Dr Fernando Martinez Estrada

Type-I interferon (IFN-I) cytokines are produced by immune cells in response to microbial infections, cancer and autoimmune diseases, and subsequently, trigger cytoprotective and antiviral responses through the activation of IFN-I stimulated genes (ISGs). The ability of intestinal microbiota to modulate innate immune responses is well known, but the mechanisms underlying such responses remain elusive. Here we report that the intracellular sensors stimulator of IFN genes (STING) and mitochondrial antiviral signaling (MAVS) are essential for the production of IFN-I in response to lactic acid bacteria (LAB), common gut commensal bacteria with beneficial properties. Using human macrophage cells we show that LAB strains that potently activate the inflammatory transcription factor NF-κB are poor inducers of IFN-I and conversely, those triggering significant amounts of IFN-I fail to activate NF-κB. This IFN-I response is also observed in human primary macrophages, which modulate CD64 and CD40 upon challenge with IFN-I-inducing LAB. Mechanistically, IFN-I inducers interact more intimately with phagocytes as compared to NF-κB-inducers, and fail to activate IFN-I in the presence of phagocytosis inhibitors. These bacteria are then sensed intracellularly by the cytoplasmic sensors STING and, to a lesser extent, MAVS. Accordingly, macrophages deficient for STING showed dramatically reduced phosphorylation of TANK-binding kinase (TBK)-1 and IFN-I activation, which resulted in lower expression of ISGs. Our findings demonstrate a major role for intracellular sensing and STING in the production of IFN-I by beneficial bacteria and the existence of bacteria-specific immune signatures, which can be exploited to promote cytoprotective responses and prevent overreactive NF-κB-dependent inflammation in the gut.

Multinucleated giant cells, formed by fusion of macrophages, are a hallmark of granulomatous inflammation. With a genetic approach, we show that signaling through the adaptor protein DAP12 (DNAX activating protein of 12 kD), its associated receptor triggering receptor expressed by myeloid cells 2 (TREM-2), and the downstream protein tyrosine kinase Syk is required for the cytokine-induced formation of giant cells and that overexpression of DAP12 potentiates macrophage fusion. We also present evidence that DAP12 is a general macrophage fusion regulator and is involved in modulating the expression of several macrophage-associated genes, including those encoding known mediators of macrophage fusion, such as DC-STAMP and Cadherin 1 . Thus, DAP12 is involved in programming of macrophages through the regulation of gene and protein expression to induce a fusion-competent state.

Oxidative stress is a common feature of obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Lung macrophages are key innate immune cells that can generate oxidants and are known to display aberrant polarization patterns and defective phagocytic responses in these diseases. Whether these characteristics are linked in one way or another and whether they contribute to the onset and severity of exacerbations in asthma and COPD remains poorly understood. Insight into oxidative stress, macrophages and their interactions may be important in fully understanding acute worsening of lung disease. This review therefore highlights the current state of the art regarding the role of oxidative stress and macrophages in exacerbations of asthma and COPD. It shows that oxidative stress can attenuate macrophage function, which may result in impaired responses towards exacerbating triggers and may contribute to exaggerated inflammation in the airways.

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.

Type-I interferon (IFN-I) cytokines are produced by immune cells in response to microbial infections, 2cancer and autoimmune diseases, and subsequently trigger cytoprotective and antiviral responses through the activation of IFN-I stimulated genes (ISGs). The ability of intestinal microbiota to modulate innate immune responses is well-known, but the mechanisms underlying such responses remain elusive. Here we report that the intracellular sensors stimulator of IFN genes (STING) and mitochondrial antiviral signalling (MAVS) are essential for the production of IFN-I in response to lactic acid bacteria (LAB), common gut commensal bacteria with beneficial properties. Using human macrophage cells we show that LAB strains that potently activate the inflammatory transcription factor NF-κB are poor inducers of IFN-I and conversely, those triggering significant amounts of IFN-I fail to activate NF-κB. This IFN-I response is also observed in human primary macrophages, which modulate CD64 and CD40 upon challenge with IFN-I-inducing LAB. Mechanistically, IFN-I inducers interact more intimately with phagocytes as compared to NF-κB-inducers, and fail to activate IFN-I in the presence of phagocytosis inhibitors. These bacteria are then sensed intracellularly by the cytoplasmic sensors STING and, to a lesser extent, MAVS. Accordingly, macrophages deficient for STING showed dramatically reduced phosphorylation of TANK-binding kinase (TBK)-1 and IFN-I activation, which resulted in lower expression of ISGs. Our findings demonstrate a major role for intracellular sensing and STING in the production of IFN-I by beneficial bacteria and the existence of bacteria-specific immune signatures, which can be exploited to promote cytoprotective responses and prevent overreactive NF-κB-dependent inflammation in the gut.

