Senescent Neutrophils Research Articles

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

Neutrophil-activating secretome characterizes palbociclib-induced senescence of breast cancer cells

Senescent cells have a profound impact on the surrounding microenvironment through the secretion of numerous bioactive molecules and inflammatory factors. The induction of therapy-induced senescence by anticancer drugs is known, but how senescent tumor cells influence the tumor immune landscape, particularly neutrophil activity, is still unclear. In this study, we investigate the induction of cellular senescence in breast cancer cells and the subsequent immunomodulatory effects on neutrophils using the CDK4/6 inhibitor palbociclib, which is approved for the treatment of breast cancer and is under intense investigation for additional malignancies. Our research demonstrates that palbociclib induces a reversible form of senescence endowed with an inflammatory secretome capable of recruiting and activating neutrophils, in part through the action of interleukin-8 and acute-phase serum amyloid A1. The activation of neutrophils is accompanied by the release of neutrophil extracellular trap and the phagocytic removal of senescent tumor cells. These findings may be relevant for the success of cancer therapy as neutrophils, and neutrophil-driven inflammation can differently affect tumor progression. Our results reveal that neutrophils, as already demonstrated for macrophages and natural killer cells, can be recruited and engaged by senescent tumor cells to participate in their clearance. Understanding the interplay between senescent cells and neutrophils may lead to innovative strategies to cope with chronic or tumor-associated inflammation.

Defective Clearance of Neutrophils Causes Pathogenic Microenvironmental Interactions and Offers a Candidate Immunotherapy in the Myeloproliferative Neoplasms

Neutrophils are the most abundant leukocytes and essential components of the immune microenvironment. Myeloproliferative neoplasms (MPNs) are characterized by excessive myelopoiesis and serious complications, such as thrombosis, myelofibrosis and leukemic transformation. Neutrophils and hematopoietic stem cells (HSCs) carrying the most common MPN driver mutation (JAK2 V617F) are protected from apoptosis. Moreover, mutant neutrophils aggregate with platelets and interact with activated endothelium, increasing thrombosis risk in MPN (Blood 107:3676; Thromb Res 129:263; Sci Transl Med 10:eaan8292; Leukemia 33:2544). However, possible pathogenic interactions between heterogeneous bone marrow (BM) niches in MPN (Nat Cancer doi:10.1038/s43018-023-00607-x) and neutrophils are understudied. Circulating HSCs and leukocytes home daily to the BM (Nature 452:442), where apoptotic/senescent neutrophils are cleared by macrophages (Immunology 125:281; Cell 153:1025). We found that senescent neutrophils carrying JAK2 V617F or CALR del52 mutation evade normal clearance and accumulate in the BM of MPN mice. The homeostatic recognition and efferocytosis of neutrophils by macrophages rely on the balance of “eat-me” and “don't-eat-me” signals. Supervised analyses of two independent human JAK2 V617F+ MPN granulocyte RNAseq datasets (Blood 123:e123; Blood 134:199) showed specific upregulation of CD24 - reportedly a don't-eat-me signal in solid tumors (Nature 572:392). CD24 expression was high in granulocytes from MPN transformed into AML and in the JAK inhibitors ruxolitinib and fedratinib-resistant HEL cells (Nat Cancer 4:108). Unlike CD274 or CD47, which are extensively studied in myeloid malignancies, our data showed that CD24 expression was high in JAK2 V617F+ human or mouse neutrophils. Functional blockade in co-culture with macrophages or after in vivo adoptive transfer showed that CD24 serves as a don't-eat-me signal in neutrophils. Given that oncogenic driver mutations lead to the constitutive activation of cytokine-regulated JAK-STAT signaling, we investigated the effect of cytokines activating this pathway. We found that high GM-CSF in MPN BM induces CD24 expression and prevents neutrophil clearance via JAK-STAT5 signaling. Consequently, ruxolitinib normalized GM-CSF-induced CD24 expression and decreased human and mouse neutrophils in MPN. Our study showed that accumulation of mutant senescent neutrophils in the BM triggered abnormal interactions within the BM microenvironment. Mutated neutrophils were found closer to Megakaryocytes (Mk) and ~30% of Mks showed neutrophils transiently invading their demarcation membrane system through a process termed “emperipolesis”, which increases thrombopoiesis and intercellular exchange between neutrophils and Mks (Elife 8:e44031) and frequently occurs in diseases associated with myelofibrosis, such as MPN (Blood 104:3573) and grey platelet syndrome (Blood 124:3624). To examine whether mutated neutrophils affect platelet production, we transferred wild-type or JAK2 V617F+ senescent neutrophils and found that JAK2 V617F+ mutated (but not WT) senescent neutrophils increased Mk emperipolesis and circulating platelets. Supervised RNA-seq analysis of CD24 hi JAK2 V617F+ human neutrophils highlighted their pro-inflammatory signature. Furthermore, CD24 expression positively correlated with neutrophil degranulation signatures and proteolytic enzymes that activate latent pro-fibrotic TGF-β in Mk, subsequently triggering myelofibrosis. Taken together, these findings suggest that accumulation of senescent neutrophils might lead to their pathogenic interactions with Mks or platelets, which may increase thrombocytosis and myelofibrosis risk in MPN. To test this hypothesis and the therapeutic potential of CD24 inhibition, MPN mice were chronically treated with CD24 blocking antibody. This treatment improved thrombocytosis, decreased platelet-neutrophil complexes, restored normal clearance of mutated senescent neutrophils and reduced emperipolesis levels which, in turn, decreased active TGF-β, ultimately improving myelofibrosis. This study reveals defective neutrophil clearance as a cause of pathogenic microenvironmental interactions that may increase thrombosis and myelofibrosis risk, and postulate CD24 as a candidate target for innate immune checkpoint in myeloid malignancies.

