Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity.

Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Hematopoietic stem cells undergo a lymphoid to myeloid switch in early stages of emergency granulopoiesis.

Emergency granulopoiesis is the enhanced and accelerated production of granulocytes that occurs during acute infection. The contribution of hematopoietic stem cells (HSCs) to this process was reported; however, how HSCs participate in emergency granulopoiesis remains elusive. Here, using a mouse model of emergency granulopoiesis we observe transcriptional changes in HSCs as early as 4 h after lipopolysaccharide (LPS) administration. We observe that the HSC identity is changed towards a myeloid-biased HSC and show that CD201 is enriched in lymphoid-biased HSCs. While CD201 expression under steady-state conditions reveals a lymphoid bias, under emergency granulopoiesis loss of CD201 marks the lymphoid-to-myeloid transcriptional switch. Mechanistically, we determine that lymphoid-biased CD201+ HSCs act as a first response during emergency granulopoiesis due to direct sensing of LPS by TLR4 and downstream activation of NF-κΒ signaling. The myeloid-biased CD201- HSC population responds indirectly during an acute infection by sensing G-CSF, increasing STAT3 phosphorylation, and upregulating LAP/LAP* C/EBPß isoforms. In conclusion, HSC subpopulations support early phases of emergency granulopoiesis due to their transcriptional rewiring from a lymphoid-biased to myeloid-biased population and thus establishing alternative paths to supply elevated numbers of granulocytes.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Microbes and the fate of neutrophils.

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults-typically 1011 per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.

The role of neutrophils in trained immunity.

The principle of trained immunity represents innate immune memory due to sustained, mainly epigenetic, changes triggered by endogenous or exogenous stimuli in bone marrow (BM) progenitors (central trained immunity) and their innate immune cell progeny, thereby triggering elevated responsiveness against secondary stimuli. BM progenitors can respond to microbial and sterile signals, thereby possibly acquiring trained immunity-mediated long-lasting alterations that may shape the fate and function of their progeny, for example, neutrophils. Neutrophils, the most abundant innate immune cell population, are produced in the BM from committed progenitor cells in a process designated granulopoiesis. Neutrophils are the first responders against infectious or inflammatory challenges and have versatile functions in immunity. Together with other innate immune cells, neutrophils are effectors of peripheral trained immunity. However, given the short lifetime of neutrophils, their ability to acquire immunological memory may lie in the central training of their BM progenitors resulting in generation of reprogrammed, that is, "trained", neutrophils. Although trained immunity may have beneficial effects in infection or cancer, it may also mediate detrimental outcomes in chronic inflammation. Here, we review the emerging research area of trained immunity with a particular emphasis on the role of neutrophils and granulopoiesis.

Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation.

Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis.

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.

Dimensions of neutrophil life and fate.

The earliest reported observations on neutrophils date from 1879 to 1880, when Paul Ehrlich utilized a set of coal tar dyes to interrogate differential staining properties of the granules from white blood cells. While acidic and basic dyes identified eosinophils and basophils respectively, neutrophils were revealed by neutral dyes. Unknowingly, his work staining blood films set the stage for one of the most exciting features of immune cells discovered in the last decade, myeloid heterogeneity. Since then, advances in live imaging and high-resolution sequencing technologies have revolutionized how we analyze and envision those cells that Ehrich fixed in blood smears. Neutrophil plasticity and heterotypic interactions with immune and non-immune compartments are increasingly appreciated as an important part of their biology. In this review, we highlight early and recent work that will help the reader to appreciate our current view of the neutrophil life cycle -from maturation to elimination-, and how neutrophils behave and dynamically modulate tissue immunity, both in steady-state and in disease.

G-CSF - A double edge sword in neutrophil mediated immunity.

Neutrophils are vital for the innate immune system's control of pathogens and neutrophil deficiency can render the host susceptible to life-threatening infections. Neutrophil responses must also be tightly regulated because excessive production, recruitment or activation of neutrophils can cause tissue damage in both acute and chronic inflammatory diseases. Granulocyte colony stimulating factor (G-CSF) is a key regulator of neutrophil biology, from production, differentiation, and release of neutrophil precursors in the bone marrow (BM) to modulating the function of mature neutrophils outside of the BM, particularly at sites of inflammation. G-CSF acts by binding to its cognate cell surface receptor on target cells, causing the activation of intracellular signalling pathways mediating the proliferation, differentiation, function, and survival of cells in the neutrophil lineage. Studies in humans and mice demonstrate that G-CSF contributes to protecting the host against infection, but conversely, it can play a deleterious role in inflammatory diseases. As such, neutrophils and the G-CSF pathway may provide novel therapeutic targets. This review will focus on understanding the role G-CSF plays in the balance between effective neutrophil mediated host defence versus neutrophil-mediated inflammation and tissue damage in various inflammatory and infectious diseases.

G-CSF, the guardian of granulopoiesis.

A considerable amount of continuous proliferation and differentiation is required to produce daily a billion new neutrophils in an adult human. Of the few cytokines and factors known to control neutrophil production, G-CSF is the guardian of granulopoiesis. G-CSF/CSF3R signaling involves the recruitment of non-receptor protein tyrosine kinases and their dependent signaling pathways of serine/threonine kinases, tyrosine phosphatases, and lipid second messengers. These pathways converge to activate the families of STAT and C/EBP transcription factors. CSF3R mutations are associated with human disorders of neutrophil production, including severe congenital neutropenia, neutrophilia, and myeloid malignancies. More than three decades after their identification, cloning, and characterization of G-CSF and G-CSF receptor, fundamental questions remain about their physiology.

Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota.

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

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.

Gasdermin D deficiency aborts myeloid calcium influx to drive granulopoiesis in lupus nephritis.

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.

Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis.

BACKGROUND: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1ß signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1ß (interleukin-1ß), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1ß is not released systemically. We hypothesize that IL-1ß is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. METHODS: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1ß secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. RESULTS: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1ß through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1ß in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. CONCLUSIONS: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1ß) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1ß release.

Type I interferon and neutrophil transcripts in lupus nephritis renal biopsies: clinical and histopathological associations.

OBJECTIVES: To investigate the expression of type I IFN (IFN-I) and neutrophil transcripts in kidney tissue from patients with different classes of LN and their association with distinct clinical and histopathological features. METHODS: Quantitation of IFN-I, defensin-α3 and formyl peptide receptor-like 1 (FPRL-1) transcripts was performed in kidney biopsy tissue from 24 patients with various classes of LN (6 class III, 14 class IV, 4 class V) and 3 control samples. Patient demographics, glomerular filtration rate (eGFR) and histopathological characteristics, including activity and chronicity indices, were analysed. RESULTS: IFNα2 and IFNß transcripts were overexpressed in renal tissues from patients with proliferative forms of LN (III/IV) compared with patients with membranous nephritis and control kidneys. Patients with LN and impaired renal function, attested by eGFR, displayed higher relative expression of IFNα2 transcripts in renal tissues compared with those with normal renal function (23.0 ± 16.2 vs 12.0 ± 14.8, P = 0.04). Defensin-α3, but not FPRL-1, transcripts were overexpressed in LN tissues, particularly those with segmental necrotizing lesions, and were correlated with higher renal pathological activity indices (r = 0.61, P = 0.02), urinary protein levels (r = 0.44, P = 0.048) and IFNα2 expression (r = 0.50, P = 0.01). CONCLUSION: IFN-I transcripts are expressed locally in kidneys from patients with proliferative LN and are associated with impaired renal function. Elevated defensin-α3 transcripts, a neutrophil product associated with neutrophil extracellular traps, may identify a driver of local IFN-I expression. These findings provide insights into the mechanisms of proliferative LN and may inform therapeutic decisions regarding selection of IFN-I pathway inhibitors.

Neutrophil sub-types in maintaining immune homeostasis during steady state, infections and sterile inflammation.

INTRODUCTION: Neutrophils are component of innate immune system and a) eliminate pathogens b) maintain immune homeostasis by regulating other immune cells and c) contribute to the resolution of inflammation. Neutrophil mediated inflammation has been described in pathogenesis of various diseases. This indicates neutrophils do not represent homogeneous population but perform multiple functions through confined subsets. Hence, in the present review we summarize various studies describing the heterogeneous nature of neutrophils and associated functions during steady state and pathological conditions. METHODOLOGY: We performed extensive literature review with key words 'Neutrophil subpopulations' 'Neutrophil subsets', Neutrophil and infections', 'Neutrophil and metabolic disorders', 'Neutrophil heterogeneity' in PUBMED. RESULTS: Neutrophil subtypes are characterized based on buoyancy, cell surface markers, localization and maturity. Recent advances in high throughput technologies indicate the existence of functionally diverse subsets of neutrophils in bone marrow, blood and tissues in both steady state and pathological conditions. Further, we found proportions of these subsets significantly vary in pathological conditions. Interestingly, stimulus specific activation of signalling pathways in neutrophils have been demonstrated. CONCLUSION: Neutrophil sub-populations differ among diseases and hence, mechanisms regulating formation, sustenance, proportions and functions of these sub-types vary between physiological and pathological conditions. Hence, mechanistic insights of neutrophil subsets in disease specific manner may facilitate development of neutrophil-targeted therapies.

A congenital CSF3R mutation in chronic neutropenia reveals a vital role for a cytokine receptor extracellular hinge motif in the response to granulocyte colony-stimulating factor.

We describe a patient with congenital neutropenia (CN) with a homozygous germline mutation in the colony-stimulating factor 3 receptor gene (CSF3R). The patient's bone marrow shows lagging neutrophil development with subtle left shift and unresponsiveness to CSF3 in in vitro colony assays. This patient illustrates that the di-proline hinge motif in the extracellular cytokine receptor homology domain of CSF3R is critical for adequate neutrophil production, but dispensable for in vivo terminal neutrophil maturation. This report underscores that CN patients with inherited CSF3R mutations should be marked as a separate clinical entity, characterized by a failure to respond to CSF3.

The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis.

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.