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1.
J Transl Med ; 22(1): 526, 2024 May 31.
Article En | MEDLINE | ID: mdl-38822352

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.


Antigens, CD34 , Neutrophils , Humans , Neutrophils/metabolism , Neutrophils/cytology , Antigens, CD34/metabolism , Cells, Cultured , Reactive Oxygen Species/metabolism , Proteomics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Gene Editing , Cell Degranulation , Stem Cells/metabolism , Stem Cells/cytology , Cytokines/metabolism , Phenotype
2.
J Matern Fetal Neonatal Med ; 37(1): 2334850, 2024 Dec.
Article En | MEDLINE | ID: mdl-38839425

OBJECTIVES: Scientific evidence provides a widened view of differences in immune response between male and female neonates. The X-chromosome codes for several genes important in the innate immune response and neonatal innate immune cells express receptors for, and are inhibited by, maternal sex hormones. We hypothesized that sex differences in innate immune responses may be present in the neonatal population which may contribute to the increased susceptibility of premature males to sepsis. We aimed to examine the in vitro effect of pro-inflammatory stimuli and hormones in neutrophils and monocytes of male and female neonates, to examine the expression of X-linked genes involved in innate immunity and the miRNA profiles in these populations. METHODS: Preterm infants (n = 21) and term control (n = 19) infants were recruited from the Coombe Women and Infants University Hospital Dublin with ethical approval and explicit consent. The preterm neonates (eight female, 13 male) were recruited with a mean gestation at birth (mean ± SD) of 28 ± 2 weeks and corrected gestation at the time of sampling was 30 + 2.6 weeks. The mean birth weight of preterm neonates was 1084 ± 246 g. Peripheral blood samples were used to analyze immune cell phenotypes, miRNA human panel, and RNA profiles for inflammasome and inflammatory genes. RESULTS: Dividing neutrophil results by sex showed no differences in baseline CD11b between sexes among either term or preterm neonates. Examining monocyte CD11b by sex shows, that at baseline, total and classical monocytes have higher CD11b in preterm females than preterm males. Neutrophil TLR2 did not differ between sexes at baseline or following lipopolysaccharide (LPS) exposure. CD11b expression was higher in preterm male non-classical monocytes following Pam3CSK treatment when compared to females, a finding which is unique to our study. Preterm neonates had higher TLR2 expression at baseline in total monocytes, classical monocytes and non-classical monocytes than term. A sex difference was evident between preterm females and term females in TLR2 expression only. Hormone treatment showed no sex differences and there was no detectable difference between males and females in X-linked gene expression. Two miRNAs, miR-212-3p and miR-218-2-3p had significantly higher expression in preterm female than preterm male neonates. CONCLUSIONS: This study examined immune cell phenotypes and x-linked gene expression in preterm neonates and stratified according to gender. Our findings suggest that the responses of females mature with advancing gestation, whereas male term and preterm neonates have very similar responses. Female preterm neonates have improved monocyte activation than males, which likely reflects improved innate immune function as reflected clinically by their lower risk of sepsis. Dividing results by sex showed changes in preterm and term infants at baseline and following LPS stimulation, a difference which is reflected clinically by infection susceptibility. The sex difference noted is novel and may be limited to the preterm or early neonatal population as TLR2 expression on monocytes of older children does not differ between males and females. The differences shown in female and male innate immune cells likely reflect a superior innate immune defense system in females with sex differences in immune cell maturation. Existing human studies on sex differences in miRNA expression do not include preterm patients, and most frequently use either adult blood or cord blood. Our findings suggest that miRNA profiles are similar in neonates of opposite sexes at term but require further investigation in the preterm population. Our findings, while novel, provide only very limited insights into sex differences in infection susceptibility in the preterm population leaving many areas that require further study. These represent important areas for ongoing clinical and laboratory study and our findings represent an important contribution to exiting literature.


Immunity, Innate , Infant, Premature , Humans , Female , Male , Infant, Newborn , Immunity, Innate/genetics , Infant, Premature/immunology , Case-Control Studies , Neutrophils/metabolism , Neutrophils/immunology , Sex Factors , Monocytes/immunology , Monocytes/metabolism , MicroRNAs/genetics , Gonadal Steroid Hormones/blood , Genes, X-Linked
3.
Oncoimmunology ; 13(1): 2363000, 2024.
Article En | MEDLINE | ID: mdl-38846085

NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor-selective killing. ß-Lapachone (ß-Lap), an NQO1 bioactivatable drug, exhibits significant antitumor effects on NQO1-positive cancer cells by inducing immunogenic cell death (ICD) and enhancing tumor immunogenicity. However, the interaction between ß-Lap-mediated antitumor immune responses and neutrophils, novel antigen-presenting cells (APCs), remains unknown. This study demonstrates that ß-Lap selectively kills NQO1-positive murine tumor cells by significantly increasing intracellular ROS formation and inducing DNA double strand breaks (DSBs), resulting in DNA damage. Treatment with ß-Lap efficiently eradicates immunocompetent murine tumors and significantly increases the infiltration of tumor-associated neutrophils (TANs) into the tumor microenvironment (TME), which plays a crucial role in the drug's therapeutic efficacy. Further, the presence of ß-Lap-induced antigen medium leads bone marrow-derived neutrophils (BMNs) to directly kill murine tumor cells, aiding in dendritic cells (DCs) recruitment and significantly enhancing CD8+ T cell proliferation. ß-Lap treatment also drives the polarization of TANs toward an antitumor N1 phenotype, characterized by elevated IFN-ß expression and reduced TGF-ß cytokine expression, along with increased CD95 and CD54 surface markers. ß-Lap treatment also induces N1 TAN-mediated T cell cross-priming. The HMGB1/TLR4/MyD88 signaling cascade influences neutrophil infiltration into ß-Lap-treated tumors. Blocking this cascade or depleting neutrophil infiltration abolishes the antigen-specific T cell response induced by ß-Lap treatment. Overall, this study provides comprehensive insights into the role of tumor-infiltrating neutrophils in the ß-Lap-induced antitumor activity against NQO1-positive murine tumors.


NAD(P)H Dehydrogenase (Quinone) , Naphthoquinones , Neutrophils , Tumor Microenvironment , Animals , Naphthoquinones/pharmacology , Naphthoquinones/therapeutic use , NAD(P)H Dehydrogenase (Quinone)/metabolism , NAD(P)H Dehydrogenase (Quinone)/genetics , Neutrophils/drug effects , Neutrophils/metabolism , Neutrophils/immunology , Mice , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Mice, Inbred C57BL , Cell Line, Tumor , Neutrophil Infiltration/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Humans , Female , Phenotype
4.
Front Immunol ; 15: 1377409, 2024.
Article En | MEDLINE | ID: mdl-38846945

Introduction: Neutrophil extracellular traps (NETs) constitute a crucial element of the immune system, and dysfunction in immune responses is implicated in the susceptibility and progression of Parkinson's disease (PD). Nevertheless, the mechanism connecting PD and NETs remains unclear. This study aims to uncover potential NETs-related immune biomarkers and elucidate their role in PD pathogenesis. Methods: Through differential gene analysis of PD and NETs in GSE7621 datasets, we identified two PD subtypes and explored potential biological pathways. Subsequently, using ClusterWGCNA, we pinpointed pertinent genes and developed clinical diagnostic models. We then optimized the chosen model and evaluated its association with immune infiltration. Validation was conducted using the GSE20163 dataset. Screening the single-cell dataset GSE132758 revealed cell populations associated with the identified gene. Results: Our findings identified XGB as the optimal diagnostic model, with CAP2 identified as a pivotal gene. The risk model effectively predicted overall diagnosis rates, demonstrating a robust correlation between infiltrating immune cells and genes related to the XGB model. Discussion: In conclusions, we identified PD subtypes and diagnostic genes associated with NETs, highlighting CAP2 as a pivotal gene. These findings have significant implications for understanding potential molecular mechanisms and treatments for PD.


Extracellular Traps , Parkinson Disease , Humans , Parkinson Disease/immunology , Parkinson Disease/diagnosis , Parkinson Disease/genetics , Extracellular Traps/immunology , Extracellular Traps/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Biomarkers , Gene Expression Profiling
5.
PeerJ ; 12: e17531, 2024.
Article En | MEDLINE | ID: mdl-38854794

