Behavior Of Neutrophils Research Articles

Perivascular Neutrophil Extracellular Traps Exacerbate Microvasospasm After Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) can lead to acute or delayed cerebral ischemia. Recent findings have revealed that spasm of microvessels, called microvasospasm, may contribute to SAH-related cerebral ischemia, and perivascular inflammation is considered important in the development of microvasospasms. However, owing to the difficulty in investigating the dynamics of vascular and perivascular events, little is known about the mechanisms underlying microvasospasms. We established an experimental system aiming to investigate the vascular and perivascular pathology of SAH by combining a SAH mouse model with intravital 2-photon imaging. SAH was induced by intracisternal blood injection, and the distribution of erythrocytes, neutrophil behavior, and morphological changes in the pial arterioles were analyzed over time by 2-photon microscopy imaging. To further explore the role of neutrophils and neutrophil extracellular traps (NETs) in microvasospasm, we performed neutrophil depletion by intraperitoneal administration of neutrophil-specific antibody or NETs removal by intracisternal administration of DNase. Erythrocytes were immediately distributed in the perivascular space of the arterioles after SAH induction; neutrophils intensively infiltrated the perivascular space within 2 days and subsequently showed NETosis; and pial arterioles in the same region developed pearl-string-like microvasospasms in the subacute phase. Neutrophil depletion significantly reduced the number of microvasospasms. Furthermore, the removal of perivascular NETs drastically reduced microvasospasms. By establishing a unique experimental system, we demonstrated that perivascular NETs could be a new therapeutic target for microvasospasms.

Stretched Vascular Endothelial Cells Polarized Neutrophils to N2 Type via TRPC1-IL13-STAT3 Axis.

VECs play a crucial role in regulating the function of neutrophils, which is essential for immune responses and inflammation. As stretch-sensitive cells, VECs sense mechanical stretch through surface mechanoreceptors, converting external mechanical stimuli into biochemical signals. This study aimed to explore the molecular mechanisms underlying the regulation of neutrophil behavior by stretched VECs. The key cytokine-inducing neutrophil N2 polarization in the conditioned medium from stretched vascular endothelial cells (CM-stretch) was validated through multifactorial matrix and flow cytometry. Additionally, the molecular mechanism underlying the response of vascular endothelial cells to stretch was systematically verified through layer-by-layer analysis using WB. IL13, not IL4, was ultimately identified as a key cytokine-inducing neutrophil N2 polarization in CM-stretch. Inhibition of the transient receptor potential channel (TRPC1) and siRNA-mediated knockdown of TRPC1 both significantly decreased IL13 production. Furthermore, neutralizing IL13 in the CM-stretch or inhibiting STAT3 phosphorylation inhibited neutrophil N2 polarization, as evidenced by reduced CD206 and VEGFA expression. These results demonstrate that stretched VECs initiate a signaling cascade that induces neutrophil N2 polarization through the TRPC1-IL13-STAT3 axis, suggesting that mechanical stretching of VECs could shift neutrophil function from a pro-inflammatory to a more regulatory and healing role.

Neutrophils under the microscope: neutrophil dynamics in infection, inflammation, and cancer revealed using intravital imaging.

Neutrophils rapidly respond to inflammation resulting from infection, injury, and cancer. Intravital microscopy (IVM) has significantly advanced our understanding of neutrophil behavior, enabling real-time visualization of their migration, interactions with pathogens, and coordination of immune responses. This review delves into the insights provided by IVM studies on neutrophil dynamics in various inflammatory contexts. We also examine the dual role of neutrophils in tumor microenvironments, where they can either facilitate or hinder cancer progression. Finally, we highlight how computational modeling techniques, especially agent-based modeling, complement experimental data by elucidating neutrophil kinetics at the level of individual cells as well as their collective behavior. Understanding the role of neutrophils in health and disease is essential for developing new strategies for combating infection, inflammation and cancer.

