Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay.

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.

Lung-resident alveolar macrophages regulate the timing of breast cancer metastasis.

Breast disseminated cancer cells (DCCs) can remain dormant in the lungs for extended periods, but the mechanisms limiting their expansion are not well understood. Research indicates that tissue-resident alveolar macrophages suppress breast cancer metastasis in lung alveoli by inducing dormancy. Through ligand-receptor mapping and intravital imaging, it was found that alveolar macrophages express transforming growth factor (TGF)-ß2. This expression, along with persistent macrophage-cancer cell interactions via the TGF-ßRIII receptor, maintains cancer cells in a dormant state. Depleting alveolar macrophages or losing the TGF-ß2 receptor in cancer cells triggers metastatic awakening. Aggressive breast cancer cells are either suppressed by alveolar macrophages or evade this suppression by avoiding interaction and downregulating the TGF-ß2 receptor. Restoring TGF-ßRIII in aggressive cells reinstates TGF-ß2-mediated macrophage growth suppression. Thus, alveolar macrophages act as a metastasis immune barrier, and downregulation of TGF-ß2 signaling allows cancer cells to overcome macrophage-mediated growth suppression.

Surprises from Intravital Imaging of the Innate Immune Response.

Successful immune responses depend on the spatiotemporal coordination of immune cell migration, interactions, and effector functions in lymphoid and parenchymal tissues. Real-time intravital microscopy has revolutionized our understanding of the dynamic behavior of many immune cell types in the living tissues of several species. Observing immune cells in their native environment has revealed many unanticipated facets of their biology, which were not expected from experiments outside a living organism. Here we highlight both classic and more recent examples of surprising discoveries that critically relied on the use of live in vivo imaging. In particular, we focus on five major cell types of the innate immune response (macrophages, microglia, neutrophils, dendritic cells, and mast cells), and how studying their dynamics in mouse tissues has helped us advance our current knowledge of immune cell-mediated tissue homeostasis, host defense, and inflammation.

Resident Macrophages Cloak Tissue Microlesions to Prevent Neutrophil-Driven Inflammatory Damage.

Neutrophils are attracted to and generate dense swarms at sites of cell damage in diverse tissues, often extending the local disruption of organ architecture produced by the initial insult. Whether the inflammatory damage resulting from such neutrophil accumulation is an inescapable consequence of parenchymal cell death has not been explored. Using a combination of dynamic intravital imaging and confocal multiplex microscopy, we report here that tissue-resident macrophages rapidly sense the death of individual cells and extend membrane processes that sequester the damage, a process that prevents initiation of the feedforward chemoattractant signaling cascade that results in neutrophil swarms. Through this "cloaking" mechanism, the resident macrophages prevent neutrophil-mediated inflammatory damage, maintaining tissue homeostasis in the face of local cell injury that occurs on a regular basis in many organs because of mechanical and other stresses. VIDEO ABSTRACT.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Dynamic landscapes and protective immunity coordinated by influenza-specific lung-resident memory CD8+ T cells revealed by intravital imaging.

Lung-tissue-resident memory (TRM) CD8+ T cells are critical for heterosubtypic immunity against influenza virus (IAV) reinfection. How TRM cells surveil the lung, respond to infection, and interact with other cells remains unresolved. Here, we used IAV infection of mice in combination with intravital and static imaging to define the spatiotemporal dynamics of lung TRM cells before and after recall infection. CD69+CD103+ TRM cells preferentially localized to lung sites of prior IAV infection, where they exhibited patrolling behavior. After rechallenge, lung TRM cells formed tight clusters in an antigen-dependent manner. Transcriptomic analysis of IAV-specific TRM cells revealed the expression of several factors that regulate myeloid cell biology. In vivo rechallenge experiments demonstrated that protection elicited by TRM cells is orchestrated in part by interferon (IFN)-γ-mediated recruitment of inflammatory monocytes into the lungs. Overall, these data illustrate the dynamic landscapes of CD103+ lung TRM cells that mediate early protective immunity against IAV infection.

T lymphocyte recruitment to melanoma brain tumors depends on distinct venous vessels.

