Neutrophil Activation During Septic Shock

Abstract

In addition to their well-known role as the cellular mediators of immunity, key other roles have been identified for neutrophils during septic shock. Importantly, neutrophils indeed play a critical role in the recently described immunothrombosis concept and in septic shock-induced coagulopathy. Septic shock is one of the most severe forms of infection, characterized by an inadequate host response to the pathogenic organism. This host response involves numerous defense mechanisms with an intense cellular activation, including neutrophil activation. Neutrophils are key cells of innate immunity through complex interactions with vascular cells and their activation may participate in systemic tissue damages. Their activation also leads to the emission of neutrophil extracellular traps, which take part in both pathogen circumscription and phagocytosis, but also in coagulation activation. Neutrophils thus stand at the interface between hemostasis and immunity, called immunothrombosis.

The present review will develop a cellular approach of septic shock pathophysiology focusing on neutrophils as key players of septic shock-induced vascular cell dysfunction and of the host response, associating immunity and hemostasis. We will therefore first develop the role of neutrophils in the interplay between innate and adaptive immunity, and will then highlight recent advances in our understanding of immunothrombosis septic shock-induced coagulopathy.

INTRODUCTION

Polymorphonuclear neutrophils (PMNs) are key players in host defense during bacterial and fungal infections ⁽¹⁾. These neutrophils are primarily innate immune cells involved in early response to infection ⁽²⁾. Rapid control of the pathogen follows the medullary release and rapid chemotactic migration of PMNs to the site of infection. Once activated, they acquire the ability to capture the pathogen by phagocytosis and to release the content of their granules ^{(3, 4)}. PMNs hence participate in microbial clearance but, at the same time, are responsible for a hyperinflammatory state contributing to the appearance of tissue lesions ⁽³⁾.

More recently, it has been shown that PMNs recruited early in response to infection participate in the regulation of the adaptive immune response, in particular by interaction with B-lymphocytes in spleen marginal zone ⁽⁵⁾.

In addition, neutrophils are also capable of releasing the content of their nucleus in the form of a meshwork of DNA fragments and modified nucleosomes, which supports enzymes such as myeloperoxidase (MPO) and neutrophil elastase (NE). These structures, called neutrophil extracellular traps (NETs), capture and engulf pathogens and circulating blood cells into their web ⁽⁶⁾. NETs thus participate in the host's immune defense. They also form a large procoagulant surface, by activating the contact phase of coagulation ⁽⁷⁾. Lastly, cytokines and pro-inflammatory mediators secreted by neutrophils alter the membrane profile of vascular cells, in particular endothelial cells, thus contributing to cellular dysfunction and septic coagulopathy ^{(3, 8)}. Through a NETosis phenomenon, PMNs may thus play a fundamental role in the interactions between immunity and thrombosis ^{(9, 10)}. The term “immunothrombosis” was introduced to describe this interaction ⁽¹¹⁾.

Immunothrombosis refers to an innate intravascular immune response responsible for thrombin generation and thrombi formation, especially in microvessels. It constitutes an independent defense mechanism, which allows the recognition, containment, and subsequent destruction of pathogens, through the activation of coagulation and the recruitment of immune cells. While immunothrombosis is involved in pathogen recognition and host defense, uncontrolled activation during septic shock can be deleterious to the host ⁽¹²⁾. As a result, there is an excessive formation of thrombin and fibrin in the microcirculation, causing bleeding, thrombosis, and coagulation factor consumption, leading to disseminated intravascular coagulation (DIC) ^{(13, 14)}.

In the present review, we will first address the role of neutrophils as main actors of innate and adaptive immunity, followed by their role in immunothrombosis. A pathophysiological approach to septic shock will hence be proposed integrating polymorphonuclear neutrophil cells as a keystone of vascular cellular dysfunction during septic shock and of the host response associating immunity and thrombosis.

POLYMORPHONUCLEAR NEUTROPHILS IN INNATE IMMUNITY

The main role of PMNs is innate immunity ⁽⁵⁾. Neutrophils are highly differentiated cells, maturing in the bone marrow before being released into the bloodstream, where they survive for 24 h ⁽²⁾. During their maturation, intracellular compartments, the granules, are formed and contain anti-inflammatory and antimicrobial proteins. PMNs are sensitive to inflammation and infection signals, which lead to their intravascular migration and subsequently their transendothelial migration by diapedesis and chemotaxis ⁽¹⁵⁾. Upon reaching the infection site, PMNs are capable of phagocytosis ⁽⁴⁾, but also of releasing the content of granules and reactive oxygen species (ROS). We will first describe the physiology of PMNs in innate immunity, then their dysfunctions during septic shock (Fig. 1).

Fig. 1: Innate immune dysfunction during septic shock.

Neutrophils, actors of innate immunity

Medullary release of neutrophils

PMNs are produced in the bone marrow by granulopoiesis, which defines the process of formation and maturation of PMNs and monocytes ⁽²⁾.

The immature cells of the granular myeloid line, or myelocytes, express different chemokine receptors (CXCR) depending on their stage of maturation. In contrast to the multiple CXC chemokines, only few CXC chemokine receptors have been shown to mediate the response to CXC chemokines in PMNs: CXCR1 and CXCR2, two receptors for IL-8 (which homolog does not exist in rodents), and CXCR4, a CXCL12 receptor ^{(16, 17)}. The less differentiated neutrophils preferentially express CXCR4, while mature PMNs express CXCR2 ^{(17, 18)}. The release of PMNs is modulated by the expression of chemokine receptors. Other receptors, such as the toll-like receptors (TLR), appear during the maturation of PMNs. CXCR2 and TLRs are thus markers of maturity and their expression is a release signal for PMNs, while CXCR4 is a medullary retention signal ⁽¹⁸⁾.

