Neutrophil Activation Markers Research Articles

Loss of Acid Ceramidase in Myeloid Cells Impairs MHC II Expression on Dendritic Cells and Macrophages to Alleviate Chronic Colitis in IL10−/− Mice

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the colon and drastically increases the risk in the development of colorectal cancer (CRC). Over expression of acid ceramidase (AC) resulting in accumulation of sphingosine-1-phosphate (S1P), which amplify inflammatory pathways, has been implicated in patients with IBD and CRC. The conditional loss of AC in myeloid cells (ACMYE) has demonstrated promise as a therapeutic target in an acute colitis model. We sought to expand the investigation of AC loss in a physiologically relevant model of IBD using IL-10 deficient mice. IL10−/− mice were crossed with AC conditional knockout mice where AC is deleted in myeloid cells to generate ACMYE/IL10−/−, with ACfl/fl/IL10−/− mice used as control. Male and female animals were collected at 8, 12, and 24 weeks of age and assessed for intestinal inflammation. Upon examination of basic parameters of colitis, ACMYE mice exhibited reduced body weight and enlarged spleens at 12 weeks of age. Histologic assessment revealed no difference between genotypes in colitis severity at 8 or 12 weeks of age. Flow cytometry analysis was utilized to define the role of AC in immune cell recruitment. ACMYE mice exhibit reduced MHC II surface expression on antigen presenting cells (APCs) as well as reductions in neutrophil activation markers in peripheral tissues and the colon at 8 weeks of age. Interestingly, at 12 weeks of age ACfl/fl mice demonstrate significant immunosuppression across various cell types in the colon compared to ACMYE mice, whose colonic immune cells demonstrate little change over time. Furthermore, CD8+ cytotoxic T cell populations were significantly elevated in peripheral tissues despite continued reductions of MHC II surface expression on APCs in ACMYE mice at 12 weeks of age. These data indicate that loss of AC may provide protection in early age while resulting in altered inflammation in later age; however, loss of AC may also disrupt MHC II expression as seen consistently in APCs, and may indicate interference with cell-to-cell cross talk. Realtime qPCR revealed that ACMYE mice exhibited significant ablation in the expression of several pro-inflammatory cytokines at 24 weeks of age. Together these data implicate AC as a potential therapeutic target in reducing inflammation in chronic IBD. This work is supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK132079 (AJS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Whole blood transcriptomics reveals the enrichment of neutrophil activation pathways during erythema nodosum leprosum reaction.

Patients with the multibacillary form of leprosy can develop reactional episodes of acute inflammation, known as erythema nodosum leprosum (ENL), which are characterized by the appearance of painful cutaneous nodules and systemic symptoms. Neutrophils have been recognized to play a role in the pathogenesis of ENL, and recent global transcriptomic analysis revealed neutrophil-related processes as a signature of ENL skin lesions. In this study, we expanded this analysis to the blood compartment, comparing whole blood transcriptomics of patients with non-reactional lepromatous leprosy at diagnosis (LL, n=7) and patients with ENL before administration of anti-reactional treatment (ENL, n=15). Furthermore, a follow-up study was performed with patients experiencing an ENL episode at the time of diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Validation in an independent cohort (ENL=8; LL=7) was performed by RT-qPCR. An enrichment of neutrophil activation and degranulation-related genes was observed in the ENL group, with the gene for the neutrophil activation marker CD177 being the most enriched gene of ENL episode when compared to its expression in the LL group. A more pro-inflammatory transcriptome was also observed, with increased expression of genes related to innate immunity. Validation in an independent cohort indicated that S100A8 expression could discriminate ENL from LL. Supernatants of blood cells stimulated in vitro with Mycobacterium leprae sonicate showed higher levels of CD177 compared to the level of untreated cells, indicating that the leprosy bacillus can activate neutrophils expressing CD177. Of note, suggestive higher CD177 protein levels were found in the sera of patients with severe/moderate ENL episodes when compared with patients with mild episodes and LL patients, highlighting CD177 as a potential systemic marker of ENL severity that deserves future confirmation. Furthermore, a follow-up study was performed with patients at the time of ENL diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Enrichment of neutrophil pathways was sustained in the transcriptomic profile of patients undergoing treatment; however, important immune targets that might be relevant to the effect of thalidomide at a systemic level, particularly NLRP6 and IL5RA, were revealed. In conclusion, our study reinforces the key role played by neutrophils in ENL pathogenesis and shed lights on potential diagnostic candidates and novel therapeutic targets that could benefit patients with leprosy.

