Normal-density Neutrophils Research Articles

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

ObjectivesLow-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties....

LDGs versus NDGs in patients with oral squamous cell carcinoma (OSCC)

BackgroundThe significant role played by neutrophils in cancer biology is indisputable; yet, their subpopulations may exhibit a contrasting role. The phenomenon of polarization of neutrophils and signaling modulators in the course of a neoplastic process has gained increased attention in recent times.The present study's objective was to quantitatively assess low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) populations including IL-17 expression in confrontation with Th17 lymphocytes in patients with oral squamous cell carcinoma (OSCC). The neutrophil to lymphocyte ratio (NLR) biomarker value was determined. Besides, the influence of rhIL-17 on the proliferation level of the squamous cell carcinoma (SCC) malignant line cells was tested. MethodsLeukocytes were isolated in the density gradient and the CD16+ population was magnetically sorted. The percentages of neutrophil subpopulations, lymphocyte Th17, and IL-17 expression in the studied cells were determined on a flow cytometer. Squamous cell carcinoma proliferation was assessed with the MTT test. ResultThe existence of two populations of human neutrophils was determined: LDNs and NDNs. A higher percentage of LDNs and Th17 was observed with the concomitant lower percentage of NDNs in patients with OSCC as compared with the control group. NLR was elevated in patients with cancer. The highest IL-17 expression was obtained in the LDNs population in these patients. However, no influence of IL-17 on SCC proliferation could be determined. ConclusionThe present study demonstrated a strong relationship between IL-17 concentration and the count of LDNs or Th17 in the course of OSCC, which may serve as a reference point for new therapies. Moreover, the obtained LDNs/NDNs and NLR values in patients with cancer prove their usefulness in diagnostic and prognostic in patients with OSCC.

ANCA autoantigen gene expression highlights neutrophil heterogeneity where expression in normal-density neutrophils correlates with ANCA-induced activation

ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.

Neutrophils-Important Communicators in Systemic Lupus Erythematosus and Antiphospholipid Syndrome.

Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are two autoimmune diseases that can occur together or separately. Insights into the pathogenesis have revealed similarities, such as development of autoantibodies targeting subcellular antigens as well as a shared increased risk of cardiovascular morbidity, potentially due to mutual pathologic mechanisms. In this review, we will address the evidence implicating neutrophils in the pathogenesis of these conditions, highlighting their shared features. The neutrophil is the most abundant leukocyte, recognized for its role in infectious and inflammatory diseases, but dysregulation of neutrophil effector functions, including phagocytosis, oxidative burst and formation of neutrophil extracellular traps (NETs) may also contribute to an autoimmune process. The phenotype of neutrophils in SLE and APS differs from neutrophils of healthy individuals, where neutrophils in SLE and APS are activated and prone to aggregate. A specific subset of low-density neutrophils with different function compared to normal-density neutrophils can also be found within the peripheral blood mononuclear cell (PBMC) fraction after density gradient centrifugation of whole blood. Neutrophil phagocytosis is required for regular clearance of cell remnants and nuclear material. Reactive oxygen species (ROS) released by neutrophils during oxidative burst are important for immune suppression and impairment of ROS production is seen in SLE. NETs mediate pathology in both SLE and APS via several mechanisms, including exposure of autoantigens, priming of T-cells and activation of autoreactive B-cells. NETs are also involved in cardiovascular events by forming a pro-thrombotic scaffolding surface. Lastly, neutrophils communicate with other cells by producing cytokines, such as Interferon (IFN) -α, and via direct cell-cell contact. Physiological neutrophil effector functions are necessary to prevent autoimmunity, but in SLE and APS these are altered.

Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus

Neutrophil dysregulation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). SLE is characterized by elevated levels of a pathogenic neutrophil subset known as low-density granulocytes (LDGs). The origin and phenotypic, functional, and pathogenic heterogeneity of LDGs remain to be systematically determined. Transcriptomics and epigenetic assessment of lupus LDGs, autologous normal-density neutrophils, and healthy control neutrophils was performed by bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. Functional readouts were compared among neutrophil subsets. SLE LDGs display significant transcriptional and epigenetic heterogeneity and comprise 2 subpopulations of intermediate-mature and immature neutrophils, with different degrees of chromatin accessibility and differences in transcription factor motif analysis. Differences in neutrophil extracellular trap (NET) formation, oxidized mitochondrial DNA release, chemotaxis, phagocytosis, degranulation, ability to harm the endothelium, and responses to type I interferon (IFN) stimulation are evident among LDG subsets. Compared with other immune cell subsets, LDGs display the highest expression of IFN-inducible genes. Distinct LDG subsets correlate with specific clinical features of lupus and with the presence and severity of coronary artery disease. Phenotypic, functional, and pathogenic neutrophil heterogeneity are prevalent in SLE and may promote immune dysregulation and prominent vascular damage characteristic of this disease.

The proportion, origin and pro-inflammation roles of low density neutrophils in SFTS disease

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a novel emerging viral infectious disease. We explored the percentage, origins and functional roles of low density neutrophils (LDNs), one of the neutrophils subsets, in SFTS.MethodsThe LDNs and normal density neutrophils (NDNs) from blood of SFTS and normal volunteers which were collected separately. The percentage, origins and the phagocytic capability of SFTS viral (SFTSV) of LDNs were investigated by flow cytometry and real time PCR. The capacity of LDNs to secrete cytokines and to damage endothelial cells was assessed by ELISA and flow cytometry.ResultsWe observed that the proportion of LDNs increased dramatically compared with the healthy donors and became the dominant circulating neutrophil population in SFTS patients. Interestingly, the NDNs from the normal donors could switch to LDNs under the SFTS environment. Moreover, SFTSV load in LDNs was significantly higher than that of NDNs in the severe SFTS patients. In addition, the LDNs secreted much higher levels of pro-inflammatory cytokines than NDNs in SFTS and could induce endothelial cell injury.ConclusionThe NDNs can be converted to LDNs. This conversion mechanism could become the source of LDNs. The LDNs in severe SFTS patient could engulf more SFTSV and exhibit pro-inflammation functions.Trial registrationThe Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IORG No: IORG0003571) gave a final APPROVAL for the study.

Abstract 609: Pro-atherosclerotic Low-density Neutrophils Are Present in Hypercholesterolemia

Introduction: Elevated cholesterol levels and increased accumulation of myeloid cells are key risk factors in the development of atherosclerosis. Whilst there is significant understanding of the impact of hypercholesterolemia on monocyte function, less is known about the impact of cholesterol on neutrophils. Neutrophils contain the necessary machinery to take up, synthesize, efflux and esterify cholesterol and unique neutrophil phenotypes have been shown in various pathologies, including low density neutrophils (LDNs) described in inflammatory diseases. Aim: Determine if cholesterol loading of neutrophils occurs in hypercholesterolemia and characterize the associated phenotypic and functional consequences. Methods: Patients (n=13) were a mix of familial hypercholesterolemia and mixed dyslipidemia all with total cholesterol > 7.0 mmol/L. Age and sex matched healthy donors were used as controls. Normal and low density neutrophils were isolated using a percoll density protocol and further purified by FACS selecting for CD66b + CD15 + cells. Results: LDNs were only present in hypercholesterolemic patients, accounting for 12.29.6% of the cells in the lower density percoll fraction (p<0.01 vs control). LDNs were morphologically similar to mature high density neutrophils and stained positive for neutral lipid. Functionally, LDNs secreted significantly more extracellular traps (NETs) in response to PMA treatment relative to normal density neutrophils isolated from the same patients or healthy controls (p<0.005). Cholesterol loaded neutrophils were more adhesive to endothelial cells compared to controls (p<0.005). Increased NETosis and neutrophil adhesion have been demonstrated to be atherogenic. Phagocytosis was increased in LDNs vs controls (p<0.05). These functional changes could all be replicated in vitro via treatment of neutrophils from healthy subjects with cholesterol or oxLDL. Conclusion: Hypercholesterolemia results in cholesterol loading and a pro-atherosclerotic phenotype in neutrophils. Our data provides evidence that this cholesterol loading occurs in the circulation. More work is now being carried out to further characterize the phenotype of LDNs and mechanisms of maturation during hypercholesterolemia.

Characterization of Circulating Low-Density Neutrophils Intrinsic Properties in Healthy and Asthmatic Horses

Low-density neutrophils (LDNs) are a subset of neutrophils first described in the bloodstream upon pathological conditions, and recently, in the blood of healthy humans. LDNs may have an enhanced pro-inflammatory (low-density granulocytes, LDGs) or an immunosuppressive (Granulocytic myeloid-derived suppressor cells, G-MDSCs) profile. Whether these characteristics are specific to LDNs or related to disease states is unknown. Thus, we sought to investigate the properties of LDNs in both health and disease states, and to compare them to those of autologous normal-density neutrophils (NDNs). We studied 8 horses with severe equine asthma and 11 healthy animals. LDNs were smaller and contained more N-formylmethionine-leucyl-phenylalanine receptors than NDNs, but the myeloperoxidase content was similar in both cell populations. They also had an increased capacity to produce neutrophil extracellular traps, and were more sensitive to activation by phorbol-12-myristate-13-acetate. This profile is suggestive of LDGs. These characteristics were similar in both healthy and diseased animals, indicating that these are intrinsic properties of LDNs. Furthermore, these results suggest that LDNs represent a population of primed and predominantly mature cells. This study is the first to characterize LDNs in health, and to compare their properties with those of NDNs and of animals with a naturally occurring disease.

Genomic alterations in abnormal neutrophils isolated from adult patients with systemic lupus erythematosus.

IntroductionPatients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell. Because a similar discrepancy in maturation status is observed in myelodysplasias, and disruption of neutrophil development is frequently associated with genomic alterations, genomic DNA isolated from autologous pairs of LDGs and normal-density neutrophils was compared for genomic changes.MethodsAlterations in copy number and losses of heterozygosity (LOH) were detected by cytogenetic microarray analysis. Microsatellite instability (MSI) was detected by capillary gel electrophoresis of fluorescently labeled PCR products.ResultsControl neutrophils and normal-density SLE neutrophils had similar levels of copy number variations, while the autologous SLE LDGs had an over twofold greater number of copy number alterations per genome. The additional copy number alterations found in LDGs were prevalent in six of the thirteen SLE patients, and occurred preferentially on chromosome 19, 17, 8, and X. These same SLE patients also displayed an increase in LOH. Several SLE patients had a common LOH on chromosome 5q that includes several cytokine genes and a DNA repair enzyme. In addition, three SLE patients displayed MSI. Two patients displayed MSI in greater than one marker, and one patient had MSI and increased copy number alterations. No correlations between genomic instability and immunosuppressive drugs, disease activity or disease manifestations were apparent.ConclusionsThe increased level of copy number alterations and LOH in the LDG samples relative to autologous normal-density SLE neutrophils suggests somatic alterations that are consistent with DNA strand break repair, while MSI suggests a replication error-prone status. Thus, the LDGs isolated have elevated levels of somatic alterations that are consistent with genetic damage or genomic instability. This suggests that the LDGs in adult SLE patients are derived from cell progenitors that are distinct from the autologous normal-density neutrophils, and may reflect a role for genomic instability in the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/ar4681) contains supplementary material, which is available to authorized users.

Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.