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Reports examining lung histopathology in coronavirus disease 2019 (COVID-19) infection provide an essential body of information for clinicians and investigators. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced lung injury is complex, involving the airways, alveoli, and pulmonary vessels. Although no anatomic marker is specific, the signature histologic lesion is diffuse alveolar damage (DAD). The biological and molecular mechanisms that drive this pattern of injury are unknown, and the relationship of SARS-CoV-2-induced DAD to physiologic alterations and clinical outcomes in COVID-19-associated acute respiratory distress syndrome is undefined. Additional histologic patterns that may be variant phenotypes have been reported.

Background Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. Objectives As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. Patients/methods Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. Results The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. Conclusions During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.

Cystic fibrosis (CF) is a multisystem disorder, but progressive inflammatory lung disease causes the greatest burden of morbidity and death. Recent translational and mechanistic studies of samples from patients, and observations in animal models, indicate that platelets may drive lung injury and contribute to dysregulated host defense in CF lung disease. In this issue of the JCI, Ortiz-Munoz and Yu et al. explored the role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in platelet-related inflammation. The authors used mouse and human model systems to show that CFTR dysfunction in platelets increased calcium entry though the transient receptor potential cation channel 6 (TRPC6), causing hyperactivation and consequent experimental lung inflammation. The study persuasively suggests that platelets are critical thromboinflammatory effector cells in CF lung disease. In the context of platelet-related organ injury seen in a variety of other diseases and syndromes, platelets may also contribute to nonpulmonary manifestations and comorbidities of CF.

There is increasing recognition that platelets have a functional role in the pathophysiology of sepsis, though this role has not been precisely defined. Whether sepsis alters the human platelet transcriptome and translational landscape has never been established. We used parallel techniques of RNA sequencing and ribosome footprint profiling to interrogate the platelet transcriptome and translatome in septic patients and healthy donors. We identified 1806 significantly differentially expressed (false discovery rate

Background The term sepsis is used to designate a systemic condition of infection and inflammation associated with hemodynamic changes that result in organic dysfunction. Gestational sepsis can impair the development of the central nervous system and may promote permanent behavior alterations in the offspring. The aim of our work was to evaluate the effects of maternal sepsis on inflammatory cytokine levels and synaptic proteins in the hippocampus, neocortex, frontal cortex, and cerebellum of neonatal, young, and adult mice. Additionally, we analyzed the motor development, behavioral features, and cognitive impairments in neonatal, young and adult offspring. Methods Pregnant mice at the 14th embryonic day (E14) were intratracheally instilled with saline 0.9% solution (control group) or Klebsiella spp. (3 × 108 CFU) (sepsis group) and started on meropenem after 5 h. The offspring was sacrificed at postnatal day (P) 2, P8, P30, and P60 and samples of liver, lung, and brain were collected for TNF-α, IL-1β, and IL-6 measurements by ELISA. Synaptophysin, PSD95, and β-tubulin levels were analyzed by Western blot. Motor tests were performed at all analyzed ages and behavioral assessments were performed in offspring at P30 and P60. Results Gestational sepsis induces a systemic pro-inflammatory response in neonates at P2 and P8 characterized by an increase in cytokine levels. Maternal sepsis induced systemic downregulation of pro-inflammatory cytokines, while in the hippocampus, neocortex, frontal cortex, and cerebellum an inflammatory response was detected. These changes in the brain immunity were accompanied by a reduction of synaptophysin and PSD95 levels in the hippocampus, neocortex, frontal cortex, and cerebellum, in all ages. Behavioral tests demonstrated motor impairment in neonates, and depressive-like behavior, fear-conditioned memory, and learning impairments in animals at P30 and P60, while spatial memory abilities were affected only at P60, indicating that gestational sepsis not only induces an inflammatory response in neonatal mouse brains, but also affects neurodevelopment, and leads to a plethora of behavioral alterations and cognitive impairments in the offspring. Conclusion These data suggest that maternal sepsis may be causatively related to the development of depression, learning, and memory impairments in the litter.

