Protein N-glycosylation in the bronchoalveolar space differs between never-smokers and long-term smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n = 5) and long-term smokers (LTS) with (LTS+, n = 4) and without COPD (LTS-, n = 8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

Short chain fatty acids released by Fusobacterium nucleatum are neutrophil chemoattractants acting via free fatty acid receptor 2 (FFAR2).

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.

Mucin Binding to Moraxella catarrhalis during Airway Inflammation Is Dependent on Sialic Acid.

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples (n = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N-acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N-acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects (n = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

Staphylococcus aureus-Derived PSMα Peptides Activate Neutrophil FPR2 but Lack the Ability to Mediate ß-Arrestin Recruitment and Chemotaxis.

Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit ß-arrestin and induce neutrophil chemotaxis, supporting the previous notion that ß-arrestin translocation is of importance for cell migration. Despite the lack of ß-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit ß-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca2+, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of ß-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.

Increased MUC1 plus a larger quantity and complex size for MUC5AC in the peripheral airway lumen of long-term tobacco smokers.

There is little information on mucins versus potential regulatory factors in the peripheral airway lumen of long-term smokers with (LTS+) and without (LTS-) chronic obstructive pulmonary disease (COPD). We explored these matters in bronchoalveolar lavage (BAL) samples from two study materials, both including LTS+ and LTS- with a very similar historic exposure to tobacco smoke, and healthy non-smokers (HNSs; n=4-20/group). Utilizing slot blot and immunodetection of processed (filtered and centrifuged), as well as unprocessed BAL samples from one of the materials, we compared the quantity and fraction of large complexes of mucins. All LTS displayed an enhanced (median) level of MUC5AC compared with HNS. LTS- displayed a higher level of large MUC5AC complexes than HNS while LTS+ displayed a similar trend. In all LTS, total MUC5AC correlated with blood leukocytes, BAL neutrophil elastase and net gelatinase activity. Large mucin complexes accounted for most MUC5B, without clear group differences. In all LTS, total MUC5B correlated with total MUC5AC and local bacteria. In the same groups, large MUC5B complexes correlated with serum cotinine. MUC1 was increased and correlated with BAL leukocytes in all LTS whereas MUC2 was very low and without clear group differences. Thus, the main part of MUC5AC and MUC5B is present as large complexes in the peripheral airway lumen and historic as well as current exposure to tobacco smoke emerge as potential regulatory factors, regardless of COPD per se. Bacteria, leukocytes and proteinases also constitute potential regulatory factors, of interest for future therapeutic strategies.

Mitocryptides from Human Mitochondrial DNA-Encoded Proteins Activate Neutrophil Formyl Peptide Receptors: Receptor Preference and Signaling Properties.

Phagocytic neutrophils express formyl peptide receptors (FPRs; FPR1 and FPR2) that distinctly recognize peptides starting with an N-formylated methionine (fMet). This is a hallmark of bacterial metabolism; similar to prokaryotes, the starting amino acid in synthesis of mitochondrial DNA-encoded proteins is an fMet. Mitochondrial cryptic peptides (mitocryptides; MCTs) with an N-terminal fMet could be identified by our innate immune system; however, in contrast to our knowledge about bacterial metabolites, very little is known about the recognition profiles of MCTs. In this study, we determined the neutrophil-recognition profiles and functional output of putative MCTs originating from the N termini of the 13 human mitochondrial DNA-encoded proteins. Six of the thirteen MCTs potently activated neutrophils with distinct FPR-recognition profiles: MCTs from ND3 and ND6 have a receptor preference for FPR1; MCTs from the proteins ND4, ND5, and cytochrome b prefer FPR2; and MCT-COX1 is a dual FPR1/FPR2 agonist. MCTs derived from ND2 and ND4L are very weak neutrophil activators, whereas MCTs from ND1, ATP6, ATP8, COX2, and COX3, do not exert agonistic or antagonistic FPR effects. In addition, the activating MCTs heterologously desensitized IL-8R but primed the response to the platelet-activating factor receptor agonist. More importantly, our data suggest that MCTs have biased signaling properties in favor of activation of the superoxide-generating NADPH oxidase or recruitment of ß-arrestin. In summary, we identify several novel FPR-activating peptides with sequences present in the N termini of mitochondrial DNA-encoded proteins, and our data elucidate the molecular basis of neutrophil activation by MCTs.

In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals.

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.

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.

