Oxidative Burst Response Research Articles

A New Approach for CRISPR/Cas9 Editing and Selection of Pathogen-Resistant Plant Cells of Wine Grape cv. 'Merlot'.

Grape is one of the most economically significant berry crops. Owing to the biological characteristics of grapes, such as the long juvenile period (5-8 years), high degree of genome heterozygosity, and the frequent occurrence of inbreeding depression, homozygosity during crossbreeding leads to loss of varietal characteristics and viability. CRISPR/Cas editing has become the tool of choice for improving elite technical grape varieties. This study provides the first evidence of a decrease in the total fraction of phenolic compounds and an increase in the concentration of peroxide compounds in grape callus cells upon the addition of chitosan to the culture medium. These previously unreported metabolic features of the grape response to chitosan have been described and used for the first time to increase the probability of selecting plant cells with MLO7 knockout characterised by an oxidative burst in response to the presence of a pathogen modulated by chitosan in the high-metabolite black grape variety 'Merlot'. This was achieved by using a CRISPR/Cas9 editing vector construction with the peroxide sensor HyPer as a reporter. This research represents the first CRISPR/Cas9 editing of 'Merlot', one of the most economically important elite technical grape varieties.

Insights into maydis leaf blight resistance in maize: a comprehensive genome-wide association study in sub-tropics of India

BackgroundIn the face of contemporary climatic vulnerabilities and escalating global temperatures, the prevalence of maydis leaf blight (MLB) poses a potential threat to maize production. This study endeavours to discern marker-trait associations and elucidate the candidate genes that underlie resistance to MLB in maize by employing a diverse panel comprising 336 lines. The panel was screening for MLB across four environments, employing standard artificial inoculation techniques. Genome-wide association studies (GWAS) and haplotype analysis were conducted utilizing a total of 128,490 SNPs obtained from genotyping-by-sequencing (GBS).ResultsGWAS identified 26 highly significant SNPs associated with MLB resistance, among the markers examined. Seven of these SNPs, reported in novel chromosomal bins (9.06, 5.01, 9.01, 7.04, 4.06, 1.04, and 6.05) were associated with genes: bzip23, NAGS1, CDPK7, aspartic proteinase NEP-2, VQ4, and Wun1, which were characterized for their roles in diminishing fungal activity, fortifying defence mechanisms against necrotrophic pathogens, modulating phyto-hormone signalling, and orchestrating oxidative burst responses. Gene mining approach identified 22 potential candidate genes associated with SNPs due to their functional relevance to resistance against necrotrophic pathogens. Notably, bin 8.06, which hosts five SNPs, showed a connection to defense-regulating genes against MLB, indicating the potential formation of a functional gene cluster that triggers a cascade of reactions against MLB. In silico studies revealed gene expression levels exceeding ten fragments per kilobase million (FPKM) for most genes and demonstrated coexpression among all candidate genes in the coexpression network. Haplotype regression analysis revealed the association of 13 common significant haplotypes at Bonferroni ≤ 0.05. The phenotypic variance explained by these significant haplotypes ranged from low to moderate, suggesting a breeding strategy that combines multiple resistance alleles to enhance resistance to MLB. Additionally, one particular haplotype block (Hap_8.3) was found to consist of two SNPs (S8_152715134, S8_152460815) identified in GWAS with 9.45% variation explained (PVE).ConclusionThe identified SNPs/ haplotypes associated with the trait of interest contribute to the enrichment of allelic diversity and hold direct applicability in Genomics Assisted Breeding for enhancing MLB resistance in maize.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhanced oxidative stress resistance in Ustilago maydis and its implications on the virulence

The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H2O2).To achieve this, we exposed U. maydis SG200 to 20 escalating H2O2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H2O2, a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1–149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H2O2; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H2O2 resistance.In summary, enhanced H2O2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H2O2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms.

OsACA9, an Autoinhibited Ca2+-ATPase, Synergically Regulates Disease Resistance and Leaf Senescence in Rice.

