Journal: Nature Communications

Article Title: Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor

doi: 10.1038/s41467-019-09915-2

Figure Lengend Snippet: Inhibition of EGFR activity suppresses C. albicans- and Candidalysin (CL)-induced protein activation and expression. Use of either Gefitinib or PD153035 EGFR inhibitor suppresses WT C. albicans -induced ( a – f ) and CL-induced ( g – l ) pEGFR (Y1068 and Y845), c-Fos and pMKP1 ( a , g ), as well as secretion of GM-CSF ( e , k ) and G-CSF ( f , l ). Both EGFR inhibitors suppressed C. albicans -induced IL-1α, IL-1β, IL-6 ( b – d ) but not CL-induced IL-1α, IL-1β, IL-6 ( h – j ). The CL-deficient ece1 Δ/Δ +ECE1 Δ184–279 strain poorly induced all proteins investigated. The data are representative images ( a , g ) or combined averages of three independent experiments ( b – f , h – l ). Protein lysates taken at 2 h post infection for western blot analysis, cytokines assessed at 24 h post infection via luminex. Solid vertical lines indicate omitted, extraneous portions of blot images. Unmatched, one-way ANOVA with Bonferroni multiple comparison’s test was used to assess statistical significance. Error bars represent SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Anti-IL-1α, IL-1β, IL-6, GM-CSF and G-CSF antibody beads were purchased from Bio-techne.

Techniques: Inhibition, Activity Assay, Activation Assay, Expressing, Infection, Western Blot, Luminex

Journal: Nature Communications

Article Title: Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor

doi: 10.1038/s41467-019-09915-2

Figure Lengend Snippet: Candidalysin-induced release of EREG and EPG contribute to subsequent EGFR-mediated signalling. Release of EREG ( a ), EPG ( b ) and NRGs 2, 3 and 4 ( c – e ) was observed following treatment of candidalysin to TR146 cells, in a dose-dependent manner with rapid onset (within 15 min). AREG was also detected in candidalysin-treated cell supernatants, but the release was gradual and accumulated over 6 h ( f ). At 24 h p.i. epiregulin (EREG) ( g ) and epigen (EPG) ( h ) were induced by candidalysin-expressing (WT and ece1 Δ/Δ +ECE1 (black bars)) but not candidalysin-deficient ( ece1 Δ/Δ and ece1 Δ/Δ +ECE1 Δ184–279 (grey bars)) C. albicans strains. While amphiregulin (AREG) was induced by all fungal strains tested, diminished potency was observed by those unable to express candidalysin ( i ). Increasing concentrations of EREG, EPG or EREG + EPG, were used to stimulate TR146 oral epithelial cells. j Phosphorylation of EGFR (at Y1068 and Y845 sites) and MKP1 proteins occurred in a dose-dependent manner in response to EREG and EREG + EPG, but not EPG alone. Induction of c-Fos by ligand stimulation was not dose-dependent ( j ). The effects of ligand exposure are not comparable to that of 70 µM candidalysin. While a dose-dependent trend of IL-6 ( m ), GM-CSF ( n ) and G-CSF ( o ) induction is observed in response to increasing concentrations of all ligand combinations, the changes are not statistically significant. IL-1α ( k ) and IL-1β ( l ) are not induced by ligand exposure. One-way ANOVA followed by a Bonferroni multiple comparison’s test was used to calculate statistical significance between groups. Graphs are an average of 3 ( a – g , i – o ) or 2 ( h ) independent experiments; one-way ANOVA with Bonferroni multiple comparison’s test used to assess statistical significance between samples from the same timepoint. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Anti-IL-1α, IL-1β, IL-6, GM-CSF and G-CSF antibody beads were purchased from Bio-techne.

Techniques: Expressing

Journal: Nature Communications

Article Title: Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor

doi: 10.1038/s41467-019-09915-2

Figure Lengend Snippet: MMP inhibition suppresses the candidalysin and C. albicans-induced response pathway. Pre-treatment of TR146 cells with Marimastat (MMP inhibitor, 10 µM) but not GI-253023X (ADAM10 inhibitor, 5 µM), significantly suppressed candidalysin-induced expression of pEGFR (Y1068 and Y845), c-Fos and pMKP1 ( a ). MMP inhibition had no suppressive effects on candidalysin-induced EREG ( b ) EPG ( c ), IL-1α ( d ), IL-1β ( e ) or IL-6 ( f ) but did suppress GM-CSF ( g ) and G-CSF ( h ) cytokine release. In Marimastat-inhibited, WT C. albicans-infected cells, significant suppression of all investigated proteins ( i–o ) except IL-6 ( p ), was observed. The candidalysin-deficient ece1 Δ/Δ +ECE1 Δ184–279 strain also failed to induce phosphorylation of EGFR or MKP1, c-Fos expression ( i ) or our panel of cytokines ( l – p ). Protein lysates were taken at 2 h post infection for western blot analysis, cytokines assessed at 24 h post infection via luminex. Solid vertical lines indicate omitted, extraneous portions of blot images. Unmatched, one-way ANOVA with Bonferroni multiple comparison’s test used to assess statistical significance. All images and graphs are representative of three independent experiments. One-way ANOVA with Bonferroni multiple comparison’s test was used to assess statistical significance. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Anti-IL-1α, IL-1β, IL-6, GM-CSF and G-CSF antibody beads were purchased from Bio-techne.

Techniques: Inhibition, Expressing, Infection, Western Blot, Luminex

Journal: Nature Communications

Article Title: Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor

doi: 10.1038/s41467-019-09915-2

Figure Lengend Snippet: Calcium is required for candidalysin-triggered immune responses and lies upstream of EGFR. Pre-treatment of TR146 cells with a calcium chelator (Bapta-AM 30 µM) reduced levels of pEGFR Y1068 and Y845, c-Fos, pMKP1 ( a ), IL-1α ( b ), IL-1β ( c ), G-CSF ( f ), EREG ( g ) and EPG ( h ), following candidalysin exposure. This is not observed when using the potassium inhibitor glibenclamide (30 µM). IL-6 ( d ) and GM-CSF ( e ) are not significantly suppressed by either inhibitor, though a reduction is observed. Protein lysates were taken at 2 h post infection for western blot analysis, cytokines assessed at 24 h post infection via luminex. Unmatched, one-way ANOVA with Bonferroni multiple comparison’s test was used to assess statistical significance. Images and graphs are representative of 2 ( a , g , h ) or 3 ( b – f ) independent experiments, respectively. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05

Article Snippet: Anti-IL-1α, IL-1β, IL-6, GM-CSF and G-CSF antibody beads were purchased from Bio-techne.

Techniques: Infection, Western Blot, Luminex