Constitutive Apoptosis Research Articles

Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate.

Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC)cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.

Neutrophil function following treatment of psoriatic arthritis patients with secukinumab: altered cytokine signalling but no impairment of host defence.

Identifying that dysfunction of the IL-23/17 axis underlies PsA has led to the development of effective targeted therapies such as the IL-17A inhibitor secukinumab. As IL-17A stimulates the secretion of neutrophil chemoattractants, such as CXCL8 (IL-8), we examined the effect of secukinumab on neutrophil function in PsA. Nineteen patients with active PsA were treated with secukinumab. Clinical response [PsA Response Criteria (PsARC) and Psoriasis Area and Severity Index (PASI)] and peripheral blood neutrophil function (apoptosis, receptor expression, phagocytosis/killing, chemotaxis and RNA expression) were measured at 12 week intervals for 48 weeks and compared with age- and sex-matched healthy controls. At 12 weeks, 12/16 (75%) patients had a PsARC response (100% at 36 weeks) and 10/14 (71%) achieved a 90% PASI response. At baseline, there were no differences in PsA neutrophil reactive oxygen species generation, constitutive or cytokine-delayed apoptosis, chemotaxis or phagocytosis of opsonized Staphylococcus aureus compared with healthy controls. Similarly, there were no differences in these functions from baseline to 12 weeks of therapy. However, surface levels of CD11b/CD18 and CD63 increased and expression of CD16 decreased during therapy. In addition, in a subgroup of early (12 week) responders to secukinumab, RNA sequencing revealed transcriptome changes predicting down-regulation of cytokine signalling and chemotaxis pathways and up-regulation of de novo gene expression pathways, including translation initiation, mRNA catabolism and translation. Complex changes in the properties of circulating neutrophils occur with secukinumab treatment in PsA that may indicate altered responsiveness to changes in both local and systemic levels of pro-inflammatory cytokines. However, host defence processes of neutrophils were unaltered.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

Treponema pallidum delays the apoptosis of human polymorphonuclear neutrophils through the intrinsic and extrinsic pathways

Treponema pallidum is a “stealth pathogen” responsible for infectious sexually transmitted diseases. Although neutrophils are usually present in skin lesions of early syphilis, the role of these cells in T. pallidum infection has barely been investigated. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis usually accelerates this process. Here, we demonstrated that human polymorphonuclear neutrophils (hPMNs) could phagocytose T. pallidum in vitro. An unexpected discovery was that T. pallidum inhibited hPMNs apoptosis markedly in an opsonin-independent manner. Furthermore, this phenomenon was not affected by bacterial viability, as detected by annexin V, morphology studies, and TUNEL staining. Exploration of the underlying mechanism showed that expression of the cleaved forms of caspase-3, −8, and −9 and effector caspase activity were diminished significantly in T. pallidum-infected hPMNs. T. pallidum also impaired staurosporine- and anti-Fas-induced signaling for neutrophil apoptosis. Of note, these effects were accompanied by inducing the autocrine production of the anti-apoptotic cytokine IL-8. Taken together, our data revealed that T. pallidum could inhibit the apoptosis of hPMNs through intrinsic and extrinsic pathways and provide new insights for understanding the pathogenicity mechanisms of T. pallidum.

Low-density neutrophils in chronic graft versus host disease (cGVHD) are primarily immature CD10- and enhance T cell activation.

Chronic graft-versus-host disease (cGVHD) is a frequent complication of allogeneic haematopoietic stem cell transplantation. Low density neutrophils (LDNs) in autoimmunity, which shares disease features with cGVHD, are proinflammatory, whereas those in cancer and sepsis suppress T cell immunity. Mature LDNs can be distinguished from immature LDNs on the basis of expression of CD10 and suppressive neutrophils can be identified using lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. The functionality of LDNs in cGVHD has not been specifically investigated. Here, we have determined the relative contribution of immature and mature neutrophils to LDNs in cGVHD and assessed whether these were suppressive or potentially proinflammatory. Peripheral blood LDNs and normal density neutrophils (NDNs) from 30cGVHD patients and NDNs from 10healthy controls (HCs) were immunophenotyped by flow cytometry. The ability of LDNs and NDNs to influence T cell proliferation and cytokine production in co-cultures was quantified. To further characterize LDNs, their propensity to undergo constitutive apoptosis and differentiate ex vivo was assessed. LDNs were elevated in cGVHD versus HCs, heterogeneous in phenotype, with a predominance of immature CD10- cells in most patients, but some mature CD10+ LOX-1+ LDNs were also detected. LDNs enhanced autologous T cell proliferation, interleukin (IL)-6 and interferon (IFN)-γ production. LDN, but not NDN, CD10 expression was inversely correlated with LOX-1, which correlated with IL-6 production. LDNs resisted apoptosis and differentiated into antigen-presenting/neutrophil-hybrid-like cells, which co-expressed major histocompatibility complex (MHC) class II HLA-DR and immuno-inhibitory programmed cell death ligand 1 (PD-L1), but did not suppress T cell proliferation. These data suggest LDNs in cGVHD are predominantly immature, proinflammatory and may have pathogenic potential.

Potential Oncogenic Effect of the MERTK-Dependent Apoptotic-Cell Clearance Pathway in Starry-Sky B-Cell Lymphoma

The histological architecture of certain aggressive B-cell lymphomas (prototypically Burkitt's lymphoma, BL) is characterized by a “starry-sky” (SS) appearance. This is caused by tumor-associated macrophages (TAMs), which appear in standard histological preparations as “stars” in a darkly stained “sky” of lymphoma cells. SS-TAMs accumulate in response to constitutive apoptosis in these tumors and are activated by the apoptotic tumor cells to a pro-oncogenic phenotype. The extent to which SS-TAMs contribute to lymphoma growth through responses generated by interactions with apoptotic tumor cells is unknown. Here, we demonstrate a role for the receptor tyrosine kinase, MERTK, in the oncogenic activity of SS-TAMs. We show that MERTK expression is largely restricted to the macrophages of human BL and of murine models of SS B-cell lymphoma and that it is upregulated in SS-TAMs as compared to the germinal center or paracortical macrophages of normal lymph nodes. Our results further demonstrate that MERTK is active in the phagocytosis of apoptotic lymphoma cells by macrophages and, most significantly, that SS lymphoma growth is markedly inhibited in Mertk−/− mice. These results point toward the MERTK apoptotic-cell clearance/response pathway playing a key role in growth of aggressive B-cell lymphoma and identifies MERTK as a novel potential antilymphoma target.

Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma.

S100A8 and S100A9 are important proteins in the pathogenesis of allergy. Asthma is an allergic lung disease, characterized by bronchial inflammation due to leukocytes, bronchoconstriction, and allergen-specific IgE. In this study, we examined the role of S100A8 and S100A9 in the interaction of cytokine release from bronchial epithelial cells, with constitutive apoptosis of neutrophils. S100A8 and S100A9 induce increased secretion of neutrophil survival cytokines such as MCP-1, IL-6 and IL-8. This secretion is suppressed by TLR4 inhibitor), LY294002, AKT inhibitor, PD98059, SB202190, SP600125, and BAY-11-7085. S100A8 and S100A9 also induce the phosphorylation of AKT, ERK, p38 MAPK and JNK, and activation of NF-κB, which were blocked after exposure to TLR4i, LY294002, AKTi, PD98059, SB202190 or SP600125. Furthermore, supernatants collected from bronchial epithelial cells after S100A8 and S100A9 stimulation suppressed the apoptosis of normal and asthmatic neutrophils. These inhibitory mechanisms are involved in suppression of caspase 9 and caspase 3 activation, and BAX expression. The degradation of MCL-1 and BCL-2 was also blocked by S100A8 and S100A9 stimulation. Essentially, neutrophil apoptosis was blocked by co-culture of normal and asthmatic neutrophils with BEAS-2B cells in the presence of S100A8 and S100A9. These findings will enable elucidation of asthma pathogenesis.

Continuum‐mechanical modelling of apoptosis

AbstractIn cancer cells, the administration of drugs, such as TRAIL, can induce a mode of cell death (apoptosis). This applied reagent shifts the cell's internal inhibition and activation reactions towards the activation of caspases, triggering the cell death. In this contribution, a macroscopic model in the framework of the well‐known Theory of Porous Media is introduced to model the apoptosis reaction for lung‐cancer metastases located in brain tissue. Therein, the density‐production terms describe the apoptosis via a set of ordinary differential equations. The model is adapted to apoptosis experiments, measuring the survival rate of lung‐cancer cells in dependency of the TRAIL concentration. Thereby, the model is compared to a reduced model version using a constitutive logarithmic apoptosis function within the production terms.

Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan.

Francisella novicida is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Francisella tularensis Data published by us and others demonstrate that F. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, F. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that F. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

Influence of Microbes on Neutrophil Life and Death.

Neutrophils are the most abundant leukocyte in humans and they are among the first white cells recruited to infected tissues. These leukocytes are essential for the innate immune response to bacteria and fungi. Inasmuch as neutrophils produce or contain potent microbicides that can be toxic to the host, neutrophil turnover and homeostasis is a highly regulated process that prevents unintended host tissue damage. Indeed, constitutive neutrophil apoptosis and subsequent removal of these cells by mononuclear phagocytes is a primary means by which neutrophil homeostasis is maintained in healthy individuals. Processes that alter normal neutrophil turnover and removal of effete cells can lead to host tissue damage and disease. The interaction of neutrophils with microbes and molecules produced by microbes often alters neutrophil turnover. The ability of microbes to alter the fate of neutrophils is highly varied, can be microbe-specific, and ranges from prolonging the neutrophil lifespan to causing rapid neutrophil lysis after phagocytosis. Here we provide a brief overview of these processes and their associated impact on innate host defense.

Delay in Human Neutrophil Constitutive Apoptosis after Infection with Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae serotype K1 is a major cause of invasive syndrome defined by liver abscess with metastatic infections at other body sites. This culprit is known to be resistant to neutrophil phagocytosis and bactericidal activity. We hypothesized that K. pneumoniae serotype K1 might regulate neutrophil apoptosis and enhance the survival of the infected neutrophils that might serve as a vector for dissemination of the bacteria. Two serotypes of K. pneumoniae, KP-M1 isolated from a patient with liver abscess and DT-X (an acapsular mutant strain of KP-M1), were used to infect human neutrophils. The infected neutrophils were examined for their cytotoxicity, annexin V staining, proteins, DNA fragmentation, cytokine production, and viability that are involved in apoptosis. We found that KP-M1 was not destroyed and the ingested bacteria survived within neutrophils. While the uninfected neutrophils became apoptotic within 10 h, the neutrophils infected with KP-M1 could survive up to 24 h post infection. Constitutive apoptosis of KP-M1-infected neutrophils was significantly delayed compared to that of DT-X-infected or uninfected neutrophils (p < 0.01). KP-M1 modulated the anti-apoptotic effects by down-regulating the ratio of Bax to Bcl-2 and Mcl-1, and then delayed caspase-3 activation in the neutrophils, which was accompanied by inducing the anti-apoptotic cytokine, IL-8. These data suggest that K. pneumoniae serotype K1 can prolong the lifespan of infected neutrophils by delaying constitutive apoptosis within the first several hours of infection.

Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.

Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.

Modulation of macrophage antitumor potential by apoptotic lymphoma cells

In aggressive non-Hodgkin’s lymphoma (NHL), constitutive apoptosis of a proportion of the tumor cell population can promote net tumor growth. This is associated with the accumulation of tumor-associated macrophages (TAMs) that clear apoptotic cells and exhibit pro-oncogenic transcriptional activation profiles characteristic of reparatory, anti-inflammatory and angiogenic programs. Here we consider further the activation status of these TAMs. We compare their transcriptomic profile with that of a range of other macrophage types from various tissues noting especially their expression of classically activated (IFN-γ and LPS) gene clusters – typically antitumor – in addition to their previously described protumor phenotype. To understand the impact of apoptotic cells on the macrophage activation state, we cocultured apoptotic lymphoma cells with classically activated macrophages (M(IFN-γ/LPS), also known as M1, macrophages). Although untreated and M(IFN-γ/LPS) macrophages were able to bind apoptotic lymphoma cells equally well, M(IFN-γ/LPS) macrophages displayed enhanced ability to phagocytose them. We found that direct exposure of M(IFN-γ/LPS) macrophages to apoptotic lymphoma cells caused switching towards a protumor activation state (often referred to as M2-like) with concomitant inhibition of antitumor activity that was a characteristic feature of M(IFN-γ/LPS) macrophages. Indeed, M(IFN-γ/LPS) macrophages exposed to apoptotic lymphoma cells displayed increased lymphoma growth-promoting activities. Antilymphoma activity by M(IFN-γ/LPS) macrophages was mediated, in part, by galectin-3, a pleiotropic glycoprotein involved in apoptotic cell clearance that is strongly expressed by lymphoma TAMs but not lymphoma cells. Intriguingly, aggressive lymphoma growth was markedly impaired in mice deficient in galectin-3, suggesting either that host galectin-3-mediated antilymphoma activity is required to sustain net tumor growth or that additional functions of galectin-3 drive key oncogenic mechanisms in NHL. These findings have important implications for anticancer therapeutic approaches aimed at polarizing macrophages towards an antitumor state and identify galectin-3 as a potentially important novel target in aggressive NHL.

Impaired anti-inflammatory action of glucocorticoid in neutrophil from patients with steroid-resistant asthma.

BackgroundSteroid resistant (SR) asthma is characterized by persistent airway inflammation that fails to resolve despite treatment with high doses of corticosteroids. Furthermore, SR patient airways show increased numbers neutrophils, which are less responsive to glucocorticoid. The present study seeks to determine whether dexamethasone (DEX) has different effect on neutrophils from steroid sensitive (SS) asthmatics compared to SR asthmatics.MethodsAdults with asthma (n = 38) were classified as SR or SS based on changes in lung FEV1% following a one-month inhaled corticosteroid (ICS) treatment. Blood samples were collected from all patients during their first visit of the study. Neutrophils isolated from the blood were cultured with dexamethasone and/or atopic asthmatic serum for 18 h. The mRNA expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), a glucocorticoid transactivation target, and glucocorticoid-induced transcript 1 (GLCCI1), an early marker of glucocorticoid-induced apoptosis whose expression was associated with the response to inhaled glucocorticoids in asthma , was determined by real-time PCR, and ELISA was used to assess the pro-inflammatory cytokine IL-8 levels in the supernatant. Constitutive neutrophil apoptosis was detected by flow cytometry.ResultsDEX significantly induced MKP-1 expression in both patients with SS and SR patients in a concentration-dependent manner, but greater induction was observed for SS patients at a low concentration (10−6 M). Asthmatic serum alone showed no MKP-1expression, and there was impaired induction of MKP-1 by DEX in SR asthma patients. The expression of GLCCI1 was not induced in neutrophils with DEX or DEX/atopic asthmatic serum combination. Greater inhibition of IL-8 production was observed in neutrophils from patients with SS asthma treated with DEX/atopic asthmatic serum combination compared with SR asthma patients, though DEX alone showed the same effect on neutrophils from SS and SR asthma patients. Meanwhile, DEX dependent inhibition of constitutive neutrophil apoptosis was similar between SS asthma and SR asthma patients.ConclusionsDEX exerted different effects on neutrophils from patients with SS asthma and SR asthma, which may contribute to glucocorticoid insensitivity.

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

House Dust Mite Allergen Inhibits Constitutive Neutrophil Apoptosis by Cytokine Secretion via PAR2/PKCδ/p38 MAPK Pathway in Allergic Lymphocytes

House Dust Mite Allergen Inhibits Constitutive Neutrophil Apoptosis by Cytokine Secretion via PAR2/PKCδ/p38 MAPK Pathway in Allergic Lymphocytes

Cytokine secreted by S100A9 via TLR4 in monocytes delays neutrophil apoptosis by inhibition of caspase 9/3 pathway

Dysregulation of neutrophil apoptosis causes pathogenesis and aggravation of allergy. S100A9 exists as one of the proteins in the neutrophils, triggering inflammatory responses by activating the immune cells. In this study, we investigated whether S100A9 affects constitutive neutrophil apoptosis by activating the monocytes in normal and allergic subjects. Supernatant from human monocytic THP-1 cells after treatment with S100A9 suppressed normal neutrophil apoptosis by inhibiting the activations of caspase 9 and caspase 3. S100A9 upregulated the release of MCP-1, IL-6, and IL-8 in THP-1 cells. An increase in cytokine was suppressed by CLI-095, a Toll-like receptor (TLR) 4 inhibitor, PP2, a Src inhibitor, rottlerin, a PKCδ inhibitor, MAP kinase inhibitors, including PD98059, SB202190, and SP600125, and BAY-11-7085, an NF-κB inhibitor. Src, PKCδ, ERK1/2, p38 MAPK, and JNK were phosphorylated by S100A9. The phosphorylation of Src and PKCδ was suppressed by CLI-095, and the activation of ERK1/2, p38 MAPK, and JNK was inhibited by CLI-095, PP2, and rottlerin. S100A9 induced NF-κB activity, and the activation was suppressed by CLI-095, PP2, rottlerin, and MAPK kinase inhibitors. In normal and allergic subjects, supernatant from normal and allergic monocytes after stimulation with S100A9 suppressed normal and allergic neutrophil apoptosis, respectively; MCP-1, IL-6, and IL-8 in the supernatant was increased by S100A9. The cytokine secretion induced by S100A9 is related to TLR4, Src, PKCδ, ERK1/2, p38 MAPK, JNK, and NF-κB. Taken together, S100A9 induces anti-apoptotic effect on normal and allergic neutrophils by increasing cytokine secretion of monocytes. These findings may help us to better understand neutrophil apoptosis regulated by S100A9 and pathogenesis of allergic diseases.

Suppressive Effect of Der p 2 on Constitutive Neutrophil Apoptosis by Cytokine Secretion of Normal and Allergic Lymphocytes

Suppressive Effect of Der p 2 on Constitutive Neutrophil Apoptosis by Cytokine Secretion of Normal and Allergic Lymphocytes

Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis

AbstractNeutrophils play an essential role in the initial stages of inflammation by balancing pro- and antiinflammatory signals. Among these signals are the production of proinflammatory cytokines and the timely initiation of antiinflammatory cell death via constitutive apoptosis. Here we identify ataxia-telangiectasia mutated (ATM) kinase as a modulator of these neutrophil functions. Ataxia-telangiectasia (AT) is a pleiotropic multisystem disorder caused by mutations in the gene-encoding ATM, a master regulator of the DNA damage response. In addition to progressive neurodegeneration and high rates of cancer, AT patients have numerous symptoms that can be linked to chronic inflammation. We report that neutrophils isolated from patients with AT overproduce proinflammatory cytokines and have a prolonged lifespan compared with healthy controls. This effect is partly mediated by increases in activation of p38 MAP kinase. Furthermore, we show that the oxidative burst, catalyzed by nicotinamide adenine dinucleotide phosphate oxidase, can activate ATM in neutrophils. Finally, activation of ATM and DNA damage signaling suppress cytokine production and can abrogate the overproduction of IL-8 in ROS-deficient cells. This reveals a novel mechanism for the regulation of cytokine production and apoptosis, establishing DNA damage as a downstream mediator of immune regulation by reactive oxygen species. We propose that deficiencies in the DNA damage response, like deficiencies in the oxidative burst seen in chronic granulomatous disease, could lead to pathologic inflammation.

House dust mite allergen suppresses neutrophil apoptosis by cytokine release via PAR2 in normal and allergic lymphocytes.

House dust mite (HDM) is an essential allergen in allergic diseases such as allergic rhinitis and asthma. The pathogenic mechanism of allergy is associated with cytokine release of lymphocytes and constitutive apoptosis of neutrophils. In this study, we examined whether HDM induces cytokine release of lymphocytes and whether the secretion of cytokines is involved in modulation of neutrophil apoptosis. In normal and allergic subjects, extract of Dermatophagoides pteronyssinus (DP) increased IL-6, IL-8, MCP-1, and GM-CSF secretion in a time-dependent manner. This secretion was suppressed by PAR2i, an inhibitor of PAR2, in a dose-dependent manner, as well as by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, and BAY-11-7085, and an inhibitor of NF-κB. DP induced ERK and NF-κB activation in a time-dependent manner. ERK activation was suppressed by PAR2i, LY294002, and AKTi, and NF-κB activation was blocked by PAR2i, LY294002, AKTi, and PD98059. Supernatants collected from normal and allergic neutrophils after DP treatment inhibited the apoptosis of normal and allergic neutrophils through suppression of caspase 9 and caspase 3 cleavage. DP inhibited neutrophil apoptosis in coculture of normal neutrophils with normal lymphocytes, similar to the anti-apoptotic effects of DP on neutrophils alone. DP more strongly inhibited apoptosis of allergic neutrophils cocultured with allergic lymphocytes than allergic neutrophils without lymphocytes. In summary, DP induces the release of cytokines through the PAR2/PI3K/Akt/ERK/NF-κB pathway, which has anti-apoptotic effects on neutrophils of normal and allergic subjects. These results will facilitate elucidation of the pathogenic mechanism of allergic diseases.