Phagocytosis: The Legacy of Metchnikoff.

This year is the centenary of the death of Elie Metchnikoff, the father of innate immunity. His work on phagocytosis has continued to flourish, particularly over the past half century.

Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages.

Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogen Cryptococcus neoformans in 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Marco-/- macrophages also showed elevated vomocytosis of a yeast-locked C. albicans strain, suggesting this to be a broadly relevant observation. We go on to show that MARCO's role in modulating vomocytosis is independent of its role as a phagocytic receptor, suggesting that this protein may play an important and hitherto unrecognised role in modulating macrophage behaviour.

Phagocytosis: An Immunobiologic Process.

It has been a century since the death of Élie Metchnikoff, who championed the role of phagocytosis in cellular immunity. Whereas others had observed the uptake of particles by cells from simple to complex organisms, he grasped its significance in the host response to injury and infection and established a firm basis for our understanding of inflammation and tissue homeostasis. The past century has brought improved tools of cellular and molecular biology to the study of phagocytosis and its contribution to physiological and pathological processes, including receptor function in innate and acquired immunity. In this review, I assess our present knowledge and consider opportunities for future research and therapeutic targeting.

DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Proteinase 3 promotes formation of multinucleated giant cells and granuloma-like structures in patients with granulomatosis with polyangiitis.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune vasculitides associated with antineutrophil cytoplasm antibodies that target proteinase 3 (PR3) or myeloperoxidase (MPO) found within neutrophils and monocytes. Granulomas are exclusively found in GPA and form around multinucleated giant cells (MGCs), at sites of microabscesses, containing apoptotic and necrotic neutrophils. Since patients with GPA have augmented neutrophil PR3 expression, and PR3-expressing apoptotic cells frustrate macrophage phagocytosis and cellular clearance, we investigated the role of PR3 in stimulating giant cell and granuloma formation. METHODS: We stimulated purified monocytes and whole peripheral blood mononuclear cells (PBMCs) from patients with GPA, patients with MPA or healthy controls with PR3 or MPO and visualised MGC and granuloma-like structure formation using light, confocal and electron microscopy, as well as measuring the cell cytokine production. We investigated the expression of PR3 binding partners on monocytes and tested the impact of their inhibition. Finally, we injected zebrafish with PR3 and characterised granuloma formation in a novel animal model. RESULTS: In vitro, PR3 promoted monocyte-derived MGC formation using cells from patients with GPA but not from patients with MPA, and this was dependent on soluble interleukin 6 (IL-6), as well as monocyte MAC-1 and protease-activated receptor-2, found to be overexpressed in the cells of patients with GPA. PBMCs stimulated by PR3 formed granuloma-like structures with central MGC surrounded by T cells. This effect of PR3 was confirmed in vivo using zebrafish and was inhibited by niclosamide, a IL-6-STAT3 pathway inhibitor. CONCLUSIONS: These data provide a mechanistic basis for granuloma formation in GPA and a rationale for novel therapeutic approaches.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.

Adipoclast: a multinucleated fat-eating macrophage.

Cell membrane fusion and multinucleation in macrophages are associated with physiologic homeostasis as well as disease. Osteoclasts are multinucleated macrophages that resorb bone through increased metabolic activity resulting from cell fusion. Fusion of macrophages also generates multinucleated giant cells (MGCs) in white adipose tissue (WAT) of obese individuals. For years, our knowledge of MGCs in WAT has been limited to their description as part of crown-like structures (CLS) surrounding damaged adipocytes. However, recent evidence indicates that these cells can phagocytose oversized lipid remnants, suggesting that, as in osteoclasts, cell fusion and multinucleation are required for specialized catabolic functions. We thus reason that WAT MGCs can be viewed as functionally analogous to osteoclasts and refer to them in this article as adipoclasts. We first review current knowledge on adipoclasts and their described functions. In view of recent advances in single cell genomics, we describe WAT macrophages from a 'fusion perspective' and speculate on the ontogeny of adipoclasts. Specifically, we highlight the role of CD9 and TREM2, two plasma membrane markers of lipid-associated macrophages in WAT, which have been previously described as regulators of fusion and multinucleation in osteoclasts and MGCs. Finally, we consider whether strategies aiming to target WAT macrophages can be more selectively directed against adipoclasts.

Plasma membrane receptors of tissue macrophages: functions and role in pathology.

The cells of the mononuclear phagocyte system (MPS) constitute a dispersed organ, which is distributed throughout the body. Macrophages in different tissues display distinctive mosaic phenotypes as resident and recruited cells of embryonic and bone marrow origin, respectively. They help to maintain homeostasis during development and throughout adult life, yet contribute to the pathogenesis of many disease processes, including inflammation, innate and adaptive immunity, metabolic disorders, and cancer. Heterogeneous tissue macrophage populations display a wide variety of surface molecules to recognise and respond to host, microbial, and exogenous ligands in their environment; their receptors mediate the uptake and destruction of effete and dying host cells and pathogens, as well as contribute trophic and secretory functions within every organ in the body. Apart from local cellular interactions, macrophage surface molecules and products serve to mobilise and coordinate systemic humoral and cellular responses. Their use as antigen markers in pathogenesis and as potential drug targets has lagged in clinical pathology and human immunotherapy. In this review, we summarise the properties of selected surface molecules expressed on macrophages in different tissues and disease processes, to provide a functional basis for diagnosis, further research, and treatment. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Common signalling pathways in macrophage and osteoclast multinucleation.

Macrophage cell fusion and multinucleation are fundamental processes in the formation of multinucleated giant cells (MGCs) in chronic inflammatory disease and osteoclasts in the regulation of bone mass. However, this basic cell phenomenon is poorly understood despite its pathophysiological relevance. Granulomas containing multinucleated giant cells are seen in a wide variety of complex inflammatory disorders, as well as in infectious diseases. Dysregulation of osteoclastic bone resorption underlies the pathogenesis of osteoporosis and malignant osteolytic bone disease. Recent reports have shown that the formation of multinucleated giant cells and osteoclast fusion display a common molecular signature, suggesting shared genetic determinants. In this Review, we describe the background of cell-cell fusion and the similar origin of macrophages and osteoclasts. We specifically focus on the common pathways involved in osteoclast and MGC fusion. We also highlight potential approaches that could help to unravel the core mechanisms underlying bone and granulomatous disorders in humans.

Innate and adaptive interferons suppress IL-1α and IL-1ß production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1ß, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1ß are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4(+) T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Alternative activation of macrophages: mechanism and functions.

The concept of an alternative pathway of macrophage activation has stimulated interest in its definition, mechanism, and functional significance in homeostasis and disease. We assess recent research in this field, argue for a restricted definition, and explore pathways by which the T helper 2 (Th2) cell cytokines interleukin-4 (IL-4) and IL-13 mediate their effects on macrophage cell biology, their biosynthesis, and responses to a normal and pathological microenvironment. The stage is now set to gain deeper insights into the role of alternatively activated macrophages in immunobiology.

Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.

Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand.

The central nervous system (CNS) is endowed with several immune-related mechanisms that contribute to its protection and maintenance in homeostasis and under pathology. Here, we discovered an additional mechanism that controls inflammatory responses within the CNS milieu under injurious conditions, involving CD200 ligand (CD200L) expressed by newly formed endothelial cells. We observed that CD200L is constitutively expressed in the mouse healthy CNS by endothelial cells of the blood-cerebrospinal fluid barrier and of the spinal cord meninges, but not by the endothelium of the blood-spinal cord barrier. Following spinal cord injury (SCI), newly formed endothelial cells, located only at the epicenter of the lesion site, expressed CD200L. Moreover, in the absence of CD200L expression by CNS-resident cells, functional recovery of mice following SCI was impaired. High throughput single-cell flow cytometry image analysis following SCI revealed CD200L-dependent direct interaction between endothelial and local CD200R+ myeloid cells, including activated microglia and infiltrating monocyte-derived macrophages (mo-MΦ). Absence of CD200L signaling, both in vitro and in vivo, resulted in a higher inflammatory response of the encountering macrophages, manifested by elevation in mRNA expression of Tnfα and Il1ß, increased intracellular TNFα immunoreactivity, and reduced expression levels of macrophage factors that are associated with resolution of inflammation, Dectin-1, CD206 (mannose receptor), and IL-4R. Collectively, our results highlight the importance of CD200-mediated immune dialogue between endothelial cells and the local resident microglia and infiltrating mo-MΦ within the lesion area, as a mechanism that contributes to regulation of inflammation following acute CNS injury. SIGNIFICANCE STATEMENT: This manuscript focuses on a novel mechanism of inflammation-regulation following spinal cord injury (SCI), orchestrated by CD200-ligand (CD200L) expressed by newly formed endothelial cells within the lesion site. Our study reveals that, in homeostasis, CD200L is expressed by endothelial cells of the mouse blood-cerebrospinal fluid barrier and of the blood-leptomeningeal barrier, but not by endothelial cells of the blood-spinal cord barrier. Following SCI, newly formed endothelial cells located within the epicenter of the lesion site were found to express CD200L at time points that were shown to be critical for repair. Our results reveal a direct interaction between CD200L+ endothelial cells and CD200R+ microglia and macrophages, resulting in attenuated inflammation, biasing macrophage phenotype toward inflammation-resolving cells, and promotion of functional recovery following SCI.

