Glucocorticoids impair T lymphopoiesis after myocardial infarction.

The thymus, where T lymphocytes develop and mature, is sensitive to insults such as tissue ischemia or injury. The insults can cause thymic atrophy and compromise T-cell development, potentially impairing adaptive immunity. The objective of this study was to investigate whether myocardial infarction (MI) induces thymic injury to impair T lymphopoiesis and to uncover the underlying mechanisms. When compared with sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, as well as less thymocytes at different developmental stages, indicative of T-lymphopoiesis impairment following MI. Accordingly, the spleen of MI mice has less T cells and recent thymic emigrants (RTEs), implying that the thymus of MI mice releases fewer mature thymocytes than sham controls. Interestingly, the secretory function of splenic T cells was not affected by MI. Further experiments showed that the reduction of thymocytes in MI mice was due to increased thymocyte apoptosis. Removal of adrenal glands by adrenalectomy (ADX) prevented MI-induced thymic injury and dysfunction, whereas corticosterone supplementation in ADX + MI mice reinduced thymic injury and dysfunction, indicating that glucocorticoids mediate thymic damage triggered by MI. Eosinophils play essential roles in thymic regeneration postirradiation, and eosinophil-deficient mice exhibit impaired thymic recovery after sublethal irradiation. Interestingly, the thymus was fully regenerated in both wild-type and eosinophil-deficient mice at day 14 post-MI, suggesting that eosinophils are not critical for thymus regeneration post-MI. In conclusion, our study demonstrates that MI-induced glucocorticoids trigger thymocyte apoptosis and impair T lymphopoiesis, resulting in less mature thymocyte release to the spleen.NEW & NOTEWORTHY The thymus is essential for maintaining whole body T-cell output. Thymic injury can adversely affect T lymphopoiesis and T-cell immune response. This study demonstrates that MI induces thymocyte apoptosis and compromises T lymphopoiesis, resulting in fewer releases of mature thymocytes to the spleen. This process is mediated by glucocorticoids secreted by adrenal glands. Therefore, targeting glucocorticoids represents a novel approach to attenuate post-MI thymic injury.

The Impact of the COVID-19 Pandemic on Older Adults Who Participate in Group Physical Exercise Program.

The COVID pandemic exposed the vulnerability of older adults in myriad ways and social service organizations faced unprecedented challenges in safely providing support for older adults. Since 2007, Hawai'i Healthy Aging Partnership (HHAP) has offered Enhance®Fitness, an evidence-based program to reduce the risk of falls and promote health among older adults. Due to the pandemic, all the Enhance®Fitness sites had to close and stop offering the program. The HHAP started to provide alternative activities remotely in May 2020. To explore the pandemic's impact, the feasibility of online exercise programs, and the support needed among older adults to stay physically active, HHAP surveyed existing Enhance®Fitness participants and received 291 responses (59% response rate). The study used frequency distributions, comparison of means, and chi-square to analyze the survey data. Findings showed that the shutdown of the group exercise program during the pandemic led to a health status decline, a reduction in physical activities, and a shift from group to individual physical activities among older adult participants. Most respondents tried the remote exercise opportunities during the pandemic and would consider joining the remote programs in the future. However, about one-fourth of the respondents did not participate in remote exercise activities due to the lack of electronic devices, internet access, or interest in remote activity formats. To ensure equitable access to physical exercise programs for older adults in the post-pandemic era, it is critical to address the access challenges and resources needed for providing multiple programming options.

C-TYPE LECTIN-2D RECEPTOR CONTRIBUTES TO HISTONE-INDUCED VASCULAR BARRIER DYSFUNCTION DURING BURN INJURY.

ABSTRACT: Severe burns are associated with massive tissue destruction and cell death where nucleus histones and other damage-associated molecular patterns are released into the circulation and contribute to the pathogenesis of multiple-organ dysfunction. Currently, there is limited information regarding the pathophysiology of extracellular histones after burns, and the mechanisms underlying histone-induced vascular injury are not fully understood. In this study, by comparing the blood samples from healthy donors and burn patients, we confirmed that burn injury promoted the release of extracellular histones into the circulation, evidenced by increased plasma levels of histones correlating with injury severity. The direct effects of extracellular histones on human endothelial monolayers were examined, and the results showed that histones caused cell-cell adherens junction discontinuity and barrier dysfunction in a dose-related manner. Like burn patients, mice subjected to a scald burn covering 25% total body surface area also displayed significantly increased plasma histones. Intravital microscopic analysis of mouse mesenteric microcirculation indicated that treatment with a histone antibody greatly attenuated burn-induced plasma leakage in postcapillary venules, supporting the pathogenic role of extracellular histones in the development of microvascular barrier dysfunction during burns. At the molecular level, intrigued by the recent discovery of C-type lectin domain family 2 member D (Clec2d) as a novel receptor of histones, we tested its potential involvement in the histone interaction with endothelial cells. Indeed, we identified abundant expression of Clec2d in vascular endothelial cells. Further proximity ligation assay demonstrated a close association between extracellular histones and endothelial expressing Clec2d. Functionally, in vivo administration of an anti-Clec2d antibody attenuated burn-induced plasma leakage across mesenteric microvessels. Consistently, Clec2d knockdown in endothelial cells partially inhibited histone-induced endothelial barrier dysfunction. Together, our data suggest that burn injury-induced increases in circulating histones contribute to microvascular leakage and endothelial barrier dysfunction via a mechanism involving the endothelial Clec2d receptor.

Determination of Solute Permeability of Microvascular Endothelial Cell Monolayers In Vitro.

The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed.

Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

BACKGROUND: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. METHODS: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain. RESULTS: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. CONCLUSIONS: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.

Lung infection by P. aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

Background: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. Methods: Pneumonia was induced in adult C57BL/6 mice by intratracheal inoculation of Pseudomonas aeruginosa (PA). Solute extravasation, histology, immunofluorescence, RT-PCR, multiphoton imaging and neurological testing were performed in this study. Results: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 hours and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. Conclusions: These results suggest that lung bacterial infection causes cerebral microvascular leakage and neuroinflammation via a mechanism involving cytokine-induced BBB injury.

Cellular mechanisms underlying the impairment of macrophage efferocytosis.

The phagocytosis and clearance of dying cells by macrophages, a process termed efferocytosis, is essential for both maintaining homeostasis and promoting tissue repair after infection or sterile injury. If not removed in a timely manner, uncleared cells can undergo secondary necrosis, and necrotic cells lose membrane integrity, release toxic intracellular components, and potentially induce inflammation or autoimmune diseases. Efferocytosis also initiates the repair process by producing a wide range of pro-reparative factors. Accumulating evidence has revealed that macrophage efferocytosis defects are involved in the development and progression of a variety of inflammatory and autoimmune diseases. The underlying mechanisms of efferocytosis impairment are complex, disease-dependent, and incompletely understood. In this review, we will first summarize the current knowledge about the normal signaling and metabolic processes of macrophage efferocytosis and its importance in maintaining tissue homeostasis and repair. We then will focus on analyzing the molecular and cellular mechanisms underlying efferocytotic abnormality (impairment) in disease or injury conditions. Next, we will discuss the potential molecular targets for enhanced efferocytosis in animal models of disease. To provide a balanced view, we will also discuss some deleterious effects of efferocytosis.

Lung infection by P. aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

Background Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. Methods Pneumonia was induced in adult C57BL/6 mice by intratracheal inoculation of Pseudomonas aeruginosa (PA). Solute extravasation, histology, immunofluorescence, RT-PCR, multiphoton imaging and neurological testing were performed in this study. Results Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 hours and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. Conclusions These results suggest that lung bacterial infection causes cerebral microvascular leakage and neuroinflammation via a mechanism involving cytokine-induced BBB injury.

Endothelial glycocalyx-associated molecules as potential serological markers for sepsis-associated encephalopathy: A systematic review and meta-analysis.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is characterized by a diffuse cerebral dysfunction that accompanies sepsis in the absence of direct central nervous system infection. The endothelial glycocalyx is a dynamic mesh containing heparan sulfate linked to proteoglycans and glycoproteins, including selectins and vascular/intercellular adhesion molecules (V/I-CAMs), which protects the endothelium while mediating mechano-signal transduction between the blood and vascular wall. During severe inflammatory states, components of the glycocalyx are shed into the circulation and can be detected in soluble forms. Currently, SAE remains a diagnosis of exclusion and limited information is available on the utility of glycocalyx-associated molecules as biomarkers for SAE. We set out to synthesize all available evidence on the association between circulating molecules released from the endothelial glycocalyx surface during sepsis and sepsis-associated encephalopathy. METHODS: MEDLINE (PubMed) and EMBASE were searched since inception until May 2, 2022 to identify eligible studies. Any comparative observational study: i) evaluating the association between sepsis and cognitive decline and ii) providing information on level of circulating glycocalyx-associated molecules was eligible for inclusion. RESULTS: Four case-control studies with 160 patients met the inclusion criteria. Meta-analysis of biomarkers ICAM-1 (SMD 0.41; 95% CI 0.05-0.76; p = 0.03; I2 = 50%) and VCAM-1 (SMD 0.55; 95% CI 0.12-0.98; p = 0.01; I2 = 82%) revealed higher pooled mean concentration in patients with SAE compared to the patients with sepsis alone. Single studies reported elevated levels of P-selectin (MD 0.80; 95% CI -17.77-19.37), E-selectin (MD 96.40; 95% Cl 37.90-154.90), heparan sulfate NS2S (MD 19.41; 95% CI 13.37-25.46), and heparan sulfate NS+NS2S+NS6S (MD 67.00; 95% CI 31.00-103.00) in patients with SAE compared to the patients with sepsis alone. CONCLUSION: Plasma glycocalyx-associated molecules are elevated in SAE and may be useful for early identification of cognitive decline in sepsis patients.

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.

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.

The Endothelial Glycocalyx as a Double-Edged Sword in Microvascular Homeostasis and Pathogenesis.

Expressed on the endothelial cell (EC) surface of blood vessels, the glycocalyx (GCX), a mixture of carbohydrates attached to proteins, regulates the access of cells and molecules in the blood to the endothelium. Besides protecting endothelial barrier integrity, the dynamic microstructure of the GCX confers remarkable functions including mechanotransduction and control of vascular tone. Recently, a novel perspective has emerged supporting the pleiotropic roles of the endothelial GCX (eGCX) in cardiovascular health and disease. Because eGCX degradation occurs in certain pathological states, the circulating levels of eGCX degradation products have been recognized to have diagnostic or prognostic values. Beyond their biomarker roles, certain eGCX fragments serve as pathogenic factors in disease progression. Pharmacological interventions that attenuate eGCX degradation or restore its integrity have been sought. This review provides our current understanding of eGCX structure and function across the microvasculature in different organs. We also discuss disease or injury states, such as infection, sepsis and trauma, where eGCX dysfunction contributes to severe inflammatory vasculopathy.

DHHC21 deficiency attenuates renal dysfunction during septic injury.

Renal dysfunction is one of the most common complications of septic injury. One critical contributor to septic injury-induced renal dysfunction is renal vascular dysfunction. Protein palmitoylation serves as a novel regulator of vascular function. Here, we examined whether palmitoyl acyltransferase (PAT)-DHHC21 contributes to septic injury-induced renal dysfunction through regulating renal hemodynamics. Multispectral optoacoustic imaging showed that cecal ligation and puncture (CLP)-induced septic injury caused impaired renal excretion, which was improved in DHHC21 functional deficient (Zdhhc21dep/dep) mice. DHHC21 deficiency attenuated CLP-induced renal pathology, characterized by tissue structural damage and circulating injury markers. Importantly, DHHC21 loss-of-function led to better-preserved renal perfusion and oxygen saturation after CLP. The CLP-caused reduction in renal blood flow was also ameliorated in Zdhhc21dep/dep mice. Next, CLP promoted the palmitoylation of vascular α1-adrenergic receptor (α1AR) and the activation of its downstream effector ERK, which were blunted in Zdhhc21dep/dep mice. Vasoreactivity analysis revealed that renal arteries from Zdhhc21dep/dep mice displayed reduced constriction response to α1AR agonist phenylephrine compared to those from wild-type mice. Consistently, inhibiting PATs with 2-bromopalmitate caused a blunted vasoconstriction response to phenylephrine in small arteries isolated from human kidneys. Therefore, DHHC21 contributes to impaired renal perfusion and function during septic injury via promoting α1AR palmitoylation-associated vasoconstriction.

Cholangiopathy After Severe COVID-19: Clinical Features and Prognostic Implications.

INTRODUCTION: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 virus, is a predominantly respiratory tract infection with the capacity to affect multiple organ systems. Abnormal liver tests, mainly transaminase elevations, have been reported in hospitalized patients. We describe a syndrome of cholangiopathy in patients recovering from severe COVID-19 characterized by marked elevation in serum alkaline phosphatase (ALP) accompanied by evidence of bile duct injury on imaging. METHODS: We conducted a retrospective study of COVID-19 patients admitted to our institution from March 1, 2020, to August 15, 2020, on whom the hepatology service was consulted for abnormal liver tests. Bile duct injury was identified by abnormal liver tests with serum ALP > 3x upper limit of normal and abnormal findings on magnetic resonance cholangiopacreatography. Clinical, laboratory, radiological, and histological findings were recorded in a Research Electronic Data Capture database. RESULTS: Twelve patients were identified, 11 men and 1 woman, with a mean age of 58 years. Mean time from COVID-19 diagnosis to diagnosis of cholangiopathy was 118 days. Peak median serum alanine aminotransferase was 661 U/L and peak median serum ALP was 1855 U/L. Marked elevations of erythrocyte sedimentation rate, C-reactive protein, and D-dimers were common. Magnetic resonance cholangiopacreatography findings included beading of intrahepatic ducts (11/12, 92%), bile duct wall thickening with enhancement (7/12, 58%), and peribiliary diffusion high signal (10/12, 83%). Liver biopsy in 4 patients showed acute and/or chronic large duct obstruction without clear bile duct loss. Progressive biliary tract damage has been demonstrated radiographically. Five patients were referred for consideration of liver transplantation after experiencing persistent jaundice, hepatic insufficiency, and/or recurrent bacterial cholangitis. One patient underwent successful living donor liver transplantation. DISCUSSION: Cholangiopathy is a late complication of severe COVID-19 with the potential for progressive biliary injury and liver failure. Further studies are required to understand pathogenesis, natural history, and therapeutic interventions.

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.

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.

Gut Ischemia Reperfusion Injury Induces Lung Inflammation via Mesenteric Lymph-Mediated Neutrophil Activation.

Gut ischemia/reperfusion (I/R) injury is a common clinical problem associated with significant mortality and morbidities that result from systemic inflammation and remote organ dysfunction, typically acute lung injury. The mechanisms underlying the dissemination of gut-derived harmful mediators into the circulation are poorly understood. The objective of our study was to determine the role of mesenteric lymphatic circulation in the systemic and pulmonary inflammatory response to gut I/R. Using a murine intestinal I/R model, we evaluated whether and how blocking mesenteric lymph flow affects the inflammatory response in local tissues (gut) and remote organs (lungs). We further explored the mechanisms of post-I/R lymph-induced systemic inflammation by examining neutrophil activity and interaction with endothelial cells in vitro. Mice subjected to intestinal I/R displayed a significant inflammatory response in local tissues, evidenced by neutrophil infiltration into mucosal areas, as well as lung inflammation, evidenced by increased myeloperoxidase levels, neutrophil infiltration, and elevated microvascular permeability in the lungs. Mesenteric lymph duct ligation (MLDL) had no effect on gut injury per se, but effectively attenuated lung injury following gut I/R. Cell experiments showed that lymph fluid from post-I/R animals, but not pre-I/R, increased neutrophil surface CD11b expression and their ability to migrate across vascular endothelial monolayers. Moreover, post-I/R lymph upregulated neutrophil expression of pro-inflammatory cytokines and chemokines, which was mediated by a mechanism involving nuclear factor (NF)-κB signaling. Consistently, gut I/R activated NF-κB in lung neutrophils, which was alleviated by MLDL. In conclusion, all these data indicate that mesenteric lymph circulation contributes to neutrophil activation and lung inflammation following gut I/R injury partly through activating NF-κB.

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.

Site-specific opening of the blood-brain barrier by extracellular histones.

BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.