Granulocyte Colony-stimulating Factor Receptor Research Articles

A JAGN1–associated severe congenital neutropenia zebrafish model revealed an altered G-CSFR signaling and UPR activation

AbstractA variety of autosomal recessive mutations in the JAGN1 gene cause severe congenital neutropenia (CN). However, the underlying pathomechanism remains poorly understood, mainly because of the limited availability of primary hematopoietic stem cells from JAGN1-CN patients and the absence of animal models. In this study, we aimed to address these limitations by establishing a zebrafish model of JAGN1-CN. We found 2 paralogs of the human JAGN1 gene, namely jagn1a and jagn1b, which play distinct roles during zebrafish hematopoiesis. Using various approaches such as morpholino-based knockdown, CRISPR/Cas9–based gene editing, and misexpression of a jagn1b harboring a specific human mutation, we successfully developed neutropenia while leaving other hematopoietic lineages unaffected. Further analysis of our model revealed significant upregulation of apoptosis and genes involved in the unfolded protein response (UPR). However, neither UPR nor apoptosis is the primary mechanism that leads to neutropenia in zebrafish. Instead, Jagn1b has a critical role in granulocyte colony-stimulating factor receptor signaling and steady-state granulopoiesis, shedding light on the pathogenesis of neutropenia associated with JAGN1 mutations. The establishment of a zebrafish model for JAGN1-CN represents a significant advancement in understanding the specific pathologic pathways underlying the disease. This model provides a valuable in vivo tool for further investigation and exploration of potential therapeutic strategies.

Potential diagnostic and drug target markers in glioblastoma

Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.

G-CSFR Is a Checkpoint of Natural Killer Cells Against Tumor

G-CSFR is a cell surface receptor that can activate cell proliferation and differentiation after binding to granulocyte colony stimulating factor(G-CSF). However, in natural killer (NK) cells, the biological role of G-CSFR has not been fully elucidated. Here we show that granulocyte colony-stimulating factor receptor, that is encoded by the CSF3R gene, is a novel and potent checkpoint in both human and mouse NK cells fighting against tumors. Previous studies have shown that NK cells under resting state with low expression of G-CSFR, but highly expressed in peripheral blood and bone marrow NK cells after G-CSF mobilization. We therefore used G-CSFR blocking agent in vitro to evaluate the effect of G-CSFR on human NK cells proliferation and cytotoxicity capacity. The results showed that G-CSFR agonist significantly down-regulated the proliferation and cytotoxicity of NK cells in vitro and this is independent of G-CSF stimulation. Since NK92 and YT cell lines highly express G-CSFR, we then knocked down G-CSFR in NK92 and YT cells. The results showed that the low expression of G-CSFR enhanced the expression of CD107a in vitro, indicating G-CSFR may be an inhibitory signaling in NK cells. To the further evaluate the specific role of G-CSFR in NK cells and we therefore constructed NK-specific CSF3R deficiency mice (CSF3R △NK/NK; CSF3Rfl/flNCR1cre, thecre group) and the control group ( CSF3Rfl/fl ( CSF3RWT/WT mice, the flox group). We then evaluated the phenotype, differentiation and functional state of NK cells between the two group. NK-specific CSF3R deficiency increased mature NK subsets in mice, and these CSF3R-deficient NK cells exhibited enhanced activation, cytotoxicity and proliferation, which is in accordance with the role in human NK cells. After constructing four tumor-bearing models, namely MC38 (colon tumor), LLC (lung tumor), MLL-AF8(acute myeloid leukemia), B16-F10(melanoma), we found that CSF3R specific depletion in NK cells reduced the tumor burden, prolonged the survival and reduced tumor metastasis in CSF3R△NK/NK mice (Figure 1A-D), indicating CSF3R depletion in NK cells promotes increased ability against tumors. To further evaluate the molecular mechanism of G-CSFR regulation of NK cell function, single cell RNA sequencing on bone marrow cells from both CSF3R△NK/NK mice and CSF3RWT/WT mice and NK cells were extracted for further subpopulation analysis. UMAP analysis revealed the clustering of bone marrow NK cells from both genotypes into ten subsets and on the basis of the key gene expression of NK cell effector molecules, transcriptional factors, maturation markers, and proliferation marker, we identified NK cell maturation program as mBM5+mBM8, mBM2+mBM3+ mBM6 and mBM0, by which immature NK cells should go through to gain optimal effector function, according to this model, CSF3R△NK/NK mice contained more NK cells belonging to the terminally mature mBM0 than CSF3RWT/WT mice. “Cell Killing” and “leukocyte mediated cytotoxicity” pathway was enriched in CSF3R△NK/NK mice, indicating enhanced activation state of NK cells. In conclusion, our results uncover a potent checkpoint in NK cells protecting against tumors, suggesting a promising approach of targeting G-CSFR for NK cell-based immunotherapies.

