Organization of immune antimicrobial response

basophils and some lymphocytes have been reported in this blood product (47). Lymphocytes are the second most abundant cells in GCs. Since they are a major player in adaptive immunity, GCs are irradiated prior to transfusion to inhibit their differentiation and proliferation. Thus, it is possible that other innate and adaptive immune cells found in GCs also contribute to the overall antimicrobial effect of GCs in vivo.

2.1 Organization of immune antimicrobial response

As previously mentioned, our immune system provides both innate and adaptive immunity.

These seemingly separate entities function together to mount a robust and efficient immune response.

Epithelial cells

The epithelium is the first barrier against pathogens and is also part of innate immunity. To prevent the entry of bacteria, fungi and viruses, epithelial cells can produce mucus to trap and eliminate pathogens for example in the respiratory track (66, 67).

Epithelial cells recognise pathogens through pathogen recognition receptors (PRR) expressed on their surface. These receptors recognize pathogen associated molecular patterns (PAMPs) through different types of PRR such as toll-like receptors (TLRs) (68). Each TLR is specialised in the recognition of different PAMPs from exogenous pathogens as well as damage associated molecular patterns (DAMPs) that can be released by endogenous damaged cells. Bacterial components such as lipopolysaccharide (LPS), a component of the surface membrane of Gram-negative bacteria, is recognized by TLR4. Components of fungal pathogens such as mannans and beta-glycans can be

recognized by TLR4, TLR1/TLR2, and TLR6/TLR2 and C-type lectin receptors such as Dectin-1 and SIGNR1 (69). Viruses are also recognized through their RNA or DNA by intracellular TLRs (TLR9, TLR7, and TLR3) at the surface of endosomes (70). Recognition of pathogens through PRRs and co-receptors causes the activation of intracellular signalling cascades involving adaptor molecules, translocation of transcription factors to the nucleus and expression of cytokines such as interleukin (IL)-8, a potent chemoattractant for neutrophils and monocytes.

Neutrophil life cycle and effector functions

Neutrophils comprise 50-70 % of all circulating leukocytes, and are one of the first leukocytes to be recruited to the site of infection (61). Neutrophils have a short life span in circulation of 8-12 hrs on average that can be extended to 5.4 days when activated (71-74). Since they were described by Elli Metchnikoff who was awarded the Nobel Prize in 1908 (75), neutrophil biology has evolved considerably. At first, neutrophils were only considered short-lived phagocytic cells often causing aggregation and inflammatory tissue damage. Neutrophils not only phagocyte pathogens and kill them by producing reactive oxygen species (ROS) and antimicrobial peptides but they also trap them extracellularly in NETs during acute infection. Furthermore, neutrophils are now considered essential in the regulation of innate and adaptive immunity through cross-talk with monocytes and macrophages to eradicate pathogens (76).They also link the innate and adaptive immune response by interacting with dendritic cells and by inducing adaptive T-cell immune response against pathogens and tumour antigens as well as the B-cell response (62, 64, 65). New features of neutrophil biology also include the emergence of neutrophil sub-populations in health and disease characterized by a pro- or anti-inflammatory phenotype (60).

Life cycle of neutrophils

Neutrophils, eosinophils, basophils and monocytes, are cells of the myeloid lineage whereas lymphocytes such as T and B cells are form the lymphoid lineage. Neutrophils originate from a self-renewing hematopoietic stem cell that in the bone marrow differentiates into a common myeloid progenitor (CMP). The CMP further differentiates into granulocyte and macrophage progenitors (GMP)

that gives rise to neutrophils and macrophage and dendritic cell (DC) progenitors (MDP) (77). GMPs differentiate into promyelocytes, myelocytes, metamyelocytes, band cells prior to becoming segmented mature neutrophils. Each step of differentiation is carefully regulated by specific growth factors and cytokines such as G-CSF (64, 71).

Neutrophils express the chemokine receptor CXCR4 at their surface to maintain their homing in the bone marrow by binding with chemokine ligand CXCL12 expressed on the surface of stromal cells. G-CSF directly stimulates the proliferation of neutrophil precursors and neutrophil release from the bone marrow by interfering with the CXCR4/CXCL12 interaction. During infection, neutrophil production is partly regulated by Th17 lymphocytes that produce IL-17. This cytokine increases the secretion of G-CSF and as a consequence, granulopoiesis and induces the release of neutrophils from the bone marrow (Fig. 4) (78-80).

Once neutrophils exit the bone marrow, their cytoplasmic granules and secretory vesicles are fully formed. The primary granules (azurophilic) are formed during the promyelocyte stage and contain myeloperoxidase (MPO), elastase (NE) and defensins. Secondary granules (specific) are formed at the myelocyte stage and contain metalloproteinase, lactofferin, cathelicidin, lipocalin 2 and olfactomedin 4. The tertiary granules (gelatinase) are the last granules to form when neutrophils differentiate into band cells and express arginase 1, gelatinase and lysozyme. Secretary vesicles are only found in mature segmented neutrophils. They contain several membrane receptors, components of the nicotinamide adenin dinucleotide phosphate (NADPH) oxidase complex as well as albumin and soluble mediators (Fig. 5) (63, 81). The release of the content of granules and secretory vesicles increases when neutrophils destroy pathogens through a process known as degranulation (81, 82).

After neutrophils complete their antimicrobial functions, they are removed from the site of infection to avoid collateral damage and to control the immune response. The interaction of neutrophils with

pathogens can result in different outcomes. Classically, neutrophils undergo spontaneous apoptosis after the internalization of pathogens by phagocytosis and are removed from the site of infection by

Figure 4

Figure 4: Regulation of neutrophils life cycle. Neutrophils mature in the bone marrow from GMP to mature segmented neutrophils and then exit the bone marrow under the action of G-CSF that inhibits the interaction between CXCL12 and the CXCR4 receptor on neutrophils. During inflammation, neutrophils produce cytokines such as CCL2 and CCL20 that promote the secretion of IL-17 by Th17 cells. The release of G-CSF by Th17 cells induces neutrophil differentiation and release from the bone marrow creating a positive regulation loop. When apoptotic neutrophils are cleared by macrophages, the production of IL-23 that stimulates Th17 cells is inhibited diminishing the production of G-CSF and consequently the release of neutrophils from the bone marrow.

Adapted from (64)

Figure 5

Figure 5: Sequential granule maturation of neutrophils. Neutrophils acquire the various granules in a sequential manner during differentiation from the promyelocyte state to mature neutrophil.

Granule formation coincides with the temporal expression by the neutrophil of the proteins stored in each type of granule. Azurophilic primary granules are the first to mature followed by specific secondary granules and gelatine tertiary granules. The secretory vesicles mature at the last stage of neutrophil differentiation. Adapted from (83)

macrophages. This process called efferocytosis, involves the death of neutrophils and the engulfed pathogen. In other situations, the phagocytosed pathogen may bypass its elimination by delaying neutrophil apoptosis which results in pathogen survival. NETosis is also a type of cell death. This phenomenon does not always guarantee the elimination of the pathogen and may cause additional damage to the tissue (84). Though G-CSF administration to the donors in GTX is exogenous, it has the same effect as endogenous G-CSF binds to G-CSF receptors expressed on immature progenitors in the bone marrow and on circulating mature neutrophils (85).

Recruitment of neutrophils

In order to kill pathogens rapidly, neutrophils have to be rapidly recruited from the circulation to the infected tissue. Neutrophil recruitment is a tightly controlled process involving their interaction with endothelial cells followed by endothelial cell activation, neutrophil adhesion to the endothelium, neutrophil rolling and extravasation, and migration to the site of inflammation. This function is specific to mature neutrophils since it is acquired in the latest stages of neutrophil functional differentiation (Fig.

6) (86).

Endothelial cells are rapidly activated by cytokines such as IL-1β, IL-17 or tumor necrosis factor (TNF)-α produced during inflammation (63). Their activation results in the expression of adhesion molecules such as P and E-selectin at their surface that are recognized by circulating neutrophils through P-selectin glycoprotein ligand (PSGL)-1, L-P-selectin (CD62L), E-P-selectin ligand (ESL)-1 and CD44 (87).

The transient interaction between neutrophils and endothelial cells allows neutrophils to roll on the surface of the endothelium. To firmly adhere to the endothelium, neutrophils have to become activated by chemokines such as IL-8 to increase their binding affinity to the endothelium through integrins.

Integrins Mac-1 (CD11b-CD18), lymphocyte function-associated antigen (LFA)-1 (CD11a-CD18) and α4β1 integrin (VLA-4) (CD49d-CD29) mediate this interaction on neutrophils to bind intracellular adhesion molecule (ICAM)-1 and ICAM-2 immunoglobulins on endothelial cells. This firm interaction slows neutrophils rolling until they fully stop prior to transmigrating into the tissue. The transmigration of neutrophils can either be paracellular of transcellular. The paracellular pathway involves the squeezing of neutrophils between the endothelial cells whereas the transcellular route involves the

Figure 6

Figure 6: The temporal acquisition of effector functions during neutrophil differentiation.

Neutrophils acquire their antimicrobial functions sequentially during their differentiation from the myeloblast state to mature neutrophils. Neutrophils first acquire the capacity to recognize and internalize pathogen before being able to eliminate them with the production of ROS and anti-microbial molecules. Chemotaxis is the last function acquired that involves the recognition of chemoattractant molecules, up regulation of adhesion receptors and the migration of neutrophils through the endothelium. Adapted from (86)

migration of neutrophils through the endothelial cells. This process is again dependent on Mac-1 and LFA-1 integrins on neutrophils and ICAM-1 and ICAM-2 on endothelial cells. These molecules mediate the formation of a protruding uropod to achieve elongation and passage of neutrophils between pericytes and across the endothelium into the tissue (88, 89) (Fig. 7).

Recruited neutrophils are more easily activated than circulating neutrophils and consequently more efficient at phagocyting pathogens and produce larger quantities of pro-inflammatory cytokines (90-92). Tissue neutrophils display a large spectrum of antimicrobial functions against bacteria, fungi and protozoa including phagocytosis, degranulation, production of ROS and cytokines and the formation of NETs.

Phagocytosis

Neutrophils must recognize and internalize pathogens to destroy them in the phagolysosome with anti-microbial peptides and ROS. Upon their activation, neutrophils also produce pro-inflammatory mediators to communicate with other cells.

Phagocytosis is the process through which neutrophils, as well as monocytes, internalize particles or pathogens larger than 0.5 µm (64). This process depends on the recognition of pathogens through receptors expressed at the surface of neutrophils including complement receptors and Fc receptors such as CD16 and CD32 that bind opsonized pathogens and also C-type lectin receptors such as Dectin-1 and Dectin-2, that bind fungal beta-glycans. Fc and complement receptors interact with pathogens through PAMPs and opsonins including antibody and complement components.

Opsonisation of pathogens facilitates their recognition by neutrophils and enhances phagocytosis. The activation of phagocytic receptors causes a cascade of intracellular signalling that reorganizes the actin cytoskeleton and the lipid membrane structure so neutrophils can complete the engulfment of the pathogens. The newly formed phagosome then undergoes a maturation process through fusion with

Figure 7

Figure 7: Sequence of events during neutrophil recruitment from the circulation to the tissue.

Neutrophil recruitment involves several tightly regulated events including neutrophil rolling on the endothelial surface, their adhesion to the endothelium, crawling and extravasation through endothelial cells and pericytes. This process is regulated by the expression of adhesion molecules on endothelial cells and neutrophils whose interaction gradually strengthens until neutrophils stop and cross the endothelial barrier to enter the affected tissue. Adapted from (60)

different vesicles and lysosomes that gradually lowers the pH and increases the cytotoxicity of the intraphagolysosomal environment (93-95).

