Cell-intrinsic metabolic regulation of mononuclear phagocyte activation: Findings from the tip of the iceberg.

We have only recently started to appreciate the extent to which immune cell activation involves significant changes in cellular metabolism. We are now beginning to understand how commitment to specific metabolic pathways influences aspects of cellular biology that are the more usual focus of immunological studies, such as activation-induced changes in gene transcription, post-transcriptional regulation of transcription, post-translational modifications of proteins, cytokine secretion, etc. Here, we focus on metabolic reprogramming in mononuclear phagocytes downstream of stimulation with inflammatory signals (such as LPS and IFNγ) vs alternative activation signals (IL-4), with an emphasis on work on dendritic cells and macrophages from our laboratory, and related studies from others. We cover aspects of glycolysis and its branching pathways (glycogen synthesis, pentose phosphate, serine synthesis, hexose synthesis, and glycerol 3 phosphate shuttle), the tricarboxylic acid pathway, fatty acid synthesis and oxidation, and mitochondrial biology. Although our understanding of the metabolism of mononuclear phagocytes has progressed significantly over the last 10 years, major challenges remain, including understanding the effects of tissue residence on metabolic programming related to cellular activation, and the translatability of findings from mouse to human biology.

Inherited CARD9 Deficiency: Invasive Disease Caused by Ascomycete Fungi in Previously Healthy Children and Adults.

Autosomal recessive CARD9 deficiency underlies life-threatening, invasive fungal infections in otherwise healthy individuals normally resistant to other infectious agents. In less than 10 years, 58 patients from 39 kindreds have been reported in 14 countries from four continents. The patients are homozygous (n = 49; 31 kindreds) or compound heterozygous (n = 9; 8 kindreds) for 22 different CARD9 mutations. Six mutations are recurrent, probably due to founder effects. Paradoxically, none of the mutant alleles has been experimentally demonstrated to be loss-of-function. CARD9 is expressed principally in myeloid cells, downstream from C-type lectin receptors that can recognize fungal components. Patients with CARD9 deficiency present impaired cytokine and chemokine production by macrophages, dendritic cells, and peripheral blood mononuclear cells and defective killing of some fungi by neutrophils in vitro. Neutrophil recruitment to sites of infection is impaired in vivo. The proportion of Th17 cells is low in most, but not all, patients tested. Up to 52 patients suffering from invasive fungal diseases (IFD) have been reported, with ages at onset of 3.5 to 52 years. Twenty of these patients also displayed superficial fungal infections. Six patients had only mucocutaneous candidiasis or superficial dermatophytosis at their last follow-up visit, at the age of 19 to 50 years. Remarkably, for 50 of the 52 patients with IFD, a single fungus was involved; only two patients had IFDs due to two different fungi. IFD recurred in 44 of 45 patients who responded to treatment, and a different fungal infection occurred in the remaining patient. Ten patients died from IFD, between the ages of 12 and 39 years, whereas another patient died at the age of 91 years, from an unrelated cause. At the most recent scheduled follow-up visit, 81% of the patients were still alive and aged from 6.5 to 75 years. Strikingly, all the causal fungi belonged to the phylum Ascomycota: commensal Candida and saprophytic Trychophyton, Aspergillus, Phialophora, Exophiala, Corynesprora, Aureobasidium, and Ochroconis. Human CARD9 is essential for protective systemic immunity to a subset of fungi from this phylum but seems to be otherwise redundant. Previously healthy patients with unexplained invasive fungal infection, at any age, should be tested for inherited CARD9 deficiency. KEY POINTS: • Inherited CARD9 deficiency (OMIM #212050) is an AR PID due to mutations that may be present in a homozygous or compound heterozygous state. • CARD9 is expressed principally in myeloid cells and transduces signals downstream from CLR activation by fungal ligands. • Endogenous mutant CARD9 levels differ between alleles (from full-length normal protein to an absence of normal protein). • The functional impacts of CARD9 mutations involve impaired cytokine production in response to fungal ligands, impaired neutrophil killing and/or recruitment to infection sites, and defects of Th17 immunity. • The key clinical manifestations in patients are fungal infections, including CMC, invasive (in the CNS in particular) Candida infections, extensive/deep dermatophytosis, subcutaneous and invasive phaeohyphomycosis, and extrapulmonary aspergillosis. • The clinical penetrance of CARD9 deficiency is complete, but penetrance is incomplete for each of the fungi concerned. • Age at onset is highly heterogeneous, ranging from childhood to adulthood for the same fungal disease. • All patients with unexplained IFD should be tested for CARD9 mutations. Familial screening and genetic counseling should be proposed. • The treatment of patients with CARD9 mutations is empirical and based on antifungal therapies and the surgical removal of fungal masses. Patients with persistent/relapsing Candida infections of the CNS could be considered for adjuvant GM-CSF/G-CSF therapy. The potential value of HSCT for CARD9-deficient patients remains unclear.

Some news from the unknown soldier, the Peyer's patch macrophage.

In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintenance of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer's patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30 years ago, MF of PP escaped any kind of phenotypic description and remained "unknown" for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions.

The multifaceted role of the renal mononuclear phagocyte system.

The kidney contains a large and complex network of mononuclear phagocytes, which includes dendritic cells (DCs) and macrophages (MØs). The distinction between these cell types is traditionally based on the expression of molecular markers and morphology. However, several classification systems are used in parallel to identify DCs and MØs, leading to considerable uncertainty about their identity and functional roles. The discovery that a substantial proportion of macrophages in tissues like the kidney are embryonically derived further complicates the situation. Recent studies have used newly identified transcription factors such as ZBTB46 and lineage tracing techniques for classifying mononuclear phagocytes. These approaches have shed new light on the functional specialization of these cells in health and disease, uncovered an influence of the renal microenvironment and revealed considerable cellular plasticity, especially in inflammatory situations. In this review, the current knowledge about the developmental origins and versatile functional roles of DCs and MØs in kidney homeostasis and disease is discussed.

The role of tissue resident cells in neutrophil recruitment.

Neutrophils are first responders of the immune system, rapidly migrating into affected tissues in response to injury or infection. To effectively call in this first line of defense, strategically placed cells within the vasculature and tissue respond to noxious stimuli by sending out coordinated signals that recruit neutrophils. Regulation of organ-specific neutrophil entry occurs at two levels. First, the vasculature supplying the organ provides cues for neutrophil egress out of the bloodstream in a manner dependent upon its unique cellular composition and architectural features. Second, resident immune cells and stromal cells within the organ send coordinated signals that guide neutrophils to their final destination. Here, we review recent findings that highlight the importance of these tissue-specific responses in the regulation of neutrophil recruitment and the initiation and resolution of inflammation.

Mononuclear phagocytes of the intestine, the skin, and the lung.

Tissues that are in direct contact with the outside world face particular immunological challenges. The intestine, the skin, and the lung possess important mononuclear phagocyte populations to deal with these challenges, but the cellular origin of these phagocytes is strikingly different from one subset to another, with some cells derived from embryonic precursors and some from bone marrow-derived circulating monocytes. Here, we review the current knowledge regarding the developmental pathways that control the differentiation of mononuclear phagocytes in these barrier tissues. We have also attempted to build a theoretical model that could explain the distinct cellular origin of mononuclear phagocytes in these tissues.

Transcriptomic analysis of mononuclear phagocyte differentiation and activation.

Monocytes and macrophages differentiate from progenitor cells under the influence of colony-stimulating factors. Genome-scale data have enabled the identification of the sets of genes that are associated with specific functions and the mechanisms by which thousands of genes are regulated in response to pathogen challenge. In large datasets, it is possible to identify large sets of genes that are coregulated with the transcription factors that regulate them. They include macrophage-specific genes, interferon-responsive genes, early inflammatory genes, and those associated with endocytosis. Such analyses can also extract macrophage-associated signatures from large cancer tissue datasets. However, cluster analysis provides no support for a signature that distinguishes macrophages from antigen-presenting dendritic cells, nor the classification of macrophage activation states as classical versus alternative, or M1 versus M2. Although there has been a focus on a small subset of lineage-enriched transcription factors, such as PU.1, more than half of the transcription factors in the genome can be expressed in macrophage lineage cells under some state of activation, and they interact in a complex network. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. The data and publication deluge related to macrophage biology require the development of new analytical tools and ways of presenting information in an accessible form.

Beyond macrophages: the diversity of mononuclear cells in tuberculosis.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), is an intracellular pathogen of mononuclear phagocytes. Although M. tuberculosis has traditionally been thought to survive and replicate in macrophages, recent work in our laboratory and others has revealed that M. tuberculosis infects multiple subsets of mononuclear phagocytes in vivo and in vitro. In experimental animals, M. tuberculosis infects no fewer than five distinct cell subsets in the lungs, including resident alveolar macrophages and 4 types of cells that recruited to the lungs in response to inflammatory signals: neutrophils, monocytes, interstitial macrophages, and dendritic cells. A characteristic of the adaptive immune response in TB is that it is delayed for several weeks following infection, and we have determined that this delay is due to prolonged residence of the bacteria in lung phagocytes prior to acquisition of the bacteria by dendritic cells. Among the mechanisms used by M. tuberculosis to delay acquisition by dendritic cells is to inhibit apoptosis of alveolar macrophages and neutrophils, which sequester the bacteria and prevent their acquisition by dendritic cells in the early stages of infection. We hypothesize that each infected cell subset makes a distinct contribution to the overall biology of M. tuberculosis and allows the bacteria to evade elimination by T-cell responses and to avoid rapid killing by antimycobacterial drugs.

Human mononuclear phagocyte system reunited.

The human mononuclear phagocyte network comprises dendritic cells (DCs), monocytes and macrophages with a range of immune functions including antigen presentation linking innate and adaptive immunity. A number of DC, monocyte and macrophage subsets have been described in lymphoid and non-lymphoid tissues of mouse and human, with increased understanding of their distinct functional properties and genetic and cellular pathways of development. More recently, through comparative biology studies, a unified nomenclature of mononuclear phagocytes has begun to emerge with the identification of homologous subsets in several species. In this review, we discuss the current classification of human mononuclear phagocytes and the parallel organization of this network in the mouse. We also review the genetic control and developmental pathway of human mononuclear phagocytes and the immunological functions of the distinct subsets in health and inflammation.

Resident renal mononuclear phagocytes comprise five discrete populations with distinct phenotypes and functions.

Recent reports have highlighted greater complexity, plasticity, and functional diversity of mononuclear phagocytes (MPCs), including monocytes, macrophages, and dendritic cells (DCs), in our organs than previously understood. The functions and origins of MPCs resident within healthy organs, especially in the kidney, are less well understood, whereas studies suggest they play roles in disease states distinct from recruited monocytes. We developed an unbiased approach using flow cytometry to analyze MPCs residing in the normal mouse kidney, and identified five discrete subpopulations according to CD11b/CD11c expression as well as F4/80, CD103, CD14, CD16, and CD64 expression. In addition to distinct marker profiles, these subpopulations have different lineages and expression of genes involved in tissue homeostasis, including angiogenesis. Among them, the CD11b(int)CD11c(int) F4/80(high) subpopulation notably exhibited high capacity to produce a representative anti-inflammatory cytokine, IL-10. Each subpopulation had different degrees of both macrophage (phagocytosis) and DC (Ag presentation) capacities, with a tendency to promote differentiation of regulatory T cells, whereas two of these showed expression of transcription factors reported to be highly expressed by classical DCs, and proclivity to exit the kidney following stimulation with LPS. In summary, resident kidney MPCs comprise discrete subpopulations, which cannot be simply classified into the conventional entities, and they produce anti-inflammatory and tissue-homeostatic factors to differing degrees.

Characterization of the mononuclear phagocyte system in zebrafish.

The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

The innate immune system of the perinatal lung and responses to respiratory syncytial virus infection.

The response of the preterm and newborn lung to airborne pathogens, particles, and other insults is initially dependent on innate immune responses since adaptive responses may not fully mature and require weeks for sufficient responses to antigenic stimuli. Foreign material and microbial agents trigger soluble, cell surface, and cytoplasmic receptors that activate signaling cascades that invoke release of surfactant proteins, defensins, interferons, lactoferrin, oxidative products, and other innate immune substances that have antimicrobial activity, which can also influence adaptive responses. For viral infections such as respiratory syncytial virus (RSV), the pulmonary innate immune responses has an essential role in defense as there are no fully effective vaccines or therapies for RSV infections of humans and reinfections are common. Understanding the innate immune response by the preterm and newborn lung may lead to preventive strategies and more effective therapeutic regimens.

Study of macrophages in BCG granulomas in different compartments of the mononuclear phagocyte system.

We studied in vitro morphological and functional properties of macrophages associated with their M1 and M2 polarization in different mononuclear phagocyte compartments during BCG-induced granuloma formation, namely expression patterns of cytokines IL-1α, GM-CSF, TNF-α, and clusters of differentiation CD36 and CD16/32. We showed the mosaic pattern of macrophage polarization in BCG granulomatosis manifested by simultaneous formation of different macrophage subpopulations with M1 and M2 phenotypes in the population of mononuclear phagocytes of BCG granulomas and various compartments of the mononuclear phagocyte system. These data clarify the role of the functional polarization of macrophages in the pathogenesis of tuberculosis infection.

The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes.

BACKGROUND: The chemokine and chemokine receptor families play critical roles in both the healthy and diseased organism mediating the migration of cells. The chemokine system is complex in that multiple chemokines can bind to one chemokine receptor and vice versa. Although chemokine receptors have been well characterised in humans, the chemokine receptor repertoire of cattle is not well characterised and many sequences are yet to be experimentally validated. RESULTS: We have identified and sequenced bovine homologs to all identified functional human chemokine receptors. The bovine chemokine receptors show high levels of similarity to their human counterparts and similar genome arrangements. We have also characterised an additional bovine chemokine receptor, not present in the available genome sequence of humans or the more closely related pigs or horses. This receptor shows the highest level of similarity to CCR1 but shows significant differences in regions of the protein that are likely to be involved in ligand binding and signalling. We have also examined the mRNA abundance levels of all identified bovine chemokine receptors in mononuclear phagocytic cells. Considerable differences were observed in the mRNA abundance levels of the receptors, and interestingly the identified novel chemokine receptor showed differing levels of mRNA abundance to its closest homolog CCR1. The chemokine receptor repertoire was shown to differ between monocytes, macrophages and dendritic cells. This may reflect the differing roles of these cells in the immune response and may have functional consequences for the trafficking of these cells in vivo. CONCLUSIONS: In summary, we have provided the first characterisation of the complete bovine chemokine receptor gene repertoire including a gene that is potentially unique to cattle. Further study of this receptor and its ligands may reveal a specific role of this receptor in cattle. The availability of the bovine chemokine receptor sequences will allow further characterisation of the function of these genes and will confer wide-reaching benefits to the study of this important aspect of the bovine immune response.

The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80. Relationship between macrophages, Langerhans cells, reticular cells, and dendritic cells in lymphoid and hematopoietic organs.

The macrophage-specific antigen F4/80 has been localized in mouse lymphoid and hematopoietic tissue and skin using immunoperoxidase staining. The antigen permits identification of early mononuclear phagocyte precursors in the bone marrow, and is present also on larger cells forming the center of hematopoietic islands and lining vascular sinuses. In thymus F4/80+ cells are numerous in both cortex and medulla and are particularly concentrated around the corticomedullary region. In spleen, lymph node, and gut-associated lymphoid areas the major F4/80+ populations are in the red pulp, the medulla and subcapsular sinus, and the adjacent lamina propria, respectively. F4/80+ cells are rarely seen in T-dependent areas of lymph nodes, spleen, or Peyer's patch, but are present in large numbers in these areas during bacillus Calmette-Guerin (BCG)-induced inflammation. Macrophage infiltration occurs also in lymph nodes from athymic nu/nu mice and is therefore T cell independent. The interdigitating cell of T-dependent areas is F4/80-, but the Langerhans cell of the epidermis of the skin, which bears some ultrastructural resemblance to the interdigitating cell, is F4/80+. We conclude that the two cell types are probably not related.