Background: Wildlife poses a significant burden for the complete eradication of bovine tuberculosis (bTB). In particular, wild boar (Sus scrofa) is one of the most important reservoirs of Mycobacterium bovis, the causal agent of bTB. Wild boar can display from mild TB lesions, usually found in head lymph nodes, to generalized TB lesions distributed in different anatomical regions; but rarely clinical signs, which complicates the diagnosis of Mycobacterium bovis infection and bTB control. Among the possibilities for this variability in lesion distribution is the influence of the host-beneficial commensal-primed immune barrier. In this respect, beneficial microbes may delay bTB dissemination as a consequence of an antagonistic competition for nutrients and phagocytes. In order to explore this possibility, we have tested whether typical commensals such as lactobacilli have the capacity to reduce the survival rate of the surrogate M. bovis strain Bacillus Calmette-Guerin (BCG); and to modulate its phagocyte intake. Results: Three Lactobacillus species, L. casei, L. plantarum, and L. salivarius, isolated from wild boar feces displayed a pH-dependent inhibitory activity against BCG and influenced its intake by porcine blood phagocytes in a species-dependent manner. All lactobacilli showed a very significant bactericidal effect against BCG at low pH, but only isolates of L. plantarum and L. casei displayed such antimycobacterial activity at neutral pH. The genomes of these isolates revealed the presence of two-peptide bacteriocins whose precursor genes up-regulate in the presence of BCG cells. Furthermore, L. plantarum reduced significantly the BCG phagocytic intake, whereas L. casei had the opposite effect. L. salivarius had no significant influence on the phagocytic response to BCG. Conclusions: Our in vitro results show that lactobacilli isolated from wild boar antagonize BCG as a consequence of their antimycobacterial activity and a competitive phagocytic response. These findings suggest that commensal bacteria could play a beneficial role in influencing the outcome of bTB dissemination. Further work with lactobacilli as a potential competitive pressure to control bTB will need to take into account the complex nature of the commensal microbiome, the specific immunity of the wild boar and the in vivo infection context with pathogenic strains of M. bovis.

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.

The chemokine CXC ligand 8 (CXCL8)/IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors.

IL-8 is an inflammatory CXC chemokine involved in neutrophil recruitment and activation in various inflammatory conditions. The transcriptional profile induced by IL-8 in human neutrophils was analyzed using high-density oligonucleotide arrays and compared with that of the prototypic phagocyte activator LPS. As expected, LPS induced a major effect on the cell transcriptome, upregulating 116 (0.93%) and downregulating 70 (0.56%) of the transcripts. IL-8 induced a less profound modulation of the cell transcriptome, with upregulation of 30 (0.25%) and downregulation of 6 (0.04%) of the transcripts. Although the two proinflammatory mediators induced partially overlapping transcriptional profiles (50.0% of IL-8-responsive genes were concordantly regulated by LPS), IL-8 also modulated a significant number of genes unresponsive to LPS, including soluble mediators, membrane receptors, signaling molecules, and regulators of transcription and translation. A set of IL-8-inducible genes was related to cell motility, possibly a strategy to prepare for migration into tissues. Analysis of the IL-8-responsive gene IL-1beta at the protein level revealed that transcript induction was not followed by protein production. Neutrophils stimulated with IL-8, however, showed a significant increase in IL-1beta secretion after subsequent exposure to LPS. Thus, the effect of IL-8 at the transcriptional level could provide a synergistic effect with microbial products for neutrophil activation.

The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors that are required for adequate heart formation. Signaling pathways regulated by WT1 that promote heart development have started to be described; however, there is little information on signaling pathways regulated by WT1 that could act in a negative manner. Transcriptome analysis of Wt1KO epicardial cells reveals an unexpected role for WT1 in repressing the expression of interferon-regulated genes that could be involved in a negative regulation of heart morphogenesis. Here, we showed that WT1 is required to repress the expression of the chemokines Ccl5 and Cxcl10 in epicardial cells. We observed an inverse correlation of Wt1 and the expression of Cxcl10 and Ccl5 during epicardium development. Chemokine receptor analyses of hearts from Wt1(gfp/+) mice demonstrate the differential expression of their chemokine receptors in GFP(+) epicardial enriched cells and GFP(-) cells. Functional assays demonstrate that CXCL10 and CCL5 inhibit epicardial cells migration and the proliferation of cardiomyocytes respectively. WT1 regulates the expression levels of Cxcl10 and Ccl5 in epicardial cells directly and indirectly through increasing the levels of IRF7. As epicardial cell reactivation after a myocardial damage is linked with WT1 expression, the present work has potential implications in adult heart repair.

After the identification of the interleukin (IL)-1 type II receptor as the prototype, decoy receptors have been identified for a number of members of the IL-1/IL-18, TNF, IL-10 and IL-13 receptor families. Moreover, the silent receptor D6 is a promiscuous decoy and scavenger receptor of inflammatory chemokines. The IL-1 decoy receptor is regulated by pro- and anti-inflammatory signals and its levels may serve as a readout of the activation of anti-inflammatory pathways, for instance by glucocorticoid hormones. Decoy receptors represent a strategy to tune inflammatory and polarized adaptive responses.

Comprehensive analysis of the gene expression profiles associated with human monocyte-to-macrophage differentiation and polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage differentiation is associated with activation of cell cycle genes, substantiating the underestimated proliferation potential of monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature of macrophage differentiation and polarization. 4) Lipid metabolism is a main category of modulated transcripts, with expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2-associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology.

Microarrays are made by immobilizing to a solid support thousands of DNA probes that detect soluble complementary target sequences using the hybridization pairing rules of nucleic acids. Receptor triggering induces a cascade of signaling events that often involves the modulation of gene expression. In the last decade, the development of microarrays has provided scientists with an innovative tool to interrogate the cell transcriptional profile at a global level and to characterize genes according to their behavior in different conditions. This chapter outlines the use of microarrays as an innovative approach to study the global effect of transmembrane-receptor triggering. The effect of formyl peptides receptors activation on the gene transcriptional program of human monocytes is described as a model.

Candida albicans is a medically important pathogen, and recognition by innate immune cells is critical for its clearance. Although a number of pattern recognition receptors have been shown to be involved in recognition and phagocytosis of this fungus, the relative role of these receptors has not been formally examined. In this paper, we have investigated the contribution of the mannose receptor, Dectin-1, and complement receptor 3; and we have demonstrated that Dectin-1 is the main non-opsonic receptor involved in fungal uptake. However, both Dectin-1 and complement receptor 3 were found to accumulate at the site of uptake, while mannose receptor accumulated on C. albicans phagosomes at later stages. These results suggest a potential role for MR in phagosome sampling; and, accordingly, MR deficiency led to a reduction in TNF-alpha and MCP-1 production in response to C. albicans uptake. Our data suggest that pattern recognition receptors sample the fungal phagosome in a sequential fashion.

Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.

Macrophages are specialized but versatile cells that participate in a range of physiological and immune related processes. The macrophage repertoire of coding and regulatory RNA provides tools to understand cell identity, cell function, role in disease, and ultimately define cell specific therapeutic targets. Modern tools make it possible to quantify and compare global RNA levels. With this vast information a neologism of the decade, the suffix “ome” has been combined with “transcript” to form “transcriptome,” a new word to define the totality of transcripts that characterize a cell. In this chapter we discuss the macrophage transcriptome and how its definition is contributing to a deeper understanding of this cell identity and function.

'There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes,' so declaimed Sir Ralph Bloomfield Bonington in The Doctor's Dilemma, Act 1, by George Bernard Shaw (1906). More often nowadays, the need is to calm the phagocytes, given their role in inflammation and tissue damage. In spite of the growth of cellular and molecular information gained from studies in macrophage cell culture, mouse models and, to a lesser extent, human investigations, and the importance of macrophages in pathogenesis in a wide range of chronic disease processes, there is still a substantial shortfall in terms of clinical applications. In this review, we summarize concepts derived from macrophage studies and suggest possible properties suitable for diagnosis, prognosis and selective targeting of macrophage pathogenic functions.

Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF-5 and interleukin-12) or M2-dominant (CD206, transglutaminase-2, and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse, livers undergoing resolution of fibrosis, the total number of CD68 + macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF-5) macrophages were almost unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1- and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used flow cytometry and gene analysis.

Glucocorticoids (GCs) have been used for more than 50 y as immunosuppressive drugs, yet their efficacy in macrophage-dominated disorders, such as chronic obstructive pulmonary disease, is debated. Little is known how long-term GC treatment affects macrophage responses in inflammatory conditions. In this study, we compared the transcriptome of human macrophages, matured in the presence or absence of fluticasone propionate (FP), and their ability to initiate or sustain classical activation, mimicked using acute LPS and chronic IFN-γ stimulation, respectively. We identified macrophage gene expression networks, modulated by FP long-term exposure, and specific patterns of IFN-γ– and LPS-induced genes that were resistant, inhibited, or exacerbated by FP. Results suggest that long-term treatment with GCs weakens adaptive immune signature components of IFN-γ and LPS gene profiles by downmodulating MHC class II and costimulatory molecules, but strengthens innate signature components by maintaining and increasing expression of chemokines involved in phagocyte attraction. In a mouse model of chronic obstructive pulmonary disease, GC treatment induced higher chemokine levels, and this correlated with enhanced recruitment of leukocytes. Thus, GCs do not generally suppress macrophage effector functions, but they cause a shift in the innate–adaptive balance of the immune response, with distinct changes in the chemokine–chemokine receptor network

Macrophages of the body represent a widely dispersed, versatile, and highly responsive homeostatic and defense system. They contribute to innate as well as adaptive immunity to infection, mediating trophic as well as injurious interactions with their local and systemic environment. The concept of macrophage activation has evolved over recent decades, from relatively simple paradigms to bewildering complexity. This chapter will review the background to present understanding of alternative activation of macrophages, consider its relevance to health and disease, and suggest questions for future studies.

Macrophages differentiated from human induced pluripotent stem cells (IPSDMs) are a potentially valuable new tool for linking genotype to phenotype in functional studies. However, at a genome-wide level these cells have remained largely uncharacterised. Here, we compared the transcriptomes of naïve and lipopolysaccharide (LPS) stimulated monocyte-derived macrophages (MDMs) and IPSDMs using RNA-Seq. The IPSDM and MDM transcriptomes were broadly similar and exhibited a highly conserved response to LPS. However, there were also significant differences in the expression of genes associated with antigen presentation and tissue remodelling. Furthermore, genes coding for multiple chemokine involved in neutrophil recruitment were more highly expressed in IPSDMs upon LPS stimulation. Additionally, analysing individual transcript expression identified hundreds of genes undergoing alternative promoter and 3′ untranslated region usage following LPS treatment representing a previously under-appreciated level of regulation in the LPS response.

Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.

Interleukin (IL)-4 and IL-13 control the phenotype of a variety of cells in health and disease. Their receptor system, signaling cascades, and functional properties have gained clarity in the past 30years. In this article, we give an overview of the production, sensing, and effects of both cytokines concluding with a discussion of the therapeutic potential of IL-4 and IL-13 inhibition or supplementation in patients.

Macrophages play a central role in tuberculosis, as the site of primary infection, inducers and effectors of inflammation, innate and adaptive immunity, as well as mediators of tissue destruction and repair. Early descriptions by pathologists have emphasized their morphological heterogeneity in granulomas, followed by delineation of T lymphocyte-dependent activation of anti-mycobacterial resistance. More recently, powerful genetic and molecular tools have become available to describe macrophage cellular properties and their role in host-pathogen interactions. In this review we discuss aspects of macrophage heterogeneity relevant to the pathogenesis of tuberculosis and, conversely, lessons that can be learnt from mycobacterial infection, with regard to the immunobiological functions of macrophages in homeostasis and disease.

In this review, we discuss the often overlooked tissue-resident fetal macrophages, Hofbauer cells, which are found within the chorionic villi of the human placenta. Hofbauer cells have been shown to have a phenotype associated with regulatory and anti-inflammatory functions. They are thought to play a crucial role in the regulation of pregnancy and in the maintenance of a homeostatic environment that is crucial for fetal development. Even though the numbers of these macrophages are some of the most abundant immune cells in the human placenta, which are sustained throughout pregnancy, there are very few studies that have identified their origin, their phenotype, and functions and why they are maintained throughout gestation. It is not yet understood how Hofbauer cells may change in function throughout normal pregnancy, and especially in those complicated by maternal gestational diabetes, preeclampsia, and viral infections, such as Zika, cytomegalovirus, and human immunodeficiency virus. We review what is known about the origin of these macrophages and explore how common complications of pregnancy dysregulate these cells leading to adverse birth outcomes in humans. Our synthesis sheds light on areas for human studies that can further define these innate regulatory cells.

This unit describes how to execute a gene expression study with human macrophages. It includes protocols for human macrophage preparation, RNA extraction, real-time PCR analysis, and microarray analysis. The unit also includes a protocol for gene silencing in human macrophages. Altering gene expression can be useful to study the contribution of the gene to macrophage function or even expression of other genes.

Glucocorticoids (GCs) have been used for more than 50 y as immunosuppressive drugs, yet their efficacy in macrophage-dominated disorders, such as chronic obstructive pulmonary disease, is debated. Little is known how long-term GC treatment affects macrophage responses in inflammatory conditions. In this study, we compared the transcriptome of human macrophages, matured in the presence or absence of fluticasone propionate (FP), and their ability to initiate or sustain classical activation, mimicked using acute LPS and chronic IFN-γ stimulation, respectively. We identified macrophage gene expression networks, modulated by FP long-term exposure, and specific patterns of IFN-γ– and LPS-induced genes that were resistant, inhibited, or exacerbated by FP. Results suggest that long-term treatment with GCs weakens adaptive immune signature components of IFN-γ and LPS gene profiles by downmodulating MHC class II and costimulatory molecules, but strengthens innate signature components by maintaining and increasing expression of chemokines involved in phagocyte attraction. In a mouse model of chronic obstructive pulmonary disease, GC treatment induced higher chemokine levels, and this correlated with enhanced recruitment of leukocytes. Thus, GCs do not generally suppress macrophage effector functions, but they cause a shift in the innate–adaptive balance of the immune response, with distinct changes in the chemokine–chemokine receptor network.

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.

The gene expression profile induced by the CC chemokine ligand (CCL) 5/RANTES in human monocytes was examined using the oligonucleotide array technology. Of 5600 transcripts examined, 42 were consistently induced by CCL5, and none were suppressed. Chemokine-inducible transcripts could be clustered in functional groups, including selected cytokines and receptors (e.g., IL-1beta, CCL2/monocyte chemotactic protein-1, and the CCL5 receptor CCR1) and molecules involved in extracellular matrix recognition and digestion (e.g., CD44 splice transcripts, urokinase-type plasminogen activator receptor, matrix metalloprotease (MMP)-9, and MMP-19). Transcript expression, confirmed by quantitative real-time PCR analysis for selected genes, was associated with protein induction for some (e.g., CCL2), but not all (e.g., IL-1beta), transcripts examined. The chemokine-induced gene profile was distinct from that activated by LPS, a prototypic phagocyte activator. Although certain transcripts were stimulated by both agonists (e.g., IL-1beta and CCL2), others were induced only by either LPS (e.g., TNF-alpha and IL-6) or CCL5 (e.g., MMP-19) or were divergently regulated (e.g., CCR1). Thus, CCL5, a prototypic CC inflammatory chemokine, activates a restricted transcriptional program in monocytes distinct from that induced by the prototypic pathogen-derived proinflammatory stimulant LPS. Chemokine-induced chemokines production could represent a novel amplification loop of leukocyte recruitment, while a subset of chemokine-inducible transcripts could be involved in monocyte extravasation and tissue invasion.

To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D(3), structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase gamma was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.

IL-4 and IL-13 are prototypic Th2 cytokines that generate an "alternatively activated" phenotype in macrophages. We used high-density oligonucleotide microarrays to investigate the transcriptional profile induced in human monocytes by IL-13. After 8-h stimulation with IL-13, 142 genes were regulated (85 increased and 57 decreased). The majority of these genes were related to the inflammatory response and innate immunity; a group of genes related to lipid metabolism was also identified, with clear implications for atherosclerosis. In addition to characteristic markers of alternatively activated macrophages, a number of novel IL-13-regulated genes were seen. These included various pattern recognition receptors, such as CD1b/c/e, TLR1, and C-type lectin superfamily member 6. Several components of the IL-1 system were regulated. IL-1RI, IL-1RII, and IL-1Ra were all up-regulated, whereas the IL-1beta-converting enzyme, caspase 1, and IRAK-M were down-regulated. LPS-inducible caspase 1 enzyme activity was also reduced in IL-13-stimulated monocytes, with a consequent decrease in pro-IL-1beta processing. These data reveal that IL-13 has a potent effect on the transcriptional profile in monocytes. The IL-13-induced modulation of genes related to IL-1 clearly highlights the tightly controlled and complex levels of regulation of the production and response to this potent proinflammatory cytokine.

