Neutrophil Apoptosis Research Articles

A Novel IL2Rα-/- Model for CAR-T Toxicity in Acute Lymphoblastic Leukemia Recapitulates Cytokine Release Syndrome and Neutropenia

Breakthrough progress in CAR-T treatment for B cell Acute lymphoblastic leukemia (B-ALL) has led to major improvements in B-ALL patient survival. However, recent clinical studies involving B-ALL patients have shown that CAR-T related adverse events have a negative impact on patient prognosis. Multiple CAR-T related cytotoxic effects such as cytokine release syndrome (CRS) and neutropenia were especially shown to co-occur and were detrimental to the survival and recovery of B-ALL patients. This has consequently fueled interest in developing novel in vivo models to emulate these conditions. Currently, there are no models that can recapitulate multiple CAR-T related cytotoxic effects in a single in vivo model. To establish a mouse model that can be used to study both CRS and Neutropenia, we explored the role of IL-2Rα in inducing CAR-T related toxicities. We hypothesized that knockout of IL2Rα will lead to severe CRS after CAR-T infusion, owing to its crucial role in the regulation of cytokine-mediated inflammation, and its abundant expression in regulatory T cells that control self -tolerance and regulate T cell expansion. To prove our hypothesis, a pre-established IL-2Rα -/- or IL-2Rα KO mouse model was used, in which exons 2 and 3 of IL-2Rα gene that encode the binding site for IL-2, is replaced by a neomycin resistance gene driven by the PKG promoter. We injected 1 million murine B-ALL cells (Eμ-ALL) a week before inoculating 1 million CD19-28z CAR-T cells in IL2Rα KO and Wild type (WT) mice, and profiled for serum cytokine levels of IL6, IFNg and TNFα, followed by complete blood profiling (CBC) for weeks 1-4. Furthermore, we repeated this study without injecting Eμ-ALL cells and with CAR-T cells only (2 million), to understand whether CAR-T alone can impact CRS and Neutropenia. We found that KO mice injected with only CAR-T cells, developed severe CRS based on their elevated serum levels of IL6 (paired t test, p=0.006) (Fig 1A), IFNg (paired t test, p=0.033) and TNFa (paired t test, p=0.047) in week 4 post CAR-T vs CAR-T untreated mice. The difference in cytokine levels in WT mice under similar conditions was not statistically significant. Histopathological analysis confirmed that the IL2Rα KO mice developed secondary hemophagocytic lymphohistiocytosis, or macrophage activation (sHLH/MAS) that manifest during CAR-T associated CRS. To validate if the characteristics observed in IL2Rα KO mice are a consequence of CRS, we treated IL2Rα KO mice with 0.3 mg anti-mouse IL6-R mAb per mouse (mouse equivalent of Tocilizumab used in CRS management), 5 hours pre-CAR-T and biweekly throughout the study. We observed a significant improvement in the survival of CAR-T infused IL2Rα KO mice that were treated with anti-mouse IL6-R mAb compared to the untreated group (Gehan-Breslow-Wilcoxon test, p=0.031), thus confirming the incidence of severe CRS in these mice. Complete blood profiling of CAR-T alone treated IL2Rα KO mice showed a significant decrease in their Neutrophil concentration (paired t test, p=0.004), which did not occur in WT mice (Fig1B). This indicated that loss of IL2Rα not only regulates CRS but is also crucial for Neutrophils. We further validated the onset of Neutropenia in the KO mice by assessing whether CAR-T affects the turnover rate (rate of proliferation, maturation and apoptosis) of Neutrophils. Neutrophils isolated from bone marrow of KOs and WTs pre-CAR-T and 1-4 weeks post CAR-T treatment, showed a decrease in BrDU+ neutrophils in CAR-T treated KO mice only (paired t-test, p<0.0001), indicative of a decrease in % Neutrophil proliferation. Similarly, CAR-T treated KO mice showed increase in Annexin V+ neutrophils (paired t test,p=0.034) that is indicative of an increase in % Neutrophil apoptosis. Furthermore, CAR-T treatment impaired the maturation status of Neutrophils (measured by the % of Ly6G+CXCR2+Neutrophils), confirming the prevalence of Neutropenia alongside CRS (paired t-test, p=0.0005) We developed a novel IL2Rα KO model that recapitulates co-occurrence of CAR-T induced CRS and neutropenia observed in B-ALL patients. Our model will help understand the crosstalk between CAR-T and host immune components such as macrophages and Neutrophils that are altered during CRS and Neutropenia. Ongoing experiments are directed towards identifying key mediators of both CRS and Neutropenia during CAR-T therapy, which will improve treatment intervention during CAR-T associated cytotoxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microbes and the fate of neutrophils.

