The clinical significance of spondin 2 eccentric expression in peripheral blood mononuclear cells in bronchial asthma

Abstract Background Bronchial asthma (BA) was a heterogeneous disease characterized by chronic airway inflammation. Spondin 2 (SPON2) was reported to be implicated in the integrin pathway, protein metabolism, and drug‐induced lupus erythematosus. The purpose of this study was to evaluate the significance of SPON2 in BA diagnosis and treatment. Methods Peripheral blood samples were obtained from 137 BA pediatric patients (61 mild‐to‐moderate BA and 76 severe BA) and 59 healthy children. Subject's information, clinical indexes, pulmonary ventilation functions were recorded in the two groups. Peripheral blood mononuclear cells (PBMCs) were isolated from patients’ samples. qRT‐PCR and ELISA assays were employed to examine the levels of SPON2 and inflammatory cytokines, respectively. Pearson's correlation analysis confirmed the association between SPON2 and inflammatory cytokines. Receiver operating characteristic (ROC) analysis was used to evaluate the potentials of SPON2 in terms of BA detection and discriminating against the severity of BA. Results Bioinformatics analysis showed that SPON2, OLFM4, XIST, and TSIX were significantly upregulated, while KDM5D and RPS4Y1 were reduced in BA. GO analysis verified that these six genes were mainly involved in neutrophil degranulation, neutrophil activation involved in immune response, neutrophil activation, and neutrophil‐mediated immunity. After isolating PBMCs, we found that SPON2 was remarkably increased in BA pediatric group compared with healthy children, and the relative levels of SPON2 were related to the severity of BA. The receiver operating characteristic (ROC) analysis revealed the high potentials of SPON2 in BA diagnosis (AUC was 0.8080) and severity distinctions (AUCs were 0.7341 and 0.8541, respectively). Also, we found that there were significant differences in fractional exhaled nitric oxide (FeNO), forced expiratory volume in 1 s (FEV1)%, FEV1/ forced vital capacity (FVC)%, immunoglobulin E (IgE), serum eosinophils, and serum neutrophils between mild‐to‐moderate BA group and severe BA group. Finally, SPON2 was negatively correlated with IL‐12 while positively associated with IL‐4, IL‐13, and IL‐17A. Conclusions SPON2 was a viable biomarker for diagnosing and degree of severity in BA, providing more insight into exploring BA and treatment's pathogenesis.


| INTRODUC TI ON
Bronchial asthma (BA) was a heterogeneous chronic respiratory disease, 1 characterized by airway inflammation, 2 associated with variable airflow obstruction, airway hyperresponsiveness, and airway wall remodeling. 3 Some studies showed that BA was deemed a multifactor disease affected by genetic and environmental factors, 4 with clinical manifestations, including recurrent chest tightness, wheezing phlegm, dyspnea, or cough. [5][6][7] However, its exact mechanism remained mostly uncertain. Hence, BA is mostly hard to diagnose. BA threatened the health of 4.3% of the world's population, which was still on the rise and increased the burden on families and health care systems. 8 More importantly, the health of children with BA was inevitably affected as well. 9 To date, the bronchial provocation test was a standard method to check airway hyperresponsiveness. 10 Due to safety considerations and the lack of reliable BA detection methods, the clinical application of children excitation tests was severely limited. 11 Therefore, it was imperative to find novel biomarkers to detect and treat BA, thereby contributing to early intervention and BA treatment.
Eosinophils (EOS) were recognized in 1879 by Paul Ehrlich. 12 However, for 100 years, the role of EOS in BA remained unknown. At the beginning of the 21st century, EOS was poorly found in peripheral blood and lung tissues in healthy people. 13 Meanwhile, in 1975, Horn et al 14 found that most BA patients' severity was positively correlated with increased EC levels in blood sputum, as did their airway responsiveness. Subsequently, some scholars also verified that once BA patients inhaled allergens, EOS levels were prominently increased in bronchoalveolar lavage fluid (BALF) and lung tissues. 15,16 BA was considered a chronic inflammatory disease dominated by mast cells (MC) and EOS infiltration. 17 According to a previous study, spondin 2 (SPON2) was confirmed to be associated with EOS. 18 SPON2, a cell adhesion protein, facilitated adhesion, and outgrowth of embryonic neurons. 19,20 Through GO analysis, SPON2 was found to be involved in the initiation of the innate immune response, 21 positive regulation of interleukin-6 (IL-6), macrophage cytokine, and tumor necrosis factor production. [22][23][24] These studies above suggested the potential of SPON2 in BA. Here, in this study, we detected the role of SPON2 in detecting pediatric BA patients from healthy children and its clinical application in treatment.

| Bioinformatics analysis
The original differentially expressed genes used in this study were downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO; https://www. ncbi.nlm.nih.gov/geo/). Gene ontology (GO) enrichment analysis was implemented using DAVID (http://david.abcc.ncifc rf.gov/). GO terms with values less than 0.05 (molecular functions, biological processes, and cellular components) were considered to be significantly enriched for differentially expressed genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was a database resource for understanding the high-level functions and effects of biological systems (http://www.genome.jp/kegg/).

| Lung function assessment and IgE level measurement
Lung function assessment was evaluated through a portable pediatric spirometer; this evaluation has been done in triplicate to get the best value. IgE levels were analyzed using a human ELISA IgE kit following the manufacturer's protocol.

| Quantitative real-time polymerase chain reaction assay (qRT-PCR)
Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen) and quantified by spectrophotometer (absorbance at 260 nm and 280 nm). Then, cDNA was transcribed and synthesized using the cDNA Synthesis Kit (Vazyme) following the manufacturer's instructions. Afterward, qRT-PCR was conducted using SYBR Green Assay Kit (Vazyme) on an ABI 7500 system. The thermal cycling conditions were as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation 94°C for 10 s, and annealing at 72°C for 10 s. The expression of SPON2 was normalized to housekeeping gene GAPDH and cal-

| Functions of differentially expressed genes in BA
Using bioinformatics analysis, we identified six abnormally expressed genes in BA (shown in Table 1). Among them, SPON2, OLFM4, XIST, and TSIX were increased while RPS4Y1 and KDM5D were decreased ( Figure 1A). Moreover, the GO analysis in Figure 1B further demonstrated that these genes were mainly implicated in neutrophil degranulation, neutrophil activation involved in immune response, neutrophil activation, and neutrophil-mediated immunity.

| Eccentric expression of SPON2 concerning the varying degree of BA
In our pre-experiments, we measured SPON2, OLFM4, XIST, TSIX, KDM5D, and RPS4Y1 expressions in BA patients and healthy children detected using qRT-PCR analysis. However, there were no significant differences among OLFM4, XIST, TSIX, KDM5D, and RPS4Y1 levels between BA patients and healthy children; hence, we chose SPON2 to conduct the following experiments.
The result in Figure 2A showed that SPON2 was dramatically increased in BA patients than in healthy controls (p < 0.01). Moreover, through FEV1% levels, BA patients were further divided into mild-tomoderate and severe groups. As shown in Figure 2B, the expression of SPON2 was gradually increased with the severity of the BA (p < 0.01).

| The diagnostic significance of SPON2 in BA
The ROC value of SPON2 in Figure 4A  Furthermore, we also conducted a ROC analysis of SPON2 to predict the severity of BA. As shown in Figure 4B,4C, the AUCs of SPON2 concerning differentiating mild-to-moderate BA and a severe BA from healthy controls were 0.7341 (95% CI = 0.6335~0.8347) and 0.8541 (95% CI = 0.7832~0.9251), respectively.

| DISCUSS ION
It was generally accepted that BA in children was caused by multiple viral and bacterial infections or exposure to specific allergens, 25 some of which may eventually develop into persistent BA, seriously affecting children's health. Since BA's etiology and pathogenesis were still unclear, its treatment was relatively chaotic, and the abuse of hormones and antibiotics remained terrible, 26 28 Hence, it was of great significance to diagnose and treat BA as early as possible to arrest BA development.
BA was reported to be associated with immune system activation, addressing a pivotal role in disease progression. 29 Previous studies verified that BA was mainly associated with IgE-related T helper type 2 regulation, 30 mast cells, 31 and eosinophil recruitment. 32 Furthermore, studies also showed the importance of B cells, T cells, macrophages, and dendritic cells in BA development. 32, was mainly produced by activated T cells 34 and had immunomodulatory effects on B cells, T cells, mast cells, and macrophages. 35,36 Another study illustrated that IL-4 could enhance the antigenic ability of B cells. 37 As a growth factor secreted by T cells, IL-4 can also  38 stimulating mast cell proliferation. 39 As reported, IL-12 was the determinant of Th1 cellular immune response, 40 which can effectively promote the production of Th1-related cytokines such as IFNγ. 41 The role of IL-12 concerning regulating Th1/Th2 balance contributed to BA treatment as well. 42 Previous studies have reported that IL-12 was significantly downregulated in BA patients than healthy. 43

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.