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.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

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.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

Lipid-loaded tumor-associated macrophages sustain tumor growth and invasiveness in prostate cancer.

Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell-derived IL-1ß enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes.

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.

COVID-19 Is a Multi-Organ Aggressor: Epigenetic and Clinical Marks.

The progression of coronavirus disease 2019 (COVID-19), resulting from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, may be influenced by both genetic and environmental factors. Several viruses hijack the host genome machinery for their own advantage and survival, and similar phenomena might occur upon SARS-CoV-2 infection. Severe cases of COVID-19 may be driven by metabolic and epigenetic driven mechanisms, including DNA methylation and histone/chromatin alterations. These epigenetic phenomena may respond to enhanced viral replication and mediate persistent long-term infection and clinical phenotypes associated with severe COVID-19 cases and fatalities. Understanding the epigenetic events involved, and their clinical significance, may provide novel insights valuable for the therapeutic control and management of the COVID-19 pandemic. This review highlights different epigenetic marks potentially associated with COVID-19 development, clinical manifestation, and progression.

CSF1R defines the mononuclear phagocyte system lineage in human blood in health and COVID-19.

Mononuclear phagocytes defend tissues, present antigens, and mediate recovery and healing. To date, we lack a marker to unify mononuclear phagocytes in humans or that informs us about their origin. Here, we reassess mononuclear phagocyte ontogeny in human blood through the lineage receptor CSF1R, in the steady state and in COVID-19. We define CSF1R as the first sensitive and reproducible pan-phagocyte lineage marker, to identify and enumerate all conventional monocytes, and the myeloid dendritic cells. In the steady state, CSF1R is sufficient for sorting and immuno-magnetic isolation. In pathology, changes in CSF1R are more sensitive than CD14 and CD16. In COVID-19, a significant drop in membrane CSF1R is useful for stratifying patients, beyond the power of cell categories published thus far, which fail to capture COVID-19 specific events. Importantly, CSF1R defines cells which are neither conventional monocytes nor DCs, which are missed in published analysis. CSF1R decrease can be linked ex vivo to high CSF1 levels. Blood assessment of CSF1R+ cells opens a developmental window to the Mononuclear Phagocyte System in transit from bone marrow to tissues, supports isolation and phenotypic characterisation, identifies novel cell types, and singles out CSF1R inhibition as therapeutic target in COVID-19 and other diseases.

Foam Cell Macrophages in Tuberculosis.

Mycobacterium tuberculosis infects primarily macrophages in the lungs. Infected macrophages are surrounded by other immune cells in well organised structures called granulomata. As part of the response to TB, a type of macrophage loaded with lipid droplets arises which we call Foam cell macrophages. They are macrophages filled with lipid laden droplets, which are synthesised in response to increased uptake of extracellular lipids, metabolic changes and infection itself. They share the appearance with atherosclerosis foam cells, but their lipid contents and roles are different. In fact, lipid droplets are immune and metabolic organelles with emerging roles in Tuberculosis. Here we discuss lipid droplet and foam cell formation, evidence regarding the inflammatory and immune properties of foam cells in TB, and address gaps in our knowledge to guide further research.

SARS-CoV-2 Variants, Vaccines, and Host Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new beta coronavirus that emerged at the end of 2019 in the Hubei province of China. SARS-CoV-2 causes coronavirus disease 2019 (COVID-19) and was declared a pandemic by the World Health Organization (WHO) on 11 March 2020. Herd or community immunity has been proposed as a strategy to protect the vulnerable, and can be established through immunity from past infection or vaccination. Whether SARS-CoV-2 infection results in the development of a reservoir of resilient memory cells is under investigation. Vaccines have been developed at an unprecedented rate and 7 408 870 760 vaccine doses have been administered worldwide. Recently emerged SARS-CoV-2 variants are more transmissible with a reduced sensitivity to immune mechanisms. This is due to the presence of amino acid substitutions in the spike protein, which confer a selective advantage. The emergence of variants therefore poses a risk for vaccine effectiveness and long-term immunity, and it is crucial therefore to determine the effectiveness of vaccines against currently circulating variants. Here we review both SARS-CoV-2-induced host immune activation and vaccine-induced immune responses, highlighting the responses of immune memory cells that are key indicators of host immunity. We further discuss how variants emerge and the currently circulating variants of concern (VOC), with particular focus on implications for vaccine effectiveness. Finally, we describe new antibody treatments and future vaccine approaches that will be important as we navigate through the COVID-19 pandemic.

Corrigendum: Foam Cells Control Mycobacterium tuberculosis Infection.

[This corrects the article DOI: 10.3389/fmicb.2020.01394.].

Foam Cells Control Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) infects macrophages and macrophage-derived foam cells, a hallmark of granulomata in tuberculous lesions. We analyzed the effects of lipid accumulation in human primary macrophages and quantified strong triglyceride and phospholipid remodeling which depended on the dietary fatty acid used for the assay. The enrichment of >70% in triglyceride and phospholipids can alter cell membrane properties, signaling and phagocytosis in macrophages. In conventional macrophage cultures, cells are heterogeneous, small or large macrophages. In foam cells, a third population of 30% of cells with increased granularity can be detected. We found that foam cell formation is heterogenous and that lipid accumulation and foam cell formation reduces the phagocytosis of Mtb. Under the conditions tested, cell death was highly prevalent in macrophages, whereas foam cells were largely protected from this effect. Foam cells also supported slower Mtb replication, yet this had no discernible impact on the intracellular efficacy of four different antitubercular drugs. Foam cell formation had a significant impact in the inflammatory potential of the cells. TNF-α, IL-1ß, and prototypical chemokines were increased. The ratio of inflammatory IL-1ß, TNF-α, and IL-6 vs. anti-inflammatory IL-10 was significantly higher in response to Mtb vs. LPS, and was increased in foam cells compared to macrophages, suggestive of increased pro-inflammatory properties. Cytokine production correlated with NF-κB activation in our models. We conclude that foam cell formation reduces the host cell avidity for, and phagocytosis of, Mtb while protecting the cells from death. This protective effect is associated with enhanced inflammatory potential of foam cells and restricted intracellular growth of Mtb.

Monocyte activation in systemic Covid-19 infection: Assay and rationale.

Mononuclear phagocytes are a widely distributed family of cells contributing to innate and adaptive immunity. Circulating monocytes and tissue macrophages participate in all stages of SARS COVID-19. They contribute to comorbidities predisposing to clinical infection, virus resistance and dissemination, and to host factors that determine disease severity, recovery and sequelae. Assays are available to detect viral infection and antibody responses, but no adequate tests have been developed to measure the activation level of monocytes and tissue macrophages, and the risk of progression to a fatal hyperinflammatory syndrome. Blood monocytes provide a window on the systemic immune response, from production to tissue recruitment, reflecting the impact of infection on the host. Ready availability of blood makes it possible to monitor severity and the risk of potentially lethal complications, by developing tests to assess the status of monocyte activation and its potential for further inflammatory dysregulation after recruitment to tissues and during recovery.

Enhanced Immunogenicity of Mitochondrial-Localized Proteins in Cancer Cells.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.

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.

Stimulation of Vibratory Urticaria-Associated Adhesion-GPCR, EMR2/ADGRE2, Triggers the NLRP3 Inflammasome Activation Signal in Human Monocytes.

EMR2/ADGRE2 is an adhesion G protein-coupled receptor differentially expressed by human myeloid cells. It modulates diverse cellular functions of innate immune cells and a missense EMR2 variant is directly responsible for vibratory urticaria. Recently, EMR2 was found to activate NLRP3 inflammasome in monocytes via interaction with FHR1, a regulatory protein of complement Factor H. However, the functional involvement of EMR2 activation and its signaling mechanisms in eliciting NLRP3 inflammasome activation remain elusive. In this study, we show that EMR2-mediated signaling plays a critical role in triggering the activation (2nd) signal for the NLRP3 inflammasome in both THP-1 monocytic cell line and primary monocytes. Stimulation of EMR2 by its agonistic 2A1 monoclonal antibody elicits a Gα16-dependent PLC-ß activation pathway, inducing the activity of downstream Akt, MAPK, NF-κB, and Ca2+ mobilization, eventually leading to K+ efflux. These results identify EMR2 and its associated signaling intermediates as potential intervention targets of NLRP3 inflammasome activation in inflammatory disorders.

The Mononuclear Phagocytic System. Generation of Diversity.

We are living through an unprecedented accumulation of data on gene expression by macrophages, reflecting their origin, distribution, and localization within all organs of the body. While the extensive heterogeneity of the cells of the mononuclear phagocyte system is evident, the functional significance of their diversity remains incomplete, nor is the mechanism of diversification understood. In this essay we review some of the implications of what we know, and draw attention to issues to be clarified in further research, taking advantage of the powerful genetic, cellular, and molecular tools now available. Our thesis is that macrophage specialization and functions go far beyond immunobiology, while remaining an essential contributor to innate as well as adaptive immunity.