Human Neutrophils In Response Research Articles

Vitamin D Promotes Pneumococcal Killing and Modulates Inflammatory Responses in Primary Human Neutrophils

Streptococcus pneumoniae is a major human pathogen and a leading cause of pneumonia, septicemia, and meningitis worldwide. Despite clinical studies linking vitamin D deficiency and pneumonia, molecular mechanisms behind these observations remain unclear. In particular, the effects of vitamin D on neutrophil responses remain unknown. Using pneumococcal strains, primary neutrophils isolated from human blood, and sera from patients with frequent respiratory tract infections (RTIs), we investigated the effects of vitamin D on neutrophil bactericidal and inflammatory responses, including pattern recognition receptors, antimicrobial peptides, and cytokine regulation. We found that vitamin D upregulated pattern recognition receptors, TLR2, and NOD2, and induced the antimicrobial human neutrophil peptides (HNP1-3) and LL-37, resulting in increased killing of pneumococci in a vitamin D receptor-dependent manner. Antibodies targeting HNP1-3 inhibited bacterial killing. Vitamin D supplementation of serum from patients with bacterial RTIs enhanced neutrophil killing. Moreover, vitamin D lowered inflammatory cytokine production by infected neutrophils via IL-4 production and the induction of suppressor of cytokine signaling (SOCS) proteins SOCS-1 and SOCS-3, leading to the suppression of NF-κB signaling. Thus, vitamin D enhances neutrophil killing of S. pneumoniae while dampening excessive inflammatory responses and apoptosis, suggesting that vitamin D could be used alongside antibiotics when treating pneumococcal infections.

The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state.

PurposePostcardiac arrest syndrome (PCAS) shares many features with sepsis including plasma cytokine elevation with dysregulation of cytokine production, and the presence of endotoxin in plasma. PCAS is closely related to ischemia-reperfusion injury. During ischemia-reperfusion injury, neutrophil, which is the first line of innate immunity, plays a major role. In this study, we investigated the inflammatory response of human neutrophils in an in vitro model which we simulated with hypoxia-normoxia and hypoxia-hyperoxia environments.MethodsAfter separation of neutrophils from the whole blood, they were divided into 3 experimental groups: normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia groups. The production of H2O2, the expression of Toll-like receptor 4 (TLR4) receptor, and the extent of apoptosis of the neutrophils were checked.ResultsThe in vitro hypoxia-normoxia and -hyperoxia models, which simulated the PCAS, showed initiation of the neutrophils' inflammatory reaction by hypoxia insult. Lipopolysaccharide amplifies such inflammation; therefore, prevention of secondary infection may be critical in postresuscitation patients. Temporary hyperoxia following hypoxic insult showed no difference in inflammatory reaction compared with hypoxia-normoxia. Rather, temporary hyperoxia may suppress or minimize inflammation by attenuation of TLR4 receptor.ConclusionIt is well known that continuous hyperoxygenation after successful cardiac arrest harms patients, but temporary hyperoxygenation with 100% O2 in a clinical situation may be helpful.

Integrin Cross-Talk Regulates the Human Neutrophil Response to Fungal β-Glucan in the Context of the Extracellular Matrix: A Prominent Role for VLA3 in the Antifungal Response.

Candida albicans infection produces elongated hyphae resistant to phagocytic clearance compelling alternative neutrophil effector mechanisms to destroy these physically large microbial structures. Additionally, all tissue-based neutrophilic responses to fungal infections necessitate contact with the extracellular matrix (ECM). Neutrophils undergo a rapid, ECM-dependent mechanism of homotypic aggregation and NETosis in response to C. albicans mediated by the β2 integrin, complement receptor 3 (CR3, CD11b/CD18, αMβ2). Neither homotypic aggregation nor NETosis occurs when human neutrophils are exposed either to immobilized fungal β-glucan or to C. albicans hyphae without ECM. The current study provides a mechanistic basis to explain how matrix controls the antifungal effector functions of neutrophils under conditions that preclude phagocytosis. We show that CR3 ligation initiates a complex mechanism of integrin cross-talk resulting in differential regulation of the β1 integrins VLA3 (α3β1) and VLA5 (α5β1). These β1 integrins control distinct antifungal effector functions in response to either fungal β-glucan or C. albicans hyphae and fibronectin, with VLA3 inducing homotypic aggregation and VLA5 regulating NETosis. These integrin-dependent effector functions are controlled temporally whereby VLA5 and CR3 induce rapid, focal NETosis early after binding fibronectin and β-glucan. Within minutes, CR3 undergoes inside-out auto-activation that drives the downregulation of VLA5 and the upregulation of VLA3 to support neutrophil swarming and aggregation. Forcing VLA5 to remain in the activated state permits NETosis but prevents homotypic aggregation. Therefore, CR3 serves as a master regulator during the antifungal neutrophil response, controlling the affinity states of two different β1 integrins, which in turn elicit distinct effector functions.

