Human Neutrophils In Response Research Articles

Calcineurin-NFAT signaling controls neutrophils´ ability of chemoattraction upon fungal infection.

Calcineurin - nuclear factor of activated T-cell (CN-NFAT) inhibitors are widely clinically used drugs for immunosuppression but besides their required T-cell response inhibition, they also undesirably affect innate immune cells. Disruption of innate immune cell function can explain the observed susceptibility of CN-NFAT inhibitors-treated patients to opportunistic fungal infections. Neutrophils play an essential role in innate immunity as a defense against pathogens, however, the effect of CN-NFAT inhibitors on neutrophil function was poorly described. Thus, we tested the response of human neutrophils to opportunistic fungal pathogens, namely Candida albicans and Aspergillus fumigatus in the presence of CN-NFAT inhibitors. Here, we report that the NFAT pathway members were expressed in neutrophils and mediated part of the neutrophil response to pathogens. Upon pathogen exposure, neutrophils underwent profound transcriptomic changes with subsequent production of effector molecules. Importantly, genes and proteins involved in the regulation of the immune response and chemotaxis, including the chemokines CCL2, CCL3, and CCL4 were significantly upregulated. The presence of CN-NFAT inhibitors attenuated the expression of these chemokines and impaired the ability of neutrophils to chemoattract other immune cells. Our results amend knowledge about the impact of CN-NFAT inhibition in human neutrophils.

The different impact of drug-resistant Leishmania on the transcription programs activated in neutrophils

Drug resistance threatens the effective control of infections, including parasitic diseases such as leishmaniases. Neutrophils are essential players in antimicrobial control, but their role in drug-resistant infections is poorly understood. Here, we evaluated human neutrophil response to clinical parasite strains having distinct natural drug susceptibility. We found that Leishmania antimony drug resistance significantly altered the expression of neutrophil genes, some of them transcribed by specific neutrophil subsets. Infection with drug-resistant parasites increased the expression of detoxification pathways and reduced the production of cytokines. Among these, the chemokine CCL3 was predominantly impacted, which resulted in an impaired ability of neutrophils to attract myeloid cells. Moreover, decreased myeloid recruitment when CCL3 levels are reduced was confirmed by blocking CCL3 in a mouse model. Collectively, these findings reveal that the interplay between naturally drug-resistant parasites and neutrophils modulates the infected skin immune microenvironment, revealing a key role of neutrophils in drug resistance.

Isolation methods determine human neutrophil responses after stimulation.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.

Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies.

Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces in vitro PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear. In this work human PMN responses were assessed in vitro in response to the spike (S) protein of two different SARS-CoV-2 variants, anti-S IgG1 antibodies or immune complexes formed by them. Production of reactive oxygen species (ROS) was measured by Diogenes-based chemiluminescence. Release of myeloperoxidase (MPO) was assessed by ELISA while secretion of a list of cytokines and growth factors was determined by high-performance multiplex cytokine assay. We show that the SARS-CoV-2 Omicron variant S protein and anti-spike IgG1, either alone or together, stimulate ROS production in human PMNs. We also observed that the SARS-CoV-2 Wuhan S protein and anti-S IgG1 antibody together trigger MPO release from PMNs. Based on the relevance of SARS-CoV-2 and influenza co-infections, we have also investigated the impact of influenza virus infection on the previous PMN responses to S proteins or anti-S antibodies. We did not detect any significant effect of influenza co-infection on ROS generation in PMNs. Our data also show that PMN stimulation by S proteins induced the release of different chemokines, growth factors, regulatory and proinflammatory cytokines. Overall, our findings show that the SARS-CoV-2 S protein, an anti-spike IgG1 antibody or their immune complex, promote oxidative responses of PMNs in a variant-dependent manner, contributing to a better understanding of the role of PMN responses during SARS-CoV-2 infection.

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.