Fibroblast-type reticular stromal cells regulate the lymph node vasculature.

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)(+) endothelial cells are less sensitive than PNAd(-) endothelial cells to VEGF blockade. Lymphotoxin beta receptor (LTbetaR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTbetaR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTbetaR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd(+) and PNAd(-) endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.

Recruited peripheral blood monocytes participate in the liver extramedullary hematopoietic milieu.

The hematopoietic microenvironment has been investigated and well defined in the bone marrow. However, there is a lack of studies on the extramedullary hematopoietic milieu such as in the liver, to which hematopoietic stem cells migrate and there commence hematopoiesis under pathological conditions such as bone marrow failure. We induced extramedullary hematopoiesis by phenylhydrazine in the adult mouse liver and investigated the immunohistochemical, ultrastructural, and molecular changes within this organ. Using an intravital lectin injection technique, we found numerous monocytes attached to the central vein prior to hematopoietic foci formation. These cells were later incorporated into the hematopoietic foci. An increase in the mRNA expressions of the monocyte attracting chemokine CCL-2 (MCP-1) was noted in the central vein region as well as in cells within the hematopoietic foci. Together with local liver components, we regard these monocytes as components of the extramedullary hematopoietic milieu. We conclude that the recruitment of extra-hepatic monocytes is an important event during extramedullary hematopoiesis in the liver and that these monocytes participate in the liver hematopoietic microenvironment.

Nano-immunoimaging.

Immunoimaging is a rapidly growing field stoked in large part by the intriguing triumphs of immunotherapy. On the heels of immunotherapy's successes, there exists a growing need to evaluate tumor response to therapy particularly immunotherapy, stratify patients into responders vs. non-responders, identify inflammation, and better understand the fundamental roles of immune system components to improve both immunoimaging and immunotherapy. Innovative nanomaterials have begun to provide novel opportunities for immunoimaging, in part due to their sensitivity, modularity, capacity for many potentially varied ligands (high avidity), and potential for multifunctionality/multimodality imaging. This review strives to comprehensively summarize the integration of nanotechnology and immunoimaging, and the field's potential for clinical applications.

Isolation of mononuclear phagocytes from the mouse gut.

The intestinal tract is home to trillions of microbes that make up the gut microbiota and is a major source of environmental antigens that can be derived from food, commensal microorganisms, and potential pathogens. Amidst this complex environment, myeloid cells, including macrophages (MPs) and dendritic cells (DCs), are key immunological sentinels that locally maintain both tissue and immune homeostasis. Recent research has revealed substantial functional and developmental heterogeneity within the intestinal DC and MP compartments, with evidence pointing to their regulation by the microbiota. DCs are classically divided into three subsets based on their CD103 and CD11b expression: CD103+CD11b-(XCR1+) cDC1s, CD103+CD11b+ cDC2s, and CD103-CD11b+ cDC2s. Meanwhile, mature gut MPs have recently been classified by their expression of Tim-4 and CD4 into a long-lived, self-maintaining Tim-4+CD4+ population and short-lived, monocyte-derived Tim-4-CD4+ and Tim-4-CD4- populations. In this chapter, we provide experimental procedures to classify and isolate these myeloid subsets from the murine intestinal lamina propria for functional characterization.

Regulating Interactions Between Targeted Nanocarriers and Mononuclear Phagocyte System via an Esomeprazole-Based Preconditioning Strategy.

PURPOSE: The mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions. METHODS: In vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected. RESULTS: In vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter's lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines. CONCLUSION: Our findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.