Increased Neutrophil Activation Research Articles

Whole blood transcriptional profiles and the pathogenesis of tuberculous meningitis.

Mortality and morbidity from tuberculous meningitis (TBM) are common, primarily due to inflammatory response to Mycobacterium tuberculosis infection, yet the underlying mechanisms remain poorly understood. We aimed to uncover genes and pathways associated with TBM pathogenesis and mortality, and determine the best predictors of death, utilizing whole-blood RNA sequencing from 281 Vietnamese adults with TBM, 295 pulmonary tuberculosis (PTB), and 30 healthy controls. Through weighted gene co-expression network analysis, we identified hub genes and pathways linked to TBM severity and mortality, with a consensus analysis revealing distinct patterns between HIV-positive and HIV-negative individuals. We employed multivariate elastic-net Cox regression to select candidate predictors of death, then logistic regression and internal bootstrap validation to choose best predictors. Increased neutrophil activation and decreased T and B cell activation pathways were associated with TBM mortality. Among HIV-positive individuals, mortality associated with increased angiogenesis, while HIV-negative individuals exhibited elevated TNF signaling and impaired extracellular matrix organization. Four hub genes-MCEMP1, NELL2, ZNF354C, and CD4-were strong TBM mortality predictors. These findings indicate that TBM induces a systemic inflammatory response similar to PTB, highlighting critical genes and pathways related to death, offering insights for potential therapeutic targets alongside a novel four-gene biomarker for predicting outcomes.

Whole blood transcriptional profiles and the pathogenesis of tuberculous meningitis

Mortality and morbidity from tuberculous meningitis (TBM) are common, primarily due to inflammatory response to Mycobacterium tuberculosis infection, yet the underlying mechanisms remain poorly understood. We aimed to uncover genes and pathways associated with TBM pathogenesis and mortality, and determine the best predictors of death, utilizing whole-blood RNA sequencing from 281 Vietnamese adults with TBM, 295 pulmonary tuberculosis (PTB), and 30 healthy controls. Through weighted gene co-expression network analysis, we identified hub genes and pathways linked to TBM severity and mortality, with a consensus analysis revealing distinct patterns between HIV-positive and HIV-negative individuals. We employed multivariate elastic-net Cox regression to select candidate predictors of death, then logistic regression and internal bootstrap validation to choose best predictors. Increased neutrophil activation and decreased T and B cell activation pathways were associated with TBM mortality. Among HIV-positive individuals, mortality associated with increased angiogenesis, while HIV-negative individuals exhibited elevated TNF signaling and impaired extracellular matrix organization. Four hub genes—MCEMP1, NELL2, ZNF354C, and CD4—were strong TBM mortality predictors. These findings indicate that TBM induces a systemic inflammatory response similar to PTB, highlighting critical genes and pathways related to death, offering insights for potential therapeutic targets alongside a novel four-gene biomarker for predicting outcomes.

7972 A Shared Phenotype, Transcriptome, and Methylome in Turner Syndrome Across Various Karyotypes with Concurrent Chronic Neutrophil Activation

Abstract Disclosure: J. Just: None. L. Ridder: None. E. Johannsen: None. H. Christensen: None. A. Skakkebæk: None. C.H. Gravholt: None. Turner syndrome (TS) is a clinical diagnosis with a karyotype containing one X chromosome and complete or partial absence of the second X chromosome. TS is typically associated with clinical manifestations such as short stature, hypergonadotropic hypogonadism and metabolic conditions. The genome-wide changes in females with TS affect both transcriptome and methylome. Historically, genomic studies have primarily focused on the 45,X karyotype, leaving the impact of other karyotypes unexplored. Only 35-45% of the entire TS population has the 45,X karyotype, while the remaining TS women have a range of other karyotypes - i.e. 45,X/46,XX, 46,X,i(Xq), 46,X,i(Xp), 45,X/46,XY, ring X. This study aimed to bridge this gap by comparing TS individuals with the standard 45,X karyotype (TS 45,X) (n = 32) to those with alternative karyotypic forms (TS other karyotypes) (n = 24), alongside age-matched 46,XX controls (n = 33). Specifically, we analyzed the DNA methylation and gene expression profiles from whole-blood samples and explored the genetic and clinical similarities between these groups. Overall findings revealed a shared phenotype and an identical autosomal transcriptome and methylome between TS 45,X and TS other karyotypes. Furthermore, TS 45,X and TS other karyotypes shared the same expression profile for all pseudoautosomal 1 (PAR1) genes on the X chromosome. In addition, the expression of XIST from the q-arm of the X chromosome did not affect the autosomal transcriptome or methylome. Thus, the X chromosomal p-arm, and perhaps in particular PAR1, appear to be a main driver influencing the genome-wide transcriptome and methylome Combining DNA methylation, gene expression and white blood cell counts, we observed a higher neutrophil count (p < 0.01), and increased neutrophil activation in the combined TS group. The increased neutrophil activity, based on increased gene expression of neutrophil activation markers (myeloperoxidase, neutrophil elastase, S100A8/9, p < 0.01), suggested a pathological activation of neutrophils with concomitant release of pro-inflammatory mediators. We further validated the increase of neutrophils in TS women in an independent cohort using flow cytometry (p < 0.01). Emerging evidence has highlighted the substantial involvement of neutrophils in chronic inflammatory processes associated with metabolic diseases such as obesity and diabetes, which triggers a sustained low-grade inflammation partially driven by activated neutrophils. The neutrophil increase and activation in TS women may offer an explanation for this phenotypic trait observed in TS women. Therefore, identifying TS women with increased neutrophil activation in order to initiate anti-inflammatory treatment earlier could potentially mitigate the progression towards more severe metabolic disturbances and associated co-morbidities. Presentation: 6/1/2024

Neutrophils in nasal polyps exhibit transcriptional adaptation and proinflammatory role depend on local polyp milieu.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory upper airway disease, divided into eosinophilic CRSwNP (eCRSwNP) and noneosinophilic CRSwNP (neCRSwNP) according to eosinophilic levels. Neutrophils are major effector cells in CRSwNP. but their role in different inflammatory environments remain largely unclear. We performed an integrated transcriptome analysis of polyp-infiltrating neutrophils from CRSwNP patients, using healthy donor blood as a control. Flow cytometry and in vitro studies showed that neutrophils are activated in both CRSwNP type. The scRNA-sequencing analysis demonstrated that neutrophils were classified into five functional subsets, with GBP5+ neutrophils occurring mainly in neCRSwNPs and a high proportion of CXCL8+ neutrophils in both subendotypes. GBP5+ neutrophils exhibited significant IFN-I pathway activity in neCRSwNPs. CXCL8+ neutrophils displayed increased neutrophil activation scores and mainly secrete Oncostatin M (OSM), which facilitates communication with other cells. In vitro experiments revealed that OSM could enhance IL-13- or IL-17-mediated immune responses in nasal epithelial cells and fibroblasts. Our findings revealed that neutrophils exhibited transcriptional plasticity and activation when exposed to polyp tissue and exert their proinflammatory role in the pathogenesis of CRSwNP by releasing OSM to interact with epithelial cells and fibroblasts in a manner dependent on the inflammatory milieu.

Blockade of the CCR3 receptor reduces neutrophil recruitment to the lung during acute inflammation.

Neutrophils represent one of the host's first lines of defense against invading pathogens. However, an aberrant activation can cause damage to the host. In the case of respiratory infections with viral or bacterial pathogens, one of the most common complications is the development of acute respiratory distress syndrome, in which neutrophil infiltration into the lung is a hallmark. Neutrophils gain expression of chemokine receptors under inflammatory conditions, and their activation can amplify the neutrophil responses. Earlier studies showed that neutrophils recruited to the lung mucosa during bacterial infection upregulate expression of CCR3 and ex vivo stimulation of CCR3 results in an increased neutrophil activation. Therefore, the modulation of effector functions or migration of neutrophils to target sites through chemokine receptors constitutes an opportunity for pharmacological intervention. We aimed to determine whether the blockade of the CCR3 using the specific antagonist SB-328437 reduces neutrophil recruitment and inflammation in the lung in the lipopolysaccharide (LPS)-induced lung injury model and influenza infection in mice. We found that neutrophils acquire CCR3 expression in the lung alveolar space. The intraperitoneal administration of SB-328437 reduced neutrophil recruitment to the lung alveolar space and reduced tissue damage in both the LPS-induced lung injury model and influenza infection. Moreover, treatment with SB-328437 reduced the percentage of neutrophils producing TNFα and neutrophil activation in the alveolar space. Together, these data suggest that CCR3 blockade might be a pharmacological strategy to prevent the aberrant neutrophil activation that results detrimental for the host but preserves sufficient effector response to control the pathogen.

Stress, Hyperglycemia, and Insulin Resistance Correlate With Neutrophil Activity and Impact Acute Myocardial Infarction Outcomes.

Introduction Acute insulin resistance (IR) and hyperglycemia are frequently observed during acute myocardial infarction (AMI), significantly influencing both immediate and long-term patient outcomes, irrespective of diabetic status. Neutrophilia and increased neutrophil activity, which are common in these scenarios, have been associated with poorer prognoses, as demonstrated in our recent findings. While it is well established that neutrophils and stress-induced hyperglycemia exacerbate inflammation and hinder recovery, the complex interplay between these factors and their combined impact on AMI prognosis remains inadequately understood. This study aims to investigate the effects of stress hyperglycemia and IR on AMI patients at the onset of the event and to elucidate the relationship between these metabolic disturbances and inflammatory markers, particularly neutrophils. Methods We conducted a longitudinal prospective study on 219 AMI patients at Elias Emergency Hospital in Bucharest, Romania, from April 2021 to September 2022. Patients were included within 24 hours of AMI with ST-segment elevation and excluded if they had acute infections or chronic inflammatory diseases. Blood samples were collected to study inflammatory biomarkers, including neutrophil extracellular traps (NETs), S100A8/A9, interleukin (IL)-1β, IL-18, and IL-6. Diabetic and pre-diabetic statuses were defined using glycated hemoglobin (HbA1c) and medical history (ADA 2019 criteria). To assess glycemic parameters, we employed the glycemia ratio (GR) and the homeostatic model assessment of insulin resistance (HOMA-IR) index, enabling a precise evaluation of stress hyperglycemia, acute IR, and their prognostic implications. Patients were stratified into groups based on GR calculations, categorized as under-average glycemia, normal glycemia, and stress hyperglycemia. Results The majority of patients in the stress hyperglycemia group exhibited an unfavorable prognosis. This group also demonstrated significantly elevated neutrophil counts and neutrophil-to-lymphocyte ratios (NLR). The GR was significantly and positively correlated with inflammation markers, including neutrophil count (Pearson's R = 0.181, P = 0.008) and NLR (Pearson's R = 0.318, P < 0.001), but showed no significant correlation with other evaluated inflammatory markers. Conclusions Our findings suggest that poor outcomes in AMI patients may be associated with stress hyperglycemia, as indicated by GR. AcuteIR, quantified by GR and HOMA-IR, exhibits a strong correlation with neutrophil count and NLR within the first 24 hours of AMI onset. However, no significant correlation was observed with other inflammatory markers, such as IL-1β, IL-18, and IL-6, underscoring the specific interplay between IR and neutrophil activity in this setting.

Blood-derived products can moderate the activity of neutrophils isolated after biomaterial implantation in a sheep model

Neutrophils play a pivotal role in both the inflammatory phase and subsequent repair processes, making their activity crucial after contact with biomaterials. Excessive neutrophil activation can lead to complications, including implant rejection, highlighting the importance of modulating their activity. This study investigates the long-term interaction of circulating ovine neutrophils with titanium (Ti) and silicon-doped layer (Si-DLC) coated implants. Additionally, the effects of platelet-rich plasma (PRP), platelet-poor plasma (PPP), and antimicrobial neutrophil crude extract (ANE) on neutrophil activity were assessed. Twelve female sheep were divided into Ti, Si-DLC implant and a control group. Neutrophils were stimulated with PRP, PPP, ovine ANE (oANE), or rabbit ANE (rANE), and their enzymatic activity and reactive oxygen and nitrogen species (RONS) generation were evaluated. Hematological parameters were also analyzed. Results showed no adverse effects at implant sites, and all hematological parameters remained within reference values. Ti and Si-DLC implants did not alter neutrophil activity. PRP significantly increased neutrophil activity and RONS production, while PPP decreased activity. oANE reduced enzymatic activity and RONS generation, indicating potential antiinflammatory effects. rANE showed varied effects on neutrophil function. Both ANEs lacked antimicrobial activity against tested pathogens. This study demonstrates the hemocompatibility of Ti and Si-DLC implants and highlights the potential of blood-derived products in modulating the inflammatory response to implants.

Defining a timeline of colon pathologies after keratin 8 loss: rapid crypt elongation and diarrhea are followed by epithelial erosion and cell exfoliation.

Keratins are epithelial intermediate filament proteins that play a crucial role in cellular stress protection, with K8 being the most abundant in the colon. The intestinal epithelial-specific K8-deficient mouse model (K8flox/flox;Villin-Cre) exhibits characteristics of inflammatory bowel disease, including diarrhea, crypt erosion, hyperproliferation, and decreased barrier function. Nevertheless, the order in which these events occur and whether they are a direct cause of K8 loss or a consequence of one event inducing another remains unexplored. Increased knowledge about early events in the disruption of colon epithelial integrity would help to understand the early pathology of inflammatory and functional colon disorders and develop preclinical models and diagnostics of colonic diseases. Here, we aimed to characterize the order of physiological events after Krt8 loss by utilizing K8flox/flox;Villin-CreERt2 mice with tamoxifen-inducible Krt8 deletion in intestinal epithelial cells, and assess stool analysis as a noninvasive method to monitor real-time gene expression changes following Krt8 loss. K8 protein was significantly decreased within a day after induction, followed by its binding partners, K18 and K19 from day 4 onward. The sequential colonic K8 downregulation in adult mice leads to immediate diarrhea and crypt elongation with activation of proliferation signaling, followed by crypt loss and increased neutrophil activity within 6-8 days, highlighting impaired water balance and crypt elongation as the earliest colonic changes upon Krt8 loss. Furthermore, epithelial gene expression patterns were comparable between colon tissue and stool samples, demonstrating the feasibility of noninvasive monitoring of gut epithelia in preclinical research utilizing Cre-LoxP-based intestinal disease models.NEW & NOTEWORTHY Understanding the order in which physiological and molecular events occur helps to recognize the onset of diseases and improve their preclinical models. We utilized Cre-Lox-based inducible keratin 8 deletion in mouse intestinal epithelium to characterize the earliest events after keratin 8 loss leading to colitis. These include diarrhea and crypt elongation, followed by erosion and neutrophil activity. Our results also support noninvasive methodology for monitoring colon diseases in preclinical models.

Abstract 13994: PKM2 Ablation Increases Pro-Inflammatory Signaling in Mice

Introduction: The predominant isoform of pyruvate kinase (PKM1) promotes oxidative metabolism in the healthy heart. We previously described a hypoxia and ischemia-mediated switch to the alternatively spliced isoform PKM2, known to promote glycolysis. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). PKM2 is also a metabolic regulator of immune cell differentiation. Hypothesis: We hypothesize that PKM2 has a role in limiting inflammatory stress by regulating the migration and activity of immune cells. Methods: PKM2 control and global KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI (n=3). Hearts were excised after 3 days and the transcriptomes and gene ontologies (GO) were then assessed by RNA-sequencing and pathway analysis. Circulating C-reactive protein (CRP) was assessed using an ELISA (n=12) and cytokines in plasma and heart tissue were assessed using a multiplex assay (n=3-5). Neutrophil myeloperoxidase secretion and differentiated cardiac macrophages were analyzed by immunofluorescent staining (n=3). Results: GO term enrichment analysis of RNA-seq results showed increased abundance of genes for neutrophil chemotaxis and apoptosis, regulation of cytokine production, and the inflammatory response in PKM2 KO hearts after MI compared to controls. PKM2 KO hearts showed increased neutrophil myeloperoxidase secretion and Ly6G/C staining for neutrophils and monocytes compared to controls. Similarly, M1 macrophages appeared to be more abundant in PKM2 KO hearts compared to controls. IL-6 levels were elevated in PKM2 KO plasma at baseline compared to controls, while CRP was elevated in PKM2 KO plasma across all conditions. IL-6 was also substantially elevated in PKM2 KO hearts after MI compared to controls. Conclusions: Loss of PKM2 increased pro-inflammatory signaling in the heart of KO mice. We saw increased cardiac and plasma IL-6 and CRP, likely related to the polarization of macrophages towards the M1 phenotype, and increased neutrophil activity in PKM2 KO hearts. We suggest that PKM2, induced by a hypoxia-related splicing event, protects the heart against the inflammation that accompanies infarction.

Genetic analysis of cuproptosis subtypes and immunological features in severe influenza

The mechanisms regulating cuproptosis in severe influenza are still unknown. We aimed to identify the molecular subtypes of cuproptosis and immunological characteristics associated with severe influenza in patients requiring invasive mechanical ventilation (IMV). The expression of cuproptosis modulatory factors and immunological characteristics of these patients were analyzed using the public datasets (GSE101702, GSE21802, and GSE111368) from the Gene Expression Omnibus (GEO). Seven cuproptotic-associated genes (ATP7B, ATP7A, FDX1, LIAS, DLD, MTF1, DBT) related to active immune responses were identified in patients suffering from severe and non-severe influenza and two cuproptosis-associated molecular subtypes were discovered in severe influenza patients. Singe-set gene set expression analysis (SsGSEA) indicated that compared with subtype 2, subtype 1 was characterized by reduced adaptive cellular immune responses and increased neutrophil activation. Gene set variation assessment revealed that cluster-specific differentially expressed genes (DEGs) in subtype 1 were involved in autophagy, apoptosis, oxidative phosphorylation, and T cell, immune, and inflammatory responses, amongst others. The random forest (RF) model revealed the most differentiating efficiency with relatively small residual and root mean square error and an increased area under the curve value (AUC = 0.857). Lastly, a five-gene-based RF model (CD247, GADD45A, KIF1B, LIN7A, HLA_DPA1) was established, which showed satisfactory efficiency in the test datasets GSE111368 (AUC = 0.819). Nomogram calibration and decision curve analysis demonstrated its accuracy for the prediction of severe influenza. This study suggests that cuproptosis might be associated with the immunopathology of severe influenza. Additionally, an efficient model for the prediction of cuproptosis subtypes was developed which will contribute to the prevention and treatment of severe influenza patients needing IMV.

Myeloperoxidase inhibition may protect against endothelial glycocalyx shedding induced by COVID-19 plasma

BackgroundSARS-CoV-2, the causative agent of COVID-19, is a threat to public health. Evidence suggests increased neutrophil activation and endothelial glycocalyx (EG) damage are independently associated with severe COVID-19. Here, we hypothesised that an increased level of blood neutrophil myeloperoxidase (MPO) is associated with soluble EG breakdown, and inhibiting MPO activity may reduce EG damage.MethodsAnalysing a subset of acute and convalescent COVID-19 plasma, 10 from severe and 15 from non-severe COVID-19 cases, and 9 from pre-COVID-19 controls, we determined MPO levels, MPO activity and soluble EG proteins (syndecan-1 and glypican-1) levels by enzyme-linked immunosorbent assay. In vitro primary human aortic endothelial cells were cultured with plasma untreated or treated with specific MPO inhibitors (MPO-IN-28, AZD5904) to determine EG shedding. We then investigated whether inhibiting MPO activity decreased EG degradation.ResultsIn COVID-19 plasma, MPO levels, MPO activity and levels of soluble EG proteins are significantly raised compared to controls, and concentrations increase in proportion to disease severity. Despite clinical recovery, protein concentrations remain significantly elevated. Interestingly, there is a trend of increasing MPO activity in convalescent plasma in both severe and non-severe groups. MPO levels and MPO activity correlate significantly with soluble EG levels and inhibiting MPO activity leads to reduced syndecan-1 shedding, in vitro.ConclusionsNeutrophil MPO may increase EG shedding in COVID-19, and inhibiting MPO activity may protect against EG degradation. Further research is needed to evaluate the utility of MPO inhibitors as potential therapeutics against severe COVID-19.

Investigation of Neutrophil Extracellular Trap (NET) Induction in Patients with Chronic Idiopathic Neutropenia Bearing Mutations in the Mediterranean Fever (MEFV) Gene

Introduction: Chronic idiopathic neutropenia (CIN) is a disorder characterized by the prolonged and unexplained reduction in the number of circulating neutrophils. The pathophysiology of CIN has been associated with an inflammatory bone marrow microenvironment consisting of activated T-cells with an oligoclonal/monoclonal profile and intermediate CD14bright/CD16+ monocytes that induce the apoptotic death of the granulocytic progenitor cells. To probe further the basis of immune cell activation in CIN we have recently investigated by next generation sequencing (NGS) the frequency of mutations in the MEFV gene encoding pyrin, in a cohort of 50 CIN patients without any signs/symptoms of Familial Mediterranean Fever (FMF). We have found that 9/50 patients displayed variants associated with typical or atypical FMF phenotype in the Greek population implicating exon 10 and/or exon 2 (Skendros P et al. Blood, 138, Sup.1, p3124;2021). Aim:MEFV mutations have been associated with increased neutrophil activation in FMF and other inflammatory diseases, neutrophil entry in autophagy and extrusion of Neutrophil Extracellular Traps (NETs) decorated with the pro-inflammatory cytokine IL-1β. The aim of the current study is to investigate the association between MEFV mutations and NET formation in neutrophils of CIN patients. Patients-Methods: We studied 20 CIN patients (17 females and 3 males) aged 27 to 78 years. Six CIN patients harbored at least one MEFV mutation with potential clinical significance (A744S homo/R202Q homo, I720M hetero, R202Q homo, K965R hetero, A744S hetero, M694V hetero) and 14 were negative for MEFV pathogenic mutations. The MEFV R202Q mutation is located in exon 2 whereas all the rest mutations are located in exon 10. We isolated neutrophils from healthy subjects (n=5), age- and sex- matched with the patients, and co-cultured them with serum from CIN patients. We used the supernatants of the cultures to quantify NETs with Sytox green staining for extracellular DNA and/or ELISA for dsDNA/MPO complexes. We also used the serum-treated neutrophils for the evaluation of NETosis by immunostaining using confocal microscopy. In addition, neutrophils isolated from CIN patients were used to detect their direct response to spontaneous NETosis induction. Statistical analysis was performed with one-way ANOVA adjusting for multiple comparisons. Results: By co-culturing patients' sera with healthy neutrophils obtained from five different healthy donors, we did not identify statistically significant differences in NETosis evaluation between the three study groups i.e. CIN patients with MEFV mutations, CIN patients negative for pathogenic MEFV mutations, and healthy individuals. On an individual basis, the patient harboring the double homozygous MEFV mutations (A744S/R202Q) displayed increased NETosis in immunostaining, Sytox and ELISA evaluation, as compared to MEFV unmutated CIN patients and healthy individuals. In addition, ex vivo culture of patients' neutrophils and microscopic observation gave us the indication that CIN patients, regardless of their MEFV gene variants, present increased NETosis compared to healthy individuals, suggesting an overactivation status of neutrophils in CIN. Conclusions: The neutrophils of CIN patients display increased NETosis irrespectively of the MEFV mutation status and may thus contribute to the inflammatory processes associated with the disease, a field that has not been investigated so far. Although in our study MEFV mutations did not appear to contribute significantly to inflammatory processes through the NETosis mechanism in CIN patients, their involvement in other aspects of immune deregulation such as through the mTOR/autophagy pathway remains to be further explored.

