Platelet soluble NOX2 as a new biomarker in patients with sepsis


 Background

Substantial evidence supports the view that NOX2 may function as a pivotal regulator in various physiological and pathological processes. However, to date, the interplay between platelet soluble NOX2 levels and sepsis remains unclear. The overall aim of this work is to obtain a better insight into whether or not platelet NOX2 is involved in sepsis.
Methods

The levels of platelet soluble NOX2, from 31 patients diagnosed with Gram-positive sepsis, 36 patients with Gram-negative sepsis and 45 healthy individuals, were measured with sandwich ELISA kits that we developed.
Results

In this work, we showed that platelet NOX2 concentrations were significantly lower in healthy individuals compared to patients with sepsis (P < 0.01), and that platelet NOX2 values were significantly correlated with the platelet activation markers, including soluble CD40L and soluble P-selectin. Interestingly, we also found that platelet NOX2 values in patients with Gram-positive sepsis were higher than those in patients with Gram-negative sepsis.
Conclusions

Platelet NOX2 levels maybe an important indicator of the pathogenesis of sepsis-induced platelet activation.


Background
Sepsis is a life-threatening condition caused by an excessive in ammatory immune response to an infection. Remarkably, it has been reported that more than 30 million people worldwide have been suffered from sepsis each year [1]. Currently, it is widely accepted that sepsis can induce the dysregulation of coagulation system, which in turn contributes to the pathogenesis of the sepsis syndrome [2][3]. Over the years, growing evidence indicates that platelets act as crucial regulator in the coagulation system, and that these anucleate cell fragments play an important role in immunity and host defense [4]. Additionally, many micro-organisms including bacteria, viruses and fungi can interact with platelets, which results in the modi cation of platelet function and contribution to the pathogenesis of bloodstream infection. For example, it has been reported that (i) platelet activation status is a pivotal determinant for platelet lysis and clearance, which leads to thrombocytopenia, in dengue infection [5], (ii) platelet activation can release platelet factor 4 (PF4), which inhibits HIV-1 infection of adjacent T cells at the stage of virus entry [6], and (iii) Staphylococcus aureus and Streptococcus spp. can interact with and activate platelets [7]. However, the precise function of platelets in sepsis remains to be established.
At present, accumulating evidence supports the notion that sepsis and platelet activation are close intertwined. This is perhaps best illustrated by the observation that Streptococcus pyogenes can induce platelet activation, including integrin activation and alpha and dense-granule release, which may facilitate bacterial infection [2]. Furthermore, a recent study has shown that a sepsis with common Gram-positive pathogens is associated with higher platelet reactivity [8]. Interestingly, Shannon and her colleagues have found that platelet reactivity is higher in early Streptococcus pyogenes-induced sepsis compared to late sepsis. This study suggests that platelet activation may be a pivotal biomarker to provide prognostic information during the pathogenesis of invasive Streptococcus pyogenes infection [9]. Therefore, examining and analyzing the platelet status may be crucial to identify patients at risk of development to sepsis, which could allow clinicians to perform early preventative treatment.
Reactive oxygen species (ROS) are a group of highly reactive molecules, which act as a double-edged sword in many physiological and pathological processes. Until now, it is widely assumed that an imbalance of ROS generation and degradation can disturb the cellular redox state and damage biomolecules, which in turn initiate and promote a variety of diseases [10]. On the other hand, at physiological low levels, ROS may serve as signaling molecules in various biological pathways [11]. Over the past few decades, a growing body of evidence also suggests that ROS are either directly or indirectly involved in the initiation and progression of platelet activation. Here, it is noteworthy that ROS generated by NAPDH oxidase 1 (NOX1) and NOX2 enhance platelet activation via the Syk/PLCγ/calcium signaling pathway [12]. Moreover, a recent study has shown that ROS production may enhance autophagy to promote oxidized low-density lipoprotein (oxLDL)-induced platelet activation via the PI3K/Akt/mTOR pathway [13]. Thus, more future studies are needed to elucidate the role of ROS in platelet activation. NOX2, encoded by the CYBB gene, is a key enzyme associated with generating superoxide anion [14]. It is currently a common belief that this enzyme is mainly present in monocytes, leukocytes, platelets and endothelial cells [15]. Importantly, Violi and his colleagues have been demonstrated that NOX2-derived platelet ROS production is associated with platelet activation ex vivo and in vivo via NO and isoprostane generation [16]. In addition, the same group also reported that a close interplay between oxidative stress and platelet activation in the intracoronary blood waste and aspirated thrombi of ST-elevation myocardial infraction (STEMI) patients. These data imply that oxidative stress act as an important role in promoting thrombus formation and growth [17]. In this respect, it is essential to mention that HIV infection is involved with increased platelet oxidative stress via enhancing platelet NOX2 activity, which in turn inducing the platelet activation in vivo [18]. Unfortunately, little is known about the role of platelet NOX2 in spesis.
In this study, we aim to investigate the changes of platelet NOX2 levels in patients with sepsis and get a better insight into the interplay between platelet activation and NOX2-mediated ROS.