Dengue is characterized as one of the most important arthropod-borne human viral diseases, representing a public health problem. Increased activation of immune cells is involved in the progression of infection to severe forms. Recently, our group demonstrated the contribution of platelet–monocyte interaction to inflammatory responses in dengue, adding to evolving evidence that platelets have inflammatory functions and can regulate different aspects of innate immune responses. Furthermore, stimuli-specific-activated platelets can promote phenotypic changes and metabolic reprogramming in monocytes. Thus, this study aimed to evaluate the roles of dengue virus (DENV)-activated platelets on immunometabolic reprogramming of monocytes in vitro, focusing on lipid droplet (LD) biogenesis. We demonstrated that platelets exposed to DENV in vitro form aggregates with monocytes and signal to LD formation and CXCL8/IL-8, IL-10, CCL2, and PGE2 secretion. Pharmacologic inhibition of LD biogenesis prevents PGE2 secretion, but not CXCL8/IL-8 release, by platelet–monocyte complexes. In exploring the mechanisms involved, we demonstrated that LD formation in monocytes exposed to DENV-activated platelets is partially dependent on platelet-produced MIF. Additionally, LD formation is higher in monocytes, which have platelets adhered on their surface, suggesting that beyond paracrine signaling, platelet adhesion is an important event in platelet-mediated modulation of lipid metabolism in monocytes. Together, our results demonstrate that activated platelets aggregate with monocytes during DENV infection and signal to LD biogenesis and the secretion of inflammatory mediators, which may contribute to dengue immunopathogenesis.

Platelets are essential cellular effectors of hemostasis and contribute to disease as circulating effectors of pathologic thrombosis. These are their most widely known biologic activities. Nevertheless, recent observations demonstrate that platelets have a much more intricate repertoire beyond these traditional functions and that they are specialized for contributions to vascular barrier integrity, organ repair, antimicrobial host defense, inflammation, and activities across the immune continuum. Paradoxically, on the basis of clinical investigations and animal models of disease, some of these newly discovered activities of platelets appear to contribute to tissue injury. Studies in the last decade indicate unique interactions of platelets and their precursor, the megakaryocyte, in the lung and implicate platelets as essential effectors in experimental acute lung injury and clinical acute respiratory distress syndrome. Additional discoveries derived from evolving work will be required to precisely define the contributions of platelets to complex subphenotypes of acute lung injury and to determine if these remarkable and versatile blood cells are therapeutic targets in acute respiratory distress syndrome.

Objective— One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. Approach and Results— We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA–DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA–DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA–DNA hybrid immunoprecipitation. Conclusions— Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA–DNA hybrid formation and identify that RNA–DNA hybrids function as a novel translational control mechanism in human platelets.

