Tertiary Granules Research Articles

Abstract Tu148: cGAS-STING pathway promotes granulopoiesis and neutrophil differentiation in acute ischemia

Contemporary evidence supports neutrophils' complex roles beyond being the simple and powerful destruction tools in MI. However, our understanding remains limited. We especially lack the knowledge of how cGAS-STING regulates neutrophil functions in acute ischemia. The cGAS-STING pathway senses cytosolic DNA to produce IFN and ISGs and is a pillar of innate immunity. Early evidence suggested neutrophils did not express cGAS. Taking advantage of the improved detecting method, we revisited this topic because this pathway can play an essential role in neutrophil function in MI. Using purified neutrophils from WT and knockout mice (as control), we detected cGAS, STING, and IRF3 in these cells. To determine if cGAS-STING regulates neutrophil function, we first generated MI model in cGAS -/- , STING -/- mice, and their WT littermates and performed flow cytometry and RNA-seq analyses. We uncovered that inhibiting cGAS-STING decreases neutrophil production in bone marrow (BM) after ischemia. Interestingly, cGAS- and STING-deficient neutrophils express significantly higher levels of primary granule molecules such as MPO, Prtn3, Elane, Cts- (s, g, b, l) but much lower levels of the secondary and tertiary granule components , including Mmp9, Mmp8, Olfm4, Cd177, Lyz2, and Lyz1. Neutrophils acquire granules sequentially during differentiation. Therefore, our data suggest inhibiting cGAS-STING leads to a stagnated neutrophil differentiation in the BM and mobilizes CD101 - immature neutrophils into the circulation and MI hearts. Consistently, cGAS- and STING-deficient neutrophils release less MMP8 and MMP9 into the ischemic area. Ischemia does not trigger the degranulation of primary granules, as indicated by the minimal changes of CD63. Our data further suggest that TF C/EBP𝜹 functions downstream of cGAS-STING to promote neutrophil differentiation. Adoptive transfer experiments suggest the neutrophil-specific function of cGAS-STING. In conclusion, we identify cGAS-STING as a novel mechanism promoting emergency granulopoiesis and neutrophil differentiation after MI and uncover its functional implications in ischemia-induced remodeling. Studies using Mrp8-Cre-mediated cGAS-STING deletion in granulocyte/macrophage progenitors are in progress.

Identification of atrial fibrillation-related genes through transcriptome data analysis and Mendelian randomization.

Atrial fibrillation (AF) is a common persistent arrhythmia characterized by rapid and chaotic atrial electrical activity, potentially leading to severe complications such as thromboembolism, heart failure, and stroke, significantly affecting patient quality of life and safety. As the global population ages, the prevalence of AF is on the rise, placing considerable strains on individuals and healthcare systems. This study utilizes bioinformatics and Mendelian Randomization (MR) to analyze transcriptome data and genome-wide association study (GWAS) summary statistics, aiming to identify biomarkers causally associated with AF and explore their potential pathogenic pathways. We obtained AF microarray datasets GSE41177 and GSE79768 from the Gene Expression Omnibus (GEO) database, merged them, and corrected for batch effects to pinpoint differentially expressed genes (DEGs). We gathered exposure data from expression quantitative trait loci (eQTL) and outcome data from AF GWAS through the IEU Open GWAS database. We employed inverse variance weighting (IVW), MR-Egger, weighted median, and weighted model approaches for MR analysis to assess exposure-outcome causality. IVW was the primary method, supplemented by other techniques. The robustness of our results was evaluated using Cochran's Q test, MR-Egger intercept, MR-PRESSO, and leave-one-out sensitivity analysis. A "Veen" diagram visualized the overlap of DEGs with significant eQTL genes from MR analysis, referred to as common genes (CGs). Additional analyses, including Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and immune cell infiltration studies, were conducted on these intersecting genes to reveal their roles in AF pathogenesis. The combined dataset revealed 355 differentially expressed genes (DEGs), with 228 showing significant upregulation and 127 downregulated. Mendelian randomization (MR) analysis identified that the autocrine motility factor receptor (AMFR) [IVW: OR = 0.977; 95% CI, 0.956-0.998; P = 0.030], leucine aminopeptidase 3 (LAP3) [IVW: OR = 0.967; 95% CI, 0.934-0.997; P = 0.048], Rab acceptor 1 (RABAC1) [IVW: OR = 0.928; 95% CI, 0.875-0.985; P = 0.015], and tryptase beta 2 (TPSB2) [IVW: OR = 0.971; 95% CI, 0.943-0.999; P = 0.049] are associated with a reduced risk of atrial fibrillation (AF). Conversely, GTPase-activating SH3 domain-binding protein 2 (G3BP2) [IVW: OR = 1.030; 95% CI, 1.004-1.056; P = 0.024], integrin subunit beta 2 (ITGB2) [IVW: OR = 1.050; 95% CI, 1.017-1.084; P = 0.003], glutaminyl-peptide cyclotransferase (QPCT) [IVW: OR = 1.080; 95% CI, 1.010-0.997; P = 1.154], and tripartite motif containing 22 (TRIM22) [IVW: OR = 1.048; 95% CI, 1.003-1.095; P = 0.035] are positively associated with AF risk. Sensitivity analyses indicated a lack of heterogeneity or horizontal pleiotropy (P > 0.05), and leave-one-out analysis did not reveal any single nucleotide polymorphisms (SNPs) impacting the MR results significantly. GO and KEGG analyses showed that CG is involved in processes such as protein polyubiquitination, neutrophil degranulation, specific and tertiary granule formation, protein-macromolecule adaptor activity, molecular adaptor activity, and the SREBP signaling pathway, all significantly enriched. The analysis of immune cell infiltration demonstrated associations of CG with various immune cells, including plasma cells, CD8T cells, resting memory CD4T cells, regulatory T cells (Tregs), gamma delta T cells, activated NK cells, activated mast cells, and neutrophils. By integrating bioinformatics and MR approaches, genes such as AMFR, G3BP2, ITGB2, LAP3, QPCT, RABAC1, TPSB2, and TRIM22 are identified as causally linked to AF, enhancing our understanding of its molecular foundations. This strategy may facilitate the development of more precise biomarkers and therapeutic targets for AF diagnosis and treatment.

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.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase (GTPase). Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin-1A (Coro1A), a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes (IC) and relate this to intracellular Coro1A localization. We used bone marrow neutrophils (BMN) from WT and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase, MPO), secondary (lactoferrin, LTF) and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of Coro1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in MPO, MMP-2, and MMP-9, but not LTF, release, along with diminished filopodia formation, CD63 polarization, and colocalization of Coro1A with CD63 in IC-stimulated Rac2-/- BMNs. Rac2 and Coro1A were found associated with granules in CB/fMLF-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on Coro1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Hydroxycarboxylic acid receptor 2 (HCA2) agonists induce NET formation and MMP-9 release from bovine polymorphonuclear leukocytes

Periparturient cows are commonly fed diets supplemented with Niacin (nicotinic acid, NA) because of its anti-lipolytic properties. NA confers its anti-lipolytic effects by activating the hydroxycarboxylic acid 2 receptor (HCA2). HCA2 is also activated by the ketone body beta-hydroxybutyrate (BHB) and circulating BHB levels are elevated in postpartum dairy cows. The HCA2 receptor is highly expressed in bovine polymorphonuclear leukocytes (PMN) and could link metabolic and innate immune responses in cattle. We investigated how HCA2 agonists affected bovine PMN function in vitro. We studied different PMN responses, such as granule release, surface expression of CD11b and CD47, generation of neutrophil extracellular traps (NETs), and apoptosis. NA, BHB, and 4,4aR,5,5aR-tetrahydro-1H-cyclopropa [4,5] cyclopenta [1,2-c] pyrazole-3-carboxylic acid (MK-1903) treatment triggered the release of matrix metalloproteinase 9 (MMP-9), a component of the tertiary granule, from neutrophils. Additionally, all HCA2 agonists induced NETs formation but did not affect surface expression of CD11b and CD47. Finally, none of the HCA2 agonists triggered apoptosis in bovine PMN. This information will give new insights into the potential role of the HCA2 receptor in the bovine innate immune response.

Abstract P3082: Emergency Granulopoiesis Increases Neutrophil Phenotypic And Functional Diversity

Ischemic injury due to myocardial infarction (MI) elicits immune cell infiltration to remove necrotic tissue and facilitate scar formation. Neutrophils are among the first infiltrating immune cells, and neutrophilia has been commonly accepted as a biomarker of adverse prognoses post-MI. The increased demand for neutrophils is met by an accelerated granulocyte production in a process known as emergency granulopoiesis. However, the impact of emergency granulopoiesis on neutrophil function remains cryptic. These experiments seek to determine the impact of emergency granulopoiesis on neutrophil phenotype and secretory function. To activate emergency granulopoiesis, C57BL/6 mice were injected with G-CSF for 3 d (100 μg/kg/day, s.c.). Neutrophil phenotype and function were evaluated 24 h after the last dose of G-CSF. We found that G-CSF injections resulted in increased neutrophils in peripheral blood, spleen, and bone marrow. Flow cytometric analysis revealed that neutrophils produced via emergency granulopoiesis showed lower expression of the classical neutrophil marker Ly6G and an increased number of CD101 Neg neutrophils in peripheral blood and spleen (n=3-6/group, p<0.05). Functional analysis in vitro showed that neutrophils produced via emergency granulopoiesis are 2-3 times more susceptible to PMA- and fMLF-induced degranulation of primary granule content (neutrophil elastase and myeloperoxidase), but release of secondary (NGAL, CHI3L1), and tertiary granules (MMP9) remained unchanged (n=5/group, p<0.05). Based off these observations, we conclude that emergency granulopoiesis results increased diversity of neutrophil phenotype and function. Future studies will elucidate the impact of emergency granulopoiesis neutrophils on post-MI scar formation and chronic ventricular remodeling.

Differential Gene Analysis of Trastuzumab in Breast Cancer Based on Network Pharmacology and Medical Images.

The purpose of this study was to use network pharmacology, biomedical images and molecular docking technology in the treatment of breast cancer to investigate the feasible therapeutic targets and mechanisms of trastuzumab. In the first place, we applied pubchem swisstarget (http://www.swisstargetprediction.ch/), (https://pubchem.ncbi.nlm.nih.gov/) pharmmapper (http://lilab-ecust.cn/pharmmapper/), and the batman-tcm (http://bionet.ncpsb.org.cn/batman-tcm/) database to collect the trastuzumab targets. Then, in NCBI-GEO, breast cancer target genes were chosen (https://www.ncbi.nlm.nih.gov/geo/). The intersection regions of drug and disease target genes were used to draw a Venn diagram. Through Cytoscape 3.7.2 software, and the STRING database, we then formed a protein-protein interaction (PPI) network. Besides, we concluded KEGG pathway analysis and Geen Ontology analysis by using ClueGO in Cytospace. Finally, the top 5 target proteins in the PPI network to dock with trastuzumab were selected. After screening trastuzumab and breast cancer in databases separately, we got 521 target genes of the drug and 1,464 target genes of breast cancer. The number of overlapping genes was 54. PPI network core genes include GAPDH, MMP9, CCNA2, RRM2, CHEK1, etc. GO analysis indicated that trastuzumab treats breast cancer through abundant biological processes, especially positive regulation of phospholipase activity, linoleic acid metabolic process, and negative regulation of endothelial cell proliferation. The molecular function is NADP binding and the cellular component is tertiary granule lumen. The results of KEGG enrichment analysis exhibited four pathways related to the formation and cure of breast cancer, containing Drug metabolism, Glutathione metabolism, Pyrimidine metabolism and PPAR signaling pathway. Molecular docking showed that trastuzumab has good binding abilities with five core target proteins (GAPDH, MMP9, CCNA2, RRM2, CHEK1). This study, through network pharmacology and molecular docking, provides new pieces of evidence and ideas to understand how trastuzumab treats breast cancer at the gene level.

Identification of hub genes and their correlation with immune infiltration in coronary artery disease through bioinformatics and machine learning methods.

BackgroundCoronary artery disease (CAD) is a multifactorial disease and its pathogenesis remains unclear. We aimed to explore the optimal feature genes (OFGs) for CAD and to investigate the function of immune cell infiltration of CAD. It will be helpful for better understanding of the pathogenesis and the development of genetic prediction of CAD.MethodsDatasets related to CAD were obtained from the Gene Expression Omnibus (GEO) database. Cases from the datasets met diagnostic criteria including clinical symptoms, electrocardiographic (ECG) and angiographic evidence. We identified differentially expressed genes (DEGs) and conducted functional enrichment analysis. OFGs were obtained from the least absolute shrinkage and selection operator (LASSO) algorithm, support vector machine recursive feature elimination (SVM-RFE) algorithm, and random forest (RF) algorithm. CIBERSORT was used to compare immune infiltration between CAD patients and normal controls, and the correlation between OFGs and immune cells was analyzed.ResultsDEGs were involved in the interleukin (IL)-17 signaling pathway, nuclear factor (NF)-kappa B signaling pathway, and tumor necrosis factor (TNF) signaling pathway. Gene Ontology (GO) analysis revealed DEGs were enriched in lipopolysaccharide (LPS), tertiary granule, and pattern recognition receptor activity. Disease Ontology (DO) analysis suggested DEGs were enriched in lung disease, arteriosclerotic cardiovascular disease (CVD). Matrix metalloproteinase 9 (MMP9), Pellino E3 ubiquitin protein ligase 1 (PELI1), thrombomodulin (THBD), and zinc finger protein 36 (ZFP36) were screened by the intersection of OFGs obtained from LASSO, SVM-REF, and RF algorithms. CAD patients had a lower proportion of memory B cells (P=0.019), CD8 T cells (P<0.001), resting memory CD4 T cells (P<0.001), regulatory T cells (P=0.028), and gamma delta T cells (P<0.001) than normal controls, while the proportion of activated memory CD4 T cells (P=0.014), resting natural killer (NK) cells (P<0.001), monocytes (P<0.001), M0 macrophages (P=0.023), activated mast cells (P<0.001), and neutrophils (P<0.001) in CAD patients were higher than normal controls. MMP9, PELI1, THBD, and ZFP36 were correlated with immune cells.ConclusionsMMP9, PELI1, THBD, and ZFP36 may be predicted biomarkers for CAD. The OFGs and association between OFGs and immune infiltration may provide potential biomarkers for CAD prediction along with the better assessment of the disease.

Myeloid-Specific Pyruvate-Kinase-Type-M2-Deficient Mice Are Resistant to Acute Lung Injury.

Infiltration of polymorphonuclear neutrophils (PMNs) plays a central role in acute lung injury (ALI). The mechanisms governing PMN inflammatory responses, however, remain incompletely understood. Based on our recent study showing a non-metabolic role of pyruvate kinase type M2 (PKM2) in controlling PMN degranulation of secondary and tertiary granules and consequent chemotaxis, here we tested a hypothesis that Pkm2-deficient mice may resist ALI due to impaired PMN inflammatory responses. We found that PMN aerobic glycolysis controlled the degranulation of secondary and tertiary granules induced by fMLP and PMA. Compared to WT PMNs, Pkm2-deficient (Pkm2-/-) PMNs displayed significantly less capacity for fMLP- or PMA-induced degranulation of secondary and tertiary granules, ROS production, and transfilter migration. In line with this, myeloid-specific Pkm2-/- mice exhibited impaired zymosan-induced PMN infiltration in the peritoneal cavity. Employing an LPS-induced ALI mouse model, LPS-treated Pkm2-/- mice displayed significantly less infiltration of inflammatory PMNs in the alveolar space and a strong resistance to LPS-induced ALI. Our results thus reveal that PKM2 is required for PMN inflammatory responses and deletion of PKM2 in PMN leads to an impaired PMN function but protection against LPS-induced ALI.

Identification of Nine mRNA Signatures for Sepsis Using Random Forest.

Sepsis has high fatality rates. Early diagnosis could increase its curating rates. There were no reliable molecular biomarkers to distinguish between infected and uninfected patients currently, which limit the treatment of sepsis. To this end, we analyzed gene expression datasets from the GEO database to identify its mRNA signature. First, two gene expression datasets (GSE154918 and GSE131761) were downloaded to identify the differentially expressed genes (DEGs) using Limma package. Totally 384 common DEGs were found in three contrast groups. We found that as the condition worsens, more genes were under disorder condition. Then, random forest model was performed with expression matrix of all genes as feature and disease state as label. After which 279 genes were left. We further analyzed the functions of 279 important DEGs, and their potential biological roles mainly focused on neutrophil threshing, neutrophil activation involved in immune response, neutrophil-mediated immunity, RAGE receptor binding, long-chain fatty acid binding, specific granule, tertiary granule, and secretory granule lumen. Finally, the top nine mRNAs (MCEMP1, PSTPIP2, CD177, GCA, NDUFAF1, CLIC1, UFD1, SEPT9, and UBE2A) associated with sepsis were considered as signatures for distinguishing between sepsis and healthy controls. Based on 5-fold cross-validation and leave-one-out cross-validation, the nine mRNA signature showed very high AUC.

Histological observations on the oocyte development in the Picnic seabream, Acanthopagrus berda (Forsskål 1775)

Histological analysis was carried out on ovarian development in the picnic seabream, Acanthopagrus berda (Forsskål 1775) caught in the estuarine waters of Calicut, Kerala, India from January to December 2016. Four developmental phases of oocyte development were identified viz., oogonia proliferation and transformation into the primary oocyte, primary growth phase, secondary growth phase and maturation phase and these are similar to those described for other species. The reproductive development process begins in a very young ovary with the formation of oogonia, the cells originally derived from special primordial germ cells formed early in the embryonic development; their multiplication and differentiation are followed by the release of mature oocytes. Oocyte development stages observed in A. berda were chromatin nucleolus, early perinucleolar, late perinucleolar, lipid vesicle stage I oocyte, lipid vesicle stage II oocyte, primary yolk granule stage, secondary yolk granule stage, tertiary yolk granule stage, hydrated oocyte and atretic oocyte. The oocytes entered the maturation phase when they attained the maximum size of 459.6±71.83 µm. Histological examination of the mature ovaries showed the presence of all the different oocyte stages in the ovary of A. berda with an asynchronous mode of ovarian development shortly before spawning, indicating that the species is a multiple spawner.

C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Neutrophil Phenotypes Result in Differential Responses to Pathogens

▪Introduction: We have previously reported that neutrophils from healthy subjects vary in their susceptibility to degranulate in response to immune complexes and bacterial ligands (Duarte 2020 [abstract]). In longitudinal testing, neutrophil responses are specific to each individual, resulting in “high” or “low” amounts of degranulation and represent a neutrophil phenotype (Duarte 2019). It is unknown if the phenotype extends to other neutrophil effector functions or if the phenotype is relevant to host-pathogen interactions.Methods/Results: To determine if the neutrophil phenotype extends to other effector functions relevant to pathogen responses, we performed a series of functional assays using whole blood and isolated neutrophils from previously phenotyped “high” and “low” subjects. Our first objective was to determine if the neutrophil degranulation phenotype is conserved in response to a broad range of pathogens. To do this, we chose Staphylococcus aureus (MRSA) strain USA300 (UAMS 1182), Escherichia coli (strain DH5alpha), and Candida albicans (strain SC5314) as model pathogens for gram-positive bacteria, gram-negative bacteria, and fungus, respectively. As shown in Figure 1, degranulation responses were preserved in response to supernatant secreted from all organisms (Fig 1A, p=0.001, p=0.001, and p=0.01 for S. aureus, E. coli, and C. albicans, respectively) and in response to the organism itself (Fig 1B, p=0.01, p=0.005, p=0.005 for respective pathogens). For all organisms, “high” subjects degranulated and released more MMP9 (representative of tertiary granules) when compared to “low” subjects. Besides exocytosis of granules, phagocytosis of pathogens is critical for host defense. To determine if the neutrophil phenotype results in differential ability to phagocytose, neutrophils were isolated from “high” and “low” subjects and uptake of fluorescently-labeled S. aureus bioparticles was measured (Invitrogen, Waltham, MA). As shown in Figure 2A, neutrophils from “high” subjects were less efficient at phagocytosis when compared to neutrophils from “low” subjects (p<0.001). These findings were confirmed by direct visualization using immunofluorescent microscopy. As shown in Figure 2B/2C, neutrophils from “high” subjects had a lower phagocytic index compared to neutrophils from “low” subjects, defined as percent of S. aureus-engulfed neutrophils (p=0.002). Finally, to determine if the neutrophil phenotype results in differential ability to kill, we performed bacterial kill assays using S. aureus. As shown in Figure 3, “high” subjects were less efficient at bacterial kill when compared to “low” subjects resulting in higher bacterial survival at 60 minutes (p<0.001).Conclusions: Taken together, these studies continue to build on our prior observations that neutrophils from healthy subjects vary in their susceptibility to activation, resulting in differential ability to degranulate, phagocytose, and kill pathogens. We demonstrate that excessive exocytosis of granules is correlated with less efficient ability to phagocytose and kill. These differences in effector function are likely relevant to host defense and the innate immune response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PKM2 controls the degranulation of secondary and tertiary granules in neutrophils by phosphorylating SNAP-23.

PKM2 controls the degranulation of secondary and tertiary granules in neutrophils by phosphorylating SNAP-23.

Proteomic analysis reveals critical molecular mechanisms involved in the macrophage anti-spinal tuberculosis process

Tuberculosis infection activates the autoimmune system. However, the role of host–pathogen interactions involved in Mycobacterium tuberculosis infection is unclear. In this study, we analyzed 6 spinal tuberculosis tissues and 6 herniated disc tissues by using liquid chromatography–tandem mass spectrometry coupled with tandem mass spectrometry, and immunohistochemical staining was performed for validating the results. We identified 42 differential immune-related proteins and 3 hub genes that are primarily localised in the tertiary granule and involved in biological processes such as cellular response to the presence of cadmium ions, regulation of ion transmembrane transport, transmembrane transport, and inflammatory responses. Genes encoding cytochrome B-245 beta chain (CYBB), matrix metallopeptidase 9 (MMP9), and C-X-C motif chemokine ligand 10 (CXCL10) were identified as the hub genes that exhibited anti-tuberculosis activity and were responsible for macrophage resistance against M. tuberculosis. In conclusion, CYBB, MMP9, and CXCL10 resist M. tuberculosis infection through chemotaxis and macrophage activation. Our results indicate that CYBB, MMP9, and CXCL10 could be considered as molecular targets for spinal tuberculosis treatment, which may significantly improve patients’ quality of life and prognosis.

Variation in Neutrophil Function Among Healthy Subjects Influences Response to Bacteria

Introduction: We have recently shown that healthy subjects have variable neutrophil degranulation responses to antigen/antibody immune complexes (ICs; Duarte ME Blood 2019). With repeated testing over 2 years, we have shown that susceptibility to neutrophil activation represents a fixed phenotype: some healthy subjects have neutrophils which robustly degranulate in response to ICs, while others have neutrophils that are minimally reactive (&amp;gt;30-fold variation in release of MMP9, representative of tertiary granules). Whether this variation in degranulation responses is specific to ICs or is applicable to other stimulants is not known. Because neutrophils are involved in the first steps of host defense against bacterial pathogens, we aimed to determine if these phenotypes are associated with differences in neutrophil effector functions which may contribute to host defense. Methods/Results: To determine if observed neutrophil phenotypes are relevant to host defense, we compared neutrophil effector responses from "high" and "low" donors after being challenged with bacterial products including the bacterial tripeptide N-formyl-met-leu-phe (fMLP), LPS, and Staphylococcus aureus (USA300) supernatant. As a model IC, we chose to use KKO-platelet factor 4/heparin ICs, as neutrophil responses to this IC have been previously characterized by our lab. As a first step, we examined several healthy subjects and compared degranulation responses to ICs, fMLP, and LPS to determine if susceptibility to degranulation was preserved. As shown in Figure 1, the quantity of MMP9 released in response to ICs was highly correlated with MMP9 released in response to fMLP (Fig 1A) and to LPS (Fig 1B, r = 0.9 for both). We then determined if neutrophil phenotypes were preserved in response to bacteria. As shown in Figure 2, neutrophils from "high" and "low" donors differentially degranulated in response to Staphylococcus aureus supernatant, with continued preservation of the neutrophil phenotype. We next aimed to determine if the neutrophil phenotype was associated with differences in other neutrophil effector functions, besides degranulation, which are relevant to pathogen defense. First, we focused on neutrophil extracellular trap (NET) release, which has an important role in host defense. As shown in Figure 3, "high" and "low" donors differed in NET release in response to ICs, as quantified by visualization using immunofluorescent microscopy. This finding is supported by our previous report that neutrophil phenotypes are correlated with differences in MPO release, a NET component (Duarte ME, et al 2019). We also examined reactive oxygen species (ROS) generation, a primary mechanism by which neutrophils eliminate pathogens. As shown in Figure 4, using DCFH-DA, a fluorescent probe, we show that "high" and "low" donors differ in ROS generation in response to PMA and fMLP. Conclusions: Taken together, our studies demonstrate that neutrophil responses to a variety of stimuli (ICs, LPS, and fMLP) are highly concordant among healthy donors with "high" or "low" neutrophil phenotypes and that these differences in neutrophil effector functions are likely relevant to host defense. Further studies are needed to determine how differences in innate neutrophil function may affect clinical outcomes during bacterial infection. Disclosures Arepally: Apotex: Consultancy, Research Funding; Biokit: Consultancy, Patents &amp; Royalties; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Annexon Biosciences: Consultancy, Other; Alexion: Other; Novartis: Consultancy.

Taurine chloramine selectively regulates neutrophil degranulation through the inhibition of myeloperoxidase and upregulation of lactoferrin.

Taurine is a free amino acid rich in neutrophils, and neutrophils play an important role in the forefront defense against infection. Upon neutrophil activation, taurine reacts with hypochlorous acid (HOCl/OCl-) produced by the myeloperoxidase (MPO) system and gets converted to taurine chloramine (Tau-Cl). Neutrophils have three types of granules, of which the primary granule MPO, secondary granule lactoferrin, and tertiary granule matrix metalloproteinase (MMP)-9 are released into the extracellular space by a process called degranulation. MPO produces hypochlorous acid to kill microorganisms, and the released MPO forms neutrophil extracellular traps (NETs) with released chromatin. Excessive secretion of MPO causes oxidative damage to the surrounding tissues. Lactoferrin exerts antioxidant activity, prevents pro-inflammatory pathway activation, sepsis, and tissue damages, and delays neutrophil apoptosis. Our experimental results show that neutrophils released small amount of granules in an inactive state, and phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionine-leucyl-phenylalanine induced neutrophil degranulation. Tau-Cl inhibited the PMA-induced degranulation of MPO and formation of NETs. While Tau-Cl increased the degranulation of lactoferrin, it had no effect on MMP-9 degranulation. MPO negatively regulated the production of macrophage inflammatory protein (MIP)-2, which stimulates the degranulation and migration of neutrophils. Tau-Cl abrogated MIP-2 expression, suggestive of its inhibitory effect on MPO release. The increase in the intracellular level of MPO may negatively regulates MIP-2 expression, thereby contributing to the further regulation of neutrophil degranulation and migration. Here, we suggest that Tau-Cl selectively inhibits MPO degranulation and stimulates lactoferrin degranulation from neutrophils, thereby protecting inflamed tissues from oxidative damage induced by excessively released MPO.

Machine Learning Unveils Proteotypic Mimicry in Genetically Defined SCN Variants

Background Novel computational algorithms for multi-omics analysis bear great potential to highlight pathomechanisms of monogenic diseases. We recently defined the in-depth proteome of primary human neutrophil granulocytes (PMID 30630937). Here, we ask the question whether proteotypic patterns differ between defined genetic subtypes associated with severe congenital neutropenia (SCN). We focus on two novel genetic variants in constituents of the signal recognition particle (SRPRA and SRP19) and previously reported SCN genotypes SRP54, HAX1, and ELANE. Methods We analyzed proteomes of highly purified neutrophil granulocytes from a total of 26 SCN patients, including 5 with homozygous splice site mutations in SRP19, one patient with a de-novo heterozygous missense mutation in SRPRA (using 5 biological replicates collected months apart) as well as 6 patients with SRP54, 8 with HAX1 and 6 with ELANE mutations. Samples of 70 healthy donors (HD) served as controls. Whole cell proteome analysis was based on data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer. Data analysis was performed in R and Cytoscape, machine learning approaches included lasso regression and random forest. Results Differential expression analysis in comparison to HD showed in all genotypes overexpression of ribosomes, the translational apparatus, mitochondria, cell-substrate junctions and response to unfolded proteins. Underexpressed proteins showed genotype specific enrichment for granule subsets. Whereas ELANE showed deficiency of primary and secretory granules, HAX1 showed deficiency of specific, tertiary and secretory granules. All SRP genotypes showed markedly reduced abundance of proteins in all granule subsets. Principal component analysis showed clear separation of healthy and diseased proteotypes on the first component, whereas the separation of patient genotypes became clear only using five dimensions. We derived genotype specific proteome signatures by lasso regression, consisting of 26 (minimal specific set) to 128 (comprehensive signature) proteins, and a signature of 48 proteins when joining the SRP genotypes as one group. This signatures allow for perfect separation of the genotypes, demonstrating a clear genotype specific effect on protein abundance levels. We asked the question if the genotypes SRP19 and SRPRA show more similar proteomic profile to SRP54 than the other genotypes (ELANE, HAX1) by training a random forest model on proteome data from SRP54, HAX1, ELANE and HD and subsequently testing if the other SRP samples get classified as SRP54. We observe only few misclassifications using either all proteins (7/10) or using the lasso derived genotype defining proteins (8/10). This strongly supports our hypothesis that mutations in different subunits of the same complex lead to similar proteotype changes, a phenomenon we propose to call "proteotypic mimicry". For a systems biology perspective we selected proteins that were exclusively regulated in the SRP genotypes and restricted a network of interactions to these proteins together with their direct interactors (based on APID level 1). The resulting network contained 464 proteins and 3587 interactions. MCODE analysis identified 16 clusters that were consequently annotated using BINGO enrichment analysis. The SRP specific network shows features of the translational apparatus, the proteasome, the septin complex, splicing and cell-metabolic processes. Further studies to dissect specific pathomechanisms are under way. Conclusion Here we provide for the first time evidence for the correlation between SCN causing genotypes and their corresponding neutrophil proteotypes. In particular, we demonstrate significant overlap of all SRP related proteotypes, indicating a phenomenon we propose to be called "proteotypic mimicry". Studies on similarities and disparities of neutrophil proteotypes will help to raise new hypothesis on distinct cellular dysfunction in defined genetic defects of neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

IPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.