Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence

Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry

Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils

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

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD

The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue <i>In Vivo</i> and Give Rise to Distinct NETs <i>In Vitro</i>

<div><p>Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. <i>In vitro</i>, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites <i>in vivo</i>, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon <i>in vivo</i> transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue <i>in vivo</i>. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.</p> </div

Presence of OLFM4 in NETs.

<p><b>A</b>) Neutrophils isolated from heparinized blood were adhered to glass coverslips and either stimulated with PMA to form NETs (+PMA) or left unstimulated (-PMA). They were then left unpermeabilized (-Perm.) or permeabilized using acetone/methanol (+Perm.), after which they were immunostained for OLFM4 (red), plasma membranes (PM) were stained using FITC-conjugated WGA (green), and DNA was stained with DAPI (blue). Confocal images show representative cells or NETs from at least three individual experiments. <b>B</b>) Adherent neutrophils were induced to form NETs as in A, and unpermeabilized samples were immunostained for OLFM4 (green) and MPO (red). DNA was stained with DAPI (blue). Confocal images show representative NETs from three individual experiments. <b>A</b>–<b>B</b>: Arrows indicate OLFM4-containing cells or NETs, while arrow heads indicate cells or NETs without OLFM4. The fluorophore conjugates used for each staining are indicated (AF = Alexa Fluor). The scale bars represent 5 µm.</p

Subcellular localization of OLFM4.

<p><b>A</b>) Neutrophils were immunofluorescently stained for OLFM4 (green) together with specific granule marker NGAL or azurophil granule marker CD63 (red), and DNA was labeled with DAPI (blue). Colocalization was analyzed by imaging flow cytometry and images show representative cells from the double positive populations (BF = brightfield). The diagram shows the mean colocalization score for the two fluorophores +SD from at least three experiments. The technical positive control was FITC-conjugated mouse anti-human CD16 antibody followed by Alexa Fluor 647-coupled goat anti-mouse secondary antibody and the technical negative control was FITC-conjugated mouse anti-human CD16 antibody together with DAPI, showing the minimum and maximum values that can be obtained by this analysis. The biological positive control was specific granule marker lactoferrin (LF) together with NGAL, and the biological negative control was lactoferrin together with CD63, showing the resolution of the analysis. <b>B</b>) Pooled neutrophils from three donors were subjected to subcellular fractionation, and the fractions containing peak content of azurophil granule marker (MPO; fraction 1), specific granule marker (lactoferrin; fractions 10-12), gelatinase granule marker (gelatinase; fractions 13-15) and secretory vesicle marker (latent alkaline phosphatase; fraction 22) were subjected to Western blot with anti-OLFM4 antibody using rOLFM4 as a positive control. One representative blot out of three is shown. Lactoferrin (specific granule marker) and gelatinase (gelatinase granule marker) blots are also shown for fractions 10-15.</p

Functional analyses of the neutrophil subpopulations.

<p><b>A</b>) Neutrophils were either fixed immediately after separation (Fresh) or allowed to enter apoptosis spontaneously (-FasL) or through the Fas pathway (+FasL) for 4 or 20 h, after which they were fixed and stained using a TUNEL assay in combination with immunostaining for OLFM4. The histogram shows fresh (blue) and apoptotic (20 h +FasL, red) neutrophils stained using TUNEL. The dot plot shows double staining of fragmented DNA (TUNEL) and OLFM4, with the quadrants set using fresh neutrophils for TUNEL and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of apoptosis +SD, based on TUNEL-positivity, in the OLFM4-positive (black) and -negative (grey) subpopulations of three donors. <b>B</b>) Freshly isolated and apoptotic neutrophils (20 h incubation without or with FasL) were subjected to immunostaining of OLFM4 (black) and NGAL (grey). The diagram shows the mean percentage of positive cells +SD for each protein from three independent experiments. <b>C</b>) Neutrophils were allowed to phagocytose <i>M. tuberculosis</i> H37Ra strain expressing GFP (<i>M</i>.<i>tb</i>-GFP), either unopsonized or serum-opsonized, at an MOI of 5 for 30 min, fixed, and immunostained for OLFM4. The histogram shows neutrophils incubated without (No prey) or with (+<i>M</i>.<i>tb</i>-GFP ops) serum-opsinized <i>M. tuberculosis</i>. The dot plot shows fluorescence intensity in the GFP and OLFM4 channels after phagocytosis of opsonized bacteria, with the quadrants set using neutrophils with no prey added for <i>M</i>.<i>tb</i>-GFP and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of phagocytosing neutrophils +SD in the OLFM4-positive (black) and -negative (grey) subpopulations from three independent experiments.</p

OLFM4 expression in transmigrated tissue neutrophils.

<p>Skin chamber neutrophils (<b>A</b>) were obtained from healthy volunteers and synovial fluid neutrophils (<b>B</b>) were collected from inflammatory arthritis patients. Blood samples were also drawn from all subjects. After fixation of tissue and blood neutrophils, OLFM4 was immunolabelled. The histograms show OLFM4 staining in blood and skin chamber (Skin ch.) or synovial fluid neutrophils from one donor each. The diagrams show the percentage of OLFM4-positive neutrophils in blood and transmigrated neutrophils from three different donors.</p