The Functional Immune Response of Patients on Extracorporeal Life Support.

Extracorporeal life support (ECLS) is a widely used lifesaving technology. Whether ECLS results in immune dysregulation is unclear. This study's aim was to examine whether ECLS affected innate immune response. All patients placed on ECLS were eligible. Blood was obtained before, during, and after ECLS. Function of the innate immune system was measured by ex vivo lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and plasma cytokine levels (interleukin [IL]-6, IL-8, IL-10, and TNF-α). Immunoparalysis was defined as ex vivo TNF-α levels less than 200 pg/ml. Nineteen patients were enrolled with twelve <18 years old. Median ECLS duration was 10 days (range: 3-108); nine patients died. After stratifying the cohort by the presence of immunoparalysis before ECLS, those immunoparalyzed showed increased response to LPS on days 1 and 3 (p = 0.016). Those without pre-ECLS immunoparalysis showed a transient decrease in response on day 3 (p = 0.008). Plasma IL-10 levels were elevated in those with pre-ECLS immunoparalysis and dropped significantly by day 1 (p = 0.031). The number treated with steroids was similar in the two groups. In conclusion, patients with immunoparalysis before ECLS showed a gradual increase in immune function during ECLS, whereas those without immunoparalysis had a transient decrease in responsiveness on day 3.

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-1ß, 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.

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/ß, 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/ß hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-ß/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/ß production compared with control BMDMs. Serum IFN-ß 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-ß-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.

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

Immunomodulatory drugs-agents regulating the immune response-are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 µL), short assay time (∼30 min), heightened sensitivity (∼20-30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment.

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.

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.

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.

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.