Neutrophil Plasma Membrane Research Articles

Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis.

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

Neutrophil and endothelial cell membranes coassembled roflumilast nanoparticles attenuate myocardial ischemia/reperfusion injury.

Aim: This study aimed to develop biomimetic nanoparticles (NPs) of roflumilast (ROF) for attenuating myocardial ischemia/reperfusion (MI/R) injury. Materials & methods: We synthesized biomimetic ROF NPs and assembled ROF NPs in neutrophil and endothelial cell membranes (NE/ROF NPs). The physical properties of NE/ROF NPs were characterized and biological functions of NE/ROF NPs were tested in vitro. Targeting characteristics, therapeutic efficacyand safety ofNE/ROF NPs were examined in mice model of MI/R. Results: NE/ROF NPs exhibited significant anti-inflammatory and antiadhesion effects. Meanwhile, theywas effective in reducing MI/R injury in mice. Furthermore, NE/ROF NPs exhibited stronger targeting capabilities and demonstrated good safety. Conclusion: NE/ROF NPs may be a versatile biomimetic drug-delivery system for attenuating MI/R injury.

A Biomimetic Nanoplatform with Improved Inflammatory Targeting Behavior for ROS Scavenging-Based Treatment of Ulcerative Colitis.

Ulcerative colitis (UC), a refractory disease, has become a global problem. Herein, a biomimetic nanoplatform (AU-LIP-CM) comprising Au cluster enzymes (AU)-loaded liposomes (AU-LIP) camouflaged with the fusion membrane (CM) consisting of neutrophil (NC) and red blood cell (RBC) membrane is designed for the treatment of UC. Briefly, revealed by second near-infrared (NIR-II) imaging through collection of fluorescence emitting >1200 nm from AU, the improved inflammatory targeting behavior contributed by CM cloaking, which inherits abilities of inflammatory targeting and immune escape from NC and RBC, respectively, promotes specific accumulation of AU within inflammatory intestines with up to ≈11.5 times higher than that of bare AU. Afterward, AU possessing superoxide dismutase- and catalase-like activities realizes high-efficiency scavenging of reactive oxygen species (ROS), leading to repair of intestinal barriers, regulation of the immune system, and modulation of gut microbiota, which surpass first-line UC drug. In addition, study of underlying therapeutic mechanism demonstrated that the treatment with AU-LIP-CM can alter the gene signature associated with response to ROS for UC mice to a profile similar to that of healthy mice, deciphering related signal pathways. The strategy developed here provides insights of learning from properties of natural bio-substances to empower biomimetic nanoplatform to confront diseases.

Sfxn5 Regulation of Actin Polymerization for Neutrophil Spreading Depends on a Citrate-Cholesterol-PI(4,5)P2 Pathway.

Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter invitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.

Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O2−), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Intrinsically bioactive and biomimetic nanoparticle-derived therapies alleviate asthma by regulating multiple pathological cells

Asthma is a serious global public health concern. Airway neutrophilic inflammation is closely related to severe asthma, for which effective and safe therapies remain to be developed. Here we report nanotherapies capable of simultaneously regulating multiple target cells relevant to the pathogenesis of neutrophilic asthma. A nanotherapy LaCD NP based on a cyclic oligosaccharide-derived bioactive material was engineered. LaCD NP effectively accumulated in the injured lungs of asthmatic mice and mainly distributed in neutrophils, macrophages, and airway epithelial cells after intravenous or inhalation delivery, thereby ameliorating asthmatic symptoms and attenuating pulmonary neutrophilic inflammation as well as reducing airway hyperresponsiveness, remodeling, and mucus production. Surface engineering via neutrophil cell membrane further enhanced targeting and therapeutic effects of LaCD NP. Mechanistically, LaCD NP can inhibit the recruitment and activation of neutrophils, especially reducing the neutrophil extracellular traps formation and NLRP3 inflammasome activation in neutrophils. Also, LaCD NP can suppress macrophage-mediated pro-inflammatory responses and prevent airway epithelial cell death and smooth muscle cell proliferation, by mitigating neutrophilic inflammation and its direct effects on relevant cells. Importantly, LaCD NP showed good safety performance. Consequently, LaCD-derived multi-bioactive nanotherapies are promising for effective treatment of neutrophilic asthma and other neutrophil-associated diseases.

The Diagnostic Value of Triggering Receptor Expressed on Myeloid Cells-1 in Post-Traumatic Bacterial Endophthalmitis.

To determine whether soluble-triggering receptor expressed on myeloid cells-1 (sTREM-1) could serve as a reliable diagnostic biomarker of post-traumatic bacterial endophthalmitis (PTBE). Thirty-two patients (32 eyes) clinically diagnosed having PTBE were further divided into a culture-positive (CP) group and a culture-negative (CN) group. Sixty-two patients (62 eyes) without traumatic endophthalmic infection were also enrolled. Twenty-one eyes from 11 donors without globe ocular injuries were included as control group. Vitreous sTREM-1 levels were detected by ELISA. The expression and tissue distribution of TREM-1 were revealed by immunohistochemistry. The diagnostic value of sTREM-1 was determined by receiver operating characteristic curve (ROC). The correlation between sTREM-1 concentration and final best-corrected visual acuity (FBCVA) and Peyman endophthalmitis score (PES) were also assessed. The vitreous sTREM-1 level in the PTBE group was higher than that in noninfected group and control group (P < 0.05). No remarkable difference was found between the CP group and the CN group in vitreous sTREM-1 levels (P > 0.05). No remarkable difference was found between the noninfected group and the control group (P > 0.05). No remarkable difference in TREM-1 level was found before and after intravitreal antibiotics (P > 0.05). TREM-1 was selectively highly expressed on the surface of cell membrane of neutrophils and monocytes/macrophages infiltrated in vitreous and uveal of the PTBE group. The area under the ROC curve (AUC) was 0.79 (>0.75), with a medium diagnostic efficiency. The sensitivity and specificity of sTREM-1 to differentiate PTBE from the noninfected intraocular condition were 62.50% and 86.25% separately. A cutoff value >524.50 pg/mL for sTREM-1 was predicted to be PTBE. Vitreous sTREM-1 levels in PTBE group were positively correlated with PES (r = 0.428, P < 0.05). However, sTREM-1 levels and FBCVA did not significantly correlate with one another (P > 0.05). The sTREM-1 was a promising diagnostic biomarker of PTBE, especially CN-PTBE. Vitreous sTREM-1 levels were linked with intraocular inflammation levels and severity of PTBE.

The allosterically modulated FFAR2 is transactivated by signals generated by other neutrophil GPCRs.

Positive allosteric modulators for free fatty acid receptor 2 (FFAR2/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFAR2 was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFAR2 from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFAR2 in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFAR2s, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFAR2 activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFAR2 activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFAR2 activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFAR2 modulator.

355: RHINOVIRUS-ASSOCIATED ANCA VASCULITIS: A RARE ASSOCIATION

Introduction: The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV) are immune mediated diseases, associated with antigen exposure and host immune response. Approximately 52–80% of patients with AAV have pulmonary abnormalities on chest CT, mainly interstitial lung disease (ILD), granuloma, or alveolar hemorrhage. Myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) has been particularly associated with interstitial lung disease and the majority of patients present with pulmonary symptoms prior to the diagnosis of ANCA vasculitis. ILD is a relatively uncommon manifestation of MPO-ANCA, but signifies a poor prognosis. We present a patient with rhinovirus infection associated ANCA vasculitis. Description: A 42-year-old female with a past medical history of COPD, bipolar disorder, and intravenous drug use presented to the emergency department in acute respiratory distress. Computed tomography of the chest revealed bilateral diffuse, patchy ground glass opacities. Respiratory PCR testing was positive for rhinovirus. The patient was admitted to the ICU for acute hypoxic respiratory failure. Her respiratory status continued to decline during the ICU stay. Serum studies including DsDNA, SSA, SSB, anticentromere, myositis, aldolase and ANCA were obtained. The studies were positive for MPO-ANCA. The patient was treated with high dose methylprednisolone. Subsequently, the oxygen demand improved and the patient made a full recovery, with plan for outpatient biopsies. Discussion: MPO-ANCA is a marker which may be induced by various drugs, dust inhalation, smoking, heavy metal exposure, or silica exposure. The hypothesized mechanism involves cytokines produced by antigens to induce MPO expression within the neutrophil cell membrane. The ANCA component binds to MPO to induce neutrophil activation which then activates the inflammatory cascade. The overproduction of cytokines causes vascular endothelial cell damage and ultimately vasculitis. One proposed mechanism of MPO-ANCA involves the alteration in degradation of neutrophil extracellular traps (NETs) likely due to infections. This mechanism leads to an increase in MPO-ANCA binding. NETs have a proliferative effect on lung fibroblasts, and thus are seen alongside fibroblasts in patients with MPO-ANCA interstitial pneumonia.

Effects of Medicinal Leech-Related Cationic Antimicrobial Peptides on Human Blood Cells and Plasma.

Cationic antimicrobial peptides (CAMPs) are considered as next-generation antibiotics with a lower probability of developing bacterial resistance. In view of potential clinical use, studies on CAMP biocompatibility are important. This work aimed to evaluate the behavior of synthetic short CAMPs (designed using bioinformatic analysis of the medicinal leech genome and microbiome) in direct contact with blood cells and plasma. Eight CAMPs were included in the study. Hemolysis and lactate dehydrogenase assays showed that the potency to disrupt erythrocyte, neutrophil and mononuclear cell membranes descended in the order pept_1 > pept_3 ~ pept_5 > pept_2 ~ pept_4. Pept_3 caused both cell lysis and aggregation. Blood plasma and albumin inhibited the CAMP-induced hemolysis. The chemiluminescence method allowed the detection of pept_3-mediated neutrophil activation. In plasma coagulation assays, pept_3 prolonged the activated partial thromboplastin time (APTT) and prothrombin time (at 50 μM by 75% and 320%, respectively). Pept_3 was also capable of causing fibrinogen aggregation. Pept_6 prolonged APTT (at 50 μM by 115%). Pept_2 was found to combine higher bactericidal activity with lower effects on cells and coagulation. Our data emphasize the necessity of investigating CAMP interaction with plasma.

Coating a Self-Assembly Nanoconstruct with a Neutrophil Cell Membrane Enables High Specificity for Triple Negative Breast Cancer Treatment.

Breast cancer is one of the most commonly diagnosed cancers in American women. Triple negative breast cancer is among the most advanced and aggressive forms of breast cancer. Treatment options are limited for such cancers, making chemotherapy a convenient and effective treatment. Although these therapies can reduce morbidity and mortality, it is often followed by systemic side effects or relapse. Nanoparticles (NPs) have been considered for drug delivery approaches due to their ability to target various disease sites. Herein, we aim to develop a biomimetic NP construct (cell membrane-cloaked NPs) that exhibits specific affinity with triple negative breast cancer cells. In this regard, we designed biomimetic supramolecular nanoconstructs composed of a poly(vinyl pyrrolidone)-tannic acid (PVP-TA NPs/ PVT NPs) core and biofunctionalized with neutrophil cell membranes (PVT-NEU NPs). In this study, we have synthesized a PVT-NEU NP construct, characterized it, and evaluated it for improved targeting and therapeutic benefits in in vitro and in vivo models. Analysis of PVT-NEU NPs confirms the presence of the core of PVP-TA NPs coated with activated human neutrophil membranes. The study results confirmed that PVT-NEU NPs demonstrated an enhanced interaction and targeting with the tumor cells, thus improving the therapeutic activity of a model therapeutic agent (paclitaxel). Altogether, this study suggests the potential of biomimetic NPs as a promising therapeutic option for targeted drug delivery for advanced-stage breast cancer and other similar diseased conditions.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, μ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent μ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 μg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

A neutrophil cell membrane-biomimetic nanoplatform based on L-arginine nanoparticles for early osteoarthritis diagnosis and nitric oxide therapy.

Osteoarthritis (OA) is a common debilitating disease affecting articular joints for which no effective disease-modifying early diagnosis or medical therapy tools are currently available. The inefficient delivery of drugs into inflamed chondrocytes has restricted the development of anti-OA medication. Evidence has shown that inflammatory neutrophils possess the property of targeting inflammation via inflammatory tissue recruiting. Herein, we report neutrophil-cell-membrane-based biomimetic nanoparticles (NM-LANPs@Ru) as an OA theranostic nanoplatform; they act as a NO delivery system, coating neutrophil cell membrane onto the surface of self-assembled PEGylated L-arginine nanoparticles (LANPs) to act as a NO donor and loading a Ru complex to act as a ROS inducer. NM-LANPs@Ru demonstrated the specific targeting of inflamed OA with low toxicity, good NO release, and excellent fluorescence/photoacoustic (FL/PA) imaging properties. We showed that NM-LANPs@Ru exhibited enhanced cellular association in inflamed chondrocyte cells (C28/I2), much higher than NO release from ROS oxidized LA, and it improved the inhibition of the apoptosis of inflamed C28/I2 cells compared with control treatments. In vivo studies demonstrated that NM-LANPs@Ru effectively targeted inflamed OA, based on real-time dual-modal FL/PA imaging, eventually exhibiting its excellent anti-inflammatory activity. Our study may provide a new approach for the early diagnosis and treatment of osteoarthritis using a neutrophil-cell-membrane-based biomimetic nanoplatform for NO or drug delivery.

C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

60P CCL2 expression and the neutrophil plasma membrane characteristics in blood in different stages of endometrial cancer

The mechanisms of neutrophils (Nph) recruitment to a tumor and their pro-tumor polarization are poorly understood. The CCL family of chemokines enhance the tumor recruitment, and the plasma membrane topography affects Nph activity. We aimed to evaluate the CCL2 expression, the stiffness and surface markers expression of Nph plasma membrane in blood in endometrial cancer (EC). Blood samples were obtained from the primary endometrioid EC patients, stage I-III FIGO (n=58), uterine myoma patients (n=20) and healthy women (n=30). The CCL2 level was determined by ELISA (JSC Vektor-Best-Volga, Russia). Circulating Nph plasma membrane stiffness was measured by atomic force microscopy (NT-MDT, Russia). The CD16, CD95 Nph surface expression was assessed by fluorescence microscopy. Statistical analysis was performed using Statistica 13. We observed that serum CCL2 level was decreased in myoma (197.6 (Q1-Q3 147.1-255.1) pg/ml) (p=0.013) and increased in stage I EC (299.6 (255.8-411.8) pg/ml) (p=0.012) compared with the control (270.1 (220.1-310.8) pg/ml). In stage II-III, serum CCL2 level was lower (236.9 (190.2-276.3) pg/ml) than that in stage I (p=0.007). The Nph membrane stiffness in stage I was reduced compared with the control (37.3±2.2 MPa vs 56.7±2.4 MPa) (p=0.051). In stage II-III, the membrane stiffness (70.5±8.4 MPa) was higher than in myoma (p=0.021) and the control (p=0.046). There was a linear regression relationship between serum CCL2 level and Nph membrane stiffness value in stage I (p=0.023), but not in stage II-III. The expression of CD95, a marker involved in immune evasion, was lower in stage II-III compared with myoma (p=0.003) and the control (p=0.006). In EC, membrane stiffness negatively correlated with CD95 expression (r=-0.478, p=0.0005). The number of active mature (CD16+) Nph was lower in stage I compared with myoma (p=0.029) and negatively correlated with serum CCL2 level (r=-0.423, p=0.0024). The serum CCL2 expression, the structure and marker status of circulating Nph membrane in stage I endometrioid EC differ from those in stage II-III, probably reflecting differences in the Nph recruitment and polarization processes.

Alpha-1 Antitrypsin Augmentation Inhibits Proteolysis of Neutrophil Membrane Voltage-Gated Proton Channel-1 in Alpha-1 Deficient Individuals.

Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.

Localization of nucleobindin2/nesfatin-1-like immunoreactivity in human lungs and neutrophils

Nucleobindin2 (NUCB2)/nesfatin-1 expression in human plasma positively correlates with the expression of pro-inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD), implicating its potential role in neutrophilic lung inflammation. There are no data on the localization of nucleobindin2 (NUCB2)/nesfatin-1 in human lungs and inflammatory cells. We examined the localization of NUCB2/nesfatin-1-immunoreactivity in normal and inflamed human lungs obtained from COPD patients and neutrophils with light and immunoelectron microscopy. Immunohistology showed localization of NUCB2/nesfatin-1-like immunoreactivity in the bronchiolar epithelium, alveolar septa, vascular endothelium and various immune cells of normal and inflamed lungs. Further, NUCB2/nesfatin-1-like immunoreactivity accumulated within 0.5 μm of the plasma membrane in human neutrophils following 90 min of 1 ng/mL LPS stimulation. NUCB2/nesfatin-1-like immunoreactivity was also found to localize in euchromatic portions of neutrophilic nuclei at five times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin-1-like immunoreactivity is predominantly cytoplasmic including that in the Golgi complex and vesicles as it localizes at two times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin-1-like immunoreactivity in lungs and neutrophils, and nuclear localization of NUCB2/nesfatin-1 also implicates its potential role in transcriptional regulation.

Neutrophil-derived extracellular vesicles modulate the phenotype of naïve human neutrophils.

Neutrophils (PMN) regulate inflammation in many ways, including communication with other immune cells via extracellular vesicles (EVs). EVs released by human neutrophils activated with N-formylmethionyl-leucyl-phenylalanine (fMLF) (PMN-fMLF EVs) had an outside-out orientation and contained functionally important neutrophil plasma membrane proteins, including flavocytochrome b558, and enzymatically active granule proteins, elastase, and myeloperoxidase. Treatment of naïve PMN with PMN-fMLF EVs primed fMLF-stimulated NADPH oxidase activity, increased surface expression of the complement receptors CD11b/CD18 and CD35, the specific granule membrane protein CD66, and flavocytochrome b558 , and promoted phagocytosis of serum-opsonized Staphylococcus aureus. The primed oxidase activity reflected increased surface expression of flavocytochrome b558 and phosphorylation of SER345 in p47phox , two recognized mechanisms for oxidase priming. Taken together, these data demonstrate that stimulated PMN released EVs that altered the phenotype of naïve phagocytes by priming of the NADPH oxidase activity and augmenting phagocytosis, two responses that are integral to optimal PMN host defense.

Immune Cell Membrane-Coated Biomimetic Nanoparticles for Targeted Cancer Therapy.

Nanotechnology has provided great opportunities for managing neoplastic conditions at various levels, from preventive and diagnostic to therapeutic fields. However, when it comes to clinical application, nanoparticles (NPs) have some limitations in terms of biological stability, poor targeting, and rapid clearance from the body. Therefore, biomimetic approaches, utilizing immune cell membranes, are proposed to solve these issues. For example, macrophage or neutrophil cell membrane coated NPs are developed with the ability to interact with tumor tissue to suppress cancer progression and metastasis. The functionality of these particles largely depends on the surface proteins of the immune cells and their preserved function during membrane extraction and coating process on the NPs. Proteins on the outer surface of immune cells can render a wide range of activities to the NPs, including prolonged blood circulation, remarkable competency in recognizing antigens for enhanced targeting, better cellular interactions, gradual drug release, and reduced toxicity in vivo. In this review, nano-based systems coated with immune cells-derived membranous layers, their detailed production process, and the applicability of these biomimetic systems in cancer treatment are discussed. In addition, future perspectives and challenges for their clinical translation are also presented.

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)