Peripheral priming induces plastic transcriptomic and proteomic responses in circulating neutrophils required for pathogen containment.

Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow are shaped within the circulation remains vague. Experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile human and murine neutrophil transcriptomes and proteomes during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to dynamic shifts dominated by conserved up-regulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB signaling-dependent up-regulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by up-regulation of distinct effector molecules upon infection. These data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation.

Calprotectin as a new inflammatory marker of abdominal aortic aneurysm: A pilot study.

Abdominal aortic aneurysm (AAA) is a relevant clinical problem due to the risk of rupture of progressively dilated infrarenal aorta. It is characterized by degradation of elastic fibers, extracellular matrix, and inflammation of the arterial wall. Though neutrophil infiltration is a known feature of AAA, markers of neutrophil activation are scarcely analyzed; hence, the main objective of this study. Plasma levels of main neutrophil activation markers were quantified in patients with AAA and a double control group (CTL) formed by healthy volunteers (HV) and patients with severe atherosclerosis submitted for carotid endarterectomy (CE). Calprotectin, a cytoplasmic neutrophil protein, was quantified, by Western blot, in arterial tissue samples from patients with AAA and organ donors. Colocalization of calprotectin and neutrophil elastase was assessed by immunofluorescence. Plasma calprotectin and IL-6 were both elevated in patients with AAA compared with CTL (p ⩽ 0.0001) and a strong correlation was found between both molecules (p < 0.001). This difference was maintained when comparing with HV and CE for calprotectin but only with HV for IL-6. Calprotectin was also elevated in arterial tissue samples from patients with AAA compared with organ donors (p < 0.0001), and colocalized with neutrophils in the arterial wall. Circulating calprotectin could be a specific AAA marker and a potential therapeutical target. Calprotectin is related to inflammation and neutrophil activation in arterial wall and independent of other atherosclerotic events.

Neutrophilic Activity Biomarkers (Plasma Neutrophil Extracellular Traps and Calprotectin) in Established Patients with Rheumatoid Arthritis Receiving Biological or JAK Inhibitors: A Clinical and Ultrasonographic Study.

This study assesses the accuracy of neutrophil activation markers, including neutrophil extracellular traps (NETs) and calprotectin, as biomarkers of disease activity in patients with established rheumatoid arthritis (RA). We also analyse the relationship between NETs and various types of therapies as well as their association with autoimmunity. Observational cross-sectional study of patients with RA receiving treatment with biological disease-modifying antirheumatic drugs or Janus kinase inhibitors (JAK-inhibitors) for at least 3months. Plasma calprotectin levels were measured using an enzyme-linked immunosorbent assay test kit and NETs by measuring their remnants in plasma (neutrophil elastase-DNA and histone-DNA complexes). We also assessed clinical disease activity, joint ultrasound findings and autoantibody status [reumatoid factor (RF), anti-citrullinated peptide/protein antibodies (ACPAs) and anti-carbamylated protein (anti-CarP)]. Associations between neutrophilic biomarkers and clinical or ultrasound scores were sought using correlation analysis. The discriminatory capacity of both neutrophilic biomarkers to detect ultrasound synovitis was analysed through receiver-operating characteristic (ROC) curves. One hundred fourteen patients were included. Two control groups were included to compare NET levels. The active control group consisted of 15 patients. The second control group consisted of 30 healthy subjects. Plasma NET levels did not correlate with clinical disease status, regardless of the clinic index analysed or the biological therapy administered. No significant correlation was observed between NET remnants and ultrasound synovitis. There was no correlation between plasma NET and autoantibodies. In contrast, plasma calprotectin positively correlated with clinical parameters (swollen joint count [SJC] rho = 0.49; P < 0.001, Clinical Disease Activity Index [CDAI] rho = 0.30; P < 0.001) and ultrasound parameters (rho > 0.50; P < 0.001). Notably, this correlation was stronger than that observed with acute phase reactants. While NET formation induced by neutrophils may play a role in RA pathogenesis, our study raises questions about the utility of NET remnants in peripheral circulation as a biomarker for inflammatory activity. In contrast, this study strongly supports the usefulness of calprotectin as a biomarker of inflammatory activity in patients with RA.

Inflammatory and neutrophil activation markers in Behcet's disease

Behcet's disease (BD) is a systemic vasculitis characterized by recurrent episodes of inflammation with aphthous stomatitis, genital ulcers, skin, joint and internal organ involvement. Currently, there are no reliable laboratory markers that can be used to monitor BD activity. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) have low sensitivity, so the search for new biomarkers continues. Neutrophil reactivity intensity (NEUT-RI), neutrophil granularity intensity (NEUT-GI), immature granulocytes (IG), neutrophil-to-lymphocyte ratio (NLR), systemic immune inflammation index (SII) are new inflammatory indicators whose role in BD is not well studied.Objective: to evaluate and compare the efficacy of the determination of NEUT-RI, NEUT-GI, IG, SII, NLR, ESR and CRP in the diagnosis of active BD.Material and methods. 84 patients with a reliable diagnosis of BD and 38 healthy controls were included in the study. BD activity was assessed using the Behcet’s Disease Current Activity Form (BDCAF). Patients with BD were divided into two groups according to activity: Group I included 41 patients with high activity (BDCAF &gt;5) and Group II included 43 patients with low activity (BDCAF ≤5). A complete blood count with determination of NEUT-RI, NEUT-GI, IG, SII and NLR was performed in all patients and healthy donors using the Sysmex XN 1000 automated haematology analyzer (Sysmex Сorp, Japan). ESR was determined using the Westergren method. High-sensitivity serum CRP level was determined in all patients with BD (normal range ≤5) by immunonephelometric method.Results and discussion. Patients with active BD had significantly higher levels of neutrophils, SII, NLR, NEUT-RI, IG, ESR and CRP compared to patients with low disease activity. ROC analysis was performed to compare the significance of these inflammatory markers in the assessment of BD activity. The SII had the largest area under the ROC curve (AUC =0.816). The sensitivity and specificity of the SII at a value of 509.75 or higher for determining the active form of BD were 79.4% and 71.8%, respectively.Conclusion. The new inflammatory parameters (SII, NLR, NEUT-RI and IG) and the traditional inflammatory parameters (ESR, CRP, neutrophils) can serve as laboratory markers for BD activity. SII is the most informative parameter to determine BD activity with optimal sensitivity and specificity.

Mast cell-derived IL-10 protects intestinal barrier integrity during malaria in mice and regulates parasite transmission to Anopheles stephensi with a female-biased immune response.

Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.

Vaginal candida infection is associated with host molecular signatures of neutrophil activation in the adjacent ectocervical mucosa in Kenyan sex workers

AbstractProblemOvergrowth of candida species in the human vaginal mucosa causes inflammation, which could render the mucosal barrier more susceptible to HIV infection. Here, we investigated whether this condition also affects the ectocervical mucosa, a potential site of HIV entry, in women at high risk of HIV infection.Method of studyRetrospective medical data and ectocervical tissue samples were obtained from a cohort of Kenyan sex workers. Among 108 women, seven had signs of vaginal candida infection by wet smear microscopy and/or the presence of characteristic discharge. Women lacking these two criteria served as controls. Host transcriptomic profiling and quantitative in situ image analysis of epithelial barrier markers and CD4+ cell distribution were performed.ResultsThe candida group had 162 differentially expressed genes out of 15 435 genes as compared with the control group. Among these 162 genes, 147 were upregulated and 15 were downregulated. Gene expression pathway analysis indicated associations with an upregulated inflammatory response, defined primarily by markers of neutrophil activation. Transcription factor analysis revealed upregulation of pathways related to RELA/REL/NFKB1, JUN and STAT1 in the candida group. In situ image analysis of ectocervical tissue samples showed no differences between groups in terms of epithelial height, expression of epithelial junction proteins (E‐cadherin, claudin‐1, zonula occludens 1, and desmoglein‐1), or epithelial CD4+ cell distribution.ConclusionsVaginal candida infection was associated with inflammation and neutrophil infiltration, but not with severe epithelial disruption or CD4+ cell infiltration, in the ectocervical mucosa.

Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease

AbstractGlycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid–binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

Intrahepatic neutrophil accumulation and extracellular trap formation are associated with posthepatectomy liver failure.

Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model. We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease. We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration. Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of &amp;lt;0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p&amp;lt;0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

A murine multiple-injury model for the study of thromboinflammation.

Neutrophil extracellular traps (NETs) contribute to trauma-induced coagulopathy. We aimed to develop a murine multiple-injury model that induces thrombo-inflammatory response, that is, NETosis and accelerated thrombin generation. Wild-type male mice (n = 10, aged 8-12 weeks) underwent multiple injuries (gastrocnemius crush, femur fracture, and laparotomy) and were compared with an uninjured control group (n = 10). Mice were euthanized by cardiac puncture performed 3 hours after injury. Whole blood samples were immediately processed to platelet poor plasma for thrombin generation kinetics (calibrated automated thrombogram), myeloperoxidase (MPO), and von Willebrand factor quantification. Immunohistochemistry of lung tissue was performed to assess for citrullinated histone 3 (CitH3) and MPO. A NETosis cluster was defined as 3+ neutrophils staining for CitH3 at 400× magnification (CitH3 cluster). Data were presented either as mean (SD) or median (interquartile range) with p < 0.05 significant. Sham and trauma treated animals were compared by the two-sample Wilcoxon rank-sum test. Animals subjected to multiple injuries had accelerated thrombin generation compared with controls with greater peak height (61.3 [41.2-73.2] vs. 28.4 [19.5-37.5] nM, p = 0.035) and shorter time to peak (3.37 [2.81-3.81] vs. 4.5 [4.08-4.75] minutes, p = 0.046). Markers of neutrophil activation were greater following multiple injuries than in controls (MPO, 961.1 [858.1-1116.8] vs. 481.3 [438.0-648.9] ng/mL; p = 0.004). NETosis, as evidenced by the aforementioned defined number of CitH3 clusters in the lung, was greater in multiple-injury animals than in controls (mean [SD], 3 [2.9] vs. 0.2 [0.7]; p = 0.009). This is the first study to demonstrate that NETosis and accelerated thrombin generation can be induced using a murine multiple-injury model, as early as 3 hours following injury.

Abstract 16734: Patterns of Inflammatory Activation in Cardiovascular Disease: Insights From the Randomized Colchicine PCI Trial

INTRODUCTION: Recent studies demonstrate that neutrophils accumulate in atherosclerotic lesions and that neutrophil counts correlate with disease severity. The randomized double-blind Colchicine-PCI study demonstrated that colchicine attenuated systemic inflammation (IL-6, CRP) after percutaneous coronary intervention compared with placebo. GOALS/AIMS: We characterized neutrophil activity in patients across a spectrum of cardiovascular phenotypes, including severity of coronary artery disease (CAD) and presentation of acute myocardial infarct (AMI). METHODS: The Colchicine-PCI biomarker sub-study enrolled 478 participants with pretreatment assessment of inflammatory biomarkers. White blood cell count and differential were performed using a hematology analyzer. Soluble markers of neutrophil activity, including neutrophil extracellular traps (NETs), and systemic inflammation were assessed using multiplex assays. Neutrophil surface markers of activity and neutrophil platelet aggregates were assessed using an Accuri C6 flow cytometer. RESULTS: We observed neutrophil concentration, adhesion, transmigration, and activation, as well as increased NETs complexes; but no difference in extent of neutrophil platelet aggregation or systemic inflammation in patients with versus without obstructive CAD. Conversely, in patients with versus without AMI, neutrophil markers of adhesion and transmigration did not differ, but systemic markers of inflammation and neutrophil activation were elevated. CONCLUSION: This single-site, prospective study is the largest neutrophil biorepository in patients with cardiovascular risk factors. Inflammatory activation, and particularly neutrophil profiles, differ in patients with obstructive CAD and in the setting of AMI.

Diagnosis of septic shock by serum measurement of human neutrophil lipocalin by a rapid homogeneous assay

BackgroundHuman neutrophil lipocalin (HNL) is a marker of neutrophil activation and has a high efficacy in diagnosing bacterial infections. In this study, we applied the AlphaLISA technique to measure the serum level of HNL, evaluate HNL's efficacy in diagnosing septic shock, and identify any association between HNL level and septic patients' prognosis. MethodsWe collected 146 serum samples from the Fifth Medical Center of Chinese PLA General Hospital. HNL was measured by AlphaLISA and results were compared with commercial ELISA kits. We studied 78 patients admitted to the ICU with sepsis and data on their clinical and physiological characteristics were recorded. Blood levels of HNL, procalcitonin (PCT), high-sensitivity C-reactive protein (hs-CRP), and lactate were measured. A receiver operating characteristic (ROC) curve was used to evaluate the performance of each marker. ResultsThe AlphaLISA assay for serum HNL had a detection range from 1.5 ng/mL to 1000 ng/mL, with a detection limit of 1 ng/mL and a detection time of approximately 25 min. The AlphaLISA assay's results were in high agreement with ELISA results (R2 = 0.9413). HNL levels were analyzed in sepsis patients, and HNL was significantly higher in sepsis patients with shock compared to sepsis patients without shock (median 356.47 ng/mL vs 158.93 ng/mL, P < 0.0001) and in the 28-day non-survivor group compared to the 28-day survivor group (median 331.83 ng/mL vs 175.17 ng/mL, P < 0.0001). ROC curve analysis was performed for the biomarkers. In differentiating the diagnosis of septic shock from sepsis patients, HNL was the most effective marker (AUC = 0.857), followed by PCT (AUC = 0.754) and hs-CRP (AUC = 0.627). In predicting the prognosis of septic patients, lactate had the best effect (AUC = 0.805), followed by HNL (AUC = 0.784), PCT (AUC = 0.721), and hs-CRP (AUC = 0.583). ConclusionsAs an assessment tool, we found that our AlphaLISA had good consistency with an ELISA and had several other advantages, including requiring a shorter processing time and detecting a wider range of serum HNL concentrations. Monitoring serum HNL levels of patients admitted to the ICU might be useful in distinguishing sepsis patients who have septic shock from other sepsis patients, indicating its value in the prediction of sepsis patient prognosis.

Neutrophil metalloproteinase driven spleen damage hampers infection control of trypanosomiasis

Recent blood transcriptomic analysis of rhodesiense sleeping sickness patients has revealed that neutrophil signature genes and activation markers constitute the top indicators of trypanosomiasis-associated inflammation. Here, we show that Trypanosoma brucei infection results in expansion and differentiation of four splenic neutrophil subpopulations, including Mki67+Birc5+Gfi1+Cebpe+ proliferation-competent precursors, two intermediate immature subpopulations and Cebpb+Spi1+Irf7+Mcl1+Csf3r+ inflammation reprogrammed mature neutrophils. Transcriptomic scRNA-seq profiling identified the largest immature subpopulation by Mmp8/9 positive tertiary granule markers. We confirmed the presence of both metalloproteinases in extracellular spleen homogenates and plasma. During infection, these enzymes digest extracellular matrix components in the absence of sufficient TIMP inhibitory activity, driving remodeling of the spleen follicular architecture. Neutrophil depletion prevents the occurrence of organ damage, resulting in increased plasma cell numbers and prolonged host survival. We conclude that trypanosomiasis-associated neutrophil activation is a major contributor to the destruction of the secondary lymphoid architecture, required for maintaining an efficient adaptive immune response.

Persistent sepsis-induced transcriptomic signatures in signaling pathways of peripheral blood leukocytes: A pilot study

Sepsis is a dysregulated immune response to infections that frequently precipitates multiple organ dysfunction and death despite intensive supportive therapy. The aim of the present study was to identify sepsis-induced alterations in the signaling transcriptome of peripheral blood leukocytes that might shed light on the elusive transition from proinflammatory to anti-inflammatory responses and underlie long-term post-sepsis immunosuppression.Peripheral blood leukocytes were collected from subjects (i) with systemic inflammation, (ii) with sepsis in the acute phase and (iii) 6 months after recovery from sepsis, corresponding to progressive stages of the disease. Transcriptomic analysis was performed with the QuantStudio 12K Flex OpenArray Human Signal Transduction Panel analyzing transcripts of 573 genes playing a significant role in signaling. Of them, 145 genes exhibited differential expression in sepsis as compared to systemic inflammation. Pathway analysis revealed enhanced expression levels of genes involved in primary immune responses (proinflammatory cytokines, neutrophil and macrophage activation markers) and signatures characteristic of immunosuppression (increased expression of anti-inflammatory cytokines and proapoptotic genes; diminished expression of T and B cell receptor dependent activating and survival pathways).Importantly, sepsis-induced expression patterns of 39 genes were not normalized by the end of the 6-month follow-up period, indicating expression aberrations persisting long after clinical recovery. Functional analysis of these transcripts revealed downregulation of the antiapoptotic Wnt and mTOR signaling pathways that might explain the post-sepsis immunosuppression commonly seen in sepsis survivors.

Quantitation of oxidized nuclear and mitochondrial DNA in plasma samples of patients with abdominal aortic aneurysm.

There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.

Increased thromboinflammatory load in hereditary angioedema.

C1 inhibitor (C1Inh) is a serine protease inhibitor involved in the kallikrein-kinin system, the complement system, the coagulation system, and the fibrinolytic system. In addition to the plasma leakage observed in hereditary angioedema (HAE), C1Inh deficiency may also affect these systems, which are important for thrombosis and inflammation. The aim of this study was to investigate the thromboinflammatory load in C1Inh deficiency. We measured 27 cytokines including interleukins, chemokines, interferons, growth factors, and regulators using multiplex technology. Complement activation (C4d, C3bc, and sC5b-C9/TCC), haemostatic markers (β-thromboglobulin (β-TG), thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), active plasminogen activator inhibitor-1 (PAI-1), and the neutrophil activation marker myeloperoxidase (MPO) were measured by enzyme immunoassays. Plasma and serum samples were collected from 20 patients with HAE type 1 or 2 in clinical remission and compared with 20 healthy age- and sex-matched controls. Compared to healthy controls, HAE patients had significantly higher levels of tumour necrosis factor (TNF), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, and IL-17A, chemokine ligand (CXCL) 8, chemokine ligand (CCL) 3, CCL4, IL-1 receptor antagonist (IL-1RA), granulocyte-macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor (FGF) 2 and platelet-derived growth factor (PDGF)-BB. HAE patients also had higher levels of TAT and F1 + 2. Although granulocyte colony-stimulating factor (G-CSF), β-TG and PAI-1 were higher in HAE patients, the differences did not reach statistical significance after correction for multiple testing. In conclusion, C1Inh deficiency is associated with an increased baseline thromboinflammatory load. These findings may reflect that HAE patients are in a subclinical attack state outside of clinically apparent oedema attacks.

