Strategies of neutrophil diversification.

Neutrophils are formidable defenders. Their vast numbers, constant production, high cytotoxicity and capacity to produce extracellular traps, underlie their ability to efficiently protect in a microorganism-rich world. However, neutrophils are much more than immune sentinels, as evidenced by the expanding repertoire of functions discovered in the context of tissue homeostasis, regeneration or chronic pathologies. In this Perspective, we discuss general functional features of the neutrophil compartment that may be relevant in most, if not all, physiological scenarios in which they participate, including specialization in naïve tissues, transcriptional noise in the bloodstream as a potential strategy for diversification and functional bias in inflammatory sites. We intentionally present the reader with more questions than answers and propose models and approaches that we hope will shed new light onto the biology of these fascinating cells and spark new directions of research.

Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

Early production of IL-17A by γδ T cells in the trachea promotes viral clearance during influenza infection in mice.

The innate immune response generated against influenza infection is critical for the inhibition of viral dissemination. The trachea contains different types of innate immune cells that protect the respiratory tract from pathogen invasion. Among them, γδ T cells have the ability to rapidly generate large amounts of pro-inflammatory cytokines to preserve mucosal barrier homeostasis during infection. However, little is known about their role during the early phase of influenza infection in the airways. In this study, we found that, early after infection, γδ T cells are recruited and activated in the trachea and outnumber αß T cells during the course of the influenza infection that follows. We also showed that the majority of the recruited γδ T cells express the Vγ4 TCR chain and infiltrate in a process that involves the chemokine receptor CXCR3. In addition, we demonstrated that γδ T cells promote the recruitment of protective neutrophils and NK cells to the tracheal mucosa. Altogether, our results highlight the importance of the immune responses mediated by Î³Î´ T cells.

Variable selection for nonlinear dimensionality reduction of biological datasets through bootstrapping of correlation networks.

Identifying the most relevant variables or features in massive datasets for dimensionality reduction can lead to improved and more informative display, faster computation times, and more explainable models of complex systems. Despite significant advances and available algorithms, this task generally remains challenging, especially in unsupervised settings. In this work, we propose a method that constructs correlation networks using all intervening variables and then selects the most informative ones based on network bootstrapping. The method can be applied in both supervised and unsupervised scenarios. We demonstrate its functionality by applying Uniform Manifold Approximation and Projection for dimensionality reduction to several high-dimensional biological datasets, derived from 4D live imaging recordings of hundreds of morpho-kinetic variables, describing the dynamics of thousands of individual leukocytes at sites of prominent inflammation. We compare our method with other standard ones in the field, such as Principal Component Analysis and Elastic Net, showing that it outperforms them. The proposed method can be employed in a wide range of applications, encompassing data analysis and machine learning.

Transformer-based spatial-temporal detection of apoptotic cell death in live-cell imaging.

Intravital microscopy has revolutionized live-cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy timelapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy timelapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in mice, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

ACME: Automatic feature extraction for cell migration examination through intravital microscopy imaging.

Cell detection and tracking applied to in vivo fluorescence microscopy has become an essential tool in biomedicine to characterize 4D (3D space plus time) biological processes at the cellular level. Traditional approaches to cell motion analysis by microscopy imaging, although based on automatic frameworks, still require manual supervision at some points of the system. Hence, when dealing with a large amount of data, the analysis becomes incredibly time-consuming and typically yields poor biological information. In this paper, we propose a fully-automated system for segmentation, tracking and feature extraction of migrating cells within blood vessels in 4D microscopy imaging. Our system consists of a robust 3D convolutional neural network (CNN) for joint blood vessel and cell segmentation, a 3D tracking module with collision handling, and a novel method for feature extraction, which takes into account the particular geometry in the cell-vessel arrangement. Experiments on a large 4D intravital microscopy dataset show that the proposed system achieves a significantly better performance than the state-of-the-art tools for cell segmentation and tracking. Furthermore, we have designed an analytical method of cell behaviors based on the automatically extracted features, which supports the hypotheses related to leukocyte migration posed by expert biologists. This is the first time that such a comprehensive automatic analysis of immune cell migration has been performed, where the total population under study reaches hundreds of neutrophils and thousands of time instances.

Circadian immune circuits.

Immune responses are gated to protect the host against specific antigens and microbes, a task that is achieved through antigen- and pattern-specific receptors. Less appreciated is that in order to optimize responses and to avoid collateral damage to the host, immune responses must be additionally gated in intensity and time. An evolutionary solution to this challenge is provided by the circadian clock, an ancient time-keeping mechanism that anticipates environmental changes and represents a fundamental property of immunity. Immune responses, however, are not exclusive to immune cells and demand the coordinated action of nonhematopoietic cells interspersed within the architecture of tissues. Here, we review the circadian features of innate immunity as they encompass effector immune cells as well as structural cells that orchestrate their responses in space and time. We finally propose models in which the central clock, structural elements, and immune cells establish multidirectional circadian circuits that may shape the efficacy and strength of immune responses and other physiological processes.

Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection.

Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nß neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nß/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nß neutrophils overexpress the α4ß1 integrin compared to Nα. Finally, by inhibiting α4ß1 integrin, we increase the Nß/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.

Immunity: Neutrophil Quorum at the Wound.

How is neutrophil swarming initiated after an injury? A new study provides evidence of exquisite coordination between these immune cells, akin to quorum sensing in unicellular microorganisms, to protect tissues from invading pathogens.

Two-Photon Intravital Imaging of Leukocytes in the Trachea During Pneumococcal Infection.

Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluorescent protein (LysM-GFP) mouse, in which neutrophils express GFP. Additionally, we used a custom-designed platform, which allowed the intubation and fixation of the trachea after surgical exposition, and we injected intravenously a fluorescently labeled dextran solution to visualize the blood vessels.

Characterization of the Dynamic Behavior of Neutrophils Following Influenza Vaccination.

Neutrophils are amongst the first cells to respond to inflammation and infection. Although they play a key role in limiting the dissemination of pathogens, the study of their dynamic behavior in immune organs remains elusive. In this work, we characterized in vivo the dynamic behavior of neutrophils in the mouse popliteal lymph node (PLN) after influenza vaccination with UV-inactivated virus. To achieve this, we used an image-based systems biology approach to detect the motility patterns of neutrophils and to associate them to distinct actions. We described a prominent and rapid recruitment of neutrophils to the PLN following vaccination, which was dependent on the secretion of the chemokine CXCL1 and the alarmin molecule IL-1α. In addition, we observed that the initial recruitment occurred mainly via high endothelial venules located in the paracortical and interfollicular regions of the PLN. The analysis of the spatial-temporal patterns of neutrophil migration demonstrated that, in the initial stage, the majority of neutrophils displayed a patrolling behavior, followed by the formation of swarms in the subcapsular sinus of the PLN, which were associated with macrophages in this compartment. Finally, we observed using multiple imaging techniques, that neutrophils phagocytize and transport influenza virus particles. These processes might have important implications in the capacity of these cells to present viral antigens.

Influenza Vaccination Induces NK-Cell-Mediated Type-II IFN Response that Regulates Humoral Immunity in an IL-6-Dependent Manner.

The role of natural killer (NK) cells in the immune response against vaccines is not fully understood. Here, we examine the function of infiltrated NK cells in the initiation of the inflammatory response triggered by inactivated influenza virus vaccine in the draining lymph node (LN). We observed that, following vaccination, NK cells are recruited to the interfollicular and medullary areas of the LN and become activated by type I interferons (IFNs) produced by LN macrophages. The activation of NK cells leads to their early production of IFNγ, which in turn regulates the recruitment of IL-6+ CD11b+ dendritic cells. Finally, we demonstrate that the interleukin-6 (IL-6)-mediated inflammation is important for the development of an effective humoral response against influenza virus in the draining LN.

Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1.

The early phase of influenza infection occurs in the upper respiratory tract and the trachea, but little is known about the initial events of virus recognition and control of viral dissemination by the immune system. Here, we report that inflammatory dendritic cells (IDCs) are recruited to the trachea shortly after influenza infection through type I interferon-mediated production of the chemokine CCL2. We further show that recruited IDCs express the C-type lectin receptor SIGN-R1, which mediates direct recognition of the virus by interacting with N-linked glycans present in glycoproteins of the virion envelope. Activation of IDCs via SIGN-R1 triggers the production of the chemokines CCL5, CXCL9 and CXCL10, which initiate the recruitment of protective natural killer (NK) cells in the infected trachea. In the absence of SIGN-R1, the recruitment and activation of NK cells is impaired, leading to uncontrolled viral proliferation. In sum, our results provide insight into the orchestration of the early cellular and molecular events involved in immune protection against influenza.

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal. Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

Leukocyte Tracking Database, a collection of immune cell tracks from intravital 2-photon microscopy videos.

Recent advances in intravital video microscopy have allowed the visualization of leukocyte behavior in vivo, revealing unprecedented spatiotemporal dynamics of immune cell interaction. However, state-of-the-art software and methods for automatically measuring cell migration exhibit limitations in tracking the position of leukocytes over time. Challenges arise both from the complex migration patterns of these cells and from the experimental artifacts introduced during image acquisition. Additionally, the development of novel tracking tools is hampered by the lack of a sound ground truth for algorithm validation and benchmarking. Therefore, the objective of this work was to create a database, namely LTDB, with a significant number of manually tracked leukocytes. Broad experimental conditions, sites of imaging, types of immune cells and challenging case studies were included to foster the development of robust computer vision techniques for imaging-based immunological research. Lastly, LTDB represents a step towards the unravelling of biological mechanisms by video data mining in systems biology.

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node.

The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-ß (IFN-ß) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b+ LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.