Emerging Approaches to Understanding Microvascular Endothelial Heterogeneity: A Roadmap for Developing Anti-Inflammatory Therapeutics

The endothelium is the inner layer of all blood vessels and it regulates hemostasis. It also plays an active role in the regulation of the systemic inflammatory response. Systemic inflammatory disease often results in alterations in vascular endothelium barrier function, increased permeability, excessive leukocyte trafficking, and reactive oxygen species production, leading to organ damage. Therapeutics targeting endothelium inflammation are urgently needed, but strong concerns regarding the level of phenotypic heterogeneity of microvascular endothelial cells between different organs and species have been expressed. Microvascular endothelial cell heterogeneity in different organs and organ-specific variations in endothelial cell structure and function are regulated by intrinsic signals that are differentially expressed across organs and species; a result of this is that neutrophil recruitment to discrete organs may be regulated differently. In this review, we will discuss the morphological and functional variations in differently originated microvascular endothelia and discuss how these variances affect systemic function in response to inflammation. We will review emerging in vivo and in vitro models and techniques, including microphysiological devices, proteomics, and RNA sequencing used to study the cellular and molecular heterogeneity of endothelia from different organs. A better understanding of microvascular endothelial cell heterogeneity will provide a roadmap for developing novel therapeutics to target the endothelium

Distinct functional neutrophil phenotypes in sepsis patients correlate with disease severity

PurposeSepsis is a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis is a highly heterogeneous syndrome with distinct phenotypes that impact immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct functional phenotypes of immune cells. In this study, we utilized our Organ-on-Chip assay to categorize sepsis patients into distinct phenotypes using patient data, neutrophil functional analysis, and proteomics.MethodsFollowing informed consent, neutrophils and plasma were isolated from sepsis patients in the Temple University Hospital ICU (n=45) and healthy control donors (n=7). Human lung microvascular endothelial cells (HLMVEC) were cultured in the Organ-on-Chip and treated with buffer or cytomix ((TNF/IL-1β/IFNγ). Neutrophil adhesion and migration across HLMVEC in the Organ-on-Chip were used to categorize functional neutrophil phenotypes. Quantitative label-free global proteomics was performed on neutrophils to identify differentially expressed proteins. Plasma levels of sepsis biomarkers and neutrophil extracellular traps (NETs) were determined by ELISA.ResultsWe identified three functional phenotypes in critically ill ICU sepsis patients based on ex vivo neutrophil adhesion and migration patterns. The phenotypes were classified as: Hyperimmune characterized by enhanced neutrophil adhesion and migration, Hypoimmune that was unresponsive to stimulation, and Hybrid with increased adhesion but blunted migration. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group demonstrated higher oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. Patients with the Hyperimmune neutrophil phenotype had significantly increased circulating neutrophils and elevated plasma levels NETs.ConclusionNeutrophils and NETs play a critical role in vascular barrier dysfunction in sepsis and elevated NETs may be a key biomarker identifying the Hyperimmune group. Our results establish significant associations between specific neutrophil functional phenotypes and disease severity and identify important functional parameters in sepsis pathophysiology that may provide a new approach to classify sepsis patients for specific therapeutic interventions

Prasugrel Metabolites Inhibit Neutrophil Functions s

ABSTRACT Clopidogrel and prasugrel belong to a thienopyridine class of oral antiplatelet drugs that, after having been metabolized in the liver, can inhibit platelet function by irreversibly antagonizing the P2Y 12 receptor. Furthermore, thienopyridines influence numerous inflammatory conditions, but their effects on neutrophils have not been evaluated, despite the important role of these cells in inflammation. Therefore, we investigated the effect of prasugrel metabolites on neutrophils to further clarify the role of thienopyridines in inflammation. Interestingly, a prasugrel metabolite mixture, produced in vitro using rat liver microsomes, significantly inhibited N-formyl-methionyl-leucylphenylalanine (fMLP)-and platelet-activating factor (PAF)-induced neutrophil activation. More specifically, prasugrel metabolites inhibited neutrophil transmigration, CD16 surface expression, and neutrophil-platelet aggregation. Moreover, prasugrel metabolite pretreatment also significantly decreased fMLP-or PAF-induced extracellular-signal-regulated kinase phosphorylation as well as calcium mobilization. To determine the target of prasugrel in neutrophils, the role of both P2Y 12 and P2Y 13 receptors was studied using specific reversible antagonists, AR-C69931MX and MRS2211, respectively. Neither antagonist had any direct effect on the agonist-induced neutrophil functional responses. Our findings indicate that prasugrel metabolites may directly target neutrophils and inhibit their activation, suggesting a possible explanation for their antiinflammatory effects previously observed. However, these metabolites do not act through either the P2Y 12 or P2Y 13 receptor in neutrophils

