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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.

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3-4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells., (© 2024. The Author(s).)

Neutrophils are considered 'first-line defence' cells as they can be rapidly recruited to the site of the immune response. As key components of non-specific immune mechanisms, neutrophils use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to fight pathogens. Recently, immunoregulatory abilities of neutrophils associated with the secretion of several mediators, including cytokines and extracellular vesicles (EVs) containing, among other components, microRNAs (miRNAs), have also been reported. EVs are small structures released by cells into the extracellular space and are present in all body fluids. Microvesicles show the composition and status of the releasing cell, its physiological state, and pathological changes. Currently, EVs have gained immense scientific interest as they act as transporters of epigenetic information in intercellular communication. This review summarises findings from recent scientific reports that have evaluated the utility of miRNA molecules as biomarkers for effective diagnostics or even as start-points for new therapeutic strategies in neutrophil-mediated immune reactions. In addition, this review describes the current state of knowledge on miRNA molecules, which are endogenous regulators of gene expression besides being involved in the regulation of the immune response., (© 2023 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Background: Thromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi., Methods: Adult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data., Results: The movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300-500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells., Conclusions: In samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen., (© 2024. The Author(s).)

Neutrophils play a pivotal role in innate immunity by releasing ROS through respiratory bursts to neutralize various pathogenic factors. However, excessive ROS release can cause tissue damage. Adenosine is an endogenous anti-inflammatory molecule inhibiting respiratory burst to protect the host. Adenosine aptamers with antibody-like properties and good stability are expected to act as adenosine antagonists with functional modulation capability. This study compares the effects of adenosine and its aptamer on the respiratory bursts of salivary polymorphonuclear leukocytes and circulating polymorphonuclear leukocytes using a programmable stopped-flow injection approach, ensuring rapid and efficient analysis while maintaining the neutrophils' viability. The results show that primed salivary polymorphonuclear leukocytes exhibit specificities that differ from circulating polymorphonuclear leukocytes. Adenosine aptamer can function as an inhibitory antagonist that distinguishes between physiologically controlled and excessive priming of neutrophils, showing potential application prospects in immunotherapy., 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 B.V. All rights reserved.)

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The research was completed prior to Dr. Doty's employment with Parse Biosciences, and the results will bring no benefit to this company., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The fields of neutrophil and endothelial cell biology are being deeply revised. While lung marginated neutrophils have been identified decades ago, their roles in the healthy adult lung are still contentious. Furthermore, while it is now clear that the lung constitutes an important immunological niche, the role of lung endothelial cells has been neglected so far. A better understanding of the role of short-lived neutrophils in contributing to lung endothelial cell physiology will improve our understanding of lung endothelial cell fate and heterogeneity under homeostasis and inflammation. Furthermore, it will provide new mechanistic insights on lung marginated neutrophil function and crosstalk with endothelial cells and provide robust foundations for devising therapeutic approaches in which endothelial cell (dys)functions are involved.

A robust, sterile inflammation underlies myocardial ischemia and reperfusion injury (MIRI). Several subsets of B cells possess the immunoregulatory capacity that limits tissue damage, yet the role of B cells in MIRI remains elusive. Here, we sought to elucidate the contribution of B cells to MIRI by transient ligation of the left anterior descending coronary artery in B cell-depleted or -deficient mice. Following ischemia and reperfusion (I/R), regulatory B cells are rapidly recruited to the heart. B cell-depleted or -deficient mice exhibited exacerbated tissue damage, adverse cardiac remodeling, and an augmented inflammatory response after I/R. Rescue and chimeric experiments indicated that the cardioprotective effect of B cells was not solely dependent on IL-10. Coculture experiments demonstrated that B cells induced neutrophil apoptosis through contact-dependent interactions, subsequently promoting reparative macrophage polarization by facilitating the phagocytosis of neutrophils by macrophages. The in vivo cardioprotective effect of B cells was undetectable in the absence of neutrophils after I/R. Mechanistically, ligand-receptor imputation identified FCER2A as a potential mediator of interactions between B cells and neutrophils. Blocking FCER2A on B cells resulted in a reduction in the percentage of apoptotic neutrophils, contributing to the deterioration of cardiac remodeling. Our findings unveil a potential cardioprotective role of B cells in MIRI through mechanisms involving FCER2A, neutrophils, and macrophages.