Naphthofuranone derivatives and other constituents from Pachira aquatica with inhibitory activity on superoxide anion generation by neutrophils

Two new naphthofuranone derivatives, 11-hydroxy-2-O-methylhibiscolactone A (1) and O-methylhibiscone D (2), have been isolated from the stems of Pachira aquatica, together with 18 known compounds (3−20). The structures of two new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, 11-hydroxy-2-O-methylhibiscolactone A (1), isohemigossylic acid lactone-7-methyl ether (4), gmelofuran (6), and 5-hydroxyauranetin (8) exhibited inhibition (IC50≤28.84μM) of superoxide anion generation by human neutrophils in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP).

Zoanthamine-Type Alkaloids from the Zoanthid Zoanthus kuroshio Collected in Taiwan and Their Effects on Inflammation.

Zoanthus kuroshio is a colorful zoanthid with a fluorescent pink oral disc and brown tentacles, which dominates certain parts of the Taiwanese and Japanese coasts. This sea anemone is a rich source of biologically active alkaloids. In the current investigation, two novel halogenated zoanthamines [5α-iodozoanthenamine (1) and 11β-chloro-11-deoxykuroshine A (2)], along with four new zoanthamines [18-epi-kuroshine A (3), 7α-hydroxykuroshine E (4), 5α-methoxykuroshine E (5), and 18-epi-kuroshine E (6)], and six known compounds were isolated from Z. kuroshio. Compounds 1 and 2 are the first examples of halogenated zoanthamine-type alkaloids isolated from nature. Compounds 3 and 6 are the first zoanthamine stereoisomers with a cis-junction of the A/B rings. All isolated compounds were evaluated for their anti-inflammatory activities by measuring their effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP.

Modulation of Human Neutrophil Responses by the Essential Oils from Ferula akitschkensis and Their Constituents.

Essential oils were obtained by hydrodistillation of the umbels+seeds and stems of Ferula akitschkensis (FAEOu/s and FAEOstm, respectively) and analyzed by gas chromatography and gas chromatography-mass spectrometry. Fifty-two compounds were identified in FAEOu/s; the primary components were sabinene, α-pinene, β-pinene, terpinen-4-ol, eremophilene, and 2-himachalen-7-ol, whereas the primary components of FAEOstm were myristicin and geranylacetone. FAEOu/s, β-pinene, sabinene, γ-terpinene, geranylacetone, isobornyl acetate, and (E)-2-nonenal stimulated [Ca(2+)]i mobilization in human neutrophils, with the most potent being geranylacetone (EC50 = 7.6 ± 1.9 μM) and isobornyl acetate 6.4 ± 1.7 (EC50 = 7.6 ± 1.9 μM). In addition, treatment of neutrophils with β-pinene, sabinene, γ-terpinene, geranylacetone, and isobornyl acetate desensitized the cells to N-formyl-Met-Leu-Phe (fMLF)- and interleukin-8 (IL-8)-induced [Ca(2+)]i flux and inhibited fMLF-induced chemotaxis. The effects of β-pinene, sabinene, γ-terpinene, geranylacetone, and isobornyl acetate on neutrophil [Ca(2+)]i flux were inhibited by transient receptor potential (TRP) channel blockers. Furthermore, the most potent compound, geranylacetone, activated Ca(2+) influx in TRPV1-transfected HEK293 cells. In contrast, myristicin inhibited neutrophil [Ca(2+)]i flux stimulated by fMLF and IL-8 and inhibited capsaicin-induced Ca(2+) influx in TRPV1-transfected HEK293 cells. These findings, as well as pharmacophore modeling of TRP agonists, suggest that geranylacetone is a TRPV1 agonist, whereas myristicin is a TRPV1 antagonist. Thus, at least part of the medicinal properties of Ferula essential oils may be due to modulatory effects on TRP channels.

Tetranortriterpenes and Limonoids from the Roots of Aphanamixis polystachya.

Phytochemical investigation of the acetone extract from the roots of Aphanamixis polystachya resulted in isolation of four new tetranortriterpenes (1–4) in addition to one protolimonoid (methyl-1ξ,7R-diacetoxy-23R,25-dihydroxy-20S,24R-21,24-epoxy-3,4-seco-apotirucall-4(28),14(15)-diene-3-oate (5)), five known limonoids (rohituka 3 (6), rohituka 7 (7), nymania 1 (8), rubrin G (9), prieurianin (10)) and a steroid (2,3-dihydroxy-5-pregnan-16-one (11)). Their structures were determined by spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Cytotoxic and anti-inflammatory activities of these compounds were evaluated. Compounds 4 and 5 showed significant inhibition against superoxide generation and elastase release by human neutrophils in response to (formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B) (FMLP/CB).

Neutrophilic superoxide production can assess pharmacological and pharmacogenetic β-adrenoreceptor effects.

Asthma can be controlled well in most patients by inhaled β-adrenoreceptor (β2 AR) agonists and steroids. Poor response to β2 AR agonists is difficult to predict, especially in young children and by lung function testing, which may be affected by multiple influences. As an alternative approach, we analyzed ex vivo neutrophilic superoxide inhibition in response to β2 AR stimulation. In 60 healthy volunteers, this assay was unaffected by sex, age, smoking, atopy or asthma status. Furthermore, we assessed effects of genetic variants in β2 AR by sequencing the ADRB2 gene in our cohort and relating genotypes to β2 AR-mediated neutrophilic superoxide inhibition. Gly16Arg genotypes correlated with minor decrease in overall adrenoresponse in this small study population. Taken together, ex vivo testing of the β2 AR response in human neutrophils represents a robust tool with good signal-to-noise ratio at physiological β2 AR agonist concentrations, and this assay may be useful to complement future pharmacogenetic studies in asthma.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing.

In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense.

Priming of the respiratory burst in neutrophils exposed to a combination of weak constant and alternating low-frequency magnetic fields in vitro

An hour-long exposure of peritoneal neutrophils of mice to a combination of a weak constant magnetic field (42 μT) and low-frequency alternating magnetic fields collinear to the weak constant magnetic field (frequencies 1, 4.4, and 16.5 Hz, total amplitude 0.86 μT) at physiological temperatures promoted a significant increase in chemiluminescence of cells in response to subsequent exposure to low concentrations of respiratory burst activators (formylated peptide N-formyl-Met–Leu–Phe or phorbol ester phorbol-12-myristate-13-acetate) in the presence of luminol. The response of human neutrophils isolated from peripheral blood to the pretreatment with combined magnetic fields followed by exposure to the activator N-formyl-Met–Leu–Phe was similar to the response of mouse neutrophils.

Identification and Actions of a Novel Third Maresin Conjugate in Tissue Regeneration: MCTR3.

Maresin conjugates in tissue regeneration (MCTR) are a new family of evolutionarily conserved chemical signals that orchestrate host responses to promote tissue regeneration and resolution of infections. Herein, we identified the novel MCTR3 and established rank order potencies and matched the stereochemistries of MCTR1, MCTR2 and MCTR3 using material prepared by total organic synthesis and mediators isolated from both mouse and human systems. MCTR3 was produced from endogenous substrate by E. coli activated human macrophages and identified in sepsis patients. Each of the three synthetic MCTR dose-dependently (1–100nM) accelerated tissue regeneration in planaria by 0.6–0.9 days. When administered at the onset or peak of inflammation, each of the MCTR promoted resolution of E. coli infections in mice. They increased bacterial phagocytosis by exudate leukocytes (~15–50%), limited neutrophil infiltration (~20–50%), promoted efferocytosis (~30%) and reduced eicosanoids. MCTR1 and MCTR2 upregulated human neutrophil and macrophage phagocytic responses where MCTR3 also proved to possess potent actions. These results establish the complete stereochemistry and rank order potencies for MCTR1, MCTR2 and MCTR3 that provide novel resolution moduli in regulating host responses to clear infections and promote tissue regeneration.

The effect of continuous low-intensity laser irradiation of the red spectrum on the changes in the functional activity and speed of NADPH-oxidase response of human peripheral blood neutrophils

Registration of the changes in the functional metabolic status of neutrophils under the influence of low-intensity laser radiation (LILR) can be useful for the choice of the exposure parameters in the studies designed to evaluate immunotropic effects. Such investigations, in their turn, are very promising in clinical terms since their results can be used to optimize the laser therapy techniques. The objective of the present study was to evaluate the degranulation potential of neutrophils and the intensity of their NADPH-oxidase reaction in response to the exposure to laser radiation as exemplified by the release of lysosomal granules in vitro. The source of radiation was the LASMIK device operated in the continuous mode at the following parameters: wavelength 635 nm, power density 0.12 mW/cm2, exposure time 10, 30, 90, 120, 150 s in the case of the automatic timer control and 100 s in the case of manual shutdown. It was shown that the maximum lysosomal activity and release of lysosomal granules took place at a 90-10 s exposure and a wavelength of 635 nm with the appearance of a plateau within 120 and 150 s after the onset of irradiation. In the case of a shorter exposure of neutrophil granulocytes to LILR (10 and 30 s) no pronounced effect was observed. It means that low-intensity laser radiation of the red spectrum with a wavelength of 635 nm is a physical stimulus reinforcing exocytosis of lysosomal granules by neutrophils in vitro. The reliable changes of NADPH-oxidase activity of neutrophil granulocytes isolated from donor peripheral blood are recorded at an optimum exposure time of 90-100 s. It is concluded that the laser therapy techniques, at least those designed to regulate neutrophils should be applied with the optimum exposure time of 90-100 s.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

3-Methyl-4,5-dihydro-oxepine, polyoxygenated seco-cyclohexenes and cyclohexenes from Uvaria flexuosa and their anti-inflammatory activity

The phytochemical investigation of the methanolic extract of Uvaria flexuosa (Annonaceae) leaves led to the isolation of seven compounds including, 3-methyl-4,5-dihydro-oxepine (flexuvaroxepine A), four polyoxygenated seco-cyclohexene (flexuvarin A–D) and two polyoxygenated cyclohexene (flexuvarol A–B) derivatives, together with four known flavones. The structures of the isolated compounds were elucidated using different spectroscopic techniques. A plausible biogenetic route of the new compounds was discussed. The anti-inflammatory activity of the isolated compounds was evaluated by superoxide anion generation and elastase release assays. Among the tested compounds, flexuvarol B and chrysin showed the most potent anti-inflammatory effect by inhibiting superoxide anion generation and elastase release from human neutrophils in response to fMLP with IC50 2.25–5.55μM. Moreover, 5-hydroxy-6,7-dimethoxy-flavone showed selective inhibitory activity on superoxide anion generation (IC50=1.19±0.34μM).

Differential changes in gene expression in human neutrophils following TNF-α stimulation: Up-regulation of anti-apoptotic proteins and down-regulation of proteins involved in death receptor signaling.

Responses of human neutrophils to TNF-α are complex and multifactorial. Exposure of human neutrophils to TNF-α in vitro primes the respiratory burst, delays apoptosis and induces the expression of several genes including chemokines, and TNF-α itself. This study aimed to determine the impact of TNF-α exposure on the expression of neutrophil genes and proteins that regulate apoptosis. Quantitative PCR and RNA-Seq, identified changes in expression of several apoptosis regulating genes in response to TNF-α exposure. Up-regulated genes included TNF-α itself, and several anti-apoptotic genes, including BCL2A1, CFLAR (cFLIP) and TNFAIP3, whose mRNA levels increased above control values by between 4-20 fold (n = 3, P < 0.05). In contrast, the expression of pro-apoptotic genes, including CASP8, FADD and TNFRSF1A and TNFRSF1B, were significantly down-regulated following TNF-α treatment. These changes in mRNA levels were paralleled by decreases in protein levels of caspases 8 and 10, TRADD, FADD, TNFRSF1A and TNFRSF1B, and increased cFLIP protein levels, as detected by western blotting. These data indicate that when neutrophils are triggered by TNF-α exposure, they undergo molecular changes in transcriptional expression to up-regulate expression of specific anti-apoptotic proteins and concomitantly decrease expression of specific proteins involved in death receptor signaling which will alter their function in TNF-α rich environments.

Activation of NLRP3 inflammasome in human neutrophils by Helicobacter pylori infection.

TLRs and NLRs participate in the immune system recognition of Helicobacter pylori. However, little is known about the mechanisms leading to inflammasome activation by H. pylori and if NLRs in neutrophils are involved in the process. We studied how NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome components are involved in IL-1β maturation in human neutrophils in response to the infection and if they are dependent on T4SS (type IV secretion system) and TLRs. Human neutrophils were cultured and infected with the 26695 or the VirD4- H. pylori strains; the IL-1β concentration was analyzed by ELISA, and we also evaluated the activation of TLRs 2 and 4. The infection of neutrophils with both strains of H. pylori induced production of IL-1β and expression of the NLRP3 inflammasome components such as apoptosis-associated speck-like protein with CARD domain and NLRP3 protein. The infection also increased the activity of caspase-1, which is required for the maturation of IL-1β. Our study shows, for the first time, that H. pylori infection induces the expression and activation of components of NLRP3 inflammasomes in human neutrophils and that the activation is independent of a functional T4SS and TLR2 and TLR4.

New Flavones, a 2-(2-Phenylethyl)-4H-chromen-4-one Derivative, and Anti-Inflammatory Constituents from the Stem Barks of Aquilaria sinensis.

In the current study, two new flavones, 4′-O-geranyltricin (1) and 3′-O-geranylpolloin (2), and a new 2-(2-phenylethyl)-4H-chromen-4-one derivative, 7-hydroxyl-6-methoxy-2-(2-phenylethyl)chromone (3), have been isolated from the stem barks of A. sinensis, together with 21 known compounds 4–24. The structures of new compounds 1–3 were determined through spectroscopic and MS analyses. Compounds 2, 3, 5, 6, and 8–10 exhibited inhibition (IC50 ≤ 12.51 μM) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3, 6, 8, 10, and 19 inhibited fMLP/CB-induced elastase release with IC50 values ≤ 15.25 μM. This investigation reveals bioactive isolates (especially 2, 3, 5, 6, 8, 9, 10, and 19) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.

Ellagitannins modulate the inflammatory response of human neutrophils ex vivo

BackgroundTannin-rich plant materials are commonly used in the traditional medicine as external anti-inflammatory, antioxidant and antimicrobial agents. Plant extracts containing significant quantities of tannins are often used in the prevention and treatment of oral cavity diseases such as periodontosis or gingivitis. The contribution of pure ellagitannins to the observed anti-inflammatory activity of tannin-rich remedies is still not resolved. PurposeThe aim of the present study the study was to establish if ellagitannins and their precursor – pentagalloylglucose (1) can modulate the inflammatory response of ex-vivo stimulated neutrophils. MethodsHuman neutrophils were isolated from the buffy coats obtained from healthy volunteers. Neutrophils were cultivated with or without tested compounds. The influence of ellagitannins and 1 on the production and release of pro-inflammatory factors such as elastase, reactive oxygen species, interleukin-8, tumour necrosis factor alpha (TNF-α) and metalloproteinase-9 was evaluated using ELISA sets or chemical methods. The effect on surface expression of toll like receptor 4 (TLR-4) and apoptosis was also checked using flow cytometry. ResultsThe results showed that ellgitannins modulate the inflammatory response of human neutrophils by the inhibition of production and release of chosen cytokines and pro-inflammatory enzymes. By the induction of TNF-α ellagitannins enhance neutrophil apoptosis, which is of interest in the case of chronic inflammation within oral cavity. Ellagitannins also decrease the surface expression of TLR-4 in activated neutrophils. ConclusionThe results support the traditional use of tannin-rich products in the prevention and treatment of oral cavity diseases. The present study proves the substantial contribution of ellagitannins to the anti-inflammatory activity of tannin-rich medicinal plant materials.

Limonoids from the Seeds of Swietenia macrophylla and Their Anti-Inflammatory Activities.

A new limonoid, swietemacrophin (1), was isolated from the seeds of Swietenia macrophylla, together with five known compounds 2–6. The structure of 1 was determined through extensive 1D/2D-NMR and mass-spectrometric analyses. Swietemacrophin (1), humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), 3-O-tigloylswietenolide (4), and swietemahonin E (5) exhibited inhibition (IC50 values ≤ 45.44 μM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Compounds 1, 4, 5, and swietenine (6) showed potent inhibition with IC50 values ≤ 36.32 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation.

Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.