Timing, initiation and function: An in-depth exploration of the interaction network among neutrophil extracellular traps related genes in acute pancreatitis

BackgroundExogenous inhibition of neutrophil extracellular traps (NETs) was believed to alleviate acute pancreatitis (AP). This study aimed to comprehensively explore the key biological behavior of NETs including timing and pathogenesis in AP by integrating of single cell RNA sequencing(scRNA-seq) and bulk RNA-seq. MethodsDifferentially expressed NETs-related genes and the hub genes of NETs were screened by bulk RNA-seq. ScRNA-seq was used to identify the cell types in pancreas of AP mice and to depict the transcriptomic maps in neutrophils. The mouse AP models were build to verify the timing of initiation of NETs and underlying pathogenesis of damage on pancreas acinar cells. ResultsTlr4 and Ccl3 were screened for hub genes by bulk RNA-seq. The trajectory analysis of neutrophils showed that high expression of Ccl3, Cybb and Padi4 can be observed in the middle stage during AP. Macrophages might be essential in the biological behavior of neutrophils and NETs. Through animal models, we presented that extensive NETs structures were formed at mid-stage of inflammation, accompanied by more serious pancreas and lung damage. NETs might promote necroptosis and macrophage infiltration in AP, and the damage on pancreatic injury could be regulated by Tlr4 pathway. Ccl3 was considered to recruit neutrophils and promote NETs formation. ConclusionThe findings explored the underlying timing and pathogenesis of NETs in AP for the first time, which provided gene targets for further studies.

Methyl Syringate: A Primary Driving Factor in Manuka Honeys Ability to Ameliorate Neutrophil Intracellular ROS Activity and NETosis.

Neutrophils use both the production of reactive oxygen species (ROS) and a specialized process called NETosis to defend the body from material deemed foreign. While these neutrophil behaviors are critical in preventing infection, a dysregulated response can lead to tissue damage and fibrosis at host-biomaterial interfaces. It was hypothesized that applying the flavonoids found in Manuka honey: chrysin, pinocembrin, and pinobanksin, and the phenolic compound methyl syringate to neutrophils exhibiting pro-inflammatory behavior will reduce ROS activity and prevent NETosis in primary human neutrophils. Using primary human neutrophils isolated from donor (n = 5) peripheral blood, concentrations between 1 nM and 10 µM of each flavonoid, 10 µM and 2 mM of methyl syringate, 0.1% v/v and 10% v/v Manuka honey, and combinations of both 1 nM-10 µM of each flavonoid and 10 µM-2 mM of methyl syringate were assayed for reductions in NETosis using Sytox orange extracellular DNA staining and reduction in intracellular ROS activity via standard dichloro-dihydro-fluorescein diacetate (DCFH-DA) oxidation assay. Compared to positive control levels, individual flavonoids showed moderate effect sizes. Higher concentrations of flavonoids, especially in combination, stimulated ROS activity by up to 105%. Whole Manuka honey reduced neutrophil extracellular trap (NET) levels by up to 91% but only reduced ROS activity by 36%. However, methyl syringate reduced NET levels by up to 68% and ROS activity by 66%. Methyl syringate and whole Manuka honey are potent inhibitors of neutrophil intracellular ROS activity and NET formation. Methyl syringate potentially drives the anti-inflammatory capabilities of Manuka honey demonstrated by previous studies.

Pneumocystis murina promotes inflammasome formation and NETosis during Pneumocystis pneumonia.

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. Isolated NETs compromised P. murina viability in vitro, highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable

BackgroundNeutrophils 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.MethodsWe 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.ResultsWe 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.ConclusionEx 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.

Improving stroke prognosis by TLR4 KO to enhance N2 neutrophil infiltration and reduce M1 macrophage polarization.

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.

The neonatal liver hosts a spontaneously occurring neutrophil population, exhibiting distinct spatial and functional characteristics from adults.

