Walker McHugh

Multiplex Serum Cytokine Immunoassay Using Nanoplasmonic Biosensor Microarrays

Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5-20 pg/mL from a 1 μL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min.

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

Mitogen-activated protein kinase phosphatase 2, MKP-2, regulates early inflammation in acute lung injury

Acute lung injury (ALI) is mediated by an early proinflammatory response resulting from either a direct or indirect insult to the lung mediating neutrophil infiltration and consequent disruption of the alveolar capillary membrane ultimately leading to refractory hypoxemia. The mitogen-activated protein kinase (MAPK) pathways are a key component of the molecular response activated by those insults triggering the proinflammatory response in ALI. The MAPK pathways are counterbalanced by a set of dual-specific phosphatases (DUSP) that deactivate the kinases by removing phosphate groups from tyrosine or threonine residues. We have previously shown that one DUSP, MKP-2, regulates the MAPK pathway in a model of sepsis-induced inflammation; however, the role of MKP-2 in modulating the inflammatory response in ALI has not been previously investigated. We utilized both MKP-2-null (MKP-2(-/-)) mice and MKP-2 knockdown in a murine macrophage cell line to elucidate the role of MKP-2 in regulating inflammation during ALI. Our data demonstrated attenuated proinflammatory cytokine production as well as decreased neutrophil infiltration in the lungs of MKP-2(-/-) mice following direct, intratracheal LPS. Importantly, when challenged with a viable pathogen, this decrease in neutrophil infiltration did not impact the ability of MKP-2(-/-) mice to clear either gram-positive or gram-negative bacteria. Furthermore, MKP-2 knockdown led to an attenuated proinflammatory response and was associated with an increase in phosphorylation of ERK and induction of a related DUSP, MKP-1. These data suggest that altering MKP-2 activity may have therapeutic potential to reduce lung inflammation in ALI without impacting pathogen clearance.

AC Electroosmosis-Enhanced Nanoplasmofluidic Detection of Ultralow-Concentration Cytokine

Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1β, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.

Profiling Inflammatory Responses with Microfluidic Immunoblotting

Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/β, NF-κB p65) in bone marrow–derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout–induced IKKα/β hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain–containing adaptor inducing IFN-β/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/β production compared with control BMDMs. Serum IFN-β levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain–containing adaptor inducing IFN-β–dependent pathways.

Protein phosphatase 2A activation attenuates inflammation in murine models of acute lung injury

Acute respiratory distress syndrome (ARDS) remains a leading cause of morbidity and mortality in both adult and pediatric intensive care units. A key event in the development of ARDS is neutrophil recruitment into the lungs leading to tissue damage and destruction. Interleukin-8 (IL-8) is the major human chemokine responsible for neutrophil recruitment into the lungs. Protein phosphatase 2A (PP2A) has been shown to be a key regulator of the mitogen-activated protein kinase (MAPK) cascades, which control the production of IL-8. Previously, our laboratory employed an in vitro model to show that inhibition of PP2A results in an increase in IL-8 production in human alveolar epithelial cells. The objective of this study was to determine whether PP2A regulated this response in vivo by investigating the impact of pharmacologic activation of PP2A on chemokine production and activation of the MAPK cascade and lung injury using endotoxin- and bacterial-challenge models of ARDS in mice. N6-cyclopentyladenosine (N6-CPA) increased PP2A activity and inhibited endotoxin-induced cytokine production in a murine alveolar macrophage cell line. N6-CPA pretreatment in mice challenged with intratracheal endotoxin decreased chemokine production, reduced neutrophil infiltration, and attenuated lung injury. Following initiation of lung injury with live Pseudomonas aeruginosa, mice that received N6-CPA 4 h following bacterial challenge showed attenuated chemokine production and reduced neutrophil infiltration compared with control mice. Pharmacologic PP2A activation both limited and prevented inflammation and tissue injury in two direct injury models of ARDS. These results suggest modulation of PP2A activity as a therapeutic target in ARDS.

1394: PERSONALIZED IMMUNOMODULATION IN HIGH-RISK SEPSIS PATIENTS IN A PEDIATRIC CRITICAL CARE UNIT.

