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Clin Exp Med
Clin Exp Med
Clinical and Experimental Medicine
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10.1007/s10238-024-01457-2
Research
Characteristic changes in the mRNA expression profile of plasma exosomes from patients with MPO-ANCA-associated vasculitis and its possible correlations with pathogenesis
Chen Yangfan 1
Zhou Dongqing 1
Qian Xin 1
Ge Shangqing 1
Shuai Zongwen amushuaizw@163.com

12
1 https://ror.org/03t1yn780 grid.412679.f 0000 0004 1771 3402 Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 China
2 grid.186775.a 0000 0000 9490 772X Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Hefei, 230032 China
17 9 2024
17 9 2024
2024
24 1 22222 6 2024
30 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
To explore the expression patterns and potential roles of mRNAs in exosomes from patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-AAV). Plasma exosomes were isolated from MPO-AAV patients and healthy controls (HCs) to screen for differential mRNA expression via exosomal mRNA sequencing. The differentially expressed mRNAs in exosomes from the 2 groups were comparatively explored by bioinformatics analysis. The six most differentially expressed mRNAs were selected and validated in larger groups of MPO-AAV patients and HCs by real-time quantitative polymerase chain reaction (RT‒qPCR). The relationships between these selected mRNAs and patient characteristics were statistically analyzed. Compared with HCs, a total of 1077 mRNAs in exosomes from MPO-AAV patients were found to be significantly upregulated, including DEPDC1B and TPST1, while NSUN4 and AK4 were significantly downregulated. Statistical analysis did not reveal any correlation between the six selected mRNAs and clinical indicators, including disease activity. GO enrichment analysis revealed that these differentially expressed genes participate in various enzyme activities, protein synthesis, etc. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs. There were significant differences in the expression of exosomal mRNAs between MPO-AAV patients and HCs, which may be related to the occurrence and development of MPO-AAV. These findings provide clues for further investigations of MPO-AAV pathogenesis and the identification of new potential therapeutic targets.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10238-024-01457-2.

Keywords

MPO-ANCA-associated vasculitis
Exosomes
mRNA
Pathogenesis
issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by the inflammatory destruction of small blood vessels, mostly resulting in multiple types of systemic damage and frequently affecting the kidney and lung [1, 2]. The common ANCAs that have been identified are self-antigens, mainly myeloperoxidase (MPO) and neutrophil proteinase 3 (PR3), referred to as MPO-ANCA and PR3-ANCA, which are associated with MPO-AAV and PR3-AAV, respectively [3]. Clinically, AAV is largely divided into three groups: microscopic polyangitis (MPA), granuloma with polyangitis (GPA), and eosinophilic granuloma with polyangitis (EGPA), in which MPA and EPGA are predominantly related to MPO-ANCA, while PR3-ANCA is mainly related to GPA [4–8] . However, genetic studies have revealed that MPO-AAV and PR3-AAV are distinct autoimmune syndromes [9, 10], implying that their pathogenesis may differ, although they may sometimes share some clinical manifestations and therefore could be classified as MPA, GPA or EGPA. Therefore, to explore their pathogenesis, MPO-AAV and PR3-AAV should be investigated separately. Research has shown an increasing trend in the incidence of AAV [11, 12]. PR3-AAV is more common than MPO-AAV in Western countries [12, 13], while MPO-AAV is the most common in East Asian countries, including China and Japan [14, 15].

ANCAs are thought to be directly involved in the pathogenesis of this disease. They can induce preactivated neutrophils to be hyperactivated, to release superoxides and lyases and to produce neutrophil extracellular traps (NETs), exacerbating vascular inflammation and injury. In addition, the alternative complement pathway and T lymphocytes play important roles in the pathogenesis of MPO-AAV [16]. Although all of these pathological processes in AAV are caused by immune intolerance, the exact mechanism that leads to the immune intolerance has remained elusive.

Exosomes are cystic vesicles secreted by living cells that carry large amounts of proteins, lipids, DNA and different forms of RNA, including messenger RNA (mRNA) and noncoding RNA (ncRNA). They play important roles in multiple biological processes, such as intracellular signaling, coagulation, angiogenesis, inflammation, antigen presentation, apoptosis, and cellular homeostasis [17–20]. In particular, exosomes are also presumably involved in pathogenic pathways by modulating the immune response [21]. Exosomes derived from T-regulated cells (Tregs) can inhibit Th1 (T helper 1) cell proliferation and cytokine production by transferring their microRNAs (miRNAs) [22]. Exosomes from B cells can express functional integrins that mediate cell adhesion during inflammatory processes [23]. In the pathological process of AAV, biological molecules such as autoantibodies (ANCAs), cytokines and signaling pathway molecules are translated from mRNAs; however, to our knowledge, the exosomal mRNAs of AAV have not yet been investigated. Here, we preliminarily explored the potential role of exosomal mRNAs in the pathogenesis of MPO-AAV and their possible influences on disease activity.

Patients and methods

Participants and heath record collection

During the screening phase, six patients with MPO-AAV and six age- and sex-matched healthy controls (HCs) were selected. To exclude the possible impact of therapeutic agents on the experimental data, all 6 patients were recruited at their first onset of illness and had not yet received any treatments. During the validation phase, 22 additional MPO-AAV patients at various disease states and 22 matched HCs were further included in this study. All the patients tested positive for MPO-ANCAs and fulfilled the AAV classification criteria released by the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) in 2022 [5, 6]. The clinical records and health check records of all the selected patients were collected. Patients with acute or chronic infections, neoplasms, other chronic diseases or patient who were pregnant were excluded. This study was performed according to the principles of the Declaration of Helsinki, and each participant provided informed consent before entering the study. The ethical committee of Anhui Medical University approved the study protocol.

Isolation and identification of peripheral blood exosomes

After referencing relevant literature on the isolation and identification of exosomes from peripheral blood, exosomes were isolated from the peripheral blood of patients with MPO-AAV and HCs by ultracentrifugation [24, 25]. To determine the morphology and size of the exosomes obtained, transmission electron microscopy and nanoparticle tracking analysis (NTA) were employed. NTA was used to detect the obtained exosomes directly.

Extraction of exosomal total RNA

Total RNA was extracted from purified exosomes using TRIzol. All procedures were carried out according to the manufacturer's instructions. A NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA) was used to determine the RNA concentration.

Exosomal mRNA bioinformatics analysis

Beijing Genomics Institute (BGI) (China) prepared, sequenced, and conducted the bioinformatics analysis of the RNA library. The BGISEQ-500 platform was used for library sequencing. DESeq2 software was used to analyze mRNAs in the samples. A threshold P value < 0.05 plus a multiple change > 2 was used to identify the upregulated and downregulated mRNAs. Cluster hierarchy and volcano maps were used to display differences in exosomal mRNA expression patterns between the patient and HC groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were subsequently performed to explore the roles of the target mRNAs.

Validation of differential mRNA expression by qRT‒PCR

During the validation stage, total RNA was extracted from the exosomes of 22 patients with MPO-AAV and 22 HCs. The data were used as original template information for real-time quantitative polymerase chain reaction (qRT‒PCR) quantification. The primer information is shown in Supplementary Table S1 online. In brief, cDNA was transcribed using PrimeScript™ RT Master Mix. PCR was performed on the Cobas 4800 PCR platform. The qPCR data were quantified using the 2 − ΔΔCt method (ΔCt = Cttarget − Ctreference, − ΔΔCt = sample ΔCT − β-actin ΔCT) [26].

Statistical analysis

All the statistical analyses were performed with SPSS version 22.0 (SPSS Inc., Chicago, IL). Categorical variables are presented as frequencies and percentages. Quantitative variables are presented as the median and 25–75th percentiles (interquartile range) or as the mean ± standard deviation (SD). Differences between two groups were assessed by t tests or nonparametric tests. The chi-square test was used to analyze the count data. Group comparisons were performed using Spearman’s correlation test. Risk factors were analyzed using logistic multiple regression. The term “significant” in this report was used to denote statistical significance (P < 0.05).

Results

The demographic and clinical characteristics of the participants

During the screening phase, 6 patients were matched with 6 HCs in terms of sex and age. All 6 patients were diagnosed with MPO-AAV for the first time and therefore had relatively short disease courses with active disease status. In the validation phase, there was no statistically significant difference in sex or age between the 22 patients and 22 HCs. All the patients had a longer course of disease, and their condition was also considered to be in an active state. In addition, in both the screening and validation phases, the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and creatinine level were significantly greater in the patients than in the controls. The demographic and disease characteristics of all the included patients and controls are listed in Table 1.Table 1 Demographic and clinical characteristics of the MPO-AAV patients and healthy controls