Background: Cerebral malaria is a highly prevalent infectious disease in Sub-Saharan Africa caused by the Plasmodium parasite. The pathogenesis of cerebral malaria results from damaged vascular endothelium induced by parasite sequestration, inflammatory cytokine production and vascular leakage, which results in increased brain permeability and death. While maladaptive responses from immune cells are thought to contribute, growing evidence suggests a crucial role of platelets in malaria pathophysiology. The mammalian target of rapamycin (mTOR) pathway is critical in regulating outcomes in malaria. Previous studies have demonstrated an mTOR specific inhibitor, rapamycin, is protective in a mouse model of experimental cerebral malaria (ECM). However, if the mTOR pathway in platelets specifically contributes to the pathogenesis of malaria is unknown. Methods: Platelet-specific mTOR-deficient (mTOR plt-/-) mice and littermate controls were subjected to a well-established model of ECM, using Plasmodium berghei ANKA. In addition, platelets isolated from human malaria patients were examined for differential regulation of the mTOR pathway using RNA-seq. Results: Platelet RNA-seq and Ingenuity Pathway Analysis from patients infected with P. vivax demonstrated enrichment of mTOR-associated pathways in platelets, such as mTOR signaling and p70S6K signaling, indicating mTOR associated genes are upregulated in human platelets during malaria infection. In mice infected with P. berghei ANKA, the mTOR pathway was activated in bone marrow-megakaryocytes and platelets based on phosphorylation of mTOR and its downstream effector, 4E-BP1. As the mTOR pathway regulates protein translation in platelets, we examined de novo protein synthesis and observed increased protein translation in platelets isolated from mice infected with P. berghei ANKA compared to uninfected controls. To study the specific role of platelet mTOR during ECM pathogenesis, mTOR plt-/- mice and wild-type controls (mTOR plt+/+), were infected with P. berghei ANKA. Platelet deficient-mTOR mice had significantly (p=0.0336) prolonged survival compared to wild-type mice. Increased survival was independent of parasitemia, suggesting platelets did not alter parasite reproduction. While thrombocytopenia and anemia were similar in both genotypes, mTOR plt-/- mice had significantly reduced brain (p=0.0067) and lung (p Conclusions: Our data demonstrates that the mTOR pathway in platelets plays a significant role in malaria pathogenesis. Deletion of platelet mTOR reduces vascular permeability and prolongs survival during ECM. We hypothesize that altered platelet-inflammatory monocyte interactions drive this phenotype. Disclosures Rondina: Platelet Transcriptomics: Patents & Royalties; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

Objective— Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine whether clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases. Approach and Results— We performed next-generation RNA sequencing on monocytes extracted from whole blood clots and using a purified plasma clot system. Numerous mRNAs were differentially expressed by monocytes embedded in clots compared with unclotted controls, and IL-8 (interleukin 8) and MCP-1 (monocyte chemoattractant protein-1) were among the upregulated transcripts in both models. Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in lipopolysaccharide-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (ie, 1–2 hours) reduced the synthesis of IL-8 and MCP-1, whereas delayed fibrinolysis was far less effective. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. Conclusions— These findings demonstrate that clots are potent inducers of monocyte gene expression and that timely fibrinolysis attenuates inflammatory responses, specifically IL-8 and MCP-1. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

Introduction Molecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18 F-fluoro-2-deoxyglucose ( 18 F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18 F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14 C-2DG uptake in activated neutrophils. Methods Lung uptake of 18 F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24 h following the intraperitoneal injection of 10 mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14 C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. Results Significant uptake of 18 F-FDG occurred by 2 h following LPS, and progressively increased to 24 h. Lung uptake of 18 F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18 F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14 C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. Conclusion Systemic endotoxin-induced ALI results in very early and progressive uptake of 18 F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14 C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. Advances in knowledge and implications for patient care 18 F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14 C-2DG uptake in activated neutrophils. 18 F-FDG PET may provide a potential mechanism for early diagnosis and therapeutic assessment of ALI/ARDS.

The acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in critically ill patients and is defined by the acute onset of noncardiogenic pulmonary edema, hypoxemia, and the need for mechanical ventilation. ARDS occurs most often in the setting of pneumonia, sepsis, aspiration of gastric contents or severe trauma, and is present in ~10% of all intensive care unit patients worldwide. Despite some improvements over the past decades, mortality remains high at 30–40% in most studies. Pathologic specimens from patients with ARDS most frequently reveal diffuse alveolar damage, and laboratory studies have demonstrated both alveolar epithelial and lung endothelial injury, resulting in accumulation of protein-rich inflammatory edema fluid in the alveolar space. Diagnosis is based on consensus syndromic criteria, with recent proposed modifications for under-resourced settings and for pediatric patients. Patient management focuses on implementing a lung-protective ventilation strategy; no specific pharmacotherapies have been identified. Long-term outcomes of patients with ARDS are increasingly recognized as important research targets, as many patients survive ARDS only to suffer ongoing functional and/or psychologic sequelae. Future directions include efforts to facilitate earlier recognition of ARDS, prognostic and/or predictive enrichment in clinical studies to identify responsive subsets, and ongoing efforts to understand fundamental mechanisms of lung injury that may respond to specific treatments.

Background: Dysregulated inflammation leads to morbidity and mortality in neonates. Neutrophil-mediated inflammation can cause inflammatory tissue damage. The mammalian Target of Rapamycin (mTOR) pathway governs IL-6Rα protein expression in human neutrophils. Shed IL-6Rα then participates in trans-signaling of IL-6/IL-6Rα to cells not otherwise sensitive to IL-6. Signaling to endothelial cells triggers efferocytosis where macrophages limit persistent inflammation by phagocytizing neutrophils. We hypothesized that preterm neonatal PMNs fail to synthesize IL-6Rα due to alterations in mTOR signaling. Methods: We studied IL-6Rα expression, PAF-receptor expression, and mTOR signaling in plasma and PAF-stimulated PMNs isolated from newborn infants and healthy adults using ELISA, real time RT-PCR, Western blotting, flow cytometry, and immunocytochemistry with phospho-specific antibodies. Results: Compared to healthy adults, plasma from neonates contains significantly less soluble IL-6Rα. IL-6Rα mRNA expression in PAF-stimulated PMNs does not differ between neonates and adults, but IL-6Rα protein expression is decreased in preterm neonatal PMNs. Rapamycin, a mTOR inhibitor, blocks IL-6Rα protein expression. mTOR signaling following PAF-stimulation is decreased in preterm neonatal PMNs. Conclusions: Preterm neonatal PMNs exhibit decreased mTOR pathway signaling leading to decreased IL-6Rα synthesis. Decreased synthesis of IL-6Rα by neonatal PMNs may result in decreased IL-6/IL-6Rα trans-signaling with prolonged inflammatory response and increased morbidity.

Platelets are anucleate circulating blood cells with canonical roles in hemostasis and thrombosis. Established and emerging data highlights that in addition to these canonical functions, platelets also have a dynamic repertoire of functions that span inflammatory and immune continuums. Moreover, in many cases there is interplay between hemostatic and inflammatory responses by platelets. In this chapter, we profile a number of inflammatory syndromes and diseases in which platelets have physiological or pathophysiological roles. We briefly review both infectious and non-infectious inflammatory settings and, where available, highlight complementary clinical and experimental systems.

HIV-infected subjects under virological control still exhibit a persistent proinflammatory state. Thus, chronic HIV infection changes the host homeostasis towards an adapted immune response that may affect the outcome of coinfections. However, little is known about the impact of HIV infection on inflammatory amplification and clinical presentation in dengue. Platelets have been shown to participate in immune response in dengue and HIV. We hypothesized that altered platelet responses in HIV-infected subjects may contribute to altered inflammatory milieu and disease progression in dengue. We prospectively followed a cohort of 84 DENV-infected patients of whom 29 were coinfected with HIV under virological control. We report that dengue and HIV coinfection progress with reduced inflammation and milder disease progression with lower risk of vascular instability. Even though the degree of thrombocytopenia and platelet activation were similar between dengue-infected and HIV plus dengue-coinfected patients, plasma levels of the platelet-derived chemokines RANTES/CCL5 and PF4/CXCL4 were lower in coinfection. Consistently, platelets from coinfected patients presented defective secretion of the stored-chemokines PF4 and RANTES, but not newly synthesized IL-1β, when cultured ex vivo. These data indicate that platelets from HIV-infected subjects release lower levels of chemokines during dengue illness, which may contribute to milder clinical presentation during coinfection.

Essentials Platelets employ proteins/signaling pathways traditionally thought reserved for nuclear niche. We determined retinoic-acid-receptor alpha (RARα) expression and function in human platelets. RARα/actin-related protein-2/3 complex (Arp2/3) interact via non-genomic signaling in platelets. RARα regulates Arp2/3-mediated actin cytoskeletal dynamics and platelet spreading. SummaryBackground Platelets utilize proteins and pathways classically reserved for the nuclear niche. Methods We determined whether human platelets express retinoic-acid-receptor family members, traditionally thought of as nuclear transcription factors, and deciphered the function of RARα. Results We found that RARα is robustly expressed in human platelets and megakaryocytes and interacts directly with actin-related protein-2/3 complex (Arp2/3) subunit 5 (Arp2/3s5). Arp2/3s5 co-localized with RARα in situ and regulated platelet cytoskeletal processes. The RARα ligand all-trans retinoic acid (atRA) disrupted RARα‒Arp2/3 interactions. When isolated human platelets were treated with atRA, rapid cytoskeletal events (e.g. platelet spreading) were inhibited. In addition, when platelets were cultured for 18 h in the presence of atRA, actin-dependent morphological changes (e.g. extended cell body formation) were similarly inhibited. Using in vitro actin branching assays, RARα and Arp2/3-regulated complex actin branch formation was demonstrated. Consistent with inhibition of cytoskeletal processes in platelets, atRA, when added to this branching assay, resulted in dysregulated actin branching. Conclusion Our findings identify a previously unknown mechanism by which RARα regulates Arp2/3-mediated actin cytoskeletal dynamics through a non-genomic signaling pathway. These findings have broad implications in both nucleated and anucleate cells, where actin cytoskeletal events regulate cell morphology, movement and division.

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDβ2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDβ2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.

Cardiovascular diseases and thrombotic events became major clinical problems in the combined antiretroviral therapy (cART) era. Although the precise mechanisms behind these clinical problems have not been fully elucidated, a persistent pro-inflammatory state plays a central role. As platelets play important roles on both, thrombus formation and inflammatory/immune response, we aimed at investigating platelet function in HIV-infected subjects virologically controlled through cART. We evaluate parameters of activation, mitochondrial function and activation of apoptosis pathways in platelets from 30 HIV-infected individuals under stable cART and 36 healthy volunteers. Despite viral control achieved through cART, HIV-infected individuals exhibited increased platelet activation as indicated by P-selectin expression and platelet spreading when adhered on fibrinogen-coated surfaces. Platelets from HIV-infected subjects also exhibited mitochondrial dysfunction and activation of apoptosis pathways. Finally, thrombin stimuli induced lower levels of P-selectin translocation and RANTES secretion, but not TXA2 synthesis, in platelets from HIV-infected individuals compared to control; and labeling of platelet alpha granules showed reduced granule content in platelets from HIV-infected individuals when compared to healthy subjects. In summary, platelets derived from HIV-infected individuals under stable cART exhibit a phenotype of increased activation, activation of the intrinsic pathway of apoptosis and undermined granule secretion in response to thrombin.

β2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDβ2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDβ2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDβ2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDβ2 in bacterial infection.

Rationale: Acute respiratory distress syndrome (ARDS) is a devastating illness with limited therapeutic options. A better understanding of early biochemical and immunological events in ARDS could inform the development of new preventive and treatment strategies. Objectives: To determine select peripheral blood lipid mediator and leukocyte responses in patients at risk for ARDS. Methods: Patients at risk for ARDS were randomized as part of a multicenter, double-blind clinical trial of aspirin versus placebo (the LIPS-A [Lung Injury Prevention Study with Aspirin] trial; NCT01504867). Plasma thromboxane B(2) (TXB(2)), aspirin-triggered lipoxin A(4) (15-epi-LXA(4), ATL), and peripheral blood leukocyte number and activation were determined on enrollment and after treatment with either aspirin or placebo. Measurements and Main Results: Thirty-three of 367 subjects (9.0%) developed ARDS after randomization. Baseline ATL levels, total monocyte counts, intermediate monocyte counts, and monocyte–platelet aggregates were associated with the development of ARDS. Peripheral blood neutrophil count and monocyte–platelet aggregates significantly decreased over time. Of note, nine subjects developed ARDS after randomization yet before study drug initiation, including seven subjects assigned to aspirin treatment. Subjects without ARDS at the time of first dose demonstrated a lower incidence of ARDS with aspirin treatment. Compared with placebo, aspirin significantly decreased TXB(2) and increased the ATL/TXB(2) ratio. Conclusions: Biomarkers of intravascular monocyte activation in at-risk patients were associated with development of ARDS. The potential clinical benefit of early aspirin for prevention of ARDS remains uncertain. Together, results of the biochemical and immunological analyses provide a window into the early pathogenesis of human ARDS and represent potential vascular biomarkers of ARDS risk. Clinical trial registered with www.clinicaltrials.gov (NCT01504867). Keywords: ARDS; monocyte–platelet aggregates; aspirin; lipoxin; thromboxane

β2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDβ2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDβ2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDβ2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDβ2 in bacterial infection.

BACKGROUND Prospective studies on the incidence of VTE during severe sepsis and septic shock remain absent, hindering efficacy assessments regarding VTE prevention strategies in sepsis. METHODS We prospectively studied 113 consecutively enrolled patients in the ICU with severe sepsis and septic shock at three hospitals. All patients provided informed consent. VTE thromboprophylaxis was recorded for all patients. Patients underwent ultrasonography and were followed for VTE prior to ICU discharge. All-cause 28-day mortality was recorded. Variables from univariate analyses that were associated with VTE (including central venous catheter [CVC] insertion, age, length of stay, and mechanical ventilation) were included in a multivariable logistic regression analysis using backward stepwise elimination to determine VTE predictors. RESULTS Mean APACHE (Acute Physiology and Chronic Health Evaluation) II score was 18.2 ± 7.0, and age was 50 ± 18 years. Despite all patients receiving guideline-recommended thromboprophylaxis, the incidence of VTE was 37.2% (95% CI, 28.3-46.8). Most VTE events were clinically significant (defined as pulmonary embolism, proximal DVT, and/or symptomatic distal DVT) and associated with an increased length of stay (18.2 ± 9.9 days vs 13.4 ± 11.5 days, P .05). Mortality was higher in patients with acute VTE but did not reach statistical significance. Insertion of a CVC and longer mechanical ventilation duration were significant VTE risk factors. VTE incidence did not differ by thromboprophylaxis type. CONCLUSIONS To our knowledge this is the first multicenter prospective study to identify a high incidence of VTE in patients with severe sepsis and septic shock, despite the use of universal, guideline-recommended thromboprophylaxis. Our findings suggest that the systemic inflammatory milieu of sepsis may uniquely predispose patients with sepsis to VTE. More effective VTE prevention strategies are necessary in patients with sepsis. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT02353910; URL: www.clinicaltrials.gov

Objective: Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine if clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases. Approach and Results: We performed Next Generation RNA sequencing on monocytes extracted from whole blood clots. Thousands of mRNAs were differentially expressed by monocytes from clotted versus unclotted whole blood, including upregulation of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in LPS-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent, but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (i.e., 1-2 hours) reduced the synthesis of IL-8 and MCP-1, while delayed fibrinolysis was far less effective. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. Conclusions: These findings demonstrate that clots are potent inducers of monocyte gene expression, and that timely fibrinolysis attenuates inflammatory responses. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically-proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

Rationale: Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Objective: Determine if clot formation and lysis regulate the expression of human monocyte-derived genes that modulate vascular diseases. Methods and Results: We performed Next Generation RNA sequencing on monocytes extracted from whole blood clots. Thousands of mRNAs were significantly differentially expressed by monocytes from clotted versus unclotted whole blood (p>0.05), including upregulation of many inflammatory cytokines such as interleukin 8 (IL-8), IL-1β, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 (MCP-1). Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in LPS-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent, but fibrin-dependent, manner. Fibrinolysis initiated shortly after plasma clot formation (i.e., 1-2 hours) reduced the global inflammatory response based on RNA-seq analysis and significantly reduced the synthesis of IL-8 and MCP-1 (p>0.05). Delayed fibrinolysis was far less effective in reducing inflammation. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. Conclusion: These findings demonstrate that clots are potent inducers of monocyte gene expression, and that timely fibrinolysis attenuates inflammatory responses. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically-proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