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

The Role of Tyrosine Phosphorylation of Protein Kinase C Delta in Infection and Inflammation

Protein Kinase C (PKC) is a family composed of phospholipid-dependent serine/threonine kinases that are master regulators of inflammatory signaling. The activity of different PKCs is context-sensitive and these kinases can be positive or negative regulators of signaling pathways. The delta isoform (PKCδ) is a critical regulator of the inflammatory response in cancer, diabetes, ischemic heart disease, and neurodegenerative diseases. Recent studies implicate PKCδ as an important regulator of the inflammatory response in sepsis. PKCδ, unlike other members of the PKC family, is unique in its regulation by tyrosine phosphorylation, activation mechanisms, and multiple subcellular targets. Inhibition of PKCδ may offer a unique therapeutic approach in sepsis by targeting neutrophil-endothelial cell interactions. In this review, we will describe the overall structure and function of PKCs, with a focus on the specific phosphorylation sites of PKCδ that determine its critical role in cell signaling in inflammatory diseases such as sepsis. Current genetic and pharmacological tools, as well as in vivo models, that are used to examine the role of PKCδ in inflammation and sepsis are presented and the current state of emerging tools such as microfluidic assays in these studies is described

Interaction entre l’article et l’adjectif dans le marquage de la focalisation au sein des propositions attributives

International audienceThis paper deals with noun phrases in French by addressing the distribution of the focalization between the noun and the adjective within the NP as well as the communicative status, more or less focalized, of the NP within the utterance. The analysis of a set of examples with attributive structure, taking into account different kinds of indicators allowing to grasp the communicative goal of the utterance, shows that an interaction exists between qualification and determination in marking the communicative status of the attributive noun phrase.Cet article aborde les syntagmes nominaux en s’intéressant à la distribution de la focalisation entre le nom et l’adjectif au sein du SN ainsi qu’au statut communicatif, plus au moins focalisé, du SN au sein de l’énoncé. L’analyse d’une sélection d’exemples avec une structure attributive, attentive à différents indicateurs permettant de saisir le but communicatif de l’énoncé, montre qu’il existe une interaction entre la qualification et la détermination dans le marquage du statut communicatif du SN attribut

Protein kinase C-delta inhibition protects blood-brain barrier from sepsis-induced vascular damage

Abstract Background Neuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKCδ in the control of individual steps in neuroinflammation during sepsis. Methods The role of PKCδ in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip (B3C) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 μM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo. Results During sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKCδ in regulating neutrophil transmigration. Conclusions The BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage

Treatment with the PKCδ inhibitor decreased sepsis-induced platelet-leukocyte aggregate formation.

<p>Blood samples were incubated with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression and cell shape and data were analyzed as a percentage of aggregates expressing both CD61 and CD11b (<b>A</b>) Representative dot plot images of percentage of neutrophil-platelet aggregates in rat blood samples of Sham, CLP plus vehicle and CLP plus PKCδ inhibitor. (<b>B</b>) Graph showing the percentage of aggregate formation of the data represented in (A) (*p <0.05 sham versus CLP, **<i>p</i> < 0.01; CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 4).</p

PKCδ inhibitor decreases sepsis-mediated activation of platelet PKCδ.

<p>Rat platelets were isolated from blood samples of the following groups: Sham, vehicle-treated CLP and CLP treated with with PKCδ inhibitor. PKD2 phosphorylation was measured by Western Blotting analysis using phospho-specific antibody against PKD2. A representative image (top) and the ratio between phospho-PKD2 and loading control band (bottom) are shown. (*<i>p</i> < 0.05; Sham versus CLP plus vehicle; CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 5).</p

Treatment with the PKCδ inhibitor alters platelet secretion into the lungs of septic rats.

<p>PF4 concentration in the BALF was analyzed by ELISA. Data are expressed as International Units (IU)/ml (*<i>p</i> < 0.05; Sham versus CLP plus vehicle and CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 6).</p