Neutrophil-mediated Inflammation Research Articles

Neutrophil-Derived Proteases in Lung Inflammation: Old Players and New Prospects

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.

Dihydrophenanthropyrans derived from the pseudobulbs of Pholidota chinensis alleviates neutrophilic inflammation by inhibiting MAPKs and calcium

Five dihydrophenanthropyrans (1–5) were isolated from the pseudobulbs of Pholidota chinensis, among which 1,3-di(4′-hydroxybenzy)-imbricatin (3) was isolated from the nature for the first time. Their structures were elucidated and established through various spectroscopic methods. These compounds exhibited a potent inhibition effect on both N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced superoxide anion generation and elastase release with IC50 values ranging from 0.23 to 7.63 μM. Furthermore, dihydrophenanthropyrans (1–3) also demonstrated a dose-dependent reactive oxygen species (ROS) scavenging effect. In addition, dihydrophenanthropyrans (2–3) exhibited a dose-dependent reduction in the intracellular Ca2+ concentration ([Ca2+]i) in fMLF-activated human neutrophils. Moreover, dihydrophenanthropyrans (1–3) selectively inhibited the phosphorylation of c-Jun N-terminal kinases (JNKs) and p38, while only dihydrophenanthropyran (1) inhibited the phosphorylation of extracellular signal-regulated kinases (ERKs) in fMLF-activated human neutrophils. Notably, dihydrophenanthropyrans (1–3) did not affect protein kinase B (AKT) activity in these cells. These findings highlight the potent anti-inflammatory capabilities of dihydrophenanthropyrans, manifested through their ability to inhibit superoxide anion generation, suppress elastase release, and selectively modulate key signaling pathways in human neutrophils. This suggests that dihydrophenanthropyrans hold significant promise as therapeutic agents for conditions associated with neutrophil-mediated inflammation.

Inhibition of interferon gamma impairs induction of experimental epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a muco-cutaneous autoimmune disease characterized and caused by autoantibodies targeting type VII collagen (COL7). The treatment of EBA is notoriously difficult, with a median time to remission of 9 months. In preclinical EBA models, we previously discovered that depletion of regulatory T cells (Treg) enhances autoantibody-induced, neutrophil-mediated inflammation and blistering. Increased EBA severity in Treg-depleted mice was accompanied by an increased cutaneous expression of interferon gamma (IFN-γ). The functional relevance of IFN-γ in EBA pathogenesis had been unknown. Given that emapalumab, an anti-IFN-γ antibody, is approved for primary hemophagocytic lymphohistiocytosis patients, we sought to assess the therapeutic potential of IFN-γ inhibition in EBA. Specifically, we evaluated if IFN-γ inhibition has modulatory effects on skin inflammation in a pre-clinical EBA model, based on the transfer of COL7 antibodies into mice. Compared to isotype control antibody, anti-IFN-γ treatment significantly reduced clinical disease manifestation in experimental EBA. Clinical improvement was associated with a reduced dermal infiltrate, especially Ly6G+ neutrophils. On the molecular level, we noted few changes. Apart from reduced CXCL1 serum concentrations, which has been demonstrated to promote skin inflammation in EBA, the expression of cytokines was unaltered in the serum and skin following IFN-γ blockade. This validates IFN-γ as a potential therapeutic target in EBA, and possibly other diseases with a similar pathogenesis, such as bullous pemphigoid and mucous membrane pemphigoid.

RLS-0071, a novel anti-inflammatory agent, significantly reduced inflammatory biomarkers in a randomised human evaluation of mechanisms and safety study

BackgroundThis study was a randomised, double-blind, placebo-controlled study intended to establish the translatability of the RLS-0071 mechanisms of action from animal disease models to humans by inhibiting neutrophil-mediated inflammation at the tissue level and major inflammatory biomarkers. We hypothesized that RLS-0071 inhibits a temporary neutrophil-mediated inflammation in the lungs induced by inhalation of low-dose LPS in healthy participants.MethodsParticipants were randomised to one of three arms to receive inhaled LPS followed by three doses of either low-dose (10 mg·kg−1) or high-dose (120 mg·kg−1loading dose followed by 2 doses of 40 mg·kg−1) RLS-0071 IV or placebo (saline) every 8 h (Q8H). Biomarkers evaluating inflammatory responses, with absolute neutrophil counts in induced sputum as the primary endpoint, were collected before and at 6 and 24 h after LPS challenge.ResultsActive treatment with RLS-0071 showed a similar safety profile to participants receiving placebo. RLS-0071 significantly decreased the numbers of neutrophils in sputum at 6 h post LPS by approximately half (p=0.04). Neutrophil effectors myeloperoxidase, neutrophil elastase, and Interleukin-1β in sputum were also significantly decreased at 6 h for RLS-0071 compared with placebo. Several biomarkers showed trends suggesting sustained decreases for RLS-0071versusplacebo at 24 h.Discussion/ConclusionThis clinical trial demonstrated that RLS-0071 was safe and well tolerated and modulated neutrophil-mediated inflammation in humans after inhaled LPS challenge, consistent with results from prior animal model studies.

Abstract 2136: The Effects Of Dual Antiplatelet Therapy On Neutrophil Function

Introduction: Dual anti-platelet therapy (DAPT) is routinely administered to inhibit platelet aggregation in acute myocardial infarction (AMI) and, in doing so, reduces the risk of recurrent adverse cardiovascular events. Alongside platelets, neutrophils are also key mediators of inflammation during AMI, both secreting cytokines and chemokines and producing Neutrophil Extracellular Traps (NETs). While DAPT is known to mediate off-target anti-inflammatory effects, the extent to which DAPT affects neutrophil function is not well understood. Objective: To investigate the effects of DAPT on neutrophil-mediated inflammation. Methods: Neutrophils were isolated from whole blood of ten healthy subjects pre- and post-DAPT (ticagrelor and aspirin) treatment and NET formation in response to classical NET-inducing agonists (PMA and ionomycin) was measured by SYTOX Green fluorescence and MPO activity. Cytokine and chemokine production following stimulation with an activating agonist (FSL-1) was measured by cytometric bead array and reported as median fluorescence intensity (MFI). DAPT was effective in inhibiting platelet aggregation in response to AA and ADP. Results: NET formation in response to ionomycin, but not PMA, was greater in DAPT-treated neutrophils compared to treatment-naïve neutrophils (69.9 % ± 30.66 SEM). Levels of inflammatory mediators were also markedly different pre- and post- DAPT treatment. For example, MFI of IL-8 was decreased by 18% ± 9.97 SEM post-DAPT compared with pre-DAPT MFI, while MFI of IL-12p and IFN-α were significantly increased (32.54% ± 12.12 SEM, 58.68% ± 18.72 SEM respectively) compared to pre-DAPT. Conclusion: DAPT primes neutrophils for inducing some, but not all, NETotic pathways and releasing some, but not all, inflammatory mediators. This suggests that our primary strategy for inhibiting platelet aggregation in AMI does not provide broad off-target anti-inflammatory effects on neutrophils. In fact, DAPT may encourage a pro-inflammatory neutrophil phenotype.

Cisplatin Toxicity Causes Neutrophil-Mediated Inflammation in Zebrafish Larvae.

Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (Danio rerio), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin (il)-12, il6, and il8 increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils.

Whole blood transcriptomics reveals granulocyte colony-stimulating factor as a mediator of cardiopulmonary bypass-induced systemic inflammatory response syndrome.

Systemic inflammatory response syndrome (SIRS) is a frequent complication of cardiopulmonary bypass (CPB). SIRS is associated with significant morbidity and mortality, but its pathogenesis remains incompletely understood, and as a result, biomarkers are lacking and treatment remains expectant and supportive. This study aimed to understand the pathophysiological mechanisms driving SIRS induced by CPB and identify novel therapeutic targets that might reduce systemic inflammation and improve patient outcomes. Twenty-one patients undergoing cardiac surgery and CPB were recruited, and blood was sampled before, during and after surgery. SIRS was defined using the American College of Chest Physicians/Society of Critical Care Medicine criteria. We performed immune cell profiling and whole blood transcriptomics and measured individual mediators in plasma/serum to characterise SIRS induced by CPB. Nineteen patients fulfilled criteria for SIRS, with a mean duration of 2.7 days. Neutrophil numbers rose rapidly with CPB and remained elevated for at least 48 h afterwards. Transcriptional signatures associated with neutrophil activation and degranulation were enriched during CPB. We identified a network of cytokines governing these transcriptional changes, including granulocyte colony-stimulating factor (G-CSF), a regulator of neutrophil production and function. We identified neutrophils and G-CSF as major regulators of CPB-induced systemic inflammation. Short-term targeting of G-CSF could provide a novel therapeutic strategy to limit neutrophil-mediated inflammation and tissue damage in SIRS induced by CPB.

Plasma Calprotectin Levels Associate With New-Onset Hypertension in the General Population: A Prospective Cohort Study.

Low-grade systemic inflammation is a relevant pathogenic mechanism underlying the development of hypertension. In this study, we hypothesized that plasma calprotectin levels, as a biomarker of neutrophil-mediated inflammation, is associated with developing new-onset hypertension in the general population. Plasma calprotectin levels were determined in 3524 participants who participated in the PREVEND (Prevention of Renal and Vascular End-Stage Disease) study, a prospective population-based cohort study. Plasma calprotectin levels were studied for associations with the risk of new-onset hypertension, defined as systolic blood pressure of at least 140 mm Hg, diastolic blood pressure of at least 90 mm Hg, or the first recorded use of antihypertensives. Participants with hypertension at baseline were excluded. Median plasma calprotectin levels were 0.48 (0.34-0.66) mg/L, and median systolic blood pressure was 117 (109-126) mm Hg. Plasma calprotectin levels were significantly associated with the risk of new-onset hypertension (hazard ratio [HR], per doubling 1.30 [95% CI, 1.21-1.41]; P<0.001), also after adjustment for age and sex (HR, 1.26 [95% CI, 1.16-1.37]; P<0.001), but not after additional adjustment for potentially confounding factors, including baseline systolic blood pressure (HR, 1.00 [95% CI, 0.90-1.11]; P=0.996). Stratified analyses showed significant effect modification by sex (Pinteraction=0.023) and urinary albumin excretion (Pinteraction=0.004), with higher HRs in men (compared with women) and in individuals with higher urinary albumin excretion (>9.3 mg per 24 hours) compared with lower urinary albumin excretion (≤9.3 mg per 24 hours). Higher plasma calprotectin levels are associated with an increased risk of new-onset hypertension in the general population. This association is dependent on baseline systolic blood pressure and is particularly prominent in men compared with women.

Identification CCL2,CXCR2,S100A9 of the immune-related gene markers and immune infiltration characteristics of inflammatory bowel disease and heart failure via bioinformatics analysis and machine learning.

Recently, heart failure (HF) and inflammatory bowel disease (IBD) have been considered to be related diseases with increasing incidence rates; both diseases are related to immunity. This study aims to analyze and identify immune-related gene (IRG) markers of HF and IBD through bioinformatics and machine learning (ML) methods and to explore their immune infiltration characteristics. This study used gene expressiondata (GSE120895, GSE21610, GSE4183) from the Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs) and compare them with IRGs from the ImmPort database to obtain differentially expressed immune-related genes (DIRGs). Functional enrichment analysis of IRGs was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, three machine models and protein-protein interactions (PPIs) were established to identify diagnostic biomarkers. The receiver operating characteristic (ROC) curves were applied to evaluate the diagnostic value of the candidate biomarkersin the validation set (GSE1145, GSE36807) and obtain their correlations with immune cells through the Spearman algorithm. Finally, the CIBERSORT algorithm was used to evaluate the immune cell infiltration of the two diseases. Thirty-four DIRGs were screened and GO and KEGG analysis results showed that these genes are mainly related to inflammatory and immune responses. CCL2, CXCR2 and S100A9 were identified as biomarkers.The immune correlation results indicated in both diseases that CCL2 is positively correlated with mast cell activation, CXCR2 is positively correlated with neutrophils and S100A9 is positively correlated with neutrophils and mast cell activation. Analysis of immune characteristics showed that macrophages M2, macrophages M0 and neutrophils were present in both diseases. CCL2, CXCR2 and S100A9 are promising biomarkers that will become potential immunogenetic biomarkers for diagnosing comorbidities of HF and IBD. macrophages M2, macrophages M0, neutrophil-mediated inflammation and immune regulation play important roles in the development of HF and IBD and may become diagnostic and therapeutic targets.

Periodontal Effects of the Reversible Dipeptidyl Peptidase 1 Inhibitor Brensocatib in Bronchiectasis.

