Takayasu arteritis (TA), which prevails in East Asia, is a highly specific vasculitis that exclusively involves the large arteries and the main branches. In its early stage, patients with TA are barely manifest specific symptoms or signs, making the diagnosis very challenging. However, during the disease progression or in its late stage, the culprit lesion can lead to severe organ ischemia, such as cerebral infarction and myocardial infarction. This may be due to that these patients with TA in early stage are not timely diagnosed and treated.

Currently, medical treatment for TA mainly includes glucocorticoids (Comarmond et al., 2017), methotrexate (Hoffman et al., 1994), and mycophenolate mofetil (Li et al., 2016; Dai et al., 2017). In recent years, biological agents such as tocilizumab (Zhou et al., 2017) and infliximab (Torp et al., 2021) have also been used as candidate drugs for TA. However, the active inflammation of some patients cannot be effectively controlled following medical treatment; thus, the stenosis of the culprit vessels continues to progress, and these patients with TA ultimately requiring surgical management, including endovascular treatment and open surgical repair (Chen et al., 2018; Diao et al., 2020).

Recently, pathological studies investigated on the role of CD4⁺ T cells and interleukin (IL)-6 signaling pathway in the development and progression of TA (Sagar et al., 1992; Saadoun et al., 2015; Misra et al., 2016). IL-6 promotes the differentiation of CD4⁺ T cells into Th17 cells, which then secrete cytokines, such as IL-17, IL-21, and IL-22, and induce an autoimmune response (Ruzt et al., 2013; Sutherland et al., 2013; Camporeale and Poli, 2018). Current medical treatment are based on these mechanisms. However, as mentioned previously, it remains unclear why the culprit lesions still progress in patients with TA requiring surgical management (TARSM), even though these patients have already accepted medical drugs.

Nowadays, single-cell RNA sequencing (scRNA-seq), a high-throughput technology, has been utilized to dissect cellular heterogeneities in many immune diseases at the single-cell level (Papalexi and Satija, 2018; See et al., 2018). This new technology may provide a more precisely method to explore immune disease in different clinical stages. In this study, we adopted this state-of-the-art technique to dissect the transcriptomes of peripheral blood mononuclear cells (PBMCs) in patients with TARSM and explore potential clinical markers for the development and progression of TA.

At present, research on the pathogenesis of TA has mainly focused on genes (Terao, 2016) and proteins (Mirault et al., 2017) of the peripheral blood of patients, especially in T cells (Regnier et al., 2020). However, it remains unclear how the other types of inflammatory cells in the peripheral blood contribute to the progression and development of TA. Kotaro found that IL-1 pathway expression was elevated in the peripheral blood of patients with refractory large vessel vasculitis by analyzing bulk-seq data, but the author failed to elucidate the relationship between the IL-1 pathway and different types of immune cells (Matsumoto et al., 2021). Compared with bulk-seq, scRNA-seq technology can analyze the transcriptome in each cell type, which provides more comprehensive information on the function of different inflammatory cells. To the best of our knowledge, this is the first time that scRNA-seq was used to detect the pathogenesis in the peripheral blood of patients with TA and identify potential cell markers.

In this study, it was revealed that the proportion of monocytes in patients with TARSM was higher in TA group than in HC group, which was similar in other immune-related diseases such as tuberculosis (Naranbhai et al., 2014), rheumatoid arthritis (Du et al., 2017), and vitiligo (Demirbas et al., 2020). This phenomenon observed in this study might be due to the following mechanism: (a) CD14⁺ monocytes trigger immune responses, and (b) CD163 can inhibit the activation and proliferation of lymphocytes and reduce the absolute value of lymphocytes (Baeten et al., 2004). CD163 is a transmembrane scavenger receptor (Law et al., 1993) and is known to be elevated in patients with systemic lupus erythematosus (Borgia et al., 2018), systemic juvenile idiopathic arthritis (Minoia et al., 2015), and Kawasaki disease (Garcia-Pavon et al., 2017). CD163 is also considered as a potential marker for macrophage activation syndrome, which promotes the transition from monocytes/macrophages to M1 proinflammatory macrophages (Porcheray et al., 2005; Avcin et al., 2006). In the present study, the elevated CD163 in APC membranes in patients with TA indicates that these cells are in an active state of inflammation, suggesting that APCs may play an important role in patients with TARSM and they are activated even earlier than CD4⁺ T cells.