Sanjad Sakati Syndrome (SSS) is categorized as a neuroendocrine-related disease due to disorders of the nervous and hormonal systems. Since hormonal changes in these patients may affect the nature and function of the immune system. Thus, in this study, cell count and phagocytotic function of neutrophils were evaluated which may be influenced by changes in the hormonal rate and growth factors. In this study, the neutrophil count value and the oxidative burst were evaluated in six patients diagnosed with SSS and six healthy individuals. There was a significant reduction in the neutrophil count observed in SSS patients compared to healthy controls (37.41±7.93 percent vs. 66.5±6.8 percent). However, there was no significant difference in neutrophil oxidative index between patients with SSS and control subjects (172.33±55.08 vs. 217.00±77.38). We concluded that in patients with SSS, the phagocytic activity of neutrophils was not affected by hormonal changes, while the number of neutrophils and neutrophil-to-lymphocyte ratio (NLR) index were decreased.

Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods. The trajectories of cell movement depend on the size, rhythm, and direction of long series of pseudopods. These pseudopod properties are regulated by internal factors such as memory of previous directions and by external factors such as gradients of chemoattractants or electric currents. Here a simple method is described that defines the X, Y time coordinates of a pseudopod at the start and the end of the extension phase. The connection between the start and end of an extending pseudopod defines a vector, which is the input of different levels of analysis that defines cell movement. The primary information of the vector is its spatial length (pseudopod size), temporal length (extension time), extension rate (size divided by time), and direction. The second layer of information describes the sequence of two (or more) pseudopods: the direction of the second pseudopod relative to the direction of the first pseudopod, the start of the second pseudopod relative to the extension phase of the first pseudopod (the second starts while the first is still extending or after the first has stopped), and the alternating right/left extension of pseudopods. The third layer of information is provided by specific and detailed statistical analysis of these data and addresses question such as: is pseudopod extension in buffer in random direction or has the system internal directional memory, and how do shallow external electrical or chemical gradients bias the intrinsic pseudopod extension. The method is described for Dictyostelium, but has been used successfully for fast-moving neutrophils, slow-moving stem cells, and the fungus B.d. chytrid., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The management of granulocytopenia-associated infections is challenging, and a high mortality rate is associated with traditional supportive therapies. Neutrophils-the primary defenders of the human immune system-have potent bactericidal capabilities. Here, we investigated the dynamic in vivo distribution of neutrophil transfusion and their impact on the treatment outcome of severe granulocytopenic infections. We transfused 89 Zr-labeled neutrophils in the C57BL/6 mice and observed the dynamic neutrophil distribution in mice for 24 h using the micro-positron emission tomography (Micro-PET) technique. The labeled neutrophils were predominantly retained in the lungs and spleen up to 4 h after injection and then redistributed to other organs, such as the spleen, liver, and bone marrow. Neutrophil transfusion did not elicit marked inflammatory responses or organ damage in healthy host mice. Notably, allogeneic neutrophils showed rapid chemotaxis to the infected area of the host within 1 h. Tail vein infusion of approximately 10 7 neutrophils substantially bolstered host immunity, ameliorated the inflammatory state, and increased survival rates in neutrophil-depleted and infected mice. Overall, massive allogeneic neutrophil transfusion had a therapeutic effect in severe infections and can have extensive applications in the future., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons., Competing Interests: Unrelated to the submitted work, PB received consulting fees from Regeneron Pharmaceuticals. 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 © 2023 Krémer, Godon, Bruhns, Jönsson and de Chaisemartin.)

Immune-derived hunger hormones restore tissue after infection.

Objective: The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis., Background: Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood., Methods: Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model., Results: Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results., Conclusions: As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage., (© 2023 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd.)

The generation of traction forces by neutrophils regulates many crucial effector functions responsible for host defense, such as attachment, spreading, migration, phagocytosis, and NETosis. The activation state of the cell is a strong determinant of the functional efficacy of the neutrophil; however, the effect of activation on traction force production has not yet been determined experimentally. Previously, the mapping of cellular-generated forces produced by human neutrophils via a Traction Force Microscopy (TFM) method has required a three-dimensional imaging modality to capture out-of-plane forces, such as confocal or multiphoton techniques. A method newly developed in our laboratories can capture out-of-plane forces using only a two-dimensional imaging modality. This novel technique-combined with a topology-based single particle tracking algorithm and finite element method calculations-can construct high spatial frequency three-dimensional traction fields, allowing for traction forces in-plane and out-of-plane to the substrate to now be differentially visualized and quantified with a standard epifluorescence microscope. Here we apply this technology to determine the effect of neutrophil activation on force generation. Sepsis is a systemic inflammatory response that causes dysregulated neutrophil activation in vivo. We found that neutrophils from septic patients produced greater total forces than neutrophils from healthy donors and that the majority of this dysregulation occurred in-plane to the substrate. Ex vivo activation of neutrophils from healthy donors showed differential consequences depending on activation stimuli with mechanosensitive force decreases observed in some cases. These findings demonstrate the feasibility of epifluorescence-based microscopy in mapping traction forces to ask biologically significant questions regarding neutrophil function., (© 2023. The Author(s).)

Airway inflammation is a defense mechanism against inhaled agents characterized by infiltration of circulating immune cells. Given the inconsistent cellular identification across pre-clinical rat model, we have developed a flow cytometry panel of six colors to characterize macrophages subsets, lymphocytes and granulocytes in bronchoalveolar lavage fluid (BAL). Rats were challenged with intratracheal instillation of lipopolysaccharide (LPS). BAL were harvested 24 h after one LPS exposure in rats. This flow cytometry panel involve the description of macrophage subsets, T and B lymphocytes and neutrophils, which are central to airway immune responses, as based on scientific literature. By using a relatively small number of parameters to identify multiple cell types, additional parameters can be used for project/disease-specific activation markers., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Purpose of Review: Recent technological advances have identified distinct subpopulations and roles of the cardiac innate immune cells, specifically macrophages and neutrophils. Studies on distinct metabolic pathways of macrophage and neutrophil in cardiac injury are expanding. Here, we elaborate on the roles of cardiac macrophages and neutrophils in concomitance with their metabolism in normal and diseased hearts., Recent Findings: Single-cell techniques combined with fate mapping have identified the clusters of innate immune cell subpopulations present in the resting and diseased hearts. We are beginning to know about the presence of cardiac resident macrophages and their functions. Resident macrophages perform cardiac homeostatic roles, whereas infiltrating neutrophils and macrophages contribute to tissue damage during cardiac injury with eventual role in repair. Prior studies show that metabolic pathways regulate the phenotypes of the macrophages and neutrophils during cardiac injury. Profiling the metabolism of the innate immune cells, especially of resident macrophages during chronic and acute cardiac diseases, can further the understanding of cardiac immunometabolism., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem-locked NETosis Reporter 1 (TNR 1 ) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR 1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR 1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR 1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design., (© 2023 Wiley-VCH GmbH.)

Neutrophils are among the fastest-moving immune cells. Their speed is critical to their function as 'first responder' cells at sites of damage or infection, and it has been postulated that the unique segmented nucleus of neutrophils functions to assist their rapid migration. Here, we tested this hypothesis by imaging primary human neutrophils traversing narrow channels in custom-designed microfluidic devices. Individuals were given an intravenous low dose of endotoxin to elicit recruitment of neutrophils into the blood with a high diversity of nuclear phenotypes, ranging from hypo- to hyper-segmented. Both by cell sorting of neutrophils from the blood using markers that correlate with lobularity and by directly quantifying the migration of neutrophils with distinct lobe numbers, we found that neutrophils with one or two nuclear lobes were significantly slower to traverse narrower channels, compared to neutrophils with more than two nuclear lobes. Thus, our data show that nuclear segmentation in primary human neutrophils provides a speed advantage during migration through confined spaces., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Skeletal muscle regeneration involves coordinated interactions between different cell types. Injection of platelet-rich plasma is circumstantially considered an aid to muscle repair but whether platelets promote regeneration beyond their role in hemostasis remains unexplored. Here, we find that signaling via platelet-released chemokines is an early event necessary for muscle repair in mice. Platelet depletion reduces the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7/PPBP. Consequently, early-phase neutrophil infiltration to injured muscles is impaired whereas later inflammation is exacerbated. Consistent with this model, neutrophil infiltration to injured muscles is compromised in male mice with Cxcl7-knockout platelets. Moreover, neo-angiogenesis and the re-establishment of myofiber size and muscle strength occurs optimally in control mice post-injury but not in Cxcl7ko mice and in neutrophil-depleted mice. Altogether, these findings indicate that platelet-secreted CXCL7 promotes regeneration by recruiting neutrophils to injured muscles, and that this signaling axis could be utilized therapeutically to boost muscle regeneration., (© 2023. The Author(s).)

Nosocomial pneumonia is a leading cause of critical illness and mortality among seriously injured trauma patients. However, the link between injury and the development of nosocomial pneumonia is still not well recognized. Our work strongly suggests that mitochondrial damage-associated molecular patterns (mtDAMPs), especially mitochondrial formyl peptides (mtFPs) released by tissue injury, play a significant role in developing nosocomial pneumonia after a serious injury. Polymorphonuclear leukocytes (neutrophils, PMN) migrate toward the injury site by detecting mtFPs through formyl peptide receptor 1 (FPR1) to fight/contain bacterial infection and clean up debris. Activation of FPR1 by mtFPs enables PMN to reach the injury site; however, at the same time it leads to homo- and heterologous desensitization/internalization of chemokine receptors. Thus, PMN are not responsive to secondary infections, including those from bacteria-infected lungs. This may enable a progression of bacterial growth in the lungs and nosocomial pneumonia. We propose that the intratracheal application of exogenously isolated PMN may prevent pneumonia coupled with a serious injury.

