Persistently higher serum sCD40L levels are associated with mortality in septic patients

Soluble CD40 ligand (sCD40L) exhibits proinammatory and procoagulant effects. Recent data indicated that sCD40L plays a signicant role in septic patients. The aim of the present study was to determine sCD40L changes in surgical patients without sepsis (SWS) and in surgical sepsis patients (SS) during the rst three days at Intensive Care Unit (ICU) admission, and to observe the association between sCD40L and mortality.


Abstract
Background Soluble CD40 ligand (sCD40L) exhibits proin ammatory and procoagulant effects. Recent data indicated that sCD40L plays a signi cant role in septic patients. The aim of the present study was to determine sCD40L changes in surgical patients without sepsis (SWS) and in surgical sepsis patients (SS) during the rst three days at Intensive Care Unit (ICU) admission, and to observe the association between sCD40L and mortality.

Methods
Time changes in sCD40L levels were assessed for 3 days after ICU admission in 49 patients with SS and compared with 19 SWS. Serum sCD40L concentration was detected by ELISA. Survival at 28-days was used as the endpoint.
Results SS had signi cantly higher sCD40L levels than SWS and control patients. Advanced age (P = 0.023) was observed in the group of nonsurviving patients compared with surviving SS. We observed an association between sCD40L levels ≥ 1028.75 pg/ml at day 2 and 28-days mortality (odds ratio = 7.888; 95% con dence interval = 1.758 to 35.395; P = 0.007).

Conclusions
Septic patients show persistently higher circulating sCD40L levels in the rst three days at ICU admission, and it is likely that sCD40L on the day 2 may have a predictive value; thus, serum sCD40L could be used as a reliable biomarker and therapeutic target in sepsis.

Background
Sepsis is the overwhelming in ammatory host response to the infectious agent causing the overexpression of in ammatory mediators (1). Moreover, sepsis is also almost associated with coagulation abnormalities, present as manifest thromboembolic disease or clinically less apparent microvascular brin deposition (2). In sepsis, in ammation and coagulation are extensive cross-talk, which promotes the progress of disease and leads to organ dysfunction(3).
CD40 ligand (CD40L) and the soluble form of CD40L (sCD40L) are members of the tumor necrosis factor (TNF) family, they expressed in a diversity of cell types including B cells, epithelial cells, broblasts, endothelial cells (ECs), as well as in platelets. CD40L and sCD40L exhibit proin ammatory and procoagulant effects (4,5). Previous studies have found higher sCD40L levels in sepsis patients and that sCD40L levels are associated with mortality(6-8). Lorente's study also showed that persistent higher of sCD40L in sepsis patients at 1, 4 and 7 days predicted poor prognosis (9).
Surgical patients suffer from endothelial cell damage after surgery, postoperative in ammatory and blood coagulation changes will happen. Therefore, the purpose of this study was to determine sCD40L changes in surgical patients without sepsis (SWS)and in surgical sepsis patients (SS) during the rst three days at ICU admission, and to observe the relationship between sCD40L and mortality.

Patient characteristics
Baseline characteristics of 49 SS, 19 SWS and 6 age-and sex-matched healthy controls were shown in Table 1. The levels of sCD40L in healthy controls were 469.50 (406.83-547.36) pg/ml, and the sCD40L levels in SWS and SS were signi cantly increased,and the difference between the two groups was statistically signi cant (Table 1, P = 0.000 vs healthy controls). In addition, compared with SWS, SS also exhibited a higher incidence of higher APACHE-II (P = 0.001), SOFA (P 0.001), ISTH (P 0.001) and JAAM (P 0.001) scores, higher PT-INR(P = 0.001), brinogen(P = 0.022), FDP(P 0.001), D-dimer (P 0.001), lactate (P = 0.006), and 28 days mortality (P = 0.001) ( Table 1).  In SS, 20 patients died and 29 patients survived, all of whose demographic and clinical features are shown in Table 2 and supply Table 1. Non-surviving had higher serum sCD40L (P = 0.009), aPTT than surviving patients at day 2 (P = 0.020) and higher lactate at day 3 (P = 0.001). besides, higher age (P = 0.023) was observed in the group of non-surviving compared with surviving SS. We couldn't discover any signi cant differences in gender, presence of septic shock and the site of infection between non-survivor and survivor. In addition, on day1, 2, 3 at ICU admission, there were no statistically signi cant differences in APACHE II score, SOFA score, ISTH score, JAAM score, PT-INR, brinogen, FDP, D-dimer, platelet and leukocytes between the non-surviving and surviving groups of SS. The area under the curve (AUC) for serum sCD40L levels at day 2 (95% con dence interval (CI) = 0.570-0.871, P = 0.009) could predict mortality at 28 days, the sensitivity and speci city approached to 60.0% and 79.3%, respectively (Fig. 1). Kaplan-Meier survival analysis showed that patients with higher serum sCD40L levels at day 1(P = 0.035), day 2(P = 0.005), day 3(P = 0.003) had a risk of death at 28 days than patients with lower levels (Fig. 2)

Discussion
It can be concluded from this study that serum sCD40L levels of SS persistently increased signi cantly in the rst three days after admission to the intensive care unit, and the circulating sCD40L higher on the second day of admission in non-surviving than in surviving group. A novel nding of this study was that SWS also has a slight increase in sCD40L, but not as much as in SS.
Several studies have reported that sCD40L level can predict the prognosis of patients with sepsis (8,11). In our study, we found that serum sCD40L levels ≥ 1028.75 pg/mL at day 2 were associated with higher death risk during the 28-days period in the multiple logistic regression analysis. Serum sCD40L levels could be used as 28-days mortality biomarker. However, we did not nd a relationship between sCD40L levels and sepsis severity criteria such as, APACHE II score, SOFA score. We only observed higher lactatemia in non-surviving than in surviving SS at day 3 admission. The serum blood samples were obtained at admission to ICU, but APACHE II or SOFA was calculated at 24 hours of admission to ICU, we were not sure if this time-gap can affect the association between the two variables.
sCD40L with a dual prothrombotic and proin ammatory role. The sCD40L connects to circulating monocytes through its receptor CD40, promoting their adhesion to vascular endothelium. The sCD40L also binds to CD40 on endothelial cell surfaces. Studies have shown that sCD40L stimulates its own expression by interacting with CD40 on the surface of these cells (12). In sepsis, EC activation induced adhesion receptors, released in ammatory mediators such as interleukin(IL)-1, IL-6 and tumor necrosis factor (13,14). sCD40L also effect on neutrophil oxidative burst and neutrophil extracellular trap (5,15). Previous studies and our studies suggested that sCD40L has no correlation with other coagulation factors except tissue factor(TF) (8). The main reason was that activated ECs initiate the exogenous coagulation pathway by up-regulating TF and down-regulating the expression of thrombomodulin(16), favoring a local procoagulant status. In experimental models, sCD40L enhanced platelet activation and aggregation and induced thrombus formation (17). All these effects contribute to the development of organ dysfunction and death (18). After intraoperative operation, the ECs were damaged, and the body produced stress response and in ammatory factors (19). Therefore, sCD40L level was also increased after operation in patients with non-sepsis. In patients with sepsis, EC damage, intravascular microthrombus formation and production of in ammatory factors are more obvious, and sCD40L level is much higher than that in patients with simple surgery.
We have not found an association between serum sCD40L levels and platelet count, although 95% of sCD40L was derived from platelets (20). CD40L is stored in α-granules in unstimulated platelets, which undergo conformational changes during platelet activation, migrates to the surface of platelets and releases into the blood (21). Soluble CD40L can enhance platelet activation, aggregation, and plateletleukocyte conjugation. Therefore, sCD40L was shown to be implicated in platelet activated (12). Activated platelets via interaction with ECs play a key role in in ammatory and pro-coagulant response to a pathogen (22).
There were some limitations in our study. First, the sample size was relatively small. Second, the subjects came from a single center. Third, we only determined sCD40L levels admission in ICU for 3 days, but did not observe it for a week or longer, unable to better conclude the time course of serum sCD40L levels in sepsis patients. And nally, there are been reported an association between sCD40L levels and the activation function of platelets; we have not examined markers of platelet activation to analyzed a relationship with sCD40L levels.

Conclusions
Septic patients exhibit persistently higher circulating sCD40L levels in the rst three days at ICU admission, and it is likely that sCD40L on the day 2 may have a predictive value; thus, serum sCD40L could be used as a reliable biomarker and therapeutic target in sepsis. Receiver operation characteristic analysis using sCD40L levels ≥ 1028.75pg/mL at day 2 as 28-days mortality predictor