G-CSF Induces MAIT Cells to Exert Anti-Intestinal Gvhd Effects Dependent on CXCR6 Mediated Chemotaxis

Background: Graft-versus-host disease (GVHD) is a major complication leading to transplantation failure and affecting overall survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Granulocyte colony-stimulating factor (G-CSF) can induce T-cell tolerance and altering graft cell components to enhance GVHD prevention in HSCT. MAIT cells are a group of innate immune T cells. It has unique biological properties, including mucosal tissue enrichment, activation of microbial riboflavin derivatives and tissue repair properties, which determine MAIT cells play an important role in mucosal immunity. Previous studies found that a high MAIT cell counts in G-CSF-induced grafts (G-grafts) is associated with a low incidence of gut acute GVHD (aGVHD). However, the effect of G-CSF mobilization on the phenotype, function and its regulatory mechanism in GVHD of MAIT cells has not been elucidated. Methods: MAIT cells in PB samples from four healthy donors before and after G-CSF treatment were isolated for Single-cell RNA sequencing (Figure A). Flow cytometry, In vitro experiment and NOD-SCID-IL-2Rg−/− mice (NSG mice) were performed to observe the phenotypic, functional changes and the dynamic distribution of G-CSF induced donor-derived MAIT cells in recipients. Results: A total of 80709 MAIT cells were divided into 14 clusters. Based on key type-associated marker genes and gene sets, MAIT0 (clusters 5 and 9), MAIT1 (cluster 7), MAIT2 (clusters 4 , 11), Cycling MAIT (clusters 12, 13), Activation (clusters 0, 8), ISG (cluster 10) and MAIT17 (clusters 1,2,3,6) cells were identified (Figure B,C). The subset frequencies and expression signatures of immunosuppression (MAIT2 and Cycling cells from cluster 12) and tissue repair (Activation and MAIT17) were both increased at both transcriptome (Figure D) and protein levels, suggesting that G-CSF enhanced the immune tolerance and tissue repair of MAIT cells. In vitro further confirmed that G-CSF-induced MAIT cells showed a stronger inhibition for T cell proliferation (Figure E). Besides, overexpressed genes related to immune regulation in G-CSF induced MAIT cells were strongly enriched in cluster 12 with high CSF3R (G-CSF receptor) expression (Figure F). The cluster 12 also showed the enrichment of IL-17 and TNF signaling pathway favoring tissue repair function, while downregulation of Graft-versus-host disease pathway upon G-CSF mobilization, indicating that G-CSF-CSF3R interaction is the potential mechanism by which G-CSF mediates the immune regulation of MAIT cells. Based on whether recipients develop gut aGVHD or not after allo-HSCT, the matched donor grafts after G-CSF mobilization were divided into two groups termed as post-G-CSF-no gut aGVHD (n=3) and post-G-CSF-gut aGVHD group (n=1), respectively. MAIT cells in post-G-CSF-no gut aGVHD group overexpressed CSF3R and its related target genes, immunosuppression and tissue repair signatures compared with post-G-CSF-gut aGVHD. Besides, MAIT cells from G-PB of recipients without gut aGVHD had a higher CD114 expression (encoded by CSF3R) compared with gut aGVHD (Figure G). These results suggest that the enhanced immunosuppression and tissue repair function of MAIT cells induced by G-CSF may exert anti-gut aGVHD effects. The NSG mice confirmed MAIT cells in G-PB to tissue chemotaxis especially gut tissues in the recipient. A higher frequency of MAIT cells was detected in gut tissues in the low GVHD score group (GVHD Score＜3) compared with the high GVHD score group on post-transplant day 14 (Figure H), suggesting recruited MAIT cells in gut tissues favor anti-gut aGVHD effect. Moreover, CXCR6 was the most differentially expressed chemotactic receptor gene between post-G-CSF-no gut aGVHD and post-G-CSF-gut aGVHD group (Figure I). At the protein level, the expression of CXCR6 were significantly upregulated upon G-CSF administration (Figure I), while that of CCR6 and CXCR6 was non-significant. These results suggest that CXCR6 is necessary for G-CSF induced MAIT cell recruitment to the gut tissue. Conclusions G-CSF-CSF3R interactions enhance immunosuppression and tissue repair functions of MAIT cells, while CXCR6 recruits these functional MAIT cells from PB to gut tissues to exert anti-gut aGVHD effect. Hence, CSF3R and CXCR6 expression in G-CSF mobilized MAIT cells is crucial to exert anti-gut aGVHD effect, and has the potential to become predictive markers of gut aGVHD.

Single Cell Analysis and Functional Features of Circulating CD34 + Cells from Patients with Sickle Cell Anaemia Unveil a Defect in the Myeloid Lineage

Polymorphonuclear neutrophils (PMN) have long been suspected to be involved in the pathophysiology of Sickle cell anemia (SCA) since early studies have reported a higher neutrophil count in patients at steady-state and its positive correlation with SCA severity. This high leukocyte count has long be considered as a peripheral phenomenon, driven by RBC hemolysis causing systemic inflammation and neutrophils recruitment. Nevertheless, several lines of evidence suggest that hematopoietic stem/progenitor cell (HSPC) function might be perturbed in SCA. Given that recent report showed an increased apoptosis during terminal erythroid maturation of SCA cells and that erythroid and myeloid differentiation derive from the same common myeloid progenitor (CMP), we investigated a possible enhanced myelopoiesis in SCA and its causes. First, to better understand when and how hyperleukocytosis and chronic inflammation begin in these patients, we analyzed longitudinal biological data from 1250 infants with SCA aged 1-24 months. We observed that the increase in neutrophils count began as early as the first months of life, and was statistically correlated with the drop in HbF rate and with the increase in reticulocytes count, much more than with the increase of the LDH level. These results seem to contradict the hypothesis of chronic inflammation driven by intravascular hemolysis. To assess whether the abnormal hematopoiesis is associated with a defect on lineage commitment of Hematopoietic Stem/Progenitor Cells (HSPCs), we evaluated the phenotype of myeloid and erythroid progenitors isolated from peripheral blood (PB) of untreated patients with SCA. While the portion of the common myeloid progenitor (CMP) was similar among both groups, we observed a significant increase of granulocyte-monocyte progenitors (GMP) in SCA patients (n=6) compared to health donors (HDs) (n=9), as well as a significant decrease of erythroid progenitors (MEP). Interestingly, HSPCs from SCA PB produced more myeloid colonies in vitro than those in HD HSPCs. In addition, colony-forming assay of sorted SCA CMP displayed an increase of granulo-monocytes colonies CFU-GM and decrease of erythroid colonies BFU-E compared to HDs, indicating an increased myeloid commitment of SCA HSPCs. To comprehensively characterize HSPC composition, we performed 10´ scRNA-seq on CD34+ HSPCs isolated from HDs (n=3) and patients with SCA (n=4). We confirmed that SCA HSPCs have a high proportion of neutrophil progenitors. More interestingly, MPP/HSC populations from SCA patients highly express several genes involved in myeloid differentiation like SELL (L-Selectin) and CSF3R (G-CSF receptor) genes, indicating an enhanced myeloid commitment of early progenitor in SCA. To better characterize the myeloid features of these progenitors, we investigated ex- vivo granulopoiesis of PB HSPCs from SCA patients (n=10) and HDs (n=9) in presence of G-CSF as CSF3R is highly expressed in early SCA progenitors. While cell proliferation was similar among both groups, unexpectedly SCA myeloid cells presented a significant delayed expression of CD11b (p<0.01). Interestingly, we also observed an overexpression of monocytic markers HLA-DR and CD36 in a subset of differentiated cells, suggesting a potential commitment of part of the myeloid progenitors to a monocyte lineage , confirmed by MGG staining. To further understand this defect, we performed RNAseq analysis of the GMP isolated from PB of SCA patients (n=4) and HD (n=4). We identified a total of 420 differentially expressed genes (DEGs). Among these DEGs, significantly downregulated genes in SCA included granulocyte differentiation program such as GFI1, C/EBPa, and JAK3. In return, significantly upregulated genes included KLF4, supporting the monocytic bias observed in the early myeloid progenitors. In conclusion, these data reveal the myeloid-primed signature in PB HSPCs of patients with SCA, likely responsible for the elevated PMN and monocyte count in these patients. This initial breakthrough in the understanding of the development of hyperleukocytosis and chronic inflammation in these patients will open up new early therapeutic options. Moreover, since CD34+ cells of the peripheral blood are the main source of HSPCs for the development of gene therapy in SCA, deciphering their functional feature and myelo-monocytic skewing could be of interest to guide the gene editing strategy.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

Elevated Functional Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of G-CSF-Mobilized Dnmt3a-Mutant Mice

Clonal hematopoiesis (CH) arising from somatic mutations in hematopoietic stem cells (HSCs) is associated with increased risk of developing several non-hematologic and hematologic diseases. In the context of stem cell transplantation, donor HSCs carrying CH-associated somatic mutations can be passed from donor to recipient during stem cell transplantation. Most literature supports that there is no negative impact on recipient survival or outcomes after allogeneic stem cell transplantation (allo-HSCT) from donors with CH. However, it is less clear how allo-HSCT donors with CH respond to common mobilization methods such as Neupogen (G-CSF). As hematopoietic stem and progenitor cells (HSPCs) with CH mutations have been found to respond to external stimuli such as inflammation in a manner distinct from wild-type HSPCs, it is plausible that CH-mutant HSPCs also distinctly respond to G-CSF mobilization. It is important to understand whether the process of mobilization differentially enriches for CH-mutant HSPCs in the mobilized peripheral blood product. To address this gap in knowledge, we used a murine model of Dnmt3a-mutant CH. After five consecutive days of Neupogen (G-CSF) administration, modeling the procedure for human HSC mobilization, we observed that Dnmt3a-mutant mice had significantly increased white blood cell, neutrophil and monocyte counts compared to control mice. We observed a significant increase in cell surface marker-defined HSPCs in the peripheral blood and spleen of G-CSF-treated Dnmt3a-mutant mice compared to G-CSF-treated control mice. Using in vitro colony-forming unit assays, the peripheral blood of G-CSF-treated Dnmt3a-mutant mice was found to contain a greater number of functional hematopoietic progenitors compared to G-CSF-treated control mice. To determine if the peripheral blood of G-CSF-treated Dnmt3a-mutant mice had a greater abundance of functional HSCs, we transplanted equal volumes of mobilized blood from Dnmt3a-mutant and control mice into primary and secondary lethally irradiated wild-type recipient mice. We observed increased long-term engraftment of mobilized blood from Dnmt3a-mutant mice compared to control mice in both primary and secondary recipient animals. At a mechanistic level, HSC mobilization is facilitated by myeloid cells that respond directly to G-CSF via the G-CSF receptor ( Csf1r). In Dnmt3a-mutant mice, we observed increased Csf1r expression in myeloid progenitor and mature myeloid cells compared to control mice, providing a putative mechanism by which increased mobilization is enabled. Together, our data support differential mobilization potential of Dnmt3a-mutant compared to control HSCs in response to G-CSF, which may have implications in the context of allo-HSCT.

Semimechanistic pharmacokinetic-pharmacodynamic model of tripegfilgrastim for pediatric patients after chemotherapy.

Tripegfilgrastim is a long-acting granulocyte colony-stimulating factor (G-CSF) that has been used to prevent chemotherapy-induced neutropenia in adults. This study aimed to establish a pharmacokinetic (PK)-pharmacodynamic (PD) model to explore the impact of chemotherapy and tripegfilgrastim on absolute neutrophil counts (ANCs) and to further propose a fixed-dose regimen in pediatric patients. Because neutrophils affect the clearance of tripegfilgrastim, the semimechanistic PK-PD model was developed simultaneously by using data from 40 healthy adults and 27 pediatric patients with solid tumors. Tripegfilgrastim PK and ANC dynamics were described with a pharmacodynamics-mediated drug disposition model assuming quasi-equilibrium with five transit compartments mimicking neutrophil granulopoiesis. The effect of chemotherapy on neutrophils was included by stimulating the elimination of the G-CSF receptor at the mitotic cells. Healthy adult and pediatric patients showed significantly different value for dissociation constant of the tripegfilgrastim-G-CSF receptor complex (Kd ) and apparent volume of distribution (Vd /F). Patients treated with chemotherapy had a higher Vd /F and 62% lower Kd than healthy adults. As the age increased, the absorption rate of tripegfilgrastim was decreased. Body weight affected the G-CSF receptor-mediated internalization of tripegfilgrastim, and the baseline ANC value impacted the production rate of G-CSF receptors. Simulations from the developed model suggested that 1.5, 2.5, 4, and 6 mg single subcutaneous tripegfilgrastim doses for the respective weight groups of 10-20, 21-30, 31-44, and more than 45 kg significantly reduced the duration of Grade 4 neutropenia similar to tripegfilgrastim weight-based treatment with 100 μg/kg.

Effects of G-CSF on hPDLSC proliferation and osteogenic differentiation in the LPS-induced inflammatory microenvironment

BackgroundPeriodontitis is a chronic infectious disease of periodontal support tissue caused by microorganisms in dental plaque, which causes alveolar bone resorption and tooth loss. Periodontitis treatment goals include prevention of alveolar bone resorption and promotion of periodontal regeneration. We previously found that granulocyte colony-stimulating factor (G-CSF) was involved in periodontitis-related alveolar bone resorption through induction of an immune response and subsequent destruction of periodontal tissue. However, the mechanisms underlying the effects of G-CSF on abnormal bone remodeling have not yet been fully elucidated. Human periodontal ligament stem cells (hPDLSCs) are major modulators of osteogenic differentiation in periodontal tissues. Thus, the aim of this study was to investigated whether G-CSF acts effects on hPDLSC proliferation and osteogenic differentiation, as well as periodontal tissue repair.MethodshPDLSCs were cultured and identified by short tandem repeat analysis. The expression patterns and locations of G-CSF receptor (G-CSFR) on hPDLSCs were detected by immunofluorescence analysis. The effects of G-CSF on hPDLSCs in a lipopolysaccharide (LPS)-induced inflammatory microenvironment were investigated. Specifically, Cell-Counting Kit 8 (CCK8) and Alizarin red staining were used to examine hPDLSC proliferation and osteogenic differentiation; reverse transcription-polymerase chain reaction was performed to detect the expression patterns of osteogenesis-related genes (alkaline phosphatase [ALP], runt-related transcription factor 2 [Runx2], and osteocalcin [OCN]) in hPDLSCs; and Western blotting was used to detect the expression patterns of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) of PI3K/Akt signaling pathway.ResultshPDLSCs exhibited a typical spindle-shaped morphology and good clonogenic ability. G-CSFR was mostly localized on the cell surface membrane. Analyses showed that G-CSF inhibited hPDLSC proliferation. Also, in the LPS-induced inflammatory microenvironment, G-CSF inhibited hPDLSC osteogenic differentiation and reduced the expression levels of osteogenesis-related genes. G-CSF increased the protein expression levels of hPDLSC pathway components p-PI3K and p-Akt.ConclusionsWe found that G-CSFR was expressed on hPDLSCs. Furthermore, G-CSF inhibited hPDLSC osteogenic differentiation in vitro in the LPS-induced inflammatory microenvironment.

Abstract A38: Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia

Abstract Chronic Neutrophilic Leukemia (CNL) is a rare BCR-ABL negative myeloproliferative neoplasm that affects approximately 1 in a million individuals. The typical age of onset and disease presentation is in elderly patients with a median age of 63 and a range of 9-84. The oncogenic driver of CSF3R, the G-CSF receptor protein, in CNL has been classified over the last decade and has improved therapies for patients with targeted Tyrosine Kinase Inhibitors, such as Ruxolitinib (a potent JAK1/2 inhibitor). Currently, the only curative option is an allogeneic hemopoietic stem cell transplant with typical median overall survival of 2 years. Because of the low occurrence rate of CNL, there have only been 3 germline cases of CSF3R classified in literature. With these patients, they exhibit a lifelong skewing of the myeloid lineage causing gross neutrophilia. Our patient presented at 13 months with leukocytosis and neutrophilia, resulting in genetic testing revealing the diagnosis of the youngest documented case of CNL. A germline CSF3R T618I constitutive activating mutation was documented in our patient and in her father. Her father had documented lifelong leukocytosis of indeterminate origin and it was not until 33 that genetic testing was done, and he was diagnosed with CNL. He underwent an allogeneic stem cell transplant after being admitted with gross hepatosplenomegaly with a WBC of 322, the highest recorded in a germline CNL patient in literature. Our patient presented to clinic with leukocytosis ranging from 45-98 and hepatosplenomegaly. After a bone marrow aspirate and biopsy was taken to determine disease stage and severity, we immediately began Ruxolitinib therapy. We began 20 mg BID dosing based on platelets and monitored response to therapy while conducting targeted RNA-seq for myelodysplastic and myeloid leukemia genes on the bone marrow, which did not detect any secondary mutations. Ruxolitinib dosing was tapered down in a stepwise fashion, until the patient arrived at the current dose of 7.5 mg BID Ruxolitinib, resulting in stable leukocyte counts around 25. As part of monitoring, we are currently conducting 6-month BM procedures and NGS for AML/MDS genes and Ruxolitinib resistance mutations alongside monthly CBCs. Our patient has been on Ruxolitinib for 9 months and has not developed any secondary contributing mutations and remains sensitive to therapy. We continue to monitor our patient’s response to Ruxolitinib to determine if germline predisposition alters the transformative nature of the disease. Within T618I germline CNL cases, we are investigating the difference in phenotype and disease burden that we observe in our patient’s family compared to literature. Based on current cases, it seems that germline CNL does not transform during childhood and adolescence into AML without the influence of a secondary mutation. Retrospective studies into previous cases of CNL to determine if there is a higher frequency of germline origin could pave the way for potential early-stage therapies and detection of CNL prior to transformation. Citation Format: Kevin Zhang, Matthew Rees, Mahsa Khanlari, Laura Rooms, Raul Ribeiro, Marcin Wlodarski. Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A38.

1556 CSL324, a G-CSF receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa

1556 CSL324, a G-CSF receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa

Antibodies to sclerostin or G-CSF receptor partially eliminate bone or marrow adipocyte loss, respectively, following vertical sleeve gastrectomy.

Vertical sleeve gastrectomy (VSG), the most utilized bariatric procedure in clinical practice, greatly reduces body weight and improves a variety of metabolic disorders. However, one of its long-term complications is bone loss and increased risk of fracture. Elevated circulating sclerostin (SOST) and granulocyte-colony stimulating factor (G-CSF) concentrations have been considered as potential contributors to VSG-associated bone loss. To test these possibilities, we administrated antibodies to SOST or G-CSF receptor and investigated alterations to bone and marrow niche following VSG. Neutralizing either SOST or G-CSF receptor did not alter beneficial effects of VSG on adiposity and hepatic steatosis, and anti-SOST treatment provided a further improvement to glucose tolerance. SOST antibodies partially reduced trabecular and cortical bone loss following VSG by increasing bone formation, whereas G-CSF receptor antibodies had no effects on bone mass. The expansion in myeloid cellularity and reductions in bone marrow adiposity seen with VSG were partially eliminated by treatment with Anti-G-CSF receptor. Taken together, these experiments demonstrate that antibodies to SOST or G-CSF receptor may act through independent mechanisms to partially block effects of VSG on bone loss or marrow niche cells, respectively.

G-CSF Is a Novel Mediator of T-Cell Suppression and an Immunotherapeutic Target for Women with Colon Cancer.

G-CSF enhances colon cancer development. This study defines the prevalence and effects of increased G-CSF signaling in human colon cancers and investigates G-CSF inhibition as an immunotherapeutic strategy against metastatic colon cancer. Patient samples were used to evaluate G-CSF and G-CSF receptor (G-CSFR) levels by IHC with sera used to measure G-CSF levels. Peripheral blood mononuclear cells were used to assess the rate of G-CSFR+ T cells and IFNγ responses to chronic ex vivo G-CSF. An immunocompetent mouse model of peritoneal metastasis (MC38 cells in C57Bl/6J) was used to determine the effects of G-CSF inhibition (αG-CSF) on survival and the tumor microenvironment (TME) with flow and mass cytometry. In human colon cancer samples, the levels of G-CSF and G-CSFR are higher compared to normal colon tissues from the same patient. High patient serum G-CSF is associated with increases in markers of poor prognosis, (e.g., VEGF, IL6). Circulating T cells from patients express G-CSFR at double the rate of T cells from controls. Prolonged G-CSF exposure decreases T cell IFNγ production. Treatment with αG-CSF shifts both the adaptive and innate compartments of the TME and increases survival (HR, 0.46; P = 0.0237) and tumor T-cell infiltration, activity, and IFNγ response with greater effects in female mice. There is a negative correlation between serum G-CSF levels and tumor-infiltrating T cells in patient samples from women. These findings support G-CSF as an immunotherapeutic target against colon cancer with greater potential benefit in women.

Systemic inflammatory regulators and proliferative diabetic retinopathy: A bidirectional Mendelian randomization study.

Increasing evidence shows that systemic inflammation is an embedded mechanism of proliferative diabetic retinopathy (PDR). However, the specific systemic inflammatory factors involved in this process remained obscure. The study aimed to identify the upstream and downstream systemic regulators of PDR by using Mendelian randomization (MR) analyses. We performed a bidirectional two-sample MR analysis implementing the results from genome-wide association studies for 41 serum cytokines from 8,293 Finnish individuals, and PDR from FinnGen consortium (2,025 cases vs. 284,826 controls) and eight cohorts of European ancestry (398 cases vs. 2,848 controls), respectively. The inverse-variance-weighted method was adopted as the main MR method, and four additional MR methods (MR-Egger, weighted-median, MR-pleiotropy residual sum and outlier (MR-PRESSO), and MR-Steiger filtering methods) were used for the sensitivity analyses. Results from FinnGen and eight cohorts were pooled into a meta-analysis. Our results showed that genetically predicted higher stem cell growth factor-β (SCGFb) and interleukin-8 were positively associated with an elevated risk of PDR, with a combined effect of one standard deviation (SD) increase in SCGFb and interleukin-8 causing 11.8% [95% confidence interval (CI): 0.6%, 24.2%]) and 21.4% [95% CI: 3.8%, 41.9%]) higher risk of PDR, respectively. In contrast, genetically predisposition to PDR showed a positive association with the increased levels of growth-regulated oncogene-α (GROa), stromal cell-derived factor-1 alpha (SDF1a), monocyte chemotactic protein-3 (MCP3), granulocyte colony-stimulating factor (GCSF), interleukin-12p70, and interleukin-2 receptor subunit alpha (IL-2ra). Our MR study identified two upstream regulators and six downstream effectors of PDR, providing opportunities for new therapeutic exploitation of PDR onset. Nonetheless, these nominal associations of systemic inflammatory regulators and PDR require validation in larger cohorts.