Recognition of pathogens in response to acute inflammation

Invasion by a pathogen causes an immediate response of the attacked organism via innate immunity. In 1989, Janeway ⁽¹⁹⁾ proposed the recognition of conserved pathogen-associated molecular patterns by host cells, including PMNs. This recognition involves the detection of danger signals, namely pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns expressed on the surface of pathogens, in particular lipopolysaccharide (LPS), as well as damage-associated molecular patterns (DAMPs), endogenous signals expressed after contact with the pathogen ⁽²⁰⁾. PAMPs and DAMPs are recognized by specific transmembrane receptors, the pattern recognition receptors (PRRs), including TLRs, expressed on the surface of immune cells such as macrophages, antigen-presenting-cells (APCs), or lymphocytes ⁽²¹⁾. The ensuing formation of the TLR-ligand complex activates various intracellular signaling pathways, responsible for the innate immune response ⁽²²⁾. This cascade results in the activation of transcription factors, such as nuclear factor-kappa B (NF-κB) ⁽²³⁾.

NF-κB activates the transcription of genes coding for pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-12 (IL-12). NF-κB is also activated via the LPS-TLR4 pathway and thus leads to the transcription of interferon β genes. Cytokines and interferon β amplify the activation loop ⁽²⁴⁾. The simultaneous activation of several TLRs is necessary for the reprogramming of immune cells to a pro-inflammatory phenotype ⁽²⁵⁾. In response to these transcriptional changes, the immune cells release antimicrobial peptides (interferon β, tumor necrosis factor α TNFα, IL-6, IL-8, IL-1, etc.), activate the complement, and recruit other circulating cells, notably neutrophils, which subsequently migrate by chemotaxis.

Chemotaxis

Subsequent to this pathogen recognition step, the PMNs migrate to the site of infection by chemotaxis or directed migration of leukocytes by means of various molecules ⁽²⁶⁾. These molecules are variable in nature: antibacterial peptides, such as formylmethionyl-leucyl-phenylalanine (fMLP), and chemokines, including IL-8, etc.

The acute phase of septic shock is hence characterized by the release of pro-inflammatory mediators (cytokines, proteases, etc.): this is called the systemic inflammatory response syndrome (SIRS), which is counterbalanced by the secretion of anti-inflammatory cytokines, the compensatory anti-inflammatory response syndrome or CARS ⁽²⁷⁾.

Neutrophils express TLRs capable of recognizing pathogens, in particular TLR-2 and TLR-4. Activation of the TLR pathways, notably the TLR-4 pathway, promotes chemotaxis. The response to chemotactic signals is a property acquired in the bone marrow. Thus, chemokines responsible for chemotaxis have membrane receptors on the surface of PMNs: for example, CXCR2 is the IL-8 receptor. Once in proximity to the site of infection, PMNs interact with the endothelium to reach the tissues: this step is called margination, followed by a rolling phase, i.e., the labile adhesion of PMNs to the endothelium, which requires leukocyte-endothelium interaction ⁽²⁸⁾. This step involves adhesion molecules, selectins, and ultimately integrins ⁽²⁸⁾. The selectins allow the rolling of the PMNs, while integrins facilitate their adhesion.

The final step, regulated by chemokine gradients, in particular fMLP, constitutes the trans-endothelial migration of PMNs through tight junctions.

Phagocytosis and degranulation

After migrating to the site of aggression, the neutrophil is capable of phagocytosis. Phagocytosis is a process that allows PMNs to engulf and subsequently destroy a pathogenic microorganism ⁽²⁹⁾. This constitutes the main function of neutrophils. The compartment in which phagocytosis occurs is the phagolysosome. PMNs are incapable of phagocytosis in the bloodstream due to the velocity of blood flow and thus acquire this function during their migration to the site of infection. PMNs possess two mechanisms of phagocytosis, which coexist in vivo: an oxygen-dependent mechanism involving nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase and an oxygen-independent mechanism ⁽²⁹⁾. During septic episodes, tissue oxygenation is dependent on oxygen supply and demand, resulting in a change in phagocytosis metabolism.

The oxygen-dependent mechanism is mediated by the reactive oxygen species (ROS). In response to the recognition of a pathogen, the NADPH oxidase complex assembles ⁽³⁰⁾: electrons are transported to electron acceptors, which allows the generation of ROS. These ROS are in turn metabolized into highly bactericidal products, such as hydrogen peroxide, and destroy the pathogens trapped in the phagolysosome ⁽³¹⁾. These molecules are naturally toxic and capable of destroying healthy tissues by interaction with the nucleic acids and proteins they constitute ⁽³²⁾.

During the oxygen-independent mechanism, the content of the granules, in particular the lysosomal enzymes, are released into the phagosome ⁽³³⁾. The phagosome becomes a phagolysosome whose maturation is accompanied by an alkalization (pH 7.8–8.0) enabling activation of the enzymes. Secondarily, the content of the phagolysosome becomes acid (pH at around 7.0) via the Na⁺/H⁺ exchanger ⁽³⁴⁾, which allows digestion of the captured bacteria. These changes in pH protect healthy tissue from the toxicity of granule enzymes.

Innate immune dysfunction of neutrophils and sepsis

During septic shock, all the previously described mechanisms are deficient and participate in organ dysfunction (Fig. 1).

Release of incompetent polymorphonuclear neutrophils

Neutrophil dysfunction can appear as early as during their medullary production. Thus, septic shock is accompanied by an increased medullary release in PMNs, linked to an increase in cytokine production, particularly G-CSF, and responsible for blood neutrophilia. This neutrophilia is associated with medullary depletion, causing the release of immature myelocytes ^{(35, 36)}. A greater than 10% release of immature forms is an inherent component of the definition of SIRS ⁽³⁷⁾. The release of immature PMNs is responsible for heterogeneity of circulating myelocytes, with the immature forms expressing different receptors (PRRs) in a diminished manner, resulting in an alteration in pathogen recognition ⁽³⁶⁾. Their phagocytic capacity is also reduced ⁽³⁸⁾. The presence of circulating immature forms, evidenced on blood smears, is associated with excess mortality during septic shock and the percentage of myelocytes and metamyelocytes is correlated with time until death ^{(39, 40)}. Immature PMNs may therefore not only represent a putative prognostic marker, but also reflect organ dysfunction ⁽⁴⁰⁾. This is explained by the rheological properties of immature myelocytes (rigid cell membrane), responsible for their stagnation in the microcirculation, particularly in the lungs. Thus, myelocytes and metamyelocytes contribute to the obstruction of microvessels, and therefore to hypoxemia and tissue hypoperfusion ⁽⁴¹⁾.

In addition, septic shock is characterized by mature PMNs with a heterogeneous phenotype due to the aging of circulating neutrophils, which can survive for up to 5 days ^{(42, 43)}. The aging PMNs modify their molecular repertoire and overexpress CXCR4 ⁽⁴⁴⁾. This overexpression causes PMNs to cease their return to the bone marrow, thereby promoting their margination (PMNs captured in the capillary networks) and their clearance via chemokines such as CXCL12 ⁽⁴⁴⁾. For example, in a mouse endotoxin shock model, PMNs present in normal tissues (kidneys, lungs) increase significantly ⁽⁴⁴⁾. Both the inflammatory environment of septic shock and the expression of TLR4 on the surface of these PMNs promote the change in the migratory potency of aging PMNs which have a predominant role in acute inflammation and phagocytosis ⁽⁴⁵⁾.

PMNs also exhibit impairment in pathogen recognition function during severe septic episodes. This is due to molecular mimicry between mitochondrial DAMPs and PAMPs which share similar PRR on the neutrophil ^{(46, 47)}. This mimicry and the expression of the same receptors lead to a lack of recognition of danger signals allowing the propagation of the unrecognized microorganism. Molecular mimicry is also responsible for inadequate “sepsis-like” responses in other, particularly traumatic, attacks ⁽⁴⁸⁾. Accordingly, mitochondrial DAMPs are the cause of PMN-mediated cell damage during septic shock by inhibiting chemotaxis in favor of a margination responsible for diffuse lesions in healthy tissues ⁽⁴⁸⁾.

Impaired neutrophil migration

During severe septic episodes, the decreased expression of TLR4, and hence of pathogen recognition, is responsible for lower neutrophil migration to the site of infection ⁽⁴⁹⁾: this is notably due to a deficiency in CXCR2 by PMNs as a result of the overproduction of nitric oxide (NO) ^{(50, 51, 50)}, and to a decrease in the response to its ligands (CXCL1,2, IL-8) which are nonetheless produced in large quantities ⁽⁵²⁾. The expression of CXCR1 is conserved ⁽⁵²⁾. In contrast, the expression of the β-integrin CD11b is increased. The inadequate recruitment of PMNs is responsible for a poor local control of the infection and promotes bacterial spread ⁽⁵⁰⁾ (Fig. 1). A decrease in chemokine-induced PMN motility is also observed in experimental models of endotoxemia in animals and humans ⁽⁵³⁾. The changes in the expression of chemotactic molecules result in a reduction in both PMN motility and the return of mature PMNs to the bone marrow ⁽⁵²⁾. The decrease in CXCR2 is associated with the severity of shock and is an unfavorable prognostic marker ⁽⁵²⁾. The ensuing modification of chemotaxis is indeed responsible for an inappropriate migration of PMNs in healthy organs including the lungs, resulting in occlusion of the microcirculation and a depletion in lymphoid organs ^{(54, 55)}. In addition to the repression of receptors normally expressed on the surface of PMNs, receptors physiologically absent from the PMN surface, such as the CCR2 chemokine receptor, are expressed and involved in multiple organ failure in septic shock: in a murine model of septic shock induced by cecal ligation and puncture, CCR2 expression in response to TLR-2 and TLR-4 stimulation was found to be associated with massive infiltration of vital organs by activated PMNs ⁽⁵⁶⁾. The expression of CCR2 thus contributes to multiple organ failure by causing occlusion of the microcirculation by PMNs, resulting in tissue hypoxemia. Accordingly, CCR2 is associated with excess mortality in the early phase of septic shock ⁽⁵⁶⁾ (Fig. 2).

Fig. 2: Neutrophils regulate adaptive immunity.

The migration of PMNs is also altered by the release of microparticles (MPs). MPs are plasma membrane submicron fragments with procoagulant properties released by stressed cells and capable of disseminating cytoplasmic and membrane bioactive molecules from parental cells to other cells ^{(57, 58)}. In both humans and animals, septic shock is associated with an excessive generation of endothelial-, monocyte-, and neutrophil-derived MPs ^(59–63). They promote the occurrence of a multiple organ failure syndrome by their modulatory effects on the inflammatory response, and notably participate in the recruitment of PMNs ^{(61, 63, 64)}. Neutrophil-derived MPs modulate their migratory potency: thus, PMN chemotaxis is increased in response to IL-8 by the expression of various selectins at their surface ⁽⁶⁵⁾. Conversely, PMN chemotaxis is inhibited in response to IL-1β by the expression of the anti-inflammatory molecule annexin 1 on the surface of neutrophil-derived MPs ⁽⁶⁶⁾.

Finally, Arraes et al. ⁽⁶⁷⁾ showed that severe sepsis might alter neutrophil ability to migrate toward chemoattractants. During severe sepsis, overstimulation of circulating neutrophils would induce G protein-coupled receptor kinase (GRK2 and GRK5) expression and receptor desensibilization. Subsequent desensibilization of circulating neutrophils thus led to reduced neutrophil chemotaxis, because of impaired chemoattractant-induced tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4 ⁽⁶⁷⁾.

Once in proximity to the site of infection, PMNs interact with the endothelium to enter the tissues. During septic states, the expression of selectins and integrins that initiate this interaction is altered. The expression of L-selectin is hence decreased while CD11b expression is increased ⁽³⁾. This alteration in cell adhesion during septic shock is secondary to vasoplegia due to excessive NO release ⁽⁴⁹⁾. In addition, the margination of neutrophils in the pulmonary microcirculation induces an endothelial dysfunction through the accumulation of cytokines and contributes to multiple organ failure associated with septic shock ^{(68, 69)}.

