Neutrophil Homeostasis Research Articles

Inhibitors of CXC chemokine receptor type 4

The CXC chemokine receptor type 4 (CXCR4), which is a G-protein coupled receptor, and its ligand CXCL12 play an important role in neutrophil homeostasis and inflammation. This review focuses on involvement of the CXCL12/CXCR4 axis in inflammation and different inflammatory diseases and depicts that blocking CXCR4 is an attractive therapeutic strategy. Binding of CXCL12 to CXCR4 retains immature neutrophils in the bone marrow and also participates in leukocyte recruitment into inflamed tissue. The CXCL12/CXCR4 axis is also involved in several inflammatory processes and diseases including the WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome, HIV, autoimmune disorders, ischemic injury, and pulmonary fibrosis. Based on these findings, blocking CXCR4 seems to be a therapeutic strategy in inflammatory diseases. Several promising CXCR4 antagonists are in different stages of development and clinical trials. Currently, only plerixafor (AMD3100) has been approved for short-term application.

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.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4-CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4-CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.

Neutrophils produce interleukin-17B in rheumatoid synovial tissue

T helper 17 (Th17) and mast cells produce IL-17A in RA and critically contribute to the pathogenesis of RA. However, the complete IL-17 cytokine profile in RA is unknown. The aim of the study was to systematically study the expression of IL-17 family cytokines in RA. The expression of all IL-17 cytokines in RA synovium and pannus as well as in the synovium of OA was determined using quantitative RT-PCR (qRT-PCR). IL-17A and IL-17B were immunostained. Peripheral blood neutrophils were analysed for IL-17B. The effect of IL-17B alone or in combination with TNF-α was tested in vitro on fibroblasts and endothelial cells. In all tissues IL-17B was the most expressed IL-17 family cytokine, found in lining but most strongly expressed in human neutrophil elastase containing polymorphonuclear cells. This pattern was distinct from that of IL-17A, which was found in mast cell tryptase immunoreactive cells. Circulating neutrophils contained IL-17B, verifying the in vivo results. Fibroblasts up-regulated the expression of IL-17RB, a putative receptor of IL-17B, after TNF-α stimulation. IL-17B significantly enhanced TNF-α-induced production of G-CSF and IL-6 in fibroblasts. IL-17B, which is present in synovium, may contribute to the pathogenesis of RA. IL-17B can enhance the effects of TNF-α on the production of cytokines and chemokines that control immune cell trafficking and neutrophil homeostasis in the inflamed tissues.

Modulation of Neutrophil Development and Homeostasis

Neutrophils are highly motile phagocytic cells that constitute the first line of defense of the innate immune system. Unlike the other innate immune cells, mature neutrophils fully equipped with an armory of granules are short lived cells which are differentiated and programmed from hematopoietic stem cells in the bone marrow. In blood, mature or nearly mature cells are devoid of proliferative potential. While the killing mechanisms of neutrophils have been the research focus in the past, attention was then directed toward granulocytopoiesis by the recent studies about the specific functional states and exact regulatory mechanisms in neutrophil development and homeostasis. Here, we focus on the recent findings regarding the mechanisms that control the generation and homeostasis of neutrophils.

Platelet CXCL7 and CXCL4 inhibit chemokine scavenging and improve innate immunity to bacterial infection (P1317)

Abstract We recently demonstrated the critical role of CXCL5 in chemokine scavenging, neutrophil homeostasis and host defense to bacterial pneumonia. Here we generated Cxcl7-/- mice and found that Cxcl7 deletion also disrupted expression of CXCL4 and CXCL5, but did not affect expression of two other neutrophil chemokines, CXCL1 and CXCL2. Cxcl4, 7, 5 form a conserved genomic locus (14.5 kb in length) in both human and mice, suggesting their importance during evolution. Heparin treatment of blood led to over 10-fold more plasma CXCL4, but comparable plasma CXCL7 as compared to PBS treatment. In addition, the inactive PPBP form of murine CXCL7 showed considerable binding affinity with red cell DARC. We further delineated the role of CXCL7 and CXCL4 by comparing the phenotypes of Cxcl7-/- and Cxcl5-/- mice. CXCL7 and CXCL4 inhibit chemokine scavenging not only in blood, but also in tissues, and platelet-derived CXCL7 is present in alveolar space and peritoneum. In CXCL1- and nebulized LPS-induced acute lung inflammation models, K. pneumoniae pneumonia and bacteremia models, as compared to Cxcl5-/- mice, Cxcl7-/- mice showed attenuated neutrophil chemokine levels in tissues and blood, decreased neutrophil influx to the lung, and impaired innate immunity. This study is the first to reveal the central role of homeostatic CXCL7 and CXCL4 in increasing chemokine levels in blood and tissues, and potentiating neutrophil transmigration during inflammation and infection.

Myeloid cell-specific TRAF3-deficient mice exhibit normal myeloid cell development and homeostasis (P1196)

Abstract Myeloid cells, including monocytes, macrophages and granulocytes, are crucial players of innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNF receptor superfamily and Toll-like receptors (TLRs), whose signals are transduced by TRAF molecules. In light of the in vitro evidence that TRAF3 is required for TLRs-induced type I interferon production and for CD40-induced IL-12 production in macrophages, we generated myeloid cell-specific TRAF3-/- (M-TRAF3-/-; TRAF3flox/floxLysM+/Cre) mice to investigate the in vivo functions of TRAF3 in myeloid cells. We examined immune cell populations in the bone marrow, spleen, and peritoneal cavity in M-TRAF3-/- mice at the age of 8-12 weeks. No significant differences in the frequencies or numbers of immune cell populations such as B cells, T cells, granulocytes and macrophages were found between M-TRAF3-/- and littermate control (LMC) mice. TRAF3-/- macrophages also expressed similar levels of MHC class II, CD80 and CD86 as compared to LMC macrophages, suggesting that no endogenous activation of macrophages occurs. Our findings indicate that specific ablation of TRAF3 in myeloid cells neither affected myeloid cell maturation nor changed the homeostasis of macrophages and neutrophils. In contrast, activation of macrophages induced by TLRs was altered in the absence of TRAF3. Our ongoing study will further elucidate the in vivo functions of TRAF3 in innate immunity and inflammation.

Impairment of Mitochondrial–Nuclear Cross Talk in Neutrophils of Patients with Type 2 Diabetes Mellitus

Increased leukocyte apoptosis is intrinsically linked to disease patho-physiology, susceptibility to and severity of infections in type 2 diabetes mellitus (T2DM) patients. A consistent defect in neutrophil function is considered central to this increased risk for infections. Although redox imbalance is considered a potential mediator of these associated complications, detailed molecular evidence in clinical samples remains largely undetected. The study consisted of three groups (n=50 each) of Asian Indians: early diagnosed diabetic patients, cases with late-onset diabetic complications and age and gender-matched healthy controls. We evaluated mitochondrial oxidative stress, levels of nuclear DNA damage and apoptosis in peripheral blood neutrophils isolated from T2DM patients. We observed that in both early and late diabetic subjects, the HbA1c levels in neutrophils were altered considerably with respect to healthy controls. Increased oxidative stress observed in both early and late diabetics imply the disentanglement of fine equilibrium of mitochondria-nuclear cross talk which eventually effected the augmentation of downstream nuclear γH2AX activation and caspase-3 expression. It would be overly naïve to refute the fact that mitochondrial deregulation in neutrophils perturbs immunological balance in type 2 diabetic conditions. By virtue of our data, we posit that maneuvering mitochondrial function might offer a prospective and viable method to modulate neutrophil function in T2DM. Nevertheless, similar investigations from other ethnic groups in conjunction with experimental evidences would be a preeminent need. Obviously, our study might aid to comprehend this complex interplay between mitochondrial dysfunction and neutrophil homeostasis in T2DM.

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

AbstractPolymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Although it has long been known that marrow cellularity is inversely correlated with G-CSF levels, the mechanism linking peripheral clearance to production remains unknown. Herein, the feedback response to antibody induced neutropenia is characterized to consist of G-CSF–dependent shifts of marrow hematopoietic progenitor populations including expansion of the lin−/Sca-1+/c-kit+ (LSK) and granulocyte macrophage progenitor (GMP) compartments at the expense of thrombopoietic and red cell precursors. Evidence is provided that positive feedback regulation is independent from commensal germs as well as T, B, and NK cells. However, in vivo feedback is impaired in TLR4−/− and TRIF−/−, but not MyD88−/− animals. In conclusion, steady-state neutrophil homeostasis is G-CSF–dependent and regulated through pattern-recognition receptors, thereby directly linking TLR-triggering to granulopoiesis.

Phosphatase Wip1 negatively regulates neutrophil development through p38 MAPK-STAT1

AbstractNeutrophils are critically involved in host defense and tissue damage. Intrinsic molecular mechanisms controlling neutrophil differentiation and activities are poorly defined. Herein we found that p53-induced phosphatase 1(Wip1) is preferentially expressed in neutrophils among immune cells. The Wip1 expression is gradually up-regulated during the differentiation of myeloid precursors into mature neutrophils. Wip1-deficient mice and chimera mice with Wip1−/− hematopoietic cells had an expanded pool of neutrophils with hypermature phenotypes in the periphery. The in vivo and in vitro studies showed that Wip1 deficiency mainly impaired the developing process of myeloid progenitors to neutrophils in an intrinsic manner. Mechanism studies showed that the enhanced development and maturation of neutrophils caused by Wip1 deficiency were mediated by p38 MAPK-STAT1 but not p53-dependent pathways. Thus, our findings identify a previously unrecognized p53-independent function of Wip1 as a cell type-specific negative regulator of neutrophil generation and homeostasis through limiting the p38 MAPK-STAT1 pathway.

Glycogen synthase kinase-3β inactivation is an intracellular marker and regulator for endotoxemic neutrophilia

Neutrophilia, defined as a large number of neutrophils in the circulating blood, is caused by increased differentiation and survival from activation-induced apoptosis. Regulation of apoptosis is essential for neutrophil homeostasis; however, the molecular signaling that regulates this process needs further investigation. Unlike TLR4 wild-type C3H/HeN mice, TLR4 mutated C3H/HeJ mice were insusceptible to LPS-induced blood neutrophilia. LPS prevented constitutive apoptosis in neutrophils and partly involved a blockade of the mitochondrial pathway including mitochondria transmembrane potential loss, myeloid cell leukemia sequence (Mcl) 1 degradation, and caspase-3 activation. In apoptotic neutrophils, glycogen synthase kinase (GSK)-3β was activated, and inhibiting GSK-3β decreased Mcl-1 degradation and apoptosis. LPS caused p38 MAPK-, JNK-, and PI3K/AKT-mediated Mcl-1 stabilization and prevented apoptosis, and LPS induced GSK-3β inactivation mainly through p38 MAPK and PI3K/AKT. Neutrophils in the neutrophilia showed increased GSK-3β inactivation and Mcl-1 stabilization accompanied by activation of p38 MAPK, JNK, and AKT. Notably, LPS-induced ROS generation can partly facilitate p38 MAPK/JNK/AKT activation to regulate GSK-3β-mediated Mcl-1 stability, apoptosis, and neutrophilia. These results demonstrate that the molecular basis of endotoxemic neutrophilia is through a direct action on neutrophils involving GSK-3β inactivation to prevent constitutive apoptosis.

Current insights into neutrophil homeostasis

Neutrophil granulocytes represent the first immunologic barrier against invading pathogens, and neutropenia predisposes to infection. However, neutrophils may also cause significant collateral inflammatory damage. Therefore, neutrophil numbers are tightly regulated by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Granulocyte colony-stimulating factor (G-CSF) is accepted to be the major determinant of neutrophil production, and G-CSF levels have, soon after its discovery, been described to be inversely correlated with neutrophil counts. A neutrophil sensor, or "neutrostat," has, therefore, been postulated. The prevailing feedback hypothesis was established in adhesion molecule-deficient mice; it includes macrophages and Th17 cells, which determine G-CSF levels in response to the number of peripherally transmigrated, apoptosing neutrophils. Recent work has deepened our understanding of homeostatic regulation of neutrophil granulopoiesis, but there are still inconsistent findings and unresolved questions when it comes to a plausible hypothesis, similar to the feedback control models of red cell or platelet homeostasis.

CXCR2 and CXCL5 Regulate the IL-17/G-CSF axis and Neutrophil Homeostasis (48.15)

Abstract Neutrophil homeostasis is critical for maintaining innate immune surveillance under normal conditions. CXCR2 plays a critical role in neutrophil homeostasis, as Cxcr2-/- mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. We found that neutrophil CXCR2 inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis, while enterocyte-derived CXCL5 in the gut, regulates the IL-17/G-CSF axis and contributes to CXCR2-regulated neutrophil homeostasis. CXCL5-regulated neutrophil homeostasis was completely dependent on G-CSF, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5-/- and Cxcr2-/- mice. Cxcr2-/- mice express IL-17A, and show increased IL-17A-producing cells in the lung, terminal ileum and spleen, which include conventional Th17 cells, γδ T cells, and innate immune cells. Most IL-17 producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment markedly decreased IL-17A and G-CSF expression, neutrophilia and marrow myeloid hyperplasia in Cxcr2-/- mice. These data provide insights regarding the critical role of CXCR2, CXCL5 and commensal bacteria in regulating the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites, which keeps the set point of innate immunity in tight control.

Balancing the books

CXCR2 and CXCL5 control neutrophil homeostasis by downregulating IL-17A and G-CSF.

Regulating neutrophil homeostasis

Regulating neutrophil homeostasis

Cxcr2 and Cxcl5 regulate the IL-17/G-CSF axis and neutrophil homeostasis in mice

Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2(-/-) mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5(-/-) and Cxcr2(-/-) mice. Cxcr2(-/-) mice constitutively expressed IL-17A and showed increased numbers of IL-17A-producing cells in the lung, terminal ileum, and spleen. Most IL-17-producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2(-/-) mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.

Coordinate regulation of neutrophil homeostasis by liver X receptors in mice

The most abundant immune cell type is the neutrophil, a key first responder after pathogen invasion. Neutrophil numbers in the periphery are tightly regulated to prevent opportunistic infections and aberrant inflammation. In healthy individuals, more than 1 × 10⁹ neutrophils per kilogram body weight are released from the bone marrow every 24 hours. To maintain homeostatic levels, an equivalent number of senescent cells must be cleared from circulation. Recent studies indicate that clearance of senescent neutrophils by resident tissue macrophages and DCs helps to set homeostatic levels of neutrophils via effects on the IL-23/IL-17/G-CSF cytokine axis, which stimulates neutrophil production in the bone marrow. However, the molecular events in phagocytes underlying this feedback loop have remained indeterminate. Liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate both lipid metabolic and inflammatory gene expression. Here, we demonstrate that LXRs contribute to the control of neutrophil homeostasis. Using gain- and loss-of-function models, we found that LXR signaling regulated the efficient clearance of senescent neutrophils by peripheral tissue APCs in a Mer-dependent manner. Furthermore, activation of LXR by engulfed neutrophils directly repressed the IL-23/IL-17/G-CSF granulopoietic cytokine cascade. These results provide mechanistic insight into the molecular events orchestrating neutrophil homeostasis and advance our understanding of LXRs as integrators of phagocyte function, lipid metabolism, and cytokine gene expression.

Regulation of neutrophil trafficking 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 highly regulated. Neutrophil number in the blood is determined by the balance between neutrophil production in the bone marrow and release from the bone marrow to blood with neutrophil clearance from the circulation. This review will focus on mechanisms regulating neutrophil release from the bone marrow. In particular, recent data demonstrating a central role for the chemokines CXCL12 and CXCL2 in regulating neutrophil egress from the bone marrow will be discussed.

Dual Roles for Rac2 in Neutrophil Motility and Active Retention in Zebrafish Hematopoietic Tissue

Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Noninvasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling invivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively active CXCR4 mutation in zebrafish warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.