Brensocatib is a reversible inhibitor of dipeptidyl peptidase 1 (cathepsin C), in development to treat chronic non-cystic fibrosis bronchiectasis. The phase 2, randomized, placebo-controlled WILLOW trial (NCT03218917) was conducted to examine whether brensocatib reduced the incidence of pulmonary exacerbations. Brensocatib prolonged the time to the first exacerbation and led to fewer exacerbations than placebo. Because brensocatib potentially affects oral tissues due to its action on neutrophil-mediated inflammation, we analyzed periodontal outcomes in the trial participants. Patients with bronchiectasis were randomized 1:1:1 to receive once-daily oral brensocatib 10 or 25 mg or placebo. Periodontal status was monitored throughout the 24-week trial in a prespecified safety analysis. Periodontal pocket depth (PPD) at screening, week 8, and week 24 was evaluated. Gingival inflammation was evaluated by a combination of assessing bleeding upon probing and monitoring the Löe-Silness Gingival Index on 3 facial surfaces and the mid-lingual surface. At week 24, mean ± SE PPD reductions were similar across treatment groups: -0.07 ± 0.007, -0.06 ± 0.007, and -0.15 ± 0.007 mm with brensocatib 10 mg, brensocatib 25 mg, and placebo, respectively. The distribution of changes in PPD and the number of patients with multiple increased PPD sites were similar across treatment groups at weeks 8 and 24. The frequencies of gingival index values were generally similar across treatment groups at each assessment. An increase in index values 0-1 and a decrease in index values 2-3 over time and at the end of the study were observed in all groups, indicating improved oral health. In patients with non-cystic fibrosis bronchiectasis, brensocatib 10 or 25 mg had an acceptable safety profile after 6 months' treatment, with no changes in periodontal status noted. Improvement in oral health at end of the study may be due to regular dental care during the trial and independent of brensocatib treatment. The results of this study suggest that 24 weeks of treatment with brensocatib does not affect periodontal disease progression. This information can be used by clinicians when considering treatment approaches for bronchiectasis and suggests that the use of brensocatib will not be limited by periodontal disease risks. Nevertheless, routine dental/periodontal care should be provided to patients irrespective of brensocatib treatment.

The Role of Cytokines in Neutrophil Development, Tissue Homing, Function and Plasticity in Health and Disease.

Neutrophils are crucial innate immune cells and comprise 50-70% of the white blood cell population under homeostatic conditions. Upon infection and in cancer, blood neutrophil numbers significantly increase because of the secretion of various chemo- and cytokines by, e.g., leukocytes, pericytes, fibroblasts and endothelial cells present in the inflamed tissue or in the tumor microenvironment (TME). The function of neutrophils in cancer has recently gained considerable attention, as they can exert both pro- and anti-tumorigenic functions, dependent on the cytokine milieu present in the TME. Here, we review the effect of cytokines on neutrophil development, tissue homing, function and plasticity in cancer and autoimmune diseases as well as under physiological conditions in the bone marrow, bloodstream and various organs like the spleen, kidney, liver, lung and lymph nodes. In addition, we address several promising therapeutic options, such as cytokine therapy, immunocytokines and immunotherapy, which aim to exploit the anti-tumorigenic potential of neutrophils in cancer treatment or block excessive neutrophil-mediated inflammation in autoimmune diseases.

Network analysis identifies a gene biomarker panel for sepsis-induced acute respiratory distress syndrome

BackgroundAcute respiratory distress syndrome (ARDS) is characterized by non-cardiogenic pulmonary edema caused by inflammation, which can lead to serious respiratory complications. Due to the high mortality of ARDS caused by sepsis, biological markers that enable early diagnosis are urgently needed for clinical treatment.MethodsIn the present study, we used the public microarray data of whole blood from patients with sepsis-induced ARDS, patients with sepsis-alone and healthy controls to perform an integrated analysis based on differential expressed genes (DEGs) and co-expression network to identify the key genes and pathways related to the development of sepsis into ARDS that may be key targets for diagnosis and treatment.ResultsCompared with controls, we identified 180 DEGs in the sepsis-alone group and 152 DEGs in the sepsis-induced ARDS group. About 70% of these genes were unique to the two groups. Functional analysis of DEGs showed that neutrophil-mediated inflammation and mitochondrial dysfunction are the main features of ARDS induced by sepsis. Gene network analysis identified key modules and screened out key regulatory genes related to ARDS. The key genes and their upstream regulators comprised a gene panel, including EOMES, LTF, CSF1R, HLA-DRA, IRF8 and MPEG1. Compared with the healthy controls, the panel had an area under the curve (AUC) of 0.900 and 0.914 for sepsis-alone group and sepsis-induced ARDS group, respectively. The AUC was 0.746 between the sepsis-alone group and sepsis-induced ARDS group. Moreover, the panel of another independent blood transcriptional expression profile dataset showed the AUC was 0.769 in diagnosing sepsis-alone group and sepsis-induced ARDS group.ConclusionsTaken together, our method contributes to the diagnosis of sepsis and sepsis-induced ARDS. The biological pathway involved in this gene biomarker panel may also be a critical target in combating ARDS caused by sepsis.

Spatial characteristics of neutrophils and eosinophils in conducting airway mucosa of mice with induced allergic airway inflammation

Daily inhaled antigens induce cellular immune response in the airways. In case of allergens, allergic airway inflammation is usually represented by eosinophils, however, neutrophil infiltration is also observed during severe asthma. Animal models contribute to investigation of the mechanisms that involve the switching to eosinophil- or neutrophil-mediated inflammation. Data about the spatial location of eosinophils and neutrophils in the airways are necessary for both the understanding of allergic airway inflammation mechanisms and the drag potential estimation, however, not completely investigated. In the present study, we characterized the model of Aspergillus fumigatus extract-induced allergic airway inflammation that allowed investigating the early stage of inflammation development. The model adequacy was confirmed according to the blood and bronchoalveolar lavage eosinophilia. Using immunohistochemical staining of conducting airway as a whole-mount and confocal laser scanning microscopy, we estimated neutrophil and eosinophil spatial location: in the luminal side of the epithelium, in the airway wall or in the submucosal compartment close to the smooth muscle layer. An allergic airway response activation was detected upon significant elevation of blood eosinophil percentage compared to intact mice. Simultaneously, the number of eosinophils in the bronchoalveolar lavage was also significantly increased compared to the intact mice. At this time point, eosinophils predominated both in bronchoalveolar lavages and in conducting airway mucosa compared to neutrophils. Spatial location of conducting airway mucosal cell analysis demonstrated that eosinophils mostly located in the submucosal compartment, in a lesser extent in the airway wall, and a few eosinophils were detected in the luminal side of the epithelium. Neutrophils mainly infiltrated the luminal side of the epithelium, and a few neutrophils were detected in the submucosal compartment, while no neutrophils were detected in the airway wall. The data suggests that in response to the further allergen challenge, evidently eosinophils but not neutrophils will migrate through the airway wall to the airway lumen. Thus, eosinophils can be expected to damage airway epithelium in allergic airway inflammation development. Simultaneously, neutrophils located in close proximity to the smooth muscle layer together with eosinophils can contribute to bronchoconstriction induction.

Association Between Soluble Intercellular Adhesion Molecule-1 and Intracerebral Hemorrhage Outcomes in the FAST Trial.

Neutrophil-mediated inflammation in the acute phase of intracerebral hemorrhage (ICH) worsens outcome in preclinical studies. sICAM-1 (soluble intercellular adhesion molecule-1), an inducible ligand for integrins and cell-cell adhesion molecules, is critical for neutrophil extravasation. We aimed to determine whether serum levels of sICAM-1 are associated with worse outcomes after ICH. We conducted a post hoc secondary analysis of an observational cohort using data from the FAST trial (Factor-VII for Acute Hemorrhagic Stroke Treatment). The study exposure was the admission serum level of sICAM-1. The coprimary outcomes were mortality and poor outcome (modified Rankin Scale score 4-6) at 90 days. Secondary radiological outcomes were hematoma expansion at 24 hours and perihematomal edema expansion at 72 hours. We used multiple linear and logistic regression analyses to test for associations between sICAM-1 and outcomes, after adjustment for demographics, ICH severity characteristics, change in the systolic blood pressure in the first 24 hours, treatment randomization arm, and the time from symptom onset to study drug administration. Of 841 patients, we included 507 (60%) with complete data. Hematoma expansion occurred in 169 (33%), while 242 (48%) had a poor outcome. In multivariable analyses, sICAM-1 was associated with mortality (odds ratio, 1.53 per SD increase [95% CI, 1.15-2.03]) and poor outcome (odds ratio, 1.34 per SD increase [CI, 1.06-1.69]). In multivariable analyses of secondary outcomes, sICAM-1 was associated with hematoma expansion (odds ratio, 1.35 per SD increase [CI, 1.11-1.66]), but was not associated with log-transformed perihematomal edema expansion at 72 hours. In additional analyses stratified by treatment assignment, similar results were noted in the recombinant activated factor-VII arm, but not in the placebo arm. Admission serum levels of sICAM-1 were associated with mortality, poor outcome, and hematoma expansion. Given the possibility of a biological interaction between recombinant activated factor-VII and sICAM-1, these findings highlight the need to further explore the role of sICAM-1 as a potential marker of poor ICH outcomes.

Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency.

The rupture of atherosclerotic plaque contributes significantly to cardiovascular disease. Plasma concentrations of bilirubin-a byproduct of heme catabolism-inversely associate with risk of cardiovascular disease, although the link between bilirubin and atherosclerosis remains unclear. To assess the role of bilirubin in atherosclerotic plaque stability, we crossed Bvra-/- with Apoe-/- mice and used the tandem stenosis model of plaque instability. Human coronary arteries were obtained from heart transplant recipients. Analysis of bile pigments, heme metabolism, and proteomics were performed by liquid chromatography tandem mass spectrometry. MPO (myeloperoxidase) activity was determined by in vivo molecular magnetic resonance imaging, liquid chromatography tandem mass spectrometry analysis, and immunohistochemical determination of chlorotyrosine. Systemic oxidative stress was evaluated by plasma concentrations of lipid hydroperoxides and the redox status of circulating Prx2 (peroxiredoxin 2), whereas arterial function was assessed by wire myography. Atherosclerosis and arterial remodeling were quantified by morphometry and plaque stability by fibrous cap thickness, lipid accumulation, infiltration of inflammatory cells, and the presence of intraplaque hemorrhage. Compared with Bvra+/+Apoe-/- tandem stenosis littermates, Bvra-/-Apoe-/- tandem stenosis mice were deficient in bilirubin, showed signs of increased systemic oxidative stress, endothelial dysfunction, as well as hyperlipidemia, and had a higher atherosclerotic plaque burden. Heme metabolism was increased in unstable compared with stable plaque of both Bvra+/+Apoe-/- and Bvra-/-Apoe-/- tandem stenosis mice and in human coronary plaques. In mice, Bvra deletion selectively destabilized unstable plaque, characterized by positive arterial remodeling and increased cap thinning, intraplaque hemorrhage, infiltration of neutrophils, and MPO activity. Proteomic analysis confirmed Bvra deletion enhanced extracellular matrix degradation, recruitment and activation of neutrophils, and associated oxidative stress in unstable plaque. Bilirubin deficiency, resulting from global Bvra deletion, generates a proatherogenic phenotype and selectively enhances neutrophil-mediated inflammation and destabilization of unstable plaque, thereby providing a link between bilirubin and cardiovascular disease risk.

Abstract WP131: Association Between Intercellular Adhesion Molecule-1 And Intracerebral Hemorrhage: A Post-hoc Analysis Of The Fast Trial

Introduction: Neutrophil-mediated inflammation in the acute phase of intracerebral hemorrhage (ICH) worsens outcome in pre-clinical studies. Intercellular adhesion molecule-1 (ICAM-1), an inducible ligand for integrins and cell-cell adhesion molecules, is critical for neutrophil extravasation. We aimed to determine whether serum levels of ICAM-1 are associated with worse outcomes after ICH. Methods: We conducted a post-hoc analysis of the randomized controlled FAST (Factor-VII-for-Acute-Hemorrhagic-Stroke-Treatment) trial. The study exposure was serum level of ICAM-1, ascertained at admission. The co-primary outcomes were mortality, and death or major disability (modified Rankin Score 4-6) at 90 days. Secondary radiological outcomes were baseline ICH volume, perihematomal edema volume (PHE), and hematoma expansion at 24 hours (ICH volume increase of ≥33% or 6mL). Multivariable linear and logistic regression analyses, adjusted for age, sex, baseline ICH volume, ICH location, and trial treatment arm, were used to test for associations between ICAM-1 and outcomes. Results: Of 841 patients, we included 507 (60%) with complete data (mean age 64 years, 37% women). Hematoma expansion occurred in 169 (34%), while 242 (48%) had a poor outcome. In multivariable analyses, ICAM-1 was associated with mortality (OR, 1.02; 95% confidence interval, CI, 1.01-1.05; p=0.02) and poor outcome (OR, 1.02; CI, 1.01-1.04; p=0.04). In multivariable analyses of radiological outcomes, ICAM-1 was associated with hematoma expansion (OR, 1.03, CI, 1.01-1.07) and baseline PHE (beta, 0.02; SE, 0.001, p=0.01), but was not associated with baseline ICH volume. Conclusions: In this post-hoc analysis of the FAST trial in patients with ICH, admission serum levels of ICAM-1 were associated with hematoma expansion, PHE, and poor outcome. These findings highlight the need to further explore the role of early inflammatory biomarkers in prognostication and as potential therapeutic targets.

