Functional neutrophil disorders: Chronic granulomatous disease and beyond.

Since their description by Metchnikoff in 1905, phagocytes have been increasingly recognized to be the entities that traffic to sites of infection and inflammation, engulf and kill infecting organisms, and clear out apoptotic debris all the while making antigens available and accessible to the lymphoid organs for future use. Therefore, phagocytes provide the gateway and the first check in host protection and immune response. Disorders in killing and chemotaxis lead not only to infection susceptibility, but also to autoimmunity. We aim to describe chronic granulomatous disease and the leukocyte adhesion deficiencies as well as myeloperoxidase deficiency and G6PD deficiency as paradigms of critical pathways.

Apoptosis and Phagocytosis as Antiviral Mechanisms.

Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.

Efferocytosis and Respiratory Disease.

Cells are the smallest units that make up living organisms, which constantly undergo the processes of proliferation, differentiation, senescence and death. Dead cells need to be removed in time to maintain the homeostasis of the organism and keep it healthy. This process is called efferocytosis. If the process fails, this may cause different types of diseases. More and more evidence suggests that a faulty efferocytosis process is closely related to the pathological processes of respiratory diseases. In this review, we will first introduce the process and the related mechanisms of efferocytosis of the macrophage. Secondly, we will propose some methods that can regulate the function of efferocytosis at different stages of the process. Next, we will discuss the role of efferocytosis in different lung diseases and the related treatment approaches. Finally, we will summarize the drugs that have been applied in clinical practice that can act upon efferocytosis, in order to provide new ideas for the treatment of lung diseases.

Quantification of Phagocytosis Using Flow Cytometry.

Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and robustness, leading to accurate quantification of phagocytosis that is comparable across experiments and systems.Here, we describe how to quantify phagocytosis using flow cytometry with a robust, high-throughput, and easy-to-use approach. The prey is first fluorescently double stained, followed by optional opsonization before being introduced to the phagocyte in a wide range of ratios. After incubation, data is acquired through flow cytometry. It can be assessed on both the population and single-cell level of the phagocytes, separating adhesion and internalization. As an example, we provide an experimental protocol for studying phagocytosis of opsonized Streptococcus pyogenes using the THP-1 cell line. This approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions.

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-ß/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and ß-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx.

The roles of bactericidal cathelicidins against bacterial infection have been extensively studied. However, the antibacterial property and mechanism of action of non-bactericidal cathelicidins are rarely known. Herein, a novel naturally occurring cathelicidin (PopuCATH) from tree frog (Polypedates puerensis) did not't show any direct anti-bacterial activity in vitro. Intriguingly, intraperitoneal injection of PopuCATH before bacterial inoculation significantly reduced the bacterial load in tree frogs and mice, and reduced the inflammatory response induced by bacterial inoculation in mice. PopuCATH pretreatment also increased the survival rates of septic mice induced by a lethal dose of bacterial inoculation or cecal ligation and puncture (CLP). Intraperitoneal injection of PopuCATH significantly drove the leukocyte influx in both frogs and mice. In mice, PopuCATH rapidly drove neutrophil, monocyte/macrophage influx in mouse abdominal cavity and peripheral blood with a negligible impact on T and B lymphocytes, and neutrophils, monocytes/macrophages, but not T and B lymphocytes, were required for the preventive efficacy of PopuCATH. PopuCATH did not directly act as chemoattractant for phagocytes, but PopuCATH obviously drove phagocyte migration when it was cultured with macrophages. PopuCATH significantly elicited chemokine/cytokine production in macrophages through activating p38/ERK mitogen-activated protein kinases (MAPKs) and NF-κB p65. PopuCATH markedly enhanced neutrophil phagocytosis via promoting the release of neutrophil extracellular traps (NETs). Additionally, PopuCATH showed low side effects both in vitro and in vivo. Collectively, PopuCATH acts as a host-based immune defense regulator that provides prophylactic efficacy against bacterial infection without direct antimicrobial effects. Our findings reveal a non-bactericidal cathelicidin which possesses unique anti-bacterial action, and highlight the potential of PopuCATH to prevent bacterial infection.

IFNγ and GM-CSF control complementary differentiation programs in the monocyte-to-phagocyte transition during neuroinflammation.

During inflammation, Ly6Chi monocytes are rapidly mobilized from the bone marrow (BM) and are recruited into inflamed tissues, where they undergo monocyte-to-phagocyte transition (MTPT). The in vivo developmental trajectories of the MTPT and the contribution of individual cytokines to this process remain unclear. Here, we used a murine model of neuroinflammation to investigate how granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFNγ), two type 1 cytokines, controlled MTPT. Using genetic fate mapping, gene targeting and high-dimensional single-cell multiomics analyses, we found that IFNγ was essential for the gradual acquisition of a mature inflammatory phagocyte phenotype in Ly6Chi monocytes, while GM-CSF was required to license interleukin-1ß (IL-1ß) production, phagocytosis and oxidative burst. These results suggest that the proinflammatory cytokine environment guided MTPT trajectories in the inflamed central nervous system (CNS) and indicated that GM-CSF was the most prominent target for the disarming of monocyte progenies during neuroinflammation.

The Apoptosis Paradox in Cancer.

Cancer growth represents a dysregulated imbalance between cell gain and cell loss, where the rate of proliferating mutant tumour cells exceeds the rate of those that die. Apoptosis, the most renowned form of programmed cell death, operates as a key physiological mechanism that limits cell population expansion, either to maintain tissue homeostasis or to remove potentially harmful cells, such as those that have sustained DNA damage. Paradoxically, high-grade cancers are generally associated with high constitutive levels of apoptosis. In cancer, cell-autonomous apoptosis constitutes a common tumour suppressor mechanism, a property which is exploited in cancer therapy. By contrast, limited apoptosis in the tumour-cell population also has the potential to promote cell survival and resistance to therapy by conditioning the tumour microenvironment (TME)-including phagocytes and viable tumour cells-and engendering pro-oncogenic effects. Notably, the constitutive apoptosis-mediated activation of cells of the innate immune system can help orchestrate a pro-oncogenic TME and may also effect evasion of cancer treatment. Here, we present an overview of the implications of cell death programmes in tumour biology, with particular focus on apoptosis as a process with "double-edged" consequences: on the one hand, being tumour suppressive through deletion of malignant or pre-malignant cells, while, on the other, being tumour progressive through stimulation of reparatory and regenerative responses in the TME.

The possibility of using xenogeneic phagocytes in wound treatment.

Metamorphosis in the insect larva is associated with disintegration, engulf and digestion of larval tissues. These processes are accompanied by a significant shift in physiological parameters like high activity of hydrolytic enzymes and decrease of pH. In the way, the metamorphosing larva resembles the processes occurring in the wound at the stage of inflammation. Based on this thesis, we put forward the idea of the possibility of using insect phagocytes in the wound treatment. The search for a suitable insect cell line and the study of its properties were the purpose of the work. The abilities of insect phagocytes to retain viability and functional activity under conditions physiological for humans were also investigated. We found that blue blowfly Calliphora vicina larvae had histolysocytes, a specialized population of professional phagocytes involved in the histolysis. In vitro, histolysocytes possess high phagocytic activity to fragments of vertebrate soft tissues and debris. These cells retain viability and functional activity for a long time under conditions that are physiological for vertebrate cells. Moreover histolysocytes can realize the humoral control over the bacteria through the synthesis of antimicrobial peptides. So histolysocytes have the potential to be used as xenogeneic phagocytes in the wound treatment. The data obtained allow proceeding to experiments on laboratory animals for studying the effect of such therapy on the wound healing process.

Synovial single-cell heterogeneity, zonation and interactions: a patchwork of effectors in arthritis.

Despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. Recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. Furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. Similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. A deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients.

High Na+ Environments Impair Phagocyte Oxidase-Dependent Antibacterial Activity of Neutrophils.

