Journal: Frontiers in Immunology

Article Title: Candida albicans Sap6 Initiates Oral Mucosal Inflammation via the Protease Activated Receptor PAR2

doi: 10.3389/fimmu.2022.912748

Figure Lengend Snippet: Secreted aspartyl protease Sap6 induces IL-1β and IL-8 in primary oral epithelial cells. (A) . Cytokine profiling of OEC supernatant treated with Candida albicans (Ca) or recombinant Sap6 (rSap6) was examined using a proteome profiler Cytokine Array Panel (A) Proinflammatory IL-1β and IL-8 cytokines were detected in both Ca or Sap6 treated OECs although Ca induced release of additional cytokines in OECs. (B) OEC culture supernatants were collected following treatment with Sap6 or Ca for 24 h, then immunoblotted to confirmed secretion of IL-8 and IL-1β. (C) Total RNA was isolated from OECs incubated with either Ca or Sap6 for 24 h, then expression levels of IL-8 and IL-1β quantitated using RTqPCR using Il-8 and IL-1β specific primers with GAPDH as a housekeeping gene. There was no significant difference in expression levels of IL-8 or IL-1β between OECs treated with Ca or Sap6. Experiments were performed at three independent time points in triplicate; and the mean ± SEM were analyzed using one-way ANOVA. ns, non significant.

Article Snippet: For arrays, Proteome Profiler Human Cytokine Array B (R&D Systems) membranes were incubated for 12 h with OEC supernatant (400 μl) at 4°C, washed extensively with array buffer supplied in the kit, then incubated with biotinylated antibody cocktail for 1 h. Streptavidin-HRP was added for 30 min followed by chemiluminescent substrate reagent (Bio-Rad).

Techniques: Recombinant, Isolation, Incubation, Expressing

Journal: Frontiers in Immunology

Article Title: Candida albicans Sap6 Initiates Oral Mucosal Inflammation via the Protease Activated Receptor PAR2

doi: 10.3389/fimmu.2022.912748

Figure Lengend Snippet: Both Sap6 protease activity and its RGD motif modulate MAPK signaling and cytokine release. OECs were treated with rSap6, heat inactivated rSap6 (Inact) and rSap6 Δ RGD for 3 h (for cell lysates) or 24 h (for culture supernatants). (A) OEC cell lysates treated with rSap6 (Inact) failed to induce p38 phosphorylation and IL-8 release without affecting MKP1 phosphorylation, c-Fos expression or IL-1β compared to active rSap6. OECs treated with rSap6 ΔRGD showed reduced MKP1 phosphorylation and IL-1β release without affecting p38 phosphorylation, c-Fos and IL-8 levels. (B) OEC culture supernatant was collected after 24 h of treatment with rSap6 (0.1-10µM), rSap6 (Inact, 10µM) or rSap6 ΔRGD (10µM) and IL-8 was quantitated by ELISA. IL-8 secretion was dose dependent with rSap6(0.1-10µM) and was significantly (P<0.001) reduced by treatment with rSap6 (Inact) compared with active rSap6 or rSap6 ΔRGD . Data are mean ± SEM and are representative of three independent experiments. Significance was calculated using two-way ANOVA compared to untreated OECs. P values are, p ≤ 0.01 (**), p ≤ 0.001 (***).

Article Snippet: For arrays, Proteome Profiler Human Cytokine Array B (R&D Systems) membranes were incubated for 12 h with OEC supernatant (400 μl) at 4°C, washed extensively with array buffer supplied in the kit, then incubated with biotinylated antibody cocktail for 1 h. Streptavidin-HRP was added for 30 min followed by chemiluminescent substrate reagent (Bio-Rad).

Techniques: Activity Assay, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay