Neutrophil Role in Early Diagnostics Of A Serious Bacterial Infection

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

FACULTY OF MEDICINE DEPARTMENT OF PEDIATRICS

Neutrophil Role in Early Diagnostics Of

A Serious Bacterial Infection

Student : Muhammed Fazil Kottilingal Farook Supervisor: Assoc. Prof.Lina Jankauskaitė MD, PhD

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3. SUMMARY

Title: Neutrophil Role In Early Diagnostics Of A Serious Bacterial Infection Author: Muhammed Fazil Kottilingal Farook

Supervisor: Assoc.Prof.Lina Jankauskaitė Md,Phd

Background: Differentiating bacterial and viral infection in primary care (emergency medicine as well) is challenging especially in early phase of infections. The initial preferred measures to differentiate between bacterial and viral infections are CRP and Complete Blood Count (CBC). To date, specific markers for distinguishing viral from bacterial infections (including serious bacterial infection (SBI) and sepsis) are lacking, particularly in the early stages of bacterial/viral infection.

Aim: The aim of this study was to analyze neutrophil role in early (up to 12 hours post first febrile fever episode) viral and bacterial infection (included SBI and sepsis) and to find platelet and neutrophil interaction (platelet induced neutrophil activation).

Methods: We performed a prospective experimental study of 68 children ranging in age from 1 month to 5 years old who presented to a pediatric emergency department (PED) 12 hours after their first episode of fever and who manifest with systemic inflammatory response syndrome (SIRS). All of the cases were classified into three categories: viral infection (n=42), bacterial infection (not SBI) (n=10), and SBI (n=16). SBI was further classified into sepsis (n=4) and no sepsis SBI groups. The results of the CBC and CRP were analyzed. NLR, NMR, PNR, and PNLR were calculated. sP-selectin was investigated in order to better understand neutrophil-platelet interaction.

Results: As expected, we observed significant difference in standard biomarkers such as leucocyte, neutrophil counts as well as CRP between the groups. Higher levels of CRP, neutrophil and leucocyte counts were observed in BI, SBI and sepsis groups. A significantly higher NLR, NMR, PNR and PNLR was observed in bacterial, and SBI compared to viral infection (p=0.0267, p=0.0019, p=0.0003 p=0.0085 respectively). However, NLR, NMR, PNR and PNLR did not differ between sepsis and no-sepsis samples. All bacterial samples (including SBI and sepsis) had significantly higher sP-selectin values than viral infections (58.13 [39.80-68.45] versus 30.12 [13.74-43.78], p=0.0003). However, other bacterial infections, on the other hand, were difficult to distinguish from sepsis.

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Conclusion: Classical biomarkers such as neutrophils, leucocytes, and CRP were found to be statistically insignificant in diagnosing virus and bacterial infection, but the majority of values were found to be within normal range when it could be attributed to both bacterial and viral infection. As a result, they are insufficiently specific for distinguishing between bacterial and viral infections and most importantly, between severe bacterial infections and sepsis. Biomarkers indicating neutrophil or neutrophil-platelet activation, such as NLR, NMR, PNR, and PNLR, were discovered to be statistically significant. Even so, they did not surpass traditional biomarkers in distinguishing between the groups. sP-selectin was shown to be higher in bacterial samples compared to viral. With the cut-off value of 59.59pg/ml, it had a likelihood ratio of 3.17 to predict sepsis from all the other cohort samples. Thus, it is a promising biomarker in discriminating sepsis from other acute bacterial and viral infections.

4. Acknowledgements

I would like to express my sincere appreciation to Dr. Lina Jankauskaite, who enabled me to complete this thesis.

5. Conflict of interest

I declare no conflict of interest.

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7. ABBREVIATION LIST

% Percentage *C Degree Celsius ~ Approximately °C Degree Celsius µm Micrometer

12 HETE 12 Hydroxyeicosatetraenoic acid ABP Arterial Blood Pressure

ATP Adenosine Triphosphate AUC Area under the curve

C1q Complement component 1q Cath G Cathepsin G

CCL C motif) Chemokine ligand CD Cluster of differentiation CLEC2 C type lectin like receptor2

CMV Cytomegalovirus

COPD Chronic obstructive pulmonary disease

CRAMP Mouse cathelicidin-related antimicrobial peptide CRP C-Reactive Proteins Csf Cerebrospinal fluid CXCL C-X-C motif ligand CXCR C-X- C chemokine receptor CXR Chest X-Ray DC Dendritic cell

DC SIGN Dendritic cell specific ICAM 3 grabbing integrin DEFA Defensin alpha

DENV Dengue virus

DNA Deoxy ribonucleic acid

dsRNA Double stranded ribonucleic acid EBP Enhancer binding protein

ECM Extracellular Matrix ED Emergency department

ELISA Enzyme linked immune sorbent assay

ENA78 Epithelial cell derived neutrophil activating peptide FBC Full blood count

fMLF formyl methionyl leucyl phenylalanine FWS Fever without a source

GBC General Blood Count

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GMPs Granulocyte monocyte progenitors Gpibα Glycoprotein Ib alpha

GROα Growth regulated oncogene α HA Haemagglutinin glycoprotein HCMV Human cytomegalovirus

HIV Human immunodeficiency virus HMGB1 High mobility group box 1 protein

HNP Human neutrophil peptide HNP1 Human Neutrophil Peptide 1

HR Heart rate

HRP Horse Radish Peroxidase HSC Hematopoietic stem cell HSV Herpes simplex virus

IAV Influenza A Virus

ICAM 1 Intercellular adhesion molecule 1 IFN Interferon

Ig Immunoglobulin IL1β Interleukin1β

iNKT invariant natural killer T cells IQR Interquartile Range

L Liter

LAD Leucocyte adhesion deficiency Leu Leucocytes

LFT Liver function test

LMPPs Lymphoid primed multipotent progenitor cells LPS Lipopolysaccharides LR Likelihood ratio LTB4 Leukotriene B4 LTC4 Leukotriene C4 LTD4 Leukotriene D4 LTE4 Leukotriene E4 Ly48 Leukosialin

MAC1 Macrophage 1 antigen

MAPK Mitogen activated protein kinase

MDA5 Melanoma differentiation associated protein 5 mg/l milligram per liter

MIP2α Macrophage inflammatory protein2α MODS Multiorgan dysfunction syndrome

MON Monocyte MPO Myeloperoxidase

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MPP Multipotent progenitor cells N number of patients

NADPH Nicotinamide adenine dinucleotide phosphate NETs Neutrophil extracellular traps

NEU Neutrophil

NF κB Nuclear factor kappa light chain enhancer of activated B cell NK cell Natural killer cell

NLR Neutrophil to Lymphocyte Ratio

NLRP3 Nucleotide binding domain leucine rich repeat containing pyrin 3 NMR Neutrophil to Monocyte Ratio

NO Nitric oxide

NOD Nucleotide binding oligomerization domain NOS Nitric oxide synthases

O2 Oxygen

ODN Oligodeoxynucleotide

oSBI Serious Bacterial Infection Other Than Sepsis P Significance level

PAF Platelet activating factor

PAMPs Pathogen associated molecular patterns PAMPs Pathogen associated molecular patterns

PCR Polymerase chain reaction Pct Procalcitonin

PED Pediatric emergency department pg/ml picograms per milliliter

PLT Platelets

PMA Phorbol myristate acetate

PNLR Platelet*Neutrophil to Lymphocyte Ratio PNR Platelet to Neutrophil Ratio

PRRs Pattern recognition receptors PSGL1 p selectin glycoprotein ligand 1

PTX3 Pentraxin 3 RBC Red blood cells

RhoA Ras homolog family member A ROC Receiver Operating Curve ROS Reactive oxygen species

RR Respiratory Rate

RSV Respiratory syncytial virus SBI Serious bacterial infections

ScpA Streptococcal secreted esterase and c5a peptidase A SD Standard Deviation

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Sda Streptodornase

SIRS Systemic inflammatory response syndrome SpO2 Blood Oxygen Saturation

SpyCEP S. pyogenes cell envelope protease

SSE Streptococcal secreted esterase T Temperature

TEM Transendothelial migration TF Tissue Factor

TGF β Transforming growth factor beta Th17 T helper cells 17

TLR4 Toll like receptor 4

TNF α Tumor Necrosis Factor alpha

TREM1 Triggering receptors expressed in myeloid cells 1 TSG TNF stimulated gene

TXA2 Thromboxane a2 TXA2 Thromboxane A2

VCAM1 Vascular cell adhesion molecule 1 VWF von Willebrand factor

WBC White blood cells α4β1(VLA4) Very late antigen

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8. TERMS

Chemotaxis – it is a tendency of cells to migrate towards the direction of gradients of chemotactic stimuli.

NETs – neutrophil extracellular traps are web-like arrangements that include strands of decondensed chromatin enriched with the contents of neutrophil granules.

NETosis- is a type of cell death distinguished by the discharge of decondensed chromatin and granular contents into the extracellular space.

NLR – is an absolute neutrophil to lymphocyte count ratio calculated from general blood count (GBC).

NMR – is an absolute neutrophil to monocyte count ratio calculated from GBC.

Phagocytosis – is the process where specific living cells known as phagocytes intake or engulf other cells or particles.

PNLR – is an absolute platelet * neutrophil to lymphocyte cell count ratio calculated from GBC.

PNR – is an absolute platelet to neutrophil count ration calculated from GBC.

SIRS – Systemic inflammatory response syndrome is an inflammatory condition that affects the entire body. It is the immune system's response to an infectious or non-infectious insult.

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9. INTRODUCTION

Pediatric sepsis remains one of the leading causes of death in infants and children around the world [1- 5]. Currently, sepsis is being diagnosed if a child presents with symptoms of systemic inflammatory response syndrome (SIRS) in accordance with a documented or suspected bacterial infection. However, it is hard to distinguish between a septic patient and a child with SIRS due to other causes based solely on clinical data. Fever, among the most important SIRS components, is indeed a very likely cause for children to visit the Emergency Department (ED) [6]. The majority of febrile children will have a self-limiting viral infection. Only some of them will have a severe (serious) bacterial infection (SBI), such as sepsis [7-8]. The early recognition of etiological factor and proper management of a febrile child especially infant has been a problem for nearly 30 years since majority of febrile children would only have a minor viral infection. While approximately 12% of infants aged <30 days or 9% of infants aged 30 to 90 days will have SBI [9]. Infants under 3 months of age are more likely to develop SBI due to their immature innate and adaptive immune systems [10]. The risk increases with a younger age, since the innate immune response develops during the initial stages of life and since the innate immune response is the first line of defensive measure against numerous microorganisms, making the infant the most vulnerable to SBI [11]. The incidence of SBI decreases with age [12]. However, the duration and height of fever are strongly associated with severe bacterial infection [13]. The treatment of children who appear ill and have an obvious site of infection is evident. However, fever without a source (FWS) is a major concern in emergency departments. Besides that, children with viral diseases, systemic autoimmune diseases or oncological diseases can present with SIRS. Moreover, it can take up to 24 - 48 hours period for a cause of SBI, such as sepsis, to be fully confirmed or ruled out with laboratory testing. When antibiotics are the primary choice of therapy, the initial diagnosis of SIRS can lead to a further treatment “precaution” measurement [14]. However, delay in diagnosis and antibiotic treatment in cases of sepsis or bacteremia can result in serious complications such as multiorgan failure or death [15-16]. Weiss et al. [17]. researched the epidemiology of severe sepsis death and noticed that it occurs between 3 and 7 days after the sepsis diagnosis, with later deaths were associated with multiple organ dysfunction syndrome (MODS), respiratory failure, or neurological disorders [17]. Until today, diagnostic pathways and algorithms are unable to determine and accurately differentiate the cause of fever, as non-infectious diseases can also present as SIRS. Additionally, over-diagnosis could result in additional stressful testing procedures such

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12 | P a g e as blood culture or lumbar puncture. Besides, FWS or SIRS are usually treated empirically without evaluating the causative factor. As a result, antibiotics are overused, contributing to the global antimicrobial resistance problem [18]. Quite a few protocols have been proposed and various biomarkers have been evaluated as soon as possible to predict the cause of SIRS and diagnose sepsis or bacteremia. However, to date, no perfect biomarker exists. Besides, markers such as white blood cells (WBC), counts of neutrophils, C-reactive protein (CRP), or procalcitonin have been shown to be poorly sensitive to discriminate between SIRS and sepsis, particularly when used alone and in an early stage of infection [19].

Neutrophils are one of the most important effector cells of the innate immune system. These cells are a homogenous population of short-lived cells with a principal function of defense against extracellular pathogens via phagocytosis and intracellular killing [20]. Under homeostatic states, neutrophils enter the circulation, move to tissues, where they fulfil their functions, and eventually are eliminated by macrophages. Infections and their respective inflammatory mechanisms are followed by a rapid influx of neutrophils from the peripheral blood to the site of inflammatory. Neutrophils take part in killing microorganisms and clear infections through a variety of mechanisms such as chemotaxis, phagocytosis, the release of reactive oxygen species (ROS), granular proteins, and the production and release of cytokines. If this local inflammatory response becomes intense, SIRS and MODS may occur such as in trauma, serious bacterial infection etc. [20].

Endothelial activation is one of the prominent early signs of SIRS, therefore sepsis. The activated endothelium stimulates leukocytes and platelets, which would then express molecules of cell adhesion on their surface. The transportation of cell adhesion molecules to the surface of the cells are in granules (the α-granules of platelets and the Weibel-Palade bodies of endothelial cells) [21]. The granule P-selectin and soluble P-selectin can be measured in the plasma as well as can be found on the surface of platelets and endothelium. During sepsis, Platelets and endothelial cells are one of the first responders which in turns activates endothelial cells and platelets, consequently releasing soluble P-selectin (sP-selectin), therefore sP-selectin is proposed as a useful biomarker for sepsis [22]. Sepsis is characterized by increased activation of platelets, which are small anucleate blood cells that play important roles in hemostasis. Platelets appear to play important roles in immunity, modulating both physiologic and pathologic responses to inflammation and infection. Platelets play an important role in regulating leukocyte function and, as a result, inflammatory immune responses. They interact easily with innate immune cells and have

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13 | P a g e immunomodulatory effects either directly through cell-cell contact or indirectly through the release of chemokines and cytokines [21].

In this study we evaluated neutrophil role in early (up to 12 hours post first febrile fever episode) viral and bacterial infection (including SBI and sepsis) and investigated possible platelet and neutrophil interaction (platelet induced neutrophil activation) measuring soluble P-selectin (sP-selectin).

We hypothesized that there will be platelet-neutrophil interaction differences between early viral and bacterial infection in children referred to ED with the early symptoms of SIRS.

10. AIM

The aim of this study was to analyze neutrophil role in early (up to 12 hours post first febrile fever episode) viral and bacterial infection (included SBI and sepsis) and to find platelet and neutrophil interaction (platelet induced neutrophil activation).

11. OBJECTIVES

1. To analyze standard blood biomarker (such as general blood count (GBC)-leucocyte count, neutrophils, and CRP) levels in children up to 5 years of age with early bacterial, viral infection or serious bacterial infection (SBI) including sepsis.

2. To analyze neutrophil activation markers and neutrophil-platelet activation markers such as NLR (neutrophil to lymphocyte ratio), NMR (neutrophil to monocyte ratio), PNR (platelet to neutrophil ratio), PNLR (platelet*neutrophil to lymphocyte ratio) in children up to 5 years of age with early bacterial and viral infections (up to 12 hours).

3. To analyze neutrophil related protein (sP-selectin) expression during early bacterial and viral infection.

4. To identify neutrophil-platelet related protein sP-selectin sensitivity and specificity in diagnosing early bacterial versus viral infection.

The object of research: Children up to 5 years of age who arrived at pediatric emergency department (PED) 12 hours after first episode of fever and who manifests with systemic

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14 | P a g e inflammatory response syndrome (SIRS). Sampling, research population, selection criteria and methods of research: the study population was collected according to inclusion and exclusion criteria. Selection criteria were based only on previously healthy children with acute viral or bacterial disease on early arrival (<12h post initial fever) to PED. The protein level analysis was performed from collected plasma samples via ELISA method. Statistical data analysis was performed with IBM Statistics SPSS 25.0. (SPSS, IBM Company, Armonk, New York, USA)

12. LITERATURE REVIEW

12.1 Neutrophils

Neutrophils (Figure 1) account for more than a half of all leukocytes and they are one of most important cells in acute inflammation. They are crucial players in defense mechanisms against invading pathogens via phagocytosis and production or release of reactive oxygen species (ROS), proteases, neutrophil extracellular traps (NETs)[23].

Neutrophils are important effector cells of innate immune system. These cells are produced in the bone marrow from stem cells (~1011_{cell per day)}_[24]_{. It makes up to} 60-70% of the whole amount of white blood cells (WBC). Neutrophil is approximately 12-14 µm in diameter. Neutrophils have a single multilobed nucleus and can have between 2 and 5 lobes[25](Figure 2). Neutrophils were thought to be a short-lived (8-12 hours) corpuscles, but recent studies suggest that these cells may live up to 5 days in circulation [26]. How neutrophils undergo aging in circulation was a hot topic for some years and researchers suggest that it happens when neutrophils increase their expression of CXCR4 (C-X-C motif receptor 4) and reduce expression of CD62L (CD62 ligand) before they return to the bone marrow following a circadian pattern [26]. They also noted that it takes 8 hours to remove aged neutrophils from circulation [26]. The lifespan can be prolonged when neutrophil is activated.

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15 | P a g e Figure 1 A blood smear containing a pair of neutrophils surrounded by normal red blood cells. Wadsworth Center, New York State Department of Health (John Oller, Stephen D Oller) [27].

Figure 2 A microscopic slide of neutrophil surrounded by red blood cells. Histology Guide © Faculty of Biological Sciences, University of Leeds [25]. (Dr Michelle Peckham: Textual & Graphical Content, Photomicroscopy, Adele Knibbs: Photomicroscopy, Steve Paxton: Design & Development) [25]

12.1.1 Neutrophil’s Life Cycle

The generation of neutrophils from committed hemopoietic progenitor cells in the bone marrow is a carefully regulated process that is controlled by various transcriptional factors such as CCAAT-enhancer-binding proteins (C/EBP). A self-renewing hematopoietic stem cell (HSC) initiates this process by differentiating into a multipotent progenitor cell (MPP), which then transforms into lymphoid-primed multipotent progenitor cells (LMPPs)

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[28]. LMPPs can eventually give rise to granulocyte–monocyte progenitors (GMPs) [29]. GMPs generate neutrophils in response to growth factors such as granulocyte colony-stimulating factor (G-CSF). This takes place in stages with developing neutrophils gradually acquiring their mature phenotype as they progress through promyelocyte, myelocyte, metamyelocyte, and finally band neutrophil stages (Figure 3) [28]. It is thought that during these steps, the expression of integrin α4β1 (VLA4-very late antigen-4) and CXCR4 (at least in mice) is downregulated, while the expression of CXCR2 (C-X-C motif) and Toll-like receptor 4 (TLR4) is enhanced [28]. During this stage of development, neutrophils acquire their nuclear lobular morphology. Between the myeloblast and promyelocyte stages, granules begin to form inside developing neutrophils, and different granules are formed at different stages of the maturation process [28]. A vast number of mature neutrophils are present in bone marrow, from which they can be quickly released into the circulation in response to infectious, inflammatory, or tissue damage-related stimuli [28].

Figure 3 Neutrophil production. Granulopoiesis or neutrophil generations take place in the bone marrow. A

self-renewing hematopoietic stem cell (HSC) differentiates into a multipotent progenitor (MPP) cell in the first step. MPP then differentiates into lymphoid-primed multipotent progenitors (LPMP), which lead to the formation granulocyte-monocyte progenitors (GMP). Mortaz et al. [28]

12.1.2 Neutrophil Access to Inflammatory Sites

Following an infection or tissue injury, neutrophils react quickly to inflammatory cues and migrate to the inflamed/damaged area [30]. Migration of neutrophils into inflamed tissue involves many steps, beginning with adhesion to the endothelial surface, afterward by

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17 | P a g e intravascular migration, extravasation, and migration in the interstitium (Figure 4) [28]. Migration of neutrophils into inflamed tissue involves many steps, beginning with adhesion to the endothelial surface, afterward by intravascular migration, extravasation, and migration in the interstitium [28]. Neutrophils are then activated by chemokines like CXCL8, that also activate G-protein coupled receptors, causing a conformational change and stimulation of neutrophil integrin molecules like VLA-4 (CD49D/CD29), Macrophage-1 antigen (MAC-1) or CD11b/CD18), and Lymphocyte function-associated antigen 1 (LFA-1 or CD11a/CD18) [28]. These results in increased affinity for Immunoglobulin (Ig)-superfamily cell adhesion ligands (such as intercellular adhesion molecule-1(ICAM-1) expressed on the endothelium, allowing neutrophils to adhere to endothelial cells under flow conditions [28]. Neutrophils then patrol the endothelial surface or migrate along a chemokine gradient in search of the site of inflammation, crossing the endothelial layer in a process known as transendothelial migration (TEM) [31].

Extravasation of neutrophils through the endothelium can take either a paracellular or a transcellular route [28]. The paracellular route involves leukocytes passing through endothelial cell junctions, whereas the transcellular route involves neutrophils passing straight via the endothelial cell body [28]. The paracellular route is used in most of the cases of neutrophil extravasation. Neutrophils move along the endothelial basement membrane till they come across a small gap among pericytes [32]. Pericytes are contractile cells found at the abluminal site of micro-vessels that regulate capillary permeability [28]. Pericytes envelop over endothelial cells and cover 22–99% of the subcellular surface of endothelial cells. These cells are also abundant in pattern recognition receptors (PRRs), which allow them to detect and respond to inflammatory cues [28]. The cells enable the extravasation process of neutrophils into tissues [28].

When a neutrophil detects a gap between pericytes, it begins to migrate through the space by developing a protruding lamellapodium [33]. Elongation and movement of the lamellapodium through the pericyte/endothelial membrane is regulated by integrins, such as MAC1, LFA-1, and VLA-3, respectively [28]. Finally, extravasating neutrophils remove their CD18 integrins via vesicles from their extended tail or uropod at the subendothelial layer, allowing the extended tail to retract [28]. This provides access to the inflamed area, where the activated neutrophil will begin interaction with microorganisms and clearing cell debris

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18 | P a g e Figure 4 A diagram of the neutrophil extravasation cascade. The process of neutrophil migration starts with

neutrophils "tethering to" the endothelium of blood vessels through the steps of (1) rolling, (2) adhesion, and (3) crawling, firm adhesion, and patrolling. (4) Transendothelial migration takes place after neutrophils reach the inflammation site where they cross the blood vessel wall in an extravasation manner in which they travel along the endothelial basement membrane until they find a small gap between pericytes. They begin their migration through the space by forming a protruding uropod that allows neutrophils to enter the inflamed area. After uropod formation, microparticle formation occurs, which has been shown to play an important role in controlling vascular permeability. Mortaz et al. [28].

12.1.3 Neutrophil Function

Neutrophils, which are effector cells of the innate immune system, are plentiful in the circulation, accounting for up to 50–70% of overall circulating leukocytes in humans. Patients with leucocyte adhesion deficiency (LAD) – I are at risk for necrotizing infections and sepsis due to insufficient neutrophil transendothelial migration to the infection site [34].

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12.2 Neutrophil Functions Related to Antimicrobial Defense

12.2.1 Chemotaxis

The controlled process of phagocytosis (engulfing of microbes) and microbe killing by neutrophils firstly need a chemotaxis towards the infection site. Chemotaxis is the tendency of cells to migrate towards the direction of gradients of chemotactic stimuli [34]. The ability to appropriately sense chemotactic gradients is one of the final abilities obtained by neutrophils during bone marrow maturation, and this feature seems to be the most sensitive to perturbations in vivo and in vitro [34]. Various mechanisms impair neutrophil chemotaxis in sepsis. Interleukin 33 (IL33) minimizes this impairment by blocking CXCR2 downregulation, and it helps improve outcome in a murine model [34].

12.2.2 Intracellular Killing

Once neutrophils have identified and recognized a pathogen, phagocytosis can take place, followed by bacterial killing in the phagolysosome [34]. Neutrophils have two distinct but intercalating antimicrobial mechanisms, one dependent on oxygen and the other not [34]. Although classification of killing mechanisms in this way provides a comprehensive understanding, it does not reflect the in vivo situation in which both systems are active at the same time [34]. Besides that, the individual significance of both killing mechanisms is likely to change during the course of inflammation [34]. This is due to oxygen demand and supply fluctuations caused by dynamic tissue perfusion and oxygenation during the inflammatory process [34]. The oxygen-dependent pathways are controlled by ROS, which are formed downstream of O2 by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. In brief, when a neutrophil is activated, either by bacterial intake or by extracellular stimuli, the NADPH oxidase complex is formed from both cytosolic and membrane-bound components [34]. The active oxidase complex transfers electrons from cytosolic NADPH from across membrane to the electron acceptor, molecular oxygen, producing superoxide anion in the process [34].

Granule products are the basis of neutrophil nonoxidative killing [35]. The azurophilic granule contains serine proteases such as neutrophil elastase, cathepsin G, proteinase 3, and azurocidin [34]. When granules fuse with a phagosome containing bacteria, these

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20 | P a g e digestive proteases are delivered into the phagolysosome [34]. The intraphagosomal pH is rigorously changed during phagolysosome maturation. The early transition of intraphagosomal pH towards an alkaline level (pH 8.5–9.5) caused by O2− dismutation provides the initial environment for appropriate activation of proteases, resulting in ideal microbicidal and digestive function of these enzymes [34]. The presence and precise operation of granules intracellularly are critical because these organelles provide neutrophils with an armory of antimicrobial mechanisms similarly. Uncontrolled stimulation of neutrophils in an inflammatory microenvironment can cause collateral tissue injury due to elevated extracellular degranulation and neutrophil protease release [34].

Recently scientists have found a different defense mechanism of neutrophils which were completely different from classic mechanisms this is cytotoxic molecules which are produced from DNA backbone of neutrophils and are called Neutrophil Extracellular Traps (NET). NET helps to catch and kill or at least immobilize various bacteria [36].

12.3 Extracellular Matrix Proteins and Neutrophils Activation

Neutrophils are highly affected by their microenvironment that also includes the involvement of extracellular matrix (ECM). The influence of ECM proteins like collagen laminin, fibronectin, and fibrinogen on inflammation has recently been investigated but it is now clear that these proteins serve a vital role in providing signals that control various stages of neutrophil recruitment, transmigration, and activation [34-38].

The cytokine-dependent respiratory burst in human neutrophils is relies upon the communication of ECM proteins with CD11/CD18 integrins [39]. Successive studies revealed that the bovine neutrophil responses to IL-8 and platelet-activating factor (PAF), such as intracellular calcium, actin polymerization, degranulation, adhesion, and oxidative burst changed significantly after selective adhesion to various ECM proteins [28]. Moreover, the connection between CD11b on neutrophils and the ECM protein fibrinogen, provided signals that increased neutrophil life span. ECM proteins indirectly regulate neutrophil apoptosis by altering tumor necrosis factor-alpha (TNF-α) expression in the local inflammatory environment [28]. On the contrary, neutrophils' ECM proteolytic activity is required for transmigration across the basement membrane [28].

Tissue damage can result from neutrophils responding to ECM protein signals in the inflammatory microenvironment [28]. For instance, in atherosclerosis, the release of matrix

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21 | P a g e modifying mediators like neutrophil elastase (NE), myeloperoxidase (MPO), and defensins leads to the development and progression of atherosclerotic plaques [28]. In chronic obstructive pulmonary (COPD), the production and discharge of oxidants in the lung as a consequence of neutrophil interactions with ECM proteins causes injury and remodeling of the extracellular matrix. ECM proteins' interaction with neutrophils can also lead to tumor metastasis [28]. Chemokines formed from tumor cells stimulate microvascular endothelial cells, causing neutrophil adhesion and activation, which is accompanied by the discharge of neutrophil oxidants and numerous other matrix remodeling mediators [28]. This leads to remodeling of the local microenvironment, allowing tumor cells easy accessibility to premetastatic sites. Increased research efforts in this area may lead to new therapeutic options for neutrophil-mediated inflammatory disorders. This leads to remodeling of the local microenvironment, allowing tumor cells easy accessibility to premetastatic sites. Extensive research initiatives in this area may give rise to new therapeutic options for neutrophil-mediated inflammatory disorders [28].

12.4 Neutrophil Functions in Infection

12.4.1 Bacterial Infection

Recognizing the pathogens and the following recruitment of neutrophils towards the places of infection are main elements of the host defense against bacterial disease. Neutrophil recruitment is a multi-stage process that requires extravasation to distal sites of infection and/or injury of bloodstream neutrophils, enhanced recruitment of neutrophils from bone marrow reserves, and increased hematopoiesis as needed. Host pattern recognition receptors (PRRs), that include Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) proteins, identify invading pathogens and their characteristic pathogen-associated molecular patterns (PAMPs) [40].

Binding to receptors stimulates the production of a variety of proinflammatory host cytokines and chemokines, like interleukin (IL)-8, IL1α, and IL-β, CXCL1 (Growth regulated oncogene α (GROα)), CXCL2 (Macrophage inflammatory protein2α(MIP2α)),CXCL5 (Epithelial cell-derived neutrophil-activating peptide(ENA78)), tumor necrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). These molecules work as chemo attractants and increase neutrophil recruitment to infected tissues [40].

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22 | P a g e Some molecules from pathogens such as N-formylated peptides (e.g., formyl-methionyl-leucyl-phenylalanine (fMLF) that are byproducts of bacterial protein synthesis also drive the neutrophil migration. Pathogens also produce molecules which decreases neutrophil recruitment. Many of these molecule's target chemo attractants for example, the peptidases such as Streptococcal secreted esterase (SSE) and C5a peptidase A (ScpA) and Streptococcus pyogenes Cell-Envelope Proteinase (ScpC/SpyCEP) that are secreted and produced by Streptococcus pyogenes degrade compliment 5a (C5a) and IL-8 [41-42]

respectively, or SSE secreted by streptococcus which inactivates platelet-activating factor (PAF) [43]. Staphylococcus aureus holds an armory of virulence factors that can counteract the primary steps of the innate immune response are some of the examples [40].

12.4.1.1 Phagocytosis

Neutrophil’s ability to eat and afterwards killing the invading microbes is necessary for the maintenance of host health. Neutrophils kill bacteria and fungi by a process known as phagocytosis. Detection of invading microbial pathogens is mediated by receptors like PRRs (e.g., TLRs) and opsonic receptors found on the neutrophil surface that identify host proteins that are present on the microbial surface. The ligation of PRRs undergoes a complex series of molecular signals which modulate the functions of the effector, such as increased phagocytosis, killing and inflammation regulation via the production of cytokines. Phagocytosis is very effective in the presence of opsonins like specific immunoglobulin G (IgG) and complement factors which directly mediate uptake (opsonophagocytosis). IgG or IgM bound to the microbial surface is identified by Complement component 1q (C1q) that also stimulates the classic complement pathway [40].

12.4.1.2 Bactericidal Activity

Oxygen-dependent and oxygen-independent processes are used by neutrophils to kill ingested microorganisms. After phagocytosis the neutrophil produces potent antimicrobial ROS, such as superoxide radicals, hydrogen peroxide, hypochlorous acid, hydroxyl radicals, and chloramines. Besides these, cytoplasmic granules blend with bacteria-containing phagosomes and fill the vacuole lumen with antimicrobial peptides and proteases. Therefore, the strong antimicrobial activity of the neutrophil is a joint effort among and complicated effort

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23 | P a g e between highly potent proteolytic and degradative enzymes, cationic molecules, and ROS

[40].

Neutrophil extracellular traps (NETs) are web-like arrangements that include strands of decondensed chromatin enriched with the contents of neutrophil granules. NETs are discharged either from live or dying cells and, most importantly, NETs have been reported to capture and kill various bacterial pathogens. Even though NETs own the potential to increase the innate host defense, many of the bacterial pathogens secrete nucleases (e.g., Sda (streptodornase) of S. pyogenes) that can kill cell-free DNA that forms NETs[40].

12.4.2 Viral Infection

Neutrophils are the one of the first immune cell population recruited to the site of infection in viral infection. The function of neutrophils has been studied mostly in case of IAV (Influenza A Virus) infection. In the earlier section, we have discussed how neutrophil functions in bacterial infections, for eliminating the virus neutrophil have a controversial story behind. Although many viruses have been found intracellularly in neutrophils such as RSV (Respiratory Syncytial Virus), HCMV (Human Cytomegalovirus), HIV, and Hepatitis B and Hepatitis C viruses, still we are not clear whether this was due to engulfing of the virus itself or by active infection and spread of the virus within the neutrophils [44].

Neutrophils have been shown to engage specifically with RSV-infected fibroblasts and epithelial cells infected with influenza viruses which present influenza haemagglutinin glycoprotein (HA) on their surface. Neutrophil surface molecule CD43 (also known as sialophorin and leukosialin (Ly48)) was then identified as binding specifically to influenza viruses. Further, studies utilizing phorbol myristate acetate (PMA) or neutrophil elastase (treatments that cleave CD43 from the neutrophil surface) indicated that CD43 was not a unique or specific binding molecule. Thus, indicated that there were other binding molecules (like, sialic acid bearing cellular proteins (e.g., sialyl-Lex antigen), CD45, and influenza HA protein, triggering receptors expressed in myeloid cells 1 (TREM-1), Pentraxin 3 (PTX3), also known as TNF-stimulated gene 14 (TSG-14)) other than CD43[45].

In a study by Ratcliffe D et al. [46] it was shown that in in vitro neutrophils can actively take up IAV [46]. Contrary, in in vivo was observed that IAV invades neutrophils by phagocytosis of infected apoptotic epithelial cells. Likewise, neutrophils were shown to phagocytose Herpes Simplex Virus (HSV) by infected fibroblasts and CMV by infected

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24 | P a g e endothelial cells, thereby contributing to viral removal or dissemination. Multiple studies have shown that post-viral infection, neutrophils have a significant ability to mediate direct antiviral effects [44]. They can immediately start an antiviral program after intracellular regulation of polyinosinic-polycytidylic acid, a synthetic Double Stranded Ribonucleic Acid (dsRNA) analogue impersonating viral replication, which produces an overabundance of antiviral genes that restrain viral proliferation and contribute to its clearance. Since neutrophils do not express Toll Like Receptor 3 (TLR3) in case of viral infection, activation of these kinds of responses is carried by cytoplasmic RNA helicases such as melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I. TLR7 (an endoplasmic TLR that recognizes viral ssRNA) may also be involved in IAV identification and activation of neutrophils [44]. The α-defensins Human Neutrophil Peptide (HNP) and HNP-2 (a type of chemokine contained in neutrophil granules) can also restrain the IAV infectivity of epithelial cells and enhance IAV uptake by neutrophils by a mechanism presumably concerning viral aggregation [44]. HNP-1 is also involved in direct inactivation of HSV-1 and -2, CMV, vesicular stomatitis virus, and IAV in vitro, this says that its effects are not limited to specific virus types. Apart from these functions, activated neutrophils can secrete several numerous cytokines and chemokines, including TNF, IL-6, IL-8, and IFN (interferon), that recruit and activate more neutrophils to the site of infection, strengthening the immune response (Figure 5, 6) [44]. This is a tool that improves the host defense against the invading pathogen; but it can also worsen inflammation and cause parallel tissue injury. Activated neutrophils also produce matrix metalloproteases 9 (MMP9). These increases extracellular protein degradation and increases the recruitment of more neutrophils to the lung infected by IAV thus increasing the damage[44].

Formation of NETs depends on direct signaling of the virus through neutrophil TLR7 and TLR8 and the guarding effect of NET formation was reversible by DNase treatment [44].

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25 | P a g e Figure 5 Anti-viral mechanism of neutrophil. (i) The antiviral program has started after neutrophils phagocytose virus or become activated by pattern recognition receptors (PRRs) such as melanoma differentiation-associated protein 5 (MDA5) and toll like receptor (TLR-7) and start innate immunity. (ii) Neutrophils also secrete many antimicrobial agents that inactivate the virus, such as myeloperoxidase (myeloperoxidase MPO) and a-defensins defensin alpha (DEFA)) (iii) Neutrophils secrete Neutrophil Extra Cellular Trap (NETs) that can capture the virus and inactivate it through antimicrobial particles attached to NETs. (iv)Neutrophils during their interaction with other cell populations, such as CD8+ T cells and Natural Killer cells (NK), can arrange antiviral responses. Galani E, Andreakos E. [44].

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26 | P a g e Figure 6 The antiviral mechanisms that have a pathological effect on the host.(i) Neutrophils can engulf virus to propagate viral infections (ii) excessive activation of neutrophils leads to increased immune activation and leading to host tissue damage. (iii) After NET formation neutrophils can trigger autoantibody production and consequent autoimmunity. (iv) Neutrophils, through troubles in their homeostasis, can intervene with the activation of NK cells and T cells and reduce antiviral cytotoxic responses. Galani IE, Andreakos E. [44]. Toll like receptor (TLR-7), myeloperoxidase (MPO), defensin alpha (DEFA), Natural Killer (NK), Cluster of differentiation (CD), Interleukin (IL)

12.5 Neutrophil Function in Serious Bacterial Infection and Sepsis

In the initial stages of sepsis, bone marrow releases numerous neutrophils in response to a variety of cytokines, bacterial products, and other inflammatory mediators. The cells that enter circulation could spread inflammation into other organs, finally leading to damage [23]. In later stages of sepsis, most of the patients undergo immune refractoriness with unobservable levels of immune pro-inflammatory cytokines but substantial amounts of anti-inflammatory cytokines and particular cytokine inhibitors [47]. Failure to keep equilibrium between excessive and inadequate inflammation has proven to be the most significant symptom of sepsis [48].

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12.5.1 Activation of Complement and Its Association with Neutrophils In Sepsis

The excessive inflammatory reaction that characterizes sepsis is usually associated with over activation of the innate immune response and the complement system. Increased discharge of complement fragment 5a (C5a) and increased expression of C5a receptor (C5aR) increase the neutrophil circulation. Decreased gene transcription for TNF-α in the presence of lipopolysaccharides (LPSs) in vitro is seen after expression of C5a[48-49].LPS is known to induce tumor necrotic factor alpha (TNF-alpha) development in human sepsis by stimulating various kinases, leading to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Degradation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha) has been considered critical in enabling NF-κB to be activated and nuclear translocated. Compared to healthy and SIRS cases, septic patients had low levels of C5aR and C5L2 expression on neutrophils, and this expression pattern was associated with severity of the disease [48].

12.5.2 Neutrophil Migration Defect in Sepsis

To control bacterial infection locally, the migration of neutrophils to the infection site is very important and this avoids bacterial spread, but the migration mechanism is weak during sepsis, resulting in poor patient prognosis [50]. But the mechanism is still not completely known. An excessive discharge of pro-inflammatory mediators is believed to account for this consequence. In response to chemokines, including CXCL8, neutrophils bind to the endothelium. CXCL8 binds to the CXCR1 and CXCR2 high-affinity receptors and elicits neutrophil chemotaxis through activation of the phosphoinositide 3-kinase – phosphatase and tensin homolog pathway. Some experiments have shown that surface expression of chemokine receptor CXCR2 and beta-integrin CD11b, chemotaxis to IL-8, has decreased relative to healthy individuals in the neutrophils of septic shock patients [48].

12.5.3 Endothelial Injuries and Neutrophil Activation in Sepsis

Sepsis is marked by reduced oxygen supply and increased tissue demand for oxygen and this trait contributes to a severe disruption in metabolic autoregulation. Increased receptor-mediated neutrophil-endothelial cell adherence results in the secretion of reactive

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28 | P a g e oxygen species, lytic enzymes, and vasoactive substances Nitric oxide (NO), endothelin, platelet-derived growth factor, and platelet-activating factor (PAF) into the extracellular environment, which can destroy endothelial cells. Cytoskeleton degradation and microvascular endothelial barrier integrity may also be caused by LPSs, partially by activation of nitric oxide synthases (NOS), Ras homolog family member A (RhoA), and NF-κB[48,51].

12.6 Neutrophil Interaction with Platelets

Platelets are essential components of our blood having a function of keeping hemostasis and preventing excessive bleeding when injured, platelets adhere on the injured site and recruit more platelets to the injured area which leads to thrombus formation, during injury along with platelets leukocytes are also recruited to the site directly and indirectly. Directly by cell-to-cell contact and indirectly by cytokines and platelet derived macrovesicles

[52].

Apart from damage of vessels, there are other factors which activate platelets like certain bacteria and viruses. Thus, more and more data reveal platelet role as crucial part in innate and adaptive immune response (Figure 7) [52].

Figure 7 Platelet interaction with viruses and virus-related pathogen-associated molecular patterns (PAMPs). Schematic description of the major receptors and pathways taking part in virus binding and

internalization, as well as the pattern recognition receptor taking part in viral PAMP recognition by platelets. Hottz ED et al [53].

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Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), dengue virus (DENV), human immunodeficiency virus 1 (HIV-1), C-type lectin-like receptor 2 (CLEC-2), hepatitis C virus (HCV), oligodeoxynucleotide (ODN), carboxyalkylpyrrole (CAP), Nucleotide-binding domain leucine rich repeat containing pyrin 3 (NLRP3).

The formation of platelet–neutrophil aggregates in vivo is supported by margination of platelets and neutrophils to the periphery of blood vessels as a result of displacement of

erythrocytes to the central part of the vessels [54,55].

The initiation of platelet-neutrophil interaction is triggered by soluble mediators which are capable of directly activating these cells. Those mediators are as following: glycoproteins (p-selectin, von Willebrand Factor (VWF) etc.), chemokines (CXCl1, CXCl5), such molecules as High mobility group box 1 protein (HMGB1), CCL7 and plasma membrane mediators

thromboxane a2 ((TXA2), platelet activating factor (PAF))[54].

Platelets bind to neutrophil via their p-selectin and P-selectin glycoprotein ligand-1

(PSGL-1)[56] as well as platelet glycoprotein Ibα interacting with neutrophil MAC-1 (Figure

8) [54].

Figure 8 The way in which platelets and neutrophils interact, as well as the consequences of this interaction. The main receptor–ligand couples taking part in the platelet–neutrophil interaction (P-selectin–

PSGL1 and GPIb–Mac-1) are portrayed, as well as the mechanisms by which platelets strengthen leukocyte activation (through the release of CCL5 and PF4) and neutrophils stimulate platelet activation (via the release of elastase and cathepsin G (CathG) Downstream outcomes of the platelet–neutrophil interaction consists of increased leucocyte phagocytic activity, increased reactive oxygen species production, enhanced transmigration of leukocytes over the endothelial cell lining, discharge of different bioactive leukotrienes, coagulation activation by tissue factor (TF), leukocyte-mediated tissue repair and neutrophil extracellular traps (NETs) production. Lisman T et al. [54].

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30 | P a g e Although neutrophils can adhere to activated endothelium like platelets but unlike

platelets neutrophil become less efficient in adhering with increasing shear [57]. Therefore,

platelets help neutrophils to adhere to activated or injured endothelium resulting in increased shear which facilitate neutrophils to transmigrate through the endothelium.

Interactions with platelets were shown to improve the Neutrophils phagocytic capability towards different bacteria in vitro [54]. Platelets can increase the release of reactive oxygen Species and myeloperoxidase from neutrophils this also make a contribution to the pathogen Killing [54].

Platelet do play important role in neutrophil NETs formation [54]. P-selectin, PsgL1

and HMGB1 are involved in the NETs formation. In vitro data showed that by blocking

platelet-derived HMGB1 NET production is inhibited [54]. Another platelet-derived factor

TXA2 activates the MAPK pathway that is involved in the production of NETs [54].

There were increasing organ perfusion and function in the absence of NETs. Also, the removal of the NETs leads to bacterial dissemination. Complete blockade of net will

induce a complete damaging effect in host as net induced pathology [54] [Figure 9].

Figure 9 NETs formation. Platelets are often activated during Gram-negative bacteria infections and express a

variety of receptors, including P-selectin, that may aid in the process of NET formation (NETosis). The chromatin, histone, and granule enzyme lattices play an important role in pathogen removal and may also stimulate thromboinflammatory responses, eventually contributing to vascular and tissue injury. Guo et al.[58].

Platelets modulate the oxidative burst of leukocyte by controlling the release of ROS and myeloperoxidase (MPO). The level of circulating platelet-leukocyte aggregates is increased during viral and bacterial infection, which could also increase the oxidative burst during infections[52]. CD40L of platelet excites neutrophils to produce ROS. Also, HMGB1 triggers the transposition of MPO into the cell membrane. Platelet’s dense granules releases

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31 | P a g e Adenosine Triphosphate ATP which down-regulate the formation of ROS and MPO from neutrophils.

Platelet and neutrophil interaction can also lead to an anti-inflammatory effect because a platelet-neutrophil aggregate formation leading to production of lipoxin A4 which decreases neutrophil adhesion and extravasation[52].

Activated platelets improve neutrophil survival via the release of TGF-β. Apoptotic neutrophils produce CCR5, which decreases the circulation of platelet-derived CCL3 and CCL5 leading to down-regulation of inflammatory responses [52]. Additionally, platelets can seize neutrophil elastase thus preventing neutrophil induced tissue damage [52].

12.7 Soluble Adhesion Molecules: From the Cell Surface to The Bloodstream

Table 1 summarizes the main characteristics of five soluble adhesion molecules associated with sepsis. Three adhesion molecules (E-selectin, L-selectin and P-selectin) that are members of the selectin superfamily and are involved in leukocyte rolling (Figure 10)

[59]. Cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) that belongs to the immunoglobin domain superfamily cell adhesion molecules which are essential for strong adhesion and transendothelial migration [59].

Table 1 Characteristic of Sepsis-Related Adhesion Molecules.

Adhesion

molecule Expression Ligands Inflammatory

mediators

Mode of

expression Specific function Sheddase E-selectin Endothelial

cells ESLG-1, PSGL-1 TNFα, LPS, IL-1 Inducible Rolling Caspase L-selectin Leukocytes GlyCAM-1,

MAdCAM-1 TNFα, LPS, IL-1, IL-6 Constitutive, inducible Rolling ADAM-17 P-selectin Endothelial cells, platelets PSGL-1 TNFα, IL-4, IL-13, histamine, thrombin Constitutive Rolling MMP ICAM-1 Endothelial

cells Mac-1, LFA-1 TNFα, LPS, IL-1 Constitutive, inducible Firm adhesion, TEM

ADAM-17, NE

VCAM-1 Endothelial

cells VLA-4 TNFα, LPS, IL-1 Constitutive, inducible Firm adhesion, TEM

ADAM-17, NE

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a disintegrin and metalloproteinase(ADAM),endothelial selectin glycoprotein ligand 1(ESGL-1),glycosylation dependent cell adhesion molecule(GlyCAM-1),intercellular adhesion molecule-1 (ICAM-1),interleukin (IL),leukocyte function antigen(LFA),lipopolysaccharide (LPS),macrophage antigen(Mac),(mucosal vascular addressin cell adhesion molecule MAdCAM),matrix metalloproteinase(MMP),neutrophil elastase(NE),platelet selectin glycoprotein ligand(PSGL),transendothelial migration(TEM), tumor necrosis factor (TNF),vascular cell adhesion molecule-1(VCAM-1),very late antigen (VLA). Zonneveld R et al. [59])

Figure 10 Migration of Leukocytes into extravascular area Zonneveld R et al. [59].

Leukocytes initially tether and roll on the endothelium, which is controlled by E-selectin, L-E-selectin, and P-selectin and their carbohydrate ligands. Activation and adhesion: leukocyte rolling mediates interaction with chemoattractants present on endothelial surfaces, resulting in leukocyte activation, which leads to firm adhesion and arrest, controlled by the integrins macrophage-1 (Mac-1) and leukocyte function antigen-1 (LFA-1) binding to their endothelial ligands intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Following that, leukocytes migrate lateral over the endothelial wall in search of a transmigratory location, guided by VCAM-1/ICAM-1-enriched transmigratory cups (asterisks in (B) and (C)) present on endothelial cells. The final step in this cascade is trans-endothelial migration or diapedesis, in which leukocytes passes the trans-endothelial barrier, either (B) paracellular, via the inter-endothelial junctions or (C) transcellular, through the formation of a transcellular pore[59].

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12.8 P-selectin

It is well accepted that activated platelets adhere to neutrophils through a rapid surface expression of a granular protein P-selectin that binds to the high affinity counter ligand PSGL-1 expressed on neutrophils. The role of P-selectin in neutrophil recruitment is still largely unknown, with several studies indicating that neutrophils do not need functional P-selectin to migrate [60-62], whereas others illustrate the opposite [63-64]. In a study by Kornerup et al. [65] was shown that in either model (Lipopolysaccharide-induced lung inflammation or Zymosan-induced peritoneal inflammation), blocking P-selectin had no effect on neutrophil recruitment. In contrast, blocking the counterreceptor PSGL-1 significantly reduced neutrophilia in Zymosan-induced peritoneal inflammation [65]. Considering PSGL1 which is expressed on both neutrophils and platelets, platelets are known to help in the recruitment of neutrophils to the lungs in cases of abdominal sepsis [66]. PSGL1 is important for neutrophil infiltration in lungs and also in lung edema formation in patients with abdominal sepsis; additionally, PSGL1-dependent neutrophil recruitment is independent of circulating platelets. As a result of these new findings, PSGL1 may be a promising target for protecting the lungs from sepsis-induced neutrophil accumulation and tissue damage [66]. P-selectin, like the other selectins, could be measured in its soluble form in cell culture supernatants and blood plasma, with higher concentrations observed in septic patient plasma [59]. P-selectin shedding mechanisms are still poorly understood, though some experimental data suggest that shedding of P-selectin may take place through cleavage by matrix metalloproteinase in patients with cardiovascular disease or hypertension [59]. The extent to which plasma soluble P-selectin (sP-selectin) is obtained from endothelial cells versus platelets is unknown. Even so, one study observed a powerful positive connection between coagulation (disseminated intravascular coagulation, fibrinogen consumption, and thrombin activation markers) and sP-selectin in septic patients, signifying an important role for platelet shedding of P-selectin. Independent of its origin, sP-selectin could negatively modify direct leukocyte– endothelial encounters and/or indirect platelet-mediated secondary capture of leukocytes on the endothelium (but both are relies on P-selectin–platelet selectin glycoprotein ligand-1 adhesion), though this has yet to be tested directly [59]. However, P-selectins role in early infection (viral or bacterial) is still unknown. It is still unclear if it plays part in early serious bacterial infections, such as sepsis.

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13. RESEARCH METHODOLOGY AND METHODS

A prospective experimental study was performed in Lithuanian University of Health Sciences Hospital Kauno Klinikos (LSMU KK) Pediatric Emergency Department (PED). The research was conducted according to the following scheme:

Parents and children were informed about the ongoing study. The informative consent was received.

↓

Inclusion criteria Exclusion criteria

-Age: 1mo – 5years

-Time of arrival to PED: up to 12 hrs from the first onset of fever (febrile)

-Children with symptoms of SIRS (Systemic inflammatory response syndrome)

-Age: >5 years of age

-Time of arrival to PED: >12 hrs from the first onset of fever

-Refusal to participate

-Children with neurological disabilities -Chronic diseases, immunodeficiencies -Children who received antibiotics prior or on arrival to PED

↓

Children with SIRS were randomly assigned (in total n=68) -viral infection (n=42)

-bacterial infection, such as tonsillitis, adenoiditis, other not complicated bacterial infections and not SBI (n=10)

-SBI (serious bacterial infection) (n=16) --sepsis (from SBI) (n=4)

↓

Children with SIRS were randomly assigned (in total n=68) -

viral infection (n=42)

-bacterial infection, such as tonsillitis, adenoiditis, other not complicated bacterial infections and not SBI (n=10)

-SBI (serious bacterial infection) (n=16) --sepsis (from SBI) (n=4)

↓

Blood sample collection (GBC, CRP) and analysis

↓

Sample centrifugation according to the local protocol of LSMU KK Laboratory. Blood plasma collection, transportation and storing at -80*C.

↓

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N-number of patients; mo-months; PED-pediatric emergency department; hrs-hours; SBI-serious bacterial infection; SIRS-systematic inflammatory response syndrome; GBC-general blood count; CRP-C-reactive protein; C-celsius; LSMU KK – Lithuanian University of Health Sciences Hospital Kauno Klinikos.

All samples were collected in a period 2018-09-01 – 2019-12-01. The confidentiality of all patients was ensured, no personal information was included. There were 68 patients randomly selected for the study according to the inclusion criteria. For all included patients, the written consent of their parents/legal guardians was received. Before blood samples were collected, further data were collected as following: case history, physiological parameters, such as heart rate (HR), arterial blood pressure (ABP), blood oxygen saturation (SpO2), respiratory rate (RR) and temperature (T).

In order to evaluate neutrophil-platelet interaction and neutrophil role in SIRS etiology (viral, bacterial or SBI), additional blood sample was collected according the criteria of inclusion (as previously described). After sample centrifugation, blood plasma was received. All blood plasma samples were further stored in -80*C until further use. In 2021 February, all blood plasma samples were analyzed via immunoassay (ELISA) in LSMU physiology and pharmacology lab.