Learning Objectives: Sepsis is the leading cause of death in children worldwide. Our approach to treating critically-ill children in the U.S. is essentially unchanged in over 30 years, and primarily relies on a one-size-fits-all approach of fluid resuscitation, hemodynamic support, and appropriate antimicrobial therapy. Our group has developed a rapid immunoassay platform that enables the rapid, simultaneous quantification of multiple serum biomarkers from a single drop of blood, enabling near real-time precision medicine in the intensive care unit. Methods: This study was approved by the University of Michigan IRB. Patients aged 0–18 admitted to the pediatric intensive care unit meeting criteria for systemic inflammatory response syndrome (SIRS) were enrolled within 12 hours of admission to the PICU following informed consent. Serum samples were obtained within 24 hours of admission. Risk stratification classification was performed using the PERSEVERE risk stratification biomarker algorithm and the concentration of targetable immune mediators (existing FDA approved or experimental therapeutics currently in clinical trials) were determined using commercially available immunoassays or our group’s rapid MicroKine assay platform. Results: 40 patients were enrolled with 7 being classified as high-risk (>40% mortality risk) and 33 being classified as low-risk (<1% mortality risk). We found statisticallysignificant increases in 28-day mortality (5/7 vs. 0/30, p<0.0001) ICU lengths of stay (median = 28.0 vs 12.3 days, p-value=0.0012), and total hospitalization charges per day (

623: MULTIPLEXED, TEMPORAL IMMUNOPHENOTYPING WITH MICROFLUIDIC-BASED LOCALIZED SURFACE PLASMON RESONANCE

20,904 vs. 12,892, p=0.0021) in patients classified as highrisk compared to low-risk. Analysis of targetable immune mediators revealed statistically-significant differences in all targets (IL-1α, IL-1β, IL-6, IL-7, IL-10, IFNγ, TNFα, G-CSF, GM-CSF) except IL-13 between high-risk and low-risk patients. Conclusions: Analysis of the targetable immune mediators in individual high-risk patients reveals significant variability and supports the need for a precision-medicine based immunomodulatory approach to impact patient outcomes.

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

Learning Objectives: Immunomodulatory agents that regulate the immune response are commonly used to treat immune system disorders and minimize host-vs.-graft disease in organ transplant recipients. At the cellular level, immunosuppressant drugs, titrated by serum levels, are used to inhibit pro-inflammatory or tissue-damaging cell responses. Few studies have precisely characterized the cellular-level effect of immunomodulatory treatment due to several challenges, among them sample volumes, assay time and sensitivity, and multiplex capacity. We hypothesized that novel methodologies would overcome such limitations. Methods: We designed a microfluidic platform integrating localized-surface plasmon resonance (LSPR) with antibody-conjugated gold nanorod arrays in order to measure simultaneous, multi-time-point levels of pro-(IL-2, IFN-, TNF-α) and anti-inflammatory (IL-10) cytokines secreted by immune cells in the presence or absence of an immunomodulating agent, tacrolimus. Results: The device achieved precise measurements with low operating sample volumes (1 μL), short assay time (30 min), heightened sensitivity (~ 20-30 pg/mL), and negligible crosstalk. The limits of detection were: 31 pg/mL, 26 pg/mL, 35 pg/mL, and 21 pg/mL for IL-2, IFN-, TNF-α, and IL-10, respectively. An excellent correlation between measurements from the LSPR biosensor assay and ELISA was obtained (R2 = 0.931) across a dynamic range of concentrations. Secretion curves for IL-2, IFN-, TNF-α, and IL-10 clearly demonstrated activated and immune suppressed states of Jurkat cells that were sensitive to different concentrations of tacrlimus. An immediate reduction of IL-2 and IFN-Υ secretion was seen after their peak value by 10 and 30 mins after 1 and 10 ng/mL, respectively while 0.1 ng/mL did not cease IL-2 cytokine secretion. Conclusions: This nanoplasmonic biosensor microarray can define temporal cytokine secretion profiles of immune cells under immunosuppressive modulation. The platform is rapid, sensitive, and affords multiplexed, multi-time-point detection to enable characterization of dynamic features of an immune cell’s functional response.