Neutrophils are the most numerous white blood cells and are the first to arrive at sites of inflammation and infection. Thus, neutrophil behavior provides a comprehensive biomarker for antimicrobial defenses. Several microfluidic tools have been developed to test neutrophil chemotaxis, phagocytosis, extrusion of extracellular traps, etc. Traditional tools rely on purified neutrophil samples, which require lengthy and expensive isolation procedures from large volumes of blood. In the absence of such isolation, visualizing neutrophils in blood is complicated by the overwhelming number of red blood cells (RBCs), which outnumber neutrophils by 1000 : 1. Recently, several microfluidic technologies have been designed to analyze neutrophils directly in blood, by separating neutrophils on selectin coated surfaces before the migration assay or blocking the advance of RBCs with the moving neutrophils. However, RBC contamination remains an issue, albeit with a reduced ratio, down to 1 : 1. Here, we present an RBC-debulking strategy for neutrophil assays based on microscale passive redirection filters (PRFs) that reduce RBC contamination down to as few as a 1 : 17 RBC to neutrophil ratio. We compare the performance of different PRF designs and measure changes in neutrophil chemotaxis velocity and directionality following immune stimulation of whole blood.

Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults-typically 10 11 per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm., (© 2022 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, hyperactivated subtype of neutrophils. Neutrophils are significantly involved in the inflammatory response to myocardial infarction (MI), although we do not know the extent to which LDNs exist, or function, in MI. This study sought to determine the frequency and phenotype of LDNs in MI patients, compared to healthy subjects (HS)., Methods: LDNs and normal-density neutrophils (NDNs) were isolated from the peripheral blood of MI subjects ( n  = 12) and HSs ( n  = 12) using density gradient centrifugation. LDNs and NDNs were analysed by flow cytometry to identify neutrophils (CD66b + CD15 + CD14 - CD3 - CD19 - cells) and examine neutrophil activation (CD11b, CD66b and CD15) and maturity (CD33 and CD16)., Results: We identified LDNs within the peripheral blood mononuclear cell (PBMC) fraction of blood, and this population is significantly enriched in MI patients (1.04 ± 0.75% of PBMCs), compared to HS (0.29 ± 0.24%, p  = .003). Across both cohorts, LDNs express significantly higher levels of CD66b and CD15, indicating a heightened state of activation compared to NDNs. In this study, LDNs were described as CD33 high CD16 low , compared to CD33 low CD16 high NDNs, indicating the immaturity of this neutrophil subtype., Conclusions: An increase in the frequency of hyperactivated, immature LDNs is an immunological feature of MI. We highlight a potential pathological role of LDNs in MI, which underscores the need to expand our current understanding of this subtype in MI and other cardiovascular diseases (CVDs).

Coronavirus disease 2019 (COVID-19) can cause life-threatening lung inflammation that is thought to be mediated by neutrophils. The aim of the present work was to evaluate a novel PET tracer for neutrophil elastase (NE). Methods: In this first-in-humans study, 4 patients with hypoxia due to COVID-19 and 2 healthy controls were investigated with PET using 11 C-NES and 15 O-water for visualization and quantification of NE and perfusion in the lungs, respectively. Results: 11 C-NES accumulated selectively in lung areas with COVID-19 opacities on CT scans, suggesting high levels of NE there. In the same areas, perfusion was severely reduced in comparison to healthy lung tissue as measured with 15 O-water. Conclusion: The data suggest that NE is associated with severe lung inflammation in COVID-19 patients and that inhibition of NE could potentially reduce the acute inflammatory process and improve the condition., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis., (© 2022. The Author(s).)

Sepsis remains one of the leading causes of death globally, in spite of advanced developments in intensive care and better understandings of pathophysiology related to sepsis. There is no special treatment or drug available for sepsis, currently. Under normal circumstances, neutrophil is a major player in acute infection control. However, during sepsis, the migration abilities and antimicrobial functions of neutrophils are impaired, resulting in a dysregulated immune response. Recent studies have indeed demonstrated that blocking or reversing neutrophil migration and impaired antibacterial function can improve the outcomes in septic animal models. This article systemically synthesized information regarding related factors and signaling involved in the functions of neutrophils in sepsis. This review also discussed the possibility that neutrophils be used as a marker for specific diagnosis and/or prediction of the outcomes of sepsis., (Copyright © 2022 Chinese Medical Association. Production and hosting by Elsevier B.V. All rights reserved.)

