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The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C . albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1 , is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C . albicans . This suggests that Candidalysin-induced cell damage is a third mechanism of C . albicans -mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae. Phagocytic cells of the innate immune system play critical roles in defence against invading pathogens including the opportunistic pathogen Candida albicans . Here the authors show that C . albicans derived Candidalysin in addition to being a cell-damaging toxin to mononuclear phagocytes is a trigger of NLRP3 inflammasome activation in these cells.

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host’s immune system. Here, we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections. The discovery of antibiotic drugs, which treat diseases caused by bacteria, has been a hugely valuable advance in modern medicine. They work by targeting specific cellular processes in bacteria, ultimately stopping them from multiplying or killing them outright. Antibiotics sometimes also affect their human hosts and can cause side-effects, such as gut problems or skin reactions. Recent evidence suggests that antibiotics also have an impact on the human immune system. This may happen either indirectly, by affecting ‘friendly’ bacteria normally present in the body, or through direct effects on immune cells. In turn, this could change the effectiveness of drug treatments. For example, if an antibiotic weakens immune cells, the body could have difficulty fighting off the existing infection – or become more vulnerable to new ones. However, even though new drugs are being introduced to combat the worldwide rise of antibiotic-resistant bacteria, their effects on immunity are still not well understood. For example, bedaquiline is an antibiotic recently developed to treat tuberculosis infections that are resistant to several drugs. Giraud-Gatineau et al. wanted to determine if bedaquiline altered the human immune response to bacterial infection independently from its direct anti-microbial effects. Macrophages engulf foreign particles like bacteria and break them down using enzymes stored within small internal compartments, or ‘lysosomes’. Initial experiments using human macrophages, grown both with and without bedaquiline, showed that the drug did not harm the cells and that they grew normally. A combination of microscope imaging and genetic analysis revealed that exposure to bedaquiline not only increased the number of lysosomes within macrophage cells, but also the activity of genes and proteins that increase lysosomes’ ability to break down foreign particles. These results suggested that bedaquiline treatment might make macrophages better at fighting infection, even if the drug itself had no direct effect on bacterial cells. Further studies, where macrophages were first treated with bedaquiline and then exposed to different types of bacteria known to be resistant to the drug, confirmed this hypothesis: in every case, the treated macrophages became efficient bacterial killers. In contrast, older anti-tuberculosis drugs did not have any such potentiating effect on the macrophages. This work sheds new light on our how antibiotic drugs can interact with the cells of the human immune system, and can sometimes even boost our innate defences. Such immune-boosting effects could one day be exploited to make more effective treatments against bacterial infections.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages ofmalaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Phagocytosis is a cellular process for ingesting and eliminating particles larger than 0.5 μm in diameter, including microorganisms, foreign substances, and apoptotic cells. Phagocytosis is found in many types of cells and it is, in consequence an essential process for tissue homeostasis. However, only specialized cells termed professional phagocytes accomplish phagocytosis with high efficiency. Macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts are among these dedicated cells. These professional phagocytes express several phagocytic receptors that activate signaling pathways resulting in phagocytosis. The process of phagocytosis involves several phases: i) detection of the particle to be ingested, ii) activation of the internalization process, iii) formation of a specialized vacuole called phagosome, and iv) maturation of the phagosome to transform it into a phagolysosome. In this review, we present a general view of our current understanding on cells, phagocytic receptors and phases involved in phagocytosis.

Key Points Neutrophils, monocytes and macrophages arise from common precursors, explaining much of their functional and phenotypical overlap. However, these cells also have complementary functions during the onset and resolution of inflammation. After sensing alarming stimuli, tissue-resident macrophages and sentinel monocytes promote an inflammatory response through release of chemokines (such as CXC-chemokine ligand 1 (CXCL1), CXCL2 and CXCL8) and pro-inflammatory lipid mediators (such as leukotrienes). Neutrophils are the 'advance guard' for inflammatory monocytes. Neutrophil-derived granule proteins (such as azurocidin and LL-37) and neutrophil protease-driven alterations of the local chemokine network promote rapid infiltration of monocytes to sites of inflammation. Monocyte-derived macrophages and apoptotic neutrophils abrogate further neutrophil influx by secreting anti-inflammatory lipid-mediators (such as lipoxins) or inactivating chemokines. Neutrophils undergoing apoptosis release signals that attract monocytes to the site of inflammation. Phagocytic clearance of apoptotic neutrophils reprogrammes monocyte-derived macrophages from a pro-inflammatory to an anti-inflammatory phenotype. The interplay between phagocyte subsets during the onset and resolution of inflammation could be targeted for therapeutic purposes. Potential targets for intervention include neutrophil-derived granule proteins, neutrophil apoptosis, or chemokines and lipid-mediators released from different phagocyte subsets. This Review article discusses how the interaction of neutrophils, monocytes and macrophages is important for both initiating and terminating an inflammatory response. The authors describe the phagocyte-derived mediators involved in these interactions and highlight the therapeutic potential of targeting them. Neutrophils, monocytes and macrophages are closely related phagocytic cells that cooperate during the onset, progression and resolution of inflammation. This Review highlights the mechanisms involved in the intimate partnership of phagocytes during each progressive phase of the inflammatory response. We describe how tissue-resident macrophages recognize tissue damage to promote the recruitment of neutrophils and the mechanisms by which infiltrating neutrophils can then promote monocyte recruitment. Furthermore, we discuss the phagocyte-derived signals that abrogate neutrophil recruitment and how the uptake of apoptotic neutrophils by macrophages leads to termination of the inflammatory response. Finally, we highlight the potential therapeutic relevance of these interactions.