Other cell markers highly expressed in the APCs of patients with TA were also analyzed, including TXNIP, THBS1, and AREG. Among these three markers, TXNIP and AREG showed consistent results in ELISA. TXNIP is a binding protein of thioredoxin (TXN) and can inhibit the antioxidant capacity of TXN and promote cell stress (Tinkov et al., 2018). TXNIP promotes the formation of reactive oxygen species (ROS)-NLRP3 inflammasomes by inhibiting the transfer of ROS by TXN, thereby increasing the concentration of IL-18 (Kim et al., 2019), which has also been shown to increase in patients with TARSM (Alibaz-Oner et al., 2015). NLRP3 inflammasomes are mainly secreted by activated macrophages (Kelley et al., 2019). At the same time, TXNIP is a negative regulator of thioredoxin, a key antioxidant protein to remove reactive oxygen and promote DNA repair (Lu and Holmgren, 2014). It means the high expression of TXNIP would results in a severe oxidative stress in the patient’s body (Yu et al., 2013). The increased expression of TXNIP in TARSM monocytes is closely related to their function.

AREG, as one of the main ligands of the EFGR pathway, mainly participates in the regulation of proliferation, apoptosis, and metastasis of various cells (Berasain and Avila, 2014). AREG expression is mainly elevated in pathological conditions, such as cirrhosis (Perugorria et al., 2008) and chronic obstructive pulmonary disease (Val et al., 2012). Recent studies on mouse models of glomerulonephritis also showed that AREG can enhance the function of Treg cells, inhibit the growth of CD4⁺ T cells, and promote the recruitment of myeloid cells, proliferation, and cytokine secretion of M1 cells (Melderis et al., 2020). The increase in AREG in patients with TARSM might serve as an activation signal for monocytes.

Recent studies have shown the important roles of IL family genes in the progression of TA. Among them, IL-6 has received the most attention (Sagar et al., 1992), and anti-IL-6R biologics have also been widely used in clinical practice (Zhou et al., 2017). In this study, IL-6 was not only highly expressed in APCs, but also in B cells and even autocrine in CD4⁺ T cells, indicating that B cells and CD4⁺ T cells may have other unknown functions in TA. Interestingly, IL-15 and its receptor gene IL-15RA, which are widely expressed in a variety of cells (Patidar et al., 2016) and play a bridge between innate immunity and adaptive immunity (Pagliari et al., 2013), were also detected in our study. IL-15 can also activate the maturation and functional expression of T cells, DCs, and NK cells (Mattei et al., 2001; Saikh et al., 2001; Ali et al., 2015). In this study, IL-15 and its receptor IL-15RA were both highly expressed in APCs, indicating that their antigen presentation function is activated. IL-18 is mainly synthesized by APCs; meanwhile, its receptor genes, IL-18Rα and IL-18Rβ, are expressed in T cells and DCs. The main function of this pathway is to activate the NF-κB pathway and promote the synthesis of IFN-γ in Th1 cells (Nakanishi et al., 2001; Dinarello et al., 2013; Turner et al., 2014). As previously mentioned, IL-18 has been proven to be elevated in patients with TA (Minoia et al., 2015). In this study, IL-18 was found to be highly expressed in the DCs of patients with TA, and its receptor gene was highly expressed in CD8⁺ T/NK cells, indicating that the IL-18 pathways may also occur in the PBMCs of patients with TARSM.

