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.

The Gut-Lung Axis in Systemic Inflammation. Role of Mesenteric Lymph as a Conduit.

Emerging evidence shows that after injury or infection, the mesenteric lymph acts as a conduit for gut-derived toxic factors to enter the blood circulation, causing systemic inflammation and acute lung injury. Neither the cellular and molecular identity of lymph factors nor their mechanisms of action have been well understood and thus have become a timely topic of investigation. This review will first provide a summary of background knowledge on gut barrier and mesenteric lymphatics, followed by a discussion focusing on the current understanding of potential injurious factors in the lymph and their mechanistic contributions to lung injury. We also examine lymph factors with antiinflammatory properties as well as the bidirectional nature of the gut-lung axis in inflammation.

Extracellular vesicles: new players in regulating vascular barrier function.

Extracellular vesicles (EVs) have attracted rising interests in the cardiovascular field not only because they serve as serological markers for circulatory disorders but also because they participate in important physiological responses to stress and inflammation. In the circulation, these membranous vesicles are mainly derived from blood or vascular cells, and they carry cargos with distinct molecular signatures reflecting the origin and activation state of parent cells that produce them, thus providing a powerful tool for diagnosis and prognosis of pathological conditions. Functionally, circulating EVs mediate tissue-tissue communication by transporting bioactive cargos to local and distant sites, where they directly interact with target cells to alter their function. Recent evidence points to the critical contributions of EVs to the pathogenesis of vascular endothelial barrier dysfunction during inflammatory response to injury or infection. In this review, we provide a brief summary of the current knowledge on EV biology and advanced techniques in EV isolation and characterization. This is followed by a discussion focusing on the role and mechanisms of EVs in regulating blood-endothelium interactions and vascular permeability during inflammation. We conclude with a translational perspective on the diagnostic and therapeutic potential of EVs in vascular injury or infectious diseases, such as COVID-19.

Citrullinated histone 3 causes endothelial barrier dysfunction.

Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy.

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 µm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.

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.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels.

OBJECTIVES: The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs. METHODS: We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 µM) or/and histamine production by α-MHD (10 µM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos. RESULTS: We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells. CONCLUSIONS: This study supports a role for histamine as an EDRF in MLVs.

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.

Protein palmitoylation regulates extracellular vesicle production and function in sepsis.

Extracellular vesicles (EVs) are bioactive membrane-encapsulated particles generated by a series of events involving membrane budding, fission and fusion. Palmitoylation, mediated by DHHC palmitoyl acyltransferases, is a lipidation reaction that increases protein lipophilicity and membrane localization. Here, we report palmitoylation as a novel regulator of EV formation and function during sepsis. Our results showed significantly decreased circulating EVs in mice with DHHC21 functional deficiency (Zdhhc21dep/dep), compared to wild-type (WT) mice 24 h after septic injury. Furthermore, WT and Zdhhc21dep/dep EVs displayed distinct palmitoyl-proteomic profiles. Ingenuity pathway analysis indicated that sepsis altered several inflammation related pathways expressed in EVs, among which the most significantly activated was the complement pathway; however, this sepsis-induced complement enrichment in EVs was greatly blunted in Zdhhc21dep/dep EVs. Functionally, EVs isolated from WT mice with sepsis promoted neutrophil adhesion, transmigration, and neutrophil extracellular trap production; these effects were significantly attenuated by DHHC21 loss-of-function. Furthermore, Zdhhc21dep/dep mice displayed reduced neutrophil infiltration in lungs and improved survival after CLP challenges. These findings indicate that blocking palmitoylation via DHHC21 functional deficiency can reduce sepsis-stimulated production of complement-enriched EVs and attenuates their effects on neutrophil activity.

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.