Neutrophil death & inflammation resolution: a focus on lipid mediators 

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Monocytes, Macrophages and Mononuclear Phagocytes

Macrophages are professional phagocytes, leading members of the so-called ‘mononuclear phagocyte system’, which also includes monocytes and monocyte-derived dendritic cells [96, 97]. These cells are not only important for the phagocytosis of pathogens but also the clean up of dead, dying and senescent cells throughout the body, and they contribute critically to tissue remodelling and repair. Thereby, monocytes and macrophages can be implicated in systemic inflammation either during its propagation or resolution. This section will introduce briefly monocytes and macrophages, and their role in inflammation will be described in the respective section Systemic inflammation.
Monocytes arise in the bone marrow from a myeloid progenitor common with neutrophils, and circulate in the blood at frequencies of about 5-10%. Macrophage colony stimulating factor (M-CSF) is the major cytokine for their development and proliferation. Following inflammatory insult or infection, monocytes are recruited from the blood to the tissues and undergo differentiation into macrophages and dendritic cells, forming specialised populations within the tissue microenvironment [96].
Tissue macrophages can also have prenatal origins, more recently confirmed by fate mapping studies. Tissue resident macrophages can derive from precursors originating in the embryonic yolk sac and seeded into the tissue before birth, and their local proliferation has a considerable role in self-renewal of resident cells [98-100]. Both blood-borne monocyte precursors and yolk-sac derived cells may be equally important to replenish tissue macrophage populations during inflammation resolution [101]. The complex nomenclature of monocyte and macrophage subsets is amplified by their differences in origin and tissue location, and the functional plasticity of these cells in response to different environmental stimuli [102, 103].
The function of macrophages in different tissues has been recently well reviewed [101, 102]. The bone marrow contains stromal macrophages, which have an important function in clearing the nuclei of erythrocyte precursors [104, 105], and osteoclasts, that work with osteoblasts to control bone resorption and remodelling, and regulate the movement of HSCs [106]. The spleen contains several diverse macrophage subsets, including F4/80+ red pulp macrophages that, along with Kupffer cells of the liver, are important for erythrocyte turnover and iron recycling [107]. Phagocytic macrophages in the marginal zone of the spleen express DC-SIGN and the scavenger receptor MARCO, important for apoptotic cell clearance and capture of blood antigens [108]. As well as lymphoid tissues, specialised macrophages are abundant in all organs including the liver (Kupffer cells), lung (alveolar macrophages), skin (Langerhans’s cells), nervous system (microglia), adipose tissue and body cavities (peritoneal and pleural). Tissue specific factors and environmental cues guide transcriptional control of gene expression and therefore the diverse functional specialisation of these cells [101, 102]. Macrophages have fundamental roles in homeostatic clearance of apoptotic cells in multiple tissues, and defects in this clearance contribute to autoimmune and chronic inflammatory diseases [109].
Circulating blood monocytes exhibit also some heterogeneity. The two major populations in humans are CD14hiCD16- and CD14lowCD16+; the latter comprises between 10% and 50% of blood monocytes, as it is expanded during inflammation, leading to the descriptor ‘inflammatory’ or ‘non-classical’ monocytes, as distinct from CD14hiCD16- ‘classical’ or ‘resident’ monocytes [110]. The analogous populations in the mouse are Ly6ChiCD43+ classical monocytes and Ly6ClowCD43hi non-classical monocytes [111], usually defined as subsets of the CD115 (CSF-1) positive population, although this monocyte marker can be down-regulated during inflammation. A splenic reservoir of undifferentiated monocytes has been also described [112]. Generally, a model has emerged whereby bone marrow derived monocytes are released into the circulation with a Ly6Chi phenotype: in the absence of inflammation these cells pass through a Ly6Cmed phenotype and switch to Ly6Clow resident monocytes, which can enter the tissues and replenish resident populations. On the other hand, both Ly6Chi and Ly6Cmed monocytes can migrate into inflamed tissues and differentiate to macrophages and dendritic cells following pro-inflammatory cues [96].
At a certain point, two main polarised macrophage subsets were delineated; M1 ‘classical’ versus M2 ‘alternatively’ activated macrophages, to reflect the nomenclature of T helper cell subsets (reviewed in [113, 114]). Prototypical inflammatory signatures elicit M1 macrophages, including the cytokine IFN and TLR4 ligation, associated with macrophage production of IL-6, TNF and IL-1 . M2-type macrophages, on the other hand, have been described to be elicited via a spectrum of alternative signatures, including IL-4, Fc R ligation, glucocorticoids and IL-10 [115]. The M1/M2 concept reflects also the metabolic programming of the cells [116]. Yet this dichotomous view of macrophage activation is at odds with the considerable amount of evidence that macrophages do not form stable subsets in vivo, and that the activation status may change according to environment and stimuli, creating complex and mixed phenotypes [115, 117]. Rather than discrete subsets, M1 and M2 signatures are not mutually exclusive and form part of a spectrum of possible activation profiles, representing a modulation of macrophage critical functions [115].

Mast cells, Basophils and Eosinophils

Mast cells, basophils and eosinophils derive from a common precursor [2], and have key roles in inflammatory responses, particularly initiated at epithelial barriers such as the skin, lung, and gastrointestinal tract. These granulocyte populations contribute to classical type 2 immunity, comprising also T helper 2 cells (Th2) and innate lymphoid cells type 2 (ILC-2), which is critical for barrier defence, particularly against parasitic helminths, or airborne pathogens, but can drive chronic inflammation in the context of allergic asthma or dermatitis. Mast cell and basophil activation is particularly implicated in the classical pathway of systemic anaphylaxis induction by IgE antibodies (see section Systemic inflammation: Anaphylaxis), whereas eosinophils are heavily implicated in allergic airway inflammation.

Mast cells

Mast cells derive from bone marrow progenitor cells that migrate into tissues wherein they complete their maturation, under the synergistic influence of stem cell factor (SCF) and locally produced cytokines [118-120]. The phenotype of the mature mast cell thereby depends upon the tissue in which differentiation occurs, and the microenvironmental signals accorded [121]. Mature mast cells reside in virtually all vascularised tissue, are particularly prevalent in the skin and mucosa of the genitourinary, respiratory and gastrointestinal tracts, and are found in close proximity to blood vessels, nerves, smooth muscle cells, epithelial cells, mucous producing glands and hair follicles [122, 123].
Tissue-resident mast cells are a long-lived population of large cells (6-12 m) identifiable by a common morphology, prominent electron-dense cytoplasmic granules, and high levels of expression of c-kit (CD117), the receptor for stem cell factor (SCF) [124] and FcεRI, the high affinity IgE receptor. Mast cell specific serine proteases (tryptase and chymase) account for the majority of protein present in mast cell granules. Beside these, mast cell granules contain preformed mediators such as histamine, proteoglycans (including heparin) and carboxypeptidase A. Histamine is a potent mediator causing bronchoconstriction, bronchial smooth muscle contraction and vasodilation, and reactions such as urticaria and itch. A wide range of ligands and cytokines can activate mast cells. FcεRI aggregation results from recognition of a polyvalent antigen by IgE bound to the surface; mast cells express also receptors for IgG (FcγR) and TLR, C5a and C3a receptors. Activation can result in degranulation: the rapid release of packaged mediators into the surrounding tissue. If activated through surface c-kit or FcεRI, mast cells can rapidly synthesise eicosanoid mediators from endogenous stores of arachidonic acid. Mast cells can produce cytokines, including TNF , IL-3, GM-CSF, IL-5, IL-6, IL-10 and IL-13 [125]. Mast cells and their products can thereby influence immune responses in diverse modulatory, stimulatory or suppressive ways [126].

Basophils

Basophils share several features of mast cells, including high levels of FcεRI expression and granulocytic morphology, yet are smaller (5-8μm) and have a lifespan of days rather than months. Contrary to mast cells, basophils mature in the bone marrow and have a predominantly intravascular location, constituting less than 1% of leukocytes in the peripheral blood [127]. IL-3 is an important basophil pro-growth and survival factor. Basophils can also be activated by FcεRI aggregation or C5a and C3a receptor ligation, and may be primed by cytokines (IL-3, IL-5 and GM-CSF). TLR2 or TLR4 ligation leads to basophil production of IL-4 and IL13 and also potentiates their activation. Histamine is the major component of basophil granules, and is packaged complexed with proteoglycans. The heparin and tryptase content of basophil granules is thought to be much lower than that of mast cells. Basophils can rapidly synthesise the three cysteinyl leukotrines LTC4, LTD4 and LTE4. Notably, basophils are a major source of IL-4, which they can rapidly secrete at high levels. [125, 128]

Eosinophils

Eosinophil development occurs in the bone marrow, and mature eosinophils are released into the circulation after IL-5 stimulation, although a prominent pool remains in the bone marrow: their half-life in circulation is akin to that of neutrophils, around 8-18h. The cationic proteins contained within their specific granules determine the unique staining properties of eosinophils. These comprise major basic protein, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neutrotoxin, and are toxic to microbes and particularly parasites. Eosinophils also contain lipid bodies, which are not vesicles but cytoplasmic structures that are major sites of eicosanoid synthesis. Unlike basophils and mast cells, eosinophils express FcεRI at very low levels, but exhibit prominent expression of IgG and IgA receptors (FcγRIIA/III and FcαRI), and crosslinking of antibody receptors is most potently effected by secretory IgA. Exocytosis can be complete or piecemeal. Like basophils, eosinophils can be primed by cytokines (IL-3, IL-5, GM-CSF), chemokines and PAF. Eosinophils can produce a very wide range of cytokines, but at low levels compared to other leukocytes, and their effector mediators are more prominently lipid-derived: LTC4, PGE2, thromboxane and PAF [125].

Neutrophil death & inflammation resolution: a focus on lipid mediators

It is now well established that controlled cell death and clean up is critical to initiate pro-resolving pathways: immunologically quiescent removal of dead cells is critical to normal development and homeostasis, as well as to inflammation resolution [129]. Neutrophil apoptosis, in particular, is an important aspect [130]. Once infiltrated into the tissue, neutrophils can reverse transmigrate or return via the lymphatics, as described above, however neutrophil apoptosis and uptake by macrophages is the dominant pathway of clearance. In the context of infection, exposure to bacterial products prolongs neutrophil survival, but phagocytosis triggers apoptosis, and apoptotic cells are taken up by both resident and recruited macrophages [131].
Macrophage uptake of aging or apoptotic neutrophils is an important mechanism to limit inflammation and potential tissue injury induced by neutrophil products, as well as to dampen the inflammatory responses of macrophages themselves. After ingestion, macrophages adopt an anti-inflammatory phenotype [132]. Indeed apoptotic cells influence phagocyte reprogramming through pleiotropic immune regulatory pathways, [133, 134]. Not only do neutrophils switch from synthesis of inflammatory to pro-resolving lipid mediators [93], lipoxin production by neutrophils promotes their uptake by macrophages, and macrophage production of specialised proresolving mediators resolvins, protectins and maresins [94].
From proinflammatory mediators to agonists that actively promote resolution: the temporal regulation of lipid mediator production has become a new paradigm of inflammation resolution. This is a critical point as we go on to consider the role of neutrophils in systemic inflammation. Firstly, that neutrophil production of lipid mediators can be associated with both inflammatory and resolving signatures; and secondly, that the initial inflammatory signature initiates biosynthesis pathways that also actively promote resolution.

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Antibodies & their receptors: conferring innate immune cells with adaptive specificity

Antibodies, or B cell-derived immunoglobulins, represent the potent humoral arm of adaptive immunity and are critical for host defence; yet the effective function of antibodies is equivalently implicated in disease pathogenesis, from autoimmunity to allergy. Antibody receptors, including receptors for IgG (FcγR) and receptors for IgE (FcεR), have diverse in vivo functions and patterns of expression, and control antibody-induced activation and/or recruitment of immune cells, which can mediate inflammation and disease progression, or resolution. One major component of this thesis is the role of IgG antibodies and their receptors in driving the systemic inflammation associated with anaphylactic shock. This section will describe the production of antibodies, their subclasses, and their receptors, with a focus on classical cell surface receptors for IgG (FcγR) and their differences between mice and humans, which informs how we can apply mouse models to study IgG-dependent inflammation.

B cells and the BCR

Adaptive immunity encompasses B and T lymphocytes with specific antigen recognition receptors, the B cell receptor (BCR) and the T cell receptor (TCR). These lymphocytes develop from a common lymphoid progenitor; T cells undergo maturation in the thymus and B cells mature in the bone marrow [135]. The antigen receptors of B and T cells develop by way of numerous enzymes which coordinate to rearrange the genes coding for the variable regions of these receptors, in a process called V(D)J recombination. Successful rearrangement of genetic elements within the immunoglobulin (Ig) or Tcr loci permits the functional expression of a BCR or the TCR, respectively, containing a variable region with a unique specificity. The power of this system is to generate an enormous diversity of receptor specificities from a small region of DNA, thereby facilitating the generation of a wide range of potential immune reactivities. Immune checkpoints and balances during cell development control the emergence of mature T and B cell clones with distinct reactivities and a tolerance to self, or host-derived molecules.
The coordinated activation of B and T cells occurs for the most part in specialized lymphoid structures, most notably the spleen and the lymph nodes. Whereas T cell activation absolutely depends on other cells presenting antigen, for example macrophages or DCs, in the context of antigen presentation molecules (major histocompatibility complex; MHC), B cells can directly interact with antigen via the BCR. Importantly, the BCR provides the direct template for subsequent antibody production. When a B cell encounters its cognate antigen, it receives activating signals, and the addition of costimulatory signals via PRRs, proinflammatory factors or cytokines permit full cellular activation. Rapid clonal expansion of activated B and T cell populations permits a full and potent adaptive immune response. Critically, however, the activation of these adaptive lymphocytes takes time, and this lag phase contrasts with the rapid activation of leukocytes of the innate immune system.
B cells can undergo additional changes to their BCR prior to differentiation to become an antibody-secreting plasma cell. This process is referred to as affinity maturation, as the affinity of the BCR becomes greater for its respective epitope, the site of antigen binding, by way of small modifications within the variable region. After receiving appropriate helper signals from interaction with a T cell of shared specificity (T-dependent antigens), or suitable PRR and cytokine signalling (T-independent antigens), B cells experience somatic hypermutation, achieved by a different set of enzymes than the original V(D)J arrangement. Within the germinal centre of a secondary lymphoid organ, after somatic hypermutation B cells test their modified BCRs on antigens presented to them by follicular dendritic cells. The population undergoes affinity maturation, as more high affinity clones are selected for with survival signals, and clones of lower affinity undergo apoptosis. B cells can undergo multiple rounds of affinity maturation, both within a single immune response and over several antigen exposures over the lifetime of the individual. A high affinity B cell clone can differentiate to become a memory B cell, and thereby preserve the fine antigen recognition for subsequent encounter; the hallmark of adaptive immunity. Most of the B cell clones, however, differentiate to become plasma cells, or antibody-producing cell factories [136, 137].

 Antibodies and their classes

Antibodies are composed of two light chains and two heavy chains, connected by disulphide bridges. Each chain contains several immunoglobulin domains: a variable domain, plus one or several constant domains. The variable domains of one light chain and one heavy chain come together to form the antigen-binding portion of the BCR or antibody molecule. Antibody diversification is not limited to the variable region, described above. Naive B cells express only IgM and IgD, but other immunoglobulin classes or subclasses (isotypes) can be elicited. Immunoglobulin class switching is the process by which B cells can change the isotype of the antibody they produce: that is, the constant region of the antibody molecule can be changed while retaining the binding specificity conferred by the variable region. While the variable regions determine the exquisite specificity of antibodies, the constant regions of the antibody molecule enable and determine the in vivo functionality.
Changing constant regions occurs by class switch recombination (CSR), a genomic modification that replaces the expressed constant region (C ) of the heavy chain with a downstream region C , C or C , thereby determining B cell production of antibody isotypes IgG, IgE or IgA. Notably, IgD is generated by alternative splicing of the germline transcript, and not by CSR. CSR is central to the maturation of the antibody response, because different immunoglobulin isotypes can promote a specialised immune response to different pathogens, engaging different effector functions. The engagement of B cell PRRs and the local production of cytokines critically determine the type of CSR that occurs. Synergy between PRR and cytokine receptors within the B cell informs the targeting of the CSR machinery [138].
IgM is secreted as pentamers or hexamers, with a high avidity for antigens with repetitive motifs, and a strong potential for activation of the complement cascade. It is effectively released in the early phases of B cell responses, where prior to affinity maturation the avidity of the antibody-antigen interaction is of greater importance. Yet IgM molecules cannot pass into the extravascular space, and do not recruit cellular effectors as efficiently as other isotypes. Conversely, monomeric IgG, IgE and IgA can be distributed systemically to tissues, to therein mediate a large variety of effector functions. IgG is the most dominant antibody subclass, exhibits the highest rate of synthesis and longest biological half-life: IgG1 antibodies are present at serum concentrations of 5-12mg/ml with a half-life of 21 days. Classically speaking, in humans, IgG1 and IgG3 are effective against viruses, IgG2 against encapsulated bacteria, IgG4 and IgE against large extracellular parasites, and IgA1 and IgA2 against pathogenic bacteria at the mucosa.

Table of contents :

1 Introduction 
1.1 Myeloid cells of the innate immune system
1.1.1 Neutrophils
1.1.2 Monocytes, Macrophages and Mononuclear Phagocytes
1.1.3 Mast cells, Basophils and Eosinophils
1.2 Neutrophil death & inflammation resolution: a focus on lipid mediators
1.3 Antibodies & their receptors: conferring innate immune cells with adaptive specificity
1.3.1 B cells and the BCR
1.3.2 Antibodies and their classes
1.3.3 IgG antibody receptors (FcgR)
1.4 Systemic Inflammation
1.4.1 Inflammation-associated circulatory shock
1.4.2 Endotoxemia
1.4.3 Anaphylaxis
2 Summary and objectives 
3 A novel model of inducible neutropenia reveals a protective role for neutrophils during systemic inflammation
3.1 PAPER I
4 Neutrophils contribute to IgG-dependent anaphylaxis in FcγR-humanised mice 
4.1 PAPER II
4.2 Considering high affinity FcγRI: the Audrey mouse
4.2.1 Audrey mice exhibit hFcγRI expression patterns comparable to that of humans, and retain hFcγRIIA/IIB/III expression of VG1543 mice
4.2.2 hFcγRI does not contribute to IVIG-PSA in Audrey mice, which proceeds via a dominant pathway involving hFcγRIIA, Neutrophils and PAF
4.2.3 Supplemental Methodology and Data
5 Discussion 
5.1 Part I: A protective role for neutrophils in LPS endotoxemia
5.2 Part II: The inflammatory effects of neutrophils: novel mouse models to study neutrophil function in vivo
5.2.1 A novel model of inducible neutropenia: PMNDTR mice
5.2.2 PMNDTR mice to study neutrophils in antibody-dependent pathologies: deciphering the contribution of neutrophils to systemic anaphylaxis
5.2.3 Audrey & humanised mouse models to study FcγR: limitations and potential
5.3 Part III: Neutrophils as protective or pathological agents of systemic inflammation
5.4 Part IV: Towards the clinic: systemic anaphylaxis to neuromuscular blocking drugs
5.4.1 Developing a mouse model of systemic anaphylaxis to Rocuronium Bromide
5.4.2 Evidence from the clinical study NASA: Neutrophil activation in systemic anaphylaxis
5.5 Final Considerations and Perspectives
6 References 
7 Annex 
7.1 IgG subclasses determine pathways of anaphylaxis in mice
7.2 In vivo effector functions of high-affinity mouse IgG receptor FcgRI in disease and therapy models
7.3 Review – Contribution of human FcγRs to disease with evidence from human polymorphisms and transgenic animal studies
7.4 Book chapter – Anaphylaxis (Immediate hypersensitivity): from old to new mechanisms

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