RAGE is a cell transmembrane receptor used to recognize and bind AGEs (Schmidt et al., 1992). AGEs refer to a kind of glycosylated molecules produced by oxidative stress or metabolism, which include not only cytokines but also some metabolites. After RAGE recognizes and binds to the corresponding ligand, it can activate the NF-κB pathway, thereby promoting the secretion of cytokines by inflammatory cells, such as IL-6 and TNF-α (Shen et al., 2020). By the way, the activation of RAGE and NFκB also send a message to reactive oxidant species (ROS), a key role in oxidative stress, by NADPH oxidase (Daffu et al., 2013). In this study, according to the GO analysis results of different cell types, it was found that APCs, such as CD14⁺ monocytes and DCs, have prominent expression of RAGE pathways, indicating that activation of this pathway may be an important reason for APC activation.

Both TXNIP and RAGE are closely related to oxidative stress. Oxidative stress has been proposed as a root cause in development of many cardiovascular diseases, including atherosclerotic (Marchio et al., 2019), aneurysm (Emeto et al., 2016) and even TA (Mahajan et al., 2010). In human vessel, endothelial cells have become the major character of antioxidative, and the secreted endothelial nitric oxide synthase (eNOS) can produce NO, a vasoprotective molecule that can resist oxidation and inhibit the immune response of blood vessel walls (Forstermann et al., 2017). However, as the body is in an immune response or infection state, NADPH oxidase produced by immune cells can directly act on endothelial cells, destroy the stability of eNOS and inhibit the activity of NO, which finally produce a large amount of superoxide anion (Ghosh et al., 2017). The high expression of TXNIP and RAGE in monocyte suggested that oxidative stress may also be a cause of vascular intimal damage, and the trigger role played by monocyte cannot be ignored.

As previously mentioned, this is the first study to explore transcriptomics using scRNA-seq techniques in patients with TARSM. However, some limitations of this study should not be ignored. First, it is hardly to find TARSM patients without medical treatment for the following reasons: (a) For these patients, they are still unable to control the inflammatory activity even after receiving medical treatment, causing the disease progressing, and the vascular lumen continues to narrow, which finally to be occluded, then surgical treatment is required. (b) For the treatment strategy, medical therapy is still the first choice for TA treatment, while surgical operation is mainly for patients who have complications such as insufficient blood supply due to vascular occlusion. Second, the number of patients in this study was also limited, both in the scRNA-seq studies and in the validation studies, such as ELISA, qPCR, and FACS. In addition, the scRNA-seq technique adopted in this study only reflected a snapshot scene for this complex disease, and how the culprit lesion evolves requires further demonstration by longitudinal studies. Another issue that remains to be elucidated is the role of ILs in TA; that is, the correlation between the observation of ILs in this study and clinical studies needs to be explored and established. A validation cohort from another center would be an important next step in future research.

In conclusion, we used single-cell RNA technology to detect peripheral blood cells in patients with TARSM. Our study showed that the proportion of CD14⁺ monocytes, function, and functional receptors increased in patients with TARSM after medical treatment, which suppressed CD4⁺ T cell function. Moreover, monocytes have become a major factor in inflammation, and the inhibition of monocyte population and function can be used as a new direction for medical treatment. We also found that TXNIP and AREG can be used as diagnostic markers for TA development and progression, and highly expressed CD163 may be an important characteristic of APCs in patients with TARSM.

The data presented in the study are deposited in the Genome Sequence Archive for Human, accession number HRA001329 (https://bigd.big.ac.cn/gsa-human/browse/HRA001329).

The studies involving human participants were reviewed and approved by the Beijing Hospital Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

GQ, LW, and LY designed the study. GQ did the experiments. WZ, YJG, and YJF analyzed the scRNA-seq data. CZ, DY, MY, GY, and JJ checked the case history. LW and LY revise the manuscript. All authors contributed to the article and approved the submitted version.

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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