Discussion
scRNA-seq can more accurately analyze each cell’s type and functions by sequencing their own transcriptome compared to bulk RNA-seq. In this study, scRNA-seq was used to detect the inflammatory cells types and cell markers in the peripheral blood of TA patients.
CD4+ T cells have always been the focus of TA research, and its differentiation subtypes, Th1 and Th17 cells are considered to be the main regulators of inflammation in TA(13). Our study found fewer CD4+ T cells in the peripheral blood of TA patients as compared to the healthy controls and can be an effect of glucocorticoids and other immunosuppressive drugs taken by the patients for TA(35, 36). We also saw an increase in cytotoxic NKT cells and NK cells in TA patients indicating that these cells have the ability to regulate inflammation and can play a major role in vascular damage associated with TA. Our study further suggests that limiting the number of these cells may effectively control the disease progression in TA patients. In recent years, monocyte/macrophages have been extensively studied for their role in immune diseases. In the present study, we see a higher proportion of monocyte/macrophages represented by CD14+ in TA patients indicating that these cells may play an important role in TA. B cells have always been less involved in TA research, but in recent years, more studies indicating the role of B cell in TA pathogenesis have been published. Many studies(37-42) have showed B cells have played an important role in the TA lesions during the courses of the disease, however, the specific role of B cells in TA is still unclear. In our study, we found that the proportion of B cells (especially naive B cells) were higher in TA patients, indicating that both cellular and humoral immunity plays a role in TA pathogenesis.
We further analyzed the cell markers of different cell types that are abnormally expressed in TA. In the monocyte/macrophage subset of TA patients, we found an increase in the expression of IL1R2, THBS1, CD163, AREG, and FKBP5. Among these 5 genes, IL1R2(43) and FKBP5(44) can be elevated due to the intake of glucocorticoids. CD163 is a transmembrane scavenger receptor that is expressed on the surface of macrophages(45) and is known to be elevated in SLE, sJIA, and Kawasaki patients, indicating that CD163 can be used as a marker for macrophage activation syndrome (MAS) indicating the transition from monocyte/macrophage to M1 proinflammatory macrophages(46, 47). In the present study, the elevated CD163 gene in TA patients indicates that these cells are in an active state of inflammation, suggesting that monocytes/macrophages play an important role in the pathogenesis of TA, suggesting that CD163 may be used as a potential diagnostic marker for TA as well. THBS1, also known as thrombospondin 1 (TSP1), is a ligand for CD47. This protein exists in the extracellular matrix and can promote tissue fibrosis by binding fibrin and collagen(48). In rheumatoid arthritis patients, THBS1 activates the inflammatory function of T cells through the CD47 receptor on the surface of T cells(49, 50), and activates macrophages via the Toll-like receptor 4 (TLR4) pathway(51, 52). This protein can also inhibit the activity of VEGF in tumor-related research, thereby inhibiting the synthesis of new blood vessels(52, 53). AREG is one of the main ligands of the EFGR pathway. The protein is mainly used to regulate the proliferation, apoptosis, and metastasis of various cells(54). Under pathological conditions, especially in patients with chronic diseases, such as cirrhosis(55), chronic obstructive pulmonary disease (COPD)(56), the expression of AREG is significantly elevated. In recent studies on mouse models of glomerulonephritis, AREG has been shown to play a completely different role in CD4+ T cells and macrophages. This protein enhances the function of Treg cells and inhibits the growth of CD4+ T cells and promotes the recruitment of myeloid cells and the proliferation and cytokine secretion of M1 cells(57).
In the T cells of TA patients, we found a significant increase in the expression of IFITM1, FKBP5, MIF, and TXNIP. Similar to IL1R2 and FKBP5, the increase in MIF is closely related to the intake of glucocorticoids(58). The patients in this study were all taking glucocorticoids, which is not discussed here. IFITM1 is an IFN-related protein with a role in inhibiting viral infection by interfering with viral protein synthesis and replication(59, 60). IFITM1 is mainly expressed on T cell surface. In addition to the above functions, IFITM1 can also promote the differentiation of naive CD4+ T cells into Th2 cells(61), indicating that this protein is involved in inflammatory regulation. TXNIP is a binding protein of thioredoxin (TXN), and can inhibit the antioxidant capacity of TXN and promote cell stress(62). In the inflammatory process, TXNIP can promote the formation of ROS-NLRP3 inflammasomes by inhibiting the transfer of reactive oxygen species (ROS) by TXN, thereby increasing the concentration of IL-18 and IL-1β(63). This inflammatory reaction process also occurs in patients with coronary artery disease(64) and diabetes-related vascular disease(65). An increase in IL-18 in peripheral blood of TA patients has been shown to promote the formation of granulomas(66).
Our findings showed a similar increase in the expression of IGHG1, IGHG3, MIF, and TXNIP in B cells of TA patients suggesting a role of TXNIP in TA pathogenesis. The immunoglobulin heavy constant G chain (IGHG) genes are known to play a crucial role in the synthesis of immunoglobulins by B cells(67). Related reports of IGHG have also been found in other autoimmune diseases(68, 69). The increased expression of this gene in PBMCs of TA patients in our study indicates the secretion of antibodies and the role of humoral immunity in the pathogenesis of TA.
According to the previous studies mentioned before, it can be found that THBS1, CD163, AREG, IFITM1, TXNIP, and IGHGs expression are all related to the differentiation and activity of inflammatory cells. Therefore, the expression level of these cell markers can reflect the state of inflammatory cells, in addition the disease activity of TA patients.
In conclusion, we used single-cell RNAseq technology to detect peripheral blood cells in TA patients. Our study showed that CD14+ monocytes, cytotoxic NKT cells, CD56dim CD16+NK cells, and B cells were elevated in the peripheral blood of TA patients suggesting CD4+ T cell and IL6 pathway are not the only key, these kinds of cells are also playing a crucial role in TA pathogenesis and the potential use of THBS1, CD163, AREG, IFITM1, TXNIP, and IGHGs expression as diagnostic markers for TA development and progression.