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults-typically 1011 per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.

Chlamydia psittaci inhibits apoptosis of human neutrophils by activating P2X7 receptor expression

This study tested the hypothesis that Chlamydia psittaci (C. psittaci) survives and multiplies in human neutrophils by activating P2X7, a nonselective cationic channel receptor expressed constitutively on the surface of these cells. Findings illustrated that P2X7 receptor expression was enhanced in C. psittaci-infected neutrophils. C. psittaci was able to inhibite spontaneous apoptosis of neutrophils through mitochondrial-induced ATP release and IL-8 production. Importantly, inhibiting ATP activation of the P2X7 receptor with AZ10606120 promotes apoptosis, while stimulating P2X7 receptor expression with BzATP delayed spontaneous apoptosis of human neutrophils, suggesting that C. psittaci inhibits apoptosis of human neutrophils by activating P2X7 receptor. This study reveals new insights into the survival advantages of the latent persistent state of C. psittaci and the mechanism by which it evades the innate immune response.

Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells.

Lung ischemia-reperfusion injury (LIRI) is associated with many diseases, including primary graft dysfunction after lung transplantation, and has no specific and effective therapies. Necroptosis contributes to the pathogenesis of ischemia-reperfusion injury. Necrostatin-1 (Nec-1), the necroptosis inhibitor targeting RIPK1, has been reported to alleviate ischemia-reperfusion injury in various organs. However, the underlying mechanism of Nec-1 in LIRI remains unclear. In this paper, an in vivo LIRI model was built up by left lung hilar clamping in mice, and an in vitro cold ischemia-reperfusion (CI/R) model using BEAS-2B cells was applied to mimic the lung transplantation setting. We found Nec-1 significantly alleviated ischemia-reperfusion-induced lung injury, cytokine releasing, and necroptosis of epithelial cells in mouse lungs. In vitro, Nec-1 also mitigated CI/R-induced cell death and inflammatory responses in BEAS-2B cells, and these protective effects were achieved by simultaneously inhibiting the formation of necrosome and RIPK1-dependent apoptosis. However, Nec-1 decreased the necrosome number but increased the apoptosis level in lung tissues after ischemia reperfusion. We further clarified that Nec-1 could also attenuate lung injury by promoting neutrophil apoptosis from flow cytometry. In conclusion, Nec-1 alleviated lung ischemia-reperfusion injury by inhibiting necroptosis and apoptosis of epithelial cells and promoting the apoptosis of neutrophils. Thus, Nec-1 could be a promising medication against primary graft dysfunction after lung transplantation.