Methods
Human subjects -From March 2016 to May 2018, 31 patients diagnosed with Gram-positive sepsis, 36 patients with Gram-negative sepsis and 45 healthy individuals were enrolled in the study. Written informed consent was obtained from each subject. Patients with sepsis were diagnosed in accordance with the guidelines on treatment of Chinese severe sepsis/septic shock (2014 version), issued by the Chinese society of Critical Care Medicine. The study was carried out according to the principles of the Declaration of Helsinki and was approved by the medical ethics committee of the Second A liated Hospital of Chongqing Medical University. The clinical characteristics of study subjects are demonstrated in Table 1. Platelet preparation -Blood samples were drawn and mixed with 3.8% sodium citrate, then processed in a centrifuge for 15 minutes at 180 g to obtain platelet-rich plasma. As described by Violi and his colleagues, only the top 75% of the platelet-rich plasma was collected to avoid leukocyte contamination [15]. Next, to acquire platelet pellets, platelet-rich plasma was centrifuged for 10 minutes at 300g. In this process, acid citrate-dextrose (1:7 v/v) was added to avoid platelet activation. Platelet pellets were suspended in HEPES buffer in presence of albumin, pH 7.35 according to Carnevale et al [16]. Supernatant was collected by centrifugation for 5 minutes at 300 g and stored at -80°C until analysis.
Analysis of NOX2, sCD40L, sP-selectin and IL-6 -Soluble NOX2-derived peptide was measured by an ELISA method as previously described by Pignatelli and his colleagues [Pignatelli et al., 2010]. Here, it should be mentioned that we modi ed this protocol to evaluate the serum levels of NOX2 in HBV patients [19]. 100 µL of samples or standard were added into each capture antibody-coated well (Anti-NOX2/gp91phox antibody; Abcam, USA) and incubated 2 hours at room temperature. After aspirating and washing each well 3 times, 100 µL of diluted detection antibody-conjugated HRP (Rabbit anti-human IgG H&G antibody; Abcam, USA) was added and incubated 1 hour at room temperature while gentle shaking. Repeating the aspiration/wash of each well, 100 µL of substrate solution was added and Statistical analysis -Statistics were performed using SPSS software, version 19.0 for windows (SPSS Inc, IL, USA). The biological and clinical characteristics of human subjects were shown as means ± SD (standard deviation). Two groups were compared with a Mann-Whitney test. Spearman's rank correlation analysis was employed to analyze association between two variables. The signi cance level was chosen to be P < 0.01.

Platelet soluble NOX2 levels in patients and healthy individuals
A number of clinical and experimental studies have revealed that serum NOX2 may act as an important mediator in development of cardiovascular diseases [20]. For example, it has been reported that serum concentrations of NOX2 in hypercholesterolemic patients are signi cantly higher than those of healthy individuals [15]. In addition, we found that serum NOX2 may serve as a new biomarker for HBV-related disorders [19]. Importantly, several recent studies have reported that there is a good relationship between platelet soluble NOX2 (sNOX2) and platelet activation in various biological processes including STEMI and HIV infection [17][18]. To gain better insight into the crosstalk between platelet sNOX2 concentrations and sepsis, we detected the platelet sNOX2 levels in patients suffered from sepsis. In this study, we showed that (i) the concentrations of platelet sNOX2 in patients with sepsis were signi cantly higher than those of healthy individuals, and (ii) the platelet sNOX2 values in patients with Gram-negative sepsis were lower those in Gram-positive sepsis patients (Fig. 1).

Platelet activation in patients and healthy individuals
It is currently a common belief that platelet activation is involved in a variety of physiological and pathological pathways. However, to date, the interplay between platelet activation and sepsis remains to be determined. In this series of studies, we explored whether or not sepsis could induce platelet activation. Here, it is informative to note that platelet soluble CD40L and platelet soluble P-selectin are markers of platelet activation [16,18]. As shown in Fig. 2A Fig. 2D).
Correlation between levels of platelet sNOX2 and platelet activation markers To further our understanding of the link between platelet sNOX2 and platelet activation in sepsis, we examined whether or not platelet sNOX2 levels are correlated with platelet markers platelet sCD40L and platelet sP-selectin. In our experiments, we found that there is a positive correlation between platelet sNOX2 and platelet sCD40L in patients with sepsis (r = 0.5982, P < 0.0001, Fig. 3A). Here, it should be pointed out that there is a strong correlation between these two factors in patients with Gram-positive sepsis (r = 0.6134, P < 0.0001) and patients with Gram-negative sepsis (r = 0.6526, P < 0.0001, Fig. 3B). In addition, we have demonstrated that the same phenomenon regarding the relationship between platelet sNOX2 and platelet sP-selectin. As shown in Fig. 4B, a signi cant correlation between platelet sNOX2 and platelet sP-selectin in Gran-positive sepsis patients (r = 0.5863, P < 0.0001) and Gram-negative sepsis patients (r = 0.6268, P < 0.0001).

Correlation between levels of platelet sNOX2 and in ammatory markers
So far, mounting evidence has been collected that in ammation plays a crucial role in the initiation and progression of sepsis-related diseases [21]. Additionally, given the fact that in ammatory markers including CRP and IL-6 can be elevated in patients suffered from sepsis, one may also question whether the observed increases of CRP and IL-6 in patients with sepsis are associated with platelet sNOX2 levels.
In this work, we showed that (i) the levels of CRP and IL-6 in patients with sepsis were signi cantly higher than those of healthy subjects, and (ii) there was no signi cant difference between patients with Grampositive sepsis and patients with Gram-negative sepsis in these two in ammatory markers (Fig. 5A, 6A). Importantly, there existed signi cant correlation between platelet sNOX2 value and in ammatory markers (CRP and IL-6) in patients with sepsis (Fig. 5B, 5C, 6B, 6C).

Platelet soluble NOX2 levels in subgroup of patients with sepsis
Finally, we investigate whether or not the concentration of platelet sNOX2 can distinguish between survivors and non-survivors. These experiments showed that platelet sNOX2 values were markedly elevated in non-survivors compared with survivors (Fig. 7A). Importantly, we found that platelet sNOX2 levels declined during the recovery phase compared to the acute phase of sepsis (P < 0.01).

