Granzyme A in Human Platelets Regulates the Synthesis of Proinflammatory Cytokines by Monocytes in Aging.

Dysregulated inflammation is implicated in the pathobiology of aging, yet platelet-leukocyte interactions and downstream cytokine synthesis in aging remains poorly understood. Platelets and monocytes were isolated from healthy younger (age <45, n = 37) and older (age ≥65, n = 30) adults and incubated together under autologous and nonautologous conditions. Synthesis of inflammatory cytokines by monocytes, alone or in the presence of platelets, was examined. Next-generation RNA-sequencing allowed for unbiased profiling of the platelet transcriptome in aging. Basal IL-8 and MCP-1 synthesis by monocytes alone did not differ between older and younger adults. However, in the presence of autologous platelets, monocytes from older adults synthesized greater IL-8 (41 ± 5 versus 9 ± 2 ng/ml, p < 0.0001) and MCP-1 (867 ± 150 versus 216 ± 36 ng/ml, p < 0.0001) than younger adults. Platelets from older adults were sufficient for upregulating the synthesis of inflammatory cytokines by monocytes. Using RNA-sequencing of platelets followed by validation via RT-PCR and immunoblot, we discovered that granzyme A (GrmA), a serine protease not previously identified in human platelets, increases with aging (∼9-fold versus younger adults, p < 0.05) and governs increased IL-8 and MCP-1 synthesis through TLR4 and caspase-1. Inhibiting GrmA reduced excessive IL-8 and MCP-1 synthesis in aging to levels similar to younger adults. In summary, human aging is associated with changes in the platelet transcriptome and proteome. GrmA is present and bioactive in human platelets, is higher in older adults, and controls the synthesis of inflammatory cytokines by monocytes. Alterations in the platelet molecular signature and signaling to monocytes may contribute to dysregulated inflammatory syndromes in older adults.

Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis.

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.

Germline mutations in NFKB2 implicate the noncanonical NF-κB pathway in the pathogenesis of common variable immunodeficiency.

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.2557C>T (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.

Current challenges in understanding immune cell functions during septic syndromes.

BACKGROUND: Sepsis is a dynamic infectious disease syndrome characterized by dysregulated inflammatory responses. RESULTS: Despite decades of research, improvements in the treatment of sepsis have been modest. These limited advances are likely due, in part, to multiple factors, including substantial heterogeneity in septic syndromes, significant knowledge gaps in our understanding of how immune cells function in sepsis, and limitations in animal models that accurately recapitulate the human septic milieu. The goal of this brief review is to describe current challenges in understanding immune cell functions during sepsis. We also provide a framework to guide scientists and clinicians in research and patient care as they strive to better understand dysregulated cell responses during sepsis. CONCLUSIONS: Additional, well-designed translational studies in sepsis are critical for enhancing our understanding of the role of immune cells in sepsis.

Bacteria differentially induce degradation of Bcl-xL, a survival protein, by human platelets.

Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-x(L), an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl-x(L) protein in platelets. Bcl-x(L) protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli that produce the pore-forming toxin α-hemolysin, a response that is markedly attenuated when the gene is deleted from E coli. We also found that nonpathogenic E coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl-x(L) protein in platelets, as do clinical Staphylococcus aureus isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-x(L) protein degradation in platelets coincubated with pathogenic E coli including α-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections.

ACE2 and TAS2R38 receptor expression in pediatric and adult patients in the nasal and oral cavity.

Objective: To investigate differences in angiotensin-converting-enzyme-2 (ACE2) and bitter taste receptor (TAS2R38) expression between patient age groups and comorbidities to characterize the pathophysiology of coronavirus 19(COVID-19) pandemic. ACE2 is the receptor implicated to facilitate SARS-CoV-2 infections and levels of expression may correlate to the severity of COVID-19 infection. TAS2R38 has many non-gustatory roles in disease, with some evidence of severe COVID-19 disease in certain receptor phenotypes. Methods: We conducted a prospective cohort study and collected nasal and lingual tissue from healthy pediatric (n = 22) and adult (n = 25) patients undergoing general anesthesia for elective procedures. RNA isolation and qPCR were performed with primers targeting ACE2 and TAS2R38. Results: A total of 25 adult (52% male; 44% obese) and 22 pediatric (50% male; 36% obese) patients were enrolled, pediatric tissue had 43% more nasal ACE2 RNA expression than adults with a median fold change of 0.69 (IQR 0.37, 0.98) in adults and 0.99 (IQR 0.74, 1.43) in children (p < .05). There were no differences between the age groups in ACE2 expression of lingual tissue (p = .14) or TAS2R38 expression collected from either nasal (p = 049) or lingual tissue (p = .49). Stratifying for obesity yielded similar differences between nasal ACE2 expression between adults and children with median fold change of 0.56 (IQR 0.32, 0.87) in adults and 1.0 (IQR 0.82, 1.52) in children (p < .05). Conclusions: ACE2 receptor expression is higher in nasal tissue collected from children compared to adults, suggesting COVID-19 infectivity is more complicated than ACE2 and TAS2R38 mRNA expression. Level of Evidence: NA.