Phase	Demographic and clinical characteristics	MPO-AAV	HC	P value	
Screening	n	6	6		
Age (years, mean ± SD)	66.7 ± 10.8	60.2 ± 5.3	0.215*	
Sex (male/female)	1:1	1:1	1.000#	
Disease duration (months, M (P25, P75))	2.8 (1.9, 5.2)	/		
ESR (mm/h, mean ± SD)	69.3 ± 25.8	3.8 ± 1.9	 < 0.001*	
CRP level (mg/L, M (P25, P75))	42.0 (19.3, 57.0)	1.3 (0.9, 1.9)	0.009#	
Creatinine level (umol/mL, M (P25, P75))	115.7 (43.3, 161.5)	67.2 (64.3, 70.3)	0.460#	
BVAS-V3 (M (P25, P75))	17.5 (16, 20.3)	/		
Validation	n	22	22		
Age (years, mean ± SD)	63.5 ± 8.7	59.5 ± 14.3	0.268*	
Sex (male/female)	1.1:1	1.3:1	0.502#	
Disease duration (months, mean ± SD)	1.2 (1.0, 3.25)	/		
ESR (mm/h, M (P25, P75))	68.1 (48.5, 89.3)	3.0 (2.0, 5.0)	 < 0.001#	
CRP level (mg/L, M (P25, P75))	22.7 (4.3, 58.5)	3.2 (1.3, 5.7)	 < 0.001#	
Creatinine level (umol/mL, M (P25, P75))	180.0 (64.0, 367.5)	64.0 (45.0, 75.5)	 < 0.001#	
BVAS-V3 (M mean ± SD)	17.7 ± 2.5	/		
*T test result of the comparison between the RA and HC groups. #: result of the nonparametric test. Abbreviations: BVAS-V3 Birmingham vasculitis activity score-version 3; CRP C-reactive protein; ESR erythrocyte sedimentation rate; HC healthy control; MPO-ANCA antineutrophil cytoplasmic antibody against myeloperoxidase; MPO-AAV MPO-ANCA associated vasculitis; SD standard deviation

Features of the acquired plasma exosomes

Transmission electron microscopy clearly revealed that the exocrine bodies were round or oval vesicles with sizes ranging from 30–100 nm. The comparison of the transmission electron microscopy images of the exocrine bodies from different sources between the two groups showed that exosomes were successfully isolated from plasma, and there was no significant difference in the morphology of the exocrine bodies between the AAV patients and the healthy control group (Fig. 1).Fig. 1 Transmission electron microscope image of the exosome morphology in patients with AAV (A) and healthy controls (B) (scale bar: 100 nm) and the size distributions of the exosomes from MPO-AAV patients (C) and healthy controls (D), which indicated that exosomes were successfully obtained in our study

Hierarchical clustering analysis of the mRNAs from exosomes

Hierarchical clustering analysis was carried out based on the exosomal mRNA results. The heatmap shows differences in the levels of plasma exosomal mRNAs between MPO-AAV patients and healthy controls (Fig. 2).Fig. 2 Heatmap showing the comparison of the expression levels of exosomal mRNAs between the MPO-AAV group and the healthy control group. The X axis shows the MPO-AAV patient and HC groups in the cluster analysis, and the Y axis represents the sample differential exosomal mRNAs. The red color represents an increase in expression, while the blue color represents a decrease in expression; the larger the value is, the darker the color

The results of the GO enrichment analysis and KEGG pathway analysis of the target genes of the identified exosomal mRNAs are shown in Fig. 3. A total of 3 GO terms (molecular function, cellular components, and biological processes) were found in the analysis. The results indicate that these differential genes are related to various enzyme activities, protein synthesis, etc., and are involved in various biological processes, such as retinal ganglion cell axon guidance, choline catabolic processes, mitotic sister chromatid segregation, spliceosomal tri-snRNP complex assembly, amyloid-beta metabolic processes, positive regulation of osteoblast differentiation, and cell responses to hepatocyte growth factor stimuli. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs.Fig. 3 GO enrichment analysis and KEGG pathway analysis of the genes associated with exosomal mRNAs. A GO enrichment analysis molecular function terms; B GO enrichment analysis cellular components terms; C GO enrichment analysis biological processes terms; D KEGG pathway analysis results