The change in physical properties of the PMNs, which become more rigid, alters their migration through endothelial tight junctions ⁽⁷⁰⁾. This excess rigidity leads to reduced deformability that is proportional to the severity of the sepsis and is responsible for capillary occlusion involved in multiple organ failure ⁽⁷⁰⁾.

During septic shock, inflammation in itself also promotes capillary sequestration by activating circulating PMNs ⁽⁷¹⁾. This activation of PMNs in the microcirculation is responsible for luminal occlusion of the small vessels as well as the migration of PMNs to vital organs and the release of cytotoxic substances (pro-inflammatory cytokines, enzymes, etc.) involved in tissue damage and multiple organ failure during septic shock ^{(3, 55)}.

Alteration of phagocytic functions

Septic shock is characterized by the association of an excessive ROS production and deficient protective antioxidant mechanisms (glutathione, selenium, etc.). These elements trigger tissue damage by immune cells, in particular PMNs, and their cytotoxic enzymes ^(72–74). The excess ROS released by the PMNs thus contributes to the vascular dysfunction during septic shock and to the concomitant vascular leakage in acute respiratory distress syndromes, particularly of inflammatory origin ⁽⁷⁵⁾. ROS production decreases with resolution of shock, while it remains elevated in non-surviving patients ⁽⁷⁶⁾. The overproduction of ROS and the persistence of high concentrations are poor prognostic factors during septic shock ⁽⁷⁶⁾.

The pH regulation of phagolysosomes is also impaired in severe septic episodes. Indeed, in severe burn patients with Staphylococcus infections, the insufficient alkalization state of PMNs explains the lack of activation of microbicidal enzymes thereby allowing bacterial survival ⁽⁷⁷⁾. In contrast, in a murine cecal ligation and puncture septic shock model, PMN acidity decreased and was associated with increased mortality ⁽⁷⁸⁾.

However, studies assessing the total phagocytic capacity of PMNs in the early phase are somewhat contradictory: some conclude to an enhanced phagocytosis ⁽⁷⁹⁾, while others conversely show a decreased phagocytic activity ⁽³⁸⁾. It is likely that the initial neutrophilia accounts for increased phagocytosis, but that the alteration of the PMN phenotype ultimately results in lower microbicidal potency and impaired pathogen recognition.

Thus, for example, the serine proteases of the PMN granules are able to cleave the complement receptors CR1 and C5aR, which have a major role in the recognition of opsonized elements to be phagocytized ⁽⁸⁰⁾. Accordingly, the deficiency in complement receptors observed during septic shock is associated with impaired phagocytosis ⁽⁸⁰⁾.

POLYMORPHONUCLEAR NEUTROPHILS IN ADAPTIVE IMMUNITY

In addition to their role in innate immunity, PMNs are capable of modulating the adaptive immune response, in particular by their interaction with other cells of the immune system, such as lymphocytes or APCs (Fig. 2). In this section, we will address the physiology of PMNs in adaptive immunity, followed by the description of their dysfunctions during septic shock.

Neutrophils and adaptive immunity

Modulation of B lymphocyte activity by PMNs

Depending on the microenvironment, PMNs are capable of stimulating or inhibiting the activity of B-lymphocytes in primary and secondary lymphoid organs. Neutrophils first interact with B-lymphocytes in the marginal zone of the spleen, located near the sinusoidal capillaries, and whose role is the rapid initiation of an adaptive T-independent immune response in response to circulating pathogens. The initiation of the T-independent response requires the presentation of circulating microorganisms to B-lymphocytes. Neutrophils are the main actors in the capture of circulating microorganisms and their transport to the spleen during sepsis in murine models ⁽⁸¹⁾. The activated PMNs express the B-cell activating cytokine (BAFF) and the bound BAFF cytokine (APRIL) in the splenic marginal zone and thus participate in T-independent B lymphocyte activation ⁽⁸²⁾. BAFF and APRIL, in combination with other cytokines specific to activated splenic PMNs such as IL-21, are necessary for the B-stimulant effect ⁽⁸³⁾. This effect is acquired by contact with microbial elements in the splenic microenvironment and is specific thereof. PMNs thus acquire a “B-helper” function and promote B lymphocyte survival, antibody production, and immunoglobulin class switching ⁽⁸³⁾. There is therefore a reprogramming of a population of circulating PMNs, thus playing a role in adaptive B-cell immune modulation (Fig. 2A).

In addition to their migration in primary lymph organs, PMNs migrate in the lymph nodes either via the bloodstream or via lymphatic vessels. The PMNs come into contact with the B lymphocytes of the medullary and inter-follicular zone of the lymph nodes and inhibit the production of antibodies by these cells, resulting in a B-inhibitory action ⁽⁸³⁾ (Fig. 2A).

Modulation of T lymphocyte activity by PMNs

T-lymphocyte activation by neutrophils is more complex. Simultaneously to the arrival of neutrophils and monocytes at the site of infection, a small number of T-lymphocytes are also recruited. This phenomenon is facilitated by the pro-inflammatory microenvironment and is notably observed during viral infections ⁽⁸⁴⁾. T-lymphocytes, non-specific to the pathogen, proliferate locally and are activated in response to the inflammatory environment. This phenomenon is called “bystander response” ⁽⁸⁵⁾. Neutrophils participate in the regulation of this “bystander response,” which contributes to the recruitment of memory T-lymphocytes and the early clearance of pathogens, in particular by interaction with other immune cells ⁽⁸⁶⁾.

A significant proportion of the T-lymphocytes recruited at the site of infection belong to the γδ-T family. These γδ-T cells are activated by neutrophils either by the release of microbial metabolites such as HMB-PP after phagocytosis or by direct cell contact ⁽⁸⁷⁾. This cellular activation also requires antigen presentation, particularly by the dendritic cells ⁽⁸⁸⁾. PMNs influence the amount of available antigen by the early clearance of the pathogen through phagocytosis ⁽⁸⁹⁾, but also by their role of direct antigen presentation, since they express the molecules necessary for the cross-priming and presentation of antigens by the major type 1 histocompatibility complex (MHC1) ⁽⁹⁰⁾. PMNs therefore act as APCs for T-lymphocytes ⁽⁹¹⁾. This function is CCR-7-dependent and is particularly involved in viral infections by antigenic presentation to CD8 T-lymphocytes ⁽⁹⁰⁾. This is notably the case in Influenzae virus infections where infected PMNs are able to present the viral antigen via MHC1 at pulmonary level ⁽⁹²⁾.

By serving as antigen-presenting cells to effector T-lymphocytes, neutrophils modulate the differentiation toward a Th1, Th2, or Th17 phenotype. For instance, NE, an enzyme of the serine protease family contained in azurophilic granules, allows the preferential differentiation of lymphocytes toward a Th1 phenotype responsible for the release of cytokines stimulating macrophage microbicidal activity and the production of antibodies (Fig. 2B).

Modulation of antigen-presenting cell activity by PMNs

Neutrophils are indirectly capable of modulating adaptive immunity, especially lymphocytic, by stimulating or repressing antigenic presentation by APCs. Interactions between APCs and PMNs remain poorly understood. It is however known that activated PMNs are capable of activating dendritic cells, and this interaction is indispensable for the functioning of both cell types ⁽⁹³⁾. In humans, the interaction between APCs and PMNs involves DC-SIGN receptors on the surface of immature dendritic cells and Mac-1 expressed by the PMNs ⁽⁹⁴⁾. While Mac-1 is an integrin expressed by several cell types, the interaction with dendritic cells is specific to PMNs due to a particular glycosylation inherent to these cells ⁽⁹⁴⁾. Through this direct interaction with APCs, PMNs suppress antigen presentation by MHC type 1, thereby contributing to the polarization of lymphocytes to a Th1 phenotype ⁽⁹⁵⁾. Nevertheless, this DC-SIGN-Mac1 interaction is not sufficient for the maturation of the dendritic cells. The latter indeed require the presence of TNFα released by neutrophils as an activating signal ^{(93, 96)}. The DC-SIGN-Mac1 interaction is nonetheless essential for recruiting APCs along with TNFα released by PMNs ⁽⁹⁴⁾.

PMNs furthermore participate in the recruitment of dendritic cells: some alarmins secreted by the PMNs (HGBM1) are chemoattractant for dendritic cells allowing their accumulation at the site of infection ⁽⁹⁵⁾. Alarmins also induce the production of chemokines such as RANTES, which in turn recruit APCs ⁽⁹⁷⁾.

Finally, dendritic cells harbor antigens derived from neutrophils having phagocytized microorganisms. However, phagocytosis limits the number of available antigens and thus limits their lymphocyte presentation ⁽⁹⁸⁾.

Adaptive immune dysfunction during septic shock

During septic episodes, PMN interactions with other cells of the immune system, including lymphocytes, are deregulated and participate in the patient's inflammatory state, as well as in a form of adaptive immunosuppression that occurs in the early phase of septic shock.

During septic shock, PMNs secrete pro-inflammatory mediators such as IL-12, responsible for T-cell differentiation to a Th-1 phenotype, and IL-4, which directs to a Th2 phenotype. The transcription factors and cytokines responsible for these two phenotypes are increased ⁽⁹⁹⁾. Th1 lymphocytes release cytokines (IL-2, IL-12, TNFα, leukotrienes, etc.) which stimulate the microbicidal activity of macrophages against the intracellular pathogens, as well as the production of antibodies targeting extracellular pathogens. Th2 lymphocytes produce humoral immunomodulatory cytokines (IL-4, IL-5, IL-9, etc.) and initiate the anti-inflammatory response. Notwithstanding, the balance between the various cytokines secreted during septic episodes is initially responsible for a Th1 phenotype ^{(72, 99)}. This lymphocytic hyper-inflammatory phenotype participates in the “cytokine storm” in the early phase of septic shock in addition to contributing to tissue damage ⁽⁷²⁾. However, septic shock is associated with Th1 and Th2 lymphopenia, a weaker transcriptional intensity and lower cytokine release than that observed during sepsis ⁽⁹⁹⁾, indicating that immunosuppression is already present, in the early phase of septic shock (Fig. 2B).

PMNs also participate in the “adaptive immunosuppression” of septic shock through lymphocyte depletion. Indeed, neutrophil activation is initially responsible for the expression of arginase-1 by certain PMNs during interphase (phase of the cell cycle during which the cell doubles all of these elements). The presence of PMNs in interphase is dependent on the degree of inflammation and is correlated with circulating IL-6 ⁽¹⁰⁰⁾. Arginase-1 is responsible for the depletion of L-arginine, an amino acid essential in the cell cycling of T lymphocytes ⁽¹⁰¹⁾. This results in lymphocyte dysfunction, since T lymphocytes are unable to continue their cell cycle (blocked at the G0-G1 phase). This blockade is secondary to a repression of the ζ chain of CD3, a coreceptor of the T-cell receptor, essential for intracellular translation ⁽¹⁰⁰⁾ (Fig. 2B). The expression of arginase-1 by PMNs in septic shock patients is correlated with the number of organ failures ⁽¹⁰⁰⁾. Immature PMNs together with their altered phenotype (decreased density) predominantly express argininase-1. L-arginine supplementation could henceforth represent a therapeutic option.

In septic shock patients, PMNs and monocytes also express a surface protein, called programmed death 1 (PD1), and its ligand PD-L1 ⁽¹⁰²⁾. The PD1-PD-L1 intracellular pathway is one of the explanatory elements of lymphocytic apoptosis responsible for post-septic immunosuppression ⁽¹⁰³⁾.

Septic shock is therefore associated with increased lymphocytic apoptosis (B and T cells). This apoptosis results in prolonged lymphopenia leading to post-septic immunosuppression, which explains the host's susceptibility to nosocomial infections that are responsible for numerous deaths in the medium term ⁽¹⁰⁴⁾. The extent of lymphopenia during septic shock is moreover associated with prognosis ⁽¹⁰⁵⁾.

In addition, apoptotic bodies derived from innate (dendritic cells, PMNs) and adaptive (lymphocytes) immune cells induce the release of anti-inflammatory cytokines, such as IL-10, at the outset of the early phase of septic shock. The increase in IL-10 concentrations during septic shock is strongly associated with fatal outcome ⁽¹⁰⁶⁾.

In contrast, PMNs have a prolonged survival during septic episodes. Under physiological conditions, PMN apoptosis is finely regulated by cell death molecules, caspases, and transcription factors of the Bcl-2 family ⁽¹⁰⁷⁾. During infectious processes, a prolonged survival of PMNs thus constitutes one of the implemented defense mechanisms. This delayed apoptosis is mediated by cell survival signals, in particular G-CSF, as well as by cytokines such as interferon γ ⁽¹⁰⁸⁾. However, while delayed PMN apoptosis is beneficial in allowing neutrophilia to persist, it also allows the survival of immature and non-fully functional neutrophils, as well as that of abnormal PMNs via their morphological properties such as low density granulocytes ⁽¹⁰⁴⁾. This prolonged survival fosters the persistence of neutrophil dysfunction in severe septic episodes.

Recent studies report another mechanism of neutrophil death, which is called autophagy ^{(107, 109)}. The latter features an autologous degradation of PMNs by their own phagosomes, a form of autophagocytosis. PMN autophagy is a caspase-independent cell death mechanism that occurs in a pro-inflammatory environment (G-CSF, inflammatory cytokines). This environment is responsible for the activation of the CD44 surface receptor ⁽¹⁰⁹⁾. This in turn activates the phosphoInositide-3-kinase (PI3K) pathway usually involved in cell survival. PI3K leads to the production of ROS by NADPH-oxidase, which allows the formation of cytoplasmic vacuoles expressing CD44. This is followed by cellular destruction by autophagocytosis. During septic episodes, this mechanism of cell death prevents the destruction of healthy tissues by the ROS ⁽¹⁰⁷⁾, although its precise role in the evolution of severe sepsis remains to be defined.

POLYMORPHONUCLEAR NEUTROPHILS IN IMMUNOTHROMBOSIS

Immunothrombosis, a concept recently described by Engelmann and Massberg ⁽¹¹⁾, refers to an innate intravascular immune response that causes thrombin generation and thrombi formation, especially in microvessels. Immunothrombosis is therefore an independent defense mechanism that allows the recognition of pathogens, their containment, and ultimately their destruction through the activation of coagulation and the recruitment of immune cells ⁽¹¹⁾. Neutrophils are key players of the immunothrombosis phenomenon, notably by the release of NETs. We will first outline the physiology of the phenomenon of immunothrombosis (Fig. 3A), which will lead us to its deregulation during severe septic episodes, responsible for both an inadequate immune response and an exaggerated activation of coagulation (Fig. 3B).

Fig. 3: Immunothrombosis in sepsis: from local beneficial defense mechanism to deregulated and excessive vascular cell activation.

Neutrophils, key players of immunothrombosis

In addition to their previously described functions, activated PMNs are capable of releasing NETs that capture and degrade pathogens and circulating blood cells ^{(6, 110)}.

Two mechanisms of NETosis are described. The first results in cell suicide and lasts a few hours, hence the first description of NETosis as a mechanism of cell death ^{(110, 111)}. The second mechanism, called “vital NETosis” ⁽¹¹²⁾, allows the PMNs to survive in an anucleated form capable of phagocytosis, a function that is independent of protein synthesis, thus not requiring a nucleus. Mitochondrial DNA can also participate in the release of NETs. “Suicidal NETosis” is dependent on the production of free radicals by NADPH oxidase 2 ⁽¹¹⁰⁾. MPO is involved in the NETosis phenomenon by the generation of halogenated ions and hypochlorite ⁽¹¹³⁾. In addition, MPO and NE potentiate chromatin decondensation and relaxation by combining with chromatin through a nonenzymatic mechanism. The most important enzymatic mechanism involved in chromatin decondensation is the citrullination of histones by peptidylarginine deiminase 4 (PAD4) with arginine as substrate ⁽¹¹⁴⁾. The “vital NETosis pathway” also appears to play a role in Gram-positive bacterial infections. This mechanism involves the TLR-2 signaling pathway, with lipoteichoic acid as ligand, as well as complement-induced opsonization phenomena ⁽¹¹²⁾. The NETs are subsequently released through vesicles.

Both NETosis mechanisms are accompanied by morphological changes described in electron microscopy after in vitro induction of NETosis by phorbol myristate acetate (PMA) ⁽¹¹⁵⁾: the chromatin first decondenses, the nucleus loses its lobules, the cytoplasm degranulates, and the NETs are released. This induction of in vitro NETosis can be likened to “suicidal NETosis.” The mechanisms involved in this process differ from the mechanisms of necrosis and apoptosis and are independent of caspases.

NETs form an extracellular trap that physically impedes the spread of microorganisms during infectious episodes. Their primary function is therefore antimicrobial (predominantly antibacterial) and antifungal. This mechanism is part of innate immunity. NETs thus mechanically oppose bacterial and fungal dissemination during septic episodes ⁽¹¹⁶⁾. In vivo, the capture of bacteria, especially Klebsiella pneumoniae, has been visualized in the lungs of septic mice ⁽¹¹⁷⁾. NETs also feature an antimicrobial action due to proteases and histone toxicity ⁽¹¹⁸⁾.

Histones are positively charged, allowing them to bind to pathogens that have an anionic membrane, leading either to the opening of membrane channels for various cytotoxic mediators or to microbial lysis. In a recent study, Abrams et al. showed the association between circulating histones after severe trauma and the occurrence of acute respiratory distress syndrome ⁽¹¹⁹⁾. This work enabled to confirm histone cytotoxicity in vitro as well as to demonstrate their toxicity in vivo in an animal model of lesional pulmonary edema. The administration of an antihistone antibody to the mice prevented the lesions ⁽¹¹⁹⁾. Histones are accordingly involved in the pathophysiology of acute respiratory distress syndrome (ARDS) and sepsis ^{(120, 121)}.

Through NETosis, neutrophils also appear to be involved in thrombotic episodes and may participate in inflammation-thrombosis cross-talk ^{(9, 10, 122)}. Thus, a second function of NETs lies in the activation of coagulation, since they naturally form an anionic surface (mainly in the form of DNA filaments) activating the contact phase of coagulation-factor XII ⁽⁷⁾. The activation of this contact system contributes to the activation of coagulation inherently associated with sepsis ⁽¹²³⁾. NETs are hence responsible for the formation of nonocclusive microthrombi, which participate in host defense during infectious episodes by containing the microorganism locally ⁽¹¹⁾.

The presence of activated platelets is essential for the formation of NETs ⁽¹²⁴⁾. The neutrophil-platelet interaction is notably mediated by the interaction between P-selectin present on the surface of PMNs and platelet P-selectin ⁽¹²⁵⁾. This mutual activation between PMNs and platelets is responsible for the activation of the extrinsic pathway of coagulation, resulting in the generation of fibrin ⁽¹²⁶⁾. Although they do not constitutively express tissue factor, PMNs could thus acquire the ability to express the tissue factor on their surface and to release MPs expressing tissue factor ^{(127, 128)}. The expression of tissue factor within the NET networks would thus favor platelet adhesion and amplification of the coagulation cascade.

In addition, NET components display procoagulant properties. Indeed, DNA amplifies serine protease activity, neutrophil elastase cleaves and inactivates the tissue factor inhibitor, while histones are potent in vitro thrombosis mediators and inactivate activated protein C ⁽¹²⁹⁾.

Neutrophils therefore deploy extracellular defense strategies to protect the host from damages caused by infection. By the emission of NETs, neutrophils have a direct antimicrobial activity and activate intrinsic and extrinsic microvascular intravascular coagulation. In this manner, PMNs propagate a distinct mechanism of intravascular immunity called immunothrombosis ⁽¹³⁰⁾.

Activation of neutrophils during septic shock is responsible for deregulation of immunothrombosis

During septic shock, neutrophils participate in the local activation of coagulation, which facilitates the recognition, containment, and destruction of circulating pathogens ⁽¹¹⁾. NETs released after pathogen recognition allow the physical entrapment of microorganisms within fibrin and DNA networks ⁽⁶⁾. Thereafter, immunothrombosis results in the formation of nonocclusive thrombi in the microvessels, particularly in the sinusoidal capillaries of the liver, forming a physical barrier against bacterial invasion into tissue ⁽¹²⁶⁾. Microthrombi also foster the local accumulation of antimicrobial, physical, and chemical strategies. Thus, fibrin enables the recruitment of other immune cells that, in turn, will enhance the immune response ⁽¹³¹⁾.

While immunothrombosis and NETs are beneficial to host defense though these various mechanisms, they can also be deleterious and participate in tissue lesions; this is the case of septic shock-induced DIC, where the regulatory mechanisms of immunothrombosis are exceeded ^{(132, 133)}. DIC has been identified as an independent factor of mortality and of multiple organ failure during septic shock ⁽¹³⁴⁾. The clinician must therefore both recognize and counter this state of exaggerated hypercoagulability, while not impeding the normal immunothrombosis which is a defense mechanism ⁽¹³⁵⁾. The monitoring of coagulation is an important element for recognizing the most severe patients and could guide the use of future therapeutic approaches ⁽¹³⁶⁾.

During septic shock, the increase in circulating free DNA concentrations released by PMNs is an independent mortality factor ⁽¹³⁷⁾. This mortality appears to be secondary to an uncontrolled activation of coagulation in the microvessels, responsible for both microcirculatory followed by macrocirculatory failure. Nucleosomes and extracellular histones released within NETs are recognized by the host as DAMPs and thereby increase the release of cytokines, as well as the recruitment of innate immune cells and the formation of microthrombi at the site of infection ⁽¹³⁸⁾. Histones and circulating free DNA also self-activate coagulation independently of the normal coagulation cascade ⁽¹³⁹⁾. This effect is amplified by the action of serine proteases, in particular neutrophil elastase, which also promote fibrinogenesis by the cleavage of regulating anticoagulant proteins, including the tissue factor pathway inhibitor (TFPI) ⁽¹²⁶⁾. Finally, while free circulating DNA does activate fibrinogenesis, it also conversely inhibits plasmin-mediated fibrinolysis ⁽¹³²⁾. As a result, the free circulating DNA activates inhibitor of plasminogen activator 1 (PAI-1) and becomes a competitive inhibitor of plasmin when its concentration increases ⁽¹⁴⁰⁾.

In this manner, histones and extracellular nucleosomes appear to play a major role in the pathophysiology of organ failure during septic shock and may represent a potential therapeutic target (antihistone antibodies, activated protein C) ⁽¹³⁸⁾. Accordingly, in a mouse model of endotoxemic shock, the presence of NETs in the circulation, quantified by free circulating DNA, was associated with the demonstration of NETs in vivo in liver sinusoids and in pulmonary capillaries, venules, and arterioles ⁽¹⁴¹⁾. At the local level, NETs come into contact with endothelial cells and platelets and cause cellular damage ⁽¹⁴²⁾. These cell contacts, at the source of leukocyte and platelet aggregates, putatively contribute to the occlusion of microvessels and thus to microcirculatory failure observed in septic mice ⁽¹⁴¹⁾.

Moreover, in a murine model of CLP-induced septic shock, the release of circulating free DNA by NETs was to shown to increase the release of thrombin–antithrombin complexes and of pro-inflammatory cytokines, especially Il-6 ⁽¹⁴³⁾. Inhibition of NETosis by deoxiribonuclease 1 (DNase 1) administration improved the outcome of the animals by reducing bacterial load and organ dysfunctions ^{(143, 144)}. Altogether, these elements suggest that septic shock is accompanied by a deregulation of immunothrombosis phenomena mediated by the neutrophils, which interact with other cells in the vascular compartment, in particular platelets, and promote the evolution toward a diffuse microvascular coagulation state, the latter of which participates in hypoperfusion and organ dysfunctions ⁽¹⁴⁴⁾.