Novel anti-bacterial activities of ß-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation.

Human ß-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of ß-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of ß-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

Scientific Advancements That Empower Us to Understand CRS Pathophysiology.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a multifactorial inflammatory condition that remains poorly understood. Over the past decade, we have witnessed impressive scientific advancements that have allowed us to better understand the molecular and cellular mechanisms that underlie the inflammatory processes in mucosal diseases including asthma, allergic rhinitis, and CRSwNP. OBJECTIVE: The present review aims to summarize and highlight the most recent scientific advancements that have enriched our understanding of CRSwNP. METHODS: A comprehensive review of the available literature on the use of new scientific techniques in CRSwNP was performed. We evaluated the most recent evidence from studies using animal models, cell cultures, and genome sequencing techniques and their impact on our understanding of CRSwNP pathophysiology. RESULTS: Our understanding of CRSwNP has rapidly progressed with the development of newer scientific techniques to interrogate various pathways involved in its pathogenesis. Animal models remain powerful tools and have elucidated the mechanisms behind esinophilic inflammation in CRSwNP; however, animal models reproducing polyp formation are relatively sparse. 3D cell cultures have significant potential to better dissect the cellular interactions with the sinonasal epithelium and other cell types in CRS. Additionally, some groups are starting to utilize single-cell RNA sequencing to investigate RNA expression in individual cells with high resolution and on a genomic scale. CONCLUSION: These emerging scientific technologies represent outstanding opportunities to identify and develop more targeted therapeutics for different pathways that lead to CRSwNP. An additional understanding of these mechanisms will be critical for developing future therapies for CRSwNP.

Clinical Presentation of Pediatric Recurrent Croup: Implications for Diagnosis.

Recurrent croup is a common clinical entity afflicting the pediatric population, but is not well-characterized in the literature. We describe the largest series of recurrent croup in North America from a single tertiary care academic center, and differentiate subpopulations based on demographic characteristics and clinical presentation. We identified 114 patients for inclusion. Common symptoms included barky cough (86.0%) and inspiratory stridor (60.5%). Many (26.3%) experienced full symptom resolution within a day, and 41.2% reported onset of symptoms at any time of the day. Male patients had a higher number of prior croup episodes at presentation compared with female patients (mean of 9.8 ± 8.0 in males vs 6.6 ± 4.8 in females, P = .03). On multivariate regression, racial/ethnic minority patients were at higher risk for dyspnea (odds ratio [OR]: 58.6; 95% confidence interval [CI]: 7.2-475.4) and upper respiratory infection prodrome (OR: 7.6; 95% CI: 1.6-35.3) compared with non-Hispanic white patients.

Cationic PAMAM dendrimers disrupt key platelet functions.

Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.

The speech perception gap in cochlear implant patients.

OBJECTIVE: To determine how commonly word recognition scores obtained using insert microphones (PB max) overestimate word recognition scores obtained through appropriately fit hearing aids (A-WRS). METHODS: Aided speech recognition tests may not be performed during routine hearing aid fittings; however, they are regularly performed for cochlear implant (CI) candidacy evaluation. Therefore, audiologic data from CI recipients were queried in a retrospective cohort study at a tertiary care center. PB max and A-WRS were obtained. The Speech Perception (SP) gap, defined as PB max minus A-WRS, was calculated for each patient and a high SP gap was defined as ≥20%. RESULTS: Analyzing 78 patients with complete data, 30 patients had PB max ≥20%. Of these, 18 (60%) had a high SP gap. Eighteen of the 78 patients had PB max ≥40%, and of these patients, 15 (83%) had a high SP gap. DISCUSSION/CONCLUSION: A Speech Perception Gap of ≥20% may exist in a sizable percentage of patients with hearing loss. Our pilot study suggests that over 80% of these patients could have Class D hearing (speech recognition <50%) using conventional hearing aids and may be better served using alternate rehabilitation strategies such as middle ear or cochlear implants. Therefore, aided speech testing should be performed as part of a verified hearing aid fit in all patients, especially those with PB Max ranging from 40% to 70%.

A Systematic Review of the Association between Cigarette Smoke Exposure and Chronic Rhinosinusitis.

Objectives Cigarette smoking and passive smoke exposure have been associated with chronic rhinosinusitis (CRS). Our goal in this systematic review was to (1) determine if there was a strong correlative effect in large population studies between cigarette smoke exposure and the prevalence of CRS, (2) investigate pathogenic mechanisms of cigarette smoke in the upper airway, and (3) determine if a history of cigarette smoking affects the medical and surgical outcomes of CRS. Data Sources MEDLINE, Embase, Cochrane CENTRAL, Web of Science SCI and CPCI-S, and websites. Methods A comprehensive literature review and quantitative meta-analysis of studies based on the PRISMA protocol and examining the relationship between cigarette smoke exposure and CRS was performed. A search strategy was developed using various terms such as sinusitis, rhinitis, rhinosinusitis, and smoking. The articles were categorized by (1) epidemiology, (2) pathophysiology, and (3) outcomes. Data regarding study design, population/setting, methods, and bias were collected. Results The initial search generated 2621 titles/abstracts with 309 articles undergoing secondary review and 112 articles for final review. We determined that there is a strong correlation between active and passive cigarette smoke with the prevalence of CRS. Cigarette smoke challenge to sinonasal epithelia results in the release of inflammatory mediators and altered ciliary beat frequency. Pediatric patients exposed to secondhand smoke appear to have particularly poor outcomes. Conclusion There is clear evidence that cigarette smoke is related to CRS, but longitudinal and mechanistic studies are required to determine a causative effect. This information is critical for greater understanding of CRS health outcomes.

Proteasome function is required for platelet production.

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

Methicillin-resistant Staphylococcus aureus-induced thrombo-inflammatory response is reduced with timely antibiotic administration.

Methicillin-resistant Staphylococcus aureus (MRSA) induces a pro-thrombotic and pro-inflammatory milieu. Although timely antibiotic administration in MRSAsepsis may improve outcomes by arresting bacterial growth, the effects of antibiotics on mitigating injurious thrombo-inflammatory cellular responses remains unexplored. Using a newly developed human whole blood model and an in vivo mouse model of MRSAinfection, we examined how antibiotics inhibit MRSAinduced thrombo-inflammatory pathways. Human whole blood was inoculated with MRSA. Thrombin generation and inflammatory cytokine synthesis was measured in the presence or absence of linezolid and vancomycin. C57BL/6 mice were injected with MRSA and the effect of vancomycin administration was examined. MRSAaccelerated thrombin generation in a time- and concentration-dependent manner andinduced the release of cytokines, including interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1. The increase in thrombin generation and inflammatory responses was mediated through the synthesis of tissue factor and cytokines, respectively, and the release of microparticles. The early administration of antibiotics restored normal thrombin generation patterns and significantly reduced the synthesis of cytokines. In contrast, when antibiotic administration was delayed, thrombin generation and cytokine synthesis were not significantly reduced. In mice infected with MRSA, early antibiotic administration reduced thrombin anti-thrombin complexes and cytokine synthesis, whereas delayed antibiotic administration did not. These data provide novel mechanistic evidence of the importance of prompt antibiotic administration in infectious syndromes.

Staphylococcus aureus α-toxin triggers the synthesis of B-cell lymphoma 3 by human platelets.

The frequency and severity of bacteremic infections has increased over the last decade and bacterial endovascular infections (i.e., sepsis or endocarditis) are associated with high morbidity and mortality. Bacteria or secreted bacterial products modulate platelet function and, as a result, affect platelet accumulation at sites of vascular infection and inflammation. However, whether bacterial products regulate synthetic events in platelets is not known. In the present study, we determined if prolonged contact with staphylococcal α-toxin signals platelets to synthesize B-cell lymphoma (Bcl-3), a protein that regulates clot retraction in murine and human platelets. We show that α-toxin induced α(IIb)ß(3)-dependent aggregation (EC(50) 2.98 µg/mL ± 0.64 µg/mL) and, over time, significantly altered platelet morphology and stimulated de novo accumulation of Bcl-3 protein in platelets. Adherence to collagen or fibrinogen also increased the expression of Bcl-3 protein by platelets. α-toxin altered Bcl-3 protein expression patterns in platelets adherent to collagen, but not fibrinogen. Pretreatment of platelets with inhibitors of protein synthesis or the mammalian Target of Rapamycin (mTOR) decreased Bcl-3 protein expression in α-toxin stimulated platelets. In conclusion, Staphylococcusaureus-derived α-toxin, a pore forming exotoxin, exerts immediate (i.e., aggregation) and prolonged (i.e., protein synthesis) responses in platelets, which may contribute to increased thrombotic events associated with gram-positive sepsis or endocarditis.

Platelet-leukocyte interactions link inflammatory and thromboembolic events in ischemic stroke.

Stroke is a common and often fatal event, and, in survivors, it is accompanied by a high risk of recurrence. Ischemic stroke is associated with abnormal platelet activity and thrombus formation. In addition to their roles in the development of acute thrombi, platelets serve as a bridge for leukocytes within the vasculature. Myeloid leukocytes are critical mediators of atherosclerosis and atherothrombosis. Interactions between platelets and leukocytes foster an inflammatory and thrombotic milieu that influences lesion progression, facilitates plaque rupture, and triggers thrombus formation and embolization. Accordingly, antiplatelet agents, including aspirin, dipyridamole, and clopidogrel, are recommended therapies for most patients with a history of stroke. In addition to mitigating thrombosis, antiplatelet drugs have direct and indirect effects on inflammation, which may translate to enhanced clinical efficacy.