Infection and inflammation can augment local Na+ abundance. These increases in local Na+ levels boost proinflammatory and antimicrobial macrophage activity and can favor polarization of T cells towards a proinflammatory Th17 phenotype. Although neutrophils play an important role in fighting intruding invaders, the impact of increased Na+ on the antimicrobial activity of neutrophils remains elusive. Here we show that, in neutrophils, increases in Na+ (high salt, HS) impair the ability of human and murine neutrophils to eliminate Escherichia coli and Staphylococcus aureus. High salt caused reduced spontaneous movement, degranulation and impaired production of reactive oxygen species (ROS) while leaving neutrophil viability unchanged. High salt enhanced the activity of the p38 mitogen-activated protein kinase (p38/MAPK) and increased the interleukin (IL)-8 release in a p38/MAPK-dependent manner. Whereas inhibition of p38/MAPK did not result in improved neutrophil defense, pharmacological blockade of the phagocyte oxidase (PHOX) or its genetic ablation mimicked the impaired antimicrobial activity detected under high salt conditions. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) overcame high salt-induced impairment in ROS production and restored antimicrobial activity of neutrophils. Hence, we conclude that high salt-impaired PHOX activity results in diminished antimicrobial activity. Our findings suggest that increases in local Na+ represent an ionic checkpoint that prevents excessive ROS production of neutrophils, which decreases their antimicrobial potential and could potentially curtail ROS-mediated tissue damage.

The Role of Intracellular Signaling Molecules in the Production of Granulocytic CSF Mononuclear Phagocytes in Stress and Cytostatic Influence.

Under conditions of steady-state hemopoiesis, nuclear factor NF-κB, in contrast to MAP kinase p38, plays an important role in the maintenance of the initial level of secretory activity of monocytes. The increase in the production of G-CSF under stress conditions (10-h immobilization) is mainly regulated by the alternative p38MARK signaling pathway via activation of p38 synthesis. It was shown that under conditions of cytostatic-induced myelosuppression, the production of protein kinase p38 in cells decreases, and it, like NF-κB, is not the main one in the production of hemopoietin by mononuclear phagocytes.

Conserved and Distinct Elements of Phagocytosis in Human and C. elegans.

Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis-the engulfment and elimination of dying cells and cell debris-are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.

MERTK on mononuclear phagocytes regulates T cell antigen recognition at autoimmune and tumor sites.

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in prediabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.

TREM2-dependent lipid droplet biogenesis in phagocytes is required for remyelination.

Upon demyelinating injury, microglia orchestrate a regenerative response that promotes myelin repair, thereby restoring rapid signal propagation and protecting axons from further damage. Whereas the essential phagocytic function of microglia for remyelination is well known, the underlying metabolic pathways required for myelin debris clearance are poorly understood. Here, we show that cholesterol esterification in male mouse microglia/macrophages is a necessary adaptive response to myelin debris uptake and required for the generation of lipid droplets upon demyelinating injury. When lipid droplet biogenesis is defective, innate immune cells do not resolve, and the regenerative response fails. We found that triggering receptor expressed on myeloid cells 2 (TREM2)-deficient mice are unable to adapt to excess cholesterol exposure, form fewer lipid droplets, and build up endoplasmic reticulum (ER) stress. Alleviating ER stress in TREM2-deficient mice restores lipid droplet biogenesis and resolves the innate immune response. Thus, we conclude that TREM2-dependent formation of lipid droplets constitute a protective response required for remyelination to occur.

Differential gene expression analysis of corneal endothelium indicates involvement of phagocytic activity in Fuchs' endothelial corneal dystrophy.

Fuchs' endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet's membrane and gradual degeneration and loss of corneal endothelial cells. The aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet's membrane (CE-DM) specimens using the Illumina HumanHT-12 v4.0 expression array. RNA from pools of FECD-affected (n = 3 per pool) and individual unaffected (n = 3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log2 fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤ 0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.

Phagocytic clearance of apoptotic, necrotic, necroptotic and pyroptotic cells.

