Self-extinguishing relay waves enable homeostatic control of human neutrophil swarming.

Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves. These waves propagate through active relay in which stimulated neutrophils trigger their neighbors to release additional swarming cues. Unlike canonical active relays, we find these waves to be self-terminating, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-terminating behavior. We observe near-constant levels of neutrophil recruitment over a wide range of starting conditions, revealing surprising robustness in the swarming process. This homeostatic control is achieved by larger and more numerous swarming waves at lower cell densities. We link defective wave termination to a broken recruitment homeostat in the context of human chronic granulomatous disease.

Resolving neutrophils through genetic deletion of TRAM attenuate atherosclerosis pathogenesis.

Systemic neutrophil dysregulation contributes to atherosclerosis pathogenesis, and restoring neutrophil homeostasis may be beneficial for treating atherosclerosis. Herein, we report that a homeostatic resolving subset of neutrophils exists in mice and humans characterized by the low expression of TRAM, correlated with reduced expression of inflammatory mediators (leukotriene B4 [LTB4] and elastase) and elevated expression of anti-inflammatory resolving mediators (resolvin D1 [RvD1] and CD200R). TRAM-deficient neutrophils can potently improve vascular integrity and suppress atherosclerosis pathogenesis when adoptively transfused into recipient atherosclerotic animals. Mechanistically, we show that TRAM deficiency correlates with reduced expression of 5-lipoxygenase (LOX5) activating protein (LOX5AP), dislodges nuclear localization of LOX5, and switches the lipid mediator secretion from pro-inflammatory LTB4 to pro-resolving RvD1. TRAM also serves as a stress sensor of oxidized low-density lipoprotein (oxLDL) and/or free cholesterol and triggers inflammatory signaling processes that facilitate elastase release. Together, our study defines a unique neutrophil population characterized by reduced TRAM, capable of homeostatic resolution and treatment of atherosclerosis.

BTK inhibitor-induced defects in human neutrophil effector activity against Aspergillus fumigatus are restored by TNF-α.

Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

Monocyte Anisocytosis Changes in Patients after Major Burn Injuries.

The recovery of patients after severe burns is a long and complex process. Recently, genomic analysis of white blood cells from burn and trauma patients revealed excessive and prolonged innate immune activation in patients with complicated outcomes. However, translating this knowledge into practical biomarkers has not been possible yet. Although several biomarkers for monitoring burn patients have been proposed, their ability to accurately distinguish between inflammation stemming from initial tissue destruction, infections, and organ failure complications is limited. Here, we focused on monocytes, critical innate immune cells in the response to burn injured tissues. We measured the monocyte anisocytosis (quantified as monocyte distribution width, MDW, a recently emerged marker of sepsis) throughout the recovery of patients from the time of burn injury until the end of the hospital stay. We observed that MDW increases in patients during the first week after major burns. Among the patients with major burns who survive, MDW starts decreasing in the second week and normalizes by the end of the hospital stay. The duration of hospital stay appears to be proportional to how fast MDW decreases during the second week after the injury. We also found that MDW decreases significantly in most patients after excision and debridement surgeries but not after allo- and auto-graft surgeries. Moreover, high MDW values correlated with a higher rate of positive microbiology blood culture samples and respiratory infections. These findings underscore the importance of monitoring MDW as a potential biomarker for the risk of complications during burn patient recovery.

Microfluidic devices for precise measurements of cell directionality reveal a role for glutamine during cell migration.

Cancer cells that migrate from tumors into surrounding tissues are responsible for cancer dissemination through the body. Microfluidic devices have been instrumental in discovering unexpected features of cancer cell migration, including the migration in self-generated gradients and the contributions of cell-cell contact during collective migration. Here, we design microfluidic channels with five successive bifurcations to characterize the directionality of cancer cell migration with high precision. We uncover an unexpected role for glutamine in epithelial cancer cell orientation, which could be replaced by alfa-keto glutarate but not glucose.

Neutrophil swarms containing myeloid-derived suppressor cells are crucial for limiting oral mucosal infection by C. albicans.

Oral mucosal colonization by C. albicans (Ca) is benign in healthy people but progresses to deeper infection known as oropharyngeal candidiasis (OPC) that may become disseminated when combined with immunosuppression. Cortisone-induced immunosuppression is a well-known risk factor for OPC, however the mechanism by which it permits infection is poorly understood. Neutrophils are the primary early sentinels preventing invasive fungal growth, and here we identify that in vivo neutrophil functional complexes known as swarms are crucial for preventing Ca invasion which are disrupted by cortisone. Neutrophil swarm function required leukotriene B4 receptor 1 (BLT1) expression, and swarms were further characterized by peripheral association of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) showing that OPC recruits PMN-MDSCs to this site of infection. Furthermore, PMN-MDSCs associated with Ca hyphae had no direct antifungal effect but showed prolonged survival times and increased autophagy. Thus in vivo neutrophil swarms are complex structures with spatially associated PMN-MDSCs that likely contribute immunoregulatory functions to resolve OPC. These swarm structures have an important function in preventing deep invasion by Ca within the oral mucosa and represent a mechanism for increased disease severity under immune deficient clinical settings.

