Neutrophil CD11b Research Articles

Effect of Neutrophil–Platelet Interactions on Cytokine-Modulated Expression of Neutrophil CD11b/CD18 (Mac-1) Integrin Complex and CCR5 Chemokine Receptor in Stable Coronary Artery Disease: A Sub-Study of SMARTool H2020 European Project

Atherosclerosis is an inflammatory disease wherein neutrophils play a key role in plaque evolution. We observed that neutrophil CD11b was associated with a higher necrotic core volume in coronary plaques. Since platelets modulate neutrophil function, we explored the influence of neutrophil–platelet conjugates on the cytokine-modulated neutrophil complex CD11b/CD18 and CCR5 receptor expression. In 55 patients [68.53 ± 7.95 years old (mean ± SD); 71% male], neutrophil positivity for CD11b, CD18 and CCR5 was expressed as Relative Fluorescence Intensity (RFI) and taken as a dependent variable. Cytokines and chemokines were assessed by ELISA. Following log-10-based logarithmic transformation, they were used as independent variables in Model 1 of multiple regression together with Body Mass Index and albumin. Model 1 was expanded with the RFI of neutrophil CD41a+ (model 2). The RFI of neutrophil CD41a+ correlated positively and significantly with CD11b, CD18, and CCR5. In Model 2, CCR5 correlated positively only with the RFI of neutrophil CD41a+. Albumin maintained its positive effect on CD11b in both models. These observations indicate the complexity of neutrophil phenotypic modulation in stable CAD. Despite limitations, these findings suggest there is a role played by neutrophil–platelet interaction on the neutrophil cytokine-modulated expression of adhesive and chemotactic receptors.

Significance of neutrophil CD11b in early diagnosis of neonatal sepsis

Significance of neutrophil CD11b in early diagnosis of neonatal sepsis

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection

ObjectivesClostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. MethodsWe stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. ResultsCD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3–7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. ConclusionsNeutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

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.

The Neutrophil Dynamic Mass Redistribution Assay as a Medium throughput Primary Cell Screening Assay.

In a typical G protein coupled receptor drug discovery campaign, an in vitro primary functional screening assay is often established in a recombinant system overexpressing the target of interest, which offers advantages with respect to overall throughput and robustness of compound testing. Subsequently, compounds are then progressed into more physiologically relevant but lower throughput ex vivo primary cell assays and finally in vivo studies. Here we describe a dynamic mass redistribution (DMR) assay that has been developed in a format suitable to support medium throughput drug screening in primary human neutrophils. Neutrophils are known to express both CXC chemokine receptor (CXCR) 1 and CXCR2 that are thought to play significant roles in various inflammatory disorders and cancer. Using multiple relevant chemokine ligands and a range of selective and nonselective small and large molecule antagonists that block CXCR1 and CXCR2 responses, we demonstrate distinct pharmacological profiles in neutrophil DMR from those observed in recombinant assays but predictive of activity in neutrophil chemotaxis and CD11b upregulation, a validated target engagement marker previously used in clinical studies of CXCR2 antagonists. The primary human neutrophil DMR cell system is highly reproducible, robust, and less prone to donor variability observed in CD11b and chemotaxis assays and thus provides a unique, more physiologically relevant, and higher throughput assay to support drug discovery and translation to early clinical trials. SIGNIFICANCE STATEMENT: Neutrophil dynamic mass redistribution assays provide a higher throughput screening assay to profile compounds in primary cells earlier in the screening cascade enabling a higher level of confidence in progressing the development of compounds toward the clinic. This is particularly important for chemokine receptors where redundancy contributes to a lack of correlation between recombinant screening assays and primary cells, with the coexpression of related receptors confounding results.

Innate immune dysregulation in multisystem inflammatory syndrome in children (MIS-C)

MIS-C is a systemic inflammation disorder with poorly characterised immunopathological mechanisms. We compared changes in the systemic immune response in children with MIS-C (n = 12, 5–13 years) to healthy controls (n = 14, 5–15 years). Analysis was done in whole blood treated with LPS. Expression of CD11b and Toll-like receptor-4 (TLR4) in neutrophils and monocytes were analysed by flow cytometry. Serum cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-Ira, TNF-α, TNF-β, IFN-Υ, VEGF, EPO and GM-CSF) and mRNA levels of inflammasome molecules (NLRP3, ASC and IL-1β) were evaluated. Subpopulations of lymphocytes (CD3+, CD19+, CD56+, CD4+, CD8+, TCR Vδ1+, TCR Vδ2+) were assessed at basal levels. Absolute counts of neutrophils and NLR were high in children with MIS-C while absolute counts of lymphocytes were low. Children with MIS-C had increased levels of IL-6, IL-10, TNF-β and VEGF serum cytokines at the basal level, and significantly increased TNF-β post-LPS, compared to controls. IL-1RA and EPO decreased at baseline and post-LPS in MIS-C patients compared to controls. The percentage of CD3+ cells, NK cells and Vδ1 was lower while B cells were higher in children with MIS-C than in controls. Dysregulated immune response in children with MIS-C was evident and may be amenable to immunomodulation.

Melatonin Alters Innate Immune Function in Infants with Neonatal Encephalopathy

Introduction: Melatonin has been suggested an adjunctive therapy in neonatal encephalopathy (NE). Melatonin reduces oxidative stress and neutrophil activation; however, the immunological effects in NE have not been studied. Methods: Infants with NE and neonatal controls were prospectively recruited. Whole blood was sampled in the first week of life. Following endotoxin and or melatonin treatment, diurnal variation was measured by RT PCR for circadian rhythm genes (brain and Muscle Arnt-Like protein [BMAL1], circadian locomotor output cycles kaput [CLOCK], Nuclear Receptor Subfamily 1 Group D Member 2 [REV Erβ], and cryptochrome circadian clock [CRY]). Neutrophil and monocyte cell surface markers of activation CD11b, reactive oxygen intermediates (ROIs), and Toll-like receptor (TLR)-4 were also examined by flow cytometry in matching samples. Results: Serum and RNA samples from forty infants were included (controls n = 20; NE n = 20) over the first week of life. Melatonin reduced neutrophil CD11b and TLR-4 expression in response to LPS in infants with NE compared to controls. There were no differences in ROIs. BMAL1 and CLOCK baseline gene expression levels were similar. BMAL1 was significantly decreased with LPS stimulation in NE. There was no significant diurnal variation in melatonin, neutrophil, and monocyte function or circadian genes. Conclusions: Melatonin alters immune function ex vivo in infants with NE. Infants with NE have altered immune circadian responses following LPS stimulation, which have potential for modulation.

Immune complex-mediated neutrophil activation in patients with polymyalgia rheumatica.

Neutrophils are important in host defence. However, neutrophils are also linked to inflammation and organ damage. The purpose of this study was to assess whether markers of neutrophil activation are increased in PMR. Levels of immune complexes (IC), calprotectin and neutrophil extracellular traps (NETs) were measured in plasma of healthy individuals (n = 30) and patients with PMR (n = 60), at flare and upon treatment with glucocorticoids using ELISA. Plasma-mediated neutrophil activation was assessed in presence of an FcγRIIA inhibitory antibody (IV.3). Plasma levels of calprotectin and NETs were elevated in PMR (P < 0.001). Mechanistically, neutrophil activation was driven by ICs, present in plasma, able to up-regulate neutrophil activation markers CD66b and CD11b (P < 0.0001) in an FcγRIIA-dependent manner (P < 0.01). Of note, circulating levels of IC correlated with plasma induced CD66b and CD11b (r = 0.51, P = 0.004, and r = 0.46, P = 0.01, respectively) and decreased after glucocorticoid therapy. In contrast to NETs, calprotectin significantly decreased after glucocorticoid therapy (P < 0.001) and was higher in PMR without overlapping GCA compared with patients with overlapping disease (P = 0.014). Interestingly, musculoskeletal involvement was associated with elevated levels of calprotectin before initiation of glucocorticoid therapy (P = 0.036). Neutrophil activation, including NET formation, is increased in PMR, through IC-mediated engagement of FcγRIIA. Clinically, neutrophil activation is associated with musculoskeletal involvement, with calprotectin, but not NETs, being a biomarker of treatment response in PMR patients. In all, IC-mediated neutrophil activation is a central process in PMR pathogenesis identifying potential novel therapeutic targets (FcγRIIA), as well as soluble markers for disease monitoring (calprotectin).

Abstract 9816: Cholesterol Efflux Capacity Mediates The Relationship Between Neutrophils And Coronary Burden In Psoriasis

Introduction: Psoriasis is a chronic inflammatory disease associated with increased levels of activated neutrophils, lower cholesterol efflux capacity (CEC), and subclinical atherosclerosis. High-density lipoprotein cholesterol efflux is the primary method of cholesterol removal from peripheral tissues and is linked to innate immunity and cardiovascular disease. Preclinical models suggest a strong relationship between CEC, neutrophil frequency and activation, and atherosclerosis, yet this relationship is understudied in clinical models. Hypothesis: (1) Psoriasis patients with high neutrophils have lower CEC and high non-calcified coronary burden (NCB); (2) CEC mediates the relationship between neutrophils and NCB. Methods: Coronary computed tomography angiography was used to quantitate NCB in one-hundred seventy-nine untreated psoriasis patients. Circulating neutrophils were measured via flow cytometry, and mean fluorescence intensity of CD62L and CD11b was determined. CEC was quantified using a cell-based ex-vivo assay measuring the ability of apolipoprotein B depleted plasma to mobilize cholesterol from lipid-loaded macrophages. Results: Patients with high neutrophils had decreased CEC (0.93 (0.15) vs 1.00 (0.17); P =0.03) and higher NCB (1.24 (0.55) vs 1.10 (0.50); P =0.003). The relationship between neutrophils and NCB was mediated by CEC beyond adjustment for Framingham risk score, smoking status, and statin treatment (20%, P =&lt;0.001) ( Figure 1 ). CEC associated with neutrophil activation markers, CD11b and CD62L (β: -0.23 P =0.04, β:0.21, P =0.04). Finally, one year of biologic therapy improved CEC (0.08 (0.17) vs (-0.009 (0.19); P =0.008) and neutrophil counts (-0.45 (-1.36- -0.45) vs -0.14 (-0.60-0.42; P =0.004). Conclusion: Patients with more neutrophils had lower CEC and higher NCB. Further, CEC mediates the relationship between neutrophils and NCB, underscoring the relationship between neutrophils and CEC in atherosclerosis.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Experimental in vitro reprogramming of transformed phenotype of neutrophil granulocyte subpopulations in women with chronic recurrent infectious and inflammatory conditions of genital tract

Chronic inflammatory diseases of the pelvic organs (CIDPO) in women represent one of the urgent and insufficiently studied problems in gynecology across the world. These disorders are followed by adverse medical and socio-economic consequences, i.e., chronic local inflammatory process, chronic pelvic pain syndrome, ectopic pregnancy, infertility. Due to increasing chronicity and recurrence rates of genital infections and inflammatory diseases, there is a need for further studying the effector and regulatory mechanisms of immune system. Of special relevance are the studies of the receptor transformation in neutrophilic granulocytes (NG), the basic population of antimicrobial defense, with further substantiation of targeted immunomodulatory therapy. Purpose of the present study was to assess transformation of neutrophilic granulocytes from CD11b+CD64-CD32+CD16+ to that CD11b+CD64+CD32+CD16+ phenotype in immunocompromised women with CIDPO exacerbation, as well as to evaluate the possibility of in vitro reprogramming the neutrophile phenotype under the action of recombinant interferon (recIFN2b). Peripheral blood neutrophils were tested in the comparison group of 10 conditionally healthy women 20 to 40 years old, and in 17 women (20-40 years old) with the CIDPO exacerbation (group 1). The in vitro effect of recIFNa2b on the blood neutrophils was evaluated for 17 women with CIDPO (group 2). Flow cytometric technique (FCT, CYTOMICS FC500, Beckman Coulter, USA) was used to determine the number of NGs and cell receptor expression levels of neutrophilic CD11b+CD64-CD32+CD16+NG and CD11b+CD64+CD32+CD16+ subpopulations. In peripheral blood of women with CIDPO exacerbation, an increased expression density of surface membrane molecules was revealed by means of FCT: in the subpopulation CD11b+CD64-CD32+CD16+NG, CD16 proved to be 91.7% higher; in CD11b+CD64+CD32+CD16+NG subpopulation, CD16 was increased by 116%, and CD32 being higher by 81% against the comparison group. In the in vitro system, during the incubation of PB with recIFN2b (group 2), we have revealed an increased number of CD11b+CD64-CD32+CD16+ subpopulation relative to the comparison group and group 1, and significantly increased expression density of CD16 (by 212%); CD11b (by 56%), and CD32 (by 83%) than in comparison group, as well as higher density of CD16 expression by 163%; CD11b (by 223%) compared to group 1. The changes in expression density of membrane molecules was also detected by FCT for the activated subpopulation CD11b+CD64+CD32+CD16+NG, i.e., an increase in CD16 by 232% against control group, and decreased expression density of CD64 by 150% against the background, along with increased density of CD16 expression (by 54%), and CD11b (by 103%), relative to group 1, thus suggesting a reprogramming of negatively transformed NC phenotype. These findings may be considered a positive immunomodulatory effect providing a basis for further research in order to develop new integrated approaches to treatment of CIDPO of various etiologies.

Neutrophil extracellular traps: A novel therapeutic target for intracranial hemorrhage

Neutrophil extracellular traps (NETs) are known to play a role in various diseases affecting coagulation.As of now, it is unclear whether NETs are present in hematoma samples collected from patients who have suffered an intracranial hemorrhage (ICH). The objective of this was to determine whether NETs are present in circulation and hematoma samples from ICH patients and to evaluate the procoagulant activity (PCA) of NETs during the ICH process. The expression of NET markers in samples from 78 ICH patients and 35 healthy donners was detected by ELISA and flow cytometry. Immunostaining for neutrophil markers (neutrophil CD66b) and NET markers (citrullinated histone H3 [H3Cit] and extracellular DNA) was performed on hematoma samples obtained from ICH patients undergoing intracranial hematoma evacuation. Our findings suggest that plasma and hematoma samples from patients with ICH showed high levels of NETs. Furthermore, using DNase I to target NETs enhanced ex vivo hematoma lysis. In conclusion, NETs play an important role in the ICH process and may be a novel therapeutic target for treatment of ICH patients.

Peripheral γδ T Cells Regulate Neutrophil Expansion and Recruitment in Experimental Psoriatic Arthritis.

This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA). In this study, we performed interleukin-23 (IL-23) gene transfer in wild-type (WT) and T cell receptor δ-deficient (TCRδ-/- ) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells. We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA-like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA-related tissues. Although significantly reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A was detected systemically in TCRδ-/- mice, no GM-CSF+/IL-17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA. Our findings do not support the notion that tissue-resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.

Neutrophil Activation/Maturation Markers in Chronic Heart Failure with Reduced Ejection Fraction.

Background: Neutrophils are critically involved in the immune response. Inflammatory stimuli alter the expression status of their surface molecule toolset, while inflammation-stimulated granulopoiesis might also influence their maturation status. Data on neutrophil status in heart failure with reduced ejection fraction (HFrEF) are scarce. The present study aims to evaluate the role of neutrophil CD11b, CD66b and CD64 expression in HFrEF. Methods: A total of 135 HFrEF patients and 43 controls were recruited. Mean fluorescence intensity of the activation/maturation markers CD11b, CD66b and CD64 was measured on neutrophils by flow cytometry. CD10 (neprilysin) expression was simultaneously determined. Results: Neutrophil CD64 expression was higher in HFrEF compared with controls, while CD11b/CD66b levels were similar. Neutrophil CD11b and CD66b showed a significant direct correlation to neutrophil CD10 expression (rs = 0.573, p < 0.001 and rs = 0.184, p = 0.033). Neutrophil CD11b and CD66b correlated inversely with heart failure severity reflected by NT-proBNP and NYHA class (NT-proBNP: rs = −0.243, p = 0.005 and rs = −0.250, p = 0.004; NYHA class: p = 0.032 and p = 0.055), whereas no association for CD64 could be found. Outcome analysis did not reveal a significant association between the expression of CD11b, CD66b and CD64 and all-cause mortality (p = ns). Conclusions: The results underline the potential role of neutrophils in HFrEF disease pathophysiology and risk stratification and should stimulate further research, characterizing subpopulations of neutrophils and searching for key molecules involved in the downward spiral of inflammation and heart failure.

S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation.

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Protective effect of green tea and epigallocatechin-3-gallate in a LPS-induced systemic inflammation model

Inflammation causes severe dysregulation of organ functions, via the development of oxidative stress and inflammation damage. Polyphenol compounds found in green tea (GTE), including the most important component epigallocatechin-3-gallate (EGCG), have a great therapeutic potential. Here, protective properties of GTE and EGCG against lipopolysaccharide (LPS)-induced inflammation are explored. To this end, the effects of GTE and EGCG were studied on LPS challenged macrophages. Mice received GTE (250 mg/kg/d/p.o) or EGCG (25 mg/kg/d/i.p.) for 7 d, before the inflammation shock was provoked with a single intraperitoneal injection of LPS. The frequencies of lymphocytes CD4+, CD8+, NK1-1+ and CD4+CD25highFOXP3+ (Treg), macrophages CD11b+F480+, monocytes CD11b+Ly6Clow/high, neutrophils CD11b+Ly6G+, MDSCs CD11b+Gr-1high, M2/N2-like phenotype CD206+ and M1-like phenotype CD86+ in spleen, bone marrow and peripheral blood were determined. In vitro studies revealed that GTE and EGCG significantly attenuated LPS-induced CD80 expression and increased the CD163 expression, showing a potential to reduce the macrophage inflammatory phenotype. In vivo, GTE and EGCG inhibited the inflammation, mainly by reducing M1-macrophages and increasing Treg cells in the bone marrow. In addition, GTE and EGCG increase M2-macrophages, N2-neutrophils and Tregs in the spleen and blood and block the migration of monocytes from the bone marrow to the peripheral blood. These findings indicate that EGCG and GTE prevent LPS-induced inflammatory damage contributing to restoring the immune system homeostasis.

Repeated Social Defeat Exaggerates Fibrin-Rich Clot Formation by Enhancing Neutrophil Extracellular Trap Formation via Platelet-Neutrophil Interactions.

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

Autoantibodies to red blood cell surface Glycophorin A impact the activation poise of circulating leukocytes.

Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto-anti-M/N, prime an activated phenotype in peripheral blood leukocytes. Leukocyte activation was assessed in whole blood from patients with auto-anti-GPA (anti-M/N) and compared to those with allo-anti-M/N and healthy subjects. Control samples from healthy subjects with no antibodies incubated in vitro with either anti-GPA or anti-Rh were analyzed for neutrophil and monocyte surface activation marker expression, reactive oxygen species (ROS) content, and formation of aggregates with RBCs. Samples incubated with an IgG1 isotype antibody served as controls. Ex vivo, neutrophil CD66b and monocyte CD63 surface expression was increased in patients with auto-anti-M/N compared to those with allo anti-M/N (p=.1757; p=.0698) and to healthy subjects (p=.0186; p=.013). In vitro, neutrophil CD66b and monocyte CD63 surface expression was increased following incubation with anti-GPA compared to anti-Rh (p=.0003; p=.0328) and isotype control (p=.000; p=.0062). Intracellular ROS content increased in both neutrophils and monocytes incubated with anti-GPA compared to anti-Rh (p=.0012; p=.0693) and isotype control (p=.001; p=.0021). Percentage of neutrophil-RBC aggregates was decreased when incubated with anti-GPA compared to isotype control (p < .01). Neutrophils and monocytes in peripheral blood exposed to an antibody directed against GPA on RBC surfaces, such as M or N antigens, may be primed towards an activated phenotype.

Diagnostic, Prognostic, Predictive, and Monitoring Role of Neutrophil CD11b and Monocyte CD14 in Neonatal Sepsis.

