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. 2015 Jan 26:5:8008.
doi: 10.1038/srep08008.

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils

Juliana D B Rocha  1 , Michelle T C Nascimento  2 , Debora Decote-Ricardo  3 , Suzana Côrte-Real  4 , Alexandre Morrot  2 , Norton Heise  1 , Marise P Nunes  4 , José Osvaldo Previato  1 , Lucia Mendonça-Previato  1 , George A DosReis  1 , Elvira M Saraiva  2 , Célio G Freire-de-Lima  1
Affiliations

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils

Juliana D B Rocha (VSports最新版本) et al. Sci Rep. .

Abstract

In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release VSports手机版. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. .

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Figure 1
Figure 1. Inhibition of NET release by Cryptococcus neoformans wild-type, and NET induction by acapsular mutant CAP67.
Human neutrophils were incubated for 90 min at 35°C in the absence (Control, CTR) or presence of 100 µM PMA, B3501 or CAP67 yeasts (at the indicated ratios), and the supernatants were evaluated for DNA content. Individual experiments from 12 donors were performed in duplicate. +p<0.05 PMA versus unstimulated control (CTR); *p<0.05 CAP67 unstimulated control (CTR).
Figure 2
Figure 2. NET release induced by acapsular mutant CAP67 and the absence of NET production by wild-type Cryptococcus neoformans.
Human neutrophils were incubated with acapsular mutant CAP67 (left column) or B3501 yeasts (right column) at a 1:5 ratio on poly-L-lysine treated coverslips, for 90 min. at 35°C. (A, F) Differential interference contrast (DIC) microscopy; (B, G) Cells were fixed and stained with DAPI; (C, H) anti-elastase staining (secondary antibody-FITC); (D, I) anti-myeloperoxidase (secondary antibody-Texas-Red); (E, J) Overlay of (B-D) and (G, H, I) images, respectively. Bars = 20 μm. Human neutrophils were incubated with C. neoformans at 1:5 ratio for 90 min. at 35°C and processed for SEM. The images show the interaction of neutrophils with C. neoformans (K) B3501 or (L) CAP67, strains. The arrows indicate NETs; N = neutrophil and Y = yeasts.
Figure 3
Figure 3. Signaling pathways associated with NET extrusion induced by the acapsular CAP67 strain.
(A) Human neutrophils were incubated with acapsular CAP67 and/or DPI or chloro-amidine (ClA). (B, C) Human neutrophils were preincubated with DPI (10 μg/mL), followed by DHR or H2DCFDA, washed and incubated with CAP67 yeast or PMA. The data are presented as arbitrary fluorescence units (AU) of three experiments. (D) Lactate dehydrogenase assay of neutrophils alone (control) or in the presence of DPI or ClA. The results represent the means ± SD of 4 donors performed in duplicate. +p<0.05 unstimulated control (CTR) versus CAP67 or PMA; *p<0.05 comparing CAP67 to CAP67 + DPI; #p<0.05 CAP67 versus CAP67 + ClA and ∧p<0.05 PMA compared to PMA + DPI.
Figure 4
Figure 4. Cryptococcus neoformans GXM inhibits NET release.
(A) Human neutrophils were incubated in the absence (control, CTR) or presence of 100 µM PMA and capsular B3501 yeasts at a 1:5 ratio. After 90 min, the supernatants were collected and assayed for DNA content. The results are presented as the means ± SD of 4 donors performed in duplicate. +p<0.05 PMA versus unstimulated control (CTR); *p<0.05 PMA versus PMA + B3501 yeasts. (B) Human neutrophils were incubated with PMA and GXM (as indicated), and the supernatants were processed as described above. The results are presented as the means ± SD of 5 donors performed in duplicate. +p<0.05 compared to unstimulated control (CTR); *p<0.05 related to PMA. (C)Human neutrophils were incubated in the absence (control, CTR) or presence of acapsular CAP67 yeasts (1:5) or purified GXM (50 µg/mL), and supernatants were processed as described above. The results are presented as the means ± SD of 4 donors performed in duplicate. +p<0.05 PMA versus unstimulated control (CTR); *p<0.05 PMA versus PMA + indicated yeast.(D)Human neutrophils incubated with H2DCFDA, were washed and stimulated with 100 µM PMA and 50 µg/mL of GXM. The data are presented as the means ± SD of 3 donors performed in duplicate. +p <0.05 PMA versus unstimulated control (CTR); *p <0.05 PMA versus PMA+GXM.
Figure 5
Figure 5. Immunofluorescence detection of NET inhibition by Cryptococcus neoformans GXM.
Human neutrophils were incubated with PMA with or without GXM (50 µg/mL) for 30 min. (A) Differential interference contrast (DIC) microscopy of PMA activated neutrophils; (B) Sytox Green staining, (C) Overlay of A and B images; (D)DIC microscopy of PMA-activated neutrophils pretreated with GXM; (E) Sytox Green staining; (F) Overlay of images D and E. Bars = 20 μm.
Figure 6
Figure 6. Cryptococcus neoformans GXMGal induces NET release.
Human neutrophils were incubated with GXMGal (50 µg/mL). (A) Differential interference contrast (DIC) microscopy; (B) Cells were fixed and stained with DAPI; (C) anti-elastase (secondary antibody-FITC); (D) anti-myeloperoxidase (secondary antibody-Texas-Red); (E) Overlay of B, C, and D images. Bars = 20 μm. Neutrophils were obtained from the same donor in both figures. (F) Human neutrophils were incubated in the absence (control, CTR) or presence of PMA and GXMGal at the indicated doses, and the supernatants were collected and evaluated for DNA content. The results are presented as the means ± SD of 5 donors performed in duplicate. *p<0.05 compared with unstimulated control (CTR). (G) Human neutrophils were incubated with H2DCFDA, followed by washing and stimulation with PMA (100 µM) and GXMGal (50 µg/mL). The data from 3 different donors performed in duplicate are shown as the mean arbitrary fluorescence units (AU) ± SD. *p<0.05 compared with unstimulated control (CTR); +p<0.05 compared with PMA.
Figure 7
Figure 7. NET microbicidal activity on Cryptococcus neoformans.
B3501 wild-type yeasts were incubated overnight in the absence or presence NET-enriched supernatants, and stained with calcofluor or the Live/Dead probe FUN®1 (live yeasts metabolize green FUN1 to a red fluorescent dye; dead yeasts remained green). (A)B3501 + control supernatant stained with calcofluor; (B, C) B3501 + control supernatant labeled with FUN1;(D) Overlay of B and C images depicting live yeasts. (E) B3501 + NET-enriched supernatant stained with calcofluor; (F, G) B3501 + NET-enriched supernatant labeled with FUN1; (H) Overlay of F and G images. Bars = 20 µm. Human neutrophils were incubated in the presence or absence of PMA, capsular B3501 or acapsular CAP67 yeasts. Supernatants were added to cultures of wild-type B3501 yeasts in the presence or absence of DNAse (I), myeloperoxidase inhibitor (J), elastase inhibitor III (K), collagenase I inhibitor (L) and anti-histone neutralizing antibody (M) and growth/viability was evaluated as colony-forming units (CFU) relative to the control. The data are presented as the mean CFU ± SD of 4 donors performed in triplicate. +p<0.05 compared with unstimulated control (PBS-CTR or DMSO); *p<0.05 compared with PMA.
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