In this study, we conducted a systematic review and meta-analysis regarding the role of the neutrophil to lymphocyte ratio (NLR) in Guillain Barré syndrome (GBS). The most recent update to the search was on July 18, 2022, through the databases of Web of Science, PubMed, Embase, and Scopus. The Newcastle-Ottawa scale was used for quality assessment of included studies. Finally, 14 studies were included in the review, and among them, ten studies were included in the meta-analysis. Our results showed that NLR levels were significantly increased in the patients with GBS compared with healthy controls (SMD = 1.05; 95%CI = 0.59 to 1.50, P < 0.001). After treatment, NLR levels were decreased to the extent that they became similar to healthy controls (SMD = -0.03, 95%CI = -0.29 to 0.22, P = 0.204). Moreover, NLR was a stable predictor of outcome or response to treatment in such patients (SMD = 1.01, 95%CI = 0.65 to 1.37, P < 0.001); the higher the NLR, the worse the outcome. In addition, patients who underwent mechanical ventilation had higher levels of NLR compared to those who did not (SMD = 0.93, 95%CI = 0.05 to 1.82, P = 0.03). However, NLR levels were not different among distinct GBS subtypes, so it could not distinguish among them. In conclusion, our analysis indicates that the NLR levels are highly elevated in patients with GBS. Therefore, the NLR has the potential to be used as a biomarker to inform diagnosis, prognosis, or treatment responses in GBS, and future studies are warranted., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Shirin Sarejloo et al.)

Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed by in vivo tracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow reduces by around 60% and reduced half-life in the blood, compared with noninflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by 2-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics., (©2022 Society for Leukocyte Biology.)

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca 2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-β/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca 2+ mobilization from intracellular stores and Ca 2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca 2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca 2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and β-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed acidic tissue. The aim of this study was to elucidate the role of microenvironmental pH in neutrophil chemotaxis. To this end, we used neutrophils from male C57BL/6J mice and combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pH i ) in varied extracellular pH (pH e ). Observational studies were complemented by biochemical analyses of leukotriene B 4 (LTB 4 ) production and activation of the Cdc42 Rho GTPase. Our data show that pH i of neutrophils dose-dependently adapts to a given pH of the extracellular milieu. Neutrophil chemotaxis toward C5a has an optimum at pH i ∼7.1, and its pH i dependency is almost parallel to that of LTB 4 production. Consequently, a shallow pH e gradient, resembling that encountered by neutrophils during extravasation from a blood vessel (pH ∼7.4) into the interstitium (pH ∼7.2), favors chemotaxis of stimulated neutrophils. Lowering pH e below pH 6.8, predominantly affects neutrophil chemotaxis, although the velocity is largely maintained. Inhibition of the Na + /H + exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil chemotaxis at the optimal pH i irrespective of the high LTB 4 production. Neutrophil migration and chemotaxis are almost completely abrogated by inhibiting LTB 4 production or blocking its receptor (BLT1). The abundance of the active GTP-bound form of Cdc42 is strongly reduced by NHE1 inhibition or pH e 6.5. In conclusion, we propose that the pH dependence of neutrophil chemotaxis toward C5a is caused by a pH i -dependent production of LTB 4 and activation of Cdc42. Moreover, it requires the activity of NHE1., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Sepsis is a series of clinical syndromes caused by immunological response to severe infection. As the most important and common complication of sepsis, acute respiratory distress syndrome (ARDS) is associated with poor outcomes and high medical expenses. However, well-described studies of analysis-based researches, especially related bioinformatics analysis on revealing specific targets and underlying molecular mechanisms of sepsis and sepsis-induced ARDS (sepsis/se-ARDS), still remain limited and delayed despite the era of data-driven medicine. In this report, weight gene co-expression network based on data from a public database was constructed to identify the key modules and screen the hub genes. Functional annotation by enrichment analysis of the modular genes also demonstrated the key biological processes and signaling pathway; among which, extensive immune-involved enrichment was remarkably associated with sepsis/se-ARDS. Based on the differential expression analysis, least absolute shrink and selection operator, and multivariable logistic regression analysis of the screened hub genes, SIGLEC9, TSPO, CKS1B and PTTG3P were identified as the candidate biomarkers for the further analysis. Accordingly, a four-gene-based model for diagnostic prediction assessment was established and then developed by sepsis/se-ARDS risk nomogram, whose efficiency was verified by calibration curves and decision curve analyses. In addition, various machine learning algorithms were also applied to develop extra models based on the four genes. Receiver operating characteristic curve analysis proved the great diagnostic and predictive performance of these models, and the multivariable logistic regression of the model was still found to be the best as further verified again by the internal test, training, and external validation cohorts. During the development of sepsis/se-ARDS, the expressions of the identified biomarkers including SIGLEC9, TSPO, CKS1B and PTTG3P were all regulated remarkably and generally exhibited notable correlations with the stages of sepsis/se-ARDS. Moreover, the expression levels of these four genes were substantially correlated during sepsis/se-ARDS. Analysis of immune infiltration showed that multiple immune cells, neutrophils and monocytes in particular, might be closely involved in the process of sepsis/se-ARDS. Besides, SIGLEC9, TSPO, CKS1B and PTTG3P were considerably correlated with the infiltration of various immune cells including neutrophils and monocytes during sepsis/se-ARDS. The discovery of relevant gene co-expression network and immune signatures might provide novel insights into the pathophysiology of sepsis/se-ARDS., 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 © 2022 Ming, Dong, Song, Li, Kong, Fang, Wang, Wu and Xia.)

