Phagocytes in Health and Disease

The intestine is the body’s largest surface exposed to the environment, which is rich in dietary and microbial stimuli for hematopoietic and non-hematopoietic systems. It hosts as many neurons (enteric-associated neurons, EANs) as the spinal cord and more immune cells than all other compartments together. EANs include intrinsic and extrinsic sensory neurons that control a variety of functions within the gastrointestinal tract. Both immune and nervous systems are equipped with sensing mechanisms that monitor the perturbations at the luminal surface. We have previously demonstrated that intestinal muscularis macrophages (MMs), which lie in close proximity to enteric neurons, are skewed towards a tissue-protective gene program upon enteric infection and that this is the direct result of extrinsic sympathetic neuron-derived norepinephrine signaling through adrenergic receptor beta 2 (β₂AR) on MMs. We addressed the functional consequences of this EAN-macrophage crosstalk using different models of enteric infections. We observed significant and prolonged neuronal loss and impaired neuronal function upon enteric infection-induced inflammation, symptomatic features that resemble post-infectious IBS. These changes were coupled with rapid alterations in MM-EAN positioning, gene expression and density. We found that these symptoms were ameliorated by triggering β₂AR signaling and aggravated in mice lacking β₂ARs in the myeloid compartment. These observations support a model in which enteric pathogens lead to activation of intestine extrinsic sympathetic innervation, triggering neuro-protection pathways on gut macrophages via β₂AR-dependent mechanisms. Overall, our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.

Barrier tissues are primary targets of environmental stressors and home to the largest number of antigen-experienced lymphocytes in the body, including commensal-specific T cells. Here, we show that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type-17 program associated with a poised type-2 state. Thus, in the context of injury and exposure to inflammatory mediators such as IL-18, these cells rapidly release type-2 cytokines, thereby acquiring contextual functions. Notably, such acquisition of a type-2 effector program promotes tissue repair. Aberrant type-2 responses can also be unleashed in the context of local defects in immunoregulation. Thus, commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury.

Coauthors: Jonathan L. Linehan[1,2], Han-Yu Shih[3], Nicolas Bouladoux[1], Seong-Ji Han[1], Margery Smelkinson[4], Shurjo K. Sen[5], Allyson L. Byrd[1,2], Michel Enamorado[1], Chen Yao[3], Samira Tamoutounour[1], Francois Van Laethem[6,7], Charlotte Hurabielle[1,8], Nicholas Collins[1], Andrea Paun[9], Rosalba Salcedo[10], John J. O’Shea[3], Yasmine Belkaid[1].

1. Metaorganism Immunology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

2. Department of Cancer Immunology, Genentech, South San Francisco.

3. Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland.

4. Biological Imaging, Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

5. Leidos Biomedical Research, Inc., Basic Science Program, Cancer and Inflammation Program, Frederick National Laboratory for Cancer Research, Bethesda, Maryland.

6. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

7. Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, Montpellier, France.

8. Inserm Unité 976, Hôpital Saint-Louis, Paris, France.

9. Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryand.

10. Cancer and Inflammation Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Macrophages are important cellular constituents of all tissues and organs. Phenotypic and functional heterogeneity (i.e. plasticity) of tissue macrophages is a prerequisite to promote homeostasis across various tissues and microenvironments. Macrophage plasticity also underlies the ability of macrophages to respond to changes in the microenvironment in ways that enables the tissue to fight infection or injury. In addition, macrophages are also critical for restoring the tissue back towards homoeostasis once injury or infection has subsided (referred to as repair or wound healing phenotype). Dysegulation of this functional plasticity is thought to play a central role in the pathogenesis of various chronic diseases. An extreme expression of this dysregulated macrophage phenotype is refractoriness of macrophages to respond to regulatory tissue factors. In such a scenario, macrophages remain arrested in a phenotype that perpetually promotes tissue injury and destruction. This complete abrogation of plasticity and subsequent failure to transition or revert back to a pro-homeostaic phenotype may underlie severe and fast progressing chronic conditions like pulmonary fibrosis. Novel therapeutic approaches must therefore aim at restoring macrophage plasticity to help resolve established fibrosis and abrogate chronic inflammation. Establishment of defined experimental assays that allow determining and manipulating discrete macrophage phenotypes during various stages of tissue injury is required to accelerate the discovery of more effective therapeutic targets. Here I will discuss the current hypotheses and experimental approaches that form a basis for the potential identification of tissue factors, inter-cellular communication pathways, and intra-cellular signaling mechanisms that regulate tissue macrophage plasticity in health and disease.

Focal exocytosis is the targeted delivery of membrane and/or cargo to specific regions on the plasma membrane which is essential for cellular processes such as cell division and migration. In immune cells, phagocytosis requires membrane addition for pseudopod extension and phagosome closure, making it a good model in which to study focal exocytosis. Protein kinase Cepsilon (PKC-ε) concentrates at the phagocytic cup and its presence is required for efficient phagocytosis. Our lab has reported that the PKC-ε that localizes at the phagosome originates from the Golgi and mediates membrane addition. How PKC-ε is transported from the Golgi and how it facilitates membrane addition is unknown. Using immunofluorescence, we are able to visualize the post-Golgi trafficking of PKC-ε.

Super-Resolution microscopy revealed that PKC-ε appear as puncta which align along microtubules and colocalize with vesicle marker Vamp3 and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). TIRF microscopy showed that PKC-ε positive vesicles fuse into the plasma membrane for membrane addition. In the absence of PKC-ε, vesicles are not delivered to the forming phagosome and membrane is not added. These data suggest that PKC-ε is transported to the phagosome on vesicles bearing VAMP3 and TNF-α and PKC-ε mediates their delivery and fusion. PKC-ε may be assisting in the targeting and phosphorylating proteins to promote vesicle fusion. Uncovering PKC-ε’s role in focal exocytosis will allow us to better understand its role in metastatic breast cancer and Alzheimer’s disease where PKC-ε is elevated and down-regulated, respectively.

This research is supported by NIH GM090325, an AMC Bridge Grant, and the Johnathan R. Vasiliou Foundation.

Coauthors: Alexander Wong, Cheryl M. Zajd, and Michelle R. Lennartz, Albany Medical College.

Autophagy is a conserved pathway by which cytosolic material is engulfed in a double membrane vesicle and targeted to the lysosome for degradation. Accumulating evidence support a central role for autophagy in balancing host defense and inflammation. Our group and others have shown that autophagy proteins are important in maintaining cellular and tissue homeostasis by targeting immune activators and dampening inflammatory responses. For example, through the removal and degradation of mitochondria, autophagy inhibits the production of reactive oxygen species and NLRP3 inflammasome activation.

Autophagy also functions as a cell autonomous defense mechanism by targeting internalized intracellular pathogens, a process known as xenophagy. The ability of autophagy to dampen immune reactions while also promoting antimicrobial immunity may explain the genetic association between a common polymorphism in the autophagy gene ATG16L1 (T300A allele) and susceptibility to Crohn’s Disease (CD), a major type of inflammatory bowel disease impacting over a million people world-wide. CD is considered a consequence of harmful immune responses to commensal bacteria in the gut that occurs in genetically susceptible individuals. The ATG16L1 T300A risk allele is present in 40–45% of population with up to 15% homozygosity, raising the possibility that this variant was maintained in the human population despite conferring susceptibility to a chronic inflammatory disorder of the gut. To better understand the link between autophagy and mucosal immunity in the gut, we examined the role of ATG16L1 during enteric bacterial infection. We found that mice harboring mutations in Atg16L1 are unexpectedly protected from disease following infection by two models of enteric pathogens, Citrobacter rodentium and Salmonella enterica Typhimurium. In both models we find an essential role for inflammatory monocytes and macrophages in mediating increased protection against these enteric pathogens.

Specifically, we find that monocytes in an autophagy deficient gut have enhanced function and wound healing capabilities. We will discuss our progress in understanding the mechanism of how these myeloid subsets mediate immunity to enteric infections in an autophagy deficient setting.