Alveolar macrophages protect mice from MERS-CoV-induced pneumonia and severe disease.

Emerging and re-emerging human coronaviruses (hCoVs) cause severe respiratory illness in humans, but the basis for lethal pneumonia in these diseases is not well understood. Alveolar macrophages (AMs) are key orchestrators of host antiviral defense and tissue tolerance during a variety of respiratory infections, and AM dysfunction is associated with severe COVID-19. In this study, using a mouse model of Middle East respiratory syndrome coronavirus (MERS-CoV) infection, we examined the role of AMs in MERS pathogenesis. Our results show that depletion of AMs using clodronate (CL) liposomes significantly increased morbidity and mortality in human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice. Detailed examination of control and AM-depleted lungs at different days postinfection revealed increased neutrophil activity but a significantly reduced MERS-CoV-specific CD4 T-cell response in AM-deficient lungs during later stages of infection. Furthermore, enhanced MERS severity in AM-depleted mice correlated with lung inflammation and lesions. Collectively, these data demonstrate that AMs are critical for the development of an optimal virus-specific T-cell response and controlling excessive inflammation during MERS-CoV infection.

Detailed Transcriptional Landscape of Peripheral Blood Points to Increased Neutrophil Activation in Treatment-Naïve Inflammatory Bowel Disease.

Background and AimsInflammatory bowel disease [IBD] is a chronic relapsing disorder of the gastrointestinal tract, which generally manifests as Crohn’s disease [CD] or ulcerative colitis [UC]. These subtypes are heterogeneous in terms of disease location and histological features, while sharing common clinical presentation, genetic associations and, thus, common immune regulatory pathways.MethodsUsing miRNA and mRNA coupled transcriptome profiling and systems biology approaches, we report a comprehensive analysis of blood transcriptomes from treatment-naïve [n = 110] and treatment-exposed [n = 177] IBD patients as well as symptomatic [n = 65] and healthy controls [n = 95].ResultsBroadly, the peripheral blood transcriptomes of CD and UC patients were similar. However, there was an extensive gene deregulation in the blood of IBD patients, while only a slight deregulation in symptomatic controls, when compared with healthy controls. The deregulated mRNAs and miRNAs are mainly involved in the innate immunity and are especially enriched in neutrophil activation-related pathways. Oxidative phosphorylation and neutrophil activation-related modules were found to be differentially co-expressed among treatment-naïve IBD as compared to healthy controls. In the deregulated neutrophil activation-related co-expression module, IL1B was identified as the central gene. Levels of co-expression among IL1B and chemosensing receptor [CXCR1/2 and FPR1/2] genes were reduced in the blood of IBD patients when compared with healthy controls.ConclusionsImmune dysregulation seen in peripheral blood transcriptomes of treatment-naïve IBD patients is mainly driven by neutrophil activation.

Abstract 9846: Association Between Neutrophil Activity and Incident Heart Failure: The Atherosclerosis Risk in Communities Study

Introduction: Inflammation-reduction with colchicine improves outcomes in coronary disease, possibly through effects on neutrophil activity. Inflammation also may contribute to incident heart failure (HF) and the association of neutrophil activity with HF is unclear. We assessed the association between 10 neutrophil-associated proteins that were altered by colchicine in two previous proteomic studies with incident HF. Methods: We studied 10,317 ARIC participants who were free of HF and had aptamer-based proteomic measurements (Somalogic, Colorado) at Visit 3 (1993-1995). Candidate proteins were included in a LASSO Cox regression model which were used to derive a neutrophil activity score. The association of the neutrophil activity score with incident HF was then assessed after adjustment for age, sex, race, study field center, hypertension, smoking history, diabetes, coronary disease, BMI, Visit 3 blood pressure, eGFR, and hsCRP and neutrophil-to-lymphocyte ratio. Results: The mean age was 60±6 years, 54% were women and 20% were black. Over a mean follow-up of 13 years, 1314 participants developed incident HF. Five of the 10 candidate proteins were associated with incident HF in multivariable uni-protein models (myeloperoxidase, suPAR, antichymotrypsin, BPIP, S100A12) at p &lt;0.05. LASSO model retained 4 proteins (suPAR, antichymotrypsin, BPIP, S100A12), from which a neutrophil activity score was developed. Higher neutrophil activity score was associated with older age, higher prevalence of cardiovascular comorbidities, and higher hsCRP. Higher neutrophil activity score was associated with greater risk of incident HF in multivariable models after adjustment for clinical risk factors, hsCRP and neutrophil-to-lymphocyte ratio ( Figure ). Conclusions: Increased neutrophil activity may contribute to incident heart failure in the community. These findings provide a putative mechanism by which treatment with colchicine may influence risk of HF.

Systemic Human Neutrophil Lipocalin Associates with Severe Acute Kidney Injury in SARS-CoV-2 Pneumonia.

Neutrophils have been suggested mediators of organ dysfunction in COVID-19. The current study investigated if systemic neutrophil activity, estimated by human neutrophil lipocalin (HNL) concentration in peripheral blood, is associated with acute kidney injury (AKI) development. A total of 103 adult patients admitted to intensive care, with PCR-confirmed SARS-CoV-2 infection, were prospectively included (Clinical Trials ID: NCT04316884). HNL was analyzed in plasma (P-HNL Dimer) and in whole blood (B-HNL). The latter after ex vivo activation with N-formyl-methionine-leucine-phenylalanine. All patients developed respiratory dysfunction and 62 (60%) were treated with invasive ventilation. Sixty-seven patients (65%) developed AKI, 18 (17%) progressed to AKI stage 3, and 14 (14%) were treated with continuous renal replacement therapy (CRRT). P-HNL Dimer was higher in patients with invasive ventilation, vasopressors, AKI, AKI stage 3, dialysis, and 30-day mortality (p < 0.001–0.046). B-HNL performed similarly with the exception of mild AKI and mortality (p < 0.001–0.004). The cohort was dichotomized by ROC estimated cutoff concentrations of 13.2 µg/L and 190 µg/L for P-HNL Dimer and B-HNL respectively. Increased cumulative risks for AKI, AKI stage 3, and death were observed if above the P-HNL cutoff and for AKI stage 3 if above the B-HNL cutoff. The relative risk of developing AKI stage 3 was nine and 39 times greater if above the cutoffs in plasma and whole blood, respectively, for CRRT eight times greater for both. In conclusion, systemically elevated neutrophil lipocalin, interpreted as increased neutrophil activity, was shown to be associated with an increased risk of severe AKI, renal replacement therapy, and mortality in COVID-19 patients with respiratory failure.

Chronic E-Cigarette Aerosol Inhalation Alters the Immune State of the Lungs and Increases ACE2 Expression, Raising Concern for Altered Response and Susceptibility to SARS-CoV-2.

Conventional smoking is known to both increase susceptibility to infection and drive inflammation within the lungs. Recently, smokers have been found to be at higher risk of developing severe forms of coronavirus disease 2019 (COVID-19). E-cigarette aerosol inhalation (vaping) has been associated with several inflammatory lung disorders, including the recent e-cigarette or vaping product use-associated lung injury (EVALI) epidemic, and recent studies have suggested that vaping alters host susceptibility to pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To assess the impact of vaping on lung inflammatory pathways, including the angiotensin-converting enzyme 2 (ACE2) receptor known to be involved in SARS-CoV-2 infection, mice were exposed to e-cigarette aerosols for 60 min daily for 1–6 months and underwent gene expression analysis. Hierarchical clustering revealed extensive gene expression changes occurred in the lungs of both inbred C57BL/6 mice and outbred CD1 mice, with 2,933 gene expression changes in C57BL/6 mice, and 2,818 gene expression changes in CD1 mice (>abs 1.25-fold change). Particularly, large reductions in IgA and CD4 were identified, indicating impairment of host responses to pathogens via reductions in immunoglobulins and CD4 T cells. CD177, facmr, tlr9, fcgr1, and ccr2 were also reduced, consistent with diminished host defenses via decreased neutrophils and/or monocytes in the lungs. Gene set enrichment (GSE) plots demonstrated upregulation of gene expression related to cell activation specifically in neutrophils. As neutrophils are a potential driver of acute lung injury in COVID-19, increased neutrophil activation in the lungs suggests that vapers are at higher risk of developing more severe forms of COVID-19. The receptor through which SARS-CoV-2 infects host cells, ACE2, was found to have moderate upregulation in mice exposed to unflavored vape pens, and further upregulation (six-fold) with JUUL mint aerosol exposure. No changes were found in mice exposed to unflavored Mod device-generated aerosols. These findings suggest that specific vaping devices and components of e-liquids have an effect on ACE2 expression, thus potentially increasing susceptibility to SARS-CoV-2. In addition, exposure to e-cigarette aerosols both with and without nicotine led to alterations in eicosanoid lipid profiles within the BAL. These data demonstrate that chronic, daily inhalation of e-cigarette aerosols fundamentally alters the inflammatory and immune state of the lungs. Thus, e-cigarette vapers may be at higher risk of developing infections and inflammatory disorders of the lungs.