To improve immunotherapy for brain tumors, it is important to determine the principal intracranial site of T cell recruitment from the bloodstream and their intracranial route to brain tumors. Using intravital microscopy in mouse models of intracranial melanoma, we discovered that circulating T cells preferably adhered and extravasated at a distinct type of venous blood vessel in the tumor vicinity, peritumoral venous vessels (PVVs). Other vascular structures were excluded as alternative T cell routes to intracranial melanomas. Anti-PD-1/CTLA-4 immune checkpoint inhibitors increased intracranial T cell motility, facilitating migration from PVVs to the tumor and subsequently inhibiting intracranial tumor growth. The endothelial adhesion molecule ICAM-1 was particularly expressed on PVVs, and, in samples of human brain metastases, ICAM-1 positivity of PVV-like vessels correlated with intratumoral T cell infiltration. These findings uncover a distinct mechanism by which the immune system can access and control brain tumors and potentially influence other brain pathologies.

Positional Stability and Membrane Occupancy Define Skin Fibroblast Homeostasis In Vivo.

Fibroblasts are an essential cellular and structural component of our organs. Despite several advances, the critical behaviors that fibroblasts utilize to maintain their homeostasis in vivo have remained unclear. Here, by tracking the same skin fibroblasts in live mice, we show that fibroblast position is stable over time and that this stability is maintained despite the loss of neighboring fibroblasts. In contrast, fibroblast membranes are dynamic during homeostasis and extend to fill the space of lost neighboring fibroblasts in a Rac1-dependent manner. Positional stability is sustained during aging despite a progressive accumulation of gaps in fibroblast nuclei organization, while membrane occupancy continues to be maintained. This work defines positional stability and cell occupancy as key principles of skin fibroblast homeostasis in vivo, throughout the lifespan of mice, and identifies membrane extension in the absence of migration as the core cellular mechanism to carry out these principles.

Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

BCL6 controls contact-dependent help delivery during follicular T-B cell interactions.

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones.

Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone.

iNKT Cells Orchestrate a Switch from Inflammation to Resolution of Sterile Liver Injury.

After traumatic injury, some cells function as detectors to sense injury and to modulate the local immune response toward a restitution phase by affecting the local cytokine milieu. Using intravital microscopy, we observed that patrolling invariant natural killer T (iNKT) cells were initially excluded from a site of hepatic injury but subsequently were strategically arrested first via self-antigens and then by cytokines, circumscribing the injured site at exactly the location where monocytes co-localized and hepatocytes proliferated. Activation of iNKT cells by self-antigens resulted in the production of interleukin-4 (IL-4) but not interferon-γ (IFN-γ). This promoted increased hepatocyte proliferation, monocyte transition (from Ly6Chi to Ly6Clo), and improved healing where IL-4 from iNKT cells was critical for these processes. Disruption of any of these mechanisms led to delayed wound healing. We have shown that self-antigen-driven iNKT cells function as sensors and orchestrators of the transformation from inflammation to tissue restitution for essential timely wound repair.

Visualizing the activation of encephalitogenic T cells in the ileal lamina propria by in vivo two-photon imaging.

Autoreactive encephalitogenic T cells exist in the healthy immune repertoire but need a trigger to induce CNS inflammation. The underlying mechanisms remain elusive, whereby microbiota were shown to be involved in the manifestation of CNS autoimmunity. Here, we used intravital imaging to explore how microbiota affect the T cells as trigger of CNS inflammation. Encephalitogenic CD4+ T cells transduced with the calcium-sensing protein Twitch-2B showed calcium signaling with higher frequency than polyclonal T cells in the small intestinal lamina propria (LP) but not in Peyer's patches. Interestingly, nonencephalitogenic T cells specific for OVA and LCMV also showed calcium signaling in the LP, indicating a general stimulating effect of microbiota. The observed calcium signaling was microbiota and MHC class II dependent as it was significantly reduced in germfree animals and after administration of anti-MHC class II antibody, respectively. As a consequence of T cell stimulation in the small intestine, the encephalitogenic T cells start expressing Th17-axis genes. Finally, we show the migration of CD4+ T cells from the small intestine into the CNS. In summary, our direct in vivo visualization revealed that microbiota induced T cell activation in the LP, which directed T cells to adopt a Th17-like phenotype as a trigger of CNS inflammation.

Imaging viral infection in vivo to gain unique perspectives on cellular antiviral immunity.

The past decade has seen near continual global public health crises caused by emerging viral infections. Extraordinary increases in our knowledge of the mechanisms underlying successful antiviral immune responses in animal models and during human infection have accompanied these viral outbreaks. Keeping pace with the rapidly advancing field of viral immunology, innovations in microscopy have afforded a previously unseen view of viral infection occurring in real-time in living animals. Here, we review the contribution of intravital imaging to our understanding of cell-mediated immune responses to viral infections, with a particular focus on studies that visualize the antiviral effector cells responding to infection as well as virus-infected cells. We discuss methods to visualize viral infection in vivo using intravital microscopy (IVM) and significant findings arising through the application of IVM to viral infection. Collectively, these works underscore the importance of developing a comprehensive spatial understanding of the relationships between immune effectors and virus-infected cells and how this has enabled unique discoveries about virus/host interactions and antiviral effector cell biology.

Heterogeneity in antiviral B cell responses: Lessons from the movies.

Humoral and cellular responses to viral infections coexist in a dynamic equilibrium that often results in efficient viral clearance. However, in some infections one of the two responses prevails, for instance when an overactivation of cytotoxic T cells is accompanied by weak and insufficient antibody responses. Although the cellular response is usually sufficient to control a primary viral infection, in some cases clearance is not complete and persistent infections ensue. In order to design effective therapeutic or vaccination strategies aiming at inducing early and potent neutralizing antibody responses, a deep knowledge of the cellular and molecular determinants of antiviral immune responses is needed. Here, we review our understanding on the spatiotemporal dynamics of antiviral humoral immune responses, with a particular focus on recent studies using intravital imaging approaches as an insightful complement to more traditional techniques.

Imaging the different timescales of germinal center selection.

Germinal centers (GCs) are the site of antibody affinity maturation, a fundamental immunological process that increases the potency of antibodies and thereby their ability to protect against infection. GC biology is highly dynamic in both time and space, making it ideally suited for intravital imaging. Using multiphoton laser scanning microscopy (MPLSM), the field has gained insight into the molecular, cellular, and structural changes and movements that coordinate affinity maturation in real time in their native environment. On the other hand, several limitations of MPLSM have had to be overcome to allow full appreciation of GC events taking place across different timescales. Here, we review the technical advances afforded by intravital imaging and their contributions to our understanding of GC biology.

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo.

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B-cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL-tumor contacts in vivo. In the therapeutic settings of anti-CD19 CAR T cells, the majority of CAR T cell-tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti-tumor responses in vivo.

Intravital microscopy visualizes innate immune crosstalk and function in tissue microenvironment.

Significant advances have been made in the field of intravital microscopy (IVM) on myeloid cells due to the growing number of validated fluorescent probes and reporter mice. IVM provides a visualization platform to directly observe cell behavior and deepen our understanding of cellular dynamics, heterogeneity, plasticity, and cell-cell communication in native tissue environments. This review outlines the current studies on the dynamic interaction and function of innate immune cells with a focus on those that are studied with IVM and covers the advances in data analysis with emerging artificial intelligence-based algorithms. Finally, the prospects of IVM on innate immune cells are discussed.

Spatial diversity of in vivo tissue immunity.

The immune system exhibits spatial diversity in in vivo tissues. Immune cells are strategically distributed within tissues to maintain the organ integrity. Advanced technologies such as intravital imaging and spatial transcriptomics have revealed the spatial heterogeneity of immune cell distribution and function within organs such as the liver, kidney, intestine, and lung. In addition, these technologies visualize nutrient and oxygen environments across tissues. Recent spatial analyses have suggested that a functional immune niche is determined by interactions between immune and non-immune cells in an appropriate nutrient and oxygen environment. Understanding the spatial communication between immune cells, environment, and surrounding non-immune cells is crucial for developing strategies to control immune responses and effectively manage inflammatory diseases.