13.1Immunoassay (ELISA)

ELISA or immunosorbent assay is based on antigen and antibody detection in the target sample. Usually, ELISA is performed in 96-well-plate. Different biological samples are present in each well. Positive and negative controls are performed as well. The doublets for calibration curve (with a concentration of known protein of interest) are always carried out. The antigen or antibody is fixed by antibody or antigen respectively. After 1-2 hours of incubation, the plate is washed to remove unbound compounds. Secondary antibodies are added to detect necessary bound antibodies or antigens. Secondary antibodies are coupled with enzymes, such as peroxidases or alkaline phosphatases. After second incubation, unbound antibodies are washed out. Further, enzyme reacts with the appropriate substrate and the reaction results in specific color. The intensity of the color indicates amount of antibody or antigen.

We performed sandwich ELISA method in this study. For the soluble P-selectin detection in plasma samples, a commercial sP-Selectin (P-Selectin (Soluble) (CD62) human ELISA kit (Invitrogen) was used according manufactures instructions. Concentration of

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sP-36 | P a g e Selectin was determined with colorimetric method (wavelength 450 nm, microplate reader Multiscan Go 1.00.40 (Thermo Fischer Scientific).

13.2 sP-Selectin ELISA protocol[67]

A serial dilution was performed to prepare standard samples (Figure 11). The concentration from 0 – 40.00 ng/ml of sP-Selectin was achieved.

Figure 11 Dilute standards – microwell plate.

Table 2 Example of the arrangement of standards, samples and blanks.

-100 µl of standard samples and 100 µl Sample Diluent has been added to the wells. -90 µl of Sample Diluent was added to each sample well.

-10 µl of plasma sample was added to each sample well.

-50 µl previously prepared HRP-Conjugate was added to all wells.

-Plate was covered with an adhesive film and incubated at the room T*C for 2 hours on the microplate shaker.

-After, plate was washed 3 times

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37 | P a g e -Plate was covered with an adhesive film and incubated at the room T*C for 30 min.

-The reaction was stopped with 100 µl Stop Solution

-The results were obtained via microwell-reader using 450 nm as the primary wavelength.

14. Statistical data analysis

All patients were divided into four categories as following: first group – patients with clear viral infections; second group – patients with bacterial infections but not SBI; third group – SBI patients; fourth group – sepsis group.

Statistical data analysis was performed using Microsoft Excel and SPSS 22.0 (IBM, USA). Quantitated data was expressed in average and percentage. Descriptive statistics methods were used as following: average or median with standard deviation (SD) or interquartile range (IQR). For data comparison Mann-Whitney U criterion was used. Fisher exact criterion was used to compare qualitative data. Spearman coefficient was used for quantitative data comparison. ANOVA was used when more than two groups were compared. Dunn’s and Kruskal-Wallis non-parametrical criteria were used to determine statistical significance between the groups. ROC analysis was performed to evaluate biomarker sensitivity and specificity. P<0.05 was considered as statistically significant.

15. RESULTS

15.1 General study population characteristics

In total, 68 children were included into the study. 37 (54.51%) were male. The age median was 2 years. Viral infection was diagnosed in 42 (61.8%) cases. Non severe bacterial infection was diagnosed in 10 patients (14.7%). SBI (such as bacterial pneumonia, pyelonephritis, bacterial gastroenteritis, meningitis or sepsis) was diagnosed in 16 (23.5%) patients. Four children were diagnosed with sepsis (Table 3).

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Table 3 General study population characteristics.

N=68 Age median Etiology

Gender: Male n=37 Female n=31 2 (0-5) Viral infection n=42 Bacterial infection n=10 SBI n=16

Sepsis (from all SBI) n=4 N,n-number of patients; SBI – serious bacterial infection

15.2 Standard biomarkers

All participants received standard blood tests according to viral/bacterial infection diagnostics algorithm. GBC and CRP were performed. We further analyzed leucocyte and neutrophil counts. Higher levels of neutrophil and leucocyte counts were observed in bacterial infection. CRP levels were higher in SBI group (Table 4).

Table 4 Standard biomarkers according to etiological factor.

Biomarker Viral infection

(n=42) Bacterial infection (n=10) SBI (n=16) P value Leu, x 10*9 /L 9.39 ± 4.14 17.35 ± 5.37 14.92 ± 7.91 0.0001 Neu, x 10*9 /L 5.16 ± 3.56 12.10 ± 4 11.01 ± 7.55 <0.0001 CRP, mg/l 6.05 ± 8.19 16.96 ± 19.71 43.15 ± 59.86 0.0017

Results are expressed with standard deviation (±SD). Leu-leucocytes; Neu-neutrophils; CRP-C-reactive protein; L-liter; mg/l-milligram per liter; p-significance level; n-number of patients; SBI-serious bacterial infection.

For a better understanding, separate sepsis group was established. This group was compared to viral, bacterial and SBI (other than sepsis). Not surprisingly, when comparing all groups together we observed a statistically significant difference in leucocyte and neutrophil count (p=0.001 and p<0.0001 respectively) (Table 5).

Table 5 Standard biomarkers compared in four different etiological groups.

Biomarker Viral infection (n=42) Bacterial infection (n=10) oSBI (n=12) Sepsis (n=4) P value Leu, x 10*9 /L 9.39 ± 4.14 17.35 ± 5.37 16.02 ± 7.08 11.61 ± 10.49 0.001 Neu, x 10*9 /L 5.16 ± 3.56 12.10 ± 4 12.05 ± 7.05 7.88 ± 9.22 <0.0001

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Results are expressed as average and standard deviations (±SD). Leu-leucocytes; Neu-neutrophils; L-liter; mg/l-milligram per liter; p-significance level; n-number of patients; oSBI-other serious bacterial infection except sepsis.

15.3 Neutrophil and neutrophil-platelet activation markers

Further calculated markers showing neutrophil or neutrophil-platelet activation were calculated. NLR (neutrophil to lymphocyte ratio), NMR (neutrophil to monocyte ration), PNR (platelet to neutrophil ration) and PNLR (platelet*neutrophil to lymphocyte ratio) were first calculated and compared between viral, bacterial infection and SBI (including sepsis) group.

All the analyzed biomarkers showed to be statistically significant when compared viral versus bacterial infection and SBI (Table 6). The same was observed when all the patients were divided into four groups (viral, bacterial, sepsis and SBI other than sepsis) (Table 7). NLR, NMR, PNR and PNRL differed significantly between the groups (p-0.0267, 0.0057, 0.0003 and 0.0085 respectively).

Table 6 Neutrophil-platelet activation markers from GBC in three different groups.

Biomarker Viral infection

(n=42) Bacterial infection (n=10) SBI (n=16) P value NLR 1.830 [0.91-5.14] 5.430 [2.14-11.37] 6.387 [1.26-10.74] 0.0231 NMR 3.44 [1.85-6.23] 7.61 [5.43-9.01] 7.32 [3.60-12.45] 0.0019 PNR 62.82 [40.80-97.93] 25.93 [20.29-30.70] 32.05 [18.02-80.25] 0.0003 PNLR 482.8 [202.1-1547] 1291 [764.8-2691] 1246 [435.2-3511] 0.0157 Results are expressed as median and interquartile range. n-sample size; SBI-serious bacterial infection; p-significance level; NLR-neutrophil to lymphocyte ratio; NMR-neutrophil to monocyte ratio; PNR-platelet to neutrophil ratio; PNLR-platelet*neutrophil to lymphocyte ratio

Neutrophil Role in Early Diagnostics Of A Serious Bacterial Infection

Neutrophil Role in Early Diagnostics Of

A Serious Bacterial Infection

TABLE OF CONTENTS

3. SUMMARY

4. Acknowledgements

5. Conflict of interest

7. ABBREVIATION LIST

8. TERMS

9. INTRODUCTION

10. AIM

11. OBJECTIVES

12. LITERATURE REVIEW

13. RESEARCH METHODOLOGY AND METHODS

↓

↓

↓

↓

↓

↓

14. Statistical data analysis

15. RESULTS