Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming.

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape-changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho-rheological (MORE) changes capable of altering cell function. We employ real-time deformability cytometry (RT-DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT-DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT-DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un-primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+ /H+ antiport-dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease

Comparative Safety of the BNT162b2 Messenger RNA COVID-19 Vaccine vs Other Approved Vaccines in Children Younger Than 5 Years.

Importance SARS-CoV-2 vaccines are authorized for use in most age groups. The safety of SARS-CoV-2 vaccines is unknown in children younger than 5 years. Objective To retrospectively evaluate the safety of the BNT162b2 vaccine used off-label in children younger than 5 years compared with the safety of non-SARS-CoV-2 vaccines in the same sample. Design, Setting, and Participants This investigator-initiated retrospective cohort study included parents or caregivers who registered children for SARS-CoV-2 vaccination in outpatient care facilities in Germany. The study was performed as an authenticated online survey. A total of 19 000 email addresses were contacted from vaccination registration databases between April 14 and May 9, 2022. Inclusion criteria were child age younger than 5 years at the first BNT162b2 vaccination and use of a correct authentication code to prove invitation. Exposures Off-label BNT162b2 vaccination and on-label non-SARS-CoV-2 vaccinations. Main Outcomes and Measures Reported short-term safety data of 1 to 3 doses of 3 to 10 μg BNT162b2 in children from birth to younger than 60 months are presented. Coprimary outcomes were the frequencies of 11 categories of symptoms after vaccination with bivariate analyses and regression models adjusting for age, sex, weight, and height. Results The study included 7806 children (median age, 3 years [IQR, 2-4 years]; 3824 [49.0%] female) who were followed up of for a mean (SD) of 91.4 (38.8) days since first BNT162b2 vaccination (survey response rate, 41.1%). A 10-μg dosage was more frequently associated with local injection-site symptoms compared with lower dosages. In the active-comparator analysis, the probability of any symptoms (odds ratio [OR], 1.62; 95% CI, 1.43-1.84), local symptoms (OR, 1.68; 95% CI, 1.38-2.05), musculoskeletal symptoms (OR, 2.55; 95% CI, 1.32-4.94), dermatologic symptoms (OR, 2.18; 95% CI, 10.7-4.45), or otolaryngologic symptoms (OR, 6.37; 95% CI, 1.50-27.09) were modestly elevated after BNT162b2 compared with non-SARS-CoV-2 vaccines, whereas the probabilities of general symptoms (OR, 0.77; 95% CI, 0.63-0.95) and fever (OR, 0.42; 95% CI, 0.32-0.55) were lower after BNT162b2. Symptoms requiring hospitalization (n = 10) were reported only at BNT162b2 dosages above 3 μg. Conclusions and Relevance In this cohort study, the symptoms reported after BNT162b2 administration were comparable overall to those for on-label non-SARS-CoV-2 vaccines in this cohort of children younger than 5 years. The present data may be used together with prospective licensure studies of BNT162b2 efficacy and safety and could help guide expert recommendations about BNT162b2 vaccinations in this age group

Streptococcus agalactiae Serotype Distribution and Antimicrobial Susceptibility in Pregnant Women in Gabon, Central Africa

Neonatal invasive disease due to Streptococcus agalactiae is life threatening and preventive strategies suitable for resource limited settings are urgently needed. Protective coverage of vaccine candidates based on capsular epitopes will relate to local epidemiology of S. agalactiae serotypes and successful management of critical infections depends on timely therapy with effective antibiotics. This is the first report on serotype distribution and antimicrobial susceptibility of S. agalactiae in pregnant women from a Central African region. Serotypes V, III, and Ib accounted for 88/109 (81%) serotypes and all isolates were susceptible to penicillin and clindamycin while 13% showed intermediate susceptibility to erythromycin

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Funder: NHLI FoundationFunder: NIHR Imperial Biomedical Research Centre; FundRef: http://dx.doi.org/10.13039/501100013342Funder: National Heart Lung and Blood InstituteFunder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: National Institute of Biomedical Imaging and Bioengineering; FundRef: http://dx.doi.org/10.13039/100000070Funder: Gates Cambridge ScholarshipFunder: NIH/OXCAM FellowshipObjectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy

One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency.

BACKGROUND: Inborn errors of immunity (IEI) present with a large phenotypic spectrum of disease, which can pose diagnostic and therapeutic challenges. Suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator of cytokine signaling, and has recently been associated with a novel IEI. Of patients described to date, it is apparent that SOCS1 haploinsufficiency has a pleiotropic effect in humans. OBJECTIVE: We sought to investigate whether dysregulation of immune pathways, in addition to STAT1, play a role in the broad clinical manifestations of SOCS1 haploinsufficiency. METHODS: We assessed impacts of reduced SOCS1 expression across multiple immune cell pathways utilizing patient cells and CRISPR/Cas9 edited primary human T cells. RESULTS: SOCS1 haploinsufficiency phenotypes straddled across the International Union of Immunological Societies classifications of IEI. We found that reduced SOCS1 expression led to dysregulation of multiple intracellular pathways in immune cells. STAT1 phosphorylation is enhanced, comparably with STAT1 gain-of-function mutations, and STAT3 phosphorylation is similarly reduced with concurrent reduction of Th17 cells. Furthermore, reduced SOCS1 E3 ligase function was associated with increased FAK1 in immune cells, and increased AKT and p70 ribosomal protein S6 kinase phosphorylation. We also found Toll-like receptor responses are increased in SOCS1 haploinsufficiency patients. CONCLUSIONS: SOCS1 haploinsufficiency is a pleiotropic monogenic IEI. Dysregulation of multiple immune cell pathways may explain the variable clinical phenotype associated with this new condition. Knowledge of these additional dysregulated immune pathways is important when considering the optimum management for SOCS1 haploinsufficient patients

The German National Pandemic Cohort Network (NAPKON): rationale, study design and baseline characteristics

Schons M, Pilgram L, Reese J-P, et al. The German National Pandemic Cohort Network (NAPKON): rationale, study design and baseline characteristics. European Journal of Epidemiology . 2022.The German government initiated the Network University Medicine (NUM) in early 2020 to improve national research activities on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. To this end, 36 German Academic Medical Centers started to collaborate on 13 projects, with the largest being the National Pandemic Cohort Network (NAPKON). The NAPKON's goal is creating the most comprehensive Coronavirus Disease 2019 (COVID-19) cohort in Germany. Within NAPKON, adult and pediatric patients are observed in three complementary cohort platforms (Cross-Sectoral, High-Resolution and Population-Based) from the initial infection until up to three years of follow-up. Study procedures comprise comprehensive clinical and imaging diagnostics, quality-of-life assessment, patient-reported outcomes and biosampling. The three cohort platforms build on four infrastructure core units (Interaction, Biosampling, Epidemiology, and Integration) and collaborations with NUM projects. Key components of the data capture, regulatory, and data privacy are based on the German Centre for Cardiovascular Research. By April 01, 2022, 34 university and 40 non-university hospitals have enrolled 5298 patients with local data quality reviews performed on 4727 (89%). 47% were female, the median age was 52 (IQR 36-62-) and 50 pediatric cases were included. 44% of patients were hospitalized, 15% admitted to an intensive care unit, and 12% of patients deceased while enrolled. 8845 visits with biosampling in 4349 patients were conducted by April 03, 2022. In this overview article, we summarize NAPKON's design, relevant milestones including first study population characteristics, and outline the potential of NAPKON for German and international research activities.Trial registration https://clinicaltrials.gov/ct2/show/NCT04768998 . https://clinicaltrials.gov/ct2/show/NCT04747366 . https://clinicaltrials.gov/ct2/show/NCT04679584. © 2022. The Author(s)

Metabolic Profiling of Human Eosinophils.

Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO2 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol-myristate-acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.This work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/H531100/1), Medical Research Council (grant number MR/J00345X/1), Alexander von-Humboldt Stiftung (Humboldt-Professorship to JG), and an ERC Starting Grant (“LightTouch”, FP7 #282060 to JG). We also thank NIHR Cambridge Biomedical Research Centre and acknowledge the support of NIHR Comprehensive Clinical Research Network

Metabolic Profiling of Human Eosinophils.

Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO2 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol-myristate-acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.This work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/H531100/1), Medical Research Council (grant number MR/J00345X/1), Alexander von-Humboldt Stiftung (Humboldt-Professorship to JG), and an ERC Starting Grant (“LightTouch”, FP7 #282060 to JG). We also thank NIHR Cambridge Biomedical Research Centre and acknowledge the support of NIHR Comprehensive Clinical Research Network

Image_2_Metabolic Profiling of Human Eosinophils.tif

<p>Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO₂ 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol–myristate–acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.</p