Figure 4. Antagonism of LYSMD3 ectodomain function impairs epithelial cell inflammatory responses induced by chitin.

(A) ELISA of IL-8 in supernatants of NHBE cells stimulated for 24 h with chitin DP7 (500 μg/mL) in the presence of soluble LYSMD3 ectodomain. IL-8 concentration in cell-free supernatant in the absence of the LYSMD3 ectodomain was normalized to 100%. See also Figure S3.

(B) ELISA of IL-8 in supernatants of BEAS-2B or A549 cells stimulated for 24 h with chitin DP7 (500 μg/mL) after pre-incubation with LYSMD3 antibody (low endotoxin, azide free) or isotype control. IL-8 concentrations in supernatants were normalized to 100% as in (A).

(C) A549-Dual cells were stimulated with chitin DP7 (500 μg/mL) or FLA-ST (300 ng/mL) for 24 h after preincubation with laminarin (1 mg/mL), curdlan (1 mg/mL), soluble WGP (1 mg/mL), chitin DP5 (200 μM), Dectin-1 blocking antibody (3 μg/mL), or corresponding isotype control. NF-μB activation was assayed by optical density (OD) at 655 nm using QUANTI-Blue. NF-κB activities were normalized to 100% in the absence of inhibitors as in (A).

(D) Relative binding of rLYSMD3 (50 μg/mL) to immobilized chitin DP7 in the presence of laminarin (100 μg/mL), curdlan (500 μg/mL), soluble WGP (500 μg/mL), chitin DP5 (200 μM), chitin DP3 (200 μM), or LPS-RS (500 μg/mL), as determined by ELISA. Binding was normalized 100% in the absence of carbohydrates as in (A).

*p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test). Data are from one representative experiment of three performed (mean and SD of four samples per group in A or three samples per group in B, C, and D).