Regulating Neutrophil Survival Research Articles

Neutrophilia in severe asthma is reduced in Ormdl3 overexpressing mice.

Genome-wide association studies have linked the ORM (yeast)-like protein isoform 3 (ORMDL3) to asthma severity. Although ORMDL3 is a member of a family that negatively regulates serine palmitoyltransferase (SPT) and thus biosynthesis of sphingolipids, it is still unclear whether ORMDL3 and altered sphingolipid synthesis are causally related to non-Th2 severe asthma associated with a predominant neutrophil inflammation and high interleukin-17 (IL-17) levels. Here, we examined the effects of ORMDL3 overexpression in a preclinical mouse model of allergic lung inflammation that is predominantly neutrophilic and recapitulates many of the clinical features of severe human asthma. ORMDL3 overexpression reduced lung and circulating levels of dihydrosphingosine, the product of SPT. However, the most prominent effect on sphingolipid levels was reduction of circulating S1P. The LPS/OVA challenge increased markers of Th17 inflammation with a predominant infiltration of neutrophils into the lung. A significant decrease of neutrophil infiltration was observed in the Ormdl3 transgenic mice challenged with LPS/OVA compared to the wild type and concomitant decrease in IL-17, that plays a key role in the pathogenesis of neutrophilic asthma. LPS decreased survival of murine neutrophils, which was prevented by co-treatment with S1P. Moreover, S1P potentiated LPS-induced chemotaxis of neutrophil, suggesting that S1P can regulate neutrophil survival and recruitment following LPS airway inflammation. Our findings reveal a novel connection between ORMDL3 overexpression, circulating levels of S1P, IL-17 and neutrophil recruitment into the lung, and questions the potential involvement of ORMDL3 in the pathology, leading to development of severe neutrophilic asthma.

Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation.

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease.

Regulation of cancer playmaker survival: Increased neutrophil life span by the action of cancer derived a2 V-ATPase

Abstract Pro-tumorigenic tumor associated neutrophils (TAN) have a long life span and are playmakers “supporter” in cancer progression. Identifying cancer associated factors that regulate neutrophil survival will provide new targets for cancer therapy. A peptide (a2NTD) derived from a2 vacuolar ATPase (a2V) in cancer cells promotes the neutrophil pro-tumorigenic properties that enhance angiogenesis and cancer invasion. Here, Immunohistochemical analysis of different human cancer tissues demonstrated a positive correlation between a2NTD expression and neutrophil count with lung cancer showing the highest correlation. Tissues that are heavily infiltrated by neutrophils are either poorly or moderately differentiated that indicates highly aggressive tumors. Furthermore, treatment of freshly isolated neutrophils from blood of healthy volunteers with recombinant a2NTD led to 2 fold increase in neutrophil survival. This enhancement of survival was dependent on NF-kB activation and partially dependent on TLR-2 activation. Further analysis revealed that a2NTD treatment increased the expression of anti-apoptotic proteins as Bcl-xL, Bcl-2-A1 and decreased the expression of pro-apoptotic proteins as Bax, Apaf-1 and caspase-3,-6, -7. Furthermore, a2NTD treatment stabilized the mitochondrial membrane potential and preserved the Cyt-C in the mitochondria that led to decreased caspase-9 activation. Autocrine secretion of pro-survival cytokines as TNF-a and IL-8 from a2NTD treated cells participated in this increased survival. These data provide evidence that cancer associated a2V is regulating the apoptosis of pro-tumorigenic neutrophils by influencing the intrinsic apoptosis pathway.

Inhibition of the sphingosine-1-phosphate pathway promotes the resolution of neutrophilic inflammation.

Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.