Discussion
NOX2, a major ROS-generating enzyme, plays an important role in cellular redox metabolism. The signi cance of NOX2 for human health and development is highlighted by the existence of severe metabolic diseases [22]. Furthermore, it has become clear that NOX2 can serve as pivotal players in various physiological and pathological pathways, including chronic granulomatous disease, myocardial infarction and systemic lupus erythematosus (SLE). This postulate about SLE was based on the observations that NOX2 is an essential component of LC3-associated phagocytosis (LAP), and that mice and human lacking NOX2 may contribute to initiate and develop the pathogenesis of SLE [23]. In addition, strong arguments have been put forward that NOX2 activity may affect infection. For example, it has been reported that (i) serum NOX2 levels maybe an important indicator for the pathogenesis of progression of HBV-related disorder [19]; (ii) NOX2 serves as a crucial player in the phosphorylation of IκBα at Ser32 and of p65 at Ser536 in Human respiratory syncytial virus (RSV) and Sendai virus, which in turn result in the induction of NF-κB activation [24]; and (iii) the replication of in uenza A virus requires the NOX2 activity, which is associated with virus-induced lung in ammation [Vlahos et al., 2011].
Recently, more works have focused on the impact of NOX2-mediated oxidative stress on platelet activation. This is perhaps best illustrated by the observation that HIV infection triggers platelet oxidative stress in vivo by upregulating NOX2 activity [18]. However, so far, the biological relevance of platelet sNOX2 levels in sepsis remains to be established. In this study, we have provided novel solid evidence that platelet sNOX2 concentrations are signi cantly more elevated in patients with sepsis than in healthy individuals. Additionally, we have shown that (i) the levels of platelet sCD40L and platelet sP-selectin, two platelet activation markers, in patient with sepsis are much higher than those in healthy group; and (ii) a signi cant correlation exists between platelet sNOX2 and these two markers in sepsis patients. Therefore, our ndings support the notion that platelet sNOX2 may be a new candidate biomarker for assessing sepsis-induced platelet activation.
Currently, it is generally believed that Gram-positive sepsis can induce platelet activation. This hypothesis is mainly based on the observations that (i) pathogens isolated from patients with Gram-positive bacteremia can trigger platelet activation from the same infected host ex vivo [26]; and (ii) S. aureus are able to directly or indirectly bind platelet via releasing toxin or using surface proteins such as protein A and clumping factor A, which result in the platelet activation in S. aureus sepsis [27]. Here, it should be mentioned that the crosstalk between Gram-negative sepsis and platelet activation remains unclear and controversial. Numerous experimental data indicated that Gram-negative bacteria such as E. coli may activate platelet via FcγRIIa-dependent manner [28]. However, this model is challenged by various research groups, who provide evidence that limited platelet activation occurs in patients with E. coli sepsis [8]. Interestingly, in this work, we demonstrated that (i) the levels of platelet activation markers including sCD40L and sP-selectin in patients with sepsis are much higher than those in healthy individuals; and (ii) no difference about platelet activation markers concentrations is observed between patients with Gram-positive sepsis and patients with Gram-negative sepsis. In addition, there is a strong correlation between platelet sNOX2 levels and the platelet activation markers values in patients with sepsis. Here, our data suggest that patients with Gram-positive sepsis display higher platelet activation due to the fact that platelet sNOX2 values are higher in these patients.
Over the years, an increasing number of observations have lent support to the concept that in ammation can function as a crucial regulator in the initiation and development of pathogenesis of sepsis. In our setting, we con rm and extend these ndings by showing that the levels of pro-in ammatory markers, including CRP and IL-6, are higher than the corresponding control. Importantly, we found that there is no difference between Gram-positive patients and Gram-negative patients. Hence, our ndings imply that in ammation maybe not a determinant factor in sepsis-induced platelet activation, and that NOX2mediated oxidative stress may serve as an important role in these processes.
Collectively, our evidence, combined with these previous results, strongly indicates that platelet NOX2 may be one of crucial mediators that contributes to the initiation and development of sepsis. These ndings open up the way to potential new candidate for assessing clinical severity in sepsis. Importantly, further research with a large number of patients also needs to be done to investigate the molecular mechanisms underlying the role of NOX2 in sepsis-related pathologies.

Conclusions
In this study, we demonstrated that platelet sNOX2 values were higher in patients with sepsis compared to healthy individuals, and that platelet sNOX2 levels were signi cantly positively correlated with platelet activation markers (soluble CD40L and soluble P-selectin) and in ammatory markers (CRP and IL-6). These results imply that platelet sNOX2 values maybe a new candidate biomarker of the pathogenesis of sepsis. Furthermore, we showed that platelet NOX2 values in patients with Gram-positive sepsis were higher than those in patients with Gram-negative sepsis. Our ndings suggest that platelet NOX2 is a useful tool for assessing clinical severity in sepsis.

Declarations Ethical Approval and consent to participate
The study was approved by the Ethical Committee of the Second A liated Hospital of Chongqing Medical University, (approval no. 39/2015).

Consent for publication
Not applicable Available of data and materials Data and materials are available from the corresponding author upon request.

Competing interests
All authors declare no con ict of interest