Background: The aim of this study is to investigate the expression levels of ephrinB2 in patients with lower extremity peripheral arterial disease (PAD) and explore its association with the severity of the disease and the risk of amputation after endovascular revascularization. Methods: During the period from March 2021 to March 2023, this study collected blood samples and clinical data from 133 patients diagnosed with lower extremity PAD and 51 healthy volunteer donors. The severity of lower extremity PAD patients was classified using the Rutherford categories. The expression of ephrin-B2 in plasma samples was detected using the Western Blotting. Results: Compared to the control group, the levels of serum ephrinB2 in patients were significantly elevated (p < 0.001). Moreover, the plasma EphrinB2 levels were positively correlated with white blood cell counts (r = 0.204, p = 0.018), neutrophil counts (r = 0.174, p = 0.045), and neutrophil-to-lymphocyte ratio (NLR) (r = 0.223, p = 0.009). Furthermore, the AUCs of plasma ephrinB2 level, NLR, and their combination as predictors for amputation events within 30 months after lower extremity PAD endovascular revascularization were 0.659, 0.730 and 0.811. In the high-ephrinB2 group, the incidence of amputation events within 30 months after endovascular revascularization was higher. Conclusions: Plasma EphrinB2 levels may be linked to lower extremity PAD development, inflammation, and postoperative amputation. Combining EphrinB2 and NLR can improve amputation prediction accuracy after endovascular revascularization in lower extremity PAD patients.


Amputation, Surgical , Endovascular Procedures , Ephrin-B2 , Peripheral Arterial Disease , Humans , Peripheral Arterial Disease/surgery , Peripheral Arterial Disease/blood , Male , Female , Ephrin-B2/metabolism , Ephrin-B2/blood , Aged , Middle Aged , Endovascular Procedures/adverse effects , Lower Extremity/blood supply , Lower Extremity/surgery , Predictive Value of Tests , Biomarkers/blood , Neutrophils/metabolism , Severity of Illness Index , Case-Control Studies , Risk Factors
6.
Cell Commun Signal ; 22(1): 308, 2024 Jun 03.
Article En | MEDLINE | ID: mdl-38831451

Gasdermin D (GSDMD) is emerging as an important player in autoimmune diseases, but its exact role in lupus nephritis (LN) remains controversial. Here, we identified markedly elevated GSDMD in human and mouse LN kidneys, predominantly in CD11b+ myeloid cells. Global or myeloid-conditional deletion of GSDMD was shown to exacerbate systemic autoimmunity and renal injury in lupus mice with both chronic graft-versus-host (cGVH) disease and nephrotoxic serum (NTS) nephritis. Interestingly, RNA sequencing and flow cytometry revealed that myeloid GSDMD deficiency enhanced granulopoiesis at the hematopoietic sites in LN mice, exhibiting remarkable enrichment of neutrophil-related genes, significant increases in total and immature neutrophils as well as granulocyte/macrophage progenitors (GMPs). GSDMD-deficient GMPs and all-trans-retinoic acid (ATRA)-stimulated human promyelocytes NB4 were further demonstrated to possess enhanced clonogenic and differentiation abilities compared with controls. Mechanistically, GSDMD knockdown promoted self-renewal and granulocyte differentiation by restricting calcium influx, contributing to granulopoiesis. Functionally, GSDMD deficiency led to increased pathogenic neutrophil extracellular traps (NETs) in lupus peripheral blood and bone marrow-derived neutrophils. Taken together, our data establish that GSDMD deletion accelerates LN development by promoting granulopoiesis in a calcium influx-regulated manner, unraveling its unrecognized critical role in LN pathogenesis.


Calcium , Lupus Nephritis , Phosphate-Binding Proteins , Lupus Nephritis/pathology , Lupus Nephritis/metabolism , Lupus Nephritis/genetics , Animals , Humans , Mice , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/deficiency , Calcium/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/deficiency , Neutrophils/metabolism , Granulocytes/metabolism , Myeloid Cells/metabolism , Mice, Inbred C57BL , Female , Extracellular Traps/metabolism , Cell Differentiation , Gasdermins
7.
J Hematol Oncol ; 17(1): 43, 2024 Jun 09.
Article En | MEDLINE | ID: mdl-38853260

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.


Inflammation , Janus Kinase 2 , Myeloproliferative Disorders , Neutrophils , Animals , Neutrophils/metabolism , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mice , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Myeloproliferative Disorders/metabolism , Humans , Inflammation/genetics , Inflammation/pathology , Calreticulin/genetics , Calreticulin/metabolism , Mice, Transgenic , Mice, Inbred C57BL , Cytokines/metabolism
8.
Lipids Health Dis ; 23(1): 179, 2024 Jun 11.
Article En | MEDLINE | ID: mdl-38862998

BACKGROUND: Dry eye disease (DED) is a complication of dyslipidemia (DLP) that is caused by metabolic syndrome and increased inflammation. This research aimed to assess leukocyte and systemic inflammation index ratios as potential biomarkers for systemic inflammation in dyslipidemia patients with dry eye disease (DLP-DED). METHODS: Several blood biomarkers were studied in 32 patients with DLP-DED (study group) and 63 patients with DLP-only (control group). The evaluated blood biomarkers included specific systemic inflammation index ratios, such as the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and neutrophil-to-lymphocyte and platelet ratio (NLPR), and lipid profiles, such as total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), triglyceride (TG), albumin (ALB), and C-reactive protein (CRP) levels. RESULTS: Lymphocyte levels were significantly greater in the DLP-DED group than in the DLP-only group (P = 0.044). In addition, a significant negative correlation between HDL and the NLPR (P = 0.007; r= -0.428) and a significant negative correlation between the serum ALB concentration and the PLR (P = 0.008; r= -0.420) were identified as potential inflammatory predictors of DLP-DED. CONCLUSION: The findings of this study suggest that patients with DLP-DED may benefit from routine blood monitoring of their elevated lipid profile and blood inflammatory biomarkers, such as CRP, leukocytes, and systemic inflammation index ratios (NLR, PLR, MLR, and NLPR), to reduce the complications of DLP on ocular health. The correlation data suggest that the NLPR, PLR, serum ALB concentration, and serum HDL concentration may be valuable inflammatory biomarkers in DLP-DED patients. More research is required to ascertain the significance of the NLR, PLR, MLR, and NLPR and the additive role that leukocytes play.


Biomarkers , Dry Eye Syndromes , Dyslipidemias , Inflammation , Humans , Dyslipidemias/blood , Male , Female , Dry Eye Syndromes/blood , Middle Aged , Inflammation/blood , Case-Control Studies , Retrospective Studies , Biomarkers/blood , Aged , Cholesterol, HDL/blood , Triglycerides/blood , C-Reactive Protein/metabolism , Leukocytes/metabolism , Lymphocytes , Neutrophils/metabolism , Cholesterol, LDL/blood , Adult , Blood Platelets/pathology , Blood Platelets/metabolism
9.
Sci Rep ; 14(1): 12747, 2024 06 03.
Article En | MEDLINE | ID: mdl-38830924

Cell directed therapy is an evolving therapeutic approach to treat organ dysfunction arising from hyperinflammation and cytokine storm by processing immune cells in an extracorporeal circuit. To investigate the mechanism of action of the Selective Cytopheretic Device (SCD), in vitro blood circuits were utilized to interrogate several aspects of the immunomodulatory therapy. SCD immunomodulatory activity is due to its effects on circulating neutrophils and monocytes in a low ionized calcium (iCa, Ca2+) blood circuit. Activated neutrophils adhere to the SCD fibers and degranulate with release of the constituents of their exocytotic vesicles. Adhered neutrophils in the low iCa environment display characteristics of apoptotic senescence. These neutrophils are subsequently released and returned back to circulation, demonstrating a clear potential for in vivo feedback. For monocytes, SCD treatment results in the selective adhesion of more pro-inflammatory subsets of the circulating monocyte pool, as demonstrated by both cell surface markers and cytokine secretory rates. Once bound, over time a subset of monocytes are released from the membrane with a less inflammatory functional phenotype. Similar methods to interrogate mechanism in vitro have been used to preliminarily confirm comparable findings in vivo. Therefore, the progressive amelioration of circulating leukocyte activation and immunomodulation of excessive inflammation observed in SCD clinical trials to date is likely due to this continuous autologous leukocyte processing.


Immunomodulation , Inflammation , Monocytes , Humans , Monocytes/immunology , Monocytes/metabolism , Inflammation/metabolism , Inflammation/immunology , Neutrophils/metabolism , Neutrophils/immunology , Cytokines/metabolism , Cell Adhesion , Cell- and Tissue-Based Therapy/methods , Calcium/metabolism
10.
Sci Rep ; 14(1): 12873, 2024 06 05.
Article En | MEDLINE | ID: mdl-38834610