Jinhua Qinggan granules attenuates acute lung injury by promotion of neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceAcute lung injury (ALI) is one of the fatal complications of respiratory virus infections such as influenza virus and coronavirus, which has high clinical morbidity and mortality. Jinhua Qinggan granules (JHQG) has been approved by China Food and Drug Administration in the treatment of H1N1 influenza and mild or moderate novel coronavirus disease 2019 (COVID-19), which is an herbal formula developed based on Maxingshigan decoction and Yinqiao powder that have been used to respiratory diseases in China for thousands of years. However, the underlying mechanism of JHQG in treating infectious diseases remains unclear. Aim of the studyThis study investigated the effects of JHQG on neutrophil apoptosis and key signaling pathways in lipopolysaccharide (LPS) -induced ALI mice in order to explore its mechanism of anti-inflammation. Materials and methodsThe effect of JHQG on survival rate was observed in septic mouse model by intraperitoneal injection of LPS (20 mg/kg). To better pharmacological evaluation, the mice received an intratracheal injection of 5 mg/kg LPS. Lung histopathological changes, wet-to-dry ratio of the lungs, and MPO activity in the lungs and total protein concentration, total cells number, TNF-α, IL-1β, IL-6, and MIP-2 levels in BALF were assessed. Neutrophil apoptosis rate was detected by Ly6G-APC/Annexin V-FITC staining. Key proteins associated with apoptosis including caspase 3/7 activity, Bcl-xL and Mcl-1 were measured by flow cytometry and confocal microscope, respectively. TLR4 receptor and its downstream signaling were analyzed by Western blot assay and immunofluorescence, respectively. ResultsJHQG treatment at either 6 or 12 g/kg/day resulted in 20% increase of survival in 20 mg/kg LPS-induced mice. In the model of 5 mg/kg LPS-induced mice, JHQG obviously decreased the total protein concentration in BALF, wet-to-dry ratio of the lungs, and lung histological damage. It also attenuated the MPO activity and the proportion of Ly6G staining positive neutrophils in the lungs, as well as the MIP-2 levels in BALF were reduced. JHQG inhibited the expression of Mcl-1 and Bcl-xL and enhanced caspase-3/7 activity, indicating that JHQG partially acted in promoting neutrophil apoptosis via intrinsic mitochondrial apoptotic pathway. The levels of TNF-α, IL-1β, and IL-6 were significantly declined in LPS-induced mice treated with JHQG. Furthermore, JHQG reduced the protein expression of TLR4, MyD88, p-p65 and the proportion of nuclei p65, suggesting that JHQG treatment inhibited TLR4/MyD88/NF-κB pathway. ConclusionJHQG reduced pulmonary inflammation and protected mice from LPS-induced ALI by promoting neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway, suggesting that JHQG may be a promising drug for treatment of ALI.

An update on the incidence, risk factors and mechanisms of rituximab-associated neutropenia.

With the increasing application of rituximab (RTB) in hematological diseases and autoimmune diseases (AIDs), we have gradually increased our awareness of the adverse reaction of rituximab-associated neutropenia (RAN), but little is known about its true incidence rate, susceptibility risk factors and exact pathogenesis. At present, research groups have conducted a large number of studies on different populations. The team found that age (> 60), advanced disease, systemic lupus erythematosus (SLE) and combined cyclophosphamide therapy were independent risk factors for RAN. However, its exact mechanism is not completely clear. Several hypotheses have been put forward to solve this question, including the production of anti-neutrophil antibodies after RTB, the generation disorder and neutrophil maturation stagnation caused by abnormal B-cell reconstruction, and the amplification of T-large granular lymphocyte population that may induce neutrophil apoptosis. However, there are still many unsolved problems in all aspects of RAN. This article is an update of the incidence rate, risk factors and mechanisms of RAN.

New evidence for dietary fatty acids in the neutrophil traffic between the bone marrow and the peripheral blood

Chronic administration of a high-fat diet in mice has been established to influence the generation and trafficking of immune cells such as neutrophils in the bone marrow, the dysregulation of which may contribute to a wide range of diseases. However, no studies have tested the hypothesis that a short-term, high-fat diet could early modulate the neutrophil release from bone marrow at fasting and at postprandial in response to a high-fat meal challenge, and that the predominant type of fatty acids in dietary fats could play a role in both context conditions. Based on these premises, we aimed to establish the effects of different fats [butter, enriched in saturated fatty acids (SFAs), olive oil, enriched in monounsaturated fatty acids (MUFAs), and olive oil supplemented with eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] on neutrophil navigation from bone marrow to blood in mice. The analysis of cellular models for mechanistic understanding and of postprandial blood samples from healthy volunteers for translational purposes was assessed. The results revealed a powerful effect of dietary SFAs in promotion the neutrophil traffic from bone marrow to blood via the CXCL2-CXCR2 axis. Dietary SFAs, but not MUFAs or EPA and DHA, were also associated with increased neutrophil apoptosis and bone marrow inflammation. Similar dietary fatty-acid-induced postprandial neutrophilia was observed in otherwise healthy humans. Therefore, dietary MUFAs might preserve bone marrow health and proper migration of bone marrow neutrophils early in the course of high-fat diets even after the intake of high-fat meals.

A mouse-adapted CVA6 strain exhibits neurotropism and triggers systemic manifestations in a novel murine model

ABSTRACT CVA6 is one of Enteroviruses causing worldwide epidemics of HFMD with neurological and systemic complications. A suitable animal model is necessary for studying the pathogenesis of CVA6 and evaluating antiviral and vaccine efficacy. In this study, we generated a mouse-adapted CVA6 strain that successfully infected 10-day-old ICR mice via oral route. All infected mice were paralyzed and died within 11 dpi. Analysis of pathological changes and virus loads in fourteen tissues showed that CVA6 triggered systematic damage similar to i.p. inoculation route. Unlike i.p. route, we detected oral and gastrointestinal lesions with the presence of viral antigens. Both specific anti-CVA6 serum and inactivated vaccines successfully generated immune protection in mice. Meanwhile, we also established a successful infection of CVA6 via i.p. and i.m. route in 10-day-old mice. After infection, mice developed remarkably neurological signs and systemic manifestations such as emaciation, polypnea, quadriplegia, depilation and even death. Through i.p. inoculation, pathological examination showed brain and spinal cord damage caused by the virus infection with neuronal reduction, apoptosis, astrocyte activation, and recruitment of neutrophils and monocytes. Following neurological manifestation, the CVA6 infection became systemic, and high viral loads were detected in multiple organs along with morphological changes and inflammation. Moreover, analysis of spleen cells by FACS indicated that CVA6 led to immune system activation, which further contributed to systemic inflammation. Taken together, our novel murine model of CVA6 provides a useful tool for studying the pathogenesis and evaluating antiviral and vaccine efficacy.

Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions.

The mechanistic role of the airway microbiome in chronic obstructive pulmonary disease (COPD) remains largely unexplored. We present a landscape of airway microbe-host interactions in COPD through an in-depth profiling of the sputum metagenome, metabolome, host transcriptome and proteome from 99 patients with COPD and 36 healthy individuals in China. Multi-omics data were integrated using sequential mediation analysis, to assess in silico associations of the microbiome with two primary COPD inflammatory endotypes, neutrophilic or eosinophilic inflammation, mediated through microbial metabolic interaction with host gene expression. Hypotheses of microbiome-metabolite-host interaction were identified by leveraging microbial genetic information and established metabolite-human gene pairs. A prominent hypothesis for neutrophil-predominant COPD was altered tryptophan metabolism in airway lactobacilli associated with reduced indole-3-acetic acid (IAA), which was in turn linked to perturbed host interleukin-22 signalling and epithelial cell apoptosis pathways. In vivo and in vitro studies showed that airway microbiome-derived IAA mitigates neutrophilic inflammation, apoptosis, emphysema and lung function decline, via macrophage-epithelial cell cross-talk mediated by interleukin-22. Intranasal inoculation of two airway lactobacilli restored IAA and recapitulated its protective effects in mice. These findings provide the rationale for therapeutically targeting microbe-host interaction in COPD.

Dysregulation of neutrophil death in sepsis.

Sepsis is a prevalent disease that has alarmingly high mortality rates and, for several survivors, long-term morbidity. The modern definition of sepsis is an aberrant host response to infection followed by a life-threatening organ dysfunction. Sepsis has a complicated pathophysiology and involves multiple immune and non-immune mediators. It is now believed that in the initial stages of sepsis, excessive immune system activation and cascading inflammation are usually accompanied by immunosuppression. During the pathophysiology of severe sepsis, neutrophils are crucial. Recent researches have demonstrated a clear link between the process of neutrophil cell death and the emergence of organ dysfunction in sepsis. During sepsis, spontaneous apoptosis of neutrophils is inhibited and neutrophils may undergo some other types of cell death. In this review, we describe various types of neutrophil cell death, including necrosis, apoptosis, necroptosis, pyroptosis, NETosis, and autophagy, to reveal their known effects in the development and progression of sepsis. However, the exact role and mechanisms of neutrophil cell death in sepsis have not been fully elucidated, and this remains a major challenge for future neutrophil research. We hope that this review will provide hints for researches regarding neutrophil cell death in sepsis and provide insights for clinical practitioners.

Intercellular model predicts mechanisms of inflammation-fibrosis coupling after myocardial infarction.

After myocardial infarction (MI), cardiac cells work together to regulate wound healing of the infarct. The pathological response to MI yields cardiac remodelling comprising inflammatory and fibrosis phases, and the interplay of cellular dynamics that underlies these phases has not been elucidated. This study developed a computational model to identify cytokine and cellular dynamics post-MI to predict mechanisms driving post-MI inflammation, resolution of inflammation, andscar formation. Additionally, this study evaluated the interdependence between inflammation and fibrosis. Our model bypassed limitations of in vivo approaches in achieving cellular specificity and performing specific perturbations such as global knockouts of chemical factors. The model predicted that inflammation is a graded response to initial infarct size that is amplified by a positive feedback loop between neutrophils and interleukin 1β (IL-1β). Resolution of inflammation was driven by degradation of IL-1β, matrix metalloproteinase 9, and transforming growth factor β (TGF-β), as well as apoptosis of neutrophils. Inflammation regulated TGFβ secretion directly through immune cell recruitment and indirectly through upregulation of macrophage phagocytosis. Lastly, we found that mature collagen deposition was an ultrasensitive switch in response to inflammation, which was amplified primarily by cardiac fibroblast proliferation. These findings describe the relationship between inflammation and fibrosis and highlight how the two responses work together post-MI. This model revealed that post-MI inflammation and fibrosis are dynamically coupled, which provides rationale for designing novel anti-inflammatory, pro-resolving or anti-fibrotic therapies that may improve the response to MI. KEY POINTS: Inflammation and matrix remodelling are two processes involved in wound healing after a heart attack. Cardiac cells work together to facilitate these processes; this is done by secreting cytokines that then regulate the cells themselves or other cells surrounding them. This study developed a computational model of the dynamics of cardiac cells and cytokines to predict mechanisms through which inflammation and matrix remodelling is regulated. We show the roles of various cytokines and signalling motifs in driving inflammation, resolution of inflammation and fibrosis. The novel concept of inflammation-fibrosis coupling, based on the model prediction that inflammation and fibrosis are dynamically coupled, provides rationale for future studies and for designing therapeutics to improve the response after a heart attack.

MiR-34a induces neutrophil apoptosis by regulating Cdc42-WASP-Arp2/3 pathway-mediated F-actin remodeling and ROS production

ABSTRACT Background The number of neutrophils is significantly reduced in myelodysplastic syndrome (MDS), but the molecular basis remains unclear. We recently found that miR-34a was significantly increased in MDS neutrophils. Therefore, this study aims to clarify the effects of aberrant miR-34a expression on neutrophil counts. Methods miR-34a mimics/inhibitor transfection were performed in neutrophil-like differentiated HL60 (dHL60) cells, and a FACSCalibur flow cytometer was used to measure ROS production and apoptosis. In addition, the Cdc42-WASP-Arp2/3 pathway inhibitor (ML141) and activator (CN02) treated the dHL60 cells, and then ROS production, apoptosis and related proteins expression were detected. And, luciferase reporter assay to verify the relationship of miR-34a and the Cdc42-WASP-Arp2/3 pathway. Results overexpression of miR-34a could induce ROS production and apoptosis, decrease the expression levels of DOCK8, p-WASP, WASP, Arp2, Arp3, and increase F-actin’s expression. Meanwhile, knockdown of miR-34a could decrease ROS production and apoptosis, increase the expression of DOCK8, p-WASP, WASP, Arp2, Arp3, and decrease F-actin’s expression. Immunofluorescence staining showed aberrant miR-34a and Cdc42-WASP-Arp2/3 pathway could induce F-actin membrane transfer. Luciferase reporter assay indicated that DOCK8 was a direct target gene of miR-34a. Conclusion These data indicates miR-34a may induce neutrophil apoptosis by regulating Cdc42-WASP-Arp2/3 pathway-mediated F-actin remodeling and ROS production.

The EGFR/ErbB inhibitor neratinib modifies the neutrophil phosphoproteome and promotes apoptosis and clearance by airway macrophages.

Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo. Here, we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration, and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.

Interferon-β regulates proresolving lipids to promote the resolution of acute airway inflammation.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-β (IFN-β) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli-evoked lung injury, and suppresses production of IFN-β and the proresolving lipid mediators 15-epi-LXA4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-β delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-β accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-β-mediated resolution. These findings point to a pivotal role of IFN-β in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-β-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Interaction between iron oxide nanoparticles (Fe3O4 NPs) and human neutrophils: Evidence that Fe3O4 NPs possess some pro-inflammatory activities

Iron oxide nanoparticles (Fe3O4 NPs) are important for different medical applications. However, potential toxicity has been reported and several parameters must still be studied to reach highest therapeutic efficacy with minimal undesired effects. Inflammation is one of the most reported undesired effects of NP exposure in a variety of inflammatory models and conflicting data exist regarding whether Fe3O4 NPs possess pro- or anti-inflammatory activities. The aim of this study was to determine the direct effect of Fe3O4 NPs on the biology of neutrophil, a key player cell in inflammation. Freshly isolated human neutrophils were incubated in vitro with Fe3O4 NPs, and several functions have been studied. Using transmission electronic microscopy, Fe3O4 NPs were found to be ingested by neutrophils. These NPs do not induce a respiratory burst by themselves, but they increase the ability of neutrophils to adhere onto human endothelial cells as well as enhance phagocytosis. An antibody array approach revealed that Fe3O4 NPs induce the production of some cytokines, including the chemokine IL-8 (CXCL8), which was confirmed by ELISA. Fe3O4NPs were found to delay spontaneous neutrophil apoptosis regardless of sex of the donor. Using a pharmacological approach, we demonstrate that Fe3O4 NPs delay apoptosis by a de novo protein synthesis-dependent mechanism and via different cell signalling pathways. The data indicate that Fe3O4 NPs can alter the biology of human neutrophils and that they possess some pro-inflammatory effects, particularly based on their capacity to delay apoptosis and to induce the production of pro-inflammatory cytokines. Therefore, Fe3O4 NPs can regulate inflammation by targeting human neutrophil functions.

Investigation of estrogen-like effects of parabens on human neutrophils

This study investigated the estrogen-like effects and mechanism of action most commonly used parabens: methyl- (MeP), ethyl- (EtP), propyl- (PrP) and butylparaben (BuP) in human neutrophils.Neutrophils were isolated from 50 blood donors, pre-incubated with antagonists of estrogen receptor α (ERα), ERβ and G-protein coupled estrogen receptor 1 (GPER), then incubated with MeP, EtP, PrP, BuP and 17β-estradiol (E2; 10 nM). Cytotoxic effect was evaluated by MTT test. Neutrophils apoptosis, necrosis and NETs formation were assessed in flow cytometry and confocal microscopy. The ability of the neutrophils for chemotaxis, phagocytosis, NADPH oxidase activity and generation of superoxide anion was assessed in Boyden's chamber, Park's method with latex, the NBT test, and reduction of cytochrome C, respectively. The total nitric oxide concentration was measured in neutrophils supernatants by the Griess reaction. The expression of cathepsin G, neutrophil elastase, proteinase 3, ERα, ERβ and GPER was assessed in Western blot method.In our research, parabens did not cause a cytotoxic effect on human neutrophils nor affect their lifespan. Parabens exposure did not change neutrophils functions (chemotaxis, phagocytosis, NETs formation and oxygen-dependent killing mechanism) and expression of estrogen receptors.Our results suggest that parabens do not cause estrogen receptor-mediated neutrophils-related effects at concentrations measured in the plasma of individuals using products preserved with parabens.

Steamed Panax notoginseng and its Saponins Inhibit the Migration and Induce the Apoptosis of Neutrophils in a Zebrafish Tail-Fin Amputation Model.

Panax notoginseng (PN) is a Chinese medicinal herb that is traditionally used to treat inflammation and immune-related diseases. Its major active constituents are saponins, the types and levels of which can be changed in the process of steaming. These differences in saponins are causally relevant to the differences in the therapeutic efficacies of raw and steamed PN. In this study, we have prepared the extracts of steamed PN (SPNE) with 70% ethanol and investigated their immunomodulatory effect using a zebrafish tail-fin amputation model. A fingerprint-effect relationship analysis was performed to uncover active constituents of SPNE samples related to the inhibitory effect on neutrophil number. The results showed that SPNE significantly inhibited the neutrophil number at the amputation site of zebrafish larvae. And SPNE extracts steamed at higher temperatures and for longer time periods showed a stronger inhibitory effect. Ginsenosides Rh1, Rk3, Rh4, 20(S)-Rg3, and 20(R)-Rg3, of which the levels were increased along with the duration of steaming, were found to be the major active constituents contributing to the neutrophil-inhibiting effect of SPNE. By additionally investigating the number of neutrophils in the entire tail of zebrafish larvae and performing TUNEL assays, we found that the decreased number of neutrophils at the amputation site was due to both the inhibition of their migration and apoptosis-inducing effects of the ginsenosides in SPNE on neutrophils. Among them, Rh1 and 20(R)-Rg3 did not affect the number of neutrophils at the entire tail, suggesting that they only inhibit the migration of neutrophils. In contrast, ginsenosides Rk3, Rh4, 20(S)-Rg3, and SPNE did not only inhibit the migration of neutrophils but also promoted neutrophilic cell death. In conclusion, this study sheds light on how SPNE, in particular the ginsenosides it contains, plays a role in immune modulation.

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.

Cell damage and neutrophils promote the infection of Mycoplasma pneumoniae and inflammatory response

Mycoplasma pneumoniae (MP) is an important respiratory pathogen of human. The infection of MP can cause direct damage and immune damage in lung, resulting in Mycoplasma pneumoniae pneumonia (MPP). In this study, we aim to investigate the pathogenesis of MPP by detecting the proliferation of MP under conditions of cell damages and neutrophils in vitro. Firstly, we found the supplements of intracellular fluid, protein and RNA derived from intracellular fluid of A549 cells contribute to the survival of MP, thereby promoting the infection of MP. Cell damage can also significantly contribute to the survival of MP without supplements. At the same time, the additions of supplements contribute to apoptosis and the expression of IL-8 and IL-1β. Further, we found live neutrophils show bactericidal activity to MP, and the phagocytosis of MP promotes apoptosis of neutrophils. When co-incubated with MP and A549 cells, the proliferation of MP in the high neutrophils proportion groups were accelerated with functional decline of neutrophils, and the level of extracellular IL-1β showed a time and dose dependent manner to neutrophils. These results suggest that the release of intracellular nutrients by damaged cells and functional decline of neutrophils can promote the infection of MP and play roles in the activation of inflammatory response. Therefore, lung damage and infiltration of neutrophils would be important factors affecting the development of MPP.

Withaferin A Inhibits Neutrophil Adhesion, Migration, and Respiratory Burst and Promotes Timely Neutrophil Apoptosis.

Neutrophils play a major role in many equine conditions, including equine asthma, laminitis, and intestinal ischemia and reperfusion injury, and therefore represent an attractive target for innovative therapeutic approaches. Novel strategies for reducing neutrophilic inflammation include modulation of neutrophil functions and lifespan. Withaferin A (WFA) is a phytochemical with well-established in vitro and in vivo anti-inflammatory properties, but its direct effects on neutrophils are largely unknown. We hypothesized that WFA would inhibit adhesion, migration, and respiratory burst by equine neutrophils and promote timely apoptosis of primed equine neutrophils. Consistent with this hypothesis, our data show that WFA causes a significant, concentration-dependent inhibition of equine neutrophil adhesion, migration, and respiratory burst in response to diverse stimuli. Further, WFA treatment increased apoptosis of equine neutrophils exposed to GM-CSF for 24 h. This pro-apoptotic effect of WFA was not observed in unprimed neutrophils, nor at the 2-h time point relevant to our functional neutrophil experiments. Our data demonstrate that WFA may reduce neutrophil-mediated inflammation through multiple mechanisms, including suppression of inflammatory responses and promotion of apoptosis. Additional research is needed to elucidate the molecular mechanisms for these effects and evaluate the potential clinical use of WFA in veterinary and human patients.