Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil's ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

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.

Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood.

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.

Detergent Alternatives: Membrane Protein Purification Using Synthetic Nanodisc Polymers.

The development of styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers provides an alternative to traditional detergent extraction of integral membrane proteins. By inserting into the membrane, these polymers can extract membrane proteins along with lipids in the form of native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. Unlike detergent solubilization, where membrane proteins may lose annular lipids necessary for proper folding and stability, native nanodiscs allow for proteins to reside in the natural lipid environment. In addition, polymer-based nanodiscs can be purified using common chromatography methods similar to protocols established with detergent solubilization purification. Here we describe the solubilization screening and purification of an integral membrane protein using several commercial copolymers.

Neutrophil dysfunction in cystic fibrosis.

BACKGROUND: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms. METHODS: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT). RESULTS: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0001). When comparing trans-epithelial migration towards Pseudomonas aeruginosa epithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05). CONCLUSIONS: Advanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.

Microfluidic arenas for war games between neutrophils and microbes.

Measurements of neutrophil activities such as cell migration and phagocytosis are generally performed using low-content bulk assays, which provide little detail activity at the single cell level, or flow cytometry methods, which have the single cell resolution but lack perspective on the kinetics of the process. Here, we present a microfluidic assay for measuring the essential functions that contribute to the antimicrobial activity of neutrophils: migration towards the target, and killing of microbes. The assay interrogates the interactions between isolated human neutrophils and populations of live, proliferating microbes. The outcome is measured in a binary mode that is reflective of in vivo infections, which are either cleared or endure the host response. The outcome of the interactions is also characterized at single cell resolution for both the neutrophils and the microbes. We applied the assay to test the response of neutrophils from intensive care patients to live Staphylococcus aureus, and observed alterations of antimicrobial neutrophil activity in patients, including those with sepsis. By directly measuring neutrophil activity against live targets at high spatial and temporal resolution, this assay provides unique insights into the life-or-death contest shaping the outcome of interactions between populations of neutrophils and microbes.

Measuring spontaneous neutrophil motility signatures from a drop of blood using microfluidics.

Neutrophils play an essential role in the protection against infection, as they are the most numerous circulating white blood cell population and the first responders to injury. Their numbers in blood are frequently measured in the clinic and used as an indicator of ongoing infections. During inflammation and sepsis, the ability of neutrophils to migrate is disrupted, which may increase the risk of infection, even when the neutrophil count is normal. However, measurements of neutrophil migration in patients are rarely performed because of the challenges of performing the migration assays in a clinical setting. Here, we describe a microfluidic assay that measures the spontaneous neutrophil migration signatures associated with sepsis. The assay uses one droplet of patient's blood in a microfluidic device, which circumvents the need for neutrophil isolation from blood. This assay may also be useful for the study of the effect of various immune modulators on neutrophil migration behavior from healthy volunteers and patients.

Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay.

Current methods for the diagnosis of sepsis have insufficient precision, causing regular misdiagnoses. Microbiological tests can help diagnose sepsis but are usually too slow to have an impact on timely clinical-decision making. Neutrophils have high sensitivity to infections, yet measurements of neutrophil surface markers, genomic changes, and phenotype alterations have had only a marginal effect on sepsis diagnosis. Here, we report a microfluidic assay that measures the spontaneous motility of neutrophils in the context of plasma, in one droplet of blood. We measured the performance of the assay in two independent cohorts of critically ill patients suspected of sepsis. In the first cohort, we developed a machine-learning-based scoring system (sepsis score) that segregated patients with sepsis from those without sepsis. In the second cohort, we validated the sepsis score in a double-blinded, prospective case-control study. For the 42 patients across the two cohorts, the assay identified sepsis patients with 97% sensitivity and 98% specificity. The neutrophil assay could potentially be used to accurately diagnose and monitor sepsis in larger populations of at-risk patients.