Lipid profiling of suction blister fluid: comparison of lipids in interstitial fluid and plasma.

BACKGROUND: Recent technical advances in the extraction of dermal interstitial fluid (ISF) have stimulated interest in using this rather unexploited biofluid as an alternative to blood for detection and prediction of disease. However, knowledge about the presence of useful biomarkers for health monitoring in ISF is still limited. In this study, we characterized the lipidome of human suction blister fluid (SBF) as a surrogate for pure ISF and compared it to that of plasma. METHODS: Plasma and SBF samples were obtained from 18 healthy human volunteers after an overnight fast. Total lipids were extracted and analyzed by liquid chromatography-tandem mass spectrometry. One hundred ninety-three lipid species covering 10 complex lipid classes were detected and quantified in both plasma and SBF using multiple reaction monitoring. A fraction of the lipid extract was subjected to alkaline transesterification and fatty acid methyl esters were analyzed by gas chromatography-mass spectrometry. RESULTS: The total concentration of lipids in SBF was 17% of the plasma lipid concentration. The molar fraction of lipid species within lipid classes, as well as total fatty acids, showed a generally high correlation between plasma and SBF. However, SBF had larger fractions of lysophospholipids and diglycerides relative to plasma, and consequently less diacylphospholipids and triglycerides. Principal component analysis revealed that the interindividual variation in SBF lipid profiles was considerably larger than the within-subject variation between plasma and SBF. CONCLUSIONS: Plasma and SBF lipid profiles show high correlation and SBF could be used interchangeably with blood for the analysis of major lipids used in health monitoring.

Neutrophils from patients with SAPHO syndrome show no signs of aberrant NADPH oxidase-dependent production of intracellular reactive oxygen species.

OBJECTIVE: We aimed to investigate if aberrant intracellular production of NADPH oxidase-derived reactive oxygen species (ROS) in neutrophils is a disease mechanism in the autoinflammatory disease SAPHO syndrome, characterized by synovitis, acne, pustulosis, hyperostosis and osteitis, as has previously been suggested based on a family with SAPHO syndrome-like disease. METHODS: Neutrophil function was explored in a cohort of four patients with SAPHO syndrome, two of whom were sampled during both inflammatory and non-inflammatory phase. Intracellular neutrophil ROS production was determined by luminol-amplified chemiluminescence in response to phorbol myristate acetate. RESULTS: Cells from all patients produced normal amounts of ROS, both intra- and extracellularly, when compared with internal controls as well as with a large collection of healthy controls assayed in the laboratory over time (showing an extensive inter-personal variability in a normal population). Further, intracellular production of ROS increased during the inflammatory phase. Neutrophil activation markers were comparable between patients and controls. CONCLUSION: Dysfunctional generation of intracellular ROS in neutrophils is not a generalizable feature in SAPHO syndrome. Secondly, serum amyloid A appears to be a more sensitive inflammatory marker than CRP during improvement and relapses in SAPHO syndrome.

CFP-10 from Mycobacterium tuberculosis selectively activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor.

Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca(2+) from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca(2+) response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca(2+) signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function.

A novel receptor cross-talk between the ATP receptor P2Y2 and formyl peptide receptors reactivates desensitized neutrophils to produce superoxide.

Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca(2+) transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP.

Increased intracellular oxygen radical production in neutrophils during febrile episodes of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome.

OBJECTIVE: Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is an autoinflammatory disease of unknown etiology that primarily affects preschool-aged children. PFAPA syndrome is characterized by recurrent attacks of fever and symptoms of inflammation consistent with the disease acronym. Since autoinflammatory diseases are, by definition, mediated by cells of the innate immune system, the aim of this study was to evaluate the functional features of neutrophils, the most abundant innate immune cell in the circulation, in children with PFAPA syndrome. METHODS: Blood polymorphonuclear leukocytes (PMNs), obtained from patients with PFAPA syndrome during both febrile and asymptomatic, afebrile phases of the disease, as well as from healthy children (afebrile controls) and children with fever and abdominal pain (febrile controls), were analyzed for 3 key neutrophil characteristics: 1) apoptosis (measured by annexin V/7-aminoactinomycin D staining), 2) production of reactive oxygen species (ROS) (measured by luminol/isoluminol-amplified chemiluminescence), and 3) priming status (measured as responsiveness to galectin-3 and up-regulation of CD11b). RESULTS: Compared to PMNs obtained from patients with PFAPA syndrome during an afebrile interval and those from febrile controls, PMNs obtained from patients during a PFAPA syndrome flare produced elevated levels of intracellular NADPH oxidase-derived ROS, had significantly diminished rates of spontaneous apoptosis, and displayed signatures of priming. In contrast, PMNs from afebrile patients with PFAPA syndrome had a significantly elevated rate of spontaneous apoptosis compared to PMNs from afebrile controls. CONCLUSION: These findings demonstrate that 3 key aspects of neutrophil innate immune function, namely, apoptosis, priming, and generation of an intracellular oxidative burst, are altered, most prominently during febrile attacks, in children with PFAPA syndrome.

Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

PURPOSE: To compare the inflammatory responses of peripheral blood mononuclear cells (PBMCs) subjected to titanium (Ti) and/or zirconia (Zr) particles while growing on Ti or Zr discs. MATERIALS AND METHODS: In total, 240 discs were fabricated at a size of 2 mm in height and 5 mm in diameter. Of the 240 discs, 120 discs were made of Ti (commercially pure [CP] grade 4), and 120 discs were made of Zr (3 mol% yttria-stabilized zirconia polycrystals [3Y-TZP]). The PBMCs were cultured on the two-disc materials, and particles with a size up to 20 mm Ti (99.5% Ti) and 0.1 to 0.2 mm Zr (3Y-TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bio-Plex assay (Bio-Rad Laboratories), and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. RESULTS: The inflammatory responses of PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were seen when cells were cultured in the presence of Ti particles compared to Zr particles when no discs were used. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-γ, and TNF-α) when particles were added to Ti discs. Higher release of neutrophil extracellular traps (NETs) from neutrophils were seen in presence of Zr particles compared to Ti particles. A reduction in cell death was observed in the presence of Zr particles compared to Ti particles and unstimulated control samples. CONCLUSIONS: The type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti discs and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

Severe chronic non-bacterial osteomyelitis in combination with total MPO deficiency and responsiveness to TNFα inhibition.

We describe a female patient suffering from severe chronic non-bacterial osteomyelitis (CNO) with systemic inflammation and advanced malnutrition and complete deficiency of myeloperoxidase (MPO). CNO is a rare autoinflammatory bone disorder associated with dysregulation of the innate immune system. MPO deficiency is a genetic disorder with partial or complete absence of the phagocyte peroxidase MPO. MPO deficiency has no established clinical phenotype but reports indicate increased susceptibility to infection and chronic inflammation. The patient's symptoms began at 10 years of age with pain in the thighs, systemic inflammation and malnutrition. She was diagnosed with CNO at 14 years of age. Treatment with nonsteroidal anti-inflammatory drugs, corticosteroids, bisphosphonates or IL1-receptor antagonists (anakinra) did not relieve the symptoms. However, the patient responded instantly and recovered from her clinical symptoms when treated with TNFα blockade (adalimumab). Three years after treatment initiation adalimumab was withdrawn, resulting in rapid symptom recurrence. When reintroducing adalimumab, the patient promptly responded and went into remission. In addition to clinical and laboratory profiles, neutrophil functions (reactive oxygen species, ROS; neutrophil extracellular traps, NETs; degranulation; apoptosis; elastase activity) were investigated both in a highly inflammatory state (without treatment) and in remission (on treatment). At diagnosis, neither IL1ß, IL6, nor TNFα was significantly elevated in serum, but since TNFα blockade terminated the inflammatory symptoms, the disease was likely TNFα-driven. All neutrophil parameters were normal both during treatment and treatment withdrawal, except for MPO-dependent intracellular ROS- and NET formation. The role of total MPO deficiency for disease etiology and severity is discussed.

Low-density granulocytes are related to shorter pregnancy duration but not to interferon alpha protein blood levels in systemic lupus erythematosus.