Streptococcus pyogenes emm98.1 variants activate inflammatory caspases in human neutrophils

ABSTRACT The human pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) is responsible for invasive disease characterized by inflammation and tissue destruction. Inflammatory symptoms of invasive disease may be attributed to the neutrophil response during the early stages of infection. Here, the human neutrophil response to GAS was characterized in vitro using emm98.1 type covS mutant GAS strain NS88.2 (isolated from invasive infection) and avirulent variant NS88.2rep. NS88.2 was shown to resist phagocytic killing and proliferate in the presence of human neutrophils, where neutrophil antimicrobial defence through the production of reactive oxygen species was reduced compared with NS88.2rep. In the presence of NS88.2, neutrophil death was delayed compared with NS88.2rep. Infection with either GAS strain induced expression of inflammatory caspases-1 and -4 in neutrophils, with increased detection of activated inflammatory caspases in response to NS88.2rep compared with NS88.2. NS88.2 infection caused differential expression of cell-surface CD66b, CD16, and CD31, when compared to NS88.2rep. We conclude that the neutrophil response to NS88.2 promotes inflammation and may be a contributing factor to the severity of invasive GAS infections. Abbreviations CovRS, control of virulence regulatory system; GAS, Group A Streptococcus; PMN, polymorphonuclear leukocyte.

The acute inflammatory response to copper(II)-doped biphasic calcium phosphates

Infection and inflammation are two key features to consider to avoid septic or aseptic loosening of bone-implanted biomaterials. In this context, various approaches to fine-tune the biomaterial's properties have been studied in order to modulate the crosstalk between immune and skeletal cells. Cation-doping strategies for tuning of calcium phosphates properties has been evidenced as a promising way to control the biomaterial-induced inflammatory process, and thus improving their osteoimmunomodulatory properties. Copper(II) ions are recognized for their antibacterial potential, but the literature on their impact on particulate material-induced acute inflammation is scarce. We synthesized copper(II) ions-doped biphasic calcium phosphate (BCP), intended to exhibit osteoimmunomodulatory properties. We addressed in vitro, for the first time, the inflammatory response of human primary polymorphonuclear neutrophils (PMNs) to copper(II) ions-doped or undoped (BCP) powders, synthesized by an original and robust wet method, in the presence or absence of LPS as a costimulant to mimic an infectious environment. ELISA and zymography allowed us to evidence, in vitro, a specific increase in IL-8 and GRO-α secretion but not MIP-1β, TNF-α, or MMP-9, by PMNs. To assess in vivo relevance of these findings, we used a mouse air pouch model. Thanks to flow cytometry analysis, we highlighted an increased PMN recruitment with the copper(II) ions-doped samples compared to undoped samples. The immunomodulatory effect of copper(II) ions-doped BCP powders and the consequent induced moderate level of inflammation may promote bacterial clearance by PMNs in addition to the antimicrobial potential of the material. Copper(II) doping provides new insights into calcium phosphate (CaP)-based biomaterials for prosthesis coating or bone reconstruction by effectively modulating the inflammatory environment.

Nor-24-homoscalaranes, Neutrophilic Inflammatory Mediators from the Marine Sponge Lendenfeldia sp.

The marine sponge Lendenfeldia sp., collected from the Southern waters of Taiwan, was subjected to chemical composition screening, resulting in the isolation of four new 24-homoscalarane compounds, namely lendenfeldaranes R-U (1-4). The structures and relative stereochemistry of the new metabolites 1-4 were assigned based on NMR studies. The absolute configurations of compounds 1-4 were determined by comparing the calculated and experimental values of specific optical rotation. The antioxidant and anti-inflammatory activities of the isolated compounds were assayed using superoxide anion generation and elastase release assays. These assays are used to determine neutrophilic inflammatory responses of respiratory burst and degranulation. Compounds 2 and 4 inhibited superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB) with IC50: 3.98-4.46 μM. Compounds 2 and 4 inhibited fMLP/CB-induced elastase release, with IC50 values ranging from 4.73 to 5.24 μM. These findings suggested that these new 24-homoscalarane compounds possess unique structures and potential anti-inflammatory activity.

