The diagnostic roles of neutrophil in bloodstream infections

Journal Pre-proof The diagnostic roles of neutrophil in bloodstream infections Shu-li Shao, Hai-yan Cong, Ming-Yi Wang, Peng Liu

PII:

S0171-2985(19)30058-0

DOI:

https://doi.org/10.1016/j.imbio.2019.10.007

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IMBIO 51858

To appear in:

Immunobiology

Received Date:

19 February 2019

Revised Date:

15 April 2019

Accepted Date:

15 October 2019

Please cite this article as: Shao S-li, Cong H-yan, Wang M-Yi, Liu P, The diagnostic roles of neutrophil in bloodstream infections, Immunobiology (2019), doi: https://doi.org/10.1016/j.imbio.2019.10.007

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The diagnostic roles of neutrophil in bloodstream infections Shu-li Shaoa, Hai-yan Conga, Ming-Yi Wang*a & Peng Liu*a a Department of Central Lab, Weihai Municipal Hospital Affiliated to Dalian Medical University, Weihai, Shandong, 264200, PR China Author for correspondence：[email protected]; [email protected]

Executive summary 

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*These authors contributed equally to this work and should be considered co-corresponding authors.

The neutrophil count related biomarkers (neutrophil-lymphocyte ratio, Immature neutrophil number, Delta neutrophil index) with limited specificity should be interpreted

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with caution for diagnosis of bloodstream infection.

Membrane receptors, such as CCR2/CXCR2/CD64, showed more precise sensitivity and specificity, and for a better diagnostic performance, they should be used by combination. Novel methods like neutrophil motility and mass spectrometry can provide many

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candidates for diagnostic purposes, but more detailed characterization of these markers

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were needed to provide additional information in search for an optimized diagnosis.

ABSTRACT

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Bloodstream infections remain a leading cause of death worldwide, despite advances in critical care and understanding of the pathophysiology and treatment strategies. No specific biomarkers or therapy are available for these conditions. Neutrophils play a critical role in controlling infection

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and it is suggested that their migration and antimicrobial activity are impaired during sepsis which contribute to the dysregulation of immune responses. Recent studies further demonstrated that interruption or reversal of the impaired migration and antimicrobial function of neutrophils

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improves the outcome of sepsis in animal models. In this review, we provide an overview of the associated diagnostic biomarkers involved neutrophils in sepsis, and discuss the potential of neutrophils as a target to specifically predict the outcome of sepsis.

Keywords：neutrophil; sepsis; bloodstream infection; diagnosis; biomarker

1. Introduction

“Sepsis is a state caused by microbial invasion from a local infectious source into the bloodstream which leads to signs of systemic illness in remote organs,” this was the first scientific definition of sepsis proposed by Dr. Schottmuller in 1914 (Reinhart et al., 2012). In 2010-2015, the International Sepsis Forum and the Third International Consensus Definitions Task Force defined sepsis as “life-threatening organ dysfunction due to a dysregulated host response to infection” (Czura, 2010; Seymour et al., 2016; Singer et al., 2016). Thus, bloodstream infection and organ dysfunction were two key conditions to the diagnosis of sepsis. Though the definition of sepsis did not change significantly over the years, the performance of clinical criteria for sepsis was more precise (Vincent et al., 2013). Early diagnosis of bacteremia is the key to prevent its progression to sepsis and septic shock, which can cause organ failure and death. Blood cultures

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are currently the gold standard to determine the presence of microbial species in the body, though it is estimated that less than one half of the patients who have signs and symptoms of sepsis have

positive blood culture (Henriquez-Camacho et al., 2014). The reason may be previous antibiotic therapy by the clinicians. Even though blood culture obtain positive results, this progress normally

takes 24-48 hours. Besides if doctors take experience treatments, unnecessary antibiotics may

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induce the emergence of highly resistant bacteria. For these reasons, low specificity and poor sensitivity of current diagnostic biomarkers often delay diagnosis and treatment, resulting in worse

outcomes for patients and a considerable financial burden on both hospitals and patients (Sharma

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et al., 2008; Kumar et al., 2006). Therefore, there is currently a large effort to detect biomarkers that can aid physicians in the precise diagnosis and prognosis of sepsis. In this review, sepsis, septicemia and bacteremia were considered to refer to the same clinical conditions as bloodstream

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infections.

Neutrophils have a pivotal role in the defense against bacterial infections. They can eliminate pathogenic bacteria effectively because of their large stores of proteolytic enzymes and rapid

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production of reactive oxygen species (ROS) (Brown et al., 2006; Pham, 2006). Neutrophils also can release a web-like structure named neutrophil extracellular traps (NETs) to immobilize and kill the extracellular microorganisms (Brinkmann, 2004). Dysfunction of neutrophil contributes to

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weak immune responses to the causative infections, as well as additional organ damage (Brown et al., 2006). Neutrophils from patients with sepsis lose the ability to respond appropriately to chemotactic signals (Butler et al., 2010; Jones et al., 2014) and have altered antimicrobial activity

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(Solomkin, 1990). Therefore, neutrophils are considered to be major players in the host's immune response to bloodstream infections (Hotchkiss et al., 2014; Smith, 1994). Neutrophils are sensitive to a diverse range of circulating factors and integrate these signals to modulate their activation state and behaviour accordingly. By measuring neutrophil behaviour, Jones N.C et al. have previously revealed changes even after the neutrophils have been isolated from blood (Jones et al. ,2014). Thus, neutrophil activation during bloodstream infections may produce potential biomarkers for infections diagnosis and prognosis, and modification of neutrophil function could lead to therapeutic benefit in patients with bloodstream infections.

2. Neutrophil count

2.1.

Abnormal numbers of blood neutrophils

In the past ten years, increasing number of people use PCT as a biomarker to assess response to antimicrobial therapy. However, the measure of PCT is slightly expensive and is not universally available. In contrast, the full blood count is a cheap, fast and ubiquitous laboratory investigation (Seymour et al., 2016). Generally speaking, the increasing total WBC count means infections and usually used as an indicator responses to infections (Singer et al., 2016). But the WBC count has inevitable limitations as a marker for sepsis. In many cases of hemopathy, because of morbid

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hematopoiesis of the bone marrow, the total WBC count or neutrophil count increases. It can also be normal or even decreased sometimes. Besides, the neutrophil count increases in many other

conditions, such as surgery or trauma. This limited specificity makes the diagnostic performance

of the WBC/neutrophil count poor (Povoa P et al., 2005; Castelli et al., 2006; Davis et al., 2006;

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Van Der Meer et al., 2006).

On this basis, the neutrophil-lymphocyte ratio (NLR) is a neutrophil-count-derived biomarker of inflammation. In the development of sepsis, B-cells and T-cells occurs apoptosis leads to

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lymphocyte depletion. So NLR can be a better index than total WBC count or neutrophil count and can reflect underlying immune function in peripheral blood (Castelli et al., 2006). However several studies have reported that an elevated peripheral NLR is associated with a poor prognosis

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in colorectal cancer (Chua et al., 2011), hepatocellular carcinoma (Halazun et al., 2009), lung carcinoma (Nakahara et al., 2005), gastric cancer (Shimada et al., 2010). Besides, elevated NLR levels are associated with trauma, surgery, pancreatitis, and rheumatic disorders (Terradas et al.,

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2012; Shah rt al.; 2014; Celikbilek et al., 2013). Therefore, the specificity of NLR for an infection could be low or high. In the study of Gonul Gurol et al. detection of NLR was associated with a moderate sensitivity (57.8%) and specificity (83.9%) for diagnosing sepsis (Gurol et al., 2015).

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In conclusion, the ready availability and inexpensive of abnormal numbers of blood neutrophils make them be a basic and helpful examination in development countries. But the limited specificity of such indexes cannot be ignored. There are disadvantages for using total

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WBC count or neutrophil count and NLR in the diagnosis of sepsis.

2.2.

Immature neutrophil

The criteria for sepsis include a neutrophil count that contains more than 10% of immature cells (Bone et al., 1992). When the hematopoietic system is activated following infection, immature leukocytes appear in the peripheral blood. The presence of increased band neutrophils

and other immature neutrophils have classically been considered a sign of infection. Nierhaus A et al. reported that the number of immature granulocyte (IG) in peripheral blood from ICU patients is a good marker to discriminate infected and non-infected patients very early during systemic inflammatory response syndrome (SIRS) (Nierhaus et al., 2013). But M. Ali Ansari-Lari’s et al. work show that the percentage of IG was neither a sensitive nor a specific assay that could be used to distinguish infected from non-infected patients or to identify patients with positive blood culture results. At the same time, the article shows that the IG count is not suitable as a prognostic marker for mortality (Ansari-Lari et al., 2003). Despite the fact that many clinicians rely strongly on the clinical usefulness of the band count, the immature cell count (left shift) and/or the I/T ratio (immature/total neutrophils) for the diagnosis of infection, only limited literatures provide objective data supporting this longstanding practice. It has been convincingly demonstrated that the diagnostic performance of the IG count is inferior, or at best equivalent, to the absolute

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neutrophil count (Hoffmann, 2009). Although IG count cannot improve the positive diagnosis rate

of infection, when IG% <2.0%，it presents a nice negative predictive value (Ayres et al., 2019). Be similar to previous studies, Ayres LS et al. have shown that IG% <2.0% are able to exclude sepsis

with a high specificity(90.9%) and a low sensitivity(38.5%). The reason why sensitivity is low may be that the increase of the IG% arouse by other comorbidities (Ayres et al., 2019). In a

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conclusion, IG may be less helpful to diagnose sepsis but is helpful to exclude sepsis before blood

Delta neutrophil index

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culture result, at some extent avoiding the abuse of antibiotics.

Recently, some researchers have introduced the delta neutrophil index (DNI), which is analyzed by an automatic counter (Nahm et al., 2008). The counter analyzes the leukocyte

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differential count by two methods, a cytochemical myeloperoxidase (MPO) reaction and a light beam reflected from the nuclear lobularity of white blood cells (WBCs). The difference between the two methods is designated as DNI, which correlates with immature granulocytes calculated by

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manual counting. DNI has a significant association with disseminated intravascular coagulation (DIC) scores, positive blood culture rates, and mortality in patients with suspected sepsis. Kim HW et al. found that DNI-72 h is a meaningful marker rather than DNI at the onset of bacteraemia.

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DNI-72 h has the power to predict mortality independently in patients with bacteraemia excluding the impact of treatment and baseline co-morbidity. This means that patients who maintain high DNI until 3 days after the start of treatment have a poor prognosis. Kim et al. also found that, the risk of mortality was higher in those patients whose DNI maintain at a high level after 3 days treatments (Kim et al., 2014). And Celik et al. showed that DNI tended to be normal after 6-10 days with effective therapy (Celik et al., 2019). Lee et al. reported that DNI 2% is associated with 7-days mortality rate after 72 hours of neonatal sepsis (Lee et al., 2013). In addition, compared with other biomarkers such as CRP and PCT, the DNI can be easily calculated through complete blood count, which does not need additional apparatus (Ahn et al., 2018). Therefore, DNI-72 h

could be an alarm to check the patients’ status once more and to consider other treatment strategies whether are effective or not (Kim et al., 2012). Many researchers suggest DNI could be generalized as a basic test among patients who was suspected sepsis (Ahn et al., 2018). But DNI has one weakness that is the result of DNI would be easily influenced by other diseases, especially for children. Ahn JG et al. found that DNI was not useful as a diagnostic tool for bacteraemia in immunocompromised children (Ahn et al., 2014). Under most immunocompromised conditions, immature granulocytes increase because of the irritation by bone marrow and DNI analysis for paediatric bacteraemia in such situations is not useful as well. In addition to patients in the immunocompromised state, neonates, pregnant women, and patients with other hematologic or bone marrow alterations may show abnormal immature granulocytosis (Park et al., 2011;

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Cornbleet, 2002). Under these conditions, DNI should be interpreted with caution.

3. Membrane receptors

CCR2 and CXCR2

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3.1.

CCR2 (CC chemokine receptor type 2) is a chemokine receptor mainly expressed in

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monocytes (Mack et al., 2001) and lymphocytes (Carr et al., 1994), and it has not been detected on the surface of resting human or mouse neutrophils (Speyer et al., 2004). Interestingly,

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acute/chronic inflammation or specific inflammatory stimuli have been shown to induce the expression of CCR2 on circulating neutrophils (Johnston et al., 1999) (Fig. 1). Moreover, neutrophils from mice that had undergone cecal ligation and puncture (CLP), a polymicrobial model of sepsis, highly expressed CCR2 receptor and signaling through this receptor is essential

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for the recruitment of this cell type into the inflammatory site (Johnston et al., 2005; Smith et al., 2004). Up-regulating expression of CCR2 in circulating neutrophils during sepsis was mediated by TLR2 and TLR4 signaling (Souto et al., 2011) (Fig. 2) CCR2 knockout mice showed reduced

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neutrophil accumulation in the lungs, kidneys and heart, which was associated with reduced organ damage (Souto et al., 2011). And CCR2 knockout mice showed higher survival rates than wild-type mice. Furthermore, CCR2 antagonist also improved survival rates after sepsis induction.

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Therefore, neutrophil CCR2 level could be a potential marker for sepsis onset and targets for preventing organ damage. While activated neutrophils express CCR2 during severe sepsis, they lose responsiveness to

CXCR2 ligands by receptor internalization, and this change is critical for neutrophil infiltrate into vital organs and for development of dysfunction in multiple organs (Speyer et al., 2004; Souto et al., 2011; Rios-Santos et al., 2007). CXCR1 and CXCR2 are the main chemokine receptors expressed on the neutrophil surface and mediate the neutrophils’ response to CXC chemokines. CXCR2 promiscuously interacts with CXCL1-3 and CXCL5-8 (Konrad and Reutershan, 2012; Cummings et al., 1999). Actually CLP induction significantly increased KC/CXCL1,

MIP-2/CXCL2, MIP-1α and MCP-1 concentration in tissue homogenates (Wang et al., 2016). Rios-Santos et al. demonstrated that CXCR2 expression was reduced in circulating neutrophils of severe septic mice and this alteration in neutrophils resulted in a reduced chemotactic response toward KC/CXCL1 and MIP-2/CXCL2 (Rios-Santos et al., 2007), suggesting that increased MIP-2/CXCL2 and KC/CXCL1 production do not mediate the activated neutrophils into remote organs during CLP-induced sepsis. Treatment with CXCR2 antagonists reduced neutrophil recruitment to the infection site and worsened the mortality rate after CLP surgery (Rios-Santos et al., 2007; Alves-Filho et al., 2010) , reinforcing the importance of neutrophil CXCR2 during sepsis.

Serum soluble urokinase-type plasminogen activator receptor (suPAR)

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3.2.

suPAR is the soluble form of urokinase-type plasminogen activator receptor (uPAR) which

can be measured in CFS, blood, urine and it is expressed on neutrophils, lymphocytes, monocytes/macrophages, endothelial cells and tumour cells and can be measured in cerebrospinal

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fluid, blood, urine. It appears to be a good prognostic marker, especially when combined with other markers. Evangelos et al. developed a risk stratification system by taking into consideration the APACHE II score (Acute Physiology and Chronic Health Evaluation II) and the serum suPAR.

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They showed that APACHE II ≥17 and suPAR ≥12 ng/ml were independently associated with high mortality (Giamarellos-Bourboulis et al., 2012). Walaa et al. investigated the diagnostic value

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of suPAR combined with serum lactate and SOFA score, and they found that combined usage of suPAR and lactate showed higher area under the receiver operating characteristic curve (Khater et al., 2016). But the study by Walaa was based on a relatively small sized sample. While the level of suPAR increases at acute diseases, but this index can not specifically increase in sepsis. Katia

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Donadello et al. ,summarizes the available data on the diagnostic value of suPAR in sepsis (Donadello et al., 2012). They also thought that suPAR has poor accuracy in diagnosing sepsis compared to CRP and PCT, making suPAR of limited value as a diagnostic marker of sepsis.

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Therefore, suPAR can not serve as a diagnostic biomarker for sepsis. Although the value of suPAR is limited as a diagnostic marker of sepsis alone, the role of suPAR plays in prognostic of sepsis can not be ignored. Huttunen et al. (Huttunen et al., 2011) found that the level of median suPAR

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measured on blood culture from one day to four days were distinctly higher in sepsis non-survivors than in survivors. The study by Zimmermann et al. showed that increased suPAR levels were connected with long-term mortality in patients with sepsis. More specifically, suPAR >8 ng/mL on admission or >13 ng/mL on Day 3 was associated with unfavourable outcome (Zimmermann et al., 2012). The level of suRAP not only can predict the outcome of sepsis but also is related to the severity of the sepsis. In previous study, researcher found that compared with general sepsis patients, patients who required VP support or mechanical ventilation showed higher suPAR (Donadello et al., 2014). In addition, suRAP has an another advantage that PCT doesn’t have. The level of suRAP is less sensitivity to other biological variables such as

physical exercise (Levy et al., 2003) but PCT could elevate in various non-bacterial infection such as massive stress and trauma (Reinhart et al., 2012). In summary, high levels of suPAR could be a prognostic biomarker in various cohorts of infected patients and an index to help distribute scarce resources to the critical patients (Hamie et al., 2018) (Fig. 1).

3.3.

Soluble triggering receptor expressed on myeloid cell-1 (sTREM-1)

Triggering receptor expressed on myeloid cell-1 (TREM-1) is a cell surface receptor expressed on monocytes/macrophages and neutrophils (Fig. 1). The TREM-1/DAP12 pathway, triggered by the interaction of TREM-1 with ligands or stimulation by bacterial lipopolysaccharide,

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can amplify host inflammation response (Chen et al., 2008). Soluble TREM-1 (sTREM-1) is a special form of TREM-1 that can be directly tested in human body fluids and could be potentially used for the early diagnosis of some infectious diseases, including sepsis, bacterial meningitis,

dengue fever and fungal infections (Giamarellos-Bourboulis et al., 2006). Evangelos et al. evaluated the role of sTREM-1 in the course of the septic process. Significantly elevated serum

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sTREM-1 was found on day 1 to day 7 in non survivor-patients compared with the survivors

(Giamarellos-Bourboulis et al., 2006). Similarly, Li et al. found significantly increased sTREM-1 levels on day 1 in patients who died (Li et al., 2014). They suggested that combination of serum

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sTREM-1 and PCT levels and SOFA score can offer a powerful prognostic utility for sepsis mortality. Moreover, Xie et al. examined the value of sTREM-1 to predict the outcome of

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early-onset stroke-associated pneumonia (Xie et al. ,2015). They found that serum sTREM-1 levels were slightly elevated in the patients who died and were decreased in the patients who survived and sTREM-1 levels were significantly higher in non-survivors than in survivors. The sensitivity and specificity of sTREM-1 to predict unfavourable outcome were 71.8% and 92.3%

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respectively. Contrarily, others showed that the prognostic utility of serum sTREM-1 in septic shock was inferior to that of PCT. Phua et al. evaluated the prognostic utility of serum sTREM-1 in patients with septic shock. No significant difference was observed on the first three days

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between survivors and nonsurvivors. Bopp et al. studied 65 patients with different stages of bloodstream-infection and 21 healthy controls. But their results showed no significant difference in sTREM-1 concentrations between survivors and non-survivors on any day of measurement

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(Bopp et al., 2009; Tziolos et al., 2015). It's worth mentioning that sTREM-1 could increase in blood samples and other body fluids (CSF, pleural fluid, urine), this would cause potential delay in obtaining samples of this particular site. And the level of sTREM-1 could elevate in various diseases (Giamarellos-Bourboulis et al., 2012). Therefore, sTREM-1 is a controversial biomarker and still need to be validated clinically.

3.4.

CD64

Neutrophil can express three classes of IgG receptors: Fcγ receptor I (FcγRI) which is the high affinity receptor for monomeric IgG1 and IgG3 and also binds aggregated IgG; Fcγ receptor II (FcγRII) and Fcγ receptor III (FcγRIII), which both bind IgG, but with low affinity (Nuutila et al., 2007). These receptors are recognized by the monoclonal antibodies CD64 (FcγRI), CD32 (FcγRII) and CD16 (FcγRIII), respectively (Zola et al., 2007). In resting mature neutrophils, CD64 is expressed at very low levels. Upon neutrophil activation it is strongly upregulated by the proinflammatory cytokines IFN-γ and granulocyte colony stimulating factor (G-CSF) which are produced during infections or exposure to endotoxin (Van Der Meer et al., 2007). The two other Fcγ receptors are constitutively expressed and are upregulated as well during neutrophil activation. However, differences in expression between resting and activated neutrophils are much higher for CD64. Therefore, the CD64 antigen is the most useful candidate marker for bloodstream infection (Davis et al., 2006; Davis, 2005). Monocytes also express CD64 and upregulate this receptor

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during activation. The longitudinal patterns of monocyte and neutrophil CD64 upregulation are strikingly similar, although the change in CD64 expression is considerably higher for neutrophils (Barth et al., 2001).

Neutrophil CD64 has many characteristics that make it clinically useful as a marker of sepsis.

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JJ Hoffmann reviewed the reasons as follows (Hoffmann, 2009). First, CD64 directly reflects

physiologic events of the inflammation response to invading microorganisms. Secondly, in resting neutrophils the level of CD64 expression is rather low. However, following activation it can

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increase up to 5–10-fold (Song et al., 2008), allowing good discrimination between health and disease. Third, neutrophil CD64 becomes positive rapidly: CD64 expression is a biphasic pattern, increasing after 2 h and more significantly after 6 h. After the stimulation was removed, CD64

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levels decreased within 48 hours (Van Der Meer et al., 2007). Further, upregulation of neutrophil CD64 is specific for bloodstream infection and few other disorders are associated with CD64 upregulation. Finally, the analysis of neutrophil CD64 is relatively simple and fast. These

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recommendations should be accepted as the general referance standard for a diagnostic biomarker. But there is a difference between two test methods about CD64. A report showed that using flow cytometry and Leuko64 kitTM to test the level of CD64 , the SROC values were 0.95 and 0.85

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respectively (p = 0.013). Furthermore, the expensive analysis of flow cytometry precludes its clinical application (Larsen and Petersen, 2017). On the basis of the meta-analysis, Luo Q et al. suggest that neutrophil CD64 is not a perfect marker for sepsis (Luo et al., 2018). Morsy et al.

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reported that the sensitivity of CD64 in diagnosing local infection was significantly lower (Morsy et al., 2008). Because sepsis is a complex, dynamic syndrome and no single test is sufficiently sensitive and specific for detecting sepsis (Luo et al., 2018). As yet, scientists have not found a biomarker with sufficient sensitivity and specificity to diagnose sepsis. However, an increasing number of studies have indicated that combinations of various markers are a useful approach to improving the accuracy of diagnosing sepsis (Luo et al., 2018). For example, Gibot et al. indicated that a combination of neutrophil CD64, sTREM-1, and PCT could have a far better diagnostic performance for sepsis (Gibot et al., 2012).

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Fig. 1. Neutrophils under normal condition and blood stream infection. During infection, peripheral neutrophils are systemically stimulated by bacterial components, resulting in up-regulation of CCR2, SuPAR, STREM, CD64, and

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down-regulation of CXCR2 on the surface. N, neutrophil; IG, immature granulocyte.

Fig. 2. CXCR2 increases neutrophil recruitment at the site of infection, which enhances the importance of neutrophil CXCR2 during sepsis.

4. Novel methods/markers

4.1.

Neutrophil motility

During bloodstream infection, circulating numbers of neutrophils are often dysfunction. Neutrophils from patients with sepsis showed impaired directional migration and chemotaxis (Butler et al., 2010; Jones et al., 2014; Alves-Filho et al., 2010; Delano et al., 2011), defects in ROS generation (Alves-Filho et al., 2008; Grailer et al., 2014) and have altered antimicrobial activity (Solomkin, 1990; Stephan et al., 2002; Amatullah et al., 2017). There are four phases in the migration of neutrophil in vivo, release from the bone marrow, margination, rolling, adherence, and transmigration, all of them are impaired during sepsis. Several studies have found that the ability of migration of neutrophils has been reduced. Not only that but after increasing the ability of infected neutrophils to adhere to endothelium, endothelial barrier function has been lost (Delano et al., 2011). Fig. 3 shows the processes that the impairment of neutrophils induced by

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sepsis. Fungal sepsis induce neutrophils swarm and cluster in lung capillary (Lee et al., 2018). Ellett et al. ,reported a microfluidic assay that measures the spontaneous motility of neutrophils from one droplet of diluted blood. The assay identified sepsis patients with 97% sensitivity and

98% specificity (n=42), but it needs to be texted in larger populations of at-risk patients (Ellett et al., 2018). The ability to use whole blood distinguishes this assay from previous ones, which could

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only probe the motility of patient neutrophils after isolation from blood. In a clinical setting, the

assay will require only the training of an operator who will prepare and load the blood sample in the device. Automated imaging and analysis of cell trajectories will then generate an infection

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score readout. Although this way has high sensitivity and specificity, but there are several questions need to be solved. First, the formula for the infection score and threshold values will have to be further refined and validated in subsequent research studies. Then neutrophils analyzed

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in peripheral blood cannot completely represent the the marginating pool in the microvasculature in vivo. Finally, how the changes of neutrophils migration are involved in the pathogenesis of sepsis, particularly lead to damage of tissue and organ failure, are still unclear (Delano et al.,

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2011).

Fig. 3. The migration of neutrophils in the body involves four distinct stages, all of which are impaired during sepsis: mobilization and release from the bone marrow, limbic and rolling, adhesion, and transmigration.The mechanism contributes to the development of sepsis-induced

Proteomics for biomarkers of sepsis

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4.2.

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neutrophil migration disorders.

Mass spectrometry is often the method of choice to detect metabolomic and proteomic changes that occur during bloodstream infection progression. This strategy allow for untargeted

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profiling of thousands of metabolites and proteins from human biological samples obtained from patients. Differential expression or modifications to these metabolites and proteins can provide a

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more reliable source of diagnostic biomarkers for bloodstream infection (Ludwig and Hummon, 2017). Hattori N et al. used animal models to study acute kidney injury (AKI), which is a frequent complication during sepsis. High levels of the proteins acidic mammalian chitinase (CHIA), chitinase 3-like protein-1 (CHI3L1), and chitinase 3-like protein 3 (CHI3L3) were detected in the urine of septic mice. Both CHI3L1 and CHI3L3 are produced by activated macrophages and neutrophils during sepsis (Hattori et al., 2009). As to human septic patients, CHI3L1 was markedly higher in urine samples than the controls. But CHIA was less successful in discriminating sepsis with AKI from sepsis without AKI (Maddens et al., 2012). Erik Malmström et al. applied a combination of mass spectrometry based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or

sepsis conditions. They identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases (Malmstrom et al., 2014). They identified eight proteins that are up-regulated (Defensin alpha-1, Low affinity immunoglobulin gamma Fc region receptor III-A CD16a, Olfactomedin-4, Myeloperoxidase, Resistin, Transcobalamin-1, Orosomucoid-1, Haptoglobin) and 12 proteins that are down-regulated (CD44 antigen, Granulins, Cysteine-rich secretory protein 3/CRISP-3, Neutrophil gelatinase-associated lipocalin/NGAL, Cathelicidin antimicrobial peptide, Integrin alpha-M/CD11b, Epididymal secretory protein E1, Thioredoxin, Catalase, Glutathione S-transferase omega-1, Transaldolase, Serotransferrin). But some of the identified proteins are not entirely neutrophil specific. The mass spectrometry approach was reproducible and there was little diversity among patients. Many proteins identified in this study, have not been described previously as candidates for diagnostic purposes and it is not known whether they can cause a

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distinct disease phenotype per se. Thus, more detailed characterization of proteins were needed to provide additional information in search for an optimized treatment.

Neutrophil extracellular traps (NETs) and NETosis

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4.3.

Neutrophil extracellular traps (NETs) are large, extracellular, web-like structures composed

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of decondensed chromatin and neutrophil antimicrobial enzymes. NETs can trap, neutralize and kill bacteria, fungi, viruses, parasites and prevent them dissemination (Brinkmann et al., 2004;

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Urban et al., 2006; Saitoh et al., 2012; Abi Abdallah et al., 2012; Walker et al., 2007). NETs are released via a cell death program named NETosis that requires reactive oxygen species (ROS), the granule proteins myeloperoxidase (MPO) and neutrophil elastase (NE) (Brinkmann et al., 2004; Metzler et al., 2011; Papayannopoulos et al., 2010). Compared to the rapid process phagocytosis,

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NETosis is a slow process that takes approximately 4h (Branzk et al., 2014). During the systemic inflammatory response of severe sepsis, neutrophils accumulate in the liver microcirculation, where they exert protective effects by releasing NETs that capture and eliminate microbes from

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the bloodstream. NET production requires platelet-neutrophil interactions and can be inhibited by platelet depletion or disruption of integrin-mediated platelet-neutrophil binding (McDonald et al., 2012). Within the septic liver, NETs may contribute to tissue damage in multiple ways. NETs are

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decorated with serine proteases, and the resulting intravascular coagulation may contribute to hypoperfusion and ischemic tissue injury (Massberg et al., 2010). Histones are directly cytopathic to endothelial cells in vitro and that immunoneutralization of histones in vivo reduced organ damage and enhanced survival in animal models of sepsis and endotoxemia (Xu et al., 2009; Saffarzadeh et al., 2012). Vascular occlusion based on NETs in vivo during sterile neutrophilia and septicemia partly caused organ damage. So molecules derived from the process of NETs production or NETosis may predict the level of organ dysfunction in sepsis patients. But we know little in this field. Fortunately, DNase1 and DNase1-like 3 provide dual host protection against deleterious effects of intravascular NETs under physiological conditions (Saffarzadeh et al., 2017).

5. Conclusion

Bloodstream infection is a systemic inflammatory syndrome resulting from the immune system response to invading micropathogens and is the leading cause of death among patients in intensive care units. Neutrophils are the key cell subpopulation against invading pathogens, and they are sensitive to a diverse range of circulating factors which neutrophil integrate to modulate their behaviour accordingly. Thus, neutrophils activation during sepsis is probably the cumulative effect of infection-related factors that are present in the circulation, including many of the putative sepsis biomarkers discussed above. By measuring neutrophil behaviour or biomarkers,

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investigators can reveal changes after bloodstream infection occur. The neutrophil count related biomarkers (neutrophil-lymphocyte ratio, Immature neutrophil number, Delta neutrophil index) with limited specificity should be interpreted with caution for diagnosis of bloodstream infection.

Membrane receptors, such as CCR2/CXCR2/CD64, showed more precise sensitivity and specificity, and for a better diagnostic performance, they should be used by combination. Novel

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methods like neutrophil motility and mass spectrometry can provide many candidates for diagnostic purposes, but more detailed characterization of these markers were needed to provide

additional information in search for an optimized diagnosis. There are main biological impact of

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mentioned examines (Table 1). Further studies into the mechanisms of neutrophil dysregulation and diagnostic targets on neutrophils during bloodstream infection were needed to get better

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outcome.

Table 1 Mentioned examines’ main biological impact. examine

biological impact

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neutrophils

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abnormal numbers of blood

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immature neutrophil

delta neutrophil index

CCR2 and CXCR2

cheap,

references

availability,

limited specificity

(Singer et al., 2016; Povoa et al., 2005; Castelli et al., 2006; Davis et al., 2006; Van Der Meer et al.,2006; Gurol et al., 2015)

less help to diagnose, helpful

(Nierhaus et al., 2013; Hoffmann,

to exclude sepsis

2009; Ayres et al., 2019)

predict

mortality,

limited

(Park et al., 2011; Cornbleet, 2002)

prevention of organ injury ,

(Souto et al., 2011; Rios-Santos et

not universally available

al., 2007; Alves-Filho et al., 2010)

application

suPAR

a prognostic biomarker

(Donadello et al., 2012; Levy et al., 2003; Hamie et al., 2018)

sTREM-1

controversial

prognostic

(Li et al., 2014)

biomarker CD64

specific

neutrophil motility

upregulation,

(Van Der Meer et al., 2007; Song et

inconsistent outcomes

al., 2008; Larsen et al., 2017)

high sensitivity and specificity,

(Delano et al., 2011)

undefined diagnosis criteria proteomics for biomarkers of

diagnostic candidates

(Ludwi et al., 2017)

NETs

possible prognostic biomarker

(Jiménez-Alcázar et al., 2017)

-p

Conflict of interest

ro of

sepsis

re

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials

na

Acknowledgement

lP

discussed in the manuscript apart from those disclosed.

This work was supported by the National Natural Science Foundation of China (81600083), Shandong Province Natural Science Foundation, China (number ZR2016HM63), Weihai science

ur

and technology development plan project (number 2016GNS029) and the 2016 Technology

Jo

Development Project of Shandong Medicine and Health Science (2016WS0635).

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