Candidate mRNAs screened by high-throughput sequencing and validated by qRT‒PCR

Differentially expressed exosomal mRNAs

An analysis of the differentially expressed extracellular mRNAs was conducted, and a total of 1,077 mRNAs in exosomes were screened from plasma samples. The statistical results of the differentially expressed exosomal mRNAs are shown in a volcano plot (Fig. 4). Then, the differentially expressed exosomal mRNAs between the AAV group and HC group were screened according to the fold change (log2, multiple expression differences). The selection criteria were as follows: Q value ≤ 0.05 or |Log2FC|≥ 2; 34 exosomal mRNAs showed significantly upregulated expression levels; and 74 exosomal mRNAs had significantly downregulated expression levels (Supplementary Table S2 online). Moreover, we randomly selected six differentially expressed exosomal mRNAs (DEPDC1B, TPST1, LSM2, NSUN4, FBXO34, and AK4) and validated them by qRT–PCR (Table 2). The expression levels of DEPDC1B and TPST1 in the exosomes from MPO-AAV patients were significantly greater than those in the exosomes from normal controls, while those of NSUN4 and AK4 were significantly lower (P < 0.05, Fig. 5) in the patient group.Fig. 4 Volcano plot revealing the differentially expressed mRNAs between the patient and HC groups. The X axis is the log2 (fold change) values, and the Y axis is − log10 (Q value) values. The gray dots represent the mRNAs with no significant differences, the red dots represent the upregulated mRNAs, and the green dots represent the downregulated mRNAs

Table 2 The expression differences of 6 candidate exosomal mRNAs in the validation phase (n = 22)

Exosomal mRNA	MPO-AAV*	HC**	P#	
DEPDC1B	1.73 (0.50, 1.97)	0.24 (0.03, 0.32)	0.002	
TPST1	1.78 (0.37, 2.58)	0.64 (0.20, 1.47)	0.008	
LSM2	1.48 (0.24, 2.34)	0.90 (0.65, 1.24)	0.088	
NSUN4	1.95 (0.63, 2.45)	4.77 (1.55, 9,53)	0.008	
FBXO34	2.78 (0.90, 4.38)	2.87 (0.20, 5.26)	0.890	
AK4	19.91(5.80, 19.14)	56.56 (20.80, 90.15)	0.013	
*Data are presented as the median and 25th and 75th percentiles. #the result of the Mann‒Whitney U test between the MPO-AAV and HC groups

HC healthy control; MPO-AAV antineutrophil cytoplasmic antibody against myeloperoxidase associated vasculitis

Fig. 5 Comparison of the expression levels of exosomal mRNAs (DEPDC1B, TPST1, FBXO34, and AK4) between the patient and HC groups (P < 0.05)

Relationships between the 6 selected mRNAs and MPO-AAV-related clinical indices

The results obtained from the univariate linear regression analysis of the 6 selected mRNAs and clinical or laboratory parameters in MPO-AAV patients are shown in Table 3. The results indicated that there were no statistically significant correlations between the expression of the 6 selected mRNAs and disease activity parameters (ESR, CRP level, and Birmingham vasculitis activity score version 3 (BVAS-V3)).Table 3 Results of the correlation analysis between the 6 candidate mRNAs and the clinical indices

Clinical index	DEPDC1B	TPST1	LSM2	NSUN4	FBXO34	AK4	
r	P	r	P	r	P	r	P	r	P	r	P	
Disease duration (months)	 − 0.11	0.96	 − 0.21	0.38	 − 0.24	0.38	 − 0.24	0.28	 − 0.18	0.43	0.15	0.52	
ESR (mm/h)	0.13	0.56	 − 0.07	0.76	0.09	0.76	0.34	0.13	0.22	0.32	 − 0.22	0.34	
CRP level (mg/L)	 − 0.10	0.66	 − 0.08	0.74	0.14	0.74	0.28	0.19	0.16	0.47	 − 0.27	0.24	
BVAS-V3	0.12	0.59	0.15	0.53	0.34	0.53	0.03	0.91	0.10	0.68	 − 0.04	0.87	
*The results of the Spearman rank correlation analysis

BVAS-V3 Birmingham vasculitis activity score-version 3; CRP C-reactive protein; ESR erythrocyte sedimentation rate

Discussion

Exosomes are widely distributed in the human body. They can carry proteins, lipids, and nucleic acids to exchange information between cells and regulate the activity of recipient cells. Studies have shown that exosomes can act as mediators of immune stimulation and regulation and participate in the regulation of a variety of immune processes, including antigen presentation, T-cell activation and polarization, and immunosuppression [27, 28]. In recent years, researchers have investigated miRNAs in AAV exosomes [29–31], but studies on the mRNAs in AAV exosomes are lacking. The relationship between plasma-derived exosomal mRNA and inflammatory injury in vascular endothelial cells in patients with AAV is not clear. Exosomes enter receptor cells through various mechanisms, such as phagocytosis, fusion and signal transduction [32]. Vascular endothelial cells can produce and absorb extracellular vesicles. Therefore, exosomes may be captured by vascular endothelial cells throughout the body and cause cell damage, leading to the onset of MPO-AAV [33–35].

In the present study, we investigated the characteristics of mRNA expression profiles in the circulating exosomes of AAV patients. We isolated exosomes from peripheral blood for mRNA sequencing analysis and then identified candidate mRNAs by comparing the mRNA expression profiles between AAV patients and healthy controls. The RNA-sequencing data revealed that mRNAs may contribute to AAV disease progression. In this study, we identified three upregulated mRNAs (DEPDC1B, TPST1, and LSM2) and three downregulated mRNAs (NSUN4, FBXO34, and AK4) and validated the differential expression of these mRNAs by qRT‒PCR in 22 samples from AAV patients. The results indicated that the expression levels of DEPDC1B and TPST1 in the exosomes of AAV patients were significantly greater than those in the exosomes of normal controls, whereas those of NSUN4 and AK4 were significantly lower. We found the four mRNAs that may reveal original and valuable information for MPO-AAV pathogenesis and treatment.

We acquired many signaling pathways related exosome mRNAs of MPO-AAV. The enrichment analysis results indicated that metabolic pathways, cell adhesion molecules, epithelial signaling, and MAPK signaling pathways are involved in AAV pathogenesis. Cell adhesion molecules and MAPK signaling pathways are considered to play crucial roles in autoimmune disease [30, 36, 37]. Studies have shown that plasma levels of extracellular vesicles increase when the vascular endothelium is activated or damaged [38, 39]. Exosomes may damage endothelium-dependent vasodilation and reduce NO, leading to endothelial dysfunction [40]. In addition, exosomes can bind to the endothelium and activate it by increasing the production of reactive oxygen species (ROS) [41]. Netosis is a special form of neutrophil death in which nuclear DNA is released from the broken nuclear membrane [42] and the broken plasma membrane forms NETs, which may lead to endothelial dysfunction through the activation of matrix metalloproteinases2 [43], leading to vasculitis. Moreover, studies have confirmed a positive correlation between proinflammatory cytokines and adhesion molecules [37].Therefore, we believe that endothelial injury and/or activation are essential features of AAV. Adhesion molecules in endothelial cells are upregulated during metabolism and inflammatory activation, which then mediate the adhesion and migration of white blood cells in blood vessels and promote inflammation and tissue damage. In general, cell adhesion molecules play a role in the pathogenesis of AAV. MAPK signaling pathways are also considered to play a crucial role in autoimmune disease [44, 45]. In the vascular wall, macrophages produce proinflammatory cytokines (IL-1β and TNF-α). These proinflammatory cytokines aggravate vascular inflammation through phosphorylation of signaling pathways, such as the MAPK pathway. During inflammation, adhesion molecules, cytokines, and chemokines are regulated by the MAPK signaling pathway. Increasing evidence suggests that the inhibition of signaling cascade phosphorylation is effective in treating inflammatory diseases [46, 47].

In AAV, ANCAs activate human neutrophils through the p38MAPK-mediated pathway, and this signaling cascade is responsible for the transfer of ANCA-specific antigens from cytoplasmic particles to the cell surface [48]. Many studies have confirmed that inflammatory damage to the vascular endothelium is caused by leukocyte infiltration in AAV [49, 50]. The patients we selected were all active AAV patients (with elevated BVAS-V3), and compared to those of healthy individuals, their ESR, CRP levels, and creatinine levels were significantly greater, indicating that the patients were experiencing a systemic inflammatory response. Considering the key role of inflammatory injury in endothelial cells in the pathogenesis of AAV, we speculate that the mRNAs from the exosomes of patients with active AAV may be absorbed by vascular endothelial cells and contribute to the induction of endothelial cell inflammation and neutrophil adhesion.

At present, there have not found the relationship between the four mRNAs and MPO-AAV. This is the first study to analyses of exosome mRNA expression profiles in MPO-AAV. An increase in the mRNAs DEPDC1B and TPST1 in AAV exosomes may promote the adhesion of neutrophils. DEPDC1B recognizes G protein-coupled receptors and regulates signaling pathways through effector and regulatory factors [51]. DEPDC1B interacts with diverse signaling molecules, including splicing regulatory molecules and transmembrane proteins. Studies have shown that DEPDC1B participates in cell adhesion, cell proliferation, and cell cycle regulation [52, 53]. Some studies have reported that the expression of the TPST1 gene may affect the tumor microenvironment and may even be related to the immunotherapy response in bladder cancer [54]. Neutrophils express TPST1 [55]. TPST1 plays a role in the production of proinflammatory cytokines in LPS-induced macrophages, and the downregulation of TPST1 inhibits LPS-induced IL-6 production [56]. The expression of TPST1, which is related to the immune response, is upregulated in the bone marrow monocytes of patients with rheumatoid arthritis [57]. TPST1 may play an important role in the development of inflammation. Therefore, We propose that the upregulation of DEPDC1B and TPST1 may contribute to the pathogenesis of autoimmune diseases (including MPO-AAV) by promoting cell adhesion and the expression of inflammatory cytokines. Additionally, there is no research on the relationship between the mRNA expression of NSUN4 and AK4 and autoimmune diseases, bue there are some studies have shown that the mRNA expression of NSUN4 and AK4 is related to tumors, but its role in autoimmune diseases needs further research [58–60].

In conclusion, The pathophysiological mechanisms of AAV are complex. According to the bioinformatics analysis of the differentially expressed mRNAs, we believe that the disease activity of AAV is related to an activation and imbalance of inflammation-related signaling pathways. this study revealed two mRNAs (DEPDC1B and TPST1) that were significantly upregulated in patients with AAV and two mRNAs (NSUN4 and AK4) that were significantly downregulated in patients with AAV. Subsequently, through correlation analysis, we found that these indicators were not related to disease activity, and we speculated that these mRNA interactions may be involved in the development or progression of AAV. These data suggest that DEPDC1B, TPST1, NSUN4 and AK4 may be key regulators of AAV. These mRNAs can partially predict the onset of AAV, targeting these mRNAs may be useful for inhibiting the inflammatory response in AAV, further studies are needed to understand the mechanisms underlying the effect of these target mRNAs on disease progression. However, the exact role of these mRNAs in the pathogenesis of AAV and their diagnostic and prognostic value for this disease are still unclear. This study detected the serum exosomal mRNAs of patients with AAV by high-throughput sequencing technology and further confirmed that exosomal mRNAs can be used for the early diagnosis and treatment of AAV. However, the sample size of this study was small, and thus should be further experimentally verified with an expanded sample size.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 17 kb)

Abbreviations

ANCA Antineutrophil cytoplasmic antibody

AAV ANCA-associated vasculitis

BVAS-V3 Birmingham vasculitis activity score-version 3

CRP C-reactive protein

EGPA Eosinophilic granuloma with polyangiitis

ESR Erythrocyte sedimentation rate

GO Gene ontology

HC Healthy control

KEGG Encyclopedia of genes and genomes

MAPK Mitogen-activated protein kinase

miRNA MicroRNA

MPO Myeloperoxidase

mRNA Messenger RNA

MPA Microscopic polyangiitis

ncRNA Noncoding RNA

NETs Neutrophil extracellular traps

PR3 Neutrophil proteinase 3

qRT‒PCR Real-time quantitative polymerase chain reaction

ROS Reactive oxygen species

SD Standard deviation

GPA Granuloma with polyangitis

Treg T-regulated cell

Th1 T helper 1

Acknowledgements

We thank all the patients for their enthusiastic participation in the study.

Author contributions

YF Chen contributed to the study design, experiment conduct, data analysis and drafting of the article. X Qian, Y Wang and SQ Ge contributed to sample collection, experiment conduct and data analysis. YF Chen and ZW Shuai reviewed and edited the article.

Funding

The current study received support from the Basic and Clinical Cooperative Research Promotion Program of Anhui Medical University (2021xkjT034).

Data availability

Sequence data that support the findings of this study have been deposited in the NCBI with the primary accession code PRJNA1114026.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval and consent to participate

All subjects signed informed consent forms in accordance with the ethical principles of the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of Anhui Medical University (PJ2020-06-11).

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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