Establishment of Biotesting System to Study Features of Innovative Multifunctional Biotextile

Abstract An established biotesting system designed to discover specific features of innovative multifunctional biotextile, encompassing integrated silica dioxide and succinite (Baltic amber) particles, is founded on reproducible, reliable, and relatively fast methods. The main idea starting this study was to create a system of test methods devoted to identification of specific features of biotextile materials designed to preserve living organisms from adverse environmental factors like enhanced electromagnetic radiation of different frequencies, without use of vivarium animals. Cultures of the freshwater macrophyte duckweed (Lemna minor) line Sta2 and fruit flies (Drosophila melanogaster) were chosen as model systems suitable for the study of the influence of electromagnetic field (EMF) radiation. The experiments showed changes of phenotypic features and growth parameters of test objects, as well as induction of point mutations of DNA (for example, insertions or deletions in chloroplast DNA and nucleotide substitutions in nuclear genes). The responses of test organisms induced by EMF were studied using microscopy, flow cytometry, and DNA sequencing methods. On the cell level, a new fast flow cytometry method for biotextile testing was developed: immature gametic (pollen) cell cultures were used as a highly sensitive model system (plant gametic cell response is comparable to human neutrophil response) to study the influence of EMF radiation. The method was based on the measurement of differences of fluorescence intensity between group of cells experimentally affected by EMF radiation and non-affected cells.

Composition and Biological Activity of the Essential Oils from Wild Horsemint, Yarrow, and Yampah from Subalpine Meadows in Southwestern Montana: Immunomodulatory Activity of Dillapiole.

Agastache urticifolia (Benth.) Kuntze (horsemint), Achillea millefolium L. (yarrow), and Perideridia gairdneri (Hook. & Arn.) Mathias (yampah) are native, culturally important plants that grow in the subalpine meadows of Montana. Analysis of the composition of essential oils extracted from these plants showed that the main components of essential oils obtained from flowers and leaves of A. urticifolia (designated as AUF/AUL) were menthone (2.7/25.7%), isomenthone (2.6/29.1%), pulegone (78.9/28.8%), and limonene (4.2/6.2%), whereas essential oils obtained from the inflorescence of A. millefolium (designated as AMI) were high in α-thujone (17.1%) and β-thujone (14.9%), 1,8-cineole (17.0%), camphor (13.0%), sabinene (7.0%), guaia-3,9-dien-11-ol (3.2%), and terpinen-4-ol (2.5%). Essential oils obtained from the inflorescence of P. gairdneri (designated as PGI) contained high amounts of dillapiole (30.3%), p-cymen-8-ol (14.1%), terpinolene (12.0%), 4-hydroxy-4-methyl-cyclohex-2-enone (6.2%), and γ-terpinene (2.4%). Evaluation of their immunomodulatory activity demonstrated that essential oils extracted from all of these plants could activate human neutrophils with varying efficacy. Analysis of individual components showed that dillapiole activated human neutrophil intracellular Ca2+ flux ([Ca2+]i) (EC50 = 19.3 ± 1.4 μM), while α-thujone, β-thujone, menthone, isomenthone, and pulegone were inactive. Since dillapiole activated neutrophils, we also evaluated if it was able to down-regulate neutrophil responses to subsequent agonist activation and found that pretreatment with dillapiole inhibited neutrophil activation by the chemoattractant fMLF (IC50 = 34.3 ± 2.1 μM). Pretreatment with P. gairdneri essential oil or dillapiole also inhibited neutrophil chemotaxis induced by fMLF, suggesting these treatments could down-regulate human neutrophil responses to inflammatory chemoattractants. Thus, dillapiole may be a novel modulator of human neutrophil function.

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.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