Tissue-Resident Macrophages in the Bone Marrow Comprise a Diverse Cellular Population Derived from Hematopoietic Stem Cells

In the bone marrow, tissue-resident macrophages serve several specific roles including engulfing senescent neutrophils, maintaining hematopoietic stem cells (HSCs), and comprising an important component of the erythroid microenvironment within erythroblastic islands (EBIs). These latter macrophages are thought to “nurture” maturing erythroblasts with iron and cytokines, and to serve as a sink for erythroblast mitochondria, while simultaneously phagocytosing newly formed pyrenocytes. Here we sought to delineate the developmental origin of marrow macrophages, as well as their cellular heterogeneity within the complex cellular milieu of the marrow. Macrophages form an extensive cellular network extending long processes throughout the marrow, which results in cellular injury during dissociation and the presence of macrophage remnants on diverse hematopoietic cell types. We visualized macrophage remnants utilizing imaging flow cytometry to optimize conditions for marrow dissociation and to develop immunophenotyping that removes contaminants to enrich for bona fide macrophages. Importantly, in vitro coculture of marrow macrophages with late-stage erythroid progenitors significantly expanded erythroid output >1.5-fold, consistent with a role of EBI-macrophages in erythroblast maturation. To better define the diversity of macrophages within the marrow we performed single cell transcriptomic analysis using the 10X Genomics Chromium platform of sorted macrophages from mice at steady state and 3 days after induction of anemia by phlebotomy, which resulted in a 50% reduction in hematocrit. Approximately 2/3 of 2,937 steady state and 2,440 anemic marrow cells clustered together in UMAP space with expression consistent with macrophages (Csf1r and genes associated with phagocytosis), while the remaining 1/3 of cells comprised other hematopoietic lineages. Integration of our data with a larger dataset of myeloid cells in the marrow confirmed that our population of macrophages was distinct from other hematopoietic lineages, including monocytes and dendritic cells. Rescaling, dimensionality reduction, and clustering of macrophage cells in UMAP space revealed 8 clusters constituting three “super-clusters”, which we termed A, B, and C. The ‘A’ supercluster was enriched in iron metabolism (ferritin, heme oxygenase, Hfe, ferroportin) and mitochondrial processing (Pink1, Tmem14c) genes. Additionally, the proportion of cells in ‘A’ increased from 25% to 73% of all marrow macrophages in response to anemia. Comparison of anemic and steady state macrophages within supercluster ‘A’ revealed that anemic macrophages were enriched in pathways associated with hypoxia and cellular response to stress, as well as inflammatory, Nf-kb, Nrf1, and Myc signaling. Taken together, these findings support the concept that EBI macrophages are enriched in supercluster ‘A’. In contrast, supercluster ‘B’ was enriched in genes associated with antigen presentation (MHCII and Cd74), while supercluster ‘C’ was enriched in genes associated with immune response (S100a9/a8 and SLC15a3), consistent with a diversity of macrophage subpopulations in the marrow. Tissue-resident macrophages can originate either from HSC-independent or from HSC-derived developmental sources, and additionally can differ in their capacities to self-sustain or be replaced. Using an MDS1-ERCre lineage tracing model with embryonic induction of HSC labeling, we previously determined that adult marrow macrophages are almost entirely HSC-derived (Zhang, et al. 2021). Consistent with these findings, ongoing studies with the FlkSwitch mouse model indicate the bone marrow macrophages are derived from HSCs via Flk2 expressing progenitors. Additionally, induction of HSC labeling in adult MDS1-ERCre mice results in labeling of 75-80% of marrow macrophages. We conclude that a diversity of tissue resident macrophage subpopulations exists in the bone marrow and that the proportion of putative EBI-macrophages rapidly expands in response to acute anemia. Surprisingly, our lineage tracking studies indicate that the vast majority of macrophages in the murine marrow are derived from HSCs, with most, but not all, being replaced on an ongoing basis.

The APOE-TREM2 Axis Mediates Senescent Neutrophils in Prostate Cancer.

Prostate tumor cell secretion of APOE promotes senescence of TREM2+ immunosuppressive neutrophils.

Senescent neutrophils-derived exosomal piRNA-17560 promotes chemoresistance and EMT of breast cancer via FTO-mediated m6A demethylation

Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of malignancies and play key roles in tumor development. However, the role of senescent neutrophils in cancer progression is presently unexplored. Here, we demonstrate that neutrophils display enhanced senescence in breast cancer patients receiving chemotherapy. The senescent neutrophils produce increased number of exosomes, which confer drug resistance to tumor cells in vitro and in vivo. Mechanistically, senescent neutrophils-derived exosomal piRNA-17560 enhances the expression of fat mass and obesity-associated protein (FTO) in breast cancer cells. The upregulation of FTO further strengthens ZEB1 transcripts stability and expression by decreasing N6-methyladenosine (m6A) RNA methylation, leading to chemoresistance and epithelial-mesenchymal transition (EMT) of tumor cells. Clinically, the level of exosomal piR-17560 correlates with poor chemotherapy response in patients with breast cancer. In addition, YTHDF2 is essential for the posttranscriptional regulation of ZEB1 by piRNA-17560/FTO signaling. Senescent neutrophils secret exosomal piR-17560 in a STAT3-dependent manner. Altogether, this study suggests that senescent neutrophils-derived exosomal piR-17560 confers chemoresistance to tumor cells and senescent neutrophils may serve as a potential therapeutic target in breast cancer.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Ionizing irradiation-induced Fgr in senescent cells mediates fibrosis

The role of cellular senescence in radiation-induced pulmonary fibrosis (RIPF) and the underlying mechanisms are unknown. We isolated radiation-induced senescent tdTOMp16 positive mesenchymal stem cells, established their absence of cell division, then measured levels of irradiation-induced expression of biomarkers of senescence by RNA-seq analysis. We identified a Log2 6.17-fold upregulation of tyrosine kinase Fgr, which was a potent inducer of biomarkers of fibrosis in target cells in non-contact co-cultures. Inhibition of Fgr by shRNA knockdown did not block radiation-induced senescence in vitro; however, both shRNA knockdown, or addition of a specific small-molecule inhibitor of Fgr, TL02-59, abrogated senescent cell induction of profibrotic genes in transwell-separated target cells. Single-cell RNA-seq (scRNAseq) analysis of mouse lungs at day 150 after 20 Gy thoracic irradiation revealed upregulation of Fgr in senescent neutrophils, and macrophages before detection of lung fibrosis. Thus, upregulated Fgr in radiation-induced senescent cells mediates RIPF and is a potential therapeutic target for the prevention of this radiation late effect.