Coauthors: Samantha L. Schuster, Elisabeth Kernbauer, and Ken Cadwell, Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York.

Targeting of macrophage netrin-1 expression promotes plaque regression and resolution of chronic inflammation in atherosclerosis. Chronic inflammation in atherosclerosis is driven by the accumulation of cholesterol-laden macrophages in the arterial wall. These pro-inflammatory macrophages persist in this site, undergo apoptosis and show a defective efforcytosis. Thereby, these macrophages contribute to the necrotic core, and sustain local and systemic inflammation. Recently, we reported that the neuronal guidance protein netrin-1 plays a pivotal role in the retention of macrophages in plaques and the progression of atherosclerosis.

To test whether targeting netrin-1 in established atherosclerotic plaques could promote the resolution of chronic inflammation in atherosclerosis and/or induce plaque regression, we developed mice in which macrophage netrin-1 expression could be inducibly deleted by tamoxifen administration (Ntn1[fl/fl]CX3CR1[CreER2+] mice). We first induced atherosclerosis in Ntn1[fl/fl]CX3CR1[CreER2+] and control Ntn1[fl/fl]CX3CR1[CreER2−] using a recombinant AAV-vector overexpressing PSCK9 and Western diet feeding. After 20 weeks, 10 mice from each group were sacrificed for baseline plaque measurements, while the remaining mice were switched to chow diet to stop further progression of atherosclerosis and treated with tamoxifen (n=14/group). After 4 weeks, mice were sacrificed to assess the effects of macrophagespecific netrin-1 deletion on plaque size and composition. Analysis of peritoneal macrophages confirmed robust deletion of netrin-1 in tamoxifen treated Ntn1[fl/fl]CX3CR1[CreER2+] but not control Ntn1[fl/fl]CX3CR1[CreER2−] mice. Macrophage-specific deletion of netrin-1 caused a 30% reduction in plaque burden in the aortic arch as measured by en face analysis, compared to control mice. This immunohistochemical analysis of aortic root cross-section, we observed that macrophage specific netrin-1 deletion further resulted in a significant reduced macrophage content in the regressing plaque. These findings correlated with a reduced retention and proliferation of netrin-1 deficient macrophages.

To gain further inside into macrophage functions, we performed scRNAseq of the aortic arches and identified a monocyte and a distinct macrophage population in which specifically migratory and phagocytosis pathway were enhanced following netrin-1 deletion. Furthermore, deletion of macrophage netrin-1 expression was associated with a reduction of systemic IL-1β levels. Collectively, these data suggest that targeting macrophage expression of netrin-1 in established atherosclerosis fosters efferocytosis and egress in distinct monocyte and macrophage populations, who promote plaque regression and resolution of atherosclerosis.

Coauthors: Martin Schlegel, Monika Sharma, Milessa Afonso, Graeme Koelwyn, Emma Corr, Coen van Solingen, Emily Brown, Lauren Beckett, Lianne Shannely, and Kathryn Moore, Marc and Ruti Bell Vascular Biology and Disease Program, Leon H. Charney Division of Cardiology, Department of Medicine, New York University Medical Center.

The classical view of immune cells in peripheral nerves after injury is that macrophages infiltrate into the distal nerve and play a vital role in the clearance of myelin and axonal debris, a process known as Wallerian degeneration. Surprisingly we have found that in CCR2 −/− mice in which this infiltration does not occur Wallerian degeneration proceeds normally in the axotomized sciatic nerve. We have found that in the absence of these infiltrating macrophages, there is an increase in the expression of the neutrophil chemokines CXCL1 and CXCL2 and an increase in neutrophils in the distal nerve segment. Experiments using Ly6G to decrease neutrophils in the blood and distal nerve led to an inhibition of myelin clearance. In the 1990s we found that in addition to infiltrating the distal nerve, macrophages enter peripheral ganglia and surround the axotomized neuronal cell bodies. At first it was puzzling as to what these phagocytes might be doing in these ganglia. In CCL2 −/− mice in which infiltration into dorsal root ganglia (DRG) is blocked we found that the growth of injured sensory neurons in response to a conditioning lesion was blocked. Furthermore, overexpressing CCL2 in the DRG in intact animals led to macrophage accumulation in the ganglia and increased the growth capacity of the neurons. Thus, we find that phagocytes are involved in two aspects of nerve regeneration. First, they phagocytose axonal debris, thus removing molecules that are inhibitory to regeneration. Second, they stimulate the growth capacity of the injured neurons.