Design and Validation of a Droplet-based Microfluidic System To Study Non-Photochemical Laser-Induced Nucleation of Potassium Chloride Solutions.

Non-photochemical laser-induced nucleation (NPLIN) has emerged as a promising primary nucleation control technique offering spatiotemporal control over crystallization with potential for polymorph control. So far, NPLIN was mostly investigated in milliliter vials, through laborious manual counting of the crystallized vials by visual inspection. Microfluidics represents an alternative to acquiring automated and statistically reliable data. Thus we designed a droplet-based microfluidic platform capable of identifying the droplets with crystals emerging upon Nd:YAG laser irradiation using the deep learning method. In our experiments, we used supersaturated solutions of KCl in water, and the effect of laser intensity, wavelength (1064, 532, and 355 nm), solution supersaturation (S), solution filtration, and intentional doping with nanoparticles on the nucleation probability is quantified and compared to control cooling crystallization experiments. Ability of dielectric polarization and the nanoparticle heating mechanisms proposed for NPLIN to explain the acquired results is tested. Solutions with lower supersaturation (S = 1.05) exhibit significantly higher NPLIN probabilities than those in the control experiments for all laser wavelengths above a threshold intensity (50 MW/cm2). At higher supersaturation studied (S = 1.10), irradiation was already effective at lower laser intensities (10 MW/cm2). No significant wavelength effect was observed besides irradiation with 355 nm light at higher laser intensities (≥50 MW/cm2). Solution filtration and intentional doping experiments showed that nanoimpurities might play a significant role in explaining NPLIN phenomena.

Infiltrating CD8+ T cells exacerbate Alzheimer's disease pathology in a 3D human neuroimmune axis model.

Brain infiltration of peripheral immune cells and their interactions with brain-resident cells may contribute to Alzheimer's disease (AD) pathology. To examine these interactions, in the present study we developed a three-dimensional human neuroimmune axis model comprising stem cell-derived neurons, astrocytes and microglia, together with peripheral immune cells. We observed an increase in the number of T cells (but not B cells) and monocytes selectively infiltrating into AD relative to control cultures. Infiltration of CD8+ T cells into AD cultures led to increased microglial activation, neuroinflammation and neurodegeneration. Using single-cell RNA-sequencing, we identified that infiltration of T cells into AD cultures led to induction of interferon-γ and neuroinflammatory pathways in glial cells. We found key roles for the C-X-C motif chemokine ligand 10 (CXCL10) and its receptor, CXCR3, in regulating T cell infiltration and neuronal damage in AD cultures. This human neuroimmune axis model is a useful tool to study the effects of peripheral immune cells in brain disease.

Monocyte anisocytosis corresponds with increasing severity of COVID-19 in children.

Introduction: Although SARS-CoV-2 infection can lead to severe COVID-19 in children, the role of biomarkers for assessing the risk of progression to severe disease is not well established in the pediatric population. Given the differences in monocyte signatures associated with worsening COVID-19 in adults, we aimed to determine whether monocyte anisocytosis early in the infectious course would correspond with increasing severity of COVID-19 in children. Methods: We performed a multicenter retrospective study of 215 children with SARS-CoV-2 infection, Multisystem Inflammatory Syndrome in Children (MIS-C), convalescent COVID-19, and healthy age-matched controls to determine whether monocyte anisocytosis, quantified by monocyte distribution width (MDW) on complete blood count, was associated with increasing severity of COVID-19. We performed exploratory analyses to identify other hematologic parameters in the inflammatory signature of pediatric SARS-CoV-2 infection and determine the most effective combination of markers for assessing COVID-19 severity in children. Results: Monocyte anisocytosis increases with COVID-19 severity and need for hospitalization. Although other inflammatory markers such as lymphocyte count, neutrophil/lymphocyte ratio, C-reactive protein, and cytokines correlate with disease severity, these parameters were not as sensitive as MDW for identifying severe disease in children. An MDW threshold of 23 offers a sensitive marker for severe pediatric COVID-19, with improved accuracy when assessed in combination with other hematologic parameters. Conclusion: Monocyte anisocytosis corresponds with shifting hematologic profiles and inflammatory markers in children with COVID-19, and MDW serves as a clinically accessible biomarker for severe COVID-19 in children.