Apoptotic intestinal epithelial cells can be sampled by lamina propria phagocytes, leading to distinct phagocyte-type-specific anti-inflammatory gene signatures and dendritic-cell-mediated induction of regulatory T cells. Apoptotic cell phagocytosis in intestinal mucosa Rapid clearance of apoptotic cells can reduce inflammatory and autoimmune consequences of their disintegration. Its disruption can trigger immune responses against molecular components and self-antigens released from degenerating apoptotic cells. Magarian Blander and colleagues show here in a mouse model that clearance of apoptotic epithelial cells by intestinal phagocytes (dendritic cells and two types of macrophages) results in the upregulation of distinct cell-type-specific anti-inflammatory gene signatures, and dendritic-cell-mediated induction of regulatory T cells. Some of the induced genes overlap with susceptibility genes for inflammatory bowel disease. Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies 1 , 2 . The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ . The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions 3 , are not merely extruded to maintain homeostatic cell numbers 4 , but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria 5 , 6 . Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4 + T-cell activation. A common ‘suppression of inflammation’ signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4 + T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease 7 . Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine. Synthetic biology can be used to create rationally designed living therapeutics. Here the authors engineer E. coli Nissle to target STING activation in antigen presenting cells for the treatment of solid tumors and demonstrate preclinical activity in murine models.

Active elimination of apoptotic cells is very important for maintaining homeostasis of early embryos. Recent observations on mouse blastocysts freshly isolated from healthy dams have shown that the majority of incidentally occurring apoptotic cells is eliminated by neighbouring embryonic cells. Some apoptotic cells escape phagocytosis, but the frequency of such processes usually does not exceed 10%. The aim of the current study was to evaluate whether the non-professional embryonic phagocytes can respond to experimentally induced increase in apoptosis by increasing the frequency of efferocytosis and whether their activity can be decreased by selective inhibition of specific component of efferoctosis machinery. Experiments were performed in vitro on cultured mouse blastocysts with a differentiated trophectoderm and inner cell mass and on the human trophoblast cell line Ac-1M88. Samples were assessed using fluorescence immunostaining: Apoptotic cells (TUNEL) internalised within the cytoplasm of non-professional embryonic phagocytes (phalloidin T membrane staining) were considered ingested; apoptotic cells co-localised with acidified phagosomes (LysoTracker) were considered digested. First, we tested the ability of embryonic phagocytes to respond to elevated incidence of apoptosis induced by actinomycin D (4 nM). The results showed that the increase in apoptosis was accompanied by a significant elevation of the phagocytosis and digestion of dead cells in both mouse blastocysts and human trophoblast cells. We then assessed the effect of selective inhibition of lysosomal acidification in embryonic phagocytes using a specific V-ATPase inhibitor bafilomycin A1. The results showed that the inhibitor at 0.1 and 0.2 nM was able to negatively affect the execution of both initiative and terminal phases of efferocytosis in mouse blastocysts, although the decrease was not as profound as expected. When compared to mouse trophectoderm cells, human hybrid cells displayed a very low sensitivity to bafilomycin A1. Higher concentrations of bafilomycin A1 had a more harmful impact on overall cell viability than on digestive activity. The results show that the ability of non-professional embryonic phagocytes to successfully execute all stages of efferocytosis is not limited by the frequency of apoptosis and is preserved even at elevated rates of the apoptotic process. The effectiveness of embryonic phagocytes can be partially decreased by selective inhibition of lysosomal acidification conducted via V-ATPase.

Apoptosis: how cells become targets Apoptosis occurs in essentially all tissues as part of normal development and homeostasis. Yet even in tissues with high cellular turnover, apoptotic cells are rarely seen; this has been attributed to the ability of apoptotic cells to advertise their presence via release of 'find-me' signals to recruit phagocytes and initiate prompt clearance. It has been unclear, however, what type of find-me signals are released by apoptotic cells and how these are sensed by phagocytes. In this paper apoptotic cells are shown to release ATP and UTP that act as a 'find me ' signal and chemoattractant for phagocytes expressing the P2Y 2 ATP/UTP receptor. The efficient removal of apoptotic cells in vivo is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes. Here, the caspase-dependent release of ATP and UTP during the early stages of apoptosis is demonstrated. ATP and UTP are found to act as chemoattractants in a process mediated through the ATP/UTP receptor P2Y 2 , which is present on monocytes and macrophages. Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo . We then identified the ATP/UTP receptor P2Y 2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y 2 -null mice 3 . The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y 2 -/- (also known as P2ry2 -/- ) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y 2 -/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y 2 -dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo .