C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.

Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment.

The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we show that microbiota signals program mononuclear phagocytes in the TME toward immunostimulatory monocytes and dendritic cells (DCs). Single-cell RNA sequencing revealed that absence of microbiota skews the TME toward pro-tumorigenic macrophages. Mechanistically, we show that microbiota-derived stimulator of interferon genes (STING) agonists induce type I interferon (IFN-I) production by intratumoral monocytes to regulate macrophage polarization and natural killer (NK) cell-DC crosstalk. Microbiota modulation with a high-fiber diet triggered the intratumoral IFN-I-NK cell-DC axis and improved the efficacy of immune checkpoint blockade (ICB). We validated our findings in individuals with melanoma treated with ICB and showed that the predicted intratumoral IFN-I and immune compositional differences between responder and non-responder individuals can be transferred by fecal microbiota transplantation. Our study uncovers a mechanistic link between the microbiota and the innate TME that can be harnessed to improve cancer therapies.

IFNγ and GM-CSF control complementary differentiation programs in the monocyte-to-phagocyte transition during neuroinflammation.

During inflammation, Ly6Chi monocytes are rapidly mobilized from the bone marrow (BM) and are recruited into inflamed tissues, where they undergo monocyte-to-phagocyte transition (MTPT). The in vivo developmental trajectories of the MTPT and the contribution of individual cytokines to this process remain unclear. Here, we used a murine model of neuroinflammation to investigate how granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFNγ), two type 1 cytokines, controlled MTPT. Using genetic fate mapping, gene targeting and high-dimensional single-cell multiomics analyses, we found that IFNγ was essential for the gradual acquisition of a mature inflammatory phagocyte phenotype in Ly6Chi monocytes, while GM-CSF was required to license interleukin-1ß (IL-1ß) production, phagocytosis and oxidative burst. These results suggest that the proinflammatory cytokine environment guided MTPT trajectories in the inflamed central nervous system (CNS) and indicated that GM-CSF was the most prominent target for the disarming of monocyte progenies during neuroinflammation.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages.

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.

A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aß particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)-/- Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases. VIDEO ABSTRACT.

Human Adaptive Immunity Rescues an Inborn Error of Innate Immunity.

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.

Clearing Your Mind: Mechanisms of Debris Clearance After Cell Death During Neural Development.

Neurodevelopment and efferocytosis have fascinated scientists for decades. How an organism builds a nervous system that is precisely tuned for efficient behaviors and survival and how it simultaneously manages constant somatic cell turnover are complex questions that have resulted in distinct fields of study. Although neurodevelopment requires the overproduction of cells that are subsequently pruned back, very few studies marry these fields to elucidate the cellular and molecular mechanisms that drive nervous system development through the lens of cell clearance. In this review, we discuss these fields to highlight exciting areas of future synergy. We first review neurodevelopment from the perspective of overproduction and subsequent refinement and then discuss who clears this developmental debris and the mechanisms that control these events. We then end with how a more deliberate merger ofneurodevelopment and efferocytosis could reframe our understanding of homeostasis and disease and discuss areas of future study.

Ontogeny and function of murine epidermal Langerhans cells.

Langerhans cells (LCs) are epidermis-resident antigen-presenting cells that share a common ontogeny with macrophages but function as dendritic cells (DCs). Their development, recruitment and retention in the epidermis is orchestrated by interactions with keratinocytes through multiple mechanisms. LC and dermal DC subsets often show functional redundancy, but LCs are required for specific types of adaptive immune responses when antigen is concentrated in the epidermis. This Review will focus on those developmental and functional properties that are unique to LCs.

Living on the Edge: Efferocytosis at the Interface of Homeostasis and Pathology.

Nearly every tissue in the body undergoes routine turnover of cells as part of normal healthy living. The majority of these cells undergoing turnover die via apoptosis, and then are rapidly removed by phagocytes by the process of efferocytosis that is anti-inflammatory. However, a number of pathologies have recently been linked to defective clearance of apoptotic cells. Perturbed clearance arises for many reasons, including overwhelming of the clearance machinery, disruptions at different stages of efferocytosis, and responses of phagocytes during efferocytosis, all of which can alter the homeostatic tissue environment. This review covers linkages of molecules involved in the different phases of efferocytosis to disease pathologies that can arise due to their loss or altered function.

Cytokine Circuits in Cardiovascular Disease.

Arterial inflammation is a hallmark of atherosclerosis, and appropriate management of this inflammation represents a major unmet therapeutic need for cardiovascular disease patients. Here, we review the diverse contributions of immune cells to atherosclerosis, the mechanisms of immune cell activation in this context, and the cytokine circuits that underlie disease progression. We discuss the recent application of these insights in the form of immunotherapy to treat cardiovascular disease and highlight how studies on the cardiovascular co-morbidity that arises in autoimmunity might reveal additional roles for cytokines in atherosclerosis. Currently, data point to interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and IL-17 as cytokines that, at least in some settings, are effective targets to reduce cardiovascular disease progression.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

Microbiota-Induced TNF-like Ligand 1A Drives Group 3 Innate Lymphoid Cell-Mediated Barrier Protection and Intestinal T Cell Activation during Colitis.

Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1+MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

Single-cell RNA profiling reveals classification and characteristics of mononuclear phagocytes in colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer mortality and a serious health problem worldwide. Mononuclear phagocytes are the main immune cells in the tumor microenvironment of CRC with remarkable plasticity, and current studies show that macrophages are closely related to tumor progression, invasion and dissemination. To understand the immunological function of mononuclear phagocytes comprehensively and deeply, we use single-cell RNA sequencing and classify mononuclear phagocytes in CRC into 6 different subsets, and characterize the heterogeneity of each subset. We find that tissue inhibitor of metalloproteinases (TIMPs) involved in the differentiation of proinflammatory and anti-inflammatory mononuclear phagocytes. Trajectory of circulating monocytes differentiation into tumor-associated macrophages (TAMs) and the dynamic changes at levels of transcription factor (TF) regulons during differentiation were revealed. We also find that C5 subset, characterized by activation of lipid metabolism, is in the terminal state of differentiation, and that the abundance of C5 subset is negatively correlated with CRC patients' prognosis. Our findings advance the understanding of circulating monocytes' differentiation into macrophages, identify a new subset associated with CRC prognosis, and reveal a set of TF regulons regulating mononuclear phagocytes differentiation, which are expected to be potential therapeutic targets for reversing immunosuppressive tumor microenvironment.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.