Abstract 130: Cancer teammate survives longer: Role of cancer-associated a2 V-ATPase in regulation of neutrophil survival

Abstract Tumor associated neutrophils (TAN) represent a significant portion of tumor leukocytes and accumulate in many types of cancer. TAN can survive for weeks in cancer tissues and have been proposed as key mediator of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity. Identifying cancer associated factors that promote the survival and protumorigenic properties of neutrophils will provide new targets for cancer therapy. Cancer cells secrete a peptide from the Nterminal domain of a2isoform vacuolar ATPase (a2NTD) that promotes the protumorigenic properties of neutrophils that enhances angiogenesis and tumor cell invasiveness. The present study attempts to decipher the role of a2NTD in the regulation of neutrophil apoptosis. The treatment of human neutrophils with recombinant a2NTD led to 2.1 fold increase in the percentage survival of neutrophils (P < 0.001). a2NTD treatment activates NF- kBp65 by activating reactive oxygen species (ROS) generation in neutrophils. Activation of NF- kB pathway leads to upregulation of the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL, c-Flip, G-CSF and downregulates the pro-apoptotic factors such as BAX. Parthenolide; NF-kB inhibitor, negated the effect of a2NTD on enhancing neutrophil survival confirming the involvement of NF-kB pathway in the a2NTD mediated increase of neutrophil survival. Further analysis showed that a2NTD treatment stabilizes the neutrophil mitochondrial membrane potential and decreases Bax translocation to the mitochondria that was associated with a decrease in caspase 9 and caspase 3 activity. Since there is a direct link between the intrinsic and the extrinsic pathways of apoptosis, our results showed that there was no change in the Fas and Fas ligand expression upon a2NTD treatment. Further analysis would uncover the effect of a2NTD treatment on the extrinsic pathway of apoptosis in neutrophils. Together, our results demonstrate a novel role of the cancer associated a2-vacuolar ATPase on retarding the mitochondrial death pathway in neutrophils by the action of a2NTD. Citation Format: Safaa A. Ibrahim, Arpita Kulshrestha, Gajendra K. Katara, Manoranjan Sahoo, Kenneth D. Beaman. Cancer teammate survives longer: Role of cancer-associated a2 V-ATPase in regulation of neutrophil survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 130.

Role of neutrophils in equine asthma

Neutrophilic bronchiolitis is the primary lesion in asthma-affected horses. Neutrophils are key actors in host defense, migrating toward sites of inflammation and infection, where they act as early responder cells toward external insults. However, neutrophils can also mediate tissue damage in various non-infectious inflammatory processes. Within the airways, these cells likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by releasing pro-inflammatory mediators, including the cytokines interleukin (IL)-8 and IL-17, neutrophil elastase, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). The mechanisms that regulate neutrophil functions in the tissues are complex and incompletely understood. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task achieved through a complex network of mechanisms that regulates neutrophil survival. The discovery and development of compounds that can help regulate ROS, NET formation, cytokine release, and clearance would be highly beneficial in the design of therapies for this disease in horses. In this review, neutrophil functions during inflammation will be discussed followed by a discussion of their contribution to airway tissue injury in equine asthma.

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival

AbstractThe lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3. Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis.

Delayed neutrophil apoptosis is regulated by cancer associated a2 isoform vacuolar ATPase

Abstract Neutrophils are short life span cells but they can live for weeks in cancer tissues and promote tumor progression. Identifying cancer derived factors that enhance neutrophil survival will provide new targets for cancer therapy. A peptide from the N-terminal domain of a2- isoform vacuolar ATPase (a2NTD) is specifically secreted from cancer cells and promotes the neutrophil protumorigenic properties which in turn, enhance tumor progression. This gives a2NTD the propensity to regulate neutrophil survival. In fact the treatment of human neutrophils with recombinant a2NTD led to 2.1 fold increase in the percentage survival of neutrophils (P < 0.001). a2NTD treatment modulates different apoptosis pathways. Executive pathway; a2NTD treated neutrophils (a2Neuφ) showed a significantly decreased gene expression of caspase-3, -6, -7, that corresponded with decreased activity of these caspases. Intrinsic pathway; a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL and downregulates the pro-apoptotic factors; BAX and Apaf-1. Extrinsic pathway; a2NTD treatment dowenregulats the gene expression and the activity of caspase-8 and upregulates the gene expression of c-Flip. a2NTD treatment also stimulates reactive oxygen species generation that activate NF-kB p65 in neutrophils. Moreover, NF-kB inhibition negated that promoted neutrophil survival that was associated with downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Thus, a2NTD enhances neutrophil survival by activating the NF-kB pathway in neutrophils. Together, these data demonstrate a novel role of the cancer associated a2-vacuolar ATPase on regulating neutrophil survival by the action of a2NTD.

Abstract 728: Enhancement of survival and migration of neutrophils by tumor associated a2 isoform vacuolar ATPase

Abstract Tumors associated neutrophils (TAN) regulate the tumor environment by directly promoting tumor progression and represent a potential therapeutic target for cancer treatment. Identifying tumor associated factors that promote neutrophil recruitment and survival will uncover new targets for cancer therapy. In tumor cells, a secreted peptide from the N­terminal domain of a2­ isoform vacuolar ATPase (a2NTD) promotes the protumorigenic properties of neutrophils which in turn, enhance tumor progression. In order to investigate the regulatory role of a2NTD on neutrophils survival, we stimulated freshly isolated neutrophils from healthy volunteers with recombinant a2NTD. a2NTD treatment delayed apoptosis in neutrophils and led to 2.1 fold increase in the percentage survival (P < 0.001). In addition, a2NTD treated neutrophils (a2Neuφ) showed a significant decreased gene expression of caspase-3, -6, -7 and -8, that was associated with decreased activity of these caspases. Further analysis of the apoptosis related molecules revealed that a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL, c-FLIP, G-CSF and downregulates the gene expression of pro-apoptotic factors; BAX and Apaf-1. Since NF-kB pathway is an important regulator of apoptosis, here we showed by immunofluorescence analysis that a2NTD induces NF-kB p65 activation in neutrophils by nuclear translocation in a reactive oxygen species generation dependent manner. Additionally, treatment of a2Neuφ with specific NF-kB inhibitor; parthenolide showed a significant abrogation in neutrophil survival as well as downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Suggesting that a2NTD enhances neutrophil survival by activating NF-kB pathway in neutrophils. Interestingly, live imaging revealed that a2NTD stimulates neutrophil polarization which was confirmed by the increased assembly of F-actin. Also, flow cytometry analysis revealed that a2NTD treatment stimulates surface expression and activation of Mac-1 integrin. Moreover, important regulators of neutrophil migration; FAK and Src Kinases were activated in a2Neuφ. Previous studies showed that IL-8; potent neutrophil chemoattractant is secreted by neutrophils. Here, we showed that a2NTD treatment led to upregulation of the gene expression and the secreted protein levels of IL-8 in a time dependent manner. This enhanced secretion of IL-8 was NF-kB dependent. Furthermore, to evaluate the influence of a2NTD on neutrophil migration we did trans-well migration assay. a2NTD stimulation caused 2.8 fold increase in neutrophil migration which decreased to 1.5 fold upon neutralizing IL-8 (P < 0.05). Together, these data demonstrate a novel role of the tumor associated a2-vacuolar ATPase on regulating neutrophil survival and migration, by the action of a2NTD. Citation Format: Safaa A. Ibrahim, Arpita Kulshrestha, Mukesh K. Jaiswal, Gajendra K. Katara, Magdy A. Amin, Kenneth D. Beaman. Enhancement of survival and migration of neutrophils by tumor associated a2 isoform vacuolar ATPase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 728.

