Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent

Bianchi, Matteo; Niemiec, Maria Joanna; Siler, Ulrich; Urban, Constantin F.; Reichenbach, Janine

doi:10.1016/j.jaci.2011.01.021

Cited by 221 publications

(205 citation statements)

References 55 publications

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“…Other reports have described the involvement of NADPH oxidase in NET formation. Thus, when NADPH oxidase activity was restored by gene transfer methodologies in neutrophils derived from the stem cells of patients suffering chronic granulomatous disease, reestablishment of NETosis and restoration of fungal defences against Aspergillus nidulans were observed (Yost et al, 2009;Bianchi et al, 2011). Additionally, neutrophils co-cultured with A. fumigatus and treated with DPI completely eliminated NET formation (Bruns et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…and Cryptococcus gattii) have been reported to activate their production (Urban et al, 2006;Bruns et al, 2010;Springer et al, 2010;Bianchi et al, 2011). NET production is induced after recognition of b-glucan present on the cell wall of Ca.…”

Section: Introductionmentioning

confidence: 99%

“…albicans and Aspergillus fumigatus hyphae. These structures are not phagocytosed owing to their large size, and NET formation may be a good strategy to arrest fungal spread (Urban et al, 2009;Bianchi et al, 2011). Although Cr.…”

Section: Introductionmentioning

confidence: 99%

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Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis

et al. 2015

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Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-c did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis

et al. 2015

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“…Although CP is known to inhibit the growth of a wide range of pathogens, the respective contributions of Mn and Zn sequestration to this broad antimicrobial activity have not been determined (10)(11)(12)(13)33). We therefore assessed the sensitivity of a panel of Gram-positive and Gram-negative bacterial pathogens including S. aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Shigella flexneri to WT CP, as well as to the ΔS1 and ΔS2 mutants.…”

Section: Inhibition Of S Aureus Superoxide Defenses Requires Sequestmentioning

confidence: 99%

“…CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10)(11)(12)(13)(14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11,13).…”

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Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

Damo

Kehl-Fie

Sugitani

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

329

594

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The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn 2+ and Zn 2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn 2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn 2+ -binding site from six histidine residues, which distinguishes CP from all other Mn 2+ -binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn 2+ and Zn 2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn 2+ sequestration. These data establish the importance of Mn 2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.bacterial pathogenesis | Staphylococcus aureus | antibiotic resistance | protein crystal structure | isothermal titration calorimetry B acterial pathogens are a significant threat to global public health. This threat is compounded by the fact that these organisms are rapidly becoming resistant to all relevant antimicrobials. Of particular note is the recent emergence of antibiotic-resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States (1) and arguably the most important threat to the public health of the developed world. Consequently, the identification of therapeutics to treat bacterial pathogens is paramount to our continued ability to limit this infectious threat.One promising area of potential therapeutic development involves targeting bacterial access to essential transition metals. This strategy is based on the fact that all bacterial pathogens require these nutrient metals to colonize their hosts (2-5). In vertebrates, the bacterial need for nutrient transition metals is counteracted by the sequestration of these metals by the host. This limitation of essential nutrients, termed "nutritional immunity," is a potent defense against infection (6). Although a variety of metals is required for microbial growth, studies of nutritional immunity have been primarily restricted to the struggle for iron (Fe) between host and pathogen (7-9).An innate immune factor, calprotectin (CP), is abundant in neutrophils and plays a key role in nutritional immunity. CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10-14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11, 13). Moreover, CP-deficient mice have increased microbial burdens following systemic challenge, und...

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SiMA: A simplified migration assay for analyzing neutrophil migration

et al. 2017

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In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and three non-asthmatic patients gives a first hint to the capability of SiMA assay in the context of migration based diagnostics. Using SiMA we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB , the effect of the matrices FN versus HEM as well as the response to different medications, that is, Prednisolone induced a change of direction of migrating neutrophils in FN but no such effect was observed in human placental matrix. In addition, neutrophils of asthmatic individuals showed an increased proportion of cells migrating toward the vehicle. With the SiMA platform we presented a simplified but yet flexible platform for cost-effective tracking and quantification of neutrophil migration. The introduced method is based on a simple microscopic video stage, standardized, commercially available, µ-fluidic migration chambers and automated image analysis, and track validation software. © 2017 International Society for Advancement of Cytometry.

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Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent

Cited by 221 publications

References 55 publications

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis

Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

SiMA: A simplified migration assay for analyzing neutrophil migration

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