Activation Of Human Neutrophils Research Articles

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches

ABSTRACT Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017–2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Abstract 170: Late CCL23 Response is Associated With Poor Outcomes After Cardiac Arrest

Introduction: Chemokines mediate the entrance and migration of leukocytes to inflamed tissue areas, contributing to the intensity of local inflammation. We found a high level of circulating chemokines, CCL2, CCL4, and CCL23, in patients resuscitated after cardiac arrest (CA). Hypothesis: The level of circulating chemokines is associated with systemic immune cell response, neurologic injury, and outcome after cardiac arrest. Goal: To characterize temporal chemokine and immune cell responses to cardiac arrest. Methods: A total of 42 patients after CA and 22 control subjects who underwent coronary artery bypass grafting (CABG) were enrolled. ELISA was used to determine chemokines in plasma at 6 hr, 24 hr, 48 hr, and 72 hr after CA and in CABG patients pre-operatively. Flow cytometry was used to determine subpopulations of immune cells. We measured changes in chemokines over time and the association of chemokine levels with outcome, markers of inflammation, and brain injury. A mouse model of CA was employed to assess the activation of human neutrophils locally in brain tissue. Results: Temporal dynamics were different for each chemokine, with early increases in CCL2 and CCL4, followed by a late elevation in CCL23 at 48 hours after resuscitation. A higher level of CCL23 was associated with a worse cerebral performance category score (r s =0.3, p=0.042) and higher mortality (r s =0.3, p=0.039). Positive correlations were found between CCL23 and an increased number of neutrophils, cellular mediators of inflammation, and levels of neuron-specific enolase. To study the potential role of neutrophil activation locally in injured brain tissue, we injected human post-CA neutrophils into the bloodstream of successfully resuscitated mice and measured the CCL23 in human neutrophils in mouse blood and brain 24 hours after resuscitation. An increased level of CCL23 production was found in mouse brain neutrophils compared to those in the peripheral circulation. Conclusion: It is well known that an early intense inflammatory response is associated with poor outcomes after CA. Our new data indicate that late activation of neutrophils in brain tissue may represent an additional mechanism contributing to ongoing brain tissue injury via the production of CCL23 after cardiac arrest.

Differential Transcriptional Responses of Human Granulocytes to Fungal Infection with Candida albicans and Aspergillus fumigatus.

Neutrophils are critical phagocytic cells in innate immunity, playing a significant role in defending against invasive fungal pathogens. This study aimed to explore the transcriptional activation of human neutrophils in response to different fungal pathogens, including Candida albicans and Aspergillus fumigatus, compared to the bacterial pathogen Escherichia coli. We identified distinct transcriptional profiles and stress-related pathways in neutrophils during fungal infections, highlighting their functional diversity and adaptability. The transcriptional response was largely redundant across all pathogens in immune-relevant categories and cytokine pathway activation. However, differences in the magnitude of differentially expressed genes (DEGs) were observed, with A. fumigatus inducing a lower transcriptional effect compared to C. albicans and E. coli. Notably, specific gene signatures associated with cell death were differentially regulated by fungal pathogens, potentially increasing neutrophil susceptibility to autophagy, pyroptosis, and neutrophil extracellular trap (NET) formation. These findings provide valuable insights into the complex immunological responses of neutrophils during fungal infections, offering new avenues for diagnostic and therapeutic strategies, particularly in the management of invasive fungal diseases.

67 Infant Health Status Impacts the Neutrophil Phenotypes and Polyunsaturated Fatty Acids Composition in Human Milk

Abstract Background The abundance of human milk leukocytes, including neutrophils, changes in response to the health status of mothers and their infants. Elevated leukocyte counts occur during infant infection and return to baseline levels upon recovery. However, it is unclear how infant health status can affect the neutrophil phenotypes and the concentration of fatty acids in their mothers’ human milk. Objectives This study investigates the association between infant health status and human milk neutrophil counts and activation state, and fatty acid levels, in human milk. Design/Methods This is a prospective cohort study of 50 healthy breastfeeding mothers recruited from St. Michael’s hospital, in Toronto, Ontario, who were followed up from 2-4 weeks until 4 months postpartum. Human milk samples were collected from participants and data regarding infant health status were collected from self-reported questionnaires completed by participants at both timepoints, then imputed based on whether infants had taken antibiotics, had been hospitalized, or had been diagnosed with a medical condition, at least 2 weeks prior to samples collection. Neutrophils were quantified using flow cytometry and labelled with a panel of antibodies to detect specific cluster of differentiation (CD) biomarkers. Fatty acids were identified and quantified using a gas chromatography-flame ionization detector (GC-FID). Linear mixed-effects models were used to evaluate the correlation between infant health status and changes in neutrophil counts, along with their expressed CD markers, and fatty acids composition in human milk during lactation. Results The CD markers were classified into 4 categories based on their function: degranulation/activation markers (CD63, CD64, and CD66a), an immunoregulation marker (CD16), adhesion markers (CD11b, CD18), and a lipopolysaccharide receptor (CD14). Human milk from mothers whose infants had a health condition had neutrophils which significantly expressed elevated levels of the CD64 biomarker (β: 85.65, p=0.009 in adjusted models), compared to human milk from mothers of healthy infants. However, there were no significant associations between infant health status, absolute hmPMN counts, andother CD biomarkers (p>0.05). There were also elevated levels of arachidonic acid (β: 0.12, p=0.013) and C22:5n-6 (β: 0.06, p=0.015) fatty acids in the human milk of mothers whose infants had a health condition, compared with human milk from mothers of healthy infants, after adjusting for maternal age, post-pregnancy body mass index, infant sex, and infant feeding pattern. Conclusion This study demonstrates that infant health status is associated with changes in human milk immunological components, suggesting an inflammatory protective response mechanism in the mammary glands triggered by infants infection. How these alterations can affect infant health outcomes in the short and longer terms need critical consideration.

A secreted sirtuin from Campylobacter jejuni contributes to neutrophil activation and intestinal inflammation during infection.

Histone modifications control numerous processes in eukaryotes, including inflammation. Some bacterial pathogens alter the activity or expression of host-derived factors, including sirtuins, to modify histones and induce responses that promote infection. In this study, we identified a deacetylase encoded by Campylobacter jejuni which has sirtuin activities and contributes to activation of human neutrophils by the pathogen. This sirtuin is secreted from the bacterium into neutrophils, where it associates with and deacetylates host histones to promote neutrophil activation and extracellular trap production. Using the murine model of campylobacteriosis, we found that a mutant of this bacterial sirtuin efficiently colonized the gastrointestinal tract but was unable to induce cytokine production, gastrointestinal inflammation, and tissue pathology. In conclusion, these results suggest that secreted bacterial sirtuins represent a previously unreported class of bacterial effector and that bacterial-mediated modification of host histones is responsible for the inflammation and pathology that occurs during campylobacteriosis.

Innate activation of human neutrophils and neutrophil-like cells by the pro-inflammatory bacterial metabolite ADP-heptose and Helicobacter pylori

Lipopolysaccharide inner core heptose metabolites, including ADP-heptose, play a substantial role in the activation of cell-autonomous innate immune responses in eukaryotic cells, via the ALPK1-TIFA signaling pathway, as demonstrated for various pathogenic bacteria. The important role of LPS heptose metabolites during Helicobacter pylori infection of the human gastric niche has been demonstrated for gastric epithelial cells and macrophages, while the role of heptose metabolites on human neutrophils has not been investigated. In this study, we aimed to gain a better understanding of the activation potential of bacterial heptose metabolites for human neutrophil cells. To do so, we used pure ADP-heptose and, as a bacterial model, H. pylori, which can transport heptose metabolites into the human host cell via the Cag Type 4 Secretion System (CagT4SS). Main questions were how bacterial heptose metabolites impact on the pro-inflammatory activation, alone and in the bacterial context, and how they influence maturation of human neutrophils. Results of the present study demonstrated that neutrophils respond with high sensitivity to pure heptose metabolites, and that global regulation networks and neutrophil maturation are influenced by heptose exposure. Furthermore, activation of human neutrophils by live H. pylori is strongly impacted by the presence of LPS heptose metabolites and the functionality of its CagT4SS. Similar activities were determined in cell culture neutrophils of different maturation states and in human primary neutrophils. In conclusion, we demonstrated that specific heptose metabolites or bacteria producing heptoses exhibit a strong activity on cell-autonomous innate responses of human neutrophils.

Anti-viral and Anti-inflammatory Isoflavonoids from Ukrainian Iris aphylla Rhizomes: Structure-Activity Relationship Coupled with ChemGPS-NP Analysis.

Dried Iris rhizomes have been used in Chinese and European traditional medicine for the treatment of various diseases such as bacterial infections, cancer, and inflammation, as well as for being astringent, laxative, and diuretic agents. Eighteen phenolic compounds including some rare secondary metabolites, such as irisolidone, kikkalidone, irigenin, irisolone, germanaism B, kaempferol, and xanthone mangiferin, were isolated for the first time from Iris aphylla rhizomes. The hydroethanolic Iris aphylla extract and some of its isolated constituents showed protective effects against influenza H1N1 and enterovirus D68 and anti-inflammatory activity in human neutrophils. The promising anti-influenza effect of apigenin (13: , almost 100% inhibition at 50 µM), kaempferol (14: , 92%), and quercetin (15: , 48%) were further confirmed by neuraminidase inhibitory assay. Irisolidone (1: , almost 100% inhibition at 50 µM), kikkalidone (5: , 93%), and kaempferol (14: , 83%) showed promising anti-enterovirus D68 activity in vitro. The identified compounds were plotted using ChemGPS-NP to correlate the observed activity of the isolated phenolic compounds with the in-house database of anti-influenza and anti-enterovirus agents. Our results indicated that the hydroethanolic Iris aphylla extract and Iris phenolics hold the potential to be developed for the management of seasonal pandemics of influenza and enterovirus infections.

Endocrine-disrupting chemical exposure augments neutrophilic inflammation in severe asthma through the autophagy pathway.

Corticosteroid resistance, progressive lung function decline, and frequent asthma exacerbations are the hallmarks of neutrophilic asthma (NA). However, the potential contributors and their mechanisms of NA aggravation have not yet been fully clarified. This study was conducted to assess the precise mechanism and inflammatory effects of endocrine-disrupting chemicals using mono-n-butyl phthalate (MnBP) on an NA model. BALB/c mice from normal control and LPS/OVA-induced NA groups were treated with or without MnBP. The effects of MnBP on the airway epithelial cells (AECs), macrophages (Mφ), and neutrophils were investigated in vitro and in vivo. NA mice exposed to MnBP had significantly increased airway hyperresponsiveness, total and neutrophil cell counts in the bronchoalveolar lavage fluid, and the percentage of M1Mφ in the lung tissues compared to those non-exposed to MnBP. In in vitro study, MnBP induced the human neutrophil activation to release neutrophil DNA extracellular traps, Mφ polarizing toward M1Mφ, and AEC damage. Treatment with hydroxychloroquine (an autophagy inhibitor) reduced the effects of MnBP in vivo and in vitro. The results of our study suggest that MnBP exposure may increase the risk of neutrophilic inflammation in severe asthma and autophagy pathway-targeted therapeutics can help control MnBP-induced harmful effects in asthma.

Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

МОДУЛЯЦИЯ ФАГОЦИТАРНОЙ АКТИВНОСТИ НЕЙТРОФИЛОВ ПРЕПАРАТАМИ ИЗ КАМЧАТСКИХ БУРЫХ ВОДОРОСЛЕЙ

In this paper, we present data on the influence of products produced from the kelp seaweeds, such as gel from Hedophyllum bongardianum and extracts from H. bongardianum, Alaria esculenta and Fucus distichus, on the modulation of phagocytic activity of human blood neutrophils. Our experiment involved 16 volunteers who took 15 g of algal gel daily for 30 days. Before and after the experiment, their blood was taken for general analysis and also to study the level of immunomodulation after priming exposure to different concentrations (0.5, 1, 2, 5%) of algal extracts. The nonspecific resistance of the experiment participants' body was assessed by the following indicators: phagocytic activity of neutrophils, phagocytic number, absolute phagocytic index, and sum of phagocytosis. Algal extracts diluted with saline to a concentration of 2% showed the highest priming effect on the blood of examined individuals taken both before and after the consumption of algal gel per os. Low concentrations (0.25 and 0.5%) did not have a stimulating effect. When algal extracts were diluted to 5% concentration, phagocytosis was noticeably inhibited. After 30 days of consuming the algal gel, the phagocytic activity of neutrophils in all participants increased by an average of 23%, phagocytic number by 10%, and absolute phagocytic index from 2.1 to 42%. The immunostimulating effect was greatest for Alaria extracts, and the lowest for Fucus extracts. The experimental results allowed us to recommend products from Kamchatka's kelp seaweeds as adaptogenic substances.

Phytochemical characterization and immunomodulatory effects of aqueous and ethanolic extracts and essential oil of Moroccan Laurus nobilis L. (Lauraceae) on human neutrophils

Introduction: The leaves of Moroccan bay laurel (Laurus nobilis L.) have been used in several forms of extracts to cure rheumatic pain due to their anti-inflammatory properties. Our work aimed to evaluate the effects of aqueous and ethanolic extracts, as well as the essential oil (EO) from laurel, on the microbicidal activity of human neutrophils when compared to the effect of eucalyptol. Methods: The extracts (ethanolic and aqueous) were subject to phytochemical profiling and high-performance liquid chromatography (HPLC) analyses. The EO obtained by hydrodistillation from laurel was analyzed by gas chromatography-mass spectrometry (GC-MS). The immunomodulatory effects on neutrophil microbicidal activity of the extracts, EO, and eugenol were carried out by 3-(4,5-diméthylthiazol-2-yl)-2,5-diphényltétrazolium (MTT) assay. Results: The phytochemical analysis of the extracts revealed the presence of flavonoids, coumarins, phenols, flavone aglycones, and tannins. HPLC analysis showed the presence of numerous phenolic molecules such as syringic acid, ferulic acid, gallic acid, caffeine, and quercetin. The chemical composition of EO revealed that the major components were eucalyptol (44.14%), α-terpinyl acetate (11.11%), and β-phellandrene (6.74%). Aqueous and ethanolic extracts and EO revealed a significant and dose-dependent ability to inhibit neutrophils microbicidal activity with maximal inhibition at 200 µg/mL concentration with 30.42%, 24.7%, and 38.13%, respectively (P<0.001). Conclusion: The obtained results revealed the immunomodulatory properties of laurel as a potential natural anti-inflammatory agent that would also allow the development of new anti-inflammatory drugs.

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice

Background and aimsKinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs).MethodsFibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study.ResultsB1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFβ, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-β and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway.ConclusionsB1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

Neutrophil Immunomodulatory Activity of Nerolidol, a Major Component of Essential Oils from Populus balsamifera Buds and Propolis.

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees. Black poplar (Populus balsamifera L.) buds are one of the primary sources of propolis. Despite their reported therapeutic properties, little is known about the innate immunomodulatory activity of essential oils from P. balsamifera and propolis. In the present studies, essential oils were isolated from the buds of P. balsamifera and propolis collected in Montana. The main components of the essential oil from P. balsamifera were E-nerolidol (64.0%), 1,8-cineole (10.8%), benzyl benzoate (3.7%), α-terpinyl acetate (2.7%), α-pinene (1.8%), o-methyl anisol (1.8%), salicylaldehyde (1.8%), and benzyl salicylate (1.6%). Likewise, the essential oil from propolis was enriched with E-nerolidol (14.4%), cabreuva oxide-VI (7.9%), α-bisabolol (7.1%), benzyl benzoate (6.1%), β-eudesmol (3.6%), T-cadinol (3.1%), 2-methyl-3-buten-2-ol (3.1%), α-eudesmol (3.0%), fokienol (2.2%), nerolidol oxide derivative (1.9%), decanal (1.8%), 3-butenyl benzene (1.5%), 1,4-dihydronaphthalene (1.5%), selina-4,11-diene (1.5%), α-cadinol (1.5%), linalool (1.4%), γ-cadinene (1.4%), 2-phenylethyl-2-methyl butyrate (1.4%), 2-methyl-2-butenol (1.3%), octanal (1.1%), benzylacetone (1.1%), and eremoligenol (1.1%). A comparison between P. balsamifera and propolis essential oils demonstrated that 22 compounds were found in both essential oil samples. Both were enriched in E-nerolidol and its derivatives, including cabreuva oxide VI and nerolidol oxides. P. balsamifera and propolis essential oils and pure nerolidol activated Ca2+ influx in human neutrophils. Since these treatments activated neutrophils, the essential oil samples were also evaluated for their ability to down-regulate the neutrophil responses to subsequent agonist activation. Indeed, treatment with P. balsamifera and propolis essential oils inhibited subsequent activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, nerolidol inhibited human neutrophil activation induced by fMLF (IC50 = 4.0 μM) and WKYMVM (IC50 = 3.7 μM). Pretreatment with the essential oils and nerolidol also inhibited human neutrophil chemotaxis induced by fMLF, again suggesting that these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Finally, reverse pharmacophore mapping predicted several potential kinase targets for nerolidol. Thus, our studies have identified nerolidol as a potential anti-inflammatory modulator of human neutrophils.

Spatiotemporal characteristics of P-selectin-induced β2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

Differential activation of human neutrophils by SARS-CoV-2 variants of concern.

The emerging SARS-CoV-2 virus has affected the entire world with over 600 million confirmed cases and 6.5 million deaths as of September 2022. Since the beginning of the pandemic, several variants of SARS-CoV-2 have emerged, with different infectivity and virulence. Several studies suggest an important role of neutrophils in SARS-Cov-2 infection severity, but data about direct activation of neutrophils by the virus is scarce. Here, we studied the in vitro activation of human neutrophils by SARS-CoV-2 variants of concern (VOCs). In our work, we show that upon stimulation with SARS-Cov-2 infectious particles, human healthy resting neutrophils upregulate activation markers, degranulate IL-8, produce Reactive Oxygen Species and release Neutrophil Extracellular Traps. Neutrophil activation was dependent on TLR7/8 and IRF3/STING. We then compared the activation potential of neutrophils by SARS-CoV-2 variants and showed a significantly increased activation by the Delta variant and a decreased activation by the Omicron variant as compared to the initial strain. In this study, we demonstrate that the SARS-Cov-2 virus can directly activate neutrophils in COVID-19 and that the different VOCs had differences in neutrophil activation intensity that mirror the differences of clinical severity. These data highlight the need to address neutrophil-virus interactions as a potential target for therapeutic intervention in SARS-CoV-2 infection.

Atovaquone attenuates experimental colitis by reducing neutrophil infiltration of colonic mucosa.

Ulcerative colitis (UC) is a chronic relapsing disease featuring aberrant accumulation of neutrophils in colonic mucosa and the luminal space. Although significant advances in UC therapy have been made with the development of novel biologics and small molecules targeting immune responses, success of most current therapies is still limited, with significant safety concerns. Thus, there is a need to develop additional safe and effective therapies for the treatment of UC. Antimalarial drugs have been safely used for many years to resolve tissue inflammation and the associated pathologies. Atovaquone is a recent FDA-approved antimalarial drug that has shown anti-viral and tumor-suppressive properties in vitro however, its role in mucosal inflammation has not been evaluated. Using pre-clinical murine DSS-induced colitis model combined with complementary in vivo peritonitis and ex vivo human neutrophil activation and chemotaxis assays we investigated functional and mechanistic impacts of atovaquone on disease resolution and neutrophil trafficking. We demonstrate that atovaquone promotes resolution of DSS-induced murine colitis by reducing neutrophil accumulation in the inflamed colonic mucosa. Mechanistically, we show that atovaquone suppressed induction of CD11b expression in neutrophils, reducing their polarization and migratory ability. Thus, our findings identify a new role of atovaquone in promoting resolution of mucosal inflammation, supporting the idea of potential repurposing of this FDA-approved drug as UC therapeutic.

Phytochemical characterization and immunomodulatory effects of aqueous, ethanolic extracts and essential oil of Syzygium aromaticum L. on human neutrophils

In Morocco, the flower buds of Syzygium aromaticum L (Clove) from Myrtaceae are essential in traditional medicine; they are used in many forms (infusion, maceration, and essential oil) and are suggested to mitigate inflammatory conditions such as muscle and dental pain as well as rheumatic diseases. This study aims to chemically characterize the aqueous and ethanolic extracts as well as the essential oil from cloves; also, we aimed to evaluate their effects on the bactericidal activity of human neutrophils compared with eugenol. The chemical composition of extracts was evaluated via qualitative phytochemical screening followed by quantitative screening using spectrophotometry and HPLC technique. The essential oil was analyzed by the GC-MS technique. The PMNs bactericidal activity of extracts, essential oil, and eugenol was carried out by MTT assay. The screening of extracts showed the presence of phenols, flavonoids, flavones aglycones, coumarins, and tannins. HPLC analysis revealed the presence of numerous phenolic compounds such as gallic acid, rutin, and quercetin while the GC-MS analysis of essential oil showed that the main components are eugenol (78.67%), eugenyl acetate (11.77%), and caryophyllene (6.85%). The aqueous, ethanolic extracts and essential oil showed an immunomodulatory activity by exerting a significant inhibition of neutrophil bactericidal activity in a dose dependent-manner reaching maximal inhibition at the concentration of 200 µg/ml with only 29.92%, 32.24%, and 48.15%, respectively (p < 0.001).Our study showed the immunomodulatory virtues of cloves as a natural anti-inflammatory agent. The strength of this effect is related to the presence of eugenol and the extraction forms used.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 μg/mL, positive control) or Cr-LAAO (50 μg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1β and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1β release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1β was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay.

Transfusion effectiveness of red blood cells (RBCs)has been associated with duration of the storage period. Storage-dependent RBC alterations lead to hemolysis and release of toxic free heme, but the increase of free hemelevels over time is largely unknown. In the current study, an apo-horseradish peroxidase (apoHRP)-based assay was applied to measure levels of free heme atregular intervals or periodically in supernatants of RBCs until a maximum storage period of 42days. Free heme levelsincreased with linear time-dependent kinetics up to day 21 and accelerated disproportionally after day 28 until day 42, as determined with the apoHRP assay. Individual timecourses of free heme in different RBC units exhibited high variability. Notably, levels of free hemoglobin, an established indicator of RBC damage, and those of total hemeincreased with continuous time-dependent linear kinetics over the entire 42day storage period, respectively. Supernatants from RBC units with high levels of free heme ledto inflammatory activation of human neutrophils. In conclusion, determining free heme in stored RBCs with the applied apoHRP assay may become feasible for testing ofRBC storage quality in clinical transfusion medicine.

The interplay between serine proteases and caspase-1 regulates the autophagy-mediated secretion of Interleukin-1 beta in human neutrophils.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1β) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1β) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1β is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1β processing in these cells. We found that although caspase-1 is required for IL-1β secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1β processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1β secretion.