Elevated risk of developing Alzheimer’s disease (AD) is associated with hypomorphic variants of a surface receptor called triggering receptor expressed on myeloid cells 2 (TREM2). My laboratory originally cloned TREM2 and demonstrated that it is required for microglial responses to amyloid-b (Ab) plaques, including proliferation, survival, clustering and phagocytosis. How TREM2 promotes such diverse responses was unknown. Recently, we found that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA-seq linked this anomalous autophagy to defective mTOR signaling, which affects ATP levels and biosynthetic pathways. Thus, TREM2 is required to sustain the increased metabolic demands of microglia during responses to Ab plaques, while defective mTOR signaling in TREM2-deficient microglia is associated with a compensatory increase of autophagy in vitro and in vivo in AD.

Our studies show that, while increased autophagy may be beneficial in reducing inflammation and Aβ load in the short-term, a defect in mTOR signaling is detrimental and severely impairs microglia fitness and capacity to respond to Aβ accumulation in the long-term. Moreover, autophagy and metabolic derailment can be offset in vitro through creatine analogs that can supply ATP. Dietary creatine analogs can temper autophagy, restore microglial clustering around plaques, and decrease plaque-adjacent neuronal dystrophy in TREM2-deficient mice with Aβ pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic, biosynthetic metabolism and preventing prolonged autophagy.

A successful immune response involves the coordinated and sequential activation of host defense mechanisms, resolution of inflammation and restitutio ad integrum of the affected tissue. Macrophages are polyfunctional immune cells critical for initiation, resolution of inflammation, and consequent tissue repair. Upon damage, tissue-resident macrophages release inflammatory cytokines and chemokines, and recruit effectors such as neutrophils. Subsequently, macrophages at the damage site adopt a tissue repair profile. A set of diverse and unrelated cues has emerged as critical signals that induce the restorative function of macrophages. These range from the recognition of apoptotic cells to the sensing of cytokines, including IL-4. How such disparate signals, known to elicit distinct phenotypic responses, achieve a degree of functional congruence to induce the tissue repair program remains unknown. Here I will discuss recent findings on the integration of IL4 receptor activation with recognition of apoptotic cells for the induction of wound healing responses.

Successful anti-tumor immunotherapeutic approaches are improving survival in many malignancies but not all patients are responding to these interventions, and several also relapse.  This may result from an immunosuppressive tumor microenvironment (TME) that contributes towards tumor growth and metastasis while dampening anti-tumor immune responses.  Natural Killer cell (NK) and dendritic cells are key elements of the innate immune system that are necessary to initiate anti-tumor immunity.  NK effector functions are diminished during cancer but the array of inhibitory receptors that underlie this process are unclear. In contrast, dysfunctional T cells have been shown to upregulate inhibitory receptors such as CTLA-4 and PD-1 that mark a state of “exhaustion”, and inhibition of signaling through these molecules has improved clinical outcome in multiple cancers. We find that NK cells are subject to an analogous process of immune exhaustion in cancers.  We examined their phenotype, functional potential, and susceptibility to ex vivo checkpoint blockade in the peripheral blood and primary tumors of more than one hundred individuals with bladder cancer (BlCa). We found that both NK and T cells significantly upregulated the inhibitory receptors Tim-3 and TIGIT in peripheral blood and tumors and were dysfunctional,  suggesting implications for normal NK maturation. We have also identified a role of cDC1 in anti-tumor immunity in human tissues and discuss approaches by which DC modulation can enhance the response to vaccination therapies.