In humans, the major role of NETs in the hypercoagulability of septic shock has been demonstrated ⁽¹⁴⁵⁾. By the way of occlusion of capillary networks, the extensive microthrombosis of small vessels participates in a decrease in tissue oxygen extraction and contributes to the multiple organ failure during septic shock. Preventive anticoagulation may have a beneficial role in the prevention of thrombotic events during septic shock ⁽¹⁴⁵⁾. Indeed, the hyper-inflammatory state observed in the early phase of septic shock could be at the origin of an inappropriate innate immune response leading to a procoagulant state, the latter being responsible for an increased incidence of deep vein thrombosis during severe sepsis and of DIC ^{(130, 145)}. Neutrophils are thus able to promote a procoagulant state by releasing NETs. The destabilization of NETs aimed at controlling the activation of intravascular coagulation could thus represent a therapeutic option in the adjuvant treatment of septic shock-induced DIC ⁽¹⁴⁴⁾. Nevertheless, the molecules proposed in animal models (DNase 1) remain unsuitable in humans due to the lack of specificity. Recent studies have furthermore shown a benefit in terms of survival by preventive anticoagulation of septic patients with heparinized derivatives ^{(135, 146)}.

Microparticles may stand as major actor of immunothrombosis. Septic shock is indeed associated with a massive release of leukocyte-derived MPs, particularly neutrophil-derived MPs, as well as endothelial- and monocyte-derived MPs ^{(63, 147)}. Accordingly, DIC is associated with an exaggerated generation of both leukocyte- and neutrophil-derived MPs ^{(62, 148)}. The role of MPs in the pathophysiology of DIC in septic shock thus emphasizes the importance of interaction between the cells in the vascular compartment.

We have recently highlighted the presence of NETs identified by the MPO-DNA complexes and nucleosomes during septic shock-induced DIC in humans ⁽¹⁴⁹⁾. Furthermore, DIC is associated with an increase in neutrophil fluorescence in flow cytometry ⁽⁶²⁾. Given that the fluorescence reflects the degree of nucleic acid condensation, it may be indicative of the phenomenon of NETosis and the increase in transcriptional activity. NETs would hence be involved in overcoming the regulatory mechanisms of coagulation during septic shock-induced DIC ⁽¹⁴⁹⁾.

Moreover, in a mouse model of endotoxemic shock using various pathogens, NETs were found to be essential for the appearance of DIC. Inhibition of NETosis (PAD4 KO mice, DNAse administration) improved microcirculation and reduced thrombin production and platelet activation ⁽¹⁴⁴⁾. This neutrophil-platelet cooperation could thus be a major factor in the regulation of immunothrombosis and represent a therapeutic strategy for the treatment of septic shock-induced DIC ⁽¹⁵⁰⁾. In a murine model of CLP-induced septic shock, the importance of neutrophils in platelet recruitment in the sinusoidal capillary networks of the liver has also been demonstrated. Such platelet aggregation in the capillaries would explain the lack of hepatic perfusion and contribute to multiple organ failure ⁽¹⁵¹⁾.

Overcoming the regulatory mechanisms of immunity and coagulation during septic shock and DIC induced-septic shock ultimately appears to be the result of the accumulation of numerous vascular cell dysfunctions. The role of NETs and MPs in septic shock-induced DIC further emphasizes the role of the host rather than the pathogen in the evolution toward DIC ^{(144, 147, 149)}. More than the failure of a particular cell type, it is the interaction between the various actors of immunity and hemostasis, in particular the neutrophils, which appears to redirect the phenomenon of immunothrombosis against the host itself. The presence of all of the cellular and molecular elements necessary for the formation of the immunothrombus and their respective spatial macromolecular organization is a sine qua none condition for the evolution toward a state of hypercoagulability during septic shock and thus allows focusing on a number of therapeutic targets. Neutrophils are the first cells to be recruited in septic and thrombotic episodes ^{(1, 152)}. By the precocity of their action and their central role in defending the host both in terms of immunity and of thrombosis, PMNs seemingly represent a cellular target of choice for the development of future therapeutic options.

Altogether future directions in both experimental and clinical research might be to elucidate whether neutrophil intracellular signaling pathways are indeed specifically and excessively activated during septic shock-induced DIC, and which specific pathways are involved in this deregulation. A genome-wide gene expression analysis of circulating neutrophils during septic shock and septic shock-induced DIC could help to answer this question. To date, most of the studies including septic shock patients have focused on all the transcribed mRNAs. Nevertheless, a transcriptomic analysis of the PMNs from six healthy subjects compared with six old and six young septic shock patients to better understand how age might modulated immune response to severe infection, suggested that there was a common genomic signature for the production of inflammatory cytokines and other conventional cell activation markers, although some major molecular pathways may be particularly affected in the elderly during sepsis ⁽¹⁵³⁾.

CONCLUSION

Cellular activation during septic shock involves neutrophils, as part of an inadequate host response to pathogen aggression. These cells partially lose their direct antimicrobial functions, beginning as early as during their medullary production up to pathogen elimination, and acquire an immunosuppressive action compared with other immune cells, particularly the lymphocytes. Neutrophils also participate in the generation of an intense coagulation activation state that can ultimately evolve into DIC. Neutrophils thus display a functional ambiguity during septic episodes: while their role in host defense is unavoidable and essential for survival, their activation can also become deleterious to the host. The release of NETs promotes the containment and destruction of the pathogen by the association of immune and thrombotic phenomena. This activation can escape regulatory systems with an uncontrolled activation of immune mechanisms and hemostasis that trigger each other. PMNs are thus at the heart of the concept of immunothrombosis and appear to be indubitable players in the pathophysiology of DIC as a manifestation of the vascular dysfunction of septic shock. A better understanding of the mechanisms of neutrophil dysfunction and their interactions with other vascular cells during DIC and septic shock could thus open new therapeutic options.