Background Sepsis is a critical medical condition that requires additional diagnostic considerations. Recently, focus has shifted to the diagnosis of sepsis using new markers to overcome the limitations of traditional laboratory diagnostic modalities. Neutrophil CD11b (nCD11b) and monocyteCD14 (mCD14) cell surface antigens have been shown to be useful in such diagnostic consideration. Aim To investigate the diagnostic, monitoring, prognostic, and predictive roles of nCD11b and mCD14 as sepsis biomarkers in comparison to each other and to traditional laboratory sepsis parameters in order to select the best fit for routine daily use in neonatal intensive care units (NICUs). Subject The study included 188 neonates from Ain Shams University Hospitals' NICUs, who were divided into two groups: the control group (n = 100) and the sepsis group (n = 88). Highly sensitive CRP (hs-CRP), complete blood count (CBC), blood culture, and nCD11b and mCD14 evaluations were all part of the laboratory sepsis evaluation (done by flow cytometry technology). Positive blood culture results (BACT/ALERT system) confirmed the sepsis diagnosis. Twenty-four enrolled sepsis neonates were subjected to follow-up assessments, and they were divided into two groups based on clinical improvement: improved sepsis and sepsis without improvement. In order to predict performance evaluation, the subjected neonates were reclassified according to their outcome into survivors' and nonsurvivors' group. Results Sepsis patients had a significant increase in mCD14 MFI values when compared to controls. With sensitivity 75.4 percent, specificity 71.9 percent, efficacy 73.3 percent, and AUC 0.703, mCD14 MFI at cutoff 9.36 could distinguish the presence of septicemia. Significant increases in both mCD14 MFI and nCD11b MFI (P = 0.001) were observed in the severe sepsis/septic shock group compared to the nonsevere sepsis group. The combined measurement of CD14 MFI at cutoff 9.97 and CD14 percent at cutoff 44.7 percent yielded the best predictive performance. Conclusion Sepsis patients had a significant increase in mCD14 MFI comparable to the controls. mCD14 MFI demonstrated better diagnostic, prognostic, and predictive results than nCD11b. hs-CRP outperformed mCD14 and nCD11b in terms of diagnostic efficacy and AUC. In the monitoring of sepsis patients, both mCD14 and nCD11b produced unsatisfactory results. Currently, the routine use of mCD14 or nCD11b as sepsis biomarkers in neonatal ICUs is not justified.

Neuromodulation of innate immunity by remote ischaemic conditioning in humans: Experimental cross-over study

Experimental animal studies on the mechanisms of remote ischaemic conditioning (RIC)-induced cardioprotection against ischaemia/reperfusion injury demonstrate involvement of both neuronal and humoral pathways. Autonomic parasympathetic (vagal) pathways confer organ protection through both direct innervation and/or immunomodulation, but evidence in humans is lacking. During acute inflammation, vagal release of acetylcholine suppresses CD11b expression, a critical β2-integrin regulating neutrophil adhesion to the endothelium and transmigration to sites of injury. Here, we tested the hypothesis that RIC recruits vagal activity in humans and has an anti-inflammatory effect by reducing neutrophil CD11b expression. Participants (age:50 ​± ​19 years; 53% female) underwent ultrasound-guided injection of local anaesthetic within the brachial plexus before applying 3 ​× ​8 min cycles of brachial artery occlusion using a blood pressure cuff (RICblock). RIC was repeated 6 weeks later without brachial plexus block. Masked analysers quantified vagal activity (heart rate, heart rate variability (HRV)) before, and 10 ​min after, the last cycle of RIC. RR-interval increased after RIC (reduced heart rate) by 40 ​ms (95% confidence intervals (95%CI):13–66; n ​= ​17 subjects; P ​= ​0.003). RR-interval did not change after brachial plexus blockade (mean difference: 20 ​ms (95%CI:-11 to 50); P ​= ​0.19). The high-frequency component of HRV was reduced after RICblock, but remained unchanged after RIC (P ​< ​0.001), indicating that RIC preserved vagal activity. LPS-induced CD16+CD11b+ expression in whole blood (measured by flow cytometry) was reduced by RIC (3615 median fluorescence units (95%CI:475-6754); P = 0.026), compared with 2331 units (95%CI:-3921 to 8582); P = 0.726) after RICblock.These data suggest that in humans RIC recruits vagal cardiac and anti-inflammatory mechanisms via ischaemia/reperfusion-induced activation of sensory nerve fibres that innervate the organ undergoing RIC.