Calcium (Ca2+) is a versatile intracellular second messenger that regulates several signaling pathways involved in growth, development, stress tolerance, and immune response in plants. Autoinhibited Ca2+-ATPases (ACAs) play an important role in the regulation of cellular Ca2+ homeostasis. Here, we systematically analyzed the putative OsACA family members in rice, and according to the phylogenetic tree of OsACAs, OsACA9 was clustered into a separated branch in which its homologous gene in Arabidopsis thaliana was reported to be involved in defense response. When the OsACA9 gene was knocked out by CRISPR/Cas9, significant accumulation of reactive oxygen species (ROS) was detected in the mutant lines. Meanwhile, the OsACA9 knock out lines showed enhanced disease resistance to both rice bacterial blight (BB) and bacterial leaf streak (BLS). In addition, compared to the wild-type (WT), the mutant lines displayed an early leaf senescence phenotype, and the agronomy traits of their plant height, panicle length, and grain yield were significantly decreased. Transcriptome analysis by RNA-Seq showed that the differentially expressed genes (DEGs) between WT and the Osaca9 mutant were mainly enriched in basal immune pathways and antibacterial metabolite synthesis pathways. Among them, multiple genes related to rice disease resistance, receptor-like cytoplasmic kinases (RLCKs) and cell wall-associated kinases (WAKs) genes were upregulated. Our results suggest that the Ca2+-ATPase OsACA9 may trigger oxidative burst in response to various pathogens and synergically regulate disease resistance and leaf senescence in rice.

Phosphopeptides P140 cause oxidative burst responses of pulmonary macrophages in an imiquimod-induced lupus model

Recent studies challenge the dogma that a 21-mer phosphopeptide P140 protects against direct cell damage in the phase-III clinical trial (NCT02504645) for lupus, involving reactive oxygen species (ROS)-dependent release of citrullinated histone H3 (H3cit)-linked neutrophil extracellular traps. An open question is the cellular location of ROS production and H3cit formation in lupus. In this study, we examined the effects of P140 peptides on ROS production and H3cit location in lupus with in vivo and situ fluorescence imaging with subcellular resolution. We developed a mouse model of the B6 strain harbouring a bioluminescent reporter under the control of the Lysozyme M promoter. Based on the imiquimod-induced disease model of B6 mice, we used bioluminescent imaging, flow cytometry analysis, and immunohistology staining to study the effects of P140 peptides in lupus. We found a profound accumulation of CX3CR1-positive macrophages in the lungs of lupus mice after the application of P140, accompanied by lung fibrosis formation. The defined P140-mediated macrophage responses were associated with an increase of H3cit in the cytosol, interleukin-1 receptor type 1 on the extracellular membrane, and intracellular production of ROS. Of interest, the disease of imiquimod-induced lupus was prevented with an antioxidant drug apocynin. This study shows that P140 peptides play a role in aggravated murine lupus in a manner dependent on ROS production and H3cit upregulation through pulmonary macrophages.

E-cigarette vapor renders neutrophils dysfunctional due to filamentous actin accumulation

Electronic cigarette (e-cigarette) use continues to rise despite concerns of long-term effects, especially the risk of developing lung diseases such as chronic obstructive pulmonary disease. Neutrophils are central to the pathogenesis of chronic obstructive pulmonary disease, with changes in phenotype and function implicated in tissue damage. We sought to measure the impact of direct exposure to nicotine-containing and nicotine-free e-cigarette vapor on human neutrophil function and phenotype. Neutrophils were isolated from the whole blood of self-reported nonsmoking, nonvaping healthy volunteers. Neutrophils were exposed to 40 puffs of e-cigarette vapor generated from e-cigarette devices using flavorless e-cigarette liquids with and without nicotine before functions, deformability, and phenotype were assessed. Neutrophil surface marker expression was altered, with CD62L and CXCR2 expression significantly reduced in neutrophils treated with e-cigarette vapor containing nicotine. Neutrophil migration to IL-8, phagocytosis of Escherichia coli and Staphylococcus aureus pHrodo bioparticles, oxidative burst response, and phorbol 12-myristate 13-acetate-stimulated neutrophil extracellular trap formation were all significantly reduced by e-cigarette vapor treatments, independent of nicotine content. E-cigarette vapor induced increased levels of baseline polymerized filamentous actin levels in the cytoplasm, compared with untreated controls. The significant reduction in effector neutrophil functions after exposure to high-power e-cigarette devices, even in the absence of nicotine, is associated with excessive filamentous actin polymerization. This highlights the potentially damaging impact of vaping on respiratory health and reinforces the urgency of research to uncover the long-term health implications of e-cigarettes.

Hyaluronan promotes intracellular ROS production and apoptosis in TNFα-stimulated neutrophils.

