Figure 6

Cytokine secretion profiles after C. albicans challenge. (A) tSNE analysis of cytokine secretion variation in response to C. albicans challenge. After staining for CD4+, CD8+ membrane-antibody markers and for IFN-γ, TNF-α, IL-17, IL-10 and IL-4 secreted cytokines of unstimulated and C. albicans-stimulated leucocytes, cells were analyzed with FlowJo 10 for doublets, debris, and dead cells exclusion before gating living cells. Total pro-inflammatory (IFN-γ, blue) and anti-inflammatory (IL-10, green) cytokine expressing cells were concatenated and analyzed in tSNE module according to post-challenge time and to UI controls, CI and PI outcomes. (B) For each cytokine the secretion was analyzed as the percentage of cytokine secreting cells among myeloid cells and T lymphocytes. Box plots depict median, minimum and maximum percentages of immune cell frequencies according to UI controls (white boxes), CI (light gray) and PI (dark gray) outcomes for all of C. albicans clinical isolates (n = 16). (C) Percentage of CD4+ and CD8+ T lymphocyte secreting IFN-γ and IL-10 two days after C. albicans challenge. (D) Secretion of IL-17 by human CD66+ neutrophils into leucocyte co-cultures, two days after challenge by Caal93, Caal121 and Caal123 clinical isolates. (E) Gating strategy and analysis of Il-17 producing human neutrophils. CD66+ neutrophils were isolated by gradient centrifugation from three independent healthy volunteers. CD66+ cells were gated among sorted neutrophils cultures, followed by exclusion of doublets using forward scatter height (FSC-H) and forward scatter width (FSC-A). Caal93, Caal121, Caal123 and SC5314 C. albicans isolates were used to stimulate sorted neutrophils for 48 hours. The RFI was the ratio of the mean of fluorescence intensity (MFI) of C. albicans-stimulated neutrophils normalized to the MFI of respective IgG-isotype controls. IL-17 in supernatants of sorted neutrophils was quantified with a ProcartaPlex multiplex immunoassay kit (eBioscience). Concentrations were expressed as the mean ± SD of IL-17 in pg/ml of stimulated neutrophils among fungal isolates and uninfected controls (n = 3). *P < 0.05; **P < 0.001; ***P < 0.0001; ****P < 0.00001; ns = not significant.