real-time-high-throughput-technique-to-quantify-neutrophil

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

National Institutes of Health (NIH)

Human leukocyte antigen (HLA) is a key genetic factor conferring risk of systemic lupus erythematosus (SLE), but precise independent localization of HLA effects is extremely challenging. As a result, the contribution of specific HLA alleles and amino-acid residues to the overall risk of SLE and to risk of specific autoantibodies are far from completely understood. Here, we dissected (a) overall SLE association signals across HLA, (b) HLA-peptide interaction, and (c) residue-autoantibody association. Classical alleles, SNPs, and amino-acid residues of eight HLA genes were imputed across 4,915 SLE cases and 13,513 controls from Eastern Asia. We performed association followed by conditional analysis across HLA, assessing both overall SLE risk and risk of autoantibody production. DR15 alleles HLA-DRB1*15:01 (P = 1.4x10-27, odds ratio (OR) = 1.57) and HLA-DQB1*06:02 (P = 7.4x10-23, OR = 1.55) formed the most significant haplotype (OR = 2.33). Conditioned protein-residue signals were stronger than allele signals and mapped predominantly to HLA-DRB1 residue 13 (P = 2.2x10-75) and its proxy position 11 (P = 1.1x10-67), followed by HLA-DRB1-37 (P = 4.5x10-24). After conditioning on HLA-DRB1, novel associations at HLA-A-70 (P = 1.4x10-8), HLA-DPB1-35 (P = 9.0x10-16), HLA-DQB1-37 (P = 2.7x10-14), and HLA-B-9 (P = 6.5x10-15) emerged. Together, these seven residues increased the proportion of explained heritability due to HLA to 2.6%. Risk residues for both overall disease and hallmark autoantibodies (i.e., nRNP: DRB1-11, P = 2.0x10-14; DRB1-13, P = 2.9x10-13; DRB1-30, P = 3.9x10-14) localized to the peptide-binding groove of HLA-DRB1. Enrichment for specific amino-acid characteristics in the peptide-binding groove correlated with overall SLE risk and with autoantibody presence. Risk residues were in primarily negatively charged side-chains, in contrast with rheumatoid arthritis. We identified novel SLE signals in HLA Class I loci (HLA-A, HLA-B), and localized primary Class II signals to five residues in HLA-DRB1, HLA-DPB1, and HLA-DQB1. These findings provide insights about the mechanisms by which the risk residues interact with each other to produce autoantibodies and are involved in SLE pathophysiology.

Amino acid signatures of HLA Class-I and II molecules are strongly associated with SLE susceptibility and autoantibody production in Eastern Asians.

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.

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

Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.

Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus.

We report on patients with STAT3 loss-of-function (LOF) mutations who developed lupus-like autoimmunity. Immune dysregulation seen in STAT3 LOF patients suggests a susceptibility to systemic autoimmunity with important implications in monitoring and management of these patients.

Lupus-like autoimmunity and increased interferon response in patients with STAT3-deficient hyper-IgE syndrome.

Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders.

Somatic Mutations in  and Severe Adult-Onset Autoinflammatory Disease.

Anti-type I interferon (IFN) autoantibodies have been reported in patients with systemic lupus erythematosus (SLE). Recently, an association of these autoantibodies with severe COVID-19 was reported in the general population. We assessed whether having pre-existing anti-IFNα autoantibodies was associated with COVID-19 infection in SLE patients.

Association between anti-interferon-alpha autoantibodies and COVID-19 in systemic lupus erythematosus.

Correspondence on 'Clinical course of coronavirus disease 2019 (COVID-19) in a series of 17 patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine'.

Dysregulation in the phenotype and function of neutrophils may play important roles in the initiation and perpetuation of autoimmune responses, including conditions affecting the skin. Neutrophils can have local and systemic effects on innate and adaptive immune cells as well as on resident cells in the skin, including keratinocytes (KCs). Aberrant formation/clearance of neutrophil extracellular traps (NETs) in systemic autoimmunity and chronic inflammatory diseases have been associated with the externalization of modified autoantigens in peripheral blood and tissues. NETs can impact the function of many cells, including macrophages, lymphocytes, dendritic cells, fibroblasts, and KCs. Emerging evidence has unveiled the pathogenic key roles of neutrophils in systemic lupus erythematosus, idiopathic inflammatory myopathies, psoriasis, hidradenitis suppurativa, and other chronic inflammatory conditions. As such, neutrophil-targeting strategies represent promising therapeutic options for these diseases.

Neutrophils as Drivers of Immune Dysregulation in Autoimmune Diseases with Skin Manifestations.

This is a review article based on the international symposium report of the "US-Japan Conference on Advances in Oncology: Cancer and Infectious Diseases" held online on June 25, 2021, which provided an update on the association between oncology and infectious disease research from cutting-edge basic science to high-impact clinical trials.

Advances in Oncology in US and Japan: Focusing on Cancer and Infectious Diseases.

Neutrophils, the most abundant white blood cell type in humans, play a crucial role in innate host defenses. Recent studies have revealed additional key roles in the pathogenesis of cancer and autoimmune diseases through multiple mechanisms including the formation of neutrophil extracellular traps (NETs). Further research to expand the understanding of neutrophils' role in health and diseases is limited by lack of techniques to quantify neutrophils undergoing NET formation in an objective, reproducible, and efficient manner. In this chapter, we describe an automated high-throughput method to quantify NETting neutrophils in real time using a two-color, live-content imaging platform, the IncuCyte™S3 (Essen BioScience, Inc.) system, coupled to membrane integrity-dependent dual-dye approach to image intracellular and extracellular DNA. Based on characteristic differences in nuclear morphology and membrane integrity, this method may also be used to distinguish between different types of neutrophil cell death. This platform can help to assess neutrophil physiology and to develop and test therapeutic targets.

Quantification of Neutrophils Undergoing NET Formation and Distinguishing Mechanisms of Neutrophil Cell Death by Use of a High-Throughput Method.

In patients with systemic lupus erythematosus (SLE), fatigue is a debilitating symptom with poorly understood pathophysiology. Cardiorespiratory dysfunction has been hypothesised as a contributor to SLE-fatigue. The purpose of this exploratory study was to examine changes in cardiorespiratory function, following an exercise training programme in women with SLE, together with patient reported outcomes and other pathophysiological measures that may underlie SLE-fatigue.

Article	Year
Amino acid signatures of HLA Class-I and II molecules are strongly associated with SLE susceptibility and autoantibody production in Eastern Asians. PLoS genetics\| PubMed ID: 31022184	2019
Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31754025	2019
Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 32094169	2020
Lupus-like autoimmunity and increased interferon response in patients with STAT3-deficient hyper-IgE syndrome. The Journal of allergy and clinical immunology\| PubMed ID: 32768442	2021
Somatic Mutations in and Severe Adult-Onset Autoinflammatory Disease. The New England journal of medicine\| PubMed ID: 33108101	2020
Association between anti-interferon-alpha autoantibodies and COVID-19 in systemic lupus erythematosus. medRxiv : the preprint server for health sciences\| PubMed ID: 33184616	2020
Correspondence on 'Clinical course of coronavirus disease 2019 (COVID-19) in a series of 17 patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine'. Annals of the rheumatic diseases\| PubMed ID: 33452005	2023
Neutrophils as Drivers of Immune Dysregulation in Autoimmune Diseases with Skin Manifestations. The Journal of investigative dermatology\| PubMed ID: 34253374	2022
Advances in Oncology in US and Japan: Focusing on Cancer and Infectious Diseases. World journal of oncology\| PubMed ID: 35059078	2021
Quantification of Neutrophils Undergoing NET Formation and Distinguishing Mechanisms of Neutrophil Cell Death by Use of a High-Throughput Method. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36087264	2022
Changes in cardiorespiratory function and fatigue following 12 weeks of exercise training in women with systemic lupus erythematosus: a pilot study. Lupus science & medicine\| PubMed ID: 36220328	2022

Shuichiro Nakabo

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