Self-extinguishing relay waves enable homeostatic control of human neutrophil swarming.

Neutrophils exhibit self-amplified swarming to sites of injury and infection. How swarming is controlled to ensure the proper level of neutrophil recruitment is unknown. Using an ex vivo model of infection, we find that human neutrophils use active relay to generate multiple pulsatile waves of swarming signals. Unlike classic active relay systems such as action potentials, neutrophil swarming relay waves are self-extinguishing, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-extinguishing behavior. Through this circuit, neutrophils adjust the number and size of swarming waves for homeostatic levels of cell recruitment over a wide range of initial cell densities. We link a broken homeostat to neutrophil over-recruitment in the context of human chronic granulomatous disease.

Fusobacterium nucleatum dissemination by neutrophils.

Recent studies uncovered that Fusobacterium nucleatum (Fn), a common, opportunistic bacterium in the oral cavity, is associated with a growing number of systemic diseases, ranging from colon cancer to Alzheimer's disease. However, the pathological mechanisms responsible for this association are still poorly understood. Here, we leverage recent technological advances to study the interactions between Fn and neutrophils. We show that Fn survives within human neutrophils after phagocytosis. Using in vitro microfluidic devices, we determine that human neutrophils can protect and transport Fn over large distances. Moreover, we validate these observations in vivo by showing that neutrophils disseminate Fn using a zebrafish model. Our data support the emerging hypothesis that bacterial dissemination by neutrophils is a mechanistic link between oral and systemic diseases. Furthermore, our results may ultimately lead to therapeutic approaches that target specific host-bacteria interactions, including the dissemination process.

Microfluidic Devices for Precise Measurements of Cell Directionality Reveal a Role for Glutamine during Cell Migration.

Cancer cells that migrate from tumors into surrounding tissues are responsible for cancer dissemination through the body. Microfluidic devices have been instrumental in discovering unexpected features of cancer cell migration, including the migration in self-generated gradients and the contributions of cell-cell contact during collective migration. Here, we design microfluidic channels with five successive bifurcations to characterize the directionality of cancer cell migration with high precision. We find that the directional decisions of cancer cells moving through bifurcating channels in response to self-generated epidermal growth factor (EGF) gradients require the presence of glutamine in the culture media. A biophysical model helps quantify the contribution of glucose and glutamine to cancer cell orientation during migration in self-generated gradients. Our study uncovers an unexpected interplay between cancer cell metabolism and cancer cell migration studies and may eventually lead to new ways to delay cancer cell invasion.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

Passive redirection filters minimize red blood cell contamination during neutrophil chemotaxis assays using whole blood.

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.

Leading edge competition promotes context-dependent responses to receptor inputs to resolve directional dilemmas in neutrophil migration.

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.

Neutrophil profiles of pediatric COVID-19 and multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory illness characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigenemia, cytokine storm, and immune dysregulation. In severe COVID-19, neutrophil activation is central to hyperinflammatory complications, yet the role of neutrophils in MIS-C is undefined. Here, we collect blood from 152 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 78 pediatric controls. We find that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism that is distinct from the neutrophil interferon-stimulated gene (ISG) response we observe in pediatric COVID-19. Moreover, we observe extensive spontaneous neutrophil extracellular trap (NET) formation in MIS-C, and we identify neutrophil activation and degranulation signatures. Mechanistically, we determine that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Our findings suggest that hyperinflammatory presentation during MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia, driven by uncontrolled neutrophil activation and NET release in the vasculature.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect.

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.

Transcellular biosynthesis of leukotriene B4 orchestrates neutrophil swarming to fungi.

Neutrophil swarming is an emergent host defense mechanism triggered by targets larger than a single neutrophil's capacity to phagocytose. Swarming synergizes neutrophil functions, including chemotaxis, phagocytosis, and reactive oxygen species (ROS) production, and coordinates their deployment by many interacting neutrophils. The potent inflammatory lipid mediator leukotriene B4 (LTB4) has been established as central to orchestrating neutrophil activities during swarming. However, the details regarding how this eicosanoid choreographs the neutrophils involved in swarming are not well explained. Here we leverage microfluidics, genetically deficient mouse cells, and targeted metabolipidomic analysis to demonstrate that transcellular biosynthesis occurs among neutrophils to generate LTB4. Furthermore, transcellular biosynthesis is an entirely sufficient means of generating LTB4 for the purposes of orchestrating neutrophil swarming. These results further our understanding of how neutrophils coordinate their activities during swarming, which will be critical in the design of eventual therapies that can harness the power of swarming behavior.