Fig 9. Temporal immune responses by BEAS-2B cells during in vitro fungal challenge assays.

Freshly harvested AF293 conidia (5 X 10⁵) were challenged against wild type and ΔNlrx1 BEAS-2B airway epithelial cells (5 X 10⁵) at 37°C at 5% CO₂. Total supernatant was harvested immediately prior to challenge (0 hrs), and at 3, 6, 9, 12 hrs post challenge. Concentration of cytokines (IL-1β, IFN-α, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33) and chemokines (MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11), TARC (CCL17), MIP-1α (CCL3), MIP-1β (CCL4), MIG (CXCL9), MIP-3α (CCL20), ENA-78 (CXCL5), GROα (CXCL1), I-TAC (CXCL11)) in culture media was determined by flow cytometry bead based array. (A-C) Differentially secreted cytokines and chemokines are presented. (DE) Western blot analysis and quantification of phosphorylated (-P) ERK1/2-P, P38-P, JNK-P, NFκB p65-P, Iκβ-α-P, Nlrx1, and GAPDH three hours post treatment with fungal PAMPs curdlan, zymosan, zymosan depleted, conidia, and control buffer. Independent experiments were run in triplicate. (FG) IL-6 secretion by wild type and ΔNlrx1 cells in response to conidia, zymosan, and hyphae at 12 hours in the presence and absence of P38 inhibitor BIRB796. Independent experiments were run in triplicate. All experiments were independently repeated, total N = 6. Error bars denote standard deviation. Statistical significance was determine using a Mann-Whitney U test and is indicated via asterisk, P < 0.05.