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In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chandrappa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Bacterial infections continue to pose a global health challenge, driven by antibiotic resistance and septicemia. This study aimed to assess the diagnostic utility of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in bacterial infections versus non-infectious causes of inflammation., Methods: A prospective study included 164 adult patients who were divided into two groups: a group of patients with confirmed bacterial infections and a second group of patients with other diagnoses (inflammatory pathologies, neoplasms, venous thromboembolic diseases, etc.). NLR and PLR values were compared between the bacterial infection group and the non-infectious causes group and the diagnostic performances of NLR and PLR for detecting bacterial infections were evaluated in comparison with other infection markers., Results: NLR and PLR were significantly higher in bacterial infections (p < 10 ^-6 ), and NLR was correlated positively with inflammation markers. NLR and PLR demonstrated significant potential in diagnosing bacterial infections, with an AUC of 0.72 and 0.60, respectively, using the following cutoff values: 4.3 for NLR and 183 for PLR., Conclusion: These findings underscore the importance of NLR and PLR as adjunctive tools for bacterial infection diagnosis., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Anoun J et al.)

Neutrophils need to migrate through tight tissue spaces to eliminate pathogens, but their movement is often hindered by their large and stiff nuclei. Neutrophil migration is impaired in sepsis patients, but it is unclear whether this defect is related to the deformability of their nuclei. Herein, we designed microfluidic devices with micron-scale narrow slits to simulate biological barriers. This setup allowed us to observe and record neutrophil movement and nuclear deformation in real-time. We also developed a method for morphological analysis to quantify nucleus deformation in numerous individual cells. Our studies showed that neutrophils from healthy individuals could adjust their nuclear shape to squeeze through these constrictions, whereas those from sepsis patients demonstrated less flexibility. Neutrophils with rigid nuclei struggled to pass through narrow gaps and were more likely to rupture under pressure. These findings suggest that the migration defects of neutrophils observed in sepsis may be attributed to the inability of neutrophils to deform their nuclei, highlighting the crucial role of microfluidic technologies in offering new insights into migration defects under pathological conditions., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

For in vitro neutrophil functional assays, neutrophils are typically isolated from whole blood, having the target cells exposed to an artificial microenvironment with altered kinetics. Isolated neutrophils exhibit limited lifespans of only a few hours ex vivo, significantly shorter than the 3-5 day lifespan of neutrophils in vivo. In addition, due to neutrophils' inherently high sensitivity, neutrophils removed from whole blood exhibit stochastic non-specific activation that contributes to assay variability. Here, a method - named "µ-Blood" - is presented that enables functional neutrophil assays using a microliter of unprocessed whole blood. µ-Blood allows multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control). In µ-Blood, neutrophils show sustained migration and limited non-specific activation kinetics (<0.1% non-specific activation) over 3-6 days. In contrast, neutrophils isolated using traditional methods show increased and divergent activation kinetics (10-70% non-specific activation) in only 3 h. Finally, µ-Blood allows the capture and quantitative comparison of distinct neutrophil functional heterogeneity between healthy donors and cancer patients in response to microbial stimuli with the preserved physiological lifespan over 6 days., (© 2024 The Author(s). Small Methods published by Wiley‐VCH GmbH.)

Background and Objective: Histologic assessment of the immune infiltrate in H&E slides is vital in diagnosing and managing inflammatory bowel diseases, but these assessments are subjective and time-consuming even for those with expertise. The development of deep learning models to aid in these assessments has been limited by the paucity of image data with reliably annotated immune cells available for training., Methods: To address these challenges, we developed a pipeline that automates the neutrophil and lymphocyte labeling in ROIs from digital H&E slides. The data included ROIs extracted from 19 digitized H&E slides and the same slides restained with immunohistochemistry. Our pipeline first delineates each nucleus in H&E ROIs. Using the colorimetric features of the immunohistochemical stains (red: neutrophils, green: lymphocytes) in the immunohistochemistry ROIs, each cell was labeled as a neutrophil, a lymphocyte, or another cell. The labels were then transferred to the corresponding H&E ROIs by image registration, and the ROI registration accuracy was assessed by the median target registration error resulting in a labeled dataset. The newly formed dataset (NeuLy-IHC) comprising 519 ROIs with 235,256 labeled cells (74,339 lymphocytes, 16,326 neutrophils and 144,591 other cells) was used to train the HoVer-Net (NeuLy) model. The performance of HoVer-Net (NeuLy) measured by DICE coefficient (segmentation accuracy) and F1-scores (classification accuracy), was compared to those achieved by HoVer-Net (MoNuSAC) and SMILE (MoNuSAC) publicly available models trained on cancer-containing ROIs from the MoNuSAC dataset with manual cell labeling and pathologists' annotations., Results: The 1.0 μm median target registration error of ROIs observed was low demonstrating robust transferring of cellular labels from immunohistochemistry ROIs to H&E ROIs. In the test set comprising 76 NeuLy-IHC and 78 MoNuSAC ROIs, the HoVer-Net (NeuLy) achieved a DICE coefficient of 0.861 and F1-sores of 0.827, 0.838, and 0.828, for neutrophils, lymphocytes, and other cells, respectively, outperforming the HoVer-Net (MoNuSAC) 's and SMILE (MoNuSAC) 's DICE coefficient and F1 scores for each cell category., Conclusions: We attribute the improved performance of HoVer-Net (NeuLy) to the larger number of immune cells in the NeuLy-IHC dataset (in total 5x more, including 21x more neutrophils) than in the MoNuSAC dataset. Despite being trained on data from inflammatory bowel disease specimens, our model maintained robust performance when tested on previously unseen data derived from cancer specimens. The NeuLy-IHC set provides opportunities for training accurate models to quantify the inflammatory infiltrate in digital histologic slides., Competing Interests: Declaration of competing interest All the data, results as well as the methodology developed are the authors’ own work and research effort. The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

