Fig 2: LYSMD3 mediates the expression of pro-inflammatory products upon stimulation with chitin or chitin containing fungi(A) ELISA of cytokines in supernatants of BEAS-2B cells stimulated for 24 h with 500 µM chitin oligomers.(B) Immunoblot of LYSMD3 in membrane protein fraction of BEAS-2B cells 48 h after transfection with control siRNA or a siRNA pool specific for LYSMD3. NaK ATPase is loading control. See also Figure S2C.(C) ELISA of cytokines in supernatants of BEAS-2B cells transfected with siRNAs as in (B), then stimulated for 24 h with chitin DP7 (500 µg/mL), chitin particles (500 µg/mL), Alternaria spores (2 × 105/mL), FLA-ST (300 ng/mL), or Poly(I:C) (3 µg/mL). See also Figures S2D and S2E.(D) Immunoblot of LYSMD3 in cell lysates of NHBE cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3. ß-Actin is loading control.(E) ELISA of IL-8 in supernatants of NHBE cells transfected with siRNA as in (D) and then stimulated for 24 h with chitin DP7 (500 µg/mL) or A. alternata spores (2 × 105/mL).(F) Immunoblot of LYSMD3 in membrane protein fraction of RAW 264.7 cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3.(G) ELISA of IL-6 in supernatants of RAW 264.7 cells transfected with siRNA as in (F) and then stimulated for 24 h with chitin DP7 (100 µg/mL) or Pam3CSK4 (100 ng/mL).(H) MTT assay 48 h after transient transfection of siRNA asin(B)in BEAS-2B cells. Cell growth rate is expressed as absorbance at 570 and was normalized to the value with control siRNA as 1.(I) LