Monocyte programmed death ligand-1 expression is an early marker for predicting infectious complications in acute pancreatitis

The study protocol was approved by the Ruijin Hospital Ethics Committee of Shanghai Jiaotong University School of Medicine, China. Formal informed consent was obtained from patients or their next of kin. Between March 2014 and December 2015, 63 patients diagnosed with AP were recruited from the general intensive care unit (ICU) or emergency ICU of Ruijin Hospital; 32 healthy volunteers matched with sex and age were included as control subjects. AP diagnosis is most often established by the presence of two of the following three criteria: (1) abdominal pain consistent with the disease, (2) serum amylase and/or lipase greater than three times the normal upper limit, and/or (3) characteristic findings based on abdominal imaging [14]. The inclusion criteria were patients with AP aged 18 years or older who were admitted to the ICU within 24 h of symptom onset. Patients were excluded if any of the following criteria were present: suspected malignancy of the pancreas or biliary tree, a medical history of immunodeficiency or receiving immunosuppressive therapy, or nonpancreatic infection or sepsis caused by a second disease. All admissions were followed until discharge from the hospital or hospital mortality. Baseline characteristics, including age, sex, possible AP etiology, and Acute Physiology and Chronic Health Evaluation II (APACHE II) score were also collected and recorded.

AP severity was categorized as mild, moderately severe (local complication or transient organ dysfunction), or severe (persistent organ dysfunction) according to the revised Atlanta classification system [15]. Infected pancreatic necrosis, bacteremia, pneumonia, infected ascites, or urosepsis during admission and/or 90-day follow-up were considered IC [16]. Infected necrosis was defined as a positive culture of peripancreatic fluid or pancreatic necrosis obtained during the first percutaneous drainage or during the first surgical intervention. Bacteremia was defined by a positive blood culture sample. Pneumonia was defined by coughing, dyspnea, chest radiograph showing infiltrative abnormalities, and lowered arterial blood gas with positive sputum culture. Infected ascites was defined as a positive bacterial culture in aspirate of intraperitoneal fluid or abdominal fluid during surgical operation. Urosepsis was defined as dysuria with bacteremia on the same day. For patients without bacterial evidence, diagnosis of IC was made by experienced clinicians on the basis of clinical symptoms and signs of patients during admission and at 90-day follow-up. All infections were weighted equally; multiple infections in the same patient were considered one endpoint.

Blood samples were obtained from 63 patients with AP at days 1 and 3 after diagnosis with AP and from 32 healthy volunteers. After lysing red blood cells with fluorescence-activated cell sorting lysing solution (BD Biosciences, San Jose, CA, USA), cells were incubated in the dark at 4 °C for 30 minutes with the following fluoresceinated monoclonal antibodies and their isotype controls: fluorescein isothiocyanate (FITC)-labeled anti-CD4 (clone A161A1), FITC-labeled anti-CD14 (clone HCD14), phycoerythrin (PE)-labeled anti-PD-1 (clone EH12.2H7), PE-labeled anti-PD-L1 (clone 29E.2A3), and PE-labeled anti-human leukocyte antigen-DR (anti-HLA-DR) (clone L243; BioLegend, San Diego, CA, USA). Stained cells were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). The percentage of PD-1-expressing CD4⁺ lymphocytes was calculated as the percentage of PD-1⁺ cells in the total CD4⁺ lymphocyte population, and the percentage of PD-L1/HLA-DR-expressing CD14⁺ monocytes was calculated as the percentage of PD-L1⁺/HLA-DR⁺ cells in the total CD14⁺ monocyte population. Interleukin (IL)-10 concentration was measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA) in accordance with the supplied instructions.

Continuous variables are presented as mean ± SEM, and categorical variables are presented as absolute numbers and percentages. All variables were tested for normal distribution using the Kolmogorov-Smirnov test. Student’s t test was used to compare the means of continuous variables and the normality of data distribution; otherwise, the Mann-Whitney U test was used. Categorical data were tested using the χ² test. Correlations between PD-1/PD-L1 expression, lymphocyte count, and IL-10 concentration were analyzed with Spearman’s rank method. Discrimination was tested using the area under the ROC curve (AUROC) to assess the ability of APACHE II score, PD-L1 expression in CD14⁺ monocytes, and HLA-DR expression in CD14⁺ monocytes to predict IC. To evaluate the predictive value of the combination of APACHE II score and HLA-DR and PD-L1 expression levels on monocytes, we constructed a predictive logistic regression model including the three variables. The coefficients for HLA-DR, PD-L1, and APACHE II score were −7.765, 9.867, and 0.323, respectively, and the constant was −3.723. On the basis of this model, we created a new variable using the formula [0.323 × APACHE II score −7.765 × PD-L1 + 9.867 × HLA-DR −3.723] to calculate the AUROC further. All statistical tests were two-tailed, and P < 0.05 was considered statistically significant. Statistical analyses were performed using STATA 12.0 for Windows software (StataCorp, College Station, TX, USA).

A total of 63 patients with AP (22 women and 41 men) were included in the study. Median age at admission was 48 years (IQR 37–60). The etiologies of AP were gallstones in 30 patients, hypertriglyceridemia in 20, alcohol abuse in 7, and other etiologies in 6. The median APACHE II score upon AP diagnosis was 10.5 (IQR 8–16.5), indicating a high level of disease severity. According to the revised Atlanta classification, 3 patients were classified as mild, 28 as moderately severe, and 32 as severe. The clinical characteristics of these patients are summarized in Table 1.

Both PD-1 expression in CD4⁺ lymphocytes and PD-L1 expression in CD14⁺ monocytes were measured in each patient on day 1 (D1) and day 3 (D3) after AP onset (Fig. 1). The percentage of PD-1-expressing CD4⁺ lymphocytes on D1 and D3 was significantly increased in patients with AP compared with healthy volunteers (D1 P < 0.001; D3 P < 0.001) (Fig. 1). Likewise, the percentage of PD-L1-expressing CD14⁺ monocytes on D1 and D3 was notably increased in patients with AP compared with healthy volunteers (D1 P < 0.001; D3 P < 0.001) (Fig. 1). In addition, the percentage of HLA-DR-expressing CD14⁺ monocytes at each time point was significantly lower in patients with AP than in healthy volunteers (D1 P < 0.001; D3 P < 0.001) (Fig. 1).

It has been reported that the PD-1/PD-L1 system is involved in lymphocyte apoptosis and IL-10 production in sepsis [12, 13]. Thus, we explored the correlation between plasma IL-10 concentration, circulating lymphocyte count, and PD-1/PD-L1 expression. We found that circulating lymphocyte counts were inversely correlated with the percentage of PD-1-expressing CD4⁺ lymphocytes on D1 and D3 (r = −0.252, P = 0.047; and r = −0.297, P = 0.018, respectively). A negative correlation was also observed between circulating lymphocyte count and the percentage of PD-L1-expressing CD14⁺ monocytes on D1 (r = −0.302, P = 0.016) but not on D3. Moreover, positive correlations were observed between IL-10 concentration and the percentage of PD-L1-expressing CD14⁺ monocytes on D1 and D3 (r = 0.296, P = 0.019; and r = 0.459, P < 0.001, respectively). The percentage of PD-1-expressing CD4⁺ lymphocytes on D1 was positively correlation with IL-10 concentration (r = 0.255, P = 0.044) but not on D3 (Table 2).

To assess the association between PD-1/PD-L1 expression and IC, patients were divided into two groups according to the presence of IC, namely IC (patients with IC, n = 25) and non-IC (patients without IC, n = 38) groups. In the patients with IC, 24% had bloodstream infection, 24% had pneumonia, and 79.2% had infected (peri)pancreatic necrosis (Table 1). There were no significant differences between these two groups in regard to sex (P > 0.05) (Table 3). Patients in the IC group were significantly older than those in the non-IC group. The APACHE II scores in the IC group were significantly higher than in the non-IC group (P < 0.05). Patients with IC showed significantly greater PD-1 expression in CD4⁺ T cells on D1 and D3 than patients without IC (D1 P < 0.05; D3 P < 0.05) (Table 3). Likewise, the percentage of PD-L1-expressing CD14⁺ monocytes on D1 and D3 was higher in the IC group than in the non-IC group (D1 P < 0.05; D3 P < 0.05) (Table 3).

Moreover, biomarkers commonly used to assess immune status were measured, and we found that the circulating lymphocyte counts and percentage of HLA-DR-expressing CD14⁺ monocytes on D1 and D3 were decreased in the IC group compared with the non-IC group. Additionally, levels of the anti-inflammatory cytokine IL-10 in the IC group were increased compared with the non-IC group (Table 3).

Univariate logistic regression analysis (Table 4) was performed to determine the predictive power of age, APACHE II score at admission, lymphocyte count on D1, percentage of HLA-DR-expressing CD14⁺ monocytes on D1, percentage of PD-1-expressing CD4⁺ lymphocytes on D1, and percentage of PD-L1-expressing CD14⁺ monocytes on D1 for IC. The variables showed significant predictive value within univariate analysis and were included in further stepwise multivariate logistic regression (Table 4). The regression analysis detected APACHE II score at admission (OR 1.305, 95% CI 1.079–1.578, P = 0.006), percentage of HLA-DR-expressing CD14⁺ monocytes on D1 (OR 0.923, 95% CI 0.869–0.981, P = 0.01), and percentage of PD-L1-expressing CD14⁺ monocytes on D1 (OR 1.098, 95% CI 1.005–1.200, P = 0.036) as independent significant predictors of IC in patients with AP.

The predictive accuracy value for IC in AP was determined by ROC curve analysis (Table 5, Fig. 2). The AUROC values were 0.772 (95% CI 0.649–0.896, P < 0.01) for APACHE II score at admission, 0.652 (95% CI 0.506–0.798, P = 0.043) for percentage of HLA-DR-expressing CD14⁺ monocytes on D1, and 0.708 (95% CI 0.573–0.842, P < 0.01) for percentage of PD-L1-expressing CD14⁺ monocytes on D1. The combination of these variables had a moderate to high accuracy for predicting IC (95% CI 0.816–0.991, P < 0.01). Table 5 shows the chosen cutoff points, sensitivity, specificity, and positive and negative predictive values of these variables for predicting development of IC.