The pandemic matrix gene is associated with increased pro-inflammatory immune responses during influenza virus infection

Abstract The 2009 H1N1 influenza pandemic (pdmH1N1) arose from multiple genetic reassortment events between three swine influenza A virus (swIAV) lineages and became established as a seasonal H1N1 human influenza virus. Through reverse-zoonotic events, the pdmH1N1 strain contributed gene segments to endemic swIAVs and the pandemic matrix gene (pdmM) completely replaced the classical swine matrix gene (swM), suggesting a fitness benefit. We assessed a panel of swIAVs in the BALB/c mouse model and demonstrated that swIAVs containing the pdmM gene caused increased morbidity, lung pathology, and mortality compared to swIAVs containing the swM gene. We hypothesized that swIAVs containing the pdmM gene induce dysregulation of the innate response, causing increased disease. To test this, mice were infected with swIAVs containing either the pdmM or the swM gene segment, and immune cell infiltrates, cytokine production, and histopathology were assessed. We found that pdmM viruses elicited increased cellular infiltration, NK cell activation and increased IFNγ production in bronchial alveolar lavage (BAL) fluid compared to mice infected with swM viruses. We also detected increased numbers of infiltrating macrophages, neutrophils, DCs, and eosinophils, as well as increased proinflammatory cytokine production. Additionally, elevated myeloperoxidase content was detected in BAL fluid, suggesting increased neutrophil activation. Finally, infection with pdmM viruses resulted in greater histologic lesion scores compared to swM viruses. We conclude the pdmM gene elicits increased innate immune responses, causing greater disease in a mouse model of infection. Future studies will identify determinants of increased disease severity.

Effects of Simulated COVID-19 Cytokine Storm on Stent Thrombogenicity

BackgroundCytokine storm-related hypercoagulation may be important in the pathogenesis of stent thrombosis in patients with SARS-CoV-2. Whether stent polymers behave differently under such conditions has never been explored. MethodsFluorinated polymer-nanocoated and uncoated COBRA stents (CeloNova), BioLinx-polymer-coated Resolute Onyx stents (Medtronic), and Synergy stents (Boston Scientific), which are abluminally coated with a bioabsorbable polymer, were exposed to human blood from healthy donors which was supplemented with 400 pg/mL IL-6 and 100 pg/mL TNF-α, similar to what is seen in cytokine storm caused by SARS-CoV-2. Platelet adhesion and neutrophil activation, assessed by immunofluorescence, were compared under cytokine storm and control conditions (untreated blood) (n = 4 experimental runs). ResultsPlatelet adhesion values, defined as %platelet-covered area x staining intensity, were significantly lower in coated and uncoated COBRA and in Resolute Onyx than in Synergy under control conditions (1.28 × 107 ± 0.43 × 107 vs. 2.92 × 107 ± 0.49 × 107 vs. 3.57 × 107 ± 0.73 × 107 vs. 9.94 × 107 ± 0.99 × 107; p ≤0.0001). In cytokine storm, platelet adhesion values remained low in coated COBRA-PzF (1.78 × 107 ± 0.38 × 107) compared to all other devices (uncoated COBRA: 5.92 × 107 ± 0.96 × 107; Resolute Onyx: 7.27 × 107 ± 1.82 × 107; Synergy: 11.28 × 107 ± 1.08 × 107; p ≤ 0.0001). Although cytokine storm conditions significantly increased neutrophil activation in all stents, it was significantly less in coated and uncoated COBRA, and in Resolute Onyx than in Synergy. ConclusionsBlood-biomaterials interactions may determine the thrombogenic potential of stents. Under simulated cytokine storm conditions, fluoropolymer-coated stents showed the most favorable anti-thrombogenic and anti-inflammatory properties.

Activated low-density granulocytes in peripheral and intervillous blood and neutrophil inflammation in placentas from SLE pregnancies

ObjectiveWomen with SLE face an increased risk of adverse pregnancy outcomes compared with healthy women, but the underlying immunological mechanisms are unknown. Given the recognised association of neutrophil activation with...