From the Editor’s Desk…

From the Editor’s Desk…

Genetic and Serum Screening for Alpha-1-Antitrypsin Deficiency in Adult Patients with Cystic Fibrosis: A Single-Center Experience.

Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two of the commonest genetic diseases affecting the Caucasian population. Neutrophil-mediated inflammation due to protease-antiprotease imbalance leads to progressive pulmonary involvement in both diseases. The aim of this study was to investigate the prevalence of AAT deficiency in CF adults. A prospective study enrolling CF adults was conducted at the Adult CF Center based in Milan from January 2018 to March 2019. Patients were tested for AAT serum protein quantification and expanded genotyping characterization of SERPINA1 during clinical stability. Genotyping characterization of SERPIN1 was compared to a control population of 2848 Caucasian individuals with the same geographical origin and similar demographic characteristics. Among 173 patients included in the study, the prevalence of AAT deficiency was 0. Genotype analysis was piMM in 166 (94.9%) patients and piMS in 9 patients (5.1%), respectively. No differences in terms of genotype characterization were found between the CF population and the control population. These data show that AAT deficiency is not common among adults with CF.

Rap1b-loss increases neutrophil lactate dehydrogenase activity to enhance neutrophil migration and acute inflammation in vivo.

Neutrophils are critical for host immune defense; yet, aberrant neutrophil tissue infiltration triggers tissue damage. Neutrophils are heterogeneous functionally, and adopt 'normal' or 'pathogenic' effector function responses. Understanding neutrophil heterogeneity could provide specificity in targeting inflammation. We previously identified a signaling pathway that suppresses neutrophilmediated inflammation via integrin-mediated Rap1b signaling pathway. Here, we used Rap1-deficient neutrophils and proteomics to identify pathways that specifically control pathogenic neutrophil effector function. We show neutrophil acidity is normally prevented by Rap1b during normal immune response with loss of Rap1b resulting in increased neutrophil acidity via enhanced Ldha activity and abnormal neutrophil behavior. Acidity drives the formation of abnormal invasive-like protrusions in neutrophils, causing a shift to transcellular migration through endothelial cells. Acidity increases neutrophil extracellular matrix degradation activity and increases vascular leakage in vivo. Pathogenic inflammatory condition of ischemia/reperfusion injury is associated with increased neutrophil transcellular migration and vascular leakage. Reducing acidity with lactate dehydrogenase inhibition in vivo limits tissue infiltration of pathogenic neutrophils but less so of normal neutrophils, and reduces vascular leakage. Acidic milieu renders neutrophils more dependent on Ldha activity such that their effector functions are more readily inhibited by small molecule inhibitor of Ldha activity, which offers a therapeutic window for antilactate dehydrogenase treatment in specific targeting of pathogenic neutrophils in vivo.

Mechanisms and Therapeutic Modulation of Neutrophil-Mediated Inflammation

Neutrophils are abundant, short-lived myeloid cells that are readily recruitable to sites of inflammation, where they serve as first-line defense against infection and other types of insult to the host. In recent years, there has been increased understanding on the involvement of neutrophils in chronic inflammatory diseases, where they may act as direct effectors of destructive inflammation. However, destructive tissue inflammation is also instigated in settings of neutrophil paucity, suggesting that neutrophils also mediate critical homeostatic functions. The activity of neutrophils is regulated by a variety of local tissue factors. In addition, systemic metabolic conditions, such as hypercholesterolemia and hyperglycemia, affect the production and mobilization of neutrophils from the bone marrow. Moreover, according to the recently emerged concept of innate immune memory, the functions of neutrophils can be enhanced through the process of trained granulopoiesis. This process may have both beneficial and potentially destructive effects, depending on context, that is, protective against infections and tumors, while destructive in the context of chronic inflammatory conditions. Although we are far from a complete understanding of the mechanisms underlying the regulation and function of neutrophils, current insights enable the development of targeted therapeutic interventions that can restrain neutrophil-mediated inflammation in chronic inflammatory diseases, such as periodontitis.