There is an ongoing need to do more with less and provide highly multiplexed analysis from limited sample volumes. Improved "sample sparing" assays would have a broad impact across pediatric and other rare sample type studies in addition to enabling sequential sampling. This capability would advance both clinical and basic research applications. Here we report the micro blood analysis technology (μBAT), a microfluidic platform that supports multiplexed analysis of neutrophils from a single drop of blood. We demonstrate the multiplexed orthogonal capabilities of μBAT including functional assays (phagocytosis, neutrophil extracellular traps, optical metabolic imaging) and molecular assays (gene expression, cytokine secretion). Importantly we validate our microscale platform using a macroscale benchmark assay. μBAT is compatible with lancet puncture or microdraw devices, and its design facilitates rapid operations without the need for specialized equipment. μBAT offers a new method for investigating neutrophil function in populations with restricted sample amounts.

Neutrophils are the most frequent immune cell type in peripheral blood, performing an essential role against pathogens. People with neutrophil deficiencies are susceptible to deadly infections, highlighting the importance of generating these cells in host immunity. Neutrophils can be generated from hematopoietic progenitor cells (HPCs) and embryonic stem cells (ESCs) using a cocktail of cytokines. In addition, induced pluripotent stem cells (iPSCs) can be differentiated into various functional cell types, including neutrophils. iPSCs can be derived from differentiated cells, such as skin and blood cells, by reprogramming them to a pluripotent state. Neutrophil generation from iPSCs involves a multistep process that can be performed through feeder cell-dependent and feeder cell-independent manners. Various cytokines and growth factors, in particular, stem cell facto, IL-3, thrombopoietin and granulocyte colony-stimulating factor (G-CSF), are used in both methods, especially, G-CSF which induces the final differentiation of neutrophils in the granulocyte lineage. iPSC-derived neutrophils have been used as a valuable tool for studying rare genetic disorders affecting neutrophils. The iPSC-derived neutrophils can also be used for disease modeling, infection research and drug discovery. However, several challenges must be overcome before iPSC-derived neutrophils can be used therapeutically in transplantation medicine. This review provides an overview of the commonly employed protocols for generating neutrophils from HPCs, ESCs and iPSCs and discusses the potential applications of the generated cells in research and medicine., Competing Interests: Declarations of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Due to high inter-observer variability the 2015 International Council for Standardization in Haematology (ICSH) recommendations state to count band neutrophils as segmented neutrophils in the white blood cell (WBC) differential. However, the inclusion of bands as a separate cell entity within the WBC differential is still widely used in hematology laboratories in Croatia. The aim of this multicentric study was to assess the degree of inter-observer variability in enumerating band neutrophils within the WBC differential among Croatian laboratories., Materials and Methods: Seven large Croatian hospital laboratories from different parts of the country participated in the study. In each of 7 participating laboratories, one blood smear, that was flagged by the analyzer as possibly having bands, was evaluated by all personnel participating in the analysis of hematology samples. Between-observer manual smear reproducibility was expressed as coefficient of variation (CV) and calculated using the following formula: CV (%) = (standard deviation (SD)/mean value) x 100%., Results: The CVs (%) and relative band neutrophil counts in participating laboratories were as follows: 15.4% (16-24), 19.2% (16-32), 19.5% (17-40), 21.1% (17-44), 35.0% (8-26), 51.9% (3-29), and remarkably high 62.4% (12-59). For segmented neutrophils CVs were lower, ranging from 7.4% to 32.2%. The CVs did not correlate with the number of staff members in each hospital (P = 0.293)., Conclusions: This study revealed very high variability in enumerating band neutrophil count in the blood smear differential among all participants, thus prompting a need for action on a national level., Competing Interests: Potential conflict of interest None declared., (Copyright Croatian Society of Medical Biochemistry and Laboratory Medicine.)

The reconstruction of bone defects has been associated with severe challenges worldwide. Nowadays, bone marrow mesenchymal stem cell (BMSC)-based cell sheets have rendered this approach a promising way to facilitate osteogenic regeneration in vivo . Extracellular vesicles (EVs) play an essential role in intercellular communication and execution of various biological functions and are often employed as an ideal natural endogenous nanomedicine for restoring the structure and functions of damaged tissues. The perception of polymorphonuclear leukocytes (neutrophils, PMNs) as indiscriminate killer cells is gradually changing, with new evidence suggesting a role for these cells in tissue repair and regeneration, particularly in the context of bone healing. However, the role of EVs derived from PMNs (PMN-EVs) in bone regeneration remains largely unknown, with limited research being conducted on this aspect. In the current study, we investigated the effects of PMN-EVs on BMSCs and the underlying molecular mechanisms as well as the potential application of PMN-EVs in bone regeneration. Toward this end, BMSC-based cell sheets with integrated PMN-EVs (BS@PMN-EVs) were developed for bone defect regeneration. PMN-EVs were found to significantly enhance the proliferation and osteogenic differentiation of BMSCs in vitro . Furthermore, BS@PMN-EVs were found to significantly accelerate bone regeneration in vivo by enhancing the maturation of the newly formed bone in rat calvarial defects; this is likely attributable to the effect of PMN-EVs in promoting the expression of key osteogenic proteins such as SOD2 and GJA1 in BMSCs. In conclusion, our findings demonstrate the crucial role of PMN-EVs in promoting the osteogenic differentiation of BMSCs during bone regeneration. Furthermore, this study proposes a novel strategy for enhancing bone repair and regeneration via the integration of PMN-EVs with BMSC-based cell sheets.

Background: In an aging population, there is an increasing need for easily accessible nutritional markers., Aims: To determine whether the neutrophil-to-lymphocyte ratio (NLR) can serve as an effective nutritional indicator compared to the Mini-Nutritional Assessment Short Form (MNA-SF) or other common markers such as albumin and body mass index (BMI)., Methods: Data were obtained from the SABE study in Ecuador, which included participants aged 60 years or older. This cross-sectional study collected comprehensive data, including demographics, health-related factors, and physical assessments. Neutrophil and lymphocyte counts were measured by complete blood count. Nutritional status was assessed by MNA-SF, and BMI was calculated. Several physical tests were performed to evaluate the participants' functional status. Confounding variables such as age, sex, and comorbidities were considered., Results: The final sample consisted of 1790 subjects (48.9% male). The overall median age was 68 years (IQR 64,76). BMI and lymphocytes were higher in females, while NLR was higher in males. MNA-SF showed a negative association with NLR. Similarly, lymphocyte count shows a positive association with MNA-SF. Physical tests, such as the Romberg test and the Five Times Sit-to-Stand test, also showed correlations with NLR and lymphocyte count, respectively., Conclusion: The study results suggest a significant relationship between NLR and lymphocytes, and nutritional status. The correlation with albumin is stronger with NLR than with BMI. The simplicity and affordability of NLR may make it suitable for routine use in several medical fields, improving our understanding of the complex relationship between nutrition, inflammation, and overall health., Competing Interests: Conflicts of Interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Cell population data (CPD) parameters may be putative biomarkers for the screening of various diseases including some infections and myelodysplastic syndrome. This study aimed to establish the age- and sex-specific reference intervals (RIs) for the CPD parameters in the Korean population., Methods: The reference population for the RIs of CPD parameters comprised 124 856 subjects aged 20-99 years. CPD parameters were obtained from Sysmex XN-2000 (Kobe, Japan) datasets from 17 health promotion centers in 13 South Korean cities. We determined significant partitions for age and sex, and calculated RIs according to Clinical and Laboratory Standards Institute C28-A3 guidelines., Results: The side scattered light intensity in the neutrophil area and the lymphocyte area did not require sex-related partitioning except in those over the age of 50, among whom the lower limit (LL) and upper limit (UL) were lower in females. However, the side scattered light distribution width in the lymphocyte area required age- and sex-related partitioning, in which LL and UL were higher in females. The LL and UL of the fluorescent light distribution width were higher in males in the neutrophil area and higher in females in the lymphocyte area, but age-related partitioning was not required. The forward scattered light intensity in the neutrophil area, lymphocyte area, and monocyte area did not require age-related partitioning in males., Conclusion: This study has determined comprehensive age- and sex-specific RIs for CPD parameters, which could help to prove the clinical significance of these parameters in the Sysmex XN-2000., (© 2024 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Context.—: Increased band neutrophils in blood smear differential counts ("bandemia") are entrenched in medicine as a flag for sepsis. However, laboratory hematology experts have long advocated for discontinuation of reporting bands separately from segmented neutrophils because of poor sensitivity and specificity, poor interobserver agreement, and availability of alternative biomarkers for sepsis., Objective.—: To describe band neutrophil reporting practices and reproducibility of band classification among laboratories participating in the College of American Pathologists (CAP) proficiency testing (PT) program., Design.—: A survey questionnaire was distributed to hematology PT participants. A subsequent morphologic challenge included 12 preselected cell identifications of segmented neutrophils, bands, and metamyelocytes, and a 100-cell manual differential count of a digitally scanned blood smear., Results.—: Among laboratories that reported manual differentials, most respondents reported bands (4554 of 5268; 86.4%). Only 3222 of 4412 respondents (73.0%) provided band reference ranges. Though participants classified "easy" band neutrophils well (78.0%-98.3%), categorization of cell identifications for "moderate" and "difficult" bands was poor (3.1%-39.0% of laboratories), with classification instead as segmented neutrophils. This pattern was seen regardless of laboratory demographic characteristics. Marked variability in band counts was observed on the 100-cell differential count for both CAP PT participants and CAP Hematology and Clinical Microscopy Committee (HCMC) members (coefficients of variation, 55.8% and 32.9%, respectively). Variability was significantly improved when segmented and band neutrophils were grouped together (coefficients of variation, 6.2% and 5.0%, respectively)., Conclusions.—: Most CAP PT-participating laboratories report band counts, many without reference ranges. The survey confirms significant interlaboratory variability of band enumeration when bands are separately identified from segmented neutrophils. This study reaffirms the CAP Hematology and Clinical Microscopy Committee's strong recommendation to group segmented and band neutrophils together in manual differential counts., Competing Interests: All authors are past or present members of the College of American Pathologists Hematology and Clinical Microscopy subcommittee., (© 2024 College of American Pathologists.)

Background: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development., Methods: We optimised a method for ex vivo production of human neutrophils from CD34 + haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors., Results: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis., Conclusion: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils., (© 2024. The Author(s).)

Myeloid-derived suppressor cells (MDSCs) play an essential role in suppressing the antitumor activity of T lymphocytes in solid tumors, thus representing an attractive therapeutic target to enhance the efficacy of immunotherapy. However, the differences in protein expression between MDSCs and their physiological counterparts, particularly polymorphonuclear neutrophils (PMNs), remain inadequately characterized, making the specific identification and targeting of MDSCs difficult. PMNs and PMN-MDSCs share markers such as CD11b+CD14-CD15+/CD66b+, and some MDSC-enriched markers are emerging, such as LOX-1 and CD84. More proteomics studies are needed to identify the signature and markers for MDSCs. Recently, we reported the induced differentiation of isogenic PMNs or MDSCs (referred to as iPMNs and iMDSCs, respectively) from the human promyelocytic cell line HL60. Here, we profiled the global proteomics and membrane proteomics of these cells with quantitative mass spectrometry, which identified a 41-protein signature ("cluster 6") that was upregulated in iMDSCs compared with HL60 and iPMN. We further integrated our cell line-based proteomics data with a published proteomics dataset of normal human primary monocytes and monocyte-derived MDSCs induced by cancer-associated fibroblasts. The analysis identified a 38-protein signature that exhibits an upregulated expression pattern in MDSCs compared with normal monocytes or PMNs. These signatures may provide a hypothesis-generating platform to identify protein biomarkers that phenotypically distinguish MDSCs from their healthy counterparts, as well as potential therapeutic targets that impair MDSCs without harming normal myeloid cells.

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P&lt;0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective 1,2 . This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury 2,3 . Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context 4-12 . However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the Na V 1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing., (© 2024. The Author(s).)

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces 1-3 . However, how polymorphonuclear structures are assembled remains unknown 4 . Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin 5 -leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Substrate anchorage is essential for cell migration, and actin polymerization at cell front and myosin contractility at cell rear are known to govern cell forward movement. Yet their differential driving strategies for neutrophil migration on distinct adhesiveness substrates and their contributions to the migration-induced trail formation remain unclear. Here we explore the morphological changes, migration dynamics, and trail formation of neutrophils on ICAM-1 and PLL substrates, with a focus on the relationships among adhesive forces, traction forces, and out-of-plane forces. Results indicate that, on ICAM-1, neutrophil migration and trail formation rely on the coordinated interactions of Arp2/3 and myosin, along with biochemical regulation (via Syk and calpain) of adhesion and de-adhesion. This pattern leads to traction forces being concentrated at relatively fewer adhesive sites, facilitating cell forward migration. On PLL, however, neutrophils primarily depend on Arp2/3-mediated actin polymerization, resulting in a broader distribution of traction forces and weaker adhesions, which allows for higher leading-edge migrating velocities. Elevated membrane tension and out-of-plane forces generated by bleb protrusions on PLL reduce the reliance on myosin-driven contraction at the trailing edge, enabling easier tail detachment through elastic recoil. This work highlights the differential impact of substrate adhesiveness on neutrophil migration and trail formation and dynamics, providing new insights into cell migration mechanisms and potential therapeutic targets for inflammatory and immune-related disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Migrating neutrophils are found to leave behind subcellular trails in vivo , but the underlying mechanisms remain unclear. Here, an in vitro cell migration test plus an in vivo observation was applied to monitor neutrophil migration on intercellular cell adhesion molecule-1 (ICAM-1) presenting surfaces. Results indicated that migrating neutrophils left behind long-lasting, chemokine-containing trails. Trail formation tended to alleviate excessive cell adhesion enhanced by the trans -binding antibody and maintain efficient cell migration, which was associated with differential instantaneous edge velocity between the cell front and rear. CD11a and CD11b worked differently in inducing trail formation with polarized distributions on the cell body and uropod. Trail release at the cell rear was attributed to membrane ripping, in which β 2 -integrin was disrupted from the cell membrane through myosin-mediated rear contraction and integrin-cytoskeleton dissociation, potentiating a specialized strategy of integrin loss and cell deadhesion to maintain efficient migration. Moreover, neutrophil trails left on the substrate served as immune forerunners to recruit dendritic cells. These results provided an insight in elucidating the mechanisms of neutrophil trail formation and deciphering the roles of trail formation in efficient neutrophil migration.

