Dietary Salicornia ramosissima improves the European seabass (Dicentrarchus labrax) inflammatory response against Photobacterium damselae piscicida.

Introduction: Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of S. ramosissima in European seabass diets. Methods: Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated Photobacterium damselae subsp. piscicida (Phdp) were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated. Results: After both feeding periods, most parameters remained unaltered by S. ramosissima inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and cluster of differentiation 8 (cd8ß) compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1ß (il1ß), while fish fed ST5 showed higher levels of ornithine decarboxylase (odc) than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and glutathione peroxidase (gpx) was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively. Discussion: In conclusion, incorporating up to 10% of the non-food fraction S. ramosissima in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, S. ramosissima inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.

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.

The Pseudomonas aeruginosa lectin LecB modulates intracellular reactive oxygen species production in human neutrophils.

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen ubiquitously present throughout nature. LecB, a fucose-, and mannose-binding lectin, is a prominent virulence factor of P. aeruginosa, which can be expressed on the bacterial surface but also be secreted. However, the LecB interaction with human immune cells remains to be characterized. Neutrophils comprise the first line of defense against infections and their production of reactive oxygen species (ROS) and release of extracellular traps (NETs) are critical antimicrobial mechanisms. When profiling the neutrophil glycome we found several glycoconjugates on granule and plasma membranes that could potentially act as LecB receptors. In line with this, we here show that soluble LecB can activate primed neutrophils to produce high levels of intracellular ROS (icROS), an effect that was inhibited by methyl fucoside. On the other hand, soluble LecB inhibits P. aeruginosa-induced icROS production. In support of that, during phagocytosis of wild-type and LecB-deficient P. aeruginosa, bacteria with LecB induced less icROS production as compared with bacteria lacking the lectin. Hence, LecB can either induce or inhibit icROS production in neutrophils depending on the circumstances, demonstrating a novel and potential role for LecB as an immunomodulator of neutrophil functional responses.

Macrophage Development and Function.

Macrophages were first described over a hundred years ago. Throughout the years, they were shown to be essential players in their tissue-specific environment, performing various functions during homeostatic and disease conditions. Recent reports shed more light on their ontogeny as long-lived, self-maintained cells with embryonic origin in most tissues. They populate the different tissues early during development, where they help to establish and maintain homeostasis. In this chapter, the history of macrophages is discussed. Furthermore, macrophage ontogeny and core functions in the different tissues are described.

Isolation and Flow Cytometry Analysis of Macrophages from the Dermis.

The dermis contains a dense network of tissue macrophages, which contribute to tissue homeostasis, inflammation, and pathogen clearance. Dermal macrophages are partly replenished by circulating monocytes, which fuel the resident population, especially in case of tissue damage or inflammation. The complexity of the tissue, containing blood and lymphoid vessels, hair bulbs, sebaceous glands, and peripheral nerves, allows for the development of distinct macrophages populations. In steady state, discrete subtypes can be distinguished due to their surface marker expression and localization within the dermis. In this chapter, we describe how to extract dermal macrophages from the skin and highlight different gating strategies to identify monocyte and macrophage populations.

Defining solute carrier transporter signatures of murine immune cell subsets.

Solute carrier (SLC) transporters are membrane-bound proteins that facilitate nutrient transport, and the movement across cellular membranes of various substrates ranging from ions to amino acids, metabolites and drugs. Recently, SLCs have gained increased attention due to their functional linkage to innate immunological processes such as the clearance of dead cells and anti-microbial defense. Further, the druggable nature of these transporters provides unique opportunities for improving outcomes in different immunological diseases. Although the SLCs represent the largest group of transporters and are often identified as significant hits in omics data sets, their role in immunology has been insufficiently explored. This is partly due to the absence of tools that allow identification of SLC expression in particular immune cell types and enable their comparison before embarking on functional studies. In this study, we used publicly available RNA-Seq data sets to analyze the transcriptome in adaptive and innate immune cells, focusing on differentially and highly expressed SLCs. This revealed several new insights: first, we identify differentially expressed SLC transcripts in phagocytes (macrophages, dendritic cells, and neutrophils) compared to adaptive immune cells; second, we identify new potential immune cell markers based on SLC expression; and third, we provide user-friendly online tools for researchers to explore SLC genes of interest (and the rest of the genes as well), in three-way comparative dot plots among immune cells. We expect this work to facilitate SLC research and comparative transcriptomic studies across different immune cells.

Histological Comparison of Porcine Small Intestine Submucosa and Bovine Type-I Collagen Conduit for Nerve Repair in a Rat Model.

Purpose: After nerve injury, macrophages and Schwann cells remove axon and myelin debris. We hypothesized that nerves repaired with different conduit materials will result in varying levels of these cell populations, which impacts Wallerian degeneration and axonal regeneration. Methods: We performed a unilateral sciatic nerve transection in 18 rats. The nerves were repaired with small intestine submucosa (SIS, n = 9) or isolated type-I collagen (CLC, n = 9) conduits. Rats were monitored for 4 weeks. Histology samples were obtained from the proximal nerve, mid-implant, and distal nerve regions. Samples were stained for total macrophages, M2 macrophages, foamy phagocytes, Schwann cells, vascular components, axon components, and collagen density. Results: Distal nerve analyses showed higher populations of total macrophages and M2 macrophages in SIS-repaired nerves and higher density of foamy phagocytes in CLC-repaired nerves. Proximal nerve, mid-implant, and distal nerve analyses showed higher Schwann cell and vascular component densities in SIS-repaired nerves. Axon density was higher in the mid-implant region of SIS-repaired nerves. Collagen staining in the mid-implant was scant, but less collagen density was observed in SIS-repaired versus CLC-repaired nerves. Conclusions: In the distal nerve, the following were observed: (1) lower total macrophages in CLC-repaired nerves, suggesting lower overall inflammation versus SIS-repaired nerves; (2) higher M2 macrophages in SIS-repaired versus CLC-repaired nerves, a driving factor for higher total macrophages and indicative of an inflammation resolution response in SIS-repaired nerves; and (3) a lower foamy phagocyte density in SIS-repaired nerves, suggesting earlier resolution of Wallerian degeneration versus CLC-repaired nerves. In the proximal nerve, mid-implant, and distal nerve, higher Schwann cell and vascular component densities were noted in SIS-repaired nerves. In the mid-implant, a higher axon component density and a lower collagen density of the SIS-repaired nerves versus CLC-repaired nerves were noted. These results indicate more robust nerve regeneration with less collagen deposition. Clinical relevance: This in vivo study evaluated two common conduit materials that are used in peripheral nerve repair. Clinical outcomes of nerves repaired with conduits may be impacted by the response to different conduit materials. These nerve repair responses include Wallerian degeneration, nerve regeneration, and nerve scarring. This study evaluated Wallerian degeneration using total macrophages, M2 macrophages, and foamy phagocytes. Nerve regeneration was evaluated using Schwann cells and axons. Nerve scarring was evaluated using vascular and collagen density.

Dectin-2 is critical for phagocyte function and resistance to Paracoccidioides brasiliensis in mice.

Germline-encoded pattern recognition receptors, particularly C-type lectin receptors (CLRs), are essential for phagocytes to sense invading fungal cells. Among CLRs, Dectin-2 (encoded by Clec4n) plays a critical role in the antifungal immune response as it recognizes high-mannose polysaccharides on the fungal cell wall, triggering phagocyte functional activities and ultimately determining adaptive responses. Here, we assessed the role of Dectin-2 on the course of primary Paracoccidioides brasiliensis systemic infection in mice with Dectin-2-targeted deletion. Paracoccidioides brasiliensis constitutes the principal etiologic agent of paracoccidioidomycosis, the most prominent invasive mycosis in Latin American countries. The deficiency of Dectin-2 resulted in shortened survival rates, high lung fungal burden, and increased lung pathology in mice infected with P. brasiliensis. Consistently, dendritic cells (DCs) from mice lacking Dectin-2 infected ex vivo with P. brasiliensis showed impaired secretion of several proinflammatory and regulatory cytokines, including TNF-α, IL-1ß, IL-6, and IL-10. Additionally, when cocultured with splenic lymphocytes, DCs were less efficient in promoting a type 1 cytokine pattern secretion (i.e., IFN-γ). In macrophages, Dectin-2-mediated signaling was required to ensure phagocytosis and fungicidal activity associated with nitric oxide production. Overall, Dectin-2-mediated signaling is critical to promote host protection against P. brasiliensis infection, and its exploitation might lead to the development of new vaccines and immunotherapeutic approaches.

We report a critical role of the innate immune receptor Dectin-2 during Paracoccidioides brasiliensis infection. Fungal sensing by Dectin-2 improved the survival of mice and lowered fungal burden. Further, Dectin-2 was required for cytokine production, phagocytosis, and fungal killing by phagocytes.

You shall not pass: how complement C5 mediated antifungal immunity blocks systemic candidiasis and preserves renal tissue barriers.

The rising prevalence of fungal infections is a significant and growing public health threat, and this risk is further underscored by our incomplete understanding of why organs like the kidney are so susceptible to systemic candidiasis. To combat the high mortality of such infections, we urgently need to advance our understanding of fungal pathogenesis and how it articulates with human immune response. Now, a recent landmark study has illuminated a crucial role of the complement system in the response to candidiasis and determined the stepwise local response of phagocytes within the kidney during infection. These fundamental discoveries provide crucial insights that can be leveraged to improve the care and outcome for patients with fungal infections.

Harnessing the immunomodulatory effects of exercise to enhance the efficacy of monoclonal antibody therapies against B-cell haematological cancers: a narrative review.

Therapeutic monoclonal antibodies (mAbs) are standard care for many B-cell haematological cancers. The modes of action for these mAbs include: induction of cancer cell lysis by activating Fcγ-receptors on innate immune cells; opsonising target cells for antibody-dependent cellular cytotoxicity or phagocytosis, and/or triggering the classical complement pathway; the simultaneous binding of cancer cells with T-cells to create an immune synapse and activate perforin-mediated T-cell cytotoxicity against cancer cells; blockade of immune checkpoints to facilitate T-cell cytotoxicity against immunogenic cancer cell clones; and direct delivery of cytotoxic agents via internalisation of mAbs by target cells. While treatment regimens comprising mAb therapy can lead to durable anti-cancer responses, disease relapse is common due to failure of mAb therapy to eradicate minimal residual disease. Factors that limit mAb efficacy include: suboptimal effector cell frequencies, overt immune exhaustion and/or immune anergy, and survival of diffusely spread tumour cells in different stromal niches. In this review, we discuss how immunomodulatory changes arising from exposure to structured bouts of acute exercise might improve mAb treatment efficacy by augmenting (i) antibody-dependent cellular cytotoxicity, (ii) antibody-dependent cellular phagocytosis, (iii) complement-dependent cytotoxicity, (iv) T-cell cytotoxicity, and (v) direct delivery of cytotoxic agents.

Host defense mechanisms against Candida auris.

INTRODUCTION: Candida auris is a pathogen of growing public health concern given its rapid spread across the globe, its propensity for long-term skin colonization and healthcare-related outbreaks, its resistance to a variety of antifungal medications, and the high morbidity and mortality associated with invasive disease. Despite that, the host immune response mechanisms that operate during C. auris skin colonization and invasive infection remains poorly understood. AREAS COVERED: In this manuscript, we review the available literature in the growing research field pertaining to C. auris host defenses and we discuss what is known about the ability of C. auris to thrive on mammalian skin, the role of lymphoid cell-mediated, IL-17-dependent defenses in controlling cutaneous colonization, and the contribution of myeloid phagocytes in curtailing systemic infection. EXPERT OPINION: Understanding the mechanisms by which the host immune system responds to and controls colonization and infection with C. auris and developing a deeper knowledge of tissue-specific host-C. auris interactions and of C. auris immune-evading mechanisms may help devise improved strategies for decolonization, prognostication, prevention, vaccination, and/or directed antifungal treatment in vulnerable patient populations.

Knockout studies using CD34+ hematopoietic cells suggest that CD44 is a physiological human neutrophil E-selectin ligand.

The recruitment of peripheral blood neutrophils at sites of inflammation involves a multistep cascade, starting with E- and P-selectin expressed on the inflamed vascular endothelium binding sialofucosylated glycans on leukocytes. As the glycoconjugate biosynthesis pathways in different cells are distinct, the precise carbohydrate ligands of selectins varies both across species, and between different immune cell populations in a given species. To study this aspect in human neutrophils, we developed a protocol to perform CRISPR/Cas9 gene-editing on CD34+ hHSCs (human hematopoietic stem/progenitor cells) as they are differentiated towards neutrophil lineage. This protocol initially uses a cocktail of SCF (stem-cell factor), IL-3 (interleukin-3) and FLT-3L (FMS-like tyrosine kinase 3 ligand) to expand the stem/progenitor cells followed by directed differentiation to neutrophils using G-CSF (granulocyte colony-stimulating factor). Microfluidics based assays were performed on a confocal microscope platform to characterize the rolling phenotype of each edited cell type in mixed populations. These studies demonstrated that CD44, but not CD43, is a major E-selectin ligand on human neutrophils. The loss of function results were validated by developing sialofucosylated recombinant CD44. This glycosylated protein supported both robust E-selectin binding in a cell-free assay, and it competitively blocked neutrophil adhesion to E-selectin on inflamed endothelial cells. Together, the study establishes important methods to study human neutrophil biology and determines that sialoflucosylated-CD44 is a physiological human E-selectin ligand.

A critical assessment of the cellular defences of the avian respiratory system: are birds in general and poultry in particular relatively more susceptible to pulmonary infections/afflictions?

In commercial poultry farming, respiratory diseases cause high morbidities and mortalities, begetting colossal economic losses. Without empirical evidence, early observations led to the supposition that birds in general, and poultry in particular, have weak innate and adaptive pulmonary defences and are therefore highly susceptible to injury by pathogens. Recent findings have, however, shown that birds possess notably efficient pulmonary defences that include: (i) a structurally complex three-tiered airway arrangement with aerodynamically intricate air-flow dynamics that provide efficient filtration of inhaled air; (ii) a specialised airway mucosal lining that comprises air-filtering (ciliated) cells and various resident phagocytic cells such as surface and tissue macrophages, dendritic cells and lymphocytes; (iii) an exceptionally efficient mucociliary escalator system that efficiently removes trapped foreign agents; (iv) phagocytotic atrial and infundibular epithelial cells; (v) phagocytically competent surface macrophages that destroy pathogens and injurious particulates; (vi) pulmonary intravascular macrophages that protect the lung from the vascular side; and (vii) proficiently phagocytic pulmonary extravasated erythrocytes. Additionally, the avian respiratory system rapidly translocates phagocytic cells onto the respiratory surface, ostensibly from the subepithelial space and the circulatory system: the mobilised cells complement the surface macrophages in destroying foreign agents. Further studies are needed to determine whether the posited weak defence of the avian respiratory system is a global avian feature or is exclusive to poultry. This review argues that any inadequacies of pulmonary defences in poultry may have derived from exacting genetic manipulation(s) for traits such as rapid weight gain from efficient conversion of food into meat and eggs and the harsh environmental conditions and severe husbandry operations in modern poultry farming. To reduce pulmonary diseases and their severity, greater effort must be directed at establishment of optimal poultry housing conditions and use of more humane husbandry practices.

HIF1α-dependent hypoxia response in myeloid cells requires IRE1α.

Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1ß, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.

Dynamics of the inhibitory immune checkpoint TIM-3 in mouse pulmonary phagocytes after silica exposure.

Long-term inhalation of silica particles in the workplace causes silicosis, which is incurable and seriously endangers the health of workers. It is believed that silicosis is caused by an imbalance of the pulmonary immune microenvironment, in which pulmonary phagocytes play a crucial role. As an emerging immunomodulatory factor, it is unclear whether T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) participate in silicosis by modulating pulmonary phagocytes function. The purpose of this study was to investigate the dynamic changes of the TIM-3 in pulmonary macrophages, dendritic cells (DCs), and monocytes during the development of silicosis in mice. The plasma levels of soluble TIM-3 in silicosis patients were also examined. Flow cytometry was used to identify alveolar macrophages (AMs), interstitial macrophages (IMs), CD11b+ DC, CD103+ DC, Ly6C+, and Ly6C- monocytes in mouse lung tissues, and further analyses were conducted on the expression of TIM-3. Results showed that soluble TIM-3 was significantly elevated in plasma of silicosis patients, and the level of which was higher in stage II and III patients than that in stage I. In silicosis mice, the protein and mRNA levels of TIM-3 and Galectin9 were significantly upregulated in lung tissues. Specific to pulmonary phagocytes, silica exposure affected TIM-3 expression in a cell-specific and dynamic manner. In macrophages, TIM-3 expression upregulated in AM after 28 days and 56 days of silica instillation, while the expression of TIM-3 in IM decreased at all observation time points. In DCs, silica exposure only caused a decrease of TIM-3 expression in CD11b+ DCs. In monocytes, TIM-3 dynamics in Ly6C+ and Ly6C- monocytes were generally consistent during silicosis development, which significant decrease after 7 and 28 days of silica exposure. In conclusion, TIM-3 may mediate the development of silicosis by regulating pulmonary phagocytes.

Mycobacterium abscessus Opsonization Allows an Escape from the Defensin Bactericidal Action in Drosophila.

Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in Drosophila that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by Drosophila macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both in vitro and in vivo and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in Drosophila. IMPORTANCE Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using Drosophila, we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by Drosophila phagocytes. Indeed, by combining in vivo and in vitro approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.

