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Among the responders to microbial invasion, neutrophils represent the earliest and perhaps the most important immune cells that contribute to host defense with the primary role to kill invading microbes using a plethora of stored anti-microbial molecules. One such process is the production of reactive oxygen species (ROS) by the neutrophil enzyme complex NADPH-oxidase, which can be assembled and active either extracellularly or intracellularly in phagosomes (during phagocytosis) and/or granules (in the absence of phagocytosis). One soluble factor modulating the interplay between immune cells and microbes is galectin-3 (gal-3), a carbohydrate-binding protein that regulates a wide variety of neutrophil functions. Gal-3 has been shown to potentiate neutrophil interaction with bacteria, including Staphylococcus aureus, and is also a potent activator of the neutrophil respiratory burst, inducing large amounts of granule-localized ROS in primed cells. Herein, the role of gal-3 in regulating S. aureus phagocytosis and S. aureus-induced intracellular ROS was analyzed by imaging flow cytometry and luminol-based chemiluminescence, respectively. Although gal-3 did not interfere with S. aureus phagocytosis per se, it potently inhibited phagocytosis-induced intracellular ROS production. Using the gal-3 inhibitor GB0139 (TD139) and carbohydrate recognition domain of gal-3 (gal-3C), we found that the gal-3-induced inhibitory effect on ROS production was dependent on the carbohydrate recognition domain of the lectin. In summary, this is the first report of an inhibitory role of gal-3 in regulating phagocytosis-induced ROS production.

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporalN-glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncatedN-glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation ofN-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophilN-glycoproteome featuring an intriguing granule-specificN-glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation.Key pointsSystems glycobiology reveals that profoundN-glycoproteome remodelling accompanies early neutrophil granulopoiesisPrecision glycoproteomics produces detailed cartography of neutrophils that exhibit site-, protein- and granule-specific N-glycosylation

The cytolytic peptide toxin candidalysin is secreted by the invasive, hyphal form of the human fungal pathogen,Candida albicans. Candidalysin is essential for inducing host cell damage during mucosal and systemicC. albicansinfections, resulting in neutrophil recruitment. Neutrophil influx toC. albicans-infected tissue is critical for limiting fungal growth and preventing the fungal dissemination. Here, we demonstrate that candidalysin secreted by hyphae promotes the stimulation of neutrophil extracellular traps (NETs), while synthetic candidalysin triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leukotoxic hypercitrullination, which is governed by protein arginine deaminase 4 activation via calcium influx and initiation of intracellular signalling events. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Interestingly, exposure to candidalysin does not immediately restrict the capability of neutrophils to produce reactive oxygen species (ROS), nor to phagocytose particles. Instead, candidalysin triggers ROS production, calcium influx and subsequent activation of downstream signalling that drive morphological alteration and the formation of NLS in a dose- and time-dependent manner. Notably, candidalysin-triggered NLS demonstrate anti-Candidaactivity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+. This study reveals thatC. albicanshyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entanglesC. albicanshyphae and inhibits their growth. Importantly, this explains discrepancies of previous studies demonstrating that neutrophil-derived extracellular chromatin structures triggered byC. albicanscan be both dependent and independent of ROS. Our data also demonstrate that while candidalysin hampers neutrophil function, the toxin also increases the capability of neutrophils to entangle hyphae and to restrict their growth, reflecting the importance of human neutrophils in controlling the dissemination ofC. albicans.

Starting from RO6800020 (1), our former γ-secretase modulator (GSM) lead compound, we utilized sequential structural replacements to improve the potency (IC50), pharmacokinetic properties including the free fraction (fraction unbound (fu)) in plasma, and in vivo efficacy. Importantly, we used novel CF3-alkoxy groups as bioisosteric replacements of a fluorinated phenyl ring and properties such as lipophilicity, solubility, metabolic stability, and free fraction could be balanced, maintaining low Pgp efflux needed for CNS penetration. In addition, by reducing aromaticity, we prevented phototoxicity. Additional substitution in the triazolopyridine core disturbed the binding to phosphatidylinositol 4-kinase, catalytic β (PIK4CB). We also introduced less lipophilic head heterocycles devoid of covalent binding (CVB) liability. After these changes, further modifications to the trifluoroethoxy bioisosteric replacement allowed rebalancing of properties, such as lipophilicity, and also potency. Our optimization strategy culminated with in vivo active RO7101556 (18B) having excellent properties and being selected as an advanced candidate.

Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40phox, but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease.

Fusobacterium nucleatum is a gram-negative and anaerobic oral commensal that is implicated in inflammatory conditions of the tooth-supporting structures, that is, periodontal diseases. One of the main characteristics of these conditions is an accumulation of neutrophil granulocytes in the gingival pockets where bacteria reside. Neutrophils are recruited to tissue-residing microbes by gradients of bacteria derived chemoattractants, and the cellular migration over the pocket epithelium into the gingival pocket is likely governed by chemoattractants released by the amino acid fermenting anaerobes typically colonising this site. However, the chemoattractants released by F. nucleatum and other oral anaerobes have long been unidentified. In the present study, we show that the major chemoattractants released during the growth of F. nucleatum are short chain fatty acids (SCFAs), primarily acetate and butyrate. These SCFAs, that are released at high levels as end-products of the metabolism of F. nucleatum, trigger chemotaxis of human neutrophils, as well as cytosolic Ca2+ signals, via free fatty acid receptor 2 (FFAR2). This finding establishes the SCFA-FFAR2 interaction as an important mechanism in the recruitment of neutrophils to the periodontal pocket, but could also be of importance in the pathogenesis of other medical conditions involving colonisation/infection of F. nucleatum.

In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177(+)and CD177(-)neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177(+)neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177(+)neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177(+)neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177(+)neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation. Funding Agencies|Swedish Research CouncilSwedish Research Council [201600982]; Swedish Heart-Lung FoundationSwedish Heart-Lung Foundation [20180218]; King Gustaf V Memorial Foundation [FAI-2017-0368]; Patent Revenue Fund Research in Preventive Odontology; Swedish state under the TUA agreement [TUAGBG-628751]

Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such asPorphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growingP. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found inP. gingivalissupernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed byP. gingivalis(and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context ofP. gingivaliscolonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoeisis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called ‘targeting-by-timing’. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand how glycosylation evolves during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation andN- andO-glycans present in each compartment were analyzed by liquid chromatography and tandem mass spectrometry. We found abundant paucimannosidicN-glycans and lack ofO-glycans in early-formed azurophil granules (AG), whereas later-formed specific and gelatinase granules (SG and GG) contained complexN- andO-glycans with remarkably elongatedN-acetyllactosamine repeats with Lewis-x and sialyl-Lewis-x epitopes. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of ‘glycosylation-by-timing’ during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Recruitment of neutrophils from blood to tissues is a cardinal event in inflammation during which neutrophils switch from a resting, naive state to a preactivated, primed phenotype; the priming process is characterized by alterations in the composition of cell surface adhesins, for example, shedding of l-selectin and mobilization of granule-stored integrins to the cell surface. Ligation of chemotactic receptors and interactions with the endothelial lining are established triggers of neutrophil priming and in line with this, in vivo transmigrated neutrophils obtained from tissues are typically highly primed. We here characterize the priming of neutrophils brought about by in vivo recruitment from blood to inflamed joints by the analyses of synovial fluid and blood from patients with inflammatory arthritis. For comparisons, we used controlled in vivo models of neutrophil transmigration to skin of healthy subjects. In contrast to the residing view and in vivo transmigrated neutrophils from skin models, neutrophils from synovial fluid were often surprisingly resting and phenotypically very similar to naive cells isolated from peripheral blood; synovial fluid cells often retained l-selectin and had undergone minimal up-regulation of integrin receptors. In complete agreement with our in vivo findings, cell-free synovial fluid was potently chemotactic without triggering alteration of surface receptors also in vitro. We conclude that tissue recruitment of neutrophils does not by default trigger l-selectin shedding and granule mobilization, and the chemoattractant(s) guiding neutrophils to synovial fluid apparently operate without inducing cellular priming.

In recent years, the therapeutic potential of antimicrobial peptides (AMPs) as immunomodulators has become generally accepted. Nevertheless, only very few AMP-based compounds have progressed into clinical trials. This paradox may be explained by the fact, that some of the intrinsic properties of natural peptides, such as proteolytic and oxidative instability, render them inconvenient as therapeutics. Therefore, substantial research efforts have been dedicated to mimic the physico-chemical properties as well as biological activities of AMPs by designing and identifying more stable peptidomimetics displaying analogous immunomodulatory activity profiles. Neutrophils play key roles in host defense as major effector cells in clearance of pathogens by phagocytosis and by regulating other processes of innate immunity as well as by promoting resolution of inflammation. Several aspects of these effects are correlated to their expression of formyl peptide receptors (FPRs) that have been shown to be targets of both natural and synthetic antimicrobial peptides. In the present review recent findings highlighting the role of FPRs in mediating immunomodulatory activities of natural and synthetic AMPs as well as of stabilized peptidomimetics are discussed, and prospects for future development of immunomodulatory therapeutics are presented.