Pathogens have developed strategies to survive phagocytosis by interfering with different parts of the phagocytic process (Fig. 8 and 9). For example, Staphylococcus aureus inhibits neutrophil effector functions such as chemotaxis (96), adhesion and phagocytosis. S. aureus produces protein A that binds to the Fc portion of IgGs to prevent their recognition by Fc receptors and thus inhibit their engulfment. It can also damage the neutrophil membrane with the production of cytolytic toxins leading to osmotic lysis of the phagocyte (97, 98). Fungal pathogens are also capable of bypassing the host defences. Candida species developed systems to evade their clearance from the host by shielding PAMPs that normally lead to their recognition by immune cells. They can also form hyphae that are less inflammatory than the yeast forms (99). Hyphae are also larger and thus diminish the ability of neutrophils to phagocyte them (100).

Degranulation

Neutrophil granules contain a variety of pre-formed microbicidal molecules ready to be released in order to kill pathogens and to reorganize the extracellular matrix of the infected tissue.

Similarly, to their formation, cytoplasmic granules and secretory vesicles are secreted sequentially (82).

Although degranulation is a quick response to kill pathogens this process is also highly cytotoxic and needs to be carefully regulated and hierarchical. Degranulation is initiated first by the interaction with integrins and then interaction with several receptors at the neutrophil surface (89, 101). Secretory vesicles and tertiary granules are the first to be released. Cytokines released by these granules are important for cellular communication and gelatinase proteins degrade the extracellular matrix. The subsequent release of secondary and primary granules requires a higher level of activation in neutrophils or a previous priming by cytokines and chemokines considering their high cytotoxicity.

Lactofferin from secondary granules controls bacterial growth whereas primary granules contain cytotoxic proteins such as MPO and NE that directly kill and digest pathogens (102). In addition,

Figure 8

Figure 8: Neutrophil fate after their interaction with pathogens. Under homeostatic conditions, neutrophils from the circulation age and are cleared by macrophages by efferocytosis. Neutrophils that phagocytose a pathogen can also be phagocytosed by macrophages which guaranties the elimination of the pathogen. The internalization of a pathogen, however, does not always ensure its elimination. Pathogens can evade neutrophil antimicrobial defences by delaying their apoptosis or by surviving the formation of NETs that ends with neutrophil lysis. Adapted from (60)

Figure 9

Figure 9: Antimicrobial defences of neutrophils in affected tissue. Neutrophils enter the tissue by following a gradient of chemoattractant molecules. Tissue neutrophils are activated and have a higher microbicidal activity than circulating neutrophils (priming). Neutrophils then recognize pathogens and phagocytose them in an intracellular vesicle called the phagosome which undergoes a maturation process involving the release of ROS and granule contents within the phagosome and the fusion with lysosome. Fully mature phagolysosomes provide a very toxic environment for the engulfed pathogen. In order to evade the neutrophil antimicrobial response, pathogens have developed different strategies to bypass the various microbial killing mechanisms of neutrophils.

Adapted from (103)

secretory vesicles also contain membrane-bound proteins such as components of the NADPH complex for the production of ROS.

Reactive oxygen species production

ROS have a microbicidal effect on bacteria since they can cross their membrane and damage their nucleic acids and proteins. ROS is produced by NADPH oxidase, a protein complex composed of membrane-bound and cytosolic subunits. Upon activation of neutrophils, the cytosolic subunits translocate to the membrane to assemble the NADPH complex and generate large amounts of superoxide, referred to as the respiratory burst (104, 105).

During phagocytosis the NADPH oxidase generate superoxide at the phagolysosomal membrane.

Superoxide has a very short half-life but is converted by superoxide dismutase into hydrogen peroxide (H2O2). H2O2 then can further react with chloride (Cl^-) under the action of MPO to form hypochlorous acid (HOCl) that has potent anti-microbial properties. The pH in the phagolysosome also drops, there is an influx of potassium (K⁺) that together with the antimicrobial peptides of the phagolysosome create a toxic environment for pathogens (105, 106).

Patients with CGD suffer from a mutation in the NADPH oxidase complex that predisposes them to life-threatening infections. CGD patients can be treated with GCs to temporarily offer neutrophils with intact NADPH oxidase activity (107).

Formation of NETs

Neutrophils are known to form NETs to capture pathogens under certain conditions such as bacterial killing during sepsis. Capturing circulating pathogens can be more challenging for neutrophils by phagocytosis (64). Neutrophils have thus evolved to expand their antimicrobial capacity.

NET formation is thought to begin with the production of ROS inside neutrophils that disturbs the nuclear envelope and causes intracellular DNA release. DNA is then decondensed under the action of the enzyme peptidyl arginine deiminase type IV (PAD4) that modifies arginines on histones to citrulline (108). Citrullinated histones are then released with the DNA backbone of NETs along with other proteases including MPO, NE and cytoskeletal proteins (109).

Neutrophils can form NETs in two different manners. The first, leads to the lysis and death of neutrophils considering that the nuclear envelope and the cellular membrane are completely compromised.

Alternatively, neutrophils also release NETs through independent vesicles in response to S. aureus infections (110) without immediately involving membrane disruption and death. Overall, NETs play an important role in limiting infection. They digest virulence factors from bacteria and fight the formation of biofilms They can capture a large variety of pathogens from Gram-positive and Gram-negative bacteria to fungal pathogens and parasites such as Leishmania amazonensis (111-114).

In patients with CGD where neutrophil dysfunction is due to impaired ROS production, the formation of NETs is also compromised. In vitro, CGD neutrophils showed impairment of NETs formation and killing of Aspergillus nudilans (115) (116). GC neutrophils thus also compensate for the lack of NET formation in CGD patients.

Neutrophils interaction with other immune cells

Neutrophil interaction with monocytes and macrophages

Similar to neutrophils, monocytes are innate immune cells that react quickly to infection (117).

Monocytes share several characteristics with neutrophils including phagocytosis, ROS and cytokine production (118). Recruited monocytes differentiate into macrophages or DCs. Macrophages exhibit extensive plasticity and can adopt a pro-inflammatory phenotype (M1) characterized by the expression of CD40, CD80 and nitric oxide synthase and the production of pro-inflammatory cytokines such as IL-6 and TNF-α. In contrast, M2 macrophages are anti-inflammatory and characterized by CD20IL-6 expression and the production of transforming-growth-factor-β (TGF-β) and anti-inflammatory

cytokines (e.g. IL-10). M1 macrophages thus execute antimicrobial functions and sustain the recruitment of immune cells by the release of pro-inflammatory mediators, while M2 macrophages are more involved in the clearance of neutrophils and cellular debris and in tissue remodelling (119-121).

Collaboration between neutrophils and monocytes/macrophages during inflammation includes their mutual recruitment to the affected site via the secretion of several cytokines and the release of the content of neutrophil granules (Fig. 10). In addition, neutrophils are indispensable to monocytes to eliminate pathogens by the coordinated surrounding of pathogens in tissue called swarm.

Recruitment and activation of immunity

Monocytes are less numerous than neutrophils in the circulation but macrophages are exclusively found in tissues where they have a higher threshold of activation that allows them to patrol the environment. When activated, M1 macrophages secrete neutrophil chemoattractants, such as the monocyte chemoattractant MCP-1 (122). Moreover, neutrophils, which are the first cells to arrive at the site of infection also secrete chemoattractant molecules to recruit monocytes/macrophages, DCs and other neutrophils. For example, neutrophils produce IL-17, mostly produced by Th17 lymphocytes that have both effect of attracting macrophages to site of infection and up regulating G-CSF production that leads to increase neutrophil viability and number. Activated macrophages by the release of TNF-α, IL-1β, G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) further enhance neutrophils recruitment and life span. (123, 124).

In addition to cytokines, the release of granule contents by neutrophils also influences

In addition to cytokines, the release of granule contents by neutrophils also influences