The anti-apoptotic Bcl-2 family protein A1/Bfl-1 regulates neutrophil survival and homeostasis and is controlled via PI3K and JAK/STAT signaling

Neutrophil granulocytes are innate effector cells of the first line of defense against pyogenic bacteria. Neutrophil lifespan is short, is prolonged by pro-inflammatory stimuli, controls functionality of the cells and can determine tissue damage. Experimental analysis of primary neutrophils is difficult because of their short lifespan and lack of possibilities of genetic manipulation. The Hoxb8 system of neutrophil differentiation from immortalized progenitor cells offers the advantage of unlimited production of neutrophils in vitro as well as easy genetic modification. We here use this system to analyze the role of the poorly characterized anti-apoptotic B-cell lymphoma protein 2 (Bcl-2) family member A1/Bfl-1 (Bcl-2-related protein A1) for survival and homeostasis of neutrophils and of neutrophil progenitors. Low constitutive mRNA and protein expression of A1 was detected, while A1 was transiently upregulated early during differentiation. Pro-inflammatory stimuli caused strong, mainly transcriptional, A1 upregulation, in contrast to posttranscriptional regulation of Mcl-1 (induced myeloid leukemia cell differentiation protein). Inhibitor studies showed that phosphoinositide-3 kinase (PI3K)/Akt and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is required for A1 expression and survival of progenitors and mature neutrophils. ShRNA-mediated constitutive A1 knockdown (KD) impaired maintenance of progenitors. ShRNA experiments further showed that A1 was required early during neutrophil differentiation as well as in mature neutrophils upon pro-inflammatory stimulation. Our data further indicate differential regulation of the two anti-apoptotic proteins A1 and Mcl-1. Relevant findings were confirmed in primary human neutrophils. Our data indicate that A1, in addition to the well-established Mcl-1, substantially contributes to neutrophil survival and homeostasis. A1 may thus be a promising target for anti-inflammatory therapy.

Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

G-CSF activation of AKT is not sufficient to prolong neutrophil survival

Neutrophils play an important role in the innate immune response against bacterial and fungal infections. They have a short lifespan in circulation, and their survival can be modulated by several cytokines, including G-CSF. Previous studies have implicated AKT as a critical signaling intermediary in the regulation of neutrophil survival. Our results demonstrate that G-CSF activation of AKT is not sufficient to prolong neutrophil survival. Neutrophils treated with G-CSF undergo apoptosis, even in the presence of high levels of p-AKT. In addition, inhibitors of AKT and downstream targets failed to alter neutrophil survival. In contrast, neutrophil precursors appear to be dependent on AKT signaling pathways for survival, whereas high levels of p-AKT inhibit proliferation. Our data suggest that the AKT/mTOR pathway, although important in G-CSF-driven myeloid differentiation, proliferation, and survival of early hematopoietic progenitors, is less essential in G-CSF suppression of neutrophil apoptosis. Whereas basal AKT levels may be required for the brief life of neutrophils, further p-AKT expression is not able to extend the neutrophil lifespan in the presence of G-CSF.

Mechanisms regulating neutrophil survival and cell death

Neutrophils not only play a critical role as a first line of defense against bacteria and fungi infections but also contribute to tissue injury associated with autoimmune and inflammatory diseases. Neutrophils are rapidly and massively recruited from the circulation into injured tissues displaying an impressive arsenal of toxic weapons. Although effective in their ability to kill pathogens, these weapons were equally effective to induce tissue damage. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task mainly achieved through a complex network of mechanisms, which regulate neutrophil survival. Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis although new forms of cell death have been characterized over the last few years. The lifespan of neutrophils can be dramatically modulated by a large variety of agents such as cytokines, pathogens, danger-associated molecular patterns as well as by pharmacological manipulation. Recent findings shed light about the complex mechanisms responsible for the regulation of neutrophil survival in different physiological, pathological, and pharmacological scenarios. Here, we provide an updated review on the current knowledge and new findings in this field and discuss novel strategies that could be used to drive the resolution of neutrophil-mediated inflammatory diseases.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

The bone marrow neutrophil reserve is regulated by serpinB1 and cathepsin G through a cell autonomous and caspase-independent death pathway (172.32)

Abstract The bone marrow (BM) neutrophil reserve is a large pool of mature neutrophils that can be rapidly mobilized to the circulation and recruited to tissues in response to danger signals. We have previously shown that serpinB1-/- mature neutrophils have a survival defect that leads to a considerable reduction of the BM reserve and failure to clear bacterial infection. SerpinB1 is one of the best inhibitors of neutrophil serine proteases including elastase (NE) and cathepsin G (CG). Here, BM chimera showed that serpinB1 deficiency in hematopoietic cells was necessary and sufficient to reproduce the bone marrow neutropenia of serpinB1-/- mice. We found that CG, but not NE, was responsible for the BM neutropenia of serpinB1-/- mice. Mice deficient in both CG and serpinB1 had a normal neutrophil reserve, whereas NE-deficiency did not rescue the BM defect of serpinB1-/- mice. Neutrophil survival was increased by pan-caspase inhibitor Q-VD-OPh in both wild-type and serpinB1-/- neutrophils in vitro compared to untreated cells. However, survival was significantly lower in serpinB1-/- neutrophils (35%) vs. wild type (80%) after 72hr. This survival defect in vitro was rescued by CG deficiency. Furthermore, neither transgenic over expression of anti-apoptotic factor BCL-2 nor G-CSF treatment of serpinB1-/- mice rescued CG-mediated neutrophil death. We conclude that CG and serpinB1 regulate neutrophil survival through a proteolytic process distinct from classical apoptotic pathways.

Targeting cytosolic proliferating cell nuclear antigen in neutrophil-dominated inflammation.

New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. The nuclear-to-cytoplasmic relocalization occurred during granulocytic differentiation and is dependent on a nuclear export sequence thus strongly suggesting that PCNA has physiologic cytoplasmic functions. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.

IL-17A as a regulator of neutrophil survival in nasal polyp disease of patients with and without cystic fibrosis

Nasal polyps in adults are characterized by a chronic inflammation of the upper airways and by the preferential activation of Th2 cells. In contrast, IL-17 producing Th17 cells dominate the inflammation in nasal polyps of cystic fibrosis (CF) patients. MethodIL-17A, IL-5, IL-6, IL-8, IL-1β, ECP, MCP-1 and myeloperoxidase expression was determined in tissue homogenates of nasal polyps of non-CF and CF patients and controls. The cellular source of IL-17A was determined by immuno-histochemistry and FACS analysis. The functional role of IL-17A in the survival of neutrophils from CF and non-CF patients was tested. ResultsA significant upregulation of IL-17A and myeloperoxidase could be observed in nasal polyps from CF-patients. The cellular sources of IL-17A in nasal polyps were mainly T-lymphocytes. IL-17A was able to modulate the survival of neutrophils in nasal polyps from non-CF patients; however the survival of neutrophils in CF patients was independent of IL-17A. ConclusionThe present study shows that IL-17A has an impact on neutrophil survival in adult nasal polyp disease, but not in nasal polyps from CF patients.

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice

The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.

Absence of functional TLR4 impairs response of macrophages afterCandida albicansinfection

Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF-α, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1β production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1β and TNF-α. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection.

New insights into the mechanisms controlling neutrophil survival

Neutrophil survival is regulated by a complex convergence of different pathways. The present review analyzes these pathways and discusses how neutrophil survival is modulated during the course of inflammatory reactions. Although apoptosis appears to be the predominant cell death pathway in the neutrophil, recent data reveal that neutrophil survival is also regulated by a number of nonconventional pathways including NETosis, autophagic cell death, and other less characterized mechanisms. This supports an even more complex picture of the mechanisms involved in the regulation of neutrophil survival than previously thought. The control of neutrophil survival is central to homoeostasis and resolution of inflammation. Cell death is usually discussed dichotomously in terms of apoptosis or necrosis. There are two main pathways responsible for the stimulation of apoptosis; a death receptor pathway triggered by Fas, tumor necrosis factor alpha, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and a mitochondrial pathway stimulated by a number of stressors such as DNA damage, growth factor deprivation, and chemotherapy drugs. Nonconventional pathways of neutrophil death include NETosis and autophagic cell death as well as a number of poorly characterized mechanisms. Understanding the integrated pathways responsible for the control of neutrophil survival holds therapeutic promise in infectious and inflammatory diseases.