Following injury and infection, neutrophils are guided to the affected site by chemoattractants released from injured tissues and invading microbes. During this process (chemotaxis), neutrophils must integrate multiple chemical signals, while also responding to physical constraints and prioritizing their directional decisions to generate an efficient immune response. In some clinical conditions, human neutrophils appear to lose the ability to chemotax efficiently, which may contribute both directly and indirectly to disease pathology. Here, a range of microfluidic designs is utilized to test the sensitivity of chemotaxing neutrophils to various perturbations, including binary decision-making in the context of channels with different chemoattractant gradients, hydraulic resistance, and angle of approach. Neutrophil migration in long narrow channels and planar environments is measured. Conditions in which neutrophils are significantly more likely to choose paths with the steepest chemoattractant gradient and the most direct approach angle, and find that migration efficiency across planar chambers is inversely correlated with chamber diameter. By sequential measurement of neutrophil binary decision-making to different chemoattractant gradients, or chemotactic index in sequential planar environments, data supporting a model of biased random walk for neutrophil chemotaxis are presented., (©2022 Society for Leukocyte Biology.)

Objective: To comprehensively characterize monocyte and neutrophil responses to E. coli and its product [lipopolysaccharide (LPS) or endotoxin] in vitro during pregnancy., Material or Subjects: Peripheral blood was collected from pregnant women during the third trimester (n = 20) and from non-pregnant women (n = 20)., Methods: The number, phagocytic activity, and reactive oxygen species (ROS) production of peripheral monocytes and neutrophils were investigated using flow cytometry. The phenotypes of peripheral monocytes and neutrophils after acute or chronic LPS stimulation were also determined using flow cytometry. Cytokine profiles were quantified for LPS-stimulated peripheral blood mononuclear cells (PBMCs) and a whole blood TruCulture ® system using a multiplex immunoassay., Results: Increased number, phagocytic activity, and ROS production capacity of monocytes and neutrophils were found in pregnant compared to non-pregnant women. Additionally, specific subsets of pro-inflammatory monocytes (IL-6 + CD14 + or MIP-1α + CD14 + cells) and neutrophils (IL-1β + CD15 + or MIP-1β + CD15 + cells) were increased in pregnant women in response to acute LPS stimulation. Moreover, distinct subsets of intermediate-activated monocytes expressing CD142, IL-6, and IL-1RA were increased in pregnant women upon chronic LPS stimulation. Last, pregnant women displayed a different cytokine profile than non-pregnant women in LPS-stimulated PBMCs and in whole blood., Conclusions: Pregnancy tailors the immune responses of circulating monocytes and neutrophils to endotoxin, a Gram-negative bacterial product., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: A dysregulated immune response is emerging as a key feature of critical illness in COVID-19. Neutrophils are key components of early innate immunity that, if not tightly regulated, contribute to uncontrolled systemic inflammation. We sought to decipher the role of neutrophil phenotypes, functions, and homeostasis in COVID-19 disease severity and outcome., Methods: By using flow cytometry, this longitudinal study compares peripheral whole-blood neutrophils from 90 COVID-19 ICU patients with those of 22 SARS-CoV-2-negative patients hospitalized for severe community-acquired pneumonia (CAP) and 38 healthy controls. We also assessed correlations between these phenotypic and functional indicators and markers of endothelial damage as well as disease severity., Results: At ICU admission, the circulating neutrophils of the COVID-19 patients showed continuous basal hyperactivation not seen in CAP patients, associated with higher circulating levels of soluble E- and P-selectin, which reflect platelet and endothelial activation. Furthermore, COVID-19 patients had expanded aged-angiogenic and reverse transmigrated neutrophil subsets-both involved in endothelial dysfunction and vascular inflammation. Simultaneously, COVID-19 patients had significantly lower levels of neutrophil oxidative burst in response to bacterial formyl peptide. Moreover patients dying of COVID-19 had significantly higher expansion of aged-angiogenic neutrophil subset and greater impairment of oxidative burst response than survivors., Conclusions: These data suggest that neutrophil exhaustion may be involved in the pathogenesis of severe COVID-19 and identify angiogenic neutrophils as a potentially harmful subset involved in fatal outcome., (© 2022. The Author(s).)

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development., (© 2022. The Author(s).)

Angiogenesis plays an important role during tumor growth and metastasis. We could previously show that Type I interferon (IFN)-deficient tumor-associated neutrophils (TANs) show strong pro-angiogenic activity, and stimulate tumor angiogenesis and growth. However, the exact mechanism responsible for their pro-angiogenic shift is not clear. Here, we set out to delineate the molecular mechanism and factors regulating pro-angiogenic properties of neutrophils in the context of Type I IFN availability. We demonstrate that neutrophils from IFN-deficient (Ifnar1 -/- ) mice efficiently release pro-angiogenic factors, such as VEGF, MMP9 or BV8, and thus significantly support the vascular normalization of tumors by increasing the maturation of perivascular cells. Mechanistically, we could show here that the expression of pro-angiogenic factors in neutrophils is controlled by the transcription factor forkhead box protein O3a (FOXO3a), which activity depends on its post-translational modifications, such as deacetylation or phosphorylation. In TANs isolated from Ifnar1 -/- mice, we observe significantly elevated SIRT1, resulting in SIRT1-mediated deacetylation of FOXO3a, its nuclear retention and activation. Activated FOXO3a supports in turn the transcription of pro-angiogenic genes in TANs. In the absence of SIRT1, or after its inhibition in neutrophils, elevated kinase MEK/ERK and PI3K/AKT activity is observed, leading to FOXO3a phosphorylation, cytoplasmic transfer and inactivation. In summary, we have found that FOXO3a is a key transcription factor controlling the angiogenic switch of neutrophils. Post-translational FOXO3a modifications regulate its transcriptional activity and, as a result, the expression of pro-angiogenic factors supporting development of vascular network in growing tumors. Therefore, targeting FOXO3a activity could provide a novel strategy of antiangiogenic targeted therapy for cancer., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The fate of infiltrating neutrophils in inflamed joints determines the development of acute gouty arthritis (AGA). GPR105 highly expressed in human neutrophils is sensitive to monosodium urate crystals (MSU); nevertheless, the roles of GPR105 in AGA remain unclear. Here, we show that GPR105 is significantly upregulated in peripheral polymorphonuclear neutrophils of AGA patients. GPR105 knockout (GPR105 -/- ) prevented NETosis and induced apoptosis of neutrophils under MSU exposure, as well as attenuating inflammatory cascades in AGA. Mechanistically, GPR105 deletion activated cAMP-PKA signals, thereby disrupting Raf-Mek1/2-Erk1/2 pathway-mediated NADPH oxidase activation, contributing to inhibition of NETosis. Whereas, cAMP-PKA activation resulting in GPR105 deficiency modulated PI3K-Akt pathway to regulate apoptosis. More importantly, suppression of cAMP-PKA pathway by SQ22536 and H-89 restored NETosis instead of apoptosis in GPR105 -/- neutrophils, promoting MSU-induced gout flares. Interestingly, lobetyolin was screened out as a potent GPR105 antagonist using molecular docking-based virtual screening and in vitro activity test, which efficiently attenuated MSU-induced inflammatory response interacting with GPR105. Taken together, our study implicated that modulating cell death patterns between NETosis and apoptosis through targeting GPR105 could be a potential therapeutic strategy for the treatment of AGA., 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 © 2022 Liu, Zhou, Jiang, Ye, Tian, Jiang, Hao, Li and Hu.)