Hyaluronan (HA) is an important structural component of the extracellular matrix and has well-described roles in maintaining tissue integrity and homeostasis. With inflammation, HA metabolism (synthesis and degradation) increases and results in higher concentrations of soluble HA. Previously, we demonstrated that (soluble) HA primed resting neutrophils for the oxidative burst in response to a secondary stimulus. Notably, HA-mediated priming was not dependent on degranulation, which is a hallmark of priming by classical agents such as TNFα. In this study, we queried the ability of HA to prime neutrophils to different stimuli and its capacity to modulate neutrophil function in the presence of TNFα. Blood neutrophils from healthy donors were stimulated ex vivo with HA in the absence and presence of classic neutrophil agonists, inclusive of TNFα. Western blotting was used to assess the activation (phosphorylation) of p38 MAPK, and key neutrophil functions associated with priming and activation, such as intracellular and extracellular ROS production, degranulation, and apoptosis, were evaluated by standard chemiluminescence assays (ROS) and flow cytometry. Hyaluronan is capable of atypical priming and, with TNFα, co-priming neutrophils for an enhanced (rate and/or magnitude) oxidative burst to various secondary stimuli. In addition, HA can augment intracellular ROS production that is directly induced by TNFα in resting neutrophils, which coincided with the activation of p38 MAPK and apoptosis. These data demonstrate that the extracellular matrix component HA is a key modulator of neutrophil function(s) in the presence of inflammatory agents such as TNFα. Moreover, it provides additional evidence for the diversity and complexity of neutrophil priming and activation during inflammation.

Mechanisms of host manipulation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae (also known as gonococcus) has been causing gonorrhoea in humans since ancient Egyptian times. Today, global gonorrhoea infections are rising at an alarming rate, in concert with an increasing number of antimicrobial-resistant strains. The gonococcus has concurrently evolved several intricate mechanisms that promote pathogenesis by evading both host immunity and defeating common therapeutic interventions. Central to these adaptations is the ability of the gonococcus to manipulate various host microenvironments upon infection. For example, the gonococcus can survive within neutrophils through direct regulation of both the oxidative burst response and maturation of the phagosome; a concerning trait given the important role neutrophils have in defending against invading pathogens. Hence, a detailed understanding of how N. gonorrhoeae exploits the human host to establish and maintain infection is crucial for combating this pathogen. This review summarizes the mechanisms behind host manipulation, with a central focus on the exploitation of host epithelial cell signaling to promote colonization and invasion of the epithelial lining, the modulation of the host immune response to evade both innate and adaptive defenses, and the manipulation of host cell death pathways to both assist colonization and combat antimicrobial activities of innate immune cells. Collectively, these pathways act in concert to enable N. gonorrhoeae to colonize and invade a wide array of host tissues, both establishing and disseminating gonococcal infection.

Reprisal of Schima superba to Mn stress and exploration of its defense mechanism through transcriptomic analysis.

One of the most diverse protein families, ATP-binding cassette (ABC) transporters, play a role in disease resistance, heavy metal tolerance, and food absorption.Differentially expressed genes contribute in the investigation of plant defense mechanisms under varying stress conditions. To elucidate the molecular mechanisms involved in Mn metal stress, we performed a transcriptomic analysis to explore the differential gene expression in Schima superba with the comparison of control. A total of 79.84 G clean data was generated and 6558 DEGs were identified in response to Mn metal stress. Differentially expressed genes were found to be involved in defense, signaling pathways, oxidative burst, transcription factors and stress responses. Genes important in metal transport were more expressive in Mn stress than control plants. The investigation of cis-acting regions in the ABC family indicated that these genes might be targeted by a large variety of trans-acting elements to control a variety of stress circumstances. Moreover, genes involved in defense responses, the mitogen-activated protein kinase (MAPK) signaling and signal transduction in S. superba were highly induced in Mn stress. Twenty ABC transporters were variably expressed on 1st, 5th, and 10th day of Mn treatment, according to the qRT PCR data. Inclusively, our findings provide an indispensable foundation for an advanced understanding of the metal resistance mechanisms. Our study will enrich the sequence information of S. superba in a public database and would provide a new understanding of the molecular mechanisms of heavy metal tolerance and detoxification.

