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.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes.

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.

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.

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.

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.

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.

Immunomodulator CD200 Promotes Neurotrophic Activity by Interacting with and Activating the Fibroblast Growth Factor Receptor.

The CD200 ligand is expressed by a variety of cell types, including vascular endothelia, kidney glomeruli, some subsets of T and B cells, and neurons in the brain and periphery. In contrast, the receptor of CD200, CD200R, has a limited expression pattern and is mainly expressed by cells of myeloid origin. A recently solved crystal structure of the CD200-CD200R ectodomain complex suggests involvement of the first immunoglobulin (Ig)-like modules in ligand-receptor binding, resulting in the inhibition of myeloid cell function. In the central nervous system, CD200 has been implicated in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR.

Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.

Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species.

AIM: Neutrophil extracellular traps (NETs) are mesh-like DNA fibers clad with intracellular proteins that are cast out from neutrophils in response to certain stimuli. The process is thought to depend on reactive oxygen species (ROS) generated by the phagocyte NADPH-oxidase and the ROS-modulating granule enzyme myeloperoxidase (MPO), but when, how, and where these factors contribute is so far uncertain. The neutrophil NADPH-oxidase can be activated at different cellular sites and ROS may be produced and processed by MPO within intracellular granules, even in situations where a phagosome is not formed, e.g., upon stimulation with phorbol myristate acetate (PMA). OBJECTIVES: We investigated the subcellular location of ROS production and processing by MPO in the context of PMA-induced NET formation. RESULTS: Complete neutralization of extracellular ROS was not sufficient to block NET formation triggered by PMA, indicating that intragranular ROS are critical for NETosis. Employing a set of novel MPO-inhibitors, inhibition of NET formation correlated with inhibition of intragranular MPO activity. Also, extracellular addition of MPO was not sufficient to rescue NET formation in completely MPO-deficient neutrophils and specific neutralization by luminol of MPO-processed ROS within intracellular granules led to a complete block of PMA-triggered NET formation. CONCLUSION: We show for the first time that inhibition of intragranular MPO activity, or neutralization of intragranular MPO-processed ROS by luminol effectively block NET formation. Our data demonstrate that ROS must be formed and processed by MPO in order to trigger NET formation, and that these events have to occur within intracellular granules.

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.

Measurement of respiratory burst products, released or retained, during activation of professional phagocytes.

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 (-)) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst 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, the absorbance change following reduction of cytochrome c, and the 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 more clinically oriented research on innate immune mechanisms and inflammation.

The human neutrophil subsets defined by the presence or absence of OLFM4 both transmigrate into tissue in vivo and give rise to distinct NETs in vitro.

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. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, 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 in vivo 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 in vivo. 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.

Inhibition of phospholipase A(2) abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils.

Upon activation of human neutrophils, superoxide can be produced at two cellular sites; either in the plasma membrane, giving extracellular release of oxidants, or in intracellular organelles, resulting in oxidants being retained in the cell. The involvement of phospholipase A(2) (PLA(2)) in phorbol myristate acetate (PMA)-induced activation of the two pools of NADPH-oxidase was investigated using a variety of PLA(2) inhibitors and the oxidase activity was measured by luminol/isoluminol-amplified chemiluminescence (CL). Two of the seven inhibitors were without effect, two inhibitors inhibited both intra- and extracellular ROS production equally, and three inhibitors inhibited intracellular but not extracellular CL. Using another technique to measure ROS, PHPA oxidation, we found that intracellular ROS production was unaltered with the three last inhibitors, indicating that PLA(2) is not involved in the NADPH-oxidase activity per se, but in the intracellular processing of the radicals necessary for the CL reaction to take place. The PLA(2) inhibitors did not abolish the activity of myeloperoxidase (MPO), an enzyme necessary for intracellular CL to occur. Instead, we suggest that these PLA(2) inhibitors block heterotypic granule fusion and prohibit the colocalization of ROS and MPO needed for intracellular CL activity.