Human neutrophils are a key target of glucocorticoids

Abstract Neutrophils play central roles in immunity and inflammation. Glucocorticoids (GCs) are the cornerstone of anti-inflammatory and immunosuppressive therapy. While GCs trigger an acute rise in circulating neutrophils, little is known about the mechanisms by which GCs modulate neutrophil biology. We studied the response of 9 primary human cells to in vitro treatment with GCs. Surprisingly, neutrophils were transcriptionally the most responsive cell type, and they express proportionally the most glucocorticoid receptor (GR) transcripts. Long-read RNA-seq revealed 42 full-length GR transcript isoforms in neutrophils, twice as many as had been reported previously in all other cell types. At the protein level, neutrophils also have a unique and diverse set of GR isoforms. In contrast to the widely accepted model of GR signaling, in which GR binds GC molecules in the cytoplasm and then translocates to the nucleus, GR localization in neutrophils is almost exclusively nuclear, even in the absence of GC treatment. To validate our findings in vivo, we then performed a study of healthy volunteers, with blood collected before and serially after GC infusion. We generated a pathway-level map of the human neutrophil transcriptome at baseline and in response to GCs. The in vivo transcriptional response of human neutrophils to GCs was as strong as that observed in vitro, with a predominance of upregulation. Among the most strongly induced genes are several that are either not expressed, or expressed at very low levels, in human neutrophils at baseline. Some of the proteins encoded by these genes may have putative effects on neutrophil function, including SAMSN1 and ZBTB16. These findings indicate that human neutrophils are a key target for GC action.

Mathematical Modelling of Neutrophil Responses to Aspergillus

Abstract Invasive aspergillosis is a dangerous infection commonly affecting immunocompromised patients. The spores of the fungal pathogen, Aspergillus, are found ubiquitously in the environment and are inhaled daily by all humans. These spores are cleared quickly and effectively healthy hosts without developing infection. On the other hand, patients with impaired immunity, can develop an invasive pneumonia with poor outcomes. Neutrophils are essential to the host defense against aspergillosis, both in direct fungal killing and as modulators of immunity against the pathogen. To better understand this role, we conducted a transcriptomic time series comparing the responses of human neutrophils to Aspergillus. Differential gene expression analysis as well as gene enrichment analysis indicated an up regulation of number of transcriptional markers and pathways including NFkB, NR4A, TNF, as well as neutrophil degranulation, cytokine-mediated signaling, chemokine signaling, and TREM1 signaling among others. Combining this data with known mechanisms and pathways from the literature, we built a mathematical model describing the intracellular pathways which determine the neutrophil’s response after detecting Aspergillus. Very few mathematical models have been built modeling intracellular mechanisms of a neutrophil and none that we know of which relates to the interaction between neutrophils and this organism. We intend to use our model to make predictions on the effects of altering different pathways within the cell. Moreover we intend to use this model in a larger systems model describing the immune system response to invasive pulmonary aspergillosis. Supported by: R01AI135128, U01EB024501, Keck Foundation Medical Research Grant 994413.

Myeloid Src-family kinases are critical for neutrophil-mediated autoinflammation in gout and motheaten models.

Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.

IL-4, IL-13 and IFN-γ -induced genes in highly purified human neutrophils

Interleukin (IL)-4 and IL-13 are related cytokines with well-known specific roles in type 2 immune response. However, their effects on neutrophils are not completely understood. For this, we studied human primary neutrophil responses to IL-4 and IL-13. Neutrophils are dose-dependently responsive to both IL-4 and IL-13 as indicated by signal transducer and activator of transcription 6 (STAT6) phosphorylation upon stimulation, with IL-4 being more potent inducer of STAT6. IL-4-, IL-13- and Interferon (IFN)-γ-stimulated gene expression in highly purified human neutrophils induced both overlapping and unique gene expression in highly purified human neutrophils. IL-4 and IL-13 specifically regulate several immune-related genes, including IL-10, tumor necrosis factor (TNF) and leukemia inhibitory factor (LIF), while type1 immune response-related IFN-γ induced gene expression related for example, to intracellular infections. In analysis of neutrophil metabolic responses, oxygen independent glycolysis was specifically regulated by IL-4, but not by IL-13 or IFN-γ, suggesting specific role for type I IL-4 receptor in this process. Our results provide a comprehensive analysis of IL-4, IL-13 and IFN-γ -induced gene expression in neutrophils while also addressing cytokine-mediated metabolic changes in neutrophils.

6]-Gingerol Facilitates CXCL8 Secretion and ROS Production in Primary Human Neutrophils by Targeting the TRPV1 Channel.

Clarifying the function of sensory active TRP (transient receptor potential) channels in non-sensory tissue is of growing interest, especially with regard to food ingredients in nutritionally relevant concentrations. The study hypothesizes the TRPV1 agonist [6]-gingerol to facilitate cellular immune responses of primary human neutrophils, after treatment with 50nM, a concentration that can be reached in the circulation after habitual dietary intake. qRT-PCR analyses reveal a high abundancy of TRP channel RNA expression in the types of primary leukocytes investigated, namely neutrophils, monocytes, NK cells, T cells, and B cells. Incubation of neutrophils with 50nM of the known TRPV1 ligand [6]-gingerol led to increased surface expression of CD11b, CD66b, and the fMLF receptor FPR1, as shown by flow cytometry. Upon subsequent stimulation with fMLF, the neutrophils display an about 30% (p < 0.05) increase in CXCL8 secretion as well as in ROS production. Pharmacological inhibition of TRPV1 by trans-tert-butylcyclohexanol abolishes the [6]-gingerol induced effects. The TRPV1 channel is functionally expressed in human neutrophils. Activation of the channel with [6]-gingerol as a food-derived ligand in nutritionally relevant concentrations leads to an enhanced responsiveness in the cells towards activating stimuli, thereby facilitating a canonical cellular immune response in human neutrophils.

Identification of a forkhead box protein transcriptional network induced in human neutrophils in response to inflammatory stimuli.

Neutrophils represent the largest proportion of circulating leukocytes and, in response to inflammatory stimuli, are rapidly recruited to sites of infection where they neutralize pathogens. We have identified a novel neutrophil transcription network induced in response to inflammatory stimuli. We performed the first RNAseq analysis of human neutrophils exposed to lipopolysaccharide (LPS), followed by a meta-analysis of our dataset and previously published studies of LPS-challenged neutrophils. This revealed a robustly enhanced transcriptional network driven by forkhead box (FOX) transcription factors. The network is enriched in genes encoding proinflammatory cytokines and transcription factors, including MAFF and ATF3, which are implicated in responses to stress, survival and inflammation. Expression of transcription factors FOXP1 and FOXP4 is induced in neutrophils exposed to inflammatory stimuli, and potential FOXP1/FOXP4 binding sites were identified in several genes in the network, all located in chromatin regions consistent with neutrophil enhancer function. Chromatin immunoprecipitation (ChIP) assays in neutrophils confirmed enhanced binding of FOXP4, but not FOXP1, to multiple sites in response to LPS. Binding to numerous motifs and transactivation of network genes were also observed when FOXP proteins were transiently expressed in HEK293 cells. In addition to LPS, the transcriptional network is induced by other inflammatory stimuli, indicating it represents a general neutrophil response to inflammation. Collectively, these findings reveal a role for the FOXP4 transcription network as a regulator of responses to inflammatory stimuli in neutrophils.

Neutrophil-Specific Syk Expression Is Crucial for Skin Disease in Experimental Epidermolysis Bullosa Acquisita

Autoantibodies against the dermal-epidermal junction component type VII collagen (C7) trigger skin disease in the inflammatory form of epidermolysis bullosa acquisita. We have previously identified the Syk tyrosine kinase as a crucial participant in anti-C7 antibody-induced experimental epidermolysis bullosa acquisita. However, it is still unclear which cellular lineage needs to express Syk during the disease process. In this study, we show that the loss of Syk, specifically from neutrophils, results in complete protection from the anti-C7 antibody-initiated skin disease both macroscopically and microscopically. Mice with a neutrophil-specific Syk deletion had decreased neutrophil accumulation and abrogated CXCL2 and IL-1β levels in the skin upon anti-C7 treatment, whereas isolated Syk-deficient neutrophils had decreased superoxide release, cell spreading, and cytokine release on C7-anti-C7 immune complex surfaces. Entospletinib and lanraplenib, two second-generation Syk-specific inhibitors, effectively abrogated immune complex-induced responses of human neutrophils and decreased the anti-C7 antibody-initiated, neutrophil-mediated exvivo dermal-epidermal separation in human skin samples. Taken together, these results point to a crucial role for Syk in neutrophils in the development and progression of epidermolysis bullosa acquisita and suggest Syk inhibition as a potential therapeutic strategy.

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.