The correlation of JKAP with risk, severity, inflammation and in-hospital mortality of severe acute pancreatitis


 Background

This study aimed to investigate the predictive value of JNK pathway-associated phosphatase (JKAP) level for severe acute pancreatitis (SAP) risk, and its association with disease severity, inflammation and in-hospital mortality in SAP patients.

Methods

Our study recruited 50 SAP patients, 50 moderate-severe acute pancreatitis (MSAP) patients, 50 mild acute pancreatitis (MAP) patients and 50 healthy controls. And the serum samples were obtained from all acute pancreatitis patients within 24 hours after admission and from health controls at their enrollment to detect JKAP level by enzyme-linked immunosorbent assay.

Results

JKAP level was decreased in SAP patients compared with healthy controls, MSAP and MAP patients. And receiver operating characteristics (ROC) curve analysis revealed that JKAP could not only distinguish SAP patients from healthy controls (AUC: 0.914, 95%CI: 0.857-0.971), but also differentiate SAP patients from MAP patients (AUC: 0.869, 95%CI: 0.802-0.937) and MSAP patients (AUC: 0.712, 95%CI: 0.610-0.813). In SAP patients, JKAP was negatively correlated with Ranson score, acute physiology and chronic health care evaluation II (APACEH II) score, sequential organ failure assessment (SOFA) score and C-reactive protein (CRP). And lower JKAP level, higher CRP level, Ranson score, APACEH II score and SOFA score were associated with increased in-hospital mortality in SAP patients. Additionally, ROC curve analysis showed that JKAP could predict decreased in-hospital mortality in SAP patients (AUC: 0.720, 95%CI: 0.526-0.914).

Conclusions

JKAP might serve as a biomarker for disease risk and management for SAP.

3 Background Acute pancreatitis (AP), a sudden inflammatory of the pancreas, is frequently caused by pancreatic ductal obstruction secondary to gallstones and alcohol misuses, which affects 34 per 100,00 person-years worldwide [1,2]. Most of the AP patients experience mild AP (MAP), while approximately 20% of AP patients develops severe AP (SAP) with a mortality rate approximately 30% [3]. SAP is characterized by multiple organ failure as well as necrosis of the pancreas and the surrounding tissues [4]. Due to the facet of rapid deterioration and high mortality of SAP, early identification of disease severity becomes essential for the evaluation of clinical treatment and the management of SAP [5]. Many multi-factoring scoring systems such as acute physiology and chronic health evaluation II (APACHE II) score and Ranson score have been widely applied for assessing SAP severity and predicting prognosis in clinical practice [3,6]. However, under some circumstances such as at the time of initial presentation of SAP, the use of APACHE II score and Ranson score may be complex and with low sensitivity [3,7]. Therefore, it is essential to explore novel sensitive biomarkers with the potential for assisting disease monitoring and improving prognosis in SAP patients.
JNK pathway-associated phosphatase (JKAP, also referred as dual-specificity phosphates (DUSP) 22) specifically activates the kinase JNK and functions as a tyrosine phosphate to dephosphorylate as well as inactivate focal adhesion kinase [8]. JKAP is abundantly expressed in various types of mammalian cells such as T cells, B cells and natural killer cells, which participates in important biological processes such as inflammatory and immune responses in human autoimmune and inflammatory disorders [9]. A few studies have displayed that the downregulation of JKAP is negatively correlated with disease severity and inflammation in inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE) and sepsis [9][10][11]. Based on the role of JKAP in the regulation of inflammation and immune response and its association with the etiology of several inflammatory disorders, we hypothesized that JKAP might be involved in the development and progression of SAP as well. However, no studies have been done in evaluating the role of JKAP in SAP until now. Therefore, this study aimed to investigate the predictive value of JKAP level for SAP risk, and its association with disease severity, inflammation and inhospital mortality in SAP patients.

Subjects
Between January 2016 and March 2019, 50 SAP patients, 50 MSAP patients and 50 MAP patients were recruited from our hospital in this study. All patients were older than 18 years and had a diagnosis of AP according to the Atlanta classification and definitions of acute pancreatitis [12]. While the patients were excluded if they were diagnosed as biliary duct-related or pancreatic-related carcinomas presenting with AP, concomitant with autoimmune diseases, complicated with malignancies, pregnant or breast feeding. The severity of AP was identified in accordance with the Atlanta classification and definitions [12]: (i) SAP was characterized by persistent organ failure (persisted for >48 h); (ii) MSAP was characterized by the presence of transient organ failure (presented for <48 h) or local complications (including peripancreatic fluid collections and acute necrotic collections) or systemic complications (exacerbations of underlying co-morbidities related to the acute pancreatitis) in the absence of persistent organ failure; (iii) MAP was characterized by the absence of organ failure and the absence of local or systemic complications. In addition, the current study also enrolled 50 healthy controls whose age and gender were matched with recruited AP patients, between April 2019 and May 2019. The general healthy status of healthy controls was confirmed by the physical examination, and all of them had no history of pancreatic-related diseases. The present study was approved by the Ethics Committee of our hospital, and all subjects provided the written informed consents.

