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.This work has been partially supported by the National Grant TEC2017-84395-P of the Spanish Ministry of Economy and Competitiveness, Madrid Regional Government and Universidad Carlos III de Madrid through the project SHARON-CM-UC3M, RTI2018- 095497-B-I00 from Ministerio de Ciencia e Innovación (MICINN) and HR17_00527 from Fundación La Caixa to A.H. M.M-M. is supported by the Spanish Ministry of Education, Culture and Sports FPU Grant FPU18/02825. M.P-S. is supported by a Federation of European Biochemical Societies long-term fellowship. J.S. is supported by a fellowship (PRE2019-089130) from MICINN.S

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

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 Nβ 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 Nβ/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 Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/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.Peer reviewe

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-β (IFN-β) 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

Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

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.M. Palomino-Segura is supported by a Federation of European Biochemical Societies long-term fellowship. A. Hidalgo is supported by the Ministerio de Ciencia e Innovacion (RTI2018-095497-B-I00), Fundación La Caixa (HR17_00527), and the Leducq Foundation Transatlantic Network of Excellence (TNE-18CVD04). The Centro Nacional de Investigaciones Cardiovasculares Carlos III is supported by the Ministerio de Ciencia e Innovacion and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (Ministerio de Ciencia e Innovacion award SEV-2015-0505).S

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.S

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.We thank members of the laboratory of A.H., P. Frenette, D. Lucas, L. Ng, G. Fernandez-Calvo, F. Sanchez-Cabo, M. Casanova-Acebes and J. M. Adrover for past and present discussion and inspiration on questions presented here. This manuscript has been possible through grants R01AI165661 from NIH/NIAD, RTI2018-095497-B-I00 from MCIN, HR17_00527 from Fundación La Caixa, the Transatlantic Network of Excellence (TNE-18CVD04) from the Leducq Foundation, and FET-OPEN (no. 861878) from the European Commission. M.P.-S. is supported by the EMBO ALTF (no. 1142-2020) long-term fellowship and from MICINN (RYC2021-033511-I). J.S. and I.B. are supported by fellowships from MICINN (PRE2019-089130 and RYC2020-029563-I, respectively). The CNIC is supported by the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S).S