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. 2013 Apr 15;190(8):4136-48.
doi: 10.4049/jimmunol.1202671. Epub 2013 Mar 15.

An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans

Angel S Byrd  1 , Xian M O'Brien, Courtney M Johnson, Liz M Lavigne, Jonathan S Reichner
Affiliations

VSports注册入口 - An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans

Angel S Byrd et al. J Immunol. .

VSports - Abstract

The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17. 2 ± 3. 4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded VSports手机版. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response. .

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Conflict of interest statement

DISCLOSURES

Conflict-of-interest disclosure: The authors claim no competing financial interests.

"VSports注册入口" Figures

Figure 1
Figure 1. Primed neutrophils in the context of Fn + β-glucan, but not Fn or β-glucan alone, form aggregates and Ab inhibition and activation of β2 integrin CR3 modulates cell aggregation
(A–C) Micrographs show PMNs that were adhered to Fn (6 µg/ml), β-glucan (1 mg/ml) or Fn + β-glucan pre-coated wells. (A) PMNs were pre-treated with 10−9 M fMLF for 20 min on ice and Mn+2 immediately before adhering cells. Cells formed aggregates in the context of Fn + β-glucan, but not Fn or β-glucan alone. Additionally, aggregation required priming with both fMLF and Mn+2 (data not shown). All experiments were incubated for 30 min at 37° C. Data represents at least ten separate experiments done using neutrophils from different individual donors. (B) Aggregation is inhibited with CR3, but not Dectin-1 blocking mAb. PMNs were pre-treated as described in (A) before adhering cells to Fn ± β-glucan coated wells for 30 min at 37° C. When PMNs were pretreated with 20 µg/ml of anti-CR3 blocking mAb (Clone 44abc), cell aggregation was prevented; anti-Dectin blocking mAb (GE2) and IgG1, which was used as an isotype control, have no effect on neutrophil aggregation; and (C) fMLF and Mn+2 primed cells were pre-treated with an anti-CR3 activating mAb (VIM12 F(ab’)2) which mimics β-glucan. Treated cells aggregate on Fn-coated wells and exhibit an exaggerated cell aggregation phenotype on wells coated with Fn + β-glucan. These data represent at least four independent experiments using neutrophils from different individual donors. All images were taken at 20× magnification (Bar=100 µm).
Figure 2
Figure 2. Phosphorylated ERK levels are increased in Fn + β-glucan treated PMNs
(A) PMNs were pre-treated as previously described in the legend for Figure 1A before they were adhered to Fn ± β-glucan pre-coated wells and incubated at 37 °C for 5, 10, 20, and 30 min. Cells were harvested at the appropriate time point, lysed and separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Membranes were immunoblotted with anti- pERK, anti-total ERK, and anti-actin mAb. White bar indicates noncontiguous samples that were run on the same gel. (B) A representative densitometric analysis was determined by scanning densitometry using Image J analysis software and expressed as phosphorylated ERK over actin. (C) Neutrophils were incubated on ice for 20 min with MEK inhibitors U0126 (50 µM) and PD 98059 (30 µM) and subsequently pre-treated as previously described in the legend for Figure 1A before they were adhered to Fn ± β-glucan pre-coated wells and incubated at 37 °C for 30 min. Inhibition of ERK phosphorylation prevented PMN aggregation when adhered to Fn + β-glucan. Images were taken at 20× magnification (Bar=100 µm). Blots were derived from the same protein samples. These results represent at least four independent experiments using neutrophils from different individual donors.
Figure 3
Figure 3. Rapid PMN NET formation of PMNs adhered to immobilized Fn + β-glucan
PMNs were pre-treated as previously described in the legend for Figure 1A before they were adhered to Fn ± β-glucan pre-coated wells and incubated at 37 °C for 30 min. (A) After aggregates formed, DNaseI was added directly to the wells, which significantly disrupted PMN aggregates. Images were taken at 20× magnification (Bar = 100 µm). (B) Sytox Green staining shows NET formation in the context of Fn + β-glucan, corresponding to PMN aggregates. Aggregates and NET formation are not seen in cells responding to Fn alone. Images were taken at 20× magnification using confocal microscopy as described (see Materials and Methods) (Bar=100 µm). Bar graph shows 17 fold increase in extracellular DNA on Fn + β-glucan as compared to Fn alone as quantified by plate fluorometer under our assay conditions using PicoGreen dsDNA staining. Error bars represent SEM; *p<0.01, paired sample Student’s t test. These results represent six independent experiments using neutrophils from at least three individual donors.
Figure 4
Figure 4. NETs formed by PMNs adhered to immobilized Fn + β-glucan decrease yeast viability
(A) Neutrophils were prepared as previously described in the legend for Figure 1A, and following glutaraldehyde fixation, samples were processed and examined with transmission electron microscopy. Visualization of changes in the nuclear envelope and NET formation (arrowhead) at a direct magnification of 7,100× (Bar=2 µm). The nuclear envelope undergoes a division of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). (i) DNA material of NETs shown extracellularly at a direct magnification of 44,000× (Bar=100 nm) and (ii) Enlarged view of cytoplasmic vesicles containing NETs shown at a direct magnification of 36,000× (Bar=500 nm) using a 80 kV voltage. Sections were stained with uranyl acetate and lead citrate (see Materials and Methods). These results represent at least four independent experiments using neutrophils from different individual donors. (B) Yeast viability by reduction of MTT. PMNs were pre-treated as described in the legend Figure 1A before they were adhered to Fn + β-glucan pre-coated wells and incubated at 37 °C for 30 min. Wells were washed and RPMI, C. albicans blastoconidia, and 10 µM cytochalasin were added to wells ± DNaseI and incubated at 37°C overnight. For comparison, blastoconidia were incubated with the hypotonic lysate of an equivalent number of PMNs. Wells were scored microscopically for yeast growth (left) and viability was quantified by MTT reduction (bar graph). * p<0.01 vs. PMN lysate; ** p<0.01 PMN NETs ± DNaseI, ANOVA full factorial, post hoc Newman-Keuls; error bars represent SEM. Results represent 6 to 24 independent experiments from at least three donors. (Bar=100 µm).