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increased cellular consumption of molecular oxygen (O2). The O2 molecules consumed are reduced by electrons delivered by a membrane localized NADPH-oxidase that initially generate one- and two electron reduced superoxide anions (O2 -) and hydrogen peroxide (H2O2), respectively. These oxidants can then be processed into other highly reactive oxygen species (ROS) that can kill microbes, but that may also cause tissue destruction and drive other immune cells into apoptosis. The development of basic techniques to measure and quantify ROS generation by phagocytes is of great importance, and a large number of methods have been used for this purpose. A selection of methods (including chemiluminescence amplified by luminol or isoluminol, absorbance change following reduction of cytochrome c, and fluorescence increase upon oxidation of PHPA) are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to diagnosis of diseases linked to the NADPH-oxidase and more clinically oriented research on innate immune mechanisms and inflammation.

We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewisx (Si-Lex) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewisx (Si-Lex) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Lex, may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Lex remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.

Periodontitis is a chronic inflammation that develops due to a destructive tissue response to prolonged inflammation and a disturbed homeostasis (dysbiosis) in the interplay between the microorganisms of the dental biofilm and the host. The infectious nature of the microbes associated with periodontitis is unclear, as is the role of specific bacterial species and virulence factors that interfere with the host defense and tissue repair. This review highlights the impact of classical virulence factors, such as exotoxins, endotoxins, fimbriae and capsule, but also aims to emphasize the often-neglected cascade of metabolic products (e.g., those generated by anaerobic and proteolytic metabolism) that are produced by the bacterial phenotypes that survive and thrive in deep, inflamed periodontal pockets. This metabolic activity of the microbes aggravates the inflammatory response from a low-grade physiologic (homeostatic) inflammation (i.e., gingivitis) into more destructive or tissue remodeling processes in periodontitis. That bacteria associated with periodontitis are linked with a number of systemic diseases of importance in clinical medicine is highlighted and exemplified with rheumatoid arthritis, The unclear significance of a number of potential “virulence factors” that contribute to the pathogenicity of specific bacterial species in the complex biofilm−host interaction clinically is discussed in this review.

The phagocyte NADPH oxidase is responsible for the neutrophil’s great capacity to produce reactive oxygen species (ROS). The NADPH oxidase can be assembled in the plasma membrane, as well as in membranes of intracellular vesicles, giving neutrophils the ability to direct ROS production to distinct subcellular sites. Neutrophil ROS contribute to microbial killing, trigger formation of neutrophil extracellular traps and appear to partake in inflammation control. Consequently, function-disrupting mutations in the NADPH oxidase lead to chronic granulomatous disease, characterized by severe infections and inflammatory disorders. Recent experimental data and description of a novel chronic granulomatous disease subtype (p40phox-deficiency) imply that ROS generated in intracellular compartments are key for NETosis and for controlling inflammatory signaling. We foresee boosted interest in intracellular ROS production. To fully understand where and how such ROS function, however, limitations of assay systems to measure ROS need to be appreciated, and the development of novel techniques/reagents would be highly useful.

Objective Women with SLE face an increased risk of adverse pregnancy outcomes compared with healthy women, but the underlying immunological mechanisms are unknown. Given the recognised association of neutrophil activation with SLE pathogenesis, we examined whether there is increased neutrophil activation and inflammation in blood and placenta in SLE relative to healthy pregnancy. Methods At delivery, peripheral blood, maternal-derived intervillous blood and placentas were collected from 12 SLE and 10 healthy control pregnancies. The proportion of low-density granulocytes (LDGs) and the activation status of LDG and normal-density granulocytes were examined with flow cytometry. The chemokines CXCL8 and CXCL1 were quantified with a cytometric bead-based assay and interferon alpha (IFN alpha) protein levels with a Simoa method. IFN alpha-stimulated maternal-derived decidual stromal cells were examined for CXCL8 gene expression with qPCR. A pathologist, blinded to the patient background, examined all placentas. Results Women with SLE had significantly higher proportions of LDG in peripheral blood compared with controls (p=0.02), and LDG in both peripheral and intervillous blood were more activated in SLE relative to healthy pregnancies (peripheral blood: p=0.002 and intervillous blood: p=0.05). There were higher levels of CXCL8 and CXCL1 in intervillous compared with peripheral blood in women with SLE (p=0.004 and p=Funding Agencies|Swedish government; county councils; ALF agreement; Swedish Rheumatism Association; Gothenburg Society of Medicine; Ingegerd Johanssons Donation; foundations of King Gustaf Vs 80th Anniversary; Queen Victorias Freemasons; Swedish Research CouncilSwedish Research CouncilEuropean Commission

Background: The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Sodertalje, Sweden. Methods: A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man. Results and Conclusions: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

Background Galectin-3 is a 32 kDa protein secreted by macrophages involved in processes such as cell activation, chemotaxis and phagocytosis. Galectin-3 has previously been shown to improve the ability of airway macrophages to ingest apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease (COPD) and may be of interest in non-eosinophilic asthma (NEA) which is also characterised by impaired efferocytosis. It was hypothesised that the addition of exogenous galectin-3 to monocyte-derived macrophages (MDMs) derived from donors with NEA would enhance their ability to engulf apoptotic granulocytes. Methods Eligible non-smoking adults with asthma (n = 19), including 7 with NEA and healthy controls (n = 10) underwent a clinical assessment, venepuncture and sputum induction. MDMs were co-cultured with apoptotic granulocytes isolated from healthy donors with or without exogenous recombinant galectin-3 (50 μg/mL) and efferocytosis was assessed by flow cytometry. Galectin-3 expression and localisation in MDMs was visualised by immunofluorescence staining and fluorescence microscopy. Galectin-3, interleukin (IL)-6 and CXCL8 secretion were measured in cell culture supernatants by ELISA and cytometric bead array. Results Baseline efferocytosis (mean (±standard deviation)) was lower in participants with asthma (33.2 (±17.7)%) compared with healthy controls (45.3 (±15.9)%; p = 0.081). Efferocytosis did not differ between the participants with eosinophilic asthma (EA) (31.4 (±19.2)%) and NEA (28.7 (±21.5)%; p = 0.748). Addition of galectin-3 significantly improved efferocytosis in asthma, particularly in NEA (37.8 (±18.1)%) compared with baseline (30.4 (±19.7)%; p = 0.012). Efferocytosis was not associated with any of the clinical outcomes but was negatively correlated with sputum macrophage numbers (Spearman r = − 0.671; p = 0.017). Galectin-3 was diffusely distributed in most MDMs but formed punctate structures in 5% of MDMs. MDM galectin-3 secretion was lower in asthma (9.99 (2.67, 15.48) ng/mL) compared with the healthy controls (20.72 (11.28, 27.89) ng/mL; p = 0.044) while IL-6 and CXCL8 levels were similar. Conclusions Galectin-3 modulates macrophage function in asthma, indicating a potential role for galectin-3 to reverse impaired efferocytosis in NEA. Electronic supplementary material The online version of this article (10.1186/s12931-018-0967-9) contains supplementary material, which is available to authorized users.

Background Neutrophils and eosinophils are multifunctional granulocytes derived from common myelocytic-committed progenitor cells. Severe congenital neutropenia 1 (SCN1) caused by ELANE mutations is a rare disease characterized by very low numbers of circulating neutrophils. Little is known about the functional characteristics of the SCN1 granulocytes, except that eosinophilia has been noticed in both bone marrow and peripheral blood. In this study, we profiled the number and function of granulocytes in patients suffering from SCN1. Methods Nine patients diagnosed with SCN1 were enrolled in this study and absolute counts of eosinophils and neutrophils from bone marrow aspirates and peripheral blood samples were analysed. In addition, Ficoll-Paque enriched granulocytes from patients and healthy controls were analysed for specific eosinophil and neutrophil markers using flow cytometry and for NADPH-oxidase activity-profile by chemiluminescence. Results Our data demonstrate a skewed granulocyte population in SCN1 patients dominated by eosinophils in both bone marrow and peripheral blood. The latter was detected only by blood smear examination, but not by automated blood analysers. Furthermore, we show that the SCN1 eosinophils exerted normal production of reactive oxygen species generated by the NADPH-oxidase, however the response was profoundly different from that of healthy control neutrophils. Conclusions SCN1 patients with ELANE mutations suffer from neutropenia yet display eosinophilia in the bone marrow and blood, as revealed by smear examination but not by automatic blood analysers. The SCN1 eosinophils are functionally normal regarding production of reactive oxygen species (ROS). However, the ROS profile produced by eosinophils differs drastically from that of neutrophils isolated from the same blood donor, implying that the eosinophilia in SCN1 cannot compensate for the loss of neutrophils regarding ROS-mediated functions.

Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.

Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.