Data collection
Demographic information including age and gender of all AP patients were documented on their recruitment. Also, the etiology, the C-reaction protein (CRP) served as inflammatory level markers, the Ranson score evaluated within 48 hours, the APACEH II score assessed within 24 hours, and the sequential organ failure assessment (SOFA) score assessed within 24 hours were recorded after patients were admitted to the hospital. And the etiologybased treatment and conventional treatment were administered to AP patients according to the IAP/APA evidence-based guidelines of for the management of acute pancreatitis [13]. Moreover, all AP patients were followed up closely until they died in the hospital or were discharged from the hospital, during which, there were 10 deaths among SAP patients, 3 deaths among MSAP patients, and no one died among MAP patients.
Accordingly, SAP patients were further analyzed by being categorized as SAP survivors and SAP deaths. Besides, demographic characteristics (age and gender) of healthy controls were also collected on their enrollment.

Blood sample collection and measurement
Venous blood samples of AP patients were collected into vacuum tubes within 24 hours after admission to the hospital. After collection, the serum was isolated from the blood samples through centrifugation at 3000 rpm for 5 min, subsequently, the serum JKAP level was measured by the enzyme-linked immunosorbent assay (ELISA) using the Human JKAP ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China), and all experiments were performed according to the protocols provided by the manufacturers. In addition, the blood samples of healthy controls were also collected on their enrolment, and the process of serum isolation and JKAP measurement were performed as same as that in AP patients.

Statistical analysis
Normality was checked for all continuous variables using Shapiro-Wilk test, and the continuous variables were described as mean and standard deviation (SD) if normally distributed, or as median and interquartile range (IQR) if non-normally distributed.
Categorical variables were displayed as count (percentage

Baseline characteristics
There were 50 healthy controls, 50 MAP patients, 50 MSAP patients and 50 SAP patients enrolled in our study (   Figure 2D),, MSAP patients from healthy controls ( Figure 2E),, MAP patients from healthy controls ( Figure 2F) as well. These data implied that JKAP could not only differentiate SAP patients from health controls, but also distinguish SAP patients from MAP and MSAP patients.

Comparison of patients' characteristics between survivors and deaths in SAP patients
SAP patients were further divided into SAP survivors (N = 40) and SAP deaths (N = 10) based on the in-hospital medical records. SAP deaths exhibited lower median JKAP (P = 0.032), higher median CRP (P = 0.032), median Ranson score (P < 0.001), median APACEH II score (P = 0.005) and median SOFA score (P = 0.010) compared with SAP survivors ( Table 2).. Furthermore, there was no difference of mean age (P = 0.589), gender (P = 0.756) or etiology (P = 0.171) between SAP survivors and SAP deaths. These disclosed that lower median JKAP, higher inflammation and disease severity scores were associated with increased in-hospital mortality in SAP patients.
Predictive value of JKAP for in-hospital mortality in SAP patients ROC curve analysis displayed that JKAP was of an acceptable value in differentiating SAP survivors from SAP deaths (AUC: 0.720, 95%CI: 0.526-0.914) (Figure 4).. Regarding commonly used assessing score systems and inflammation marker, Ranson score was of a good value in distinguishing SAP survivors from SAP deaths (AUC: 0.886, 95%CI: 0.756-1.000). And APACEH II score (AUC: 0.783, 95%CI: 0.607-0.914), SOFA score (AUC: 0.761, 95%CI: 0.600-0.923) and CRP (AUC: 0.720, 95%CI: 0.522-0.920) were with a fair value in differentiating SAP survivors from SAP deaths. By comparison, the predictive value of JKAP for in-hospital mortality was similar with that of APACEH II score, SOFA score and CRP numerically, while was inferior to that of Ranson score. These data suggested that JKAP level was of an acceptable value for predicting decreased in-hospital mortality in SAP patients.

Discussion
In this study, we discovered that: (1) JKAP could not only differentiate SAP patients from health controls, but also distinguish SAP patients from MAP and MSAP patients. (2) JKAP level was negatively correlated with disease severity and inflammation in SAP patients. (3) JKAP was with an acceptable value in predicting decreased in-hospital mortality in SAP patients.
SAP is a serious and often lethal disorder with complex pathophysiology, in which autoimmune responses and inflammation are the major contributors [14]. The deterioration of SAP occurs rapidly within a few hours or days after the onset of symptoms [5]. It starts with systemic inflammatory response through mediating the release of proinflammatory mediators, resulting in pulmonary, cardiovascular and renal insufficiency [15][16][17]. And the late deterioration of organ dysfunction or failure mainly results from secondary infection of pancreatic or peripancreatic necrosis [18]. Thus, it is important to investigate new biomarkers for determining the SAP risk and severity as well as forecasting the mortality of SAP patients.
JKAP is a member of the DUSPs family, which specifically mediates the JNK pathway and subsequent cytokine-induced JNK activation [19]. Besides, JKAP also inhibits T-cell receptor signaling through inactivating Lck, resulting in suppression of T-cell-medicated immunity and autoimmunity [8,9]. Previous studies have revealed that the downregulation of JKAP is involved in the development and progression of several autoimmune and inflammatory diseases such as SLE and sepsis [10,11]. For instance, JKAP expression in peripheral blood T cells is lower in SLE patients than that in health volunteers, and the downregulation of JKAP expression in T cells is associated with higher daily urinary protein amounts and poor renal outcomes in SLE patients [10]. Another study illuminates that the serum JKAP is downregulated in sepsis patients compared with healthy volunteers, and the downregulation of JKAP is correlated with advanced disease severity (APACHE II score and SOFA score) and systemic inflammation (CRP, PCT, TNF-α, IL-1β, IL-6 and IL-17) in sepsis patients [11]. Considering that SAP is an extremely severe immune and inflammatory disease, we hypothesized that JKAP might be dysregulated and play an Previous studies have elucidated that downregulation of JKAP is associated with poor prognosis in IBD and sepsis [9,11]. For example, low JKAP level predicts active status of Crohn's disease and ulcerative colitis [9]. Another study reports that JKAP presents a good value in differentiating deaths from survivors in sepsis patients [11]. However, no study reports the association of JKAP level with prognosis in SAP patients. Our study exhibited that JKAP level was of an acceptable value in predicting decreased in-hospital mortality (AUC: 0.720, 95%CI: 0.526-0.914) in SAP patients. These results might be explained by that: (1) JKAP inhibited immune and autoimmune response, subsequently suppressed inflammatory responses and decreased SAP severity, thus, led to lower in-hospital mortality in SAP patients. (2) Higher JKAP level was correlated with lower SOFA score (decreased severity of organ dysfunction) and CRP level (decreased inflammation, immune response and disease severity), thus, contributed to lower in-hospital mortality in SAP patients. Additionally, we also observed that the predictive value of JKAP for in-hospital mortality was similar compared with APACEH II score, SOFA score and CRP, while inferior compared with Ranson score. These suggested that JKAP might serve as an additional biomarker to assist commonly applied multi-factorial scoring systems for predicting prognosis in SAP patients.
There were some limitations in our study: (1) The sample size was relatively small, which might reduce the statistical power. (2) The mortality of all SAP patients was only measured during their hospitalization, thus, the long-term predictive value of JKAP for prognosis needed to be further studied. (3) The detailed mechanism of JKAP in regulating the development and progression of SAP was not explored, thus, further experiments in investigating the detailed mechanism of JKAP in SAP should be carried out in the future.

Conclusions
In conclusion, the downregulation of JKAP level is correlated with increased risk, disease severity, inflammation and in-hospital mortality of SAP, which implies that JKAP exhibits the potential as a biomarker for disease monitoring and prognosis in SAP patients.     Ranson score, APACEH II score, SOFA score and CRP was analyzed by the Spearman's rank correlation test. P < 0.05 was considered significant. JKAP, JNK pathway-associated phosphatase; SAP, severe acute pancreatitis; APACEH II score, acute physiology and chronic health evaluation II score; SOFA score, sequential organ failure assessment score; CRP, C-reactive protein. ROC curve analysis of JKAP, Ranson score, APACEH II score, SOFA score and CRP for predicting in-hospital mortality. The ability of JKAP, Ranson score, APACEH II score, SOFA score and CRP in differentiating survivors from deaths in SAP patients. The performance of JKAP, Ranson score, APACEH II score, SOFA score and CRP in predicting in-hospital mortality was evaluated by ROC curve and AUC with 95% CI. ROC, receiver operating characteristic; JKAP, JNK pathway-associated phosphatase; APACEH II score, acute physiology and chronic health evaluation II