BACKGROUND: An increased risk of pregnancy complications is seen in women with systemic lupus erythematosus (SLE), but the specific immunopathological drivers are still unclear. Hallmarks of SLE are granulocyte activation, type I interferon (IFN) overproduction, and autoantibodies. Here we examined whether low-density granulocytes (LDG) and granulocyte activation increase during pregnancy, and related the results to IFNα protein levels, autoantibody profile, and gestational age at birth. METHODS: Repeated blood samples were collected during pregnancy in trimesters one, two, and three from 69 women with SLE and 27 healthy pregnant women (HC). Nineteen of the SLE women were also sampled late postpartum. LDG proportions and granulocyte activation (CD62L shedding) were measured by flow cytometry. Plasma IFNα protein concentrations were quantified by single molecule array (Simoa) immune assay. Clinical data were obtained from medical records. RESULTS: Women with SLE had higher LDG proportions and increased IFNα protein levels compared to HC throughout pregnancy, but neither LDG fractions nor IFNα levels differed during pregnancy compared to postpartum in SLE. Granulocyte activation status was higher in SLE relative to HC pregnancies, and it was increased during pregnancy compared to after pregnancy in SLE. Higher LDG proportions in SLE were associated with antiphospholipid positivity but not to IFNα protein levels. Finally, higher LDG proportions in trimester three correlated independently with lower gestational age at birth in SLE. CONCLUSION: Our results suggest that SLE pregnancy results in increased peripheral granulocyte priming, and that higher LDG proportions late in pregnancy are related to shorter pregnancy duration but not to IFNα blood levels in SLE.

Receptor-dependent and -independent immunomodulatory effects of phenol-soluble modulin peptides from Staphylococcus aureus on human neutrophils are abrogated through peptide inactivation by reactive oxygen species.

The virulence and pathogenesis mechanisms of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains depend on a newly described group of phenol-soluble modulin (PSM) peptides (the PSMα peptides) with cytolytic activity. These toxins are α-helical peptides with a formyl group at the N terminus, and they activate neutrophils through formyl peptide receptor 2 (FPR2), a function closely correlated to the capacity of staphylococcal species to cause invasive infections. The effects of two synthetic PSMα peptides were investigated, and we show that they utilize FPR2 and promote neutrophils to produce reactive oxygen species (ROS) which in turn trigger inactivation of the peptides. Independently of FPR2, the PSMα peptides also downregulate the neutrophil response to other stimuli and exert a cytolytic effect to which apoptotic neutrophils are more sensitive than viable cells. The novel immunomodulatory functions of the PSMα peptides were sensitive to ROS generated by the neutrophil myeloperoxidase (MPO)-H(2)O(2) system, suggesting a role for this enzyme system in counteracting bacterial virulence.

Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis.

Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associated with azurophile granules in neutrophils indicative of A1AT expression being restricted to the promyelocyte stage. We examined the localization and production of A1AT in healthy donor neutrophils and found A1AT to be a constituent of all granule subtypes and to be released from neutrophils following stimulation. A1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor. Neutrophils from patients with A1AT-deficiency carrying the (PI)ZZ mutation in the A1AT gene appeared structurally and functionally normal, but A1AT produced in leukocytes of these patients lacked the ability to bind proteases efficiently. We conclude that A1AT generation and release from neutrophils add significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema.

Immunological response of human leucocytes after exposure to lipopolysaccharides from Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative bacterium and an important etiologic agent of periodontitis. P. gingivalis releases outer membrane vesicles containing lipopolysaccharides (LPS), which can penetrate periodontal tissues. Once in the periodontal tissues and in contact with immune cells, it may participate in the destructive innate host response associated with the disease. The exact mechanism of P. gingivalis LPS in the disease process is not clear, but it is known to affect a variety of immune responses. OBJECTIVES: To investigate how LPS from P. gingivalis affect neutrophil extracellular trap (NET) formation, cell death and production of cytokines from human neutrophils and peripheral mononuclear blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Isolated neutrophils and PBMCs were cultured with LPS from P. gingivalis or Escherichia coli (E. coli) (control). The NET formation was measured using Sytox green stain. Cell death of neutrophils and PBMCs was analyzed using flow cytometry or Sytox green stain. Cytokine production was measured using enzyme-linked immunosorbent assay (ELISA) kit or Bio-Plex assay. RESULTS: Exposure to LPS from P. gingivalis and E. coli caused significantly lower cell death in neutrophils. NETs were formed after exposure to the two different LPS. In PBMCs, exposure to P. gingivalis and E. coli LPS caused increased levels of IL-1ß and IL-6 compared to unstimulated controls. Increased cell death in PBMCs after exposure to LPS from E. coli in comparison to LPS from P. gingivalis and unstimulated controls was also observed. CONCLUSIONS: LPS from P. gingivalis has the ability to affect both human neutrophils and PBMCs with regard to cytokine production, cell death and production of NETs. LPS from P. gingivalis could be involved in the pathogenesis of periodontitis, and our results may contribute information regarding possible markers for diagnosis and targets for treatment of periodontal disease.