Neutrophil and remnant clearance in immunity and inflammation

Neutrophil-centred inflammation and flawed clearance of neutrophils cause and exuberate multiple pathological conditions. These most abundant leukocytes exhibit very high daily turnover in steady-state and stress conditions. Various armours including oxidative burst, NETs and proteases function against pathogens, but also dispose neutrophils to spawn pro-inflammatory responses. Neutrophils undergo death through different pathways upon ageing, infection, executing the intruder's elimination. These include non-lytic apoptosis and other lytic deaths including NETosis, necroptosis and pyroptosis with distinct disintegration of the cellular membrane. This causes release and presence of different intracellular cytotoxic, and tissue-damaging content as cell remnants in the extracellular environment. The apoptotic cells and apoptotic bodies get cleared with non-inflammatory outcomes, while lytic deaths associated remnants including histones and cell-free DNA cause pro-inflammatory responses. Indeed, the enhanced frequencies of neutrophil-associated proteases, cell-free DNA and autoantibodies in diverse pathologies including sepsis, asthma, lupus and rheumatoid arthritis, imply disturbed neutrophil resolution programmes in inflammatory and autoimmune diseases. Thus, the clearance mechanisms of neutrophils and associated remnants are vital for therapeutics. Though studies focused on clearance mechanisms of senescent or apoptotic neutrophils so far generated a good understanding of the same, clearance of neutrophils undergoing distinct lytic deaths, including NETs, are being the subjects of intense investigations. Here, in this review, we are providing the current updates in the clearance mechanisms of apoptotic neutrophils and focusing on not so well-defined recognition, uptake and degradation of neutrophils undergoing lytic death and associated remnants that may provide new therapeutic approaches in inflammation and autoimmunity.

Defensin Promotes the Binding of Lipoprotein(a) to Vascular Matrix

AbstractRetention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that α-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that α-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Defensin Promotes the Binding of Lipoprotein(a) to Vascular Matrix

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Flippase and scramblase for phosphatidylserine exposure.

In various biological processes, phosphatidylserine (PtdSer) that is normally sequestered to the inner leaflet of the plasma membrane (PM) is exposed to the cell surface. When platelets are activated, they expose PtdSer to activate the blood-clotting factors. Cells undergoing apoptosis and senescent neutrophils expose PtdSer that is recognized as an 'eat me' signal by phagocytes for clearance. The PtdSer-exposure and its internalization are mediated by phospholipid scramblases and flippases, respectively. Both have recently been molecularly identified, and their functional mechanism and physiological roles are being elucidated.

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.

Abstract 17039: Neutrophil Subset in Blood Contributes to Acute Oxidant Signals in the Bone Marrow, Which Regulate Innate Immune Responses After Ischemic Tissue Damage

Blood neutrophils are short-lived and have distinct subsets. Aged (senescent) neutrophils are cleared by macrophages in multiple organs including bone marrow (BM), at least, in the steady state. It is unknown, after ischemic tissue damage, how these aged-type neutrophils regulate bone marrow microenvironment and impact on downstream bone marrow responses. In a mouse model of critical limb ischemia, we found that CD62L lo aged-type neutrophils that return to the BM, measured by in vivo circulating cell-labeling, are increased (2.1-fold) after acute limb ischemia, which is corresponded with acute increase in CXCR4, a major chemokine receptor promoting BM returning of blood neutrophils (31%). Interestingly, neutrophil uptake by macrophages is reduced (by 76%) in the BM after limb ischemia, suggesting increased retention of blood-derived CD62L lo neutrophils in BM microenvironment after ischemia. We also found, in the BM, that blood-derived neutrophils have higher reactive oxygen species (ROS) than other neutrophils (61% in MFI) and that limb ischemia indeed increases ROS (34%) and oxidized lipid microparticles (61%) in BM plasma in a Nox2 NADPH oxidase-dependent manner. These suggest that increased returning and retention of blood-derived CD62L lo neutrophils in the BM may contribute to oxidant signals in the BM after limb ischemia. Indeed, using aged neutrophils generated ex vivo and adoptive transfer of these cells, we found that aged neutrophils modulate BM innate immune responses after limb ischemia through ROS—reduced activation of hematopoietic stem progenitor cells (50%); increased acute activation of myeloid progenitor cells (4%); and increased acute but inhibited later Ly6C hi inflammatory monocytes/macrophages in the ischemic tissue (72% and 45%, respectively)—resulting in improved tissue recovery from ischemic damage (2.3-fold). In summary, an aged-type neutrophil subset in blood contributes to acute oxidant signals in the BM, which coordinate innate immune responses to promote tissue recovery from ischemic damage.

CXCR4, the master regulator of neutrophil trafficking in homeostasis and disease.

BackgroundChemokines play a critical role in orchestrating the distribution and trafficking of neutrophils in homeostasis and disease.ResultsThe CXCR4/CXCL12 chemokine axis has been identified as a central regulator of these processes.ConclusionIn this review, we focus on the role of CXCR4/CXCL12 chemokine axis in regulating neutrophil release from the bone marrow and the trafficking of senescent neutrophils back to the bone marrow for clearance under homeostasis and disease. We also discuss the role of CXCR4 in fine‐tuning neutrophil responses in the context of inflammation.

Lupus-like autoimmune disease caused by a lack of Xkr8, a caspase-dependent phospholipid scramblase

Apoptotic cells expose phosphatidylserine (PtdSer) on their cell surface and are recognized by macrophages for clearance. Xkr8 is a scramblase that exposes PtdSer in a caspase-dependent manner. Here, we found that among the three Xkr members with caspase-dependent scramblase activity, mouse hematopoietic cells express only Xkr8. The PtdSer exposure of apoptotic thymocytes, splenocytes, and neutrophils was strongly reduced when Xkr8 was absent. While wild-type apoptotic lymphocytes and neutrophils were efficiently engulfed in vitro by phagocytes expressing Tim4 and MerTK, Xkr8-deficient apoptotic cells were hardly engulfed by these phagocytes. Accordingly, the number of apoptotic thymocytes in the thymus and neutrophils in the peritoneal cavity of the zymosan-treated mice was significantly increased in Xkr8-deficient mice. The percentage of CD62Llo senescent neutrophils was increased in the spleen of Xkr8-null mice, especially after the treatment with granulocyte colony-stimulating factor. Xkr8-null mice on an MRL background showed high levels of autoantibodies, splenomegaly with high levels of effector CD4 T cells, and glomerulonephritis development with immune-complex deposition at glomeruli. These results indicate that the Xkr8-mediated PtdSer exposure in apoptotic lymphocytes and aged neutrophils is essential for their clearance, and its defect activates the immune system, leading to lupus-like autoimmune disease.

Aging of Hematopoietic Stem Cells Is Driven By Regional Specialization of Marrow Macrophages

While hematopoietic stem cells (HSCs)-intrinsic effects of aging have been explored, less is known about how HSC support is altered by the aged bone marrow microenvironment (BMME). To assess the role of the BMME in HSC aging, we compared the BMME in young (6-12 weeks) and aged (20-24 months) male mice and young (<50 years old; YO) and aged (>50 YO) human volunteers. Aged mice had remodeling of the BMME, with expansion of the marrow cavity and vascular volume compared to young mice. BMME constituents were redistributed within two distinct anatomic regions, namely endosteal bone-associated (BA) and marrow-associated (MA) cells. BA cells in aged mice contained fewer phenotypic mesenchymal/osteoblastic progenitors, with reduction in their ability to constitute colony forming units (CFUs). CFU loss was also observed in aged human volunteers. Aged murine MA had significant expansion of dysfunctional mesenchymal stem cells (MSCs) and activated macrophages (MΦ). Increased MΦ were also detected in aged human marrows.Following this in vivo characterization, we developed an ex vivo co-culture system to determine if aged murine BMME cells could impart aging characteristics to young HSCs. Young murine HSCs co-cultured with aged MA cells acquired phenotypic properties of aged HSCs, including increased CD41+ expression. Single cell RNA sequencing of Long Term-HSCs (LT-HSCs) from young and aged mice also identified upregulation of integrin-β3 (CD61) as a novel marker of aged LT-HSCs. Subsequent flow cytometry analysis confirmed the increase in CD61+ expression in vivo in aged HSCs. Importantly, aged MA - but not BA cells - also increased CD61+ expression in young HSCs ex vivo, highlighting the region-specific remodeling of the BMME that occurs with age.We then used a reductionist approach to identify targetable cellular and molecular regulators of the region-specific BMME-induced HSC aging. CD45+ and Ter119+ depletion in aged MA cells did not induce CD41+ expression in young HSCs, suggesting that a critical BMME component responsible for non-cell-autonomous HSC aging is present within the hematopoietic pool. Since marrow MΦ can regulate HSCs, we co-cultured aged MA MΦ with young MA and found that aged MΦ were sufficient to increase CD41+ expression in young HSCs. The addition of aged MΦ also expanded young MSCs, demonstrating that MΦ orchestrate both BMME remodeling and HSC aging.We next aimed to explore mechanisms by which aged MA MΦ impart aging characteristics to HSCs. Transcriptional analysis of murine MA MΦ demonstrated an increase in inflammatory activation in aged mice compared to young mice. This finding was also present in aged human MΦs. Among the inflammatory signals, interleukin-1β (IL-1β) was identified to be necessary and sufficient to mediate the aging effect of aged MA MΦ on young HSCs. Transcriptional analysis also revealed downregulation of phagocytic programs in aged MA MΦ compared to young MA MΦ. Supporting the transcriptional data, aged MA MΦs cultured in vitro demonstrated impaired ability to engulf senescent neutrophils compared to young MA MΦ. Bone marrow MΦ continuously remove large quantities of senescent neutrophils through phagocytosis, a process also known as efferocytosis. Complementing the in vitro findings, in vivo testing demonstrated that young MA MΦ are primarily responsible for engulfing senescent neutrophils and that aged MA MΦ had reduced engulfment of senescent neutrophils. No phagocytic defect was identified in aged BA MΦ, highlighting the regionalization of MΦ function within the BMME that is differentially impacted with age. Consistent with the systemic impact of the efferocytic defect of aged MA MΦ, aged mice had increased levels of circulating senescent neutrophils and. Moreover, neutrophils from aged mice had increased caspase-1 activity, a signal required for IL-1β activation.Together, these data provide evidence that aging differentially remodels two anatomically distinct BMMEs. Regional specialization of marrow MΦ was differentially impacted by aging and induced aging characteristics in HSCs. We propose that impaired removal of senescent neutrophils by aged MA MΦ increases IL-1β production, leading to local inflammation and disrupted BMME and HSC function in aged mice. Strategies aimed at restoring healthy efferocytic activity as well as diminishing IL-1β production or function could therefore reduce the aging effect on HSCs by rejuvenating the BMME. DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Mechanisms of Neutrophil Release from the Bone Marrow

Neutrophils are an essential component of the innate immune response and a major contributor to inflammation. Consequently, neutrophil homeostasis in the blood is tightly regulated. This is achieved by balancing neutrophil production and release from the bone marrow with clearance of senescent neutrophils from the circulation. Here, we highlight recent studies elucidating the signals that regulate neutrophil homeostasis. Constitutive chemokine expression by bone marrow stromal cells plays a key role in regulating neutrophil egress. Specifically, CXCL12 from CXCL12-abundant reticular (CAR) cells and possibly endothelial cells serves to retain neutrophils in the bone marrow. Conversely, CXCL1/2 expression from endothelial cells may promote neutrophil egress. Consistent with these observations, gain-of-function mutations of CXCR4 (the major receptor for CXCL12) or loss-of-function mutations of CXCR2 (the major receptor for CXCL1/2) are associated with myelokathexis in humans. G-CSF promotes neutrophil release from the bone marrow, in large part, by decreasing CXCL12 expression in bone marrow stromal cells, while increasing CXCL2 expression in bone marrow endothelial cells. CXCL12 production from bone marrow stromal cells may be a target of a feedback loop involving the clearance of senescent neutrophils. Specifically, macrophage engulfment of senescent neutrophils in the bone marrow attenuates CXCL12 expression, thereby facilitating neutrophil egress. Recent data suggest that signals from gut microbiota may play a significant role in regulating neutrophil homeostasis. In particular, mice raised under germ-free conditions display marked neutropenia. Gut microbiota appear to regulate neutrophil homeostasis, at least in part through toll-like receptor signaling. These data suggest new pathways that might be targeted therapeutically to modulate neutrophil number in the blood. Disclosures:No relevant conflicts of interest to declare.

Neutrophil Homeostasis and Periodontal Health in Children and Adults

This review summarizes the current state of knowledge on neutrophil basic biology and discusses how the breakdown of neutrophil homeostasis affects periodontal health. The homeostasis of neutrophils is tightly regulated through coordinated bone marrow production, release into the circulation, transmigration to and activation in peripheral tissues, and clearance of senescent neutrophils. Dysregulation of any of these homeostatic mechanisms at any age can cause severe periodontitis in humans and animal models. Accordingly, both impaired and excessive neutrophil activity (in terms of numbers or immune function) can precipitate periodontitis. Neutrophil defects of congenital origin (e.g., congenital neutropenia, leukocyte adhesion deficiency, and Chediak-Higashi syndrome) are associated with cutaneous and systemic infections and early-onset forms of periodontitis affecting both the primary and permanent dentitions of children. However, the strong association between congenital neutrophil disorders and early-onset periodontitis is not currently adequately explained mechanistically. This suggests the operation of as-yet-unknown molecular mechanisms, although the available body of evidence leaves no doubt that neutrophils are integral to periodontal tissue homeostasis and health.

Role of cytosolic glucocorticoid receptor and Annexin‐A1 on neutrophil traffic from bone marrow into blood: SDF‐ 1alpha/CXCR‐4/CXCR‐2 axis

AimsInvestigate the role of the glucocorticoid cytosolic receptor (GCR) and ANXA1 on neutrophil traffic between bone marrow (BM) and blood (PB), focusing on SDF1alpha/CXCR‐4/CXCR2 axis.MethodsBM perfusate and PB were collected from ANXA1‐ null (ANXA1−/−) and Balb/C wild‐type mice. Balb/C were pretreated with the GCR antagonist (RU38486; RU). Leukocyte numbers, CXCR‐2, CXCR‐4 and ANXA1 expressions, SDF‐1alpha and G‐CSF levels, chemotaxis, and phagocytosis of senescent neutrophils by BM macrophages were quantified.ResultsRU and ANXA1−/− presented accelerated granulocyte maturation in BM and neutrophilia, and ANXA1−/− showed increased number of mature granulocytes in BM. ANXA1 expression on BM neutrophils is reduced in RU. RU and ANXA1−/− presented increased CXCR‐4+ and reduced CXCR‐2+ cells in BM and PB, respectively. These cells presented reduced migration to SDF‐1alpha. BM SDF‐1‐alpha levels were reduced in ANXA1−/− and BM GCSF levels were reduced in RU and ANXA1−/−. BM macrophages collected from RU or ANXA1−/− phagocyted lesser number of senescent neutrophils.ConclusionEG, via GCR, and ANXA1 modulates the granulocyte maturation, neutrophil locomotion between BM/PB/BM and the phagocytosis of senescent neutrophils by BM macrophages.