Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting.

Cardiac exophers are membrane-bound extracellular vesicles released by cardiomyocytes with varied content and an average diameter of 3.5 µm. Here, we provide a detailed protocol to enable the identification and purification of cardiomyocyte-derived exophers by using fluorescence-activated cell sorting for downstream cellular and molecular analysis. This protocol requires the use of mouse strains expressing fluorescent proteins in cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Nicolás-Ávila et al. (2020).

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Mitochondrial Adaptations in the Growing Heart.

The heart pumps blood throughout the whole life of an organism, without rest periods during which to replenish energy or detoxify. Hence, cardiomyocytes, the working units of the heart, have mechanisms to ensure constitutive production of energy and detoxification to preserve fitness and function for decades. Even more challenging, the heart must adapt to the varying conditions of the organism from fetal life to adulthood, old age, and pathological stress. Mitochondria are at the nexus of these processes by producing not only energy but also metabolites and oxidative byproducts that can activate alarm signals and be toxic to the cell. We review basic concepts about cardiac mitochondria with a focus on their remarkable adaptations, including elimination, throughout the mammalian lifetime.

Locally renewing resident synovial macrophages provide a protective barrier for the joint.

Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1- mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease.

Specialized functions of resident macrophages in brain and heart.

The functions of macrophages in healthy tissues extend beyond their well-established roles as immune sentinels and effectors. Among tissues, cells of the brain and heart possess unique excitatory properties that likely demand special support. Accordingly, existing evidence demonstrates that microglia in the brain has an active role in synaptic organization, control of neuronal excitability, phagocytic removal of debris, and trophic support during brain development. In the heart, recent studies suggest that cardiac macrophages are involved in the regulation of heart homeostasis by phagocytosis, production of trophic, and immune-related factors, and by forming direct contacts with cardiomyocytes to regulate electrical conduction. In this review, we discuss mechanisms associated with the high degree of specialization of resident macrophages in both tissues, their origin and heterogeneity, and their contributions in regulating homeostasis under steady-state and pathological conditions.

Neutrophils in Homeostasis, Immunity, and Cancer.

Neutrophils were among the first leukocytes described and visualized by early immunologists. Prominent effector functions during infection and sterile inflammation classically placed them low in the immune tree as rapid, mindless aggressors with poor regulatory functions. This view is currently under reassessment as we uncover new aspects of their life cycle and identify transcriptional and phenotypic diversity that endows them with regulatory properties that extend beyond their lifetime in the circulation. These properties are revealing unanticipated roles for neutrophils in supporting homeostasis, as well as complex disease states such as cancer. We focus this review on these emerging functions in order to define the true roles of neutrophils in homeostasis, immunity, and disease.