Background: Neutrophilic inflammation in the airway is a hallmark of bronchiectasis. Neutrophil extracellular traps (NETs) have been reported to play an important role in the occurrence and development of bronchiectasis. Neutrophil side fluorescence is one of the characteristics of neutrophils that can reflect the activation of neutrophils and the formation of NETs., Objective: To explore the relationship between the values of neutrophil side fluorescence (NEUT-SFL) in the peripheral blood of bronchiectasis patients, and the severity of the disease., Methods: 82 patients with bronchiectasis from the Department of Respiratory and Critical Medicine, at the Third Affiliated Hospital of Southern Medical University and were scored with Bronchiectasis Severity Index (BSI) (2019-2021). The clinical data such as the value of NEUT-SFL, neutrophil count, C-reactive protein, and procalcitonin levels were collected and retrospectively analyzed. NEUT-SFL values neutrophil count from 28 healthy subjects were also used to ascertain cut-off values., Results: Based on the BSI scores, patients were divided into three categories as mild (32%), moderate (29%), and severe (39%). Our results showed that the values of NEUT-SFL were higher in bronchiectasis patients compared to healthy controls. The levels of NEUT-SFL positively correlated with the high BSI scores in patients (P = 0.037, r = 0.23) and negatively correlated with the lung function in these patients (r = - 0.35, P = 0.001). The area under the ROC curve was 0.813, the best cut-off was 42.145, indicating that NEUT-SFL values > 42.145 can potentially predict the severity of bronchiectasis., Conclusions: The values of NEUT-SFL in the peripheral blood can be used for predicting the severity of bronchiectasis., (© 2022. The Author(s).)

Abstract: Vagus nerve stimulation has been shown to exert anti-inflammation activities in sepsis. However, surgical implantation of stimulation devices is performed under general anesthesia, which limits its clinical application. Auricular vagus nerve stimulation (AVNS) is a minimal invasive technique that delivers electrical currents to the auricular branch of the vagus nerve. The purpose of this study was to determine the effects of AVNS on systemic inflammation, lung injury, neutrophil infiltration, and neutrophil extracellular traps (NETs) formation in the lung. In a LPS challenge lung-injury mice model, AVNS was applied to bilateral ears. Twelve hours after LPS administration, samples of blood, bronchoalveolar lavage fluid (BALF), and lung tissues were processed for investigations. We found that the treatment with AVNS significantly attenuated histopathological changes and neutrophil infiltration in the lung tissue, inhibited inflammatory cytokine elevations in serum and BALF, and decreased protein concentrations in BALF. Besides, AVNS decreased leukocyte and neutrophil accounts in BALF. Furthermore, colocalization of citrullination of histone H3 and myeloperoxidase expressions (highly specific marker of NETs) was reduced in AVNS mice. In conclusion, AVNS reduced systemic inflammation, attenuated lung edema, and inhibited neutrophil infiltration and NETs formation in the lung in LPS mice., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by the Shock Society.)

Background and Aim: Anti-tumor necrosis factor-α (anti-TNF-α) agents have been used for inflammatory bowel disease; however, it has up to 30% nonresponse rate. Identifying molecular pathways and finding reliable diagnostic biomarkers for patient response to anti-TNF-α treatment are needed., Methods: Publicly available transcriptomic data from inflammatory bowel disease patients receiving anti-TNF-α therapy were systemically collected and integrated. In silico flow cytometry approaches and Metascape were applied to evaluate immune cell populations and to perform gene enrichment analysis, respectively. Genes identified within enrichment pathways validated in neutrophils were tracked in an anti-TNF-α-treated animal model (with lipopolysaccharide-induced inflammation). The receiver operating characteristic curve was applied to all genes to identify the best prediction biomarkers., Results: A total of 449 samples were retrieved from control, baseline, and after primary anti-TNF-α therapy or placebo. No statistically significant differences were observed between anti-TNF-α treatment responders and nonresponders at baseline in immune microenvironment scores. Neutrophil, endothelial cell, and B-cell populations were higher in baseline nonresponders, and chemotaxis pathways may contribute to the treatment resistance. Genes related to chemotaxis pathways were significantly upregulated in lipopolysaccharide-induced neutrophils, but no statistically significant changes were observed in neutrophils treated with anti-TNF-α. Interleukin 13 receptor subunit alpha 2 (IL13RA2) is the best predictor (receiver operating characteristic curve: 80.7%, 95% confidence interval: 73.8-87.5%), with a sensitivity of 68.13% and specificity of 84.93%, and significantly higher in nonresponders compared with responders (P < 0.0001)., Conclusions: Hyperactive neutrophil chemotaxis influences responses to anti-TNF-α treatment, and IL13RA2 is a potential biomarker to predict anti-TNF-α treatment response., (© 2021 The Authors. Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1β, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.)

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro . We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI., 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 © 2022 Dölling, Eckstein, Singh, Schauer, Schoen, Shan, Bozec, Knopf, Schett, Muñoz and Herrmann.)