Figure 5
Figure 5. CR3 blockade attenuates neutrophil aggregation and rapid NET formation of PMNs adhered to immobilized Fn + β-glucan
PMNs were pretreated as previously described in the legend for Figure 1A. PMNs additionally pre-treated with 20 µg/ml of anti-CR3 blocking mAb (Clone 44abc) did not aggregate. Sytox Green staining after the samples were incubated at 37°C for 30 min demonstrated attenuation of NET formation; IgG1, which was used as an isotype control, has no effect on neutrophil aggregation or NET formation (Bar=100 µm). Bar graph shows significant inhibition of NET production on Fn + β-glucan when cells are pretreated with 20 µg/ml of anti-CR3 blocking mAb (Clone 44abc), as quantified by plate fluorometer using PicoGreen dsDNA staining. Error bars represent SEM; *p<0.01 untreated vs. anti-CR3 blocking mAb; nd no significant difference vs. untreated ANOVA full factorial, post-hoc Newman-Kuels. These results represent at least three independent experiments using neutrophils from different individual donors.
Figure 6
Figure 6. C. albicans induced rapid PMN NETs that are inhibited by anti-CR3 blocking mAb
C. albicans hyphae were grown on culture dishes coated with 40 µg/ml Fn. Neutrophils were pre-treated as previously described in Figure 1A and blocked with anti-CR3 mAb (Clone 44abc) for 20 min on ice before adding them to the dishes containing yeast hyphae and incubated at 37 °C for 30–50 min. Neutrophils conform to the hyphae and confocal microscopy shows NET formation by Sytox Green staining except when inhibited by anti-CR3 mAb indicating a functional role for CR3. Images were taken at 20× magnification using confocal microscopy as described (see Materials and Methods) (Bar=100 µm). These results represent at least five independent experiments using neutrophils from different individual donors.
Figure 7
Figure 7. Rapid PMN NET formation in response to C. albicans decreases hyphal viability, is matrix-dependent, and is inhibited by a β-glucan-specific mAb
(A) Hyphal viability by reduction of MTT. Neutrophils were pre-treated as previously described in Figure 1A and added to lightly seeded C. albicans hyphae grown on Fn coated wells and incubated at 37 °C for 30 min. Wells were washed and RPMI ± DNaseI was added and incubated at 37°C overnight. Wells were scored microscopically for yeast growth (left) and viability was quantified by MTT reduction (bar graph). Error bars represent SEM; * p<0.01 vs. hyphae + PMNs, paired sample Student’s t test. These data represent eight independent experiments with at least three donors. (B–C) C. albicans hyphae were grown on coverslips coated with 40 µg/ml Fn. Neutrophils were pre-treated as previously described in Figure 1A, added to the hyphae and visualized by Sytox Green staining. (B) PMN NET formation is induced when hyphae and neutrophils are adhered to Fn, but not poly-L-lysine. (C) Neutrophil NET formation is prevented when fungal β-glucan is pre-blocked by the β-glucan-specific mAb BFDiv before PMNs were added to hyphae and incubated at 37 °C for 30 min. Samples were stained and visualized at 20× magnification (Bar=100 µm). These data represent four independent experiments using neutrophils from different individual donors.
Figure 8
Figure 8. Neutrophil aggregation and rapid PMN NET formation in the context of immobilized Fn + β-glucan are independent of the PMN respiratory burst
(A–B) PMNs and yeast hyphae were prepared as previously described in Figure 6. Cells were pretreated with DPI (6.25 µM) on ice for 20 min before they were adhered to Fn ± β-glucan pre-coated wells and incubated at 37 °C for 30–50 min. (A) Inhibition of the respiratory burst with DPI does not prevent PMN aggregation. Images were taken at 10× magnification (Bar=100µM). (B) Inhibition of the respiratory burst with DPI does not attenuate NET formation in the context of Fn + β-glucan. Sytox Green was added to the sample after aggregate formation to assess NET formation. Images were taken at 20× magnification (Bar=100 µm). (C) DPI inhibits the PMN respiratory burst to PMA. Error bars represent SD. These results represent at least four independent experiments using neutrophils from different individual donors.
Figure 9
Figure 9. Rapid PMN NET formation in the context of C. albicans is independent of the PMN respiratory burst
(A–C) Neutrophils and yeast hyphae were prepared as previously described in Figure 6. (A) Cells were pretreated with DPI (6.25 µM) on ice for 20 min before adding them to coverslips containing yeast hyphae and incubated at 37 °C for 30–50 min. Inhibition of the respiratory burst with DPI does not attenuate NET formation in the context of C. albicans hyphae. NET formation was visualized at 10× magnification (Bar=100 µm). (B) Neutrophils were incubated the MEK inhibitor U0126 for 20 min on ice before adding them to hyphae to prevent ERK phosphorylation. This treatment resulted in a significant decrease in NET formation in response to C. albicans hyphae. Sytox Green was added after the samples were incubated at 37°C for 30–50 min. NET formation was visualized at 20× magnification (Bar=100 µm). (C) PMNs were loaded with the respiratory burst indicator CM-H2DCFDA, treated with U0126, and then added to hyphae. Inhibition of ERK phosphorylation with U0126 does not inhibit the respiratory burst (top) but it does inhibit the formation of NETs (bottom) as visualized by Sytox Green staining. NET formation was visualized at 10× magnification (Bar=100 µm). Data represent four independent experiments with neutrophils from different individual donors.
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References

    1. Aratani Y, Koyama H, Nyui S, Suzuki K, Kura F, Maeda N. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. Infect Immun. 1999;67:1828–1836. - PMC - PubMed
    1. Bassetti M, Righi E, Costa A, Fasce R, Molinari MP, Rosso R, Pallavicini FB, Viscoli C. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis. 2006;6:21–26. - PMC - PubMed
    1. Cheng MF, Yang YL, Yao TJ, Lin CY, Liu JS, Tang RB, Yu KW, Fan YH, Hsieh KS, Ho M, Lo HJ. Risk factors for fatal candidemia caused by Candida albicans and non-albicans candida species. BMC Infect Dis. 2005;5:22–26. - "VSports在线直播" PMC - PubMed
    1. Fraser VJ, Jones M, Dunkel J, Storfer S, Medoff G, Dunagan WC. Candidemia in a tertiary care hospital: Epidemiology, risk factors, and predictors of mortality. Clin Infect Dis. 1992;15:414–421. - PubMed
    1. Metzler KD, Fuchs TA, Nauseef WM, Reumaux D, Roesler J, Schulze I, Wahn V, Papayannopoulos V, Zychlinsky A. Myeloperoxidase is required for neutrophil extracellular trap formation: Implications for innate immunity. Blood. 2011;117:953–959. - PMC - PubMed

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