Burn Injury-Induced Extracellular Vesicle Production and Characteristics.

ABSTRACT: Extracellular vesicles (EVs) are nano-sized membrane-bound particles containing biologically active cargo molecules. The production and molecular composition of EVs reflect the physiological state of parent cells, and once released into the circulation, they exert pleiotropic functions via transferring cargo contents. Thus, circulating EVs not only serve as biomarkers, but also mediators in disease processes or injury responses. In the present study, we performed a comprehensive analysis of plasma EVs from burn patients and healthy subjects, characterizing their size distribution, concentration, temporal changes, cell origins, and cargo protein contents. Our results indicated that burn injury induced a significant increase in circulating EVs, the response peaked at the time of admission and declined over the course of recovery. Importantly, EV production correlated with injury severity, as indicated by the total body surface area and depth of burn, requirement for critical care/ICU stay, hospitalization length, wound infection, and concurrence of sepsis. Burn patients with inhalation injury showed a higher level of EVs than those without inhalation injury. We also evaluated patient demographics (age and sex) and pre-existing conditions (hypertension, obesity, and smoking) and found no significant correlation between these conditions and overall EV production. At the molecular level, flow cytometric analysis showed that the burn-induced EVs were largely derived from leukocytes and endothelial cells (ECs), which are known to be activated postburn. Additionally, a high level of zona-occludens-1 (ZO-1), a major constituent of tight junctions, was identified in burn EV cargos, indicative of injury in tissues that form barriers via tight junctions. Moreover, when applied to endothelial cell monolayers, burn EVs caused significant barrier dysfunction, characterized by decreased transcellular barrier resistance and disrupted cell-cell junction continuity. Taken together, these data suggest that burn injury promotes the production of EVs containing unique cargo proteins in a time-dependent manner; the response correlates with injury severity and worsened clinical outcomes. Functionally, burn EVs serve as a potent mediator capable of reducing endothelial barrier resistance and impairing junction integrity, a pathophysiological process underlying burn-associated tissue dysfunction. Thus, further in-depth characterization of circulating EVs will contribute to the development of new prognostic tools or therapeutic targets for advanced burn care.

Circulating lymphocyte trafficking to the bone marrow contributes to lymphopenia in myocardial infarction.

Some patients with myocardial infarction (MI) exhibit lymphopenia, a reduction in blood lymphocyte count. Moreover, lymphopenia inversely correlates with patient prognosis. The objective of this study was to elucidate the underlying mechanisms that cause lymphopenia after MI. Multiparameter flow cytometric analysis demonstrated that MI induced profound B and T lymphopenia in a mouse model, peaking at day 1 post-MI. The finding that non-MI control and MI mice exhibited similar apoptotic rate for blood B and T lymphocytes argues against apoptosis being essential for MI-induced lymphopenia. Interestingly, the bone marrow in day 1 post-MI mice contained more B and T cells but showed less B- and T-cell proliferation compared with day 0 controls. This suggests that blood lymphocytes may travel to the bone marrow after MI. This was confirmed by adoptive transfer experiments demonstrating that MI caused the loss of transferred lymphocytes in the blood, but the accumulation of transferred lymphocytes in the bone marrow. To elucidate the underlying signaling pathways, ß2-adrenergic receptor or sphingosine-1-phosphate receptor type 1 (S1PR1) was pharmacologically blocked, respectively. ß2-receptor inhibition had no significant effect on blood lymphocyte count, whereas S1PR1 blockade aggravated lymphopenia in MI mice. Furthermore, we discovered that MI-induced glucocorticoid release triggered lymphopenia. This was supported by the findings that adrenalectomy (ADX) completely prevented mice from MI-induced lymphopenia, and supplementation with corticosterone in adrenalectomized MI mice reinduced lymphopenia. In conclusion, our study demonstrates that MI-associated lymphopenia involves lymphocyte redistribution from peripheral blood to the bone marrow, which is mediated by glucocorticoids.NEW & NOTEWORTHY Lymphopenia, a reduction in blood lymphocyte count, is known to inversely correlate with the prognosis for patients with myocardial infarction (MI). However, the underlying mechanisms by which cardiac ischemia induces lymphopenia remain elusive. This study provides the first evidence that MI activates the hypothalamic-pituitary-adrenal (HPA) axis to increase glucocorticoid secretion, and elevated circulating glucocorticoids induce blood lymphocytes trafficking to the bone marrow, leading to lymphopenia.

Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation.

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Protein Palmitoylation in Leukocyte Signaling and Function.

Palmitoylation is a post-translational modification (PTM) based on thioester-linkage between palmitic acid and the cysteine residue of a protein. This covalent attachment of palmitate is reversibly and dynamically regulated by two opposing sets of enzymes: palmitoyl acyltransferases containing a zinc finger aspartate-histidine-histidine-cysteine motif (PAT-DHHCs) and thioesterases. The reversible nature of palmitoylation enables fine-tuned regulation of protein conformation, stability, and ability to interact with other proteins. More importantly, the proper function of many surface receptors and signaling proteins requires palmitoylation-meditated partitioning into lipid rafts. A growing number of leukocyte proteins have been reported to undergo palmitoylation, including cytokine/chemokine receptors, adhesion molecules, pattern recognition receptors, scavenger receptors, T cell co-receptors, transmembrane adaptor proteins, and signaling effectors including the Src family of protein kinases. This review provides the latest findings of palmitoylated proteins in leukocytes and focuses on the functional impact of palmitoylation in leukocyte function related to adhesion, transmigration, chemotaxis, phagocytosis, pathogen recognition, signaling activation, cytotoxicity, and cytokine production.

Non-muscle Mlck is required for ß-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1ß-mediated barrier dysfunction in brain endothelial cells.

Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.

Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels.

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ∼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.