Clinical value of serum JKAP in acute ischemic stroke patients

Abstract Background Jun N‐terminal kinase pathway‐associated phosphatase (JKAP) regulates neuronal function, T helper (Th) 1/2/17 cell differentiation, and inflammatory process, but its clinical role in acute ischemic stroke (AIS) patients remains unclear. Hence, this study intended to evaluate JKAP level and its relationship with disease severity, Th1, 2, 17 secreted cytokines, adhesion molecules, and prognosis of AIS patients. Methods Serum JKAP of 122 AIS patients and 50 controls was detected by ELISA. For AIS patients only, Th1, 2, 17 secreted cytokines IFN‐γ, IL‐4, IL‐17; TNF‐α, ICAM‐1, and VCAM‐1 were also detected by ELISA. Results JKAP was decreased in AIS patients compared with controls (46.350 (interquartile range (IQR): 34.250–59.875) pg/ml vs. 84.500 (IQR: 63.175–113.275) pg/ml, p < 0.001), which could distinguish AIS patients from controls (area under curve (AUC): 0.810, 95% confidence interval (CI): 0.732–0.888). In AIS patients, JKAP negatively linked with the National Institutes of Health Stroke Scale (NIHSS) score (rs = −0.342, p < 0.001); besides, it was positively related to IL‐4 (rs = 0.213, p = 0.018) and negatively associated with IL‐17 (rs = −0.270, p = 0.003) but not related to IFN‐γ (rs = −0.146, p = 0.109). Furthermore, elevated JKAP associated with declined TNF‐α (rs = −0.219, p = 0.015) and ICAM‐1 (rs = −0.235, p = 0.009) but not related to VCAM‐1 (rs = −0.156, p = 0.085). Besides, declined JKAP was linked with 2‐year recurrence (p = 0.027) and 3‐year recurrence (p = 0.010) in AIS patients; while JKAP was not related to 1‐year recurrence or death risk (both p > 0.050). Conclusion JKAP may sever as a candidate prognostic biomarker in AIS patients, indicating its potency for AIS management.

Conclusion: JKAP may sever as a candidate prognostic biomarker in AIS patients, indicating its potency for AIS management.

K E Y W O R D S
acute ischemic stroke, disease severity, inflammation cytokines, Jun N-terminal kinase pathway-associated phosphatase, prognosis of AIS is still a large challenge with high recurrence rate (1-year recurrence rate ranging from 7% to 20%), fatality rate (around 21% 1month death rate), and disability rate (approximately 36%-71%). 5,[8][9][10] Hence, aiming to improve the management of AIS, it is meaningful to explore biomarkers which could help clinicians identify high-risk AIS patients and monitor their prognosis.
Jun N-terminal kinase pathway associated phosphatase (JKAP), also named dual-specificity phosphatase 22 (DUSP22), is a tyrosine-specific protein that dephosphorylates mitogenactivated protein (MAP) kinase. [11][12][13][14] According to previous studies, JKAP not only regulates neuronal function but also moderates immune and inflammatory process in several neurological and inflammation-mediated diseases (including Parkinson's disease, Alzheimer's disease, sepsis, etc.). [15][16][17] For instance, one study finds that JKAP inhibits CD4 + T-cell activation as well as its differentiation into T helper (Th) 1 and Th17 cells in Parkinson's disease patients, but JKAP is not related to Th2 cells. 15 Besides, JKAP also associates with stenosis of blood vessels and involves in ameliorating the neointimal hyperplasia induced by cessation of blood flow. 18,19 Combining above aspects, we speculated that JKAP might participate in AIS pathogenesis and have potential to work as a biomarker in AIS patients, while there is no clinical study reporting relevant findings.
Therefore, this study detected serum JKAP level in 122 AIS patients and 50 controls, aiming to evaluate its correlation with disease severity, Th1, 2, 17 secreted cytokines, adhesion molecules, and prognosis of AIS patients.

| Participants
From January 2017 to January 2018, 122 newly diagnosed AIS patients were serially included in this study. The inclusion criteria were as follows: (i) newly confirmed as AIS in accordance with the guideline issued by American Stroke Association (ASA) 20 ; (ii) aged over 18 years; (iii) absent of intracranial hemorrhage; (iv) willing to provide peripheral blood (PB) samples; (v) willing to be followed up regularly. The patients conforming to the following conditions were excluded from the study: (i) complicated with severe infection, inflammatory disease or immune system disease; (ii) had history of solid tumor or hematological disease. In addition, between January 2017 and January 2018, the study also included 50 subjects with at least 2 of the high-risk factors (smoke, hypertension, hyperlipidemia, hyperuricemia, diabetes mellitus, and chronic kidney disease (CKD)) as controls. For study analysis, the age range and gender ratio of controls were matched to those of AIS patients: age range, 50-80 years old; gender ratio, 3:2 (male vs. female). The controls who had a prior history of stroke were ineligible for the study, and the exclusion conditions for AIS patients were also appropriate for the controls. The study was approved by the Ethics Committee. All subjects provided the written informed consents.

| Data document and sample preparation
Demographics and underlying diseases of all subjects were documented for study analysis, and the National Institutes of Health Stroke Scale (NIHSS) score of AIS patients were obtained for the assessment of disease severity. Additionally, PB samples were collected from all AIS patients after admission, as well as from all controls after recruitment, then serum was isolated for the further detections.

| Sample assessment
All collected serum samples were applied to evaluate JKAP level by enzyme-linked immunosorbent assay (ELISA) using commercial

| Thrombolysis treatment
For AIS patients within 3 h of onset, intravenous thrombolytic therapy was implemented; for AIS patients within 6 h of onset, arterial thrombectomy, and vascular intervention were adopted; for AIS patients within 9 h of onset, mechanical thrombectomy was performed under imaging guidance.

| Follow-up
All AIS patients were followed up regularly until loss to follow-up, death, or up to 36 months. The final date of follow-up was January 31, 2021. During 36-month follow-up, disease recurrence and patient's death were recorded for the further analysis.

| Statistics
Graphics were constructed using GraphPad Prism V.8.1.1 (GraphPad Software Inc.) and R V.4.0.5 (available at: https://www.r-proje ct.org/), and statistical analyses were completed using SPSS V.24.0 (IBM). Difference of JKAP between AIS patients and controls was compared using Wilcoxon rank sum test, and the abilities of JKAP in distinguishing AIS patients from controls, recurrence patients form non-recurrence patients, and death patients form survivors were evaluated using receiver operating characteristic (ROC) curves.
Among AIS patients, correlation of JKAP with NIHSS score, cytokines, and adhesions molecules was determined using Spearman's rank correlation test. Correlations between JKAP and underlying diseases were analyzed using Wilcoxon rank sum test. Comparisons of JKAP between the patients with recurrence and without recurrence as well as between the dead patients and the survivors were analyzed using Wilcoxon rank sum test. Multivariate Cox's regression analysis with forward stepwise method was conducted to investigate the potential factors affecting recurrence risk and death risk in AIS patients.

| Characteristics of AIS patients
Moreover, the clinical characteristics of the controls were listed in Table S1.   (Table S2).  In order to further verify whether JKAP was independent factor of recurrence risk and death risk of AIS, the multivariate Cox's regression analysis was conducted, which exhibited that higher JKAP

presses the differentiation of T cells into Th17 cells in inflamma-
tory bowel disease patients. 24 Another study exhibits that JKAP is negatively correlated with Th1 and Th17 cell proportion in sepsis patients. 25 However, related studies conducted on AIS patients are limited. In this study, upregulated JKAP was associated with Several evidences illustrate that JKAP involves in the inflammation process in Crohn's disease, rheumatoid arthritis, and other inflammation-mediated diseases. 14,27 For instance, one study shows that downregulated JKAP level is related to exacerbated inflammation level in Crohn's disease. 14 Besides, it is also reported that JKAP limits  In conclusion, serum JKAP correlates with disease severity, Th2 and Th17 secreted cytokines, ICAM-1, and recurrence in AIS patients, indicating its potency to realize the risk stratification and consequently help clinicians provide individual treatment to each AIS patient.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.