The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a subset (1-70%) of human neutrophils and the Olfm4-high (Olfm4-H) proportion has been found to correlate with septic shock severity. The aim of this study was to decipher proteomic differences between the subsets in healthy individuals, hypothesizing that Olfm4-H neutrophils have a proteomic profile distinct from that of Olfm4 low (Olfm4-L) neutrophils. We then extended the investigation to septic shock. A novel protocol for the preparation of fixed, antibody-stained, and sorted neutrophils for LC-MS/MS was developed. In healthy individuals, 39 proteins showed increased abundance in Olfm4-H, including the small GTPases Rab3d and Rab11a. In Olfm4-L, 52 proteins including neutrophil defensin alpha 4, CXCR1, Rab3a, and S100-A7 were more abundant. The data suggest differences in important neutrophil proteins that might impact immunological processes. However, in vitro experiments revealed no apparent difference in the ability to control bacteria nor produce oxygen radicals. In subsets isolated from patients with septic shock, 24 proteins including cytochrome b-245 chaperone 1 had significantly higher abundance in Olfm4-H and 30 in Olfm4-L, including Fc receptor proteins. There was no correlation between Olfm4-H proportion and septic shock severity, but plasma Olfm4 concentration was elevated in septic shock. Thus, the Olfm4-H and Olfm4-L neutrophils have different proteomic profiles, but there was no evident functional significance of the differences in septic shock., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Monocyte chemotactic protein-1-induced protein-1 (MCPIP-1) is highly expressed in activated immune cells and negatively regulates immune responses, while the mechanisms underlying the immunoregulation of neutrophils in acute bacterial infection and liver injury remain elusive. Here, we examined the role of MCPIP-1 in regulating neutrophil functions during acute bacterial peritonitis and liver injury. Mice with myeloid cell-specific overexpression (McpipMye-tg) or knockout (McpipΔMye) of MCPIP-1 were generated. We found that reactive oxygen species and myeloperoxidase production, formation of neutrophil extracellular traps, and migratory capacity were deficient in McpipMye-tg neutrophils but enhanced in McpipΔMye neutrophils. The recruitment of neutrophils and pathogen clearance were markedly suppressed in McpipMye-tg mice following intraperitoneal infection with Salmonella typhimurium while intensified in McpipΔMye mice. Severe acute S. typhimurium-infected peritonitis and liver injury occurred in McpipMye-tg mice but were alleviated in McpipΔMye mice. RNA sequencing, RNA-binding protein immunoprecipitation and qPCR analysis revealed that MCPIP-1 downregulated the protective functions of neutrophils via degrading the mRNA of cold inducible RNA-binding protein. Consistently, MCPIP-1 was highly expressed in neutrophils of patients with acute infectious diseases, especially in those with liver injury. Collectively, we uncover that MCPIP-1 negatively regulates the antibacterial capacities of neutrophils, leading to exacerbating severe acute bacterial peritonitis and liver injury. It may serve as a candidate target for maintaining neutrophil homeostasis to control acute infectious diseases., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

The heterogeneity of the tumour immune microenvironment (TIME), organized by various immune and stromal cells, is a major contributing factor of tumour metastasis, relapse and drug resistance 1-3 , but how different TIME subtypes are connected to the clinical relevance in liver cancer remains unclear. Here we performed single-cell RNA-sequencing (scRNA-seq) analysis of 189 samples collected from 124 patients and 8 mice with liver cancer. With more than 1 million cells analysed, we stratified patients into five TIME subtypes, including immune activation, immune suppression mediated by myeloid or stromal cells, immune exclusion and immune residence phenotypes. Different TIME subtypes were spatially organized and associated with chemokine networks and genomic features. Notably, tumour-associated neutrophil (TAN) populations enriched in the myeloid-cell-enriched subtype were associated with an unfavourable prognosis. Through in vitro induction of TANs and ex vivo analyses of patient TANs, we showed that CCL4 + TANs can recruit macrophages and that PD-L1 + TANs can suppress T cell cytotoxicity. Furthermore, scRNA-seq analysis of mouse neutrophil subsets revealed that they are largely conserved with those of humans. In vivo neutrophil depletion in mouse models attenuated tumour progression, confirming the pro-tumour phenotypes of TANs. With this detailed cellular heterogeneity landscape of liver cancer, our study illustrates diverse TIME subtypes, highlights immunosuppressive functions of TANs and sheds light on potential immunotherapies targeting TANs., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O -linked N-acetylglucosamine ( O -GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all- trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O -GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O -GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12 . The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O -GlcNAc-reducing ATRA and DON while O -GlcNAc-elevating thiamet G ( O -GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O -GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.