Polysaccharides from tropical green seaweed Chaetomorpha antennina induces non-specific immune responses and improves antioxidative activities in common carp (Cyprinus carpio) leukocyte culture cell line

Polysaccharides from seaweeds are promising biomolecules that could sustainably improve the health status of cultured fish species. In this study, water-soluble polysaccharides extracted from Chaetomorpha antennina were investigated to determine their physicochemical characteristics and biological activities. C. antennina water-soluble polysaccharides (CAWP) comprised mainly of carbohydrate (69.0 ± 2.3 %), sulphate (12.6 ± 0.5 %), uronic acid (8.8 ± 0.1 %) and protein (3.5 ± 0.2 %). High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) analysis revealed that CAWP contained mostly arabinose (17.1 ± 2.3 mg/L), galactose (9.3 ± 1.0 mg/L), glucose (5.1 ± 0.6 mg/L), xylose/mannose (1.4 ± 0.6 mg/L) and a small proportion of fucose (0.3 ± 0.1 mg/L). CAWP, with intrinsic viscosity of 0.66 dL/g and estimated molecular weight of 318 kDa, were non-toxic to Carp leukocyte culture (CLC) cell line at concentrations ≤25 μg/mL. CAWP at concentrations of 12.5 and 25 μg/mL stimulated CLC to release significant amount of nitric oxide (NO), reactive oxygen species (ROS) and augmented phagocytic activity. Intracellular antioxidant activities indicated that CAWP did not induce oxidative damage in CLC but could improve superoxide dismutase activity (SOD), total glutathione (tGSH) and decrease malondialdehyde (MDA) level. The findings from our study provide a foundation for the applicability of water-soluble polysaccharides from C. antennina in the aquaculture industry as a potent immunomodulatory agent. • Chaetomorpha antennina is a rich source of sulphated polysaccharides. • Induction of oxidative burst response in Carp leucocytes • Phagocytosis of Red Zymosan® bioparticles by Carp leucocytes • Improved intracellular antioxidant activities

Glycolysis and the Pentose Phosphate Pathway Promote LPS-Induced NOX2 Oxidase- and IFN-β-Dependent Inflammation in Macrophages.

Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-β expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-β response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-β increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.

Comparison of the oxidative respiratory burst and mitogen-induced leukocyte responses of camels, goats, sheep, and cows.

Background and Aim:The reports from the Ministry of Agriculture and Fisheries suggest that camels suffer less compared to goats, sheep, and cows from a number of common infectious diseases in Oman. However, there is no immunological evidence to substantiate this claim. This present study is, therefore, an attempt to study the immunological responses of camels, goats, sheep, and cows by comparing their oxidative respiratory burst of peripheral blood leukocytes (PBLs) as a marker of innate immunity occurring during phagocytosis and the mitogenic responses of their peripheral blood mononuclear leukocytes (PBMLs) as a marker of their adaptive immune response.Materials and Methods:Ten female adult animals (n = 10) were selected from each species (goats, sheep, and cows). The goats, sheep, and cows were maintained at the Agricultural Experiment Station, while camels were kept at the Royal Camel Corps (RCC). Blood samples were collected from the jugular vein in 7 mL of heparin and ethylenediaminetetraacetic acid vacutainer tubes. The oxidative respiratory burst of PBLs was measured using a chemiluminescence (CL) assay. Reactants consisted of 75 mL of whole blood diluted (1:50), 75 mL of luminol/isoluminol, and 75 mL of zymosan opsonized with non-heat inactivated serum/heat-inactivated serum or non-opsonized zymosan. CL responses were measured as relative light units and expressed as the mean count per minute and peak CL values. The mitogenic response of PBMLs to concanavalin A (Con-A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) was tested using a WST-8 assay and read spectrophotometrically at 450 nm.Results:The present findings showed that camel PBLs generate significantly higher CL responses, both intracellularly as well as extracellularly, with zymosan opsonized with autologous serum. Camel PBLs demonstrated a significantly higher (p = 0.001) response when stimulated with zymosan opsonized with heat-inactivated serum compared to those of goat, sheep, and cow lymphocytes from camels exhibited significantly higher (p = 0.001) stimulation indices (SI) with Con-A, PHA, and PWM.Conclusion:The present study suggests that camels are capable of mounting both superior innate as well as adaptive immune responses and provide immunological evidence supporting the belief of some authors, who have proposed that camels are less susceptible to a number of common infectious diseases than other domesticated ruminants.

Impairment of neutrophil functions and homeostasis in COVID-19 patients: association with disease severity

BackgroundA dysregulated immune response is emerging as a key feature of critical illness in COVID-19. Neutrophils are key components of early innate immunity that, if not tightly regulated, contribute to uncontrolled systemic inflammation. We sought to decipher the role of neutrophil phenotypes, functions, and homeostasis in COVID-19 disease severity and outcome.MethodsBy using flow cytometry, this longitudinal study compares peripheral whole-blood neutrophils from 90 COVID-19 ICU patients with those of 22 SARS-CoV-2-negative patients hospitalized for severe community-acquired pneumonia (CAP) and 38 healthy controls. We also assessed correlations between these phenotypic and functional indicators and markers of endothelial damage as well as disease severity.ResultsAt ICU admission, the circulating neutrophils of the COVID-19 patients showed continuous basal hyperactivation not seen in CAP patients, associated with higher circulating levels of soluble E- and P-selectin, which reflect platelet and endothelial activation. Furthermore, COVID-19 patients had expanded aged-angiogenic and reverse transmigrated neutrophil subsets—both involved in endothelial dysfunction and vascular inflammation. Simultaneously, COVID-19 patients had significantly lower levels of neutrophil oxidative burst in response to bacterial formyl peptide. Moreover patients dying of COVID-19 had significantly higher expansion of aged-angiogenic neutrophil subset and greater impairment of oxidative burst response than survivors.ConclusionsThese data suggest that neutrophil exhaustion may be involved in the pathogenesis of severe COVID-19 and identify angiogenic neutrophils as a potentially harmful subset involved in fatal outcome.Graphic

Influence of Flagellin Polymorphisms, Gene Regulation, and Responsive Memory on the Motility of Xanthomonas Species That Cause Bacterial Spot Disease ofSolanaceous Plants.

Increasingly, new evidence has demonstrated variability in the epitope regions of bacterial flagellin, including in regions harboring the microbe-associated molecular patterns flg22 and flgII-28 that are recognized by the pattern recognition receptors FLS2 and FLS3, respectively. Additionally, because bacterial motility is known to contribute to pathogen virulence and chemotaxis, reductions in or loss of motility can significantly reduce bacterial fitness. In this study, we determined that variations in flg22 and flgII-28 epitopes allow some but not all Xanthomonas spp. to evade both FLS2- and FLS3-mediated oxidative burst responses. We observed variation in the motility for many isolates, regardless of their flagellin sequence. Instead, we determined that past growth conditions may have a significant impact on the motility status of isolates, because we could minimize this variability by inducing motility using chemoattractant assays. Additionally, motility could be significantly suppressed under nutrient-limited conditions, and bacteria could "remember" its prior motility status after storage at ultracold temperatures. Finally, we observed larger bacterial populations of strains with flagellin variants predicted not to be recognized by either FLS2 or FLS3, suggesting that these bacteria can evade flagellin recognition in tomato plants. Although some flagellin variants may impart altered motility and differential recognition by the host immune system, external growth parameters and gene expression regulation appear to have more significant impacts on the motility phenotypes for these Xanthomonas spp.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin.

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.

Elicitation of defense response by transglycosylated chitooligosaccharides in rice seedlings

Long-chain chitooligosaccharides (COS) with degree of polymerization (DP) more than 4 are known to have potential biological activities. A hyper-transglycosylating mutant of an endo-chitinase from Serratia proteamaculans (SpChiD-Y28A) was used to synthesize COS with DP6 and DP7 using COS DP5 as substrate. Purified COS with DP5-7 were tested to elicit the defense response in rice seedlings. Among the COS used, DP7 strongly induced oxidative burst response as well as peroxidase, and phenylalanine ammonia lyase activites. A few selected marker genes in salicylic acid (SA)- and jasmonic acid-dependent pathways were evaluated by real-time PCR. The expression levels of pathogenesis-related (PR) genes PR1a and PR10 and defense response genes (chitinase1, peroxidase and β -1,3-glucanase) were up regulated upon COS treatment in rice seedlings. The DP7 induced Phenylalanine ammonia lyase and Isochorismate synthase 1 genes, with concomitant increase of Mitogen-activated protein kinase 6 and WRKY45 transcription factor genes indicated the possible role of phosphorylation in the transmission of a signal to induce SA-mediated defense response in rice.

CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti-Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4+ and CD8+ T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA.