Mature Neutrophils Research Articles

Androgen exposure impairs neutrophil maturation and function within the infected kidney.

Urinary tract infections (UTIs) in men are uncommon yet carry an increased risk for severe pyelonephritis and other complications. In models of Escherichia coli UTI, C3H/HeN mice develop high-titer pyelonephritis (most with renal abscesses) in a testosterone-dependent manner, but the mechanisms underlying this phenotype are unknown. Here, using female mouse models, we show that androgen exposure impairs neutrophil maturation in the upper and lower urinary tract, compounded by a reduction of neutrophil function within the infected kidney, enabling persistent high-titer infection and promoting abscess formation. Following intravesical inoculation with uropathogenic E. coli (UPEC), kidneys of androgen-exposed C3H mice showed delayed local pro-inflammatory cytokine responses while robustly recruiting neutrophils. These were enriched for an end-organ-specific population of aged but immature neutrophils (CD49d+, CD101-). Compared to their mature counterparts, these aged immature kidney neutrophils exhibited reduced function in vitro, including impaired degranulation and diminished phagocytic activity, while splenic, bone marrow, and bladder neutrophils did not display these alterations. Furthermore, aged immature neutrophils manifested little phagocytic activity within intratubular UPEC communities in vivo. Experiments with B6 conditional androgen receptor (AR)-deficient mice indicated rescue of the maturation defect when AR was deleted in myeloid cells. We conclude that the recognized enhancement of UTI severity by androgens is attributable, at least in part, to local impairment of neutrophil maturation in the urinary tract (largely via cell-intrinsic AR signaling) and a kidney-specific reduction in neutrophil antimicrobial capacity.IMPORTANCEAlthough urinary tract infections (UTIs) predominantly occur in women, male UTIs carry an increased risk of morbidity and mortality. Pyelonephritis in androgen-exposed mice features robust neutrophil recruitment and abscess formation, while bacterial load remains consistently high. Here, we demonstrate that during UTI, neutrophils infiltrating the urinary tract of androgen-exposed mice exhibit reduced maturation, and those that have infiltrated the kidney have reduced phagocytic and degranulation functions, limiting their ability to effectively control infection. This work helps to elucidate mechanisms by which androgens enhance UTI susceptibility and severity, illuminating why male patients may be predisposed to severe outcomes of pyelonephritis.

Disease Activity and Tendency to Relapse in ANCA-Associated Vasculitis Are Reflected in Neutrophil and Intermediate Monocyte Frequencies.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases with inflammation affecting small blood vessels and includes granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). In this study, we investigated granulocyte and monocyte subsets in a large cohort of AAV patients with emphasis on disease activity and tendency to relapse. A cohort of 105 patients with GPA or MPA and 126 healthy controls (HCs) were included. Clinical and laboratory data were collected for all patients, including disease activity, tendency to relapse, and pharmacological treatment. Using flow cytometry, circulating eosinophils, basophils, neutrophils, and monocytes were assessed. The monocytes were subdivided into classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14-CD16+) monocytes. Mature (CD16high) or newly released (CD16dim) neutrophils were defined, as well as the frequency of CD177+ neutrophils. AAV patients displayed increased frequencies of intermediate monocytes, mature and newly released neutrophils, and an expanded population of CD177+ neutrophils compared to HC. MPA patients differed from GPA patients in terms of lower frequency of classical monocytes. No differences in cell frequencies regarding ANCA phenotype were observed. Paired data from 23 patients demonstrated that active disease was associated with an increased frequency of mature neutrophils and a decreased frequency of monocytes, in particular intermediate monocytes. Moreover, GPA patients with a tendency to relapse displayed an increased frequency of mature neutrophils with increased expression of CD177+. Relapsing MPA patients, on the other hand, showed decreased frequency of intermediate monocytes. Finally, rituximab treatment was associated with increased frequencies of classical and intermediate monocytes. In conclusion, AAV patients exhibit a skewing of different neutrophil and monocyte subpopulations that are associated with disease subtypes, disease activity, rituximab treatment, and propensity to relapse. These changes may contribute to the inflammatory process and could potentially be used as biomarkers for relapse prediction.

ВЛИЯНИЕ РОНКОЛЕЙКИНА НА МОРФОМЕТРИЧЕСКИЕ И ГЕМАТОЛОГИЧЕСКИЕ ПОКАЗАТЕЛИ И ФАГОЦИТАРНУЮ И ГЕМАТОЛОГИЧЕСКИЕ ПОКАЗАТЕЛИ И ФАГОЦИТАРНУЮ

Cultivated hydrobionts under intensive aquaculture conditions are exposed to stress factors associated with artificial habitats, increased planting density, periodic fishing, etc. At the same time, they experience a decrease in resistance and resilience and an increase in morbidity. A good solution to this problem is the use of immunomodulators. This article presents the results of a study of the effect on fish of the immunomodulator Roncoleukin, which is a dosage form of recombinant human interleukin-2 (IL-2) isolated and purified from yeast cells (Saccharomyces cerevisiae). The subject of the study was the three-year-old Arctic char (Salvelinus alpinus). The evaluation was based on morphometric, hematological (erythro- and leukogram) and cytochemical (lysosomal cation test) indicators. The coefficient of variation of the size and weight parameters of the treated fish was low compared to the con- trol group. This indicates uniform growth of fish fed Roncoleukin, which is a great advantage in fish farming and provides more efficient use of feed. Blood for analysis was taken from the tail vein of the fish in vivo. The results showed a positive effect of the drug on the activation of erythropoiesis and leukopoiesis. There was a significant increase in the proportion of mature segmented neutrophils (the main blood microphages belonging to the non-specific link of cellular immunity) in the leukocyte formula due to a decrease in lymphocytes (specific immunity). An increase in the content of nonenzymatic lysosomal cationic proteins (defensins) in blood neutrophils can indicate a high level of cellular protection of microphages (neutrophils). The results of the study showed a positive effect of the drug Roncoleukin, which can be recommended for use in the ferming of Arctic char.

Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs.

CD80, a co-stimulatory molecule required for optimal T cell activation, is expressed on antigen-presenting cells, including monocytes and dendritic cells, in dogs and humans. We hypothesized that CD80 would be expressed on tumor cells in dogs from acute myeloid leukemia (AML) but not dogs with lymphoid neoplasms. We first evaluated the cellular staining pattern of a hamster anti-murine CD80 antibody (clone 16-10A1, ThermoFisher Scientific Cat# 17-0801-82, RRID: AB_469417) in blood and bone marrow aspirates from healthy dogs. Using flow cytometric analysis and examination of modified Wright's-stained cytologic smears of unsorted and flow cytometric or immunomagnetic bead-sorted leukocytes, we show that the antibody binds to mature and immature neutrophils and monocytes, but not lymphocytes or eosinophils, in blood and bone marrow. We then added the antibody to routine flow cytometric panels for immunophenotyping hematopoietic neoplasms in dogs. We found that the antibody labeled tumor cells in 72% of 39 dogs with AML and 36% of 11 dogs with acute leukemia expressing lymphoid and myeloid markers ("mixed lineage") but none of the dogs with B (n = 37) or T (n = 35) lymphoid neoplasms. A higher proportion of tumor cells in dogs with AML were labeled with the anti-CD80 antibody vs antibodies against other myeloid-associated antigens, including CD4 (36%, p = 0.003), CD11b (44%), CD11c (46%), CD14 (38%, p = 0.006) and CD18 (59%, clone YFC118). In contrast, antibodies against CD11b and CD11c bound to tumor cells in 8-32% of the lymphoid neoplasms. We show that CD80, as detected by antibody clone 16-10A1, is a sensitive and specific marker for AML and would be useful to include in flow cytometric immunophenotyping panels in dogs.

Diagnosis of acute appendicitis by modified Alvarado score vs. ultrasound based on histopathological findings: A comparative study

Background: Acute appendicitis commonly causes emergency surgery. Clinical examination accuracy ranges from 71-97%; despite ultrasound diagnostic improvements, accuracy depends mainly on operator experience. This study uses a modified version of the Alvarado score, excluding one laboratory finding (shift to the left of neutrophil maturation). As differential count is not routinely undertaken in the study site (Erbil), patients were scored out of 9 rather than 10 points. This study compares the diagnostic accuracy of ultrasound and modified Alvarado score in the diagnosis of acute appendicitis, to reduce appendicitis mortality and morbidity, and reduce rates of negative appendicitis. Methodology: The study design was a prospective cross-sectional comparative study, which took place in the Surgery, Radiology and Histopathology Department of Rizgary Teaching Hospital and Rozhalat Emergency Hospital, Erbil, from January 2019 to December 2021. All patients who presented to the emergency room with signs and symptoms of acute appendicitis were clinically evaluated. Patients who had a modified Alvarado score > 8 were considered positive for modified Alvarado, and those scoring 6-7 were considered negative for modified Alvarado and were considered for ultrasound examination; among the latter, those with positive ultrasound results were included in the study. Results: Among the 468 patients, 257 (54.9%) were male and 211 (45.1%) were female (1.22:1 male: female ratio), with a mean age of 23.45 ± 2.1 years (ranging from 12 to 56 years). Modified ultrasonography has a sensitivity of 82% and an accuracy of 79.9%; the modified Alvarado score had a sensitivity of 95.2% and an accuracy of 87%. There was no association between the mean age of male and female patients with the histopathological results. The most commonly affected age group was the cohort 21-30 years (51.7% of all patients). The number of patients with positive histopathology was 411; negative histopathology was recorded for 57 patients, with no association between histopathological results and gender. There was a significant association between symptoms (cough signs, localized tenderness signs, and pointing signs) and positive histopathology findings. Conclusion: Modified Alvarado score has higher sensitivity than ultrasound, while ultrasound has a higher specificity. Neither tool is superior to the other, nor both need to be used together to reduce negative appendectomy rates.

The RNA m6A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression.

Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor (G-CSF) through its receptor G-CSFR are essential for antibacterial innate defense. However, the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear. N6-methyladenosine (m6A) RNA modification and the related demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, but their roles in neutrophil production and mobilization are still unknown. We used cecal ligation and puncture (CLP)-induced polymicrobial sepsis to model systemic bacterial infection, and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization. ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice. In addition, Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation, which led to fewer neutrophils at the infection site than in their wild-type littermates. During bacterial infection, ALKBH5 imprinted production- and mobilization-promoting transcriptome signatures in both mouse and human neutrophils. Mechanistically, ALKBH5 erased m6A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR, consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils. The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA, and the binding strength declined upon bacterial infection, accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils. Considering these results collectively, we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m6A demethylation-dependent posttranscriptional regulation, indicating that m6A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.

TRPV1 controls innate immunity during Citrobacter rodentium enteric infection.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of TRPV1 in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

Features of phagocytic and chemiluminescent activity of neutrophils when exposed to polyprenols <i>in vitro</i>

Aim: Studying of the features of phagocytic activity and the state of the respiratory burst of neutrophils under the influence of polyprenols in vitro.
 Materials and methods. The study involved 30 relatively healthy donors aged 23-59 years from whose peripheral blood neutrophilic granulocytes were isolated to determine phagocytic and chemiluminescent activity. The isolated cells were incubated for 30 minutes with polyprenols in vitro. The level of phagocytosis of immature and mature neutrophils was determined by flow cytometry using FITC-labeled staphylococcal protein A. The state of the respiratory burst was assessed using chemiluminescent analysis.
 Results. It was found that the levels of phagocytic index (PI) and number (PN) of mature neutrophils were reduced after incubation with polyprenols relative to initial and control values. The values of PI and PN of immature neutrophils under the influence of polyprenols and during control incubation were decreased relative to the initial values. The intensity maximum and area under the curve of spontaneous and zymosan-induced lucigenin- and luminol-dependent chemiluminescence of the neutrophils were reduced after incubation with polyprenols.
 Conclusion. The effect of polyprenols on neutrophilic granulocytes in vitro leads to a decrease in activity in respiratory burst cells, which is realized in blocking by polyprenols the interaction of primary and secondary ROS with chemiluminescent indicators by the mechanism of primary antioxidants. The binding of ROS by polyprenols also manifests itself in a decrease in the phagocytic activity of mature neutrophils while maintaining the phagocytic activity of immature cells at the level of control incubation. In general, polyprenols exhibit anti-inflammatory activity when exposed in vitro to neutrophilic granulocytes.

Type 1 diabetes mellitus: Roles of neutrophils in the pathogenesis.

Circulating neutrophil counts are reduced both in healthy autoantibody-positive individuals and in patients with type 1 diabetes, which may be related on cell-specific autoimmunity. This paper was written to give an update on roles of neutrophils in the pathogenesis of type 1 diabetes mellitus. Different research search engines like PubMed Central, Scopus, Web of Science, Researchgate, Google Scholar etc were utilised for writing this paper. A drop in blood neutrophil counts in type 1 diabetes may be caused by decreased neutrophil generation and maturation, tissue maintenance, consumption, or peripheral damage. Neutrophil count variations between studies may be explained by results from various stages of diabetes or by ethnic groups. Neutrophils can induce type 1 diabetes by colonizing pancreatic islets and interacting with other immune cells, according to exciting findings that shed new light on their role in the pathogenesis of the disease. Knowing more about the function of neutrophils in the pathogenesis of type 1 diabetes will help in early diagnosis, treatment, and even prevention of the disease.

The methylome of buccal epithelial cells is influenced by age, sex, and physiological properties.

Epigenetic modifications, particularly DNA methylation, have emerged as regulators of gene expression and are implicated in various biological processes and disease states. Understanding the factors influencing the epigenome is essential for unraveling its complexity. In this study, we aimed to identify how the methylome of buccal epithelial cells, a noninvasive and easily accessible tissue, is associated with demographic and health-related variables commonly used in clinical settings, such as age, sex, blood immune composition, hemoglobin levels, and others. We developed a model to assess the association of multiple factors with the human methylome and identify the genomic loci significantly impacted by each trait. We demonstrated that DNA methylation variation is accurately modeled by several factors. We confirmed the well-known impact of age and sex and unveiled novel clinical factors associated with DNA methylation, such as blood neutrophils, hemoglobin, red blood cell distribution width, high-density lipoprotein cholesterol, and urea. Genomic regions significantly associated with these traits were enriched in relevant transcription factors, drugs, and diseases. Among our findings, we showed that neutrophil-impacted loci were involved in neutrophil functionality and maturation. Similarly, hemoglobin-influenced sites were associated with several diseases, including aplastic anemia, and the genomic loci affected by urea were related to congenital anomalies of the kidney and urinary tract. Our findings contribute to a better understanding of the human methylome plasticity and provide insights into novel factors shaping DNA methylation patterns, highlighting their potential clinical implications as biomarkers and the importance of considering these physiological traits in future medical epigenomic investigations.NEW & NOTEWORTHY We have developed a quantitative model to assess how the human methylome is associated with several factors and to identify the genomic loci significantly impacted by each trait. We reported novel health-related factors driving DNA methylation patterns and new site-specific regulations that further elucidate methylome dynamics. Our study contributes to a better understanding of the plasticity of the human methylome and unveils novel physiological traits with a potential role in future medical epigenomic investigations.

APOE‐4 dependent dysfunctional neutrophil‐microglia crosstalk accelerates Alzheimer’s disease

AbstractBackgroundAPOE4 is the strongest genetic risk factor for late‐onset Alzheimer’s disease (AD). The role of human APOE variants in AD has been studied extensively in the regulation of brain cells but not in peripheral immunity. APOE is also expressed in neutrophils and controls their activation. Moreover, neutrophils have been shown to play a negative role in AD mice via the induction of neutrophil extracellular traps (NETs)MethodHere we aimed to dissect the impact of APOE4‐neutrophils on AD pathology, using Ly6g‐CRE mice crossed to APOE‐KI(E3 and E4)fl/fl:APP/PS1.ResultWe identified a cell intrinsic role of APOE3 in controlling neutrophil maturation and degranulation, which is exacerbated in APOE4‐KI neutrophils and reduced in APOE4‐KO neutrophils. Moreover, APOE4 neutrophils accumulated in the brains of AD mice and in AD patients, associated with microglia at sites of plaque pathology. Here we show that genetic deletion of APOE4 in neutrophils suppressed their activation and reduced their recruitment to the brains of APP/PS1 mice. RNAseq analysis showed increased proportion of disease associated microglia (DAM/MGnD) in APP/PS1: Ly6g‐CRE:APOE4fl/fl mice, associated with reduced plaque pathology. Furthermore, we show impaired induction of MGnD signature in AD brains of APOE4 carriers. Our findings show that APOE4‐induced inflammatory neutrophils are recruited to the diseased brain and impair MGnD response leading to accelerated AD pathology.ConclusionTaken together, these findings identify a cell‐intrinsic role of APOE4 in the induction of hyperactivated neutrophils recruited to the brain, which may provide new molecular targets to modulate and restore functional microglia in AD.

Absence of Neutropenia in Patient with an Exon 2 Nonsense Mutation in ELANE: Clinical Evidence to Support Gene Therapy Efforts for Severe Congenital Neutropenia

Introduction Severe congenital neutropenia (SCN) is an inherited bone marrow failure condition most often caused by heterozygous mutations in the ELANE gene. While granulocyte colony stimulating factor (G-CSF) is effective in increasing neutrophil counts in the majority of patients, approximately 15% of patients do not respond, necessitating the creation of alternative treatment strategies. Due to its association with significant, life-threatening infections and heightened risk of progression to myelodysplastic syndrome and/or acute myeloid leukemia, SCN has been the focus of recent gene therapy efforts by multiple groups (Nasri et al., 2020; Rao et al., 2021). Pathogenic variants in ELANE most often cause missense mutations or in-frame indels which lead to the production and expression of mutant neutrophil elastase (NE) proteins. Frameshift mutations resulting in out-of-frame stop codons or nonsense mutations are also seen in the third through fifth (final) exons of ELANE due to their ability to circumvent nonsense-mediated decay (NMD) and generate mutant NE. In contrast, frameshift and nonsense mutations in the first and second exons have not been shown to cause neutropenia, most likely because NMD prevents the translation of mutant NE (Rao et al., 2021; Horwitz, 2021). Collectively, these observations suggest that the pathogenicity of ELANE mutations may rely on gain-of-function alterations in mutant NE rather than loss-of-function alterations in normal NE. Because the absence of one copy of ELANE does not appear to be deleterious, one theory posits that therapeutic inactivation of mutant copies of ELANE may have corrective potential (Horwitz et al., 2019; Nasri et al., 2020; Rao et al., 2021). Thus, the focus of several gene editing efforts has been CRISPR/Cas9-mediated knockout in early exons of ELANE in hematopoietic stem and progenitor cells (HSPCs) of patients with ELANE-mutated SCN for allogeneic stem cell transplant (Nasri et al., 2020; Rao et al., 2021). With this study, we sought to understand whether an inherited ELANE mutation analogous to those utilized in gene therapy would cause neutropenia. Case Presentation Here, we present a six-month-old Caucasian male with an ELANE mutation whose clinical findings support this model. The patient was born at full term to a 35-year-old G6P2 mother. The patient's constellation of complex symptoms, including polymicrogyria, corpus callosum dysgenesis, hypotonia, seizures, and feeding difficulties prompted clinical trio whole genome sequencing (WGS) at approximately two months of age. The patient was incidentally found to have a de novo nonsense variant in exon 2 of the ELANE gene (NM_001972.2:c.151G>T, p.[G51*]), predicted to result in protein truncation or NMD. WGS also identified variants of uncertain significance in CHRNB2 and HFE. Importantly, although the ELANE variant was classified as likely pathogenic in the clinical genetics report, the patient has not had clinically significant neutropenia. Absolute neutrophil counts have ranged from 1.27 - 10.95 K cells/μL, with mean and median values of 4.09 and 2.95 K cells/μL, respectively (n=10). The patient has not had a significant infectious history, nor has he required any treatment for hematologic concerns. Discussion The absence of a neutropenia pathology in this patient, at least in his first six months of life, provides evidence in support of gene therapy efforts which seek to ameliorate pathogenic ELANE mutations with gene editing to produce frameshifts in early exons. Most likely, in this patient, NMD of mutant ELANE mRNA prevented its translation, thus allowing for ample production and maturation of neutrophils. A similar pattern was observed in another report which examined patients with whole gene deletions of ELANE (Horwitz et al., 2019), but this is the first report of a nonsense variant analogous to those proposed to be introduced by gene or base editing for gene therapy of SCN. Thus, the present case adds to a growing body of evidence which suggests that ELANE knockout may have therapeutic potential for SCN.

Severe Chronic Idiopathic Neutropenia Managed with Chronic Filgrastim: A Case Report

Myelodysplastic syndrome (MDS) represents a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis and abnormal blood cell production. Chronic neutropenia is one such MDS and is classified into mild (Absolute Neutrophil Count (ANC): 1,000 to 1,500/µL), moderate (ANC: 500 to 1,000/µL), or severe (ANC: below 500/µL), with the latter posing the highest risk of infection-related complications. In an attempt to alleviate neutropenia, patients with MDS are often treated with colony stimulating factors (CSFs), such as filgrastim to promote growth and maturation of neutrophils. The target ANC is 1,500 to 2,000/µL for adequate protection against most infections. However, CSF treatments have the potential to stimulate the transformation of MDS into acute myeloid leukemia (AML) so CSF is often used with caution. Presented below is a case of severe chronic idiopathic neutropenia responsive to chronic filgrastim that has yet to show evidence of transformation. A 79 year-old female with a past medical history of distant tobacco use, hypertension, acute cerebrovascular accident, and Hashimoto's thyroiditis presents to the clinic for routine follow-up of her now stable chronic idiopathic neutropenia being managed with weekly filgrastim injections for three weeks with one week off in between cycles. She had originally presented to medical attention in 2004 complaining of chronic mouth sores and ulcers with leukopenia and a severely decreased ANC of <500/µL. Continued monitoring with more than 3 complete blood counts (CBC) with a differential consistently noted a decreased ANC in the setting of persisting oral mucosal erosion. Leukocytes were not noted to be dysplastic, quantitative IgG, IgA, IgM levels were noted to be within normal limits, anti-nuclear antibodies/ anti-neutrophil antibodies were noted to be negative argued against an etiology of large granular lymphocytic leukemia, immunodeficiency, and/or autoimmune disorders, respectively. A trial of glucocorticoids did not significantly increase her ANC which led to her ultimately receiving a bone marrow biopsy (BMB) in 2008. A mildly hypercellular marrow with trilineage hematopoiesis was found, with maturation of all lines, negative flow cytometry, and myelodysplasia. Comforted by the lack of MDS pathologically, the patient was started on filgrastim with significant improvement. Over the course of years the frequency and duration of filgrastim was optimized to her current regimen of weekly filgrastim injections for 3 weeks followed by one week off which allowed her to maintain an adequate ANC and avoid oral ulcers to this day. Despite the feared complication of kickstarting the transformation of MDS into AML with CSF therapy, patients such as the one mentioned here can benefit greatly from judicious use of CSF when blood work can be monitored for complications. The use of G-CSF in severe chronic neutropenia to raise levels from below 500/µL to the ideal target ANC is 1,500 to 2,000 cells per microliter (µL) for adequate protection is in line with our current understanding that even long-term use of G-CSF does not appear to increase the risk of AML (Dale et al., 2022). G-CSF should continue to be considered in the management of severe chronic neutropenia as the risk for transformation may be insignificant compared to the clinical benefit of its use when properly monitored.

Base Edited HSPCs Are Shielded from Targeted CD33 Therapy but Preserve CD33 Expression

Targeted therapy for AML remains challenging as there is broad overlap between targetable surface proteins on transformed cells and healthy hematopoietic stem and progenitor cells (HSPC). CD33 is expressed in about 90% of patients with AML but also on healthy myeloid cells with strong expression on progenitor cells and only weak expression on mature neutrophils. Given the higher expression on leukemic blasts compared to their healthy counterparts, CD33 is an attractive target in AML. However, targeting CD33 e.g. with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin often leads to prolonged cytopenia due to suppression of normal myelopoiesis. Transplantation of genetically engineered CD33 knock-out allogenic HSPCs protects reconstituting hematopoietic cells from targeted immunotherapy administered for disease control after transplantation. Early clinical trials explore this option but the long-term effect of loss-of-function of CD33 remains unknown. We recently demonstrated that single amino acid substitutions engineered into CD45, CD117 or CD123 protect HSPCs from targeted therapies against these antigens while preserving their function. Here, we describe the identification of CD33 shielding amino acid substitutions that can be engineered by base editing and preserve CD33 expression on HSPCs. We identified binding sites of two CD33 antibodies by alanine scanning of the entire extracellular domain. The most favorable amino acid substitutions at key residues were analyzed computationally and binding reduction was subsequently validated by flow cytometry. 23 individual amino acid substitutions at 3 positions were tested. Eleven retained similar expression, purity and biophysical properties as wildtype CD33. Those variants are shielded from antibody 1 even at high concentrations when assessed by biolayer interferometry. In addition, three variants are shielded from antibody 2 binding. We used the ABE8e_SpRY base editor to engineer HSPCs and tiled 21 sgRNAs centered around the intended edit. Five days after electroporation, HSPCs were evaluated for editing efficiency (Sanger sequencing) and binding to antibody 1 (flow cytometry). sgRNA4 showed high editing efficiency of about 80% as well as loss of binding to antibody 1 (Figure A) while binding to the control antibody that binds a different epitope was maintained. Importantly, Sanger sequencing did not show off-target editing of the closely related SIGLEC22P pseudogene. Edited (sgRNA4) and non-edited control (NTC) HSPCs formed comparable numbers of myeloid and erythroid colonies (Figure B). Furthermore, in vitro differentiation showed no difference in frequency of CD14, CD15, CD19, CD33, CD41 and GlyA positive cells indicating that the edited HSPCs did not display major functional alterations. In summary, CD33 variants that can be engineered into HSPCs with high editing efficiency by base editing protected from binding to antibody 1 but preserved CD33 expression. These edited HSPCs could be therapeutically useful, in particular for continued immunotherapy after allogenic HSC transplantation. Similar to HSPCs devoid of CD33 these engineered cells could enable tumor-selective immunotherapy using ADCs or CAR T cells but with preserved CD33 expression and function. Furthermore, since base editing is suitable for multiplexing, they may in the future be multiplexed to protect CD33 and other targets for combination immunotherapy.

In Vivo CRISPR Screening Platform for Studying Myeloid Inflammation and Clonal Dynamics

Previous experimental approaches using CRISPR to study factors governing hematopoiesis in vivo have been hampered by heterogeneity in cellular expansion during hematopoietic reconstitution. To control for such ‘cellular jackpotting’, we developed an in vivo hematopoietic stem and progenitor (HSPC)-based CRISPR screening system with unique molecular identifiers (UMIs) and applied these techniques to study myeloid inflammation and clonal expansion of Tet2-mutated HSPCs. Transplantation of Cas9-expressing Lin -Sca1 +c-Kit + (LSK) cells transduced with high-diversity UMI-labeled guide-RNAs (gRNAs) enabled sufficient gRNA representation at a genome-wide level after lentiviral integration of one gRNA per cell and controlled for 10 log-fold differences in cellular expansion during hematopoietic reconstitution. Simultaneous comparison of barcoded CRISPR screening with non-barcoded screening revealed that barcoding decreases false-positive biological processes related to cell division while improving sensitivity for detecting biological processes related to transcriptional events and cellular differentiation. To study inflammatory mediators, we applied this CRISPR screening tool towards studying the recruitment of CD101 +Ly6G +CD11b + mature neutrophils during an inflammatory insult, which revealed the majority of genes involved in human congenital neutropenia (Figure A). To demonstrate the reproducibility of this screening system, we performed a targeted transcription factor (TF)-based CRISPR screen, which revealed concordance between the genome-wide screen and the targeted screen. Next, we also applied this screening system to studying dynamics of Tet2-mutated clonal hematopoiesis. To study master regulators of clonal expansion in Tet2 KO, we performed a UMI-based CRISPR screen using chromatic and transcription factors, which revealed Tet3 and Bcor, genes previously identified to cooperate with Tet2 KO to promote myeloproliferation (Figure B). Validation of additional novel genetic hits is currently ongoing. We hypothesize that this screening platform will be useful for dissecting many aspects of hematopoiesis including inflammatory mediators and clonal dynamics of HSPCs.

A Multiomic Single-Cell Atlas of Human Myelopoiesis Reveals Cellular and Molecular Drivers of Immunomodulatory Drug-Induced Neutropenia

Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes are less well characterised. Understanding this is important as neutropenia can be caused by dysregulation of the genome regulatory events that orchestrate neutrophil maturation. A good example is the use of immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) for the treatment of multiple myeloma (MM) which are now standard-of-care, but their use is frequently complicated by neutropenia because of neutrophil maturation impairment. This is due to Cereblon (CRBN)-driven substrate ubiquitination and Ikaros degradation, causing PU.1 downregulation. However, restoration of Ikaros levels cannot fully alleviate the IMiD-induced myeloid differentiation block suggesting other mechanisms might also contribute. Our aim was to build a multiomic atlas of human myelopoiesis and use this to interrogate the molecular drivers of IMiD-induced neutropenia. We developed an ex vivo myeloid cell differentiation assay using human mobilized peripheral blood CD34 + cells from healthy donors and optimised methods for single cell multiomic (combined RNA-seq & ATAC-seq) analysis. This platform was validated orthogonally (immunophenotype, morphology & functional) and employed to construct a multi-omic single-cell landscape of normal human neutrophil differentiation and it's perturbation by IMiDs. Single cell transcriptomes from >110,000 cells from untreated and IMiD-treated samples were analyzed, capturing the full spectrum of human myelopoiesis spanning from early myeloid progenitors through to neutrophil populations (band, segmented, mature) (Fig.1A-left). Ex vivo differentiated neutrophils retained transcriptional signatures reflective of effector functions (granule biogenesis, chemotactic activity, respiratory burst) and mapped to previously described human neutrophil populations in vivo. Ex vivo generated neutrophils showed expected functional properties of phagocytosis and neutrophil extracellular trap formation upon stimulation. Exposure to IMiDs caused a maturation arrest with a 40-50% (p<0.001) decrease in abundance of differentiating metamyelocytes, and neutrophil populations. Crucially, this maturation arrest was due to an accumulation of transcriptionally distinct myeloid precursors that differentiated though an aberrant pathway (population 6, Fig 1A). Furthermore, immature erythroid clusters representing 10% of live cells accumulated specifically in IMiD-treated samples (p<0.001) in absence of erythropoiesis-stimulating cytokines. Simultaneous profiling of gene expression and chromatin accessibility in individual cells confirmed the presence of distinct phenotypes for untreated and IMiD-treated myeloid cells (Fig.1A) enabling the characterisation of the genome landscape of myelopoiesis, which has previously proven challenging. As expected, we observed dysregulation of Ikaros targets including PU.1 upon IMiD treatment (Fig. 1B-frame). The ability to study distinct and aberrant cellular populations at transcriptional and genome regulation level in parallel allowed us to identify several novel regulators of normal and perturbed neutrophil development. For example, we observed perturbed activation patterns of the transcription factors (TFs) zinc finger protein (ZNF467) and interferon regulatory factor 5 (IRF5) and the expression of their downstream targets as illustrated in Fig.1B. ZNF467 orchestrates a transcriptional program supporting terminal neutrophil differentiation in untreated cells (Fig.1B-top right), whilst the regulatory repertoire of IRF5, a hallmark of neutrophil activation is confined primarily to treatment-specific immature populations (Fig.1B-bottom right). We present a multiomic atlas of normal myelopoiesis which we believe will provide an important resource for the study or normal and perturbed neutrophil development. We use this platform to study IMiD-induced neutropenia and identify two aberrant cellular progenitor populations (aberrant myeloid precursors & erythroid progenitors) with distinct molecular properties as the likely cause of myelosuppression.

Granulocyte Colony-Stimulating Factor Use in Patients with Hemophagocytic Lymphohistiocytosis: A Single Center Experience

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening syndrome caused by activation of T cells that results in extreme inflammation. HLH is commonly associated with underlying conditions such as infection, autoimmune disease, or malignancy. HLH management includes treatment directed at the specific trigger, suppression of maladaptive cytokines, and inhibition of T cell and macrophage proliferation. Granulocyte colony-stimulating factor (GCSF) is a myeloid growth factor that stimulates the production, maturation, and activation of neutrophils. GCSF may theoretically promote production of inflammatory cytokines and enhance hemophagocytosis, thereby intensifying HLH. By contrast, GCSF could play a vital role in the treatment of granulocytopenia caused by HLH and thus limit the development of infections. There is a paucity of data examining the role of GCSF in patients with HLH. Current literature is limited to case reports of HLH exacerbated by GCSF administration. Practice patterns of GCSF use in HLH are highly variable, both within and between institutions. Aim: To compare the clinical course and overall survival of patients with HLH who received GCSF treatment and those who did not. Methods: We performed a retrospective, single-center cohort study of patients admitted with a diagnosis code of HLH from July 1997 through February 2023. Patients at least 18 years old with active HLH were included. Each case was reviewed to confirm the diagnosis of HLH based on available diagnostic evaluation and the final discharge diagnoses as documented by the primary treating team. In cases indeterminate for HLH, a second independent reviewer performed additional evaluation to reach consensus. The primary outcome was overall survival as determined by the Kaplan Meier method with statistical comparison using the log-rank test. Other examined outcomes included: incidence of infection, length of hospital stay (LOS), incidence of intensive care unit (ICU) admissions, length of ICU stay, incidence of 30-day re-admissions due to HLH, and time to first change in HLH-directed therapy. Results: Database analysis revealed 130 patients with a diagnosis code of HLH, of which 68 patients met inclusion criteria. The study cohort comprised 26 patients that received GCSF treatment and 42 patients that did not. The median follow-up time was 3.1 months (interquartile range [IQR] 0.6-29.4). The most common HLH triggers were infection (30.8% GCSF group vs 45.2% control, p=0.31) and malignancy (65.5% GCSF group vs 19% control, p<0.001). A median of 10 doses (IQR 6-16) of GCSF were administered per patient in the GCSF group, and the median time between diagnosis and the first administered dose was 6.5 days (IQR 2.25-18.75). Median overall survival in the GCSF group was 122.6 months compared to 1.5 months in the control group (p=0.02, Figure 1). There was a higher but statistically insignificant difference in infection rate in the GCSF arm (92.3%) compared to the control arm (71.4%, p=0.06). The median LOS was higher in the GCSF group (38 days vs 15 days, p<0.001). Rates of ICU admission (53.4% vs 62%, p=0.69), intubation and mechanical ventilation (34.6% vs 33.3%, p=1.0), and initiation of renal replacement therapy (23.1% vs 16.7%, p=0.74) were similar across the GCSF and control groups, respectively. Two patients in each group were re-admitted for HLH within 30 days of initial discharge. The median time to change in HLH-directed therapy was not reached in either group, and there was no significant difference in incidence of therapy change between groups at 1, 3, or 6 months (Figure 2). Conclusions: In this single center, retrospective cohort study of patients with HLH, treatment with GCSF was not associated with inferior overall survival. While over-representation of a malignancy-associated trigger in the GCSF group may confound interpretation of this result, it nevertheless reflects an important clinical finding that contradicts previously published case report-level evidence. This study highlights the need for further prospective investigation of GCSF administration in HLH.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.

309. Optimized hematopoietic stem cell source for the ex vivo production of mature human neutrophils as a potential cell-based therapy for neutropenia

Abstract Background Neutrophils or granulocytes are the most common innate immune cell and are critical for eliminating invasive pathogens. Patients with low neutrophil counts face an increased risk of fatal opportunistic infections. Allogeneic granulocyte transfusions offer a solution to supplement endogenous neutrophil counts but are challenging due to insufficient preservation methods and donation feasibility. Here, we compare the capacity of three different hematopoietic stem cell (HSC) sources; 1) cord blood (CB), 2) live adult bone marrow (LABM), and 3) cadaveric adult bone marrow (CABM), to expand and generate an ex vivo neutrophil source. Methods HSCs were expanded ex vivo with or without UM729 then differentiated and evaluated for neutrophil yield and purity. Ex vivo differentiated neutrophils were characterized by flow cytometry for activated phenotype surface markers. Finally, the functional capacity of ex vivo neutrophils, including phagocytosis, ectodomain shedding, and reactive oxygen species (ROS) production, was measured in response to the fungal pathogen Candida albicans. Results CB-derived HSCs were found to be superior in expansion capacity compared to LABM- or CABM-derived HSCs by 3-fold at day six. The use of UM729 reduced the purity of neutrophil differentiation by 44% for both live and deceased BM marrow-derived HSCs. UM729 had no observable effect on the purity of neutrophil differentiation for CB-derived HSCs. Additionally, due to enhanced Fc gamma receptor I expression, ex vivo neutrophils from all sources were ten times more sensitive to recognizing IgG-opsonized C. albicans than primary neutrophils. Ex vivo neutrophils were capable of phagocytosis, ROS production, and ectodomain shedding to a similar capacity as primary donor neutrophils. Conclusion We find that cadaveric bone marrow-derived HSCs are a viable source for HSC-generated neutrophils. That said, CB-derived HSC were superior in expansion capacity. We also highlight the unique opsonin-activating features of ex vivo generated neutrophils which warrant further study in granulocyte transfusion animal models of systemic infection. These data underscore the significance of neutrophil cellular therapeutics as a novel modality for preventing infection in high-risk neutropenic patients. Disclosures Nathan E. Jeffries, BA, Safi Biotherapeutics: Salary Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support

Abstract 167: Neutrophil Maturity Following Resuscitation From Cardiac Arrest

Introduction: Immature neutrophils mobilized from bone marrow in response to injury do not express CD10 and are characterized as pro-inflammatory. Maturity status of neutrophils has not yet been described in patients after the global ischemic-reperfusion injury following cardiac arrest (CA). Hypothesis: Immature neutrophils predominate the systemic cellular response after CA and are associated with mortality. Goal: To characterize neutrophils and their associations with clinical parameters in patients following resuscitation from CA. Methods: After informed consent, 100 subjects were enrolled. Blood samples were collected at 6, 12, 24, 48, 72, and 168 hours after return of spontaneous circulation (ROSC). White blood cells (WBC) were isolated and stained for flow cytometry. Data analysis was performed with FlowJo and GraphPad Prism; a P-value of <0.05 was deemed statistically significant. Results: In our cohort of 51 non-survivors and 49 survivors, there were no significant differences in WBC count at any time point. At 72 hours after ROSC, we found a higher absolute number (6.8 vs. 5.0; p=0.018) and percentage ( 87.1 vs. 80.3; p=0.030) of neutrophils in the WBC population in non-survivors compared to survivors. Analysis of neutrophil CD10 subpopulations revealed a higher number of immature CD10 - neutrophils in non-survivors at 72 hours after ROSC (6.1 vs. 4.4; p=0.007) compared to survivors. There was no significant difference between non-survivors and survivors in mature CD10 + neutrophil count or temporal changes. The CD10 - /CD10 + neutrophil ratio was significantly higher in non-survivors at 24 hours (4.7 vs. 3.1; p=0.036) and 48 hours (11.5 vs. 6.6; p=0.040) after ROSC. Conclusions: Neutrophils are important to inflammation resolution after injury; however certain subpopulations of immune cells may be involved in and perpetuate tissue damage after CA. The levels and temporal changes of pro-inflammatory, immature (CD10 - ) neutrophils in non-survivors point to the need for further research including functional studies. The identification of neutrophil marker targets that may impact the balance of pro- and anti-inflammatory immune cells and their related signaling pathways could offer opportunities to improve outcomes after cardiac arrest.