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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, CX CR1. 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 CX CR1, 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.

Key Points Different immune cell subsets use diverse metabolic pathways. In general, inflammatory and suppressive cells each utilize glycolysis and oxidative phosphorylation for distinct purposes. Mitochondrial metabolism produces a variety of signalling molecules (such as mitochondrial reactive oxygen species (mROS) and acetyl-CoA) that can drive changes in immune cell function through the regulation of transcription factors and epigenetics. mROS are produced by the mitochondrial electron transport chain as a signal to increase interleukin-2 (IL-2) production in T cells and IL-1β production in macrophages. Acetyl-CoA produced by fatty acid oxidation or pyruvate oxidation in mitochondria can be transported by the citrate shuttle into the cytoplasm, where it can be used for fatty acid synthesis or acetylation reactions. These pathways have crucial roles in immune cell function. M1 macrophages use an altered tricarboxylic acid (TCA) cycle and reverse electron transport to drive inflammation through increased succinate and mROS levels. M2 macrophages have an intact TCA cycle and require the function of the hexosamine branch of glycolysis. Cellular metabolism can be altered by drugs that target mitochondria, such as metformin and mitochondria-targeted antioxidants. Does mitochondrial metabolism simply support the bioenergetic and biosynthetic needs of committed immune cells, or does it also control their fate? In this Review, Chandel and colleagues explore variations in mitochondrial metabolism across different immune cells and discuss how mitochondria can act as important signalling organelles to dictate immune cell function. Mitochondria are important signalling organelles, and they dictate immunological fate. From T cells to macrophages, mitochondria form the nexus of the various metabolic pathways that define each immune cell subset. In this central position, mitochondria help to control the various metabolic decision points that determine immune cell function. In this Review, we discuss how mitochondrial metabolism varies across different immune cell subsets, how metabolic signalling dictates cell fate and how this signalling could potentially be targeted therapeutically.

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.

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.

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.

The California purple sea urchin, , relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the ( ) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine . A recombinant (r)SpTrf-E1-Ec protein produced by also binds but does not augment phagocytosis. To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.

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.