Rationale : Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives : Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods : Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results : Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration ( p = 0.0397) and NETosis ( p = 0.0332), and impaired phagocytosis ( p = 0.0036) associated with impaired ROS generation ( p < 0.0001). The percentage of CD54+ neutrophils ( p < 0.001) was significantly increased, while surface expression of CD11b ( p = 0.0014) and PD-L1 ( p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA ( p = 0.0396) and impaired DNAse activity ( p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils ( p = 0.0129). Conclusions : COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity.

With the concept of the gut-lung axis reinforced in recent years, emerging evidence has shown that intestinal homeostasis is vital for lung health. Nevertheless, the impacts of lung homeostasis on intestinal tracts and their mechanism are rarely studied. Our results showed that papain-induced asthmatic mice exhibited apparent colonic injuries compared with controls, including increased intestinal permeability, neutrophil and Th17 infiltration in the colonic lamina propria. Moreover, the intranasal administration of papain aggravated such colonic injuries in mice with dextran sulfate sodium-induced colitis, as evidenced by increased occult blood scores, shortened colon length, and accumulated neutrophils. The level of IL-17A was also higher in the serum of asthmatic mice than wild-type mice. Interestingly, the pathologic scores, the proportion of Th17 cells, and neutrophil infiltration in the colon were markedly reduced after IL-17A blocking. Similarly, longer length, lower pathologic scores, and fewer neutrophils were also observed in the colon of IL-17-deficient asthmatic mice. More importantly, we demonstrated that severe gastrointestinal symptoms could accompany clinical asthmatics. The frequencies of Th17 cells and the mRNA expression of IL-17A in the peripheral blood of these patients were significantly enhanced. Besides, the gastrointestinal symptom rating scale scores positively correlated with the frequencies of Th17 in asthmatics. These findings enlighten that IL-17A aggravates asthma-induced intestinal immune injury by promoting neutrophil trafficking, which facilitates the exploration of new potential biomarkers to treat asthma., (©2022 Society for Leukocyte Biology.)

Reading, interpreting and crawling along gradients of chemotactic cues is one of the most complex questions in cell biology. In this issue, Georgantzoglou et al. (2022. J. Cell. Biol.https://doi.org/10.1083/jcb.202103207) use in vivo models to map the temporal sequence of how neutrophils respond to an acutely arising gradient of chemoattractant., (© 2022 Stopp and Sixt.)

The liver is one of the most-favored distant metastatic sites for solid tumors, and interactions between cancer cells and components of the hepatic microenvironment are essential for liver metastasis (LM). Although sex is one of the determinants for primary liver cancer, sexual dimorphism in LM (SDLM) and the underlying mechanisms remain unclear. We herein demonstrate a significant male-biased SDLM, which is attributed to host androgen/androgen receptor (Ar) signaling that promotes hepatic seeding of tumor cells and subsequent outgrowth in a neutrophil-dependent manner. Mechanistically, androgen/Ar signaling promotes hepatic accumulation of neutrophils by promoting proliferation and development of neutrophil precursors in the bone marrow, as well as modulating hepatic recruitment of neutrophils and their functions. Antagonizing the androgen/Ar/neutrophil axis significantly mitigates LM in males. Our data thus reveal an important role of androgen in LM and suggest that androgen/Ar modulation represents a promising target for LM therapy in men., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) are emerging haematological inflammatory biomarkers. However, their significance in retinal vein occlusion (RVO) and its subtypes, branch and central RVO (BRVO and CRVO, respectively), is uncertain. This systematic review and meta-analysis aimed to clarify the association of NLR and PLR with RVO. We searched MEDLINE (Ovid), EMBASE (Ovid) and the Cochrane Library for studies investigating the association of NLR and PLR with RVO from inception to 2 December 2020. We used random-effects inverse-variance modelling to generate pooled effect measures. We used bivariate Bayesian modelling to meta-analyse the ability of NLR and PLR to differ between individuals with and without RVO and performed meta-regression and sensitivity analyses to explore inter-study heterogeneity. Eight studies published encompassing 1059 patients were included for analysis. Both NLR and PLR were significantly elevated in RVO, with pooled mean differences of 0.63 (95% confidence interval (CI) 0.31-0.95) and 21.49 (95% CI 10.03-32.95), respectively. The pooled sensitivity, specificity and area under the Bayesian summary receiver operating characteristic curve were, respectively, 0.629 (95% credible interval (CrI) 0.284-0.872), 0.731 (95% CrI 0.373-0.934) and 0.688 (95% CrI 0.358-0.872) for NLR; and 0.645 (95% CrI 0.456-0.779), 0.616 (95% CrI 0.428-0.761) and 0.621 (95% CrI 0.452-0.741) for PLR. Mean and variability of age and diabetes mellitus prevalence partially explained between-study heterogeneity. NLR and PLR are significantly elevated in RVO. Future research is needed to investigate the potential prognostic value and independence of these findings., (© 2021 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Here we report the identification of human CD66b - CD64 dim CD115 - neutrophil-committed progenitor cells (NCPs) within the SSC lo CD45 dim CD34 + and CD34 dim/- subsets in the bone marrow. NCPs were either CD45RA + or CD45RA - , and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b + neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-β1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-β1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-β1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-β, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-β1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-β1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-β1 can reduce the adhesive properties of these cells; however, direct effects of TGF-β1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited β-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited β-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel β-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable β-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: B.F.V. and F.C.P. have ownership interests in Protein Foundry and XLock Biosciences.

