SjS treatment.

Mononuclear phagocytes including monocytes are the main antigen presenting cells (APCs) and can initiate protective immune processes against pathogens, but they can also initiate autoimmune processes in autoimmune diseases (30–32). Recent studies have proven the existence of distinct monocyte subsets and they have critical roles in the development of some rheumatic diseases such as SLE and rheumatoid arthritis (RA), and targeting monocytes is a potential treatment for those diseases (17, 33). There are also some studies focusing on the roles of monocytes in SjS, and some have shown that aberrant changes in immunophenotypes, intracellular functional pathways and epigenetics exist in SjS patients (20–24). Those outcomes suggest the key roles of monocytes in the development and progression of SjS, though the underlying mechanisms are largely elusive.

Monocytes have several subsets with functionally distinct phenotypes, and peripheral monocytes have been calcified as proinflammatory or classic monocytes (CD14⁺⁺CD16⁻), intermediate monocytes (CD14⁺⁺CD16⁺) and nonclassic monocytes (CD14⁺CD16⁺⁺) (34). Though many studies have explored the roles of monocytes in human diseases, findings are inconsistent in both the immunophenotypes of monocyte subsets and their functions. The development of scRNA-seq has provided new opportunities in uncovering monocyte subsets and defining their disturbances in those diseases (14, 35–37). In the present study, we tried to identify changes in monocyte subsets and transcriptome in SjS patients via scRNA-seq analyses. A total of 11 monocyte subsets were identified in the scRNA-seq analyses of monocytes, and a new monocyte subset characterized by higher expressions of VNN2 (GPI-80) and S100A12 (Monocyte cluster 3) was found to increase in SjS patients. However, owing to the limited samples in current study and the minimal heterogeneity among those mononuclear phagocyte subsets, the features genes of those monocyte subsets identified in our study were not highly specific. scRNA-seq transcriptome analyses with limited number of cells or samples could undoubtedly result in high difficulty in defining unique subpopulations with specific features genes. Therefore, the changes in monocyte subsets among SjS patients still need to be explored by further scRNA-seq analyses of larger number of samples. These studies may provide new perspectives in the landscape of mononuclear phagocytes and uncover the potential key pathogenic subset in SjS.

This study suggested that IFN-related signaling and virus infection-associated pathways were key up-regulated pathways in the monocytes of SjS patients and they were involved in SjS pathogenesis. Currently, the role of IFN-α pathway in SjS pathogenesis has long been clearly defined, and therapies targeting IFN-α may be a candidate treatment strategy for SjS (38–40). Besides, there are some published literatures which could confirm the up-regulation of IFN-related signaling in the monocytes of SjS patients. A study by Brkic et al. reported that type I IFN inducible genes such as IFI44L, IFI44, IFIT3, LY6E and MX1 were systematically up-regulated in monocytes of SjS patients and were associated with high disease activity (41). Wildenberg et al. also reported that there was an upregulation of IFN-related genes such as IFI27, IFITM1, IFIT4, and IFI44 in monocytes of SiS patients (42). Sialic acid binding Ig like lectin 1 (Siglec-1), a biomarker of the activation of type I IFN pathway, was highly expressed in monocytes of SjS patients and was positively correlated with the EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) score (43). Pertovaara et al. found increased cytokine-induced STAT1 activation in monocytes of SjS patients via flow cytometry (44). Data from another 2 studies also supported the up-regulation of molecules related to the activation of IFN-related signaling in the monocytes of SjS patients (45, 46). Therefore, there is good evidence supporting the up-regulation of IFN-related signaling in the monocytes of SjS patients.

Virus infection such as Epstein-Barr virus (EBV) infection has long been studied as an important environmental risk factor of SjS, but definite conclusion on its pathogenic role in SjS is still lacking (47–50). The findings from this study support virus infection as an important player in SjS pathogenesis. However, the molecular mechanisms underlying the pathogenic roles of virus infection in SjS are still not clear, and need to be elucidated in future studies. Apart from IFN-related pathways and virus infection-associated pathways, neutrophil activation-associated pathways were also identified to be up-regulated pathways in the monocytes of SjS patients. There is accumulating evidence implicating those neutrophils as key players in the pathogeneses of autoimmune or rheumatic diseases (51–53). The hyperactivation of neutrophils have been implicated in the pathogeneses of rheumatic diseases such as systemic lupus erythematosus (SLE) (54–56). An early research reported that neutrophil adhesion was enhanced in SjS patients, which indicated an increased activation of neutrophils in SjS patients (57). The up-regulation of neutrophil activation-associated pathways in the monocytes of SjS patients suggests that a possible role of monocytes-neutrophils cross-talk in the pathogenesis of SjS. Previous studies have revealed that monocytes can mediate neutrophil activation via multiple distinct mechanisms and is involved in diseases such as SLE (58–60). However, studies focusing on the roles of neutrophil activation or monocytes-neutrophils cross-talk in SjS are still limited, and further studies are needed.

Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) is a key cytokine of the TNF superfamily, and has significant roles in regulating immunity. Previous studies have identified abnormal changes in TNFSF10 (TRAIL) among patients with distinct autoimmune and rheumatic diseases such as SLE (61–63). Another study found that TRAIL⁺ monocytes played critical roles in lung damage (64). However, the roles of TNFSF10 ^high/+ monocytes in common autoimmune and rheumatic diseases have not been clearly defined. In present study, the increased expression of TNFSF10 (TRAIL) in the monocytes of SjS patients was identified by both scRNA-seq transcriptome analyses (Figure 2B) and bulk transcriptome analyses of monocytes (Figure 3A). The outcomes above suggested TNFSF10 ^high/+ monocytes as a potential key player in SjS pathogenesis and a promising target for SjS treatment. Besides, more studies exploring the roles and potential mechanisms of TNFSF10 ^high/+ monocytes in SjS development are needed.

A limitation of this study was the small size of samples of SjS patients and controls especially in the scRNA-seq transcriptome analyses. In the scRNA-seq analyses, there were only 5 SjS cases and 4 controls, which could undoubtedly cause impaired statistical power in detecting differences across distinct monocyte subsets. The total number of monocytes in the scRNA-seq analyses was also limited, which could result in suboptimal analyses of monocyte subsets and their functions. Therefore, further scRNA-seq analyses with larger sample size are recommended in future studies, which may provide much deeper insights into the pathogenesis of SjS.

In summary, this study uncovered the abnormal changes in monocyte subsets and their transcriptomic changes in SjS patients. Both scRNA-seq and bulk RNA-seq transcriptomic analyses identified increased expression of TNFSF10 (TRAIL) in monocytes among SjS patients, suggesting TNFSF10 ^high/+ monocytes as a potential key player in SjS pathogenesis and a promising target for SjS treatment. Further work is needed to explore the roles and underlying mechanisms of monocytes in SjS development.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE157278 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173670.

The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Xiamen University. The patients/participants provided their written informed consent to participate in this study.

YH, YL, and GS contributed to conception and design of the study. YH, RC, MZ, and BW analyzed the data. YH, BW, and ZL wrote the manuscript. RC, YL, and GS reviewed and edited the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82171779) to GS, and the Fujian Provincial Health Commission (Grant No. 2021GGB025) to YL.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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