Acute Respiratory Distress Syndrome (ARDS) is a critical form of Acute Lung Injury (ALI), challenging clinical diagnosis and severity assessment. This study evaluates the potential utility of various hematological markers in burn-mediated ARDS, including Neutrophil-to-Lymphocyte Ratio (NLR), Mean Platelet Volume (MPV), MPV-to-Lymphocyte Ratio (MPVLR), Platelet count, and Platelet Distribution Width (PDW). Employing a retrospective analysis of data collected over 12 years, this study focuses on the relationship between these hematological markers and ARDS diagnosis and severity in hospitalized patients. The study establishes NLR as a reliable systemic inflammation marker associated with ARDS severity. Elevated MPV and MPVLR also emerged as significant markers correlating with adverse outcomes. These findings suggest these economical, routinely measured markers can enhance traditional clinical criteria, offering a more objective approach to ARDS diagnosis and severity assessment. Hematological markers such as NLR, MPV, MPVLR, Platelet count, and PDW could be invaluable in clinical settings for diagnosing and assessing ARDS severity. They offer a cost-effective, accessible means to improve diagnostic accuracy and patient stratification in ARDS. However, further prospective studies are necessary to confirm these findings and investigate their integration with other diagnostic tools in diverse clinical settings.


Biomarkers , Burns , Respiratory Distress Syndrome , Severity of Illness Index , Humans , Respiratory Distress Syndrome/blood , Respiratory Distress Syndrome/diagnosis , Retrospective Studies , Female , Male , Biomarkers/blood , Middle Aged , Adult , Burns/blood , Burns/complications , Neutrophils/metabolism , Mean Platelet Volume , Platelet Count , Lymphocytes/metabolism , Aged
11.
FASEB J ; 38(11): e23697, 2024 Jun 15.
Article En | MEDLINE | ID: mdl-38842874

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.


Antimicrobial Cationic Peptides , Cathelicidins , Diabetic Retinopathy , Extracellular Traps , Mice, Inbred C57BL , Neutrophils , Receptors, Formyl Peptide , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Extracellular Traps/metabolism , Animals , Receptors, Formyl Peptide/metabolism , Receptors, Formyl Peptide/genetics , Humans , Neutrophils/metabolism , Mice , Antimicrobial Cationic Peptides/metabolism , Male , Receptors, Lipoxin/metabolism , Receptors, Lipoxin/genetics , Diabetes Mellitus, Experimental/metabolism , Signal Transduction , Reactive Oxygen Species/metabolism , Female , Middle Aged
12.
Front Immunol ; 15: 1377817, 2024.
Article En | MEDLINE | ID: mdl-38868781

Background: Sepsis, causing serious organ and tissue damage and even death, has not been fully elucidated. Therefore, understanding the key mechanisms underlying sepsis-associated immune responses would lead to more potential therapeutic strategies. Methods: Single-cell RNA data of 4 sepsis patients and 2 healthy controls in the GSE167363 data set were studied. The pseudotemporal trajectory analyzed neutrophil clusters under sepsis. Using the hdWGCNA method, key gene modules of neutrophils were explored. Multiple machine learning methods were used to screen and validate hub genes for neutrophils. SCENIC was then used to explore transcription factors regulating hub genes. Finally, quantitative reverse transcription-polymerase chain reaction was to validate mRNA expression of hub genes in peripheral blood neutrophils of two mice sepsis models. Results: We discovered two novel neutrophil subtypes with a significant increase under sepsis. These two neutrophil subtypes were enriched in the late state during neutrophils differentiation. The hdWGCNA analysis of neutrophils unveiled that 3 distinct modules (Turquoise, brown, and blue modules) were closely correlated with two neutrophil subtypes. 8 machine learning methods revealed 8 hub genes with high accuracy and robustness (ALPL, ACTB, CD177, GAPDH, SLC25A37, S100A8, S100A9, and STXBP2). The SCENIC analysis revealed that APLP, CD177, GAPDH, S100A9, and STXBP2 were significant associated with various transcriptional factors. Finally, ALPL, CD177, S100A8, S100A9, and STXBP2 significantly up regulated in peripheral blood neutrophils of CLP and LPS-induced sepsis mice models. Conclusions: Our research discovered new clusters of neutrophils in sepsis. These five hub genes provide novel biomarkers targeting neutrophils for the treatment of sepsis.


Biomarkers , Neutrophils , Sepsis , Sepsis/immunology , Sepsis/genetics , Sepsis/blood , Sepsis/diagnosis , Neutrophils/immunology , Neutrophils/metabolism , Animals , Humans , Mice , Artificial Intelligence , Disease Models, Animal , Male , Machine Learning , Gene Expression Profiling , Mice, Inbred C57BL , Gene Regulatory Networks , Computational Biology/methods , Transcriptome , Multiomics
13.
Nat Commun ; 15(1): 4724, 2024 Jun 03.
Article En | MEDLINE | ID: mdl-38830855

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.


Galectin 3 , Lipopolysaccharides , Mice, Inbred C57BL , Neutrophils , Pseudomonas Infections , Pseudomonas aeruginosa , Animals , Galectin 3/metabolism , Galectin 3/genetics , Neutrophils/immunology , Neutrophils/metabolism , Humans , Mice , Pseudomonas Infections/immunology , Male , Female , Respiratory Insufficiency/metabolism , Mice, Knockout , Phagocytosis , Immunity, Innate , Galectins/metabolism , Galectins/genetics
14.
Front Immunol ; 15: 1393173, 2024.
Article En | MEDLINE | ID: mdl-38779679

Glioma is a malignant tumor of the central nervous system (CNS). Currently, effective treatment options for gliomas are still lacking. Neutrophils, as an important member of the tumor microenvironment (TME), are widely distributed in circulation. Recently, the discovery of cranial-meningeal channels and intracranial lymphatic vessels has provided new insights into the origins of neutrophils in the CNS. Neutrophils in the brain may originate more from the skull and adjacent vertebral bone marrow. They cross the blood-brain barrier (BBB) under the action of chemokines and enter the brain parenchyma, subsequently migrating to the glioma TME and undergoing phenotypic changes upon contact with tumor cells. Under glycolytic metabolism model, neutrophils show complex and dual functions in different stages of cancer progression, including participation in the malignant progression, immune suppression, and anti-tumor effects of gliomas. Additionally, neutrophils in the TME interact with other immune cells, playing a crucial role in cancer immunotherapy. Targeting neutrophils may be a novel generation of immunotherapy and improve the efficacy of cancer treatments. This article reviews the molecular mechanisms of neutrophils infiltrating the central nervous system from the external environment, detailing the origin, functions, classifications, and targeted therapies of neutrophils in the context of glioma.


Brain Neoplasms , Glioma , Immunotherapy , Neutrophils , Tumor Microenvironment , Humans , Tumor Microenvironment/immunology , Glioma/immunology , Glioma/therapy , Glioma/pathology , Neutrophils/immunology , Neutrophils/metabolism , Immunotherapy/methods , Brain Neoplasms/immunology , Brain Neoplasms/therapy , Brain Neoplasms/pathology , Animals , Blood-Brain Barrier/immunology , Neutrophil Infiltration/immunology
15.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 49(2): 279-285, 2024 Feb 28.
Article En, Zh | MEDLINE | ID: mdl-38755724

OBJECTIVES: Bone morphogenetic protein-4 (BMP4) has been proved to be an important regulatory factor for the pathological process of atherosclerosis (AS). However, there are few related clinical studies. This study aims to investigate the levels of plasma BMP4 in patients suffering from the arterial occlusive diseases (ACD) characterized by AS, and further to test the relationship between BMP4 and inflammation and vascular injury. METHODS: A total of 38 ACD patients (the ACD group) and 38 healthy people for the physical examination (the control group) were enrolled. The plasma in each subject from both groups was obtained to test the levels of BMP4, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-10, and vascular endothelial cadherin (VE-cadherin), and the relationship between BMP4 and the detected indicators above were further analyzed. RESULTS: Compared with the control group, the patients in the ACD group displayed significant elevations in the neutrophil to lymphocyte ratio [NLR, 1.63 (1.26, 1.91) vs 3.43 (2.16, 6.61)] and platelet to lymphocyte ratio [PLR, 6.37 (5.26, 7.74) vs 15.79 (7.97, 20.53)], while decrease in the lymphocyte to monocyte ratio [LMR, 5.67 (4.41, 7.14) vs 3.43 (2.07, 3.74)] (all P<0.05). Besides, the ACD patients displayed significant elevations in plasma BMP4 [581.26 (389.85, 735.64) pg/mL vs 653.97(510.95, 890.43) pg/mL], TNF-α [254.16 (182.96, 340.70) pg/mL vs 293.29(238.90, 383.44) pg/mL], and VE-cadherin [1.54 (1.08, 2.13) ng/mL vs 1.85 (1.30, 2.54) ng/mL], and decrease in IL-10 [175.89 (118.39, 219.25) pg/mL vs 135.92 (95.80, 178.04) pg/mL] (all P<0.05). While the levels of IL-1ß remained statistically comparable between the 2 groups (P=0.09). Furthermore, the plasma BMP4 levels were further revealed to be positively correlated with the levels of IL-1ß (r=0.35), TNF-α (r=0.31) and VE-cadherin (r=0.47), while they were negatively correlated with the levels of IL-10 (r=-0.37; all P<0.01). CONCLUSIONS: After ACD occurrence, the patients' plasma concentrations of BMP4 would be upregulated, which may serve as a candidate to indicate the levels of inflammation and vascular injury.


Arterial Occlusive Diseases , Bone Morphogenetic Protein 4 , Inflammation , Interleukin-10 , Tumor Necrosis Factor-alpha , Humans , Bone Morphogenetic Protein 4/blood , Inflammation/blood , Male , Female , Tumor Necrosis Factor-alpha/blood , Arterial Occlusive Diseases/blood , Interleukin-10/blood , Interleukin-1beta/blood , Cadherins/blood , Case-Control Studies , Middle Aged , Antigens, CD/blood , Vascular System Injuries/blood , Neutrophils/metabolism , Atherosclerosis/blood , Aged , Adult , Lymphocytes/metabolism
16.
Cell Biochem Funct ; 42(4): e4059, 2024 Jun.
Article En | MEDLINE | ID: mdl-38773900

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.


Macrophages , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration , Toll-Like Receptor 4 , Animals , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/deficiency , Mice , Male , Macrophages/metabolism , Macrophages/immunology , Prognosis , Stroke/metabolism , Stroke/pathology , Disease Models, Animal , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Neutrophils/metabolism , Neutrophils/immunology
17.
Gen Physiol Biophys ; 43(3): 209-219, 2024 May.
Article En | MEDLINE | ID: mdl-38774921

Atrial fibrillation (AF) is the most common cardiac arrhythmia and can cause serious complications. Several studies have shown that neutrophils may influence AF progression. However, the key genes related to neutrophils in AF have not been fully elucidated. Here, we downloaded microarray expression data of AF, and screened differentially expressed genes. Key immune cells in AF were identified by immune cell infiltration analysis. Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) analysis were used to construct gene co-expression modules and identify hub genes. The association between key genes and neutrophils was then verified. Our results showed that 303 differentially expressed genes (DEGs) were screened in AF and sinus rhythm (SR), of which 194 were up-regulated and 109 were down-regulated. DEGs were mainly enriched in functions and pathways of neutrophil activation and biological functions of neutrophil activation-mediated immune response. Immune infiltration analysis revealed elevated levels of neutrophil infiltration in AF. WGCNA analysis revealed that the modules in dark red were associated with neutrophils. PPI analysis of these modules yielded 10 hub genes. S100A12, FCGR3B and S100A8 are 3 potential key genes related to neutrophils in AF, which are significantly positively correlated with neutrophils. These genes deserve further investigation and may be potential therapeutic targets for AF.


Atrial Fibrillation , Neutrophils , Atrial Fibrillation/genetics , Atrial Fibrillation/immunology , Neutrophils/metabolism , Neutrophils/immunology , Humans , Protein Interaction Maps/genetics , Gene Regulatory Networks , Gene Expression Profiling
18.
Front Immunol ; 15: 1382449, 2024.
Article En | MEDLINE | ID: mdl-38745657

Background: Acute Respiratory Distress Syndrome (ARDS) or its earlier stage Acute lung injury (ALI), is a worldwide health concern that jeopardizes human well-being. Currently, the treatment strategies to mitigate the incidence and mortality of ARDS are severely restricted. This limitation can be attributed, at least in part, to the substantial variations in immunity observed in individuals with this syndrome. Methods: Bulk and single cell RNA sequencing from ALI mice and single cell RNA sequencing from ARDS patients were analyzed. We utilized the Seurat program package in R and cellmarker 2.0 to cluster and annotate the data. The differential, enrichment, protein interaction, and cell-cell communication analysis were conducted. Results: The mice with ALI caused by pulmonary and extrapulmonary factors demonstrated differential expression including Clec4e, Retnlg, S100a9, Coro1a, and Lars2. We have determined that inflammatory factors have a greater significance in extrapulmonary ALI, while multiple pathways collaborate in the development of pulmonary ALI. Clustering analysis revealed significant heterogeneity in the relative abundance of immune cells in different ALI models. The autocrine action of neutrophils plays a crucial role in pulmonary ALI. Additionally, there was a significant increase in signaling intensity between B cells and M1 macrophages, NKT cells and M1 macrophages in extrapulmonary ALI. The CXCL, CSF3 and MIF, TGFß signaling pathways play a vital role in pulmonary and extrapulmonary ALI, respectively. Moreover, the analysis of human single-cell revealed DCs signaling to monocytes and neutrophils in COVID-19-associated ARDS is stronger compared to sepsis-related ARDS. In sepsis-related ARDS, CD8+ T and Th cells exhibit more prominent signaling to B-cell nucleated DCs. Meanwhile, both MIF and CXCL signaling pathways are specific to sepsis-related ARDS. Conclusion: This study has identified specific gene signatures and signaling pathways in animal models and human samples that facilitate the interaction between immune cells, which could be targeted therapeutically in ARDS patients of various etiologies.


Acute Lung Injury , Cell Communication , Gene Expression Profiling , Animals , Acute Lung Injury/genetics , Acute Lung Injury/immunology , Mice , Humans , Cell Communication/immunology , Transcriptome , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/genetics , Disease Models, Animal , Single-Cell Analysis , Mice, Inbred C57BL , Neutrophils/immunology , Neutrophils/metabolism , COVID-19/immunology , COVID-19/genetics , Signal Transduction , Male , Macrophages/immunology , Macrophages/metabolism
19.
Mol Cancer ; 23(1): 107, 2024 May 18.
Article En | MEDLINE | ID: mdl-38760815

Neutrophils play a Janus-faced role in the complex landscape of cancer pathogenesis and immunotherapy. As immune defense cells, neutrophils release toxic substances, including reactive oxygen species and matrix metalloproteinase 9, within the tumor microenvironment. They also modulate the expression of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand, augmenting their capacity to induce tumor cell apoptosis. Their involvement in antitumor immune regulation synergistically activates a network of immune cells, bolstering anticancer effects. Paradoxically, neutrophils can succumb to the influence of tumors, triggering signaling cascades such as JAK/STAT, which deactivate the immune system network, thereby promoting immune evasion by malignant cells. Additionally, neutrophil granular constituents, such as neutrophil elastase and vascular endothelial growth factor, intricately fuel tumor cell proliferation, metastasis, and angiogenesis. Understanding the mechanisms that guide neutrophils to collaborate with other immune cells for comprehensive tumor eradication is crucial to enhancing the efficacy of cancer therapeutics. In this review, we illuminate the underlying mechanisms governing neutrophil-mediated support or inhibition of tumor progression, with a particular focus on elucidating the internal and external factors that influence neutrophil polarization. We provide an overview of recent advances in clinical research regarding the involvement of neutrophils in cancer therapy. Moreover, the future prospects and limitations of neutrophil research are discussed, aiming to provide fresh insights for the development of innovative cancer treatment strategies targeting neutrophils.


Immunotherapy , Neoplasms , Neutrophils , Tumor Microenvironment , Humans , Neutrophils/immunology , Neutrophils/metabolism , Neoplasms/immunology , Neoplasms/therapy , Neoplasms/metabolism , Neoplasms/pathology , Immunotherapy/methods , Tumor Microenvironment/immunology , Animals , Signal Transduction
20.
PeerJ ; 12: e17382, 2024.
Article En | MEDLINE | ID: mdl-38766483

Background: Preoperative inflammatory factors and nutritional status are strongly associated with the prognosis of a variety of cancers. We explored the relationship between preoperative lymphocytes, neutrophils and albumin (LANR) and progression-free survival in breast cancer patients. Methods: The clinical and follow-up data of 200 breast cancer patients were retrospectively analyzed in this study, and the value of LANR was determined as follows: LANR, lymphocytes × albumin/neutrophils. ROC curves, COX proportional risk regression analysis and subgroup analysis were used to assess the prognostic value of LANR in progression-free survival of breast cancer patients. Results: The median age of the patients was 55.5 years (range 50-62 years). The median follow-up time was 46 months (range 33-55 months). In progression-free survival, the area under the LANR curve was 0.748 and the HR (95% CI) was 0.035 (0.679-0.817). LANR was associated with age (p = 0.02), positive axillary lymph nodes (p < 0.001), TNM stage (p < 0.001) and human epidermal growth factor receptor 2(p = 0.004). The results indicated that preoperative LANR may be a reliable predictor of progression-free survival in patients with operable breast cancer. Conclusion: LANR may be an essential predictor for breast cancer patients and provides a therapeutic basis for clinicians and patients.


Breast Neoplasms , Lymphocytes , Neutrophils , Humans , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Breast Neoplasms/mortality , Female , Middle Aged , Retrospective Studies , Neutrophils/metabolism , Neutrophils/pathology , Prognosis , Lymphocytes/pathology , Lymphocytes/metabolism , Preoperative Period , Progression-Free Survival , Serum Albumin/analysis , Serum Albumin/metabolism , ROC Curve
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