Immunohistochemical expression of myeloperoxidase in the equine endometrium suffering from endometrosis

Myeloperoxidase (MPO) is a pro-oxidant enzyme with potent bactericidal activity playing an important role in the innate immune response against microbial pathogens. MPO is a marker of neutrophil activation, but it is also expressed by epithelial cells. In the endometrium of mares in physiological conditions, MPO is immunohistochemically expressed in a hormonal-dependent fashion with maximal expression in mid and basal glands during estrus (Parrilla Hernández et al. Animals. 2023;13(3):375). This suggests that MPO, as a part of the uterine mucosal immune system, contributes to the prevention of equine endometrial contamination and inflammation. Thus, an alteration of the MPO expression may have an impact on uterine immune defense. A deviation in the endometrial expression pattern of some other proteins has already been associated with endometrosis (Hoffmann et al. Theriogenology. 2009;71:264-274) and it has been proposed as one of the reasons for the reduced fertility in mares suffering of this condition. Therefore, the aim of this study was to investigate the immunohistochemical expression pattern of MPO in the estrous equine endometrium affected by endometrosis. Endometrial biopsies (n=7) obtained from mares in estrus with histological detection of three or less neutrophils per field(400x) and presenting a moderate endometrial fibrosis were studied. Immunohistochemical analysis using MPO-specific horse antibody was performed. The uterine glandular epithelium within fibrotic foci mostly presented a non-steroidal dependent immunostaining pattern reaction. In comparison to unaffected glands, most glandular epithelial cells surrounded by periglandular fibrosis exhibited no or decreased MPO immunostaining. No differences were observed between single glands or nests. These results are in accordance with previous studies for other endometrial expressed proteins. This decrease of MPO expression in the presence of periglandular fibrosis may be produced by a lack of paracrine communication between stromal and epithelial cells, which is necessary for the regulation of uterine secretory proteins by sex hormones (Pierro et al. Biology of reproduction. 2001;64(3):831-838). Although further investigation with different degrees of endometrosis is necessary, the reduced expression of MPO compared to that expected in estrus we observed in this study suggests that endometrosis may lead to disturbance in the uterine immune environment, which in turn may predispose to an increase of susceptibility to uterine infections and endometritis in mares.

Increased renal dysfunction and neutrophil infiltration in a mouse model of psoriasis

Psoriasis is a chronic inflammatory skin disease affecting 2-5% of the global population. As an autoimmune disease, psoriasis is characterized by T cell-mediated hyperproliferation of epidermal keratinocytes. Interestingly, most of the morbidity and mortality from psoriasis is related to increased risk of cardiovascular and renal disease. For example, moderate to severe psoriasis is associated with an approximately 1.5-fold increased risk of chronic kidney disease. Neutrophils are increased in the skin and circulation of patients with psoriasis, and markers of neutrophil activation are associated with extra-cutaneous manifestations of psoriasis including vascular inflammation. However, the molecular mechanisms linking psoriasis and kidney disease remain unclear. We hypothesized that increased neutrophil infiltration of the kidney through enhanced granulopoiesis promotes renal dysfunction in psoriasis. Using a KC-Tie2 mouse model of psoriasis with keratinocyte-specific overexpression of the angiopoietin receptor Tie2, we found that KC-Tie2 mice compared to littermate controls exhibit significantly increased urine albumin, suggestive of renal dysfunction. KC-Tie2 mice also exhibit significantly increased kidney/body weight and heart/body weight ratios compared to littermate controls. To determine a mechanism for the increased renal dysfunction in these mice, we assessed skin and renal immune cell content using flow cytometry. KC-Tie2 mice had increased neutrophils in the skin, as well as an approximately 10-fold increase in neutrophils in the kidneys. This increase in renal neutrophils in KC-Tie2 mice was relatively selective as there were no differences in the number of renal dendritic cells, lymphocytes, or monocytes and a modest but significant increase in macrophages. We also observed a selective increase in neutrophils in the spleen of KC-Tie2 mice. Given these findings and prior reports of increased circulating neutrophils in KC-Tie2 mice, we assessed mediators of granulopoiesis in the serum and found a significant increase in granulocyte colony stimulating factor (GCSF) in KC-Tie2 mice. Taken together, these results provide evidence for increased renal dysfunction in a mouse model of psoriasis coincident with marked increases in renal neutrophils that may represent a novel mechanism linking skin inflammation to renal dysfunction through enhanced granulopoiesis. MRA is funded by NIH K08HL153786-01. JM is funded by 2T32GM007569. NW is funded by NIH R01 AR075777, NIH R01 AR073196, NIH P50 CORT AR070590, and National Psoriasis Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.