Novel Insights into the Role of Kras in Myeloid Differentiation: Engaging with Wnt/β-Catenin Signaling.

Cells of the HL-60 myeloid leukemia cell line can be differentiated into neutrophil-like cells by treatment with dimethyl sulfoxide (DMSO). The molecular mechanisms involved in this differentiation process, however, remain unclear. This review focuses on the differentiation of HL-60 cells. Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knockout mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults. The Wnt/β-catenin signaling pathway plays a crucial role in regulating the homeostasis of hematopoietic cells. The protein β-catenin is a key player in the Wnt/β-catenin signaling pathway. A great deal of evidence shows that the Wnt/β-catenin signaling pathway is deregulated in malignant tumors, including hematological malignancies. Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane, and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3β phosphorylation, resulting in the release of unphosphorylated β-catenin from the β-catenin destruction complex and its accumulation in the cytoplasm. The accumulated β-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity. Thus, upon treatment of HL-60 cells with DMSO, wild-type Kras reacts with the Wnt/β-catenin pathway, thereby regulating the granulocytic differentiation of HL-60 cells. Wild-type Kras and the Wnt/β-catenin signaling pathway are activated sequentially, increasing the levels of expression of C/EBPα, C/EBPε, and granulocyte colony-stimulating factor (G-CSF) receptor.

Leukemia-associated truncation of granulocyte colony-stimulating factor receptor impacts granulopoiesis throughout the life-course.

The granulocyte colony-stimulating factor receptor (G-CSFR), encoded by the CSF3R gene, is involved in the production and function of neutrophilic granulocytes. Somatic mutations in CSF3R leading to truncated G-CSFR forms are observed in acute myeloid leukemia (AML), particularly those subsequent to severe chronic neutropenia (SCN), as well as in a subset of patients with other leukemias. This investigation introduced equivalent mutations into the zebrafish csf3r gene via genome editing and used a range of molecular and cellular techniques to understand the impact of these mutations on immune cells across the lifespan. Zebrafish harboring truncated G-CSFRs showed significantly enhanced neutrophil production throughout successive waves of embryonic hematopoiesis and a neutrophil maturation defect in adults, with the mutations acting in a partially dominant manner. This study has elucidated new insights into the impact of G-CSFR truncations throughout the life-course and created a bone fide zebrafish model for further investigation.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Exploring the Effects of G-CSF Mobilization on the Immune Function of Mucosal-Associated Invariant T Cell Subsets By Single-Cell RNA-Seq

Background: Mucosal-associated invariant T cells (MAITs) are a group of mucosal-enriched unconventional T cells that can exert a variety of immune functions including pro-inflammatory, cytotoxic and immunomodulatory effects, whereas with the characteristics of innate immune cells. A few studies including our previous work have confirmed the relationship between MAITs and gut acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). In addition, we further proved that specific gut microbiota can promote MAIT17 subset activation, and MAITs has a direct immunosuppressive effect on T cells in vitro, which may potentially explain our results that higher number of MAITs in granulocyte colony-stimulating factor (G-CSF) mobilized grafts is associated with lower incidence of gut aGVHD. G-CSF has been widely used to mobilize hematopoietic stem/progenitor cells before HSCT. It also plays an important immunomodulatory role in inducing immune tolerance by regulating both innate and adaptive immune cells, especially T cells. Therefore, we used single-cell RNA sequencing to further explore the different effects of G-CSF mobilization on the immune function of each subset of MAITs. Methods & Cases: We isolated MAITs from the peripheral blood (PB) of 4 healthy donors pre- and post-G-CSF mobilization by flow cytometry pre-transplantation. 8 samples were performed by single-cell RNA-seq (Figure 1A). One of these recipients who received these 4 healthy donor grafts developed gut aGVHD after transplantation. Through single-cell bioinformatics analysis, we compared the sequencing data from pre- and post-G-CSF-mobilized MAITs of doners, and from G-CSF-mobilized donors in the group of recipients with or without gut aGVHD after transplantation. Results: By performing UMAP cluster analysis on these 8 samples, according to the expression of key cell type-associated marker genes of each cluster, MAITs were annotated into 14 subsets (Figure 1A and 1B). Among these subsets, Cluster 1(CD8_effector_EOMES), 4(CD4_effector_CCR6), 5(CD4_immature_CCR7), 6(CD8_effector_RORC), 12(CD4_immunomodulatory) increased significantly after G-CSF mobilization (Figure 1C). By comparing the bioinformatical data of MAITs of pre- and post-G-CSF mobilization, the results showed that molecular markers responsible for MAIT cell hyporesponsiveness were upregulated, including potential direct or indirect modulation mediated by the IL-4/IL-13 signaling pathway (FOS, JUNB, DDIT4, NFKBIA, MT2A, VIM, etc.) (Figure 1D) or ligand receptor (G-CSF-CSF3R) interactions (Figure 1E). Besides, a specific subset of MAITs defined as Cluster 12-CD4_immunomodulatory MAITs (Figure 1A) was found that played the most important role in immune regulation after G-CSF stimulation, which specifically expressed G-CSF receptor CSF3R. Furthermore, IL-17 signaling pathway was up-regulated and GVHD pathway was down-regulated in Cluster 12 post-G-CSF mobilization (Figure 1F), which suggested the Cluster 12 is an important subset of MAITs anti-GVHD. Compared to the sequencing data of donors in the group of recipients with gut aGVHD after G-CSF mobilization (post-G-CSF-gut aGVHD), we also observed the higher expression of immunosuppressive genes (JUN, DUSP2, JUNB, VIM, etc.) of doner MAITs in group without gut aGVHD after mobilization (post-G-CSF-no gut aGVHD). In addition, in the post-G-CSF-no gut aGVHD group, the expression of SOCS3, STAT3, CCR6 (Figure 1G) and other key markers that may potentially explain the anti-GVHD effect of MAITs was upregulated, as well as the ability of tissue repair and gut-directed migration of MAITs was upregulated (Figure 1H), while its cytotoxic effector functions were not significantly affected. Conclusions: Our results indicated that G-CSF mobilization can enhance the immunosuppressive effect of MAITs. Cluster 12 (CD4_immunomodulatory) is an important immunoregulatory and anti-GVHD subset of MAITs, contributing to the differentiation of MAITs towards MAIT17. The upregulation of markers such as SOCS3, CCR6, STAT3 that involved in CSF3R-JAK-STAT pathway, as well as markers related to mucosal tissue repair may partially explain the protective effect from gut aGVHD of G-CSF mobilized MAITs. These results also enlighten us that gut aGVHD might be prevented by effectively expanding the specific immunosuppressive subset of MAIT cells in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Human neutrophil development and functionality are enabled in a humanized mouse model.

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system invivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

Macrophage polarization in placenta accreta and macrophage-trophoblast interactions.

Placenta accreta (PA) is defined by an abnormal invasion of placental trophoblasts into the myometrium, which can lead to serious postpartum complications. Macrophages play an important role in the regulation of trophoblast function. Both granulocyte colony-stimulating factor (G-CSF) and its receptor (granulocyte colony-stimulating factor receptor, G-CSFR) have effects on trophoblast invasion. However, the current understanding of G-CSF secretion, G-CSFR expression, abnormal polarization of decidual macrophages (dMϕ) in PA and the abnormal invasion of placental trophoblasts into the myometrium are limited. The polarization of dMϕ in PA was analyzed by flow cytometry (FCM), and the expression of G-CSFR in placental trophoblasts in PA was evaluated by immunohistochemistry. In an in vitro co-culture model, we investigated the effects of HTR-8/SVneo trophoblasts cell line (HTR-8) on macrophage human monocyte cell line (THP-1) polarization and G-CSF secretion, and we also analyzed the effects of THP-1 cells, especially M2-like subtype, on primary trophoblasts and HTR-8 proliferation, invasion, and adhesion. FCM, transwell assays, adhesion assays, and proliferation assays were used in the above model. Compared with controls (n = 9), dMϕ showed significantly lower levels of M1 markers CD80 and CD86 and higher levels of the M2 markers CD163 and CD206, and G-CSFR expression of placental trophoblasts was increased in PA (n = 5). In vitro experiments showed that the trophoblast HTR-8 cell line induced polarization of THP-1 cells to an M2-like subtype and increased their secretion of G-CSF. Furthermore, IL-4/IL-13-induced M2-like THP-1 macrophages were able to increase the expression of G-CSFR, proliferation, invasion and adhesion of both primary trophoblasts and HTR-8 trophoblasts. There is an altered immune imbalance at the maternal-fetal interface in PA, which further may lead to abnormal trophoblast function. G-CSF and its receptors may play important roles in abnormal polarization of macrophages and abnormal invasion of trophoblasts.