Microvascular and proteomic signatures overlap in COVID-19 and bacterial sepsis: the MICROCODE study.

AIMS: Although coronavirus disease 2019 (COVID-19) and bacterial sepsis are distinct conditions, both are known to trigger endothelial dysfunction with corresponding microcirculatory impairment. The purpose of this study was to compare microvascular injury patterns and proteomic signatures in COVID-19 and bacterial sepsis patients. METHODS AND RESULTS: This multi-center, observational study included 22 hospitalized adult COVID-19 patients, 43 hospitalized bacterial sepsis patients, and 10 healthy controls from 4 hospitals. Microcirculation and glycocalyx dimensions were quantified via intravital sublingual microscopy. Plasma proteins were measured using targeted proteomics (Olink). Coregulation and cluster analysis of plasma proteins was performed using a training-set and confirmed in a test-set. An independent external cohort of 219 COVID-19 patients was used for validation and outcome analysis. Microcirculation and plasma proteome analysis found substantial overlap between COVID-19 and bacterial sepsis. Severity, but not disease entity explained most data variation. Unsupervised correlation analysis identified two main coregulated plasma protein signatures in both diseases that strictly counteract each other. They were associated with microvascular dysfunction and several established markers of clinical severity. The signatures were used to derive new composite biomarkers of microvascular injury that allow to predict 28-day mortality or/and intubation (area under the curve 0.90, p < 0.0001) in COVID-19. CONCLUSION: Our data imply a common biological host response of microvascular injury in both bacterial sepsis and COVID-19. A distinct plasma signature correlates with endothelial health and improved outcomes, while a counteracting response is associated with glycocalyx breakdown and high mortality. Microvascular health biomarkers are powerful predictors of clinical outcomes.

Data-Driven Kidney Transplant Phenotyping as a Histology-Independent Framework for Biomarker Discovery.

BACKGROUND: In transplant medicine, clinical decision making largely relies on histology of biopsy specimens. However, histology suffers from low specificity, sensitivity, and reproducibility, leading to suboptimal stratification of patients. We developed a histology-independent immune framework of kidney graft homeostasis and rejection. METHODS: We applied tailored RNA deconvolution for leukocyte enumeration and coregulated gene network analysis to published bulk human kidney transplant RNA transcriptomes as input for unsupervised, high-dimensional phenotype clustering. We used framework-based graft survival analysis to identify a biomarker that was subsequently characterized in independent transplant biopsy specimens. RESULTS: We found seven immune phenotypes that confirm known rejection types and uncovered novel signatures. The molecular phenotypes allow for improved graft survival analysis compared with histology, and identify a high-risk group in nonrejecting transplants. Two fibrosis-related phenotypes with distinct immune features emerged with reduced graft survival. We identified lysyl oxidase-like 2 (LOXL2)-expressing peritubular CD68+ macrophages as a framework-derived biomarker of impaired allograft function. These cells precede graft fibrosis, as demonstrated in longitudinal biopsy specimens, and may be clinically useful as a biomarker for early fibrogenesis. CONCLUSIONS: This study provides a comprehensive, data-driven atlas of human kidney transplant phenotypes and demonstrates its utility to identify novel clinical biomarkers.

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B100-Reactive CD4+ T-Regulatory Cells.

BACKGROUND: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. METHODS: To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. RESULTS: We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. CONCLUSIONS: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.

Microvascular dysfunction in COVID-19: the MYSTIC study.

RATIONALE: Pre-clinical and autopsy studies have fueled the hypothesis that a dysregulated vascular endothelium might play a central role in the pathogenesis of ARDS and multi-organ failure in COVID-19. OBJECTIVES: To comprehensively characterize and quantify microvascular alterations in patients with COVID-19. METHODS: Hospitalized adult patients with moderate-to-severe or critical COVID-19 (n = 23) were enrolled non-consecutively in this prospective, observational, cross-sectional, multi-center study. Fifteen healthy volunteers served as controls. All participants underwent intravital microscopy by sidestream dark field imaging to quantify vascular density, red blood cell velocity (VRBC), and glycocalyx dimensions (perfused boundary region, PBR) in sublingual microvessels. Circulating levels of endothelial and glycocalyx-associated markers were measured by multiplex proximity extension assay and enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: COVID-19 patients showed an up to 90% reduction in vascular density, almost exclusively limited to small capillaries (diameter 4-6 µm), and also significant reductions of VRBC. Especially, patients on mechanical ventilation showed severe glycocalyx damage as indicated by higher PBR values (i.e., thinner glycocalyx) and increased blood levels of shed glycocalyx constituents. Several markers of endothelial dysfunction were increased and correlated with disease severity in COVID-19. PBR (AUC 0.75, p = 0.01), ADAMTS13 (von Willebrand factor-cleaving protease; AUC 0.74, p = 0.02), and vascular endothelial growth factor A (VEGF-A; AUC 0.73, p = 0.04) showed the best discriminatory ability to predict 60-day in-hospital mortality. CONCLUSIONS: Our data clearly show severe alterations of the microcirculation and the endothelial glycocalyx in patients with COVID-19. Future therapeutic approaches should consider the importance of systemic vascular involvement in COVID-19.

Migratory and Dancing Macrophage Subsets in Atherosclerotic Lesions.

RATIONALE: Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging. OBJECTIVE: Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes. METHODS AND RESULTS: Apoe-/-Cx3cr1GFPCd11cYFP mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques. CONCLUSIONS: The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.

Symmetric dimethylarginine in dysfunctional high-density lipoprotein mediates endothelial glycocalyx breakdown in chronic kidney disease.

Dysfunctional high-density lipoprotein (d-HDL) in chronic kidney disease is known to have a change in composition towards an endothelial-damaging phenotype, amongst others, via the accumulation of symmetric dimethylarginine. The endothelial glycocalyx, a carbohydrate-rich layer lining the endothelial luminal surface, is a first line defense against vascular diseases including atherosclerosis. Here we conducted a translational, cross-sectional study to determine the role of symmetric dimethylarginine in d-HDL as a mediator of glycocalyx damage. Using confocal and atomic force microscopy, intact HDL from healthy donors was found to maintain the glycocalyx while isolated HDL from hemodialysis patients and exogenous symmetric dimethylarginine caused significant damage to the glycocalyx in endothelial cells in vitro in a dose-dependent manner. Symmetric dimethylarginine triggered glycocalyx deterioration via molecular pathways mediated by toll-like-receptor 2 and matrix metalloprotease-9. Corresponding intravital microscopy revealed that exogenous symmetric dimethylarginine and d-HDL from hemodialysis patients caused glycocalyx breakdown, which subsequently contributed to alterations in leukocyte rolling. Biologically effective HDL, which estimates the functionality of HDL, was calculated from circulating HDL-cholesterol and symmetric dimethylarginine, as described in the literature. Biologically effective HDL was the only parameter that could independently predict glycocalyx damage in vivo. Thus, our data suggest that symmetric dimethylarginine in d-HDL mediates glycocalyx breakdown in chronic kidney disease.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. METHODS AND RESULTS: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. CONCLUSIONS: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Inflammatory Pathways Regulated by Tumor Necrosis Receptor-Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced Obesity.

RATIONALE: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. OBJECTIVE: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor-associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-α, IL (interleukin)-1ß, and TLRs (toll-like receptors). METHODS AND RESULTS: Mice deficient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1-/- mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance-an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1-enabled thermogenesis. TRAF-1-dependent catabolic and proinflammatory cues were synergistically driven by ß3-adrenergic and inflammatory signaling and required the presence of both TRAF-1-deficient adipocytes and macrophages. In human obesity, TRAF-1-dependent genes were upregulated. CONCLUSIONS: Enhancing TRAF-1-dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These findings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism.

Endothelial Protective Monocyte Patrolling in Large Arteries Intensified by Western Diet and Atherosclerosis.

RATIONALE: Nonclassical mouse monocyte (CX3CR1high, Ly-6Clow) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. OBJECTIVE: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. METHODS AND RESULTS: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 µm/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1+/GFP/apoE-/- mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and α4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1-/- apoE-/- mice leads to pronounced endothelial apoptosis. CONCLUSIONS: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.

The TWEAK/Fn14 pathway is required for calcineurin inhibitor toxicity of the kidneys.

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis.

Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling.

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo.

L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting. FROM THE CLINICAL EDITOR: During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.

Cross-species single-cell RNA sequencing reveals divergent phenotypes and activation states of adaptive immunity in human carotid and experimental murine atherosclerosis.

BACKGROUND AND AIMS: The distinct functions of immune cells in atherosclerosis have been mostly defined by preclinical mouse studies. Contrastingly, the immune cell composition of human atherosclerotic plaques and their contribution to disease progression is only poorly understood. It remains uncertain whether genetic animal models allow for valuable translational approaches. METHODS AND RESULTS: Single cell RNA-sequencing (scRNA-seq) was performed to define the immune cell landscape in human carotid atherosclerotic plaques. The human immune cell repertoire demonstrated an unexpectedly high heterogeneity and was dominated by cells of the T-cell lineage, a finding confirmed by immunohistochemistry. Bioinformatical integration with 7 mouse scRNA-seq data sets from adventitial and atherosclerotic vascular tissue revealed a total of 51 identities of cell types and differentiation states, of which some were only poorly conserved between species and exclusively found in humans. Locations, frequencies, and transcriptional programs of immune cells in mouse models did not resemble the immune cell landscape in human carotid atherosclerosis. In contrast to standard mouse models of atherosclerosis, human plaque leukocytes were dominated by several T-cell phenotypes with transcriptional hallmarks of T-cell activation and memory formation, T-cell receptor-, and pro-inflammatory signaling. Only mice at the age of 22 months partially resembled the activated T-cell phenotype. In a validation cohort of 43 patients undergoing carotid endarterectomy, the abundance of activated immune cell subsets in the plaque defined by multi-color flow cytometry associated with the extend of clinical atherosclerosis. CONCLUSIONS: Integrative scRNA-seq reveals a substantial difference in the immune cell composition of murine and human carotid atherosclerosis - a finding that questions the translational value of standard mouse models for adaptive immune cell studies. Clinical associations suggest a specific role for T-cell driven (auto-) immunity in human plaque formation and -instability.

Plasma protein signatures reflect systemic immunity and allograft function in kidney transplantation.

Kidney transplantation causes large perturbations of the immune system. While many studies focus on the allograft, insights into systemic effects are largely missing. Here, we analyzed the systemic immune response in 3 cohorts of kidney transplanted patients. Using serum proteomics, laboratory values, mass cytometry, histological and clinical parameters, inter-patient heterogeneity was leveraged for multi-omic co-variation analysis. We identified circulating immune modules (CIM) that describe extra-renal signatures of co-regulated plasma proteins. CIM are present in nontransplanted controls, in transplant conditions and during rejection. They are enriched in pathways linked to kidney function, extracellular matrix, signaling, and cellular activation. A complex leukocyte response in the blood during allograft quiescence and rejection is associated with CIM activity and CIM-specific cytokines. CIM activity correlates with kidney function including a 2-month prediction. Together, the data suggest a systemic and multi-layered response of transplant immunity that might be insightful for understanding allograft dysfunction and developing translational biomarkers.

The transmembrane domains of L-selectin and CD44 regulate receptor cell surface positioning and leukocyte adhesion under flow.

During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.

Improved survival and reduced vascular permeability by eliminating or blocking 12/15-lipoxygenase in mouse models of acute lung injury (ALI).

Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15(-/-) mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15(-/-) mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.

C4d Deposition after Allogeneic Renal Transplantation in Rats Is Involved in Initial Apoptotic Cell Clearance.

In the context of transplantation, complement activation is associated with poor prognosis and outcome. While complement activation in antibody-mediated rejection is well-known, less is known about complement activation in acute T cell-mediated rejection (TCMR). There is increasing evidence that complement contributes to the clearance of apoptotic debris and tissue repair. In this regard, we have analysed published human kidney biopsy transcriptome data clearly showing upregulated expression of complement factors in TCMR. To clarify whether and how the complement system is activated early during acute TCMR, experimental syngeneic and allogeneic renal transplantations were performed. Using an allogeneic rat renal transplant model, we also observed upregulation of complement factors in TCMR in contrast to healthy kidneys and isograft controls. While staining for C4d was positive, staining with a C3d antibody showed no C3d deposition. FACS analysis of blood showed the absence of alloantibodies that could have explained the C4d deposition. Gene expression pathway analysis showed upregulation of pro-apoptotic factors in TCMR, and apoptotic endothelial cells were detected by ultrastructural analysis. Monocytes/macrophages were found to bind to and phagocytise these apoptotic cells. Therefore, we conclude that early C4d deposition in TCMR may be relevant to the clearance of apoptotic cells.

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.

Classical monocyte transcriptomes reveal significant anti-inflammatory statin effect in women with chronic HIV.

AIMS: During virally suppressed chronic HIV infection, persistent inflammation contributes to the development of cardiovascular disease (CVD), a major comorbidity in people living with HIV (LWH). Classical blood monocytes (CMs) remain activated during antiretroviral therapy and are a major source of pro-inflammatory and pro-thrombotic factors that contribute to atherosclerotic plaque development and instability. METHODS AND RESULTS: Here, we identify transcriptomic changes in circulating CMs in peripheral blood mononuclear cell samples from participants of the Women's Interagency HIV Study, selected by HIV and subclinical CVD (sCVD) status. We flow-sorted CM from participants of the Women's Interagency HIV Study and deep-sequenced their mRNA (n = 92). CMs of HIV+ participants showed elevated interleukin (IL)-6, IL-1ß, and IL-12ß, overlapping with many transcripts identified in sCVD+ participants. In sCVD+ participants LWH, those reporting statin use showed reduced pro-inflammatory gene expression to a level comparable with healthy (HIV-sCVD-) participants. Statin non-users maintained an elevated inflammatory profile and increased cytokine production. CONCLUSION: Statin therapy has been associated with a lower risk of cardiac events, such as myocardial infarction in the general population, but not in those LWH. Our data suggest that women LWH may benefit from statin therapy even in the absence of overt CVD.

Rolling neutrophils form tethers and slings under physiologic conditions in vivo.

Human and mouse neutrophils are known to form tethers when rolling on selectins in vitro. Tethers are ∼0.2 µm thin, ∼5-10 µm-long structures behind rolling cells that can swing around to form slings that serve as self-adhesive substrates. Here, we developed a mouse intravital imaging method, where the neutrophil surface is labeled by injecting fluorescently labeled mAb to Ly-6G. Venules in the cremaster muscle of live mice were imaged at a high frame rate using a confocal microscope equipped with a fast resonant scanner. We observed 270 tethers (median length 3.5 µm) and 31 slings (median length 6.9 µm) on 186 neutrophils of 15 mice. Out of 199 tether break events, 123 were followed by immediate acceleration of the rolling cell, which shows that tethers are load-bearing structures in vivo. In venules with a high wall shear stress (WSS; > 12 dyn/cm2 ), median rolling velocity was higher (19 µm/s), and 43% of rolling neutrophils had visible tethers. In venules with WSS < 12 dyn/cm2 , only 26% of rolling neutrophils had visible tethers. We conclude that neutrophil tethers are commonly present and stabilize rolling in vivo.