Although millions of cells in the human body will undergo programmed cell death each day, dying cells are rarely detected under homeostatic settings in vivo. The swift removal of dying cells is due to the rapid recruitment of phagocytes to the site of cell death which then recognise and engulf the dying cell. Apoptotic cell clearance - the engulfment of apoptotic cells by phagocytes - is a well-defined process governed by a series of molecular factors including 'find-me', 'eat-me', 'don't eat-me' and 'good-bye' signals. However, in recent years with the rapid expansion of the cell death field, the removal of other necrotic-like cell types has drawn much attention. Depending on the type of death, dying cells employ different mechanisms to facilitate engulfment and elicit varying functional impacts on the phagocyte, from wound healing responses to inflammatory cytokine secretion. Nevertheless, despite the mechanism of death, the clearance of dying cells is a fundamental process required to prevent the uncontrolled release of pro-inflammatory mediators and inflammatory disease. This mini-review summarises the current understandings of: (i) apoptotic, necrotic, necroptotic and pyroptotic cell clearance; (ii) the functional consequences of dying cell engulfment and; (iii) the outstanding questions in the field.

Inborn errors of immunity and metabolic disorders: current understanding, diagnosis, and treatment approaches.

Inborn errors of metabolism consist of a heterogeneous group of disorders with various organ systems manifestations, and some metabolic diseases also cause immunological disorders or dysregulation. In this review, metabolic diseases that affect the immunological system and particularly lead to primary immune deficiency will be reviewed. In a patient with frequent infections and immunodeficiency, the presence of symptoms such as growth retardation, abnormal facial appearance, heart, skeletal, lung deformities, skin findings, arthritis, motor developmental retardation, seizure, deafness, hepatomegaly, splenomegaly, impairment of liver function tests, the presence of anemia, thrombocytopenia and eosinophilia in hematological examinations should suggest metabolic diseases for the underlying cause. In some patients, these phenotypic findings may appear before the immunodeficiency picture. Metabolic diseases leading to immunological disorders are likely to be rare but probably underdiagnosed. Therefore, the presence of recurrent infections or autoimmune findings in a patient with a suspected metabolic disease should suggest that immune deficiency may also accompany the picture, and diagnostic examinations in this regard should be deepened.

I'm Infected, Eat Me! Innate Immunity Mediated by Live, Infected Cells Signaling To Be Phagocytosed.

Innate immunity against pathogens is known to be mediated by barriers to pathogen invasion, activation of complement, recruitment of immune cells, immune cell phagocytosis of pathogens, death of infected cells, and activation of the adaptive immunity via antigen presentation. Here, we propose and review evidence for a novel mode of innate immunity whereby live, infected host cells induce phagocytes to phagocytose the infected cell, thereby potentially reducing infection. We discuss evidence that host cells, infected by virus, bacteria, or other intracellular pathogens (i) release nucleotides and chemokines as find-me signals, (ii) expose on their surface phosphatidylserine and calreticulin as eat-me signals, (iii) release and bind opsonins to induce phagocytosis, and (iv) downregulate don't-eat-me signals CD47, major histocompatibility complex class I (MHC1), and sialic acid. As long as the pathogens of the host cell are destroyed within the phagocyte, then infection can be curtailed; if antigens from the pathogens are cross-presented by the phagocyte, then an adaptive response would also be induced. Phagocytosis of live infected cells may thereby mediate innate immunity.

Assays of Eosinophil Apoptosis and Phagocytic Uptake.

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure of phosphatidylserine on the cell membrane. A greater understanding of apoptotic mechanisms, subsequent phagocytosis (efferocytosis), and regulation of these processes is critical to understanding disease pathogenesis and development of potential novel therapeutic agents. Release of soluble factors and alterations to surface marker expression by eosinophils undergoing apoptosis aid them in signaling their presence to the immediate environment, and their subsequent recognition by phagocytic cells such as macrophages. Uptake of apoptotic cells usually suppresses inflammation by restricting proinflammatory responses and promoting anti-inflammatory and tissue repair responses. This, in turn, promotes resolution of inflammation. Defects in the apoptotic or efferocytosis mechanisms perpetuate inflammation, resulting in chronic inflammation and enhanced disease severity. This can be due to increased eosinophil life span or cell necrosis characterized by loss of cell membrane integrity and release of toxic intracellular mediators. In this chapter, we detail some of the key assays that are used to assess eosinophil apoptosis, as well as the intracellular signaling pathways involved and phagocytic clearance of these cells.