The secreted or plasma form of platelet-activating factor acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 (Lp-PLA2) or phospholipase A2 group 7 (PLA2G7), is a member of the PLA2 superfamily of enzymes that circulates in blood in association with lipoproteins, and is found in atherosclerotic lesions [reviewed in (2, 3)]. This enzyme was discovered in the early 1980s based on its ability to hydrolyze the pro-inflammatory glycerophospholipid PAF, and was thus proposed to have anti-inflammatory properties. In subsequent years, it was recognized that PAF-AH hydrolyzes glycerophospholipids containing short and/or oxidized functionalities at the sn-2 position, with no preference for the type of linkage at the sn-1 position, i.e., alkyl versus acyl. Substrate hydrolysis catalyzed by PAF-AH generates lysoPAF/lyso phosphatidylcholine (lysoPC) and short and/or oxidized fatty acids, many of which also have been reported to have pro-inflammatory and pro-oxidative activities (4). These observations fueled multiple investigations that led to controversial views regarding the role of PAF-AH in human physiology and disease (1, 4–6). Notably, the hypothesis that PAF-AH might actively contribute to vascular inflammation during atherogenesis owing to its ability to generate pro-inflammatory substances (7) led to the proposition that inhibition of the activity could offer vascular protection in addition to that afforded by cholesterol lowering agents. A number of reversible PAF-AH inhibitors were developed in the pharmaceutical industry and one of them, darapladib, has been extensively tested in vitro and in vivo (8–14). Moreover, GlaxoSmithKline sponsored three darapladib clinical trials that yielded relatively consistent results (11, 15–20).

Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations, from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet–monocyte interactions to inflammatory responses in dengue have not been addressed. Patients with dengue were investigated for platelet–monocyte aggregate formation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro. We observed increased levels of platelet–monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1β, IL-8, IL-10 and MCP-1, whereas exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet–monocyte aggregates. Moreover, IL-10 secretion required platelet–monocyte contact but not phagocytosis. Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue.

Aging-related changes in platelet and monocyte interactions may contribute to adverse outcomes in sepsis but remain relatively unexamined. We hypothesized that differential platelet-monocyte aggregate (PMA) formation in older septic patients alters inflammatory responses and mortality.We prospectively studied 113 septic adults admitted to the intensive care unit with severe sepsis or septic shock. Patients were dichotomized a priori into one of two groups: older (age ≥ 65 years, n = 28) and younger (age65 years, n = 85). PMA levels were measured in whole blood via flow cytometry within 24 hours of admission. Plasma levels of IL-6 and IL-8, proinflammatory cytokines produced by monocytes upon PMA formation, were determined by commercial assays. Patients were followed for the primary outcome of 28-day, all-cause mortality.Elevated PMA levels were associated with an increased risk of mortality in older septic patients (hazard ratio for mortality 5.64, 95% confidence interval 0.64-49.61). This association remained after adjusting for potential confounding variables in multivariate regression. Receiver operating curve analyses demonstrated that PMA levels greater than or equal to 8.43% best predicted 28-day mortality in older septic patients (area under the receiver operating curve 0.82). Plasma IL-6 and IL-8 levels were also significantly higher in older nonsurvivors. In younger patients, neither PMA levels nor plasma monokines were significantly associated with mortality.Increased PMA formation, and associated proinflammatory monokine synthesis, predicts mortality in older septic patients. Although larger studies are needed, our findings suggest that heightened PMA formation in older septic patients may contribute to injurious inflammatory responses and an increased risk of mortality.

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557CT (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.