Phagocyte extracellular traps formation contributes to host defense against Clostridium perfringens infection.

Clostridium perfringens (C. perfringens) is an important Gram-positive anaerobic spore-forming pathogen that provokes life-threatening gas gangrene and acute enterotoxaemia, although it colonizes as a component of the symbiotic bacteria in humans and animals. However, the mechanisms by which C. perfringens is cleared from the host remains poorly understood, thereby impeding the development of novel strategies for control this infection. Here, we uncover a beneficial effect of extracellular traps (ETs) formation on bacterial killing and clearance by phagocytes. C. perfringens strain ATCC13124, and wild-type isolates CP1 and CP3 markedly trigger ETs formation in macrophages and neutrophils. As expected, visualization of DNA decorated with histone, myeloperoxidase (MPO) and neutrophils elastase (NE) in C. perfringens-triggered classical ETs structures. Notably, the bacteria-induced ETs formation is an ERK1/2-, P38 MAPK-, store-operated calcium entry (SOCE)-, NADPH oxidase-, histone-, NE-, and MPO-dependent process, and is independent of LDH activity. Meanwhile, the defect of bactericidal activity is mediated by impairing ETs formation in phagocytes. Moreover, In vivo studies indicated that degradation of ETs by DNase I administration leads to a defect in the protection against experimental gas gangrene, with higher mortality rates, exacerbated tissue damage, and more bacterial colonization. Together, these results suggest that phagocyte ETs formation is essential for the host defense against C. perfringens infection.

Microglia subtypes in acute, subacute, and chronic multiple sclerosis.

The study was designed to examine microglia morphology in early and late forms of multiple sclerosis (MS). Archival paraffin embedded tissue samples from 25 cases were examined immunohistochemically. Pío del Río Hortega reported that phagocytes in acute focal destructive CNS lesions develop from microglia with no early contribution from infiltrating monocytes. In this study, we were unable to identify the changes cited by del Río Hortega in support of his theory. Instead, myelin phagocytes in MS appear to originate chiefly from infiltrating monocytes. In 4 cases, walls composed of MHC class II antigen-positive "wall microglia" were observed at plaque margins separating demyelinated and bordering myelinated tissue. Wall microglia in 2 plaques were accompanied by AQP4-positive fiber-forming astrocytes. In chronic but not early disease MS cases, microglia were seen to interact with infiltrating monocytes to form microglial nodules of several types. Also, MHC II-positive "activated" microglia in bordering intact tissue were exceptionally prominent where there was little evidence of ongoing myelin loss. It is concluded that myelin phagocytes in MS derive entirely from infiltrating MRP14-positive monocytes and not from resident microglia and that Río Hortega's microglia play an anti-inflammatory role in MS and not the destructive role favored by the current literature.

Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes.

Professional phagocytic cells, such as macrophages, ingest large particles into a specialized endocytic compartment, the phagosome, which eventually turns into a phagolysosome and degrades its contents. This phagosome "maturation" is governed by successive fusion of the phagosome with early sorting endosomes, late endosomes, and lysosomes. Further changes occur by fission of vesicles from the maturing phagosome and by on-and-off cycling of cytosolic proteins. We present here a detailed protocol which allows to reconstitute in a cell-free system the fusion events between phagosomes and the different endocytic compartments. This reconstitution can be used to define the identity of, and interplay between, key players of the fusion events.

An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates.

Phagosomes are formed when phagocytic cells take up large particles, and they develop into phagolysosomes where the particles are degraded. The transformation of nascent phagosomes into phagolysosomes is a complex multi-step process, and the precise timing of these steps depends at least in part on phosphatidylinositol phosphates (PIPs). Some such-called "intracellular pathogens" are not delivered to microbicidal phagolysosomes and manipulate the PIP composition of the phagosomes they reside in. Studying the dynamic changes of the PIP composition of inert-particle phagosomes will help to understand why the pathogens' manipulations reprogram phagosome maturation.We here describe a method to detect and to follow generation and degradation of PIPs on purified phagosomes. To this end, phagosomes formed around inert latex beads are purified from J774E macrophages and incubated in vitro with PIP-binding protein domains or PIP-binding antibodies. Binding of such PIP sensors to phagosomes indicates presence of the cognate PIP and is quantified by immunofluorescence microscopy. When phagosomes are incubated with PIP sensors and ATP at a physiological temperature, the generation and degradation of PIPs can be followed, and PIP-metabolizing enzymes can be identified using specific inhibitory agents.