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.