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

To develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES). Human embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. hES monocytes (esMCs) were analyzed by microscopy, flow cytometry, transcriptome analysis, and tested for the ability to differentiate into macrophages. hES monocyte–derived macrophages (esMDM) were analyzed for phagocytosis and endocytosis by microscopy and flow cytometry, cytokine secretion by multiplex cytokine assay, and for interferon (IFN)-γ and IL-4 activation by flow cytometry. Homogeneous esMCs (>90% CD14-positive) that did not require any additional purification steps were produced after 18.7 ± 7.7 days (mean ± SD, n = 19). Production continued for several months when growth factors were replaced, with a total yield of 3.4 × 105 ± 2.0 esMCs (mean ± SD, n = 9) per EB. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood–derived monocytes and spleen tissue samples but not with other tissue samples tested. We found that esMCs and esMDMs expressed well-defined markers of the mononuclear phagocyte system including PU-1, C/EBPα, EMR1, and EMR2, MPEG1, CD1c, CD4, CD18, CD32, CD33, CD68, cathepsins and serine carboxypeptidase. Finally, esMCs differentiated into functional macrophages that could endocytose acetylated low-density lipoprotein, phagocytose opsonized yeast particles, secrete specific cytokines in response to lipopolysaccharide, and be activated differentially with IFN-γ and IL-4. We have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology.

Macrophages are innate immune cells with well-established roles in the primary response to pathogens, but also in tissue homeostasis, coordination of the adaptive immune response, inflammation, resolution, and repair. These cells recognize danger signals through receptors capable of inducing specialized activation programs. The classically known macrophage activation is induced by IFN-gamma, which triggers a harsh proinflammatory response that is required to kill intracellular pathogens. Macrophages also undergo alternative activation by IL-4 and IL-13, which trigger a different phenotype that is important for the immune response to parasites. Here we review the cellular sources of these cytokines, receptor signaling pathways, and induced markers and gene signatures. We draw attention to discrepancies found between mouse and human models of alternative activation. The evidence for in vivo alternative activation of macrophages is also analyzed, with nematode infection as prototypic disease. Finally, we revisit the concept of macrophage activation in the context of the immune response.

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.

Macrophages are ubiquitous phagocytes that can constitute up to 15% of the cellular content of tissues. These heterogeneous cells of the innate immune system perform important functions during health and disease. Equipped with receptors for the T helper cell cytokines INF-γ and IL-4, macrophages undergo specific activation programs during Th1 or Th2 immune responses. These activation profiles, termed classical (M1) or alternative (M2) activation respectively, are further tuned by the presence and recognition of microbial-associated molecular patterns, other cytokines, lipids, and even adhesion to the substratum. The activation of macrophages also relies on the maturation background of the cells, elicitation of complicated intracellular signalling cascades, and the crosstalk between the different signalling elements. Of interest, not all genes participating in the activation-related signalling cascades are equally important for the elicitation of functional profiles and a regulator gene hierarchy is emerging for the different types of activation. In this issue of the European Journal of Immunology, two papers add to our understanding of how cellular kinases and phosphatases, related to the PI3K pathway, regulate M1 or M2 activation programmes in macrophages.

Macrophages are endowed with a variety of receptors for lineage-determining growth factors, T helper (Th) cell cytokines, and B cell, host, and microbial products. In tissues, macrophages mature and are activated in a dynamic response to combinations of these stimuli to acquire specialized functional phenotypes. As for the lymphocyte system, a dichotomy has been proposed for macrophage activation: classic vs. alternative, also M1 and M2, respectively. In view of recent research about macrophage functions and the increasing number of immune-relevant ligands, a revision of the model is needed. Here, we assess how cytokines and pathogen signals influence their functional phenotypes and the evidence for M1 and M2 functions and revisit a paradigm initially based on the role of a restricted set of selected ligands in the immune response.

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.

HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

BackgroundRecent investigation of human tissue and cells from positional tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of tendon disease. These mechanisms remain poorly understood in disease of energy-storing tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture.MethodsWe studied Achilles tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers.ResultsTendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring tendon cells.ConclusionsTissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles tendon disease.