All cells must die at some point, and the dogma is that they do it either silently via apoptosis or via pro-inflammatory, lytic forms of death. Amongst these lytic cell death pathways, pyroptosis is one of the best characterized. Pyroptosis depends on inflammatory caspases which activate members of the gasdermin family of proteins, and it is associated with the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Pyroptosis is an essential component of innate immunity, it initiates and amplifies inflammation and it removes the replication niche for intracellular pathogens. Most of the literature on pyroptosis focuses on monocytes and macrophages. However, the most abundant phagocytes in humans are neutrophils. This review addresses whether neutrophils undergo pyroptosis and the underlying mechanisms. Furthermore, I discuss how and why neutrophils might be able to resist pyroptosis., Competing Interests: Conflict of interest G.S. declares no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell., (© 2021 Brunetti et al.)

Aim: To evaluate the association between the value of neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), monocyte to high-density lipoprotein ratio (MHR) and the development of retinal artery occlusion (RAO) and retinal vein occlusion (RVO)., Methods: This retrospective study assessed 41 RAO, 50 RVO and 50 control (age and gender matched senile cataract) participants. The NLR, PLR and MHR parameters of patients' peripheral blood were analyzed. A receiver operating characteristics (ROC) curve analysis and the best cutoff value were used to specify the predictive value of NLR, PLR and MHR in RAO and RVO., Results: The NLR, PLR and MHR were significantly higher in RAO group compared to the control group (p<0.001, p<0.001 and p = 0.008; respectively). The NLR, PLR and MHR were also significantly higher in the RVO group compared to the control group (p<0.001, p = 0.001 and p = 0.012, respectively). The NLR and PLR were significantly higher in the RAO group compared to the RVO group (p<0.001 and p = 0.022, respectively). The optimal cut-off value of NLR to predict RAO was >2.99, with 90.2% sensitivity and 100% specificity. The PLR to predict RAO was > 145.52, with 75.6% sensitivity and 80.0% specificity., Conclusion: Higher NLR, PLR and MHR are related to the occurrence of RAO and RVO. NLR and PLR are more prominent in RAO compared to RVO., Competing Interests: The authors have declared that no competing interests exist.

Inhibitory and activating immune receptors play a key role in modulating the amplitude and duration of immune responses during infection and in maintaining immune balance in homeostatic conditions. The CD200 Receptor (CD200R) gene family in humans encodes one inhibitory receptor, CD200R1, and one putative activating member, CD200R1 Like (CD200R1L). It is demonstrated that CD200R1L is endogenously expressed by human neutrophils and activates cellular functions such as reactive oxygen species (ROS) production via Syk, PI3Kβ, PI3Kδ, and Rac GTPase signaling. Phylogenetic analysis shows that CD200R1L is present in many species among vertebrates, ranging from birds to primates, suggesting that evolutionary conservation of this receptor is critical for protection against co-evolving pathogens. The duplication event that generated CD200R1L from CD200R occurred several times throughout evolution, supporting convergent evolution of CD200R1L. In our phylogenetic trees, CD200R1L has longer branch lengths than CD200R1 in most species, suggesting that CD200R1L is evolving faster than CD200R1. It is proposed that CD200R1L represents a hitherto uncharacterized activating receptor on human neutrophils., (©2021 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Infection with the SARS-CoV2 virus can vary from asymptomatic, or flu-like with moderate disease, up to critically severe. Severe disease, termed COVID-19, involves acute respiratory deterioration that is frequently fatal. To understand the highly variable presentation, and identify biomarkers for disease severity, blood RNA from COVID-19 patient in an intensive care unit was analyzed by whole transcriptome RNA sequencing. Both SARS-CoV2 infection and the severity of COVID-19 syndrome were associated with up to 25-fold increased expression of neutrophil-related transcripts, such as neutrophil defensin 1 (DEFA1), and 3-5-fold reductions in T cell related transcripts such as the T cell receptor (TCR). The DEFA1 RNA level detected SARS-CoV2 viremia with 95.5% sensitivity, when viremia was measured by ddPCR of whole blood RNA. Purified CD15+ neutrophils from COVID-19 patients were increased in abundance and showed striking increases in nuclear DNA staining by DAPI. Concurrently, they showed >10-fold higher elastase activity than normal controls, and correcting for their increased abundance, still showed 5-fold higher elastase activity per cell. Despite higher CD15+ neutrophil elastase activity, elastase activity was extremely low in plasma from the same patients. Collectively, the data supports the model that increased neutrophil and decreased T cell activity is associated with increased COVID-19 severity, and suggests that blood DEFA1 RNA levels and neutrophil elastase activity, both involved in neutrophil extracellular traps (NETs), may be informative biomarkers of host immune activity after viral infection., Competing Interests: TM and IT have an equity interest in True Bearing Diagnostics, Inc., a diagnostics company developing RNA biomarkers for various diseases, including coronary artery disease and internal infections. TM is seeking patent protection for technology related to the current studies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors declare there are no competing interests.

Neutrophils (Neu) migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis, including the intestinal epithelia injuries and immune responses. Although their important roles in these diseases, they are challenging to study due to their short life span and the inability to cryopreserve or expand them in vitro . Moreover, the standard cell culturing on plastic plates (two-dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. In this study, we developed a new three-dimensional (3D) culture system for human and mouse peripheral blood Neu, which is made of hydrogel. The Neu showed much better cell integrity and less cell debris in the 3D culture system compared to that in 2D culture system. Moreover, the 3D culture system was more suitable for the observation of neutrophil extracellular traps (NETs) stimulated by the classical stimulation phorbol ester (PMA), and other damage associated molecular patterns (DAMPs) such as Lipopolysaccharide (LPS)/ATP, interleukin-1 β (IL-1β) and tumor necrosis factor α (TNFα) than the 2D culture system. Moreover, NETs phenomenon in 3D culture system is similar to that in vivo . In addition, the 3D culture system was evaluated to co-culturing Neu and other parenchymal cells, such as colon mucosal epithelial cell lines. In conclusion, the 3D culture system could maintain better properties of Neu than that in 2D culture system and it may reduce the gap between in vitro an in vivo experimentations., Competing Interests: YW was employed by Guangzhou Darui Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Wang, Huang, He, Peng, Wan and Ma.)

Objective: The association between neutrophil-to-lymphocyte ratio (NLR) with subclinical macrovascular and microvascular diseases has been less investigated. We sought to examine the association between NLR and new-onset subclinical macrovascular and microvascular abnormalities in the Chinese population., Methods: From a community cohort, we included 6,430 adults aged ≥ 40 years without subclinical macrovascular and microvascular diseases at baseline. We measured subclinical macrovascular and microvascular abnormalities separately using the ankle-brachial index (ABI), brachial-ankle pulse wave velocity (baPWV), and albuminuria., Results: During a mean follow-up of 4.3 years, 110 participants developed incident abnormal ABI, 746 participants developed incident elevated baPWV, and 503 participants developed incident albuminuria. Poisson regression analysis indicated that NLR was significantly associated with an increased risk of new-onset abnormal ABI, elevated baPWV, and albuminuria. Compared to overweight/obese participants, we found a much stronger association between NLR and subclinical vascular abnormalities in participants with normal weight. Furthermore, we found an interaction between the NLR and body mass index (BMI) on the risk of new-onset abnormal ABI ( P for interaction: 0.01)., Conclusion: NLR was associated with subclinical macrovascular and microvascular diseases in the Chinese population. Furthermore, in participants with normal weight, the association between NLR and subclinical vascular abnormalities was much stronger., (Copyright © 2022 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Objective: Severe COVID-19 is characterized by a dysregulated immune response in which neutrophils play a critical role. Calprotectin reflects neutrophil activation and is involved in the self-amplifying thrombo-inflammatory storm in severe COVID-19. We aimed to evaluate the role of calprotectin in early prediction of severity in COVID-19 patients., Methods: This was a multicenter prospective observational study enrolling consecutive adult COVID-19 patients. On arrival to emergency department, blood samples were collected for laboratory tests, including serum calprotectin. The primary outcome was severe respiratory failure requiring invasive mechanical ventilation and the secondary outcome was need for Intensive Care Unit (ICU) admission., Results: Study population included 395 patients, 57 (14.4%) required invasive mechanical ventilation and 100 (25.3%) were admitted to ICU. Median serum calprotectin levels were significantly higher in intubated (3.73 mg/L vs. 2.63 mg/L; p < 0.001) and ICU patients (3.48 mg/L vs. 2.60 mg/L; p = 0.001). Calprotectin showed a significant accuracy to predict the need for invasive mechanical ventilation (ROC AUC 0.723) and ICU admission (ROC AUC 0.650). In multivariate analysis, serum calprotectin was an independent predictor of invasive mechanical ventilation (OR 1.161) and ICU admission (OR 1.068)., Conclusion: Serum calprotectin can be used as an early predictor of severity in COVID-19 patients., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes., (©2021 Society for Leukocyte Biology.)

Currently, there are a number of therapeutic schemes used for the treatment of various types of musculoskeletal disorders. However, despite the use of new treatment options, therapeutic failure remains common due to impaired and delayed healing, or implant rejection. Faced with this challenge, in recent years regenerative medicine started looking for alternative solutions that could additionally support tissue regeneration. This review aims to outline the functions and possible clinical applications of, and future hopes associated with, using autologous or heterologous products such as antimicrobial peptides (AMPs), microvesicles (MVs), and neutrophil degranulation products (DGP) obtained from circulating neutrophils. Moreover, different interactions between neutrophils and platelets are described. Certain products released from neutrophils are critical for interactions between different immune cells to ensure adequate tissue repair. By acting directly and indirectly on host cells, these neutrophil-derived products can modulate the body's inflammatory responses in various ways. The development of new formulations based on these products and their clinically proven success would give hope for significant progress in regenerative therapy in human and veterinary medicine.

Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kraus and Gruber.)

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae , is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro . Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCE Shigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella 's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.