9 research outputs found
Cell wall integrity sensing and remodeling are involved in the fungal response to neutrophil attack.
<p>(A-D) Streptavidin-Alexa 647 labeled <i>C</i>. <i>albicans</i> hyphae of the indicated strains were incubated with neutrophils or alone. Neutrophils were not lysed and samples were stained with CFW and sDectin-1-Fc. (A) Representative images of the <i>HOG1</i> strain set. Images were analyzed by scoring cells for localized chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (B) The scoring data was normalized based on the frequency of cell wall changes in the wildtype strain plus neutrophils and represents the mean ± SEM from three independent experiments. (C) Representative images of the chitin synthase strain set. Images were analyzed by scoring cells for increased chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (D) Data is presented as the percent of total cells with the phenotypes and represents the mean ± SEM from three independent experiments. The Chs3-YFP strain was incubated with neutrophils in an imaging dish and timelapses were taken. (E) Panels taken from a timelapse of the Chs3-YFP strain after neutrophil attack. (F) The JC94-2 strain was incubated with neutrophils for the indicated time in an imaging dish before being imaged. Representative images from the 3 hour timepoint. The red dotted line represents the outline of a neutrophil. (G) Images were analyzed by obtaining the mean fluorescent intensity of GFP at sites with or without chitin deposition and the data is presented as the mean MFI ± SEM at sites from three pooled experiments except the 1h timepoint, which represents two pooled experiments. (H-I) The Sur7-GFP strain was incubated with neutrophils. Following incubation, neutrophils were lysed and fungi were stained with sDectin-1-Fc and CFW. Data represents the MFI of GFP or Cy3 staining at sites with or without chitin deposition or from the no neutrophil control and is presented as the mean MFI ± SEM from three pooled experiments. * p-value of ≤0.05, ** p-value of ≤0.01 and *** p-value of ≤0.001. For I, * p≤0.05 comparing Sur7-GFP at chitin deposition sites vs no chitin deposition or no PMN control. (¥) p≤0.001 comparing the WT and <i>hog1/HOG1</i> plus neutrophil groups to the no neutrophil control groups. (≠) p≤ 0.01 comparing the <i>hog1</i> plus neutrophils group to the no neutrophil control groups. (‡) p≤ 0.01 comparing sDectin-1-Fc at chitin deposition sites vs no chitin deposition sites or no neutrophil controls. (Student’s T-test for D and G or one way ANOVA with Tukey’s post-test for B and I). n.s. means non-significant. Scale bar represents 10 μm.</p
Neutrophil Attack Triggers Extracellular Trap-Dependent <i>Candida</i> Cell Wall Remodeling and Altered Immune Recognition
<div><p>Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen <i>Candida albicans</i>, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope β-glucan in its cell wall from host recognition. It has been demonstrated previously that β-glucan becomes exposed during infection <i>in vivo</i> but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 β-glucan receptor <i>in vitro</i>. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes <i>in vivo</i>. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the <i>C</i>. <i>albicans</i> cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.</p></div
<i>C</i>. <i>albicans</i> induces an enhanced IL-6 response after neutrophil attack.
<p>RAW-blue cells were treated as indicated and incubated for 6 hours. (A-B) ELISA was conducted on supernatants from RAW-Blue cells to detect (A) IL-6 or (B) TNF-α. A representative experiment is shown for each cytokine. Comparisons were done by two way ANOVA with Tukey’s post-test. ** p-value of ≤0.01 and *** p-value of ≤0.001. (C) Schematic representation of the macrophage experiment. 1.) Neutrophils are incubated with <i>C</i>. <i>albicans</i> overnight. 2.) During this incubation, neutrophils attack <i>C</i>. <i>albicans</i>, initiating cell wall remodeling and resulting in β-glucan unmasking and chitin deposition. 3.) Following incubation, neutrophils are lysed and samples are treated with DNase 1 to reduce activation by neutrophil debris. 4.) Samples are UV inactivated. 5.) Samples are then incubated with macrophages. Macrophages recognize the exposed fungal epitopes and produce cytokines in response. The attacked fungi with increased epitope unmasking elicit more IL-6 in comparison to the controls.</p
NETs are critical for immune attack to cause β-glucan unmasking.
<p>(A-B) Neutrophils from C57BL/6J mice were incubated with WT-FarRed670 hyphae. Neutrophils were not lysed and samples were stained for MPO or citrullinated H3. Representative images of MPO (A) and citrullinated H3 staining (B). Scale bar represents 20 μm. (C-D) Neutrophils were pretreated with DNase I, DPI or ABAH and then incubated with WT-FarRed670 for 2.5 hours. Neutrophils were not lysed and then samples were stained with CFW, sDectin-1-Fc and Sytox Green. Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic far red expression (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005644#sec010" target="_blank">Methods</a>). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT or DMSO treated to other groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT or DMSO treated to other groups. * p-value ≤ 0.01 when comparing the overlap group from WT or DMSO treated to other groups. Comparisons done with one way ANOVA and Tukey’s post-test. (E-F) Neutrophils from C57BL/6J or gp91<sup>phox-/-</sup> mice were incubated with Streptavidin-Alexa 647 labelled SC5314-GFP hyphae. Neutrophils were lysed and fungi were stained with sDectin-1-Fc and Calcofluor White. (E) A representative set of images are shown for each group. (F) Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic GFP expression (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005644#sec010" target="_blank">Methods</a>). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT to either gp91<sup>phox-/-</sup> or no neutrophil groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT to either gp91<sup>phox-/-</sup> or no neutrophil groups. * p-value ≤ 0.01 when comparing the overlap group from WT to either gp91<sup>phox-/-</sup> or no neutrophil groups. Comparisons done with one way ANOVA and Tukey’s post-test. (G-K) C57BL/6J or gp91<sup>phox-/-</sup> mice were injected in the tail vein with SC5314-GFP and kidneys were harvested on day 5 post infection. (G-H) Representative images of kidney homogenates stained with sDectin-1-Fc and CFW Bottom panels show homogenates treated with secondary antibody only as a control. (I) Representative images of an overnight culture of SC5314-GFP grown in RPMI and then stained with sDectin-1-Fc and CFW. (J-K) Quantification of sDectin-1-Fc staining (J) and chitin staining (K). Data is presented as the mean ± SEM from two pooled experiments, except for the RPMI group which represents a single experiment. *** p-value ≤0.001 (Kruskal-Wallis with Dunn’s post-test). n.s means non-significant. Scale bar represents 10 μm.</p
Neutrophil attachment results in rapid cell wall damage to <i>C</i>.<i>albicans</i>.
<p>(A-D) Streptavidin-Alexa 647-labeled SC5314-GFP was incubated with or without neutrophils. (A-C) Representative timelapse images at one minute intervals for (A) attacked, (B) unattacked and (C) control hyphal segments. (D) Relative streptavidin mean fluorescence intensity (MFI) at attacked and unattacked sites. Data represents the pooled average of thirteen cells measured in three experiments ± SEM. Scale bar = 10 μm. (E-I). Streptavidin-Alexa 647-labeled Hwp1-GFP fungi were incubated with or without neutrophils. (E-G) Representative time-lapse images for (E) attacked, (F) unattacked and (G) control hyphal segments. (H) Relative streptavidin intensity for twenty three cells and Hwp1-GFP intensity for thirty three cells imaged in three independent experiments was measured and the pooled average ± SEM is shown. (I) Relative intensity at the 2 minute post-attachment (T+2) time point is shown for the pooled data as box plots to illustrate the full dataset. Box plot whiskers represent the 1.5 interquartile range either below or above the lower or upper quartile ** p ≤0.01 and *** p≤0.001 (one-way ANOVA with Tukey’s post-test).</p
Neutrophils are critical for unmasking <i>C</i>. <i>albicans</i> β-glucan in the kidney during disseminated candidiasis.
<p>(A-E) BALB/cJ mice were injected in the tail vein with SC5314-GFP. They were then treated with either 1A8 antibody to induce neutropenia or IgG2a isotype control antibody via i.p. injection at day two post infection and sacrificed at day five post infection. (A-B) Representative images of kidney homogenates stained with sDectin-1-Fc and Calcofluor White. Bottom panels show homogenates treated with secondary antibody only as a control. (C) Representative images of an overnight culture of SC5314-GFP grown in RPMI. Scale bar represents 10 μm. (D-E) Quantification of sDectin-1-Fc staining (D) and chitin staining (E). Data is presented as the mean ± SEM from three pooled experiments. ** p-value ≤0.01 and *** p-value ≤0.001 (Kruskal-Wallis with Dunn’s post-test).</p
Chitin deposition and β-glucan unmasking result from an active fungal process.
<p>(A-B) Streptavidin-Alexa 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005644#ppat.1005644.g001" target="_blank">Fig 1D</a>. (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated <i>C</i>. <i>albicans</i>. Scale bars represent 10 μm. * p-value of < 0.05 and *** p-value of ≤ 0.001 (one way ANOVA with Tukey’s post-test). n.s. means non-significant.</p
Neutrophils cause β-glucan unmasking and disrupt cell wall architecture.
<p>(A-C) SC5314-GFP cells were biotinylated, labeled with Streptavidin-Alexa647 and incubated with neutrophils or alone. Neutrophils were lysed and fungi were stained with sDectin-1-Fc and Calcofluor White. (A) A representative set of images for pre-challenge streptavidin labeling. (B-C) Images were analyzed by obtaining the mean fluorescent intensity in the blue, red and far red channels at sites of β-glucan exposure, at sites without β-glucan exposure and from sites in the no neutrophil control, limited to viable cell segments. (B) Streptavidin versus CFW fluorescence for individual sites from three pooled experiments, broken down into sites with sDectin-1-Fc staining or not. (C) Streptavidin fluorescence at sites with sDectin-1-Fc staining or no sDectin-1-Fc staining. Mean MFI ± SEM of three independent experiments. Scale bar represents 10 μm. * p ≤0.05 (one-way ANOVA with Tukey’s post-test).</p