Tissue macrophages: heterogeneity and functions.

Macrophages are present in all vertebrate tissues, from mid-gestation throughout life, constituting a widely dispersed organ system. They promote homeostasis by responding to internal and external changes within the body, not only as phagocytes in defence against microbes and in clearance of dead and senescent cells, but also through trophic, regulatory and repair functions. In this review, we describe macrophage phenotypic heterogeneity in different tissue environments, drawing particular attention to organ-specific functions.

Macrophage heterogeneity in tissues: phenotypic diversity and functions.

During development and throughout adult life, macrophages derived from hematopoietic progenitors are seeded throughout the body, initially in the absence of inflammatory and infectious stimuli as tissue-resident cells, with enhanced recruitment, activation, and local proliferation following injury and pathologic insults. We have learned a great deal about macrophage properties ex vivo and in cell culture, but their phenotypic heterogeneity within different tissue microenvironments remains poorly characterized, although it contributes significantly to maintaining local and systemic homeostasis, pathogenesis, and possible treatment. In this review, we summarize the nature, functions, and interactions of tissue macrophage populations within their microenvironment and suggest questions for further investigation.

Physiological roles of macrophages.

Macrophages are present in mammals from midgestation, contributing to physiologic homeostasis throughout life. Macrophages arise from yolk sac and foetal liver progenitors during embryonic development in the mouse and persist in different organs as heterogeneous, self-renewing tissue-resident populations. Bone marrow-derived blood monocytes are recruited after birth to replenish tissue-resident populations and to meet further demands during inflammation, infection and metabolic perturbations. Macrophages of mixed origin and different locations vary in replication and turnover, but are all active in mRNA and protein synthesis, fulfilling organ-specific and systemic trophic functions, in addition to host defence. In this review, we emphasise selected properties and non-immune functions of tissue macrophages which contribute to physiologic homeostasis.

CD14++CD16+ Monocytes Are Enriched by Glucocorticoid Treatment and Are Functionally Attenuated in Driving Effector T Cell Responses.

Human peripheral monocytes have been categorized into three subsets based on differential expression levels of CD14 and CD16. However, the factors that influence the distribution of monocyte subsets and the roles that each subset plays in autoimmunity are not well studied. In this study, we show that circulating monocytes from patients with autoimmune uveitis exhibit a skewed phenotype toward intermediate CD14(++)CD16(+) cells, and that this is associated with glucocorticoid therapy. We further demonstrate that CD14(++)CD16(+) monocytes from patients and healthy control donors share a similar cell-surface marker and gene expression profile. Comparison of the effects of intermediate CD14(++)CD16(+) monocytes with classical CD14(++)CD16(-) and nonclassical CD14(+)CD16(++) monocytes revealed that the intermediate CD14(++)CD16(+) subset had an attenuated capacity to promote both naive CD4(+) T cell proliferation and polarization into a Th1 phenotype, and memory CD4(+) T cell proliferation and IL-17 expression. Furthermore, CD14(++)CD16(+) cells inhibit CD4(+) T cell proliferation induced by other monocyte subsets and enhance CD4(+) T regulatory cell IL-10 expression. These data demonstrate the impact of glucocorticoids on monocyte phenotype in the context of autoimmune disease and the differential effects of monocyte subsets on effector T cell responses.

Monocyte and macrophage heterogeneity.

Heterogeneity of the macrophage lineage has long been recognized and, in part, is a result of the specialization of tissue macrophages in particular microenvironments. Circulating monocytes give rise to mature macrophages and are also heterogeneous themselves, although the physiological relevance of this is not completely understood. However, as we discuss here, recent studies have shown that monocyte heterogeneity is conserved in humans and mice, allowing dissection of its functional relevance: the different monocyte subsets seem to reflect developmental stages with distinct physiological roles, such as recruitment to inflammatory lesions or entry to normal tissues. These advances in our understanding have implications for the development of therapeutic strategies that are targeted to modify particular subpopulations of monocytes.