The elusive nature of the liver immune system in newborns remains an important challenge, casting a shadow over our understanding of how to effectively treat and prevent diseases in children. Therefore, deeper exploration into the intricacies of neonatal immunology might be crucial for improved pediatric healthcare. Using liver intravital microscopy, we unveiled a significant population of granulocytes in the hepatic parenchyma of fetuses and newborns. Utilizing high-dimensional immunophenotyping, we showed dynamic alterations predominantly in granulocytes during neonatal development. Liver intravital microscopy from birth through adulthood captures real-time dynamics, showing a substantial presence of Ly6G + cells that persisted significantly up to 2 weeks of age. Using CyTOF, we characterized neonatal Ly6G + cells as neutrophils, confirmed by morphology and immunohistochemistry. Surprisingly, the embryonic liver hosts a distinct population of neutrophils established as early as the second gestational week, challenging conventional notions about their origin. Additionally, we observed that embryonic neutrophils occupy preferentially the extravascular space, indicating their early establishment within the liver. Hepatic neutrophils in embryos and neonates form unique cell clusters, persisting during the initial days of life, while reduced migratory capabilities in neonates are observed, potentially compensating with increased reactive oxygen species (ROS) release in response to stimuli. Finally, in vivo imaging of acute neutrophil behavior in a newborn mouse, subjected to focal liver necrosis, unveils that neonatal neutrophils exhibit a reduced migratory response. The study provides unprecedented insights into the intricate interplay of neutrophils within the liver, shedding light on their functional and dynamic characteristics during development.

Neutrophils in cancer: dual roles through intercellular interactions.

Neutrophils, the most abundant immune cells in human blood, play crucial and diverse roles in tumor development. In the tumor microenvironment (TME), cancer cells regulate the recruitment and behaviors of neutrophils, transforming some of them into a pro-tumor phenotype. Pro-tumor neutrophils interact with cancer cells in various ways to promote cancer initiation, growth, and metastasis, while anti-tumor neutrophils interact with cancer cells to induce senescence and death. Neutrophils can also interact with other cells in TME, including T cells, macrophages, stromal cells, etc. to exert anti- or pro-tumor functions. In this review, we will analyze the anti- and pro-tumor intercellular interactions mediated by neutrophils, with a focus on generalizing the mechanisms underlying the interaction of neutrophils with tumor cells and T cells. Furthermore, we will provide an overview of cancer treatment strategies targeting neutrophil-mediated cellular interactions.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: (i) Short-term storage (< 8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; (ii) Neutrophils from long-term storage (> 24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. (iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

Neutrophils-biology and diversity.

Neutrophils, the most abundant white blood cells in the human circulation, play crucial roles in various diseases, including kidney disease. Traditionally viewed as short-lived pro-inflammatory phagocytes that release reactive oxygen species, cytokines and neutrophil extracellular traps, recent studies have revealed their complexity and heterogeneity, thereby challenging this perception. Neutrophils are now recognized as transcriptionally active cells capable of proliferation and reverse migration, displaying phenotypic and functional heterogeneity. They respond to a wide range of signals and deploy various cargo to influence the activity of other cells in the circulation and in tissues. They can regulate the behavior of multiple immune cell types, exhibit innate immune memory, and contribute to both acute and chronic inflammatory responses while also promoting inflammation resolution in a context-dependent manner. Here, we explore the origin and heterogeneity of neutrophils, their functional diversity, and the cues that regulate their effector functions. We also examine their emerging role in infectious and non-infectious diseases with a particular emphasis on kidney disease. Understanding the complex behavior of neutrophils during tissue injury and inflammation may provide novel insights, thereby paving the way for potential therapeutic strategies to manage acute and chronic conditions. By deciphering their multifaceted role, targeted interventions can be developed to address the intricacies of neutrophil-mediated immune responses and improve disease outcomes.

Proteomic Profiling and Cytokine Analysis Reveals Molecular Signatures and Inflammatory Factors Associated with Thrombosis in JAK2-V617F Positive Myeloproliferative Neoplasm (MPN) Cohorts

Background: Myeloproliferative neoplasms (MPNs), a hematological malignancy commonly associated with the JAK2-V617F mutation, exhibit a considerable burden of comorbidities resulting from inflammation-mediated processes. The JAK2-V617F malignant clones instigate systemic inflammation by upregulating various pro-inflammatory cytokines. This leads to dysregulated immune and inflammatory responses, thereby driving a process called thrombo-inflammation. Neutrophils play a crucial role as inflammatory mediators and effectors of pro-thrombotic activity in MPNs. We, therefore, hypothesize that perturbations in the neutrophil proteome may alter the molecular communication networks within neutrophils, thereby potentially contributing to the pathological thrombus formation in veins and arteries. Aim: The study was aimed to elucidate (i) the pathological significance of dysregulated protein expression profile of neutrophils carrying the JAK2-V617F mutation, and (ii) the impact of pro-inflammatory cytokines on neutrophils during thrombo-inflammation. Methods: In this study, which was conducted at the University Hospital Magdeburg, Germany, 11 patients carrying the JAK2-V617F mutation (6 with thrombosis; 5 without thrombosis) and 9 healthy volunteers were enrolled. Neutrophils were isolated from freshly collected whole blood using a negative selection kit. The isolated neutrophils were then lysed, and protein extraction was performed. High-resolution liquid chromatography - mass spectrometry (LC-MS/MS) was employed to investigate the protein inventory of neutrophils. Furthermore, platelet-poor plasma was isolated from the same cohort to analyse plasma cytokine/chemokine levels using a bead-based multiplex assay. Results: LC-MS/MS identified 2,006 proteins in neutrophils, which were then used for quantitative comparative analysis. Amongst these, 28 and 26 proteins exhibited significant up-regulation in the JAK2-V617F positive patients ‘with thrombosis’ and ‘without thrombosis’, respectively. Notably, ATP2A3 and ROCK1 were significantly upregulated in the JAK2-V617F thrombotic group, ranking among the top 10-upregulated proteins. Of note, ROCK1 activation has previously been implicated in cell proliferation, survival and cell cycle control. Functional annotation analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that upregulated proteins in the JAK2-V617F cohorts were significantly enriched for proteins involved in the calcium and TGF-ß signaling pathways. Furthermore, we assessed the levels of 60 cytokines/chemokines and soluble adhesion molecules in the same cohort, wherein significantly increased levels of ALCAM, ICAM-2 and VCAM-1 were observed in the JAK2-V617F thrombotic group. These molecules are known to play a critical role in vasculature-related inflammation and endothelial cell activation. Additionally, the JAK2-V617F thrombotic group exhibited significant upregulation of free active TGF-ß and pro-inflammatory cytokines such as IL-6 and IL-8 compared to both healthy subjects and JAK2-V617F non-thrombotic patients, indicating their contribution to the thrombo-inflammatory response. Moreover, the thrombotic cohort displayed elevated plasma levels of CXC chemokines and myeloperoxidase (MPO), a key enzyme linked to NETosis for neutrophil extracellular trap (NET) formation. Conclusions: The proteomic profile of neutrophils in MPN patients exhibit significant alterations, particularly between JAK2-V617F patients with and without thrombosis. The observed elevated expression of markers related to NETosis and calcium signaling pathways point towards the underlying mechanisms that could potentially contribute to the differential behavior of neutrophils, thereby facilitating the development of a thrombotic state in MPN patients.

Route of administration significantly affects particle deposition and cellular recruitment.

Lung exposures to dusts, pollutants, and other aerosol particulates are known to be associated with pulmonary diseases such as asthma and Chronic Obstructive Pulmonary Disease. These health impacts are attributed to the ability of aerosol components to induce pulmonary inflammation, which promotes tissue remodeling, including fibrosis, tissue degradation, and smooth muscle proliferation. Consequently, the distribution of these effects can have a significant impact on the physiologic function of the lung. In order to study the impact of distribution of inhaled particulates on lung pathogenesis, we compared the effect of different methods of particle delivery. By comparing intranasal versus aerosol delivery of fluorescent microspheres, we observed strikingly distinct patterns of particle deposition; intranasal delivery provided focused deposition concentrated on larger airways, while aerosol delivery showed unform deposition throughout the lung parenchyma. Recognizing that the impacts of inflammatory cells are contingent upon their recruitment and behavior, we postulate that these variations in distribution patterns can result in significant alterations in biological responses. To elucidate the relevance of these findings in terms of biological representation, we subsequently conducted an investigation into the responses elicited by the administration of endotoxin (bacterial Lipopolysaccharide, or LPS) in a transgenic neutrophil reporter mouse model. As with the microsphere results, patterns of recruited neutrophil inflammatory responses matched the delivery method; that is, despite the active migratory behavior of neutrophils, inflammatory histopathology patterns were either focused on large airways (intranasal administration) or diffusely throughout the parenchyma (aerosol). These results demonstrate the importance of modes of aerosol delivery as different patterns of inflammation and tissue remodeling will have distinct impacts on lung physiology.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

Phenotypic screening of novel probiotics with potential anti-neuroinflammation activity based on cell and zebrafish models

Neuroinflammation, a major pathological factor contributing to various neurodegenerative diseases, is closely associated with gut microbiota imbalance. The aim of this study was to establish a rapid and efficient screening method to obtain potential anti-neuroinflammatory strains. Firstly, 94 strains isolated from different sources were screened against neuroinflammation in lipopolysaccharide (LPS)-activated BV-2 cells model to identify potential strains quickly. In vitro cellular experiments revealed that 21 strains significantly inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity, which were selected for further evaluation of gastrointestinal tolerance and adhesion properties. Among them, 18 strains with promising probiotic properties were further confirmed for anti-neuroinflammatory effects in vivo using the CuSO4-induced zebrafish model. The results showed that eight strains significantly mitigated the migratory behavior of zebrafish neutrophils and the mRNA expression level of pro-inflammatory cytokines IL-1β compared to other strains. Additionally, Lactiplantibacillus plantarum and Limosilactobacillus fermentum were identified as the main species by sequencing their 16S rDNA. In conclusion, this cell-zebrafish model is promising for high-throughput screening of novel probiotics with potential anti-neuroinflammation activity.

Dual wave of neutrophil recruitment determines the outcome of C. albicans infection.

Candida albicans is a ubiquitous fungus that can cause superficial and systemic infections in humans. Neutrophils play a crucial role in controlling C. albicans infections. When C. albicans enters the bloodstream, it tends to get trapped in capillary vessels. However, the behavior of neutrophils in combating capillary-residing fungi has not been fully characterized. In this study, we used transgenic mice and whole mount imaging to investigate the growth of C. albicans and its interaction with innate immune cells in different organs. We observed that C. albicans rapidly grows hyphae within hours of infection. Following intravenous infection, we observed two waves of neutrophil recruitment, both of which significantly contributed to the elimination of the fungi. The first wave of neutrophils was induced by complement activation and could be prevented by C5aR blockade. Interestingly, we discovered that the fungicidal effect in the lungs was independent of adhesion molecules such as Mac-1, LFA-1, and ICAM-1. However, these molecules played a more significant role in the optimal killing of C. albicans in the kidney. Importantly, the initial difference in killing efficiency resulted in significantly reduced survival in knockout mice lacking these adhesion molecules. We identified a second wave of neutrophil recruitment associated with hyphal growth and tissue damage, which was independent of the aforementioned adhesion molecules. Overall, this study elucidates the dual wave of neutrophil recruitment during C. albicans infection and highlights the importance of early fungal clearance for favorable disease outcomes.

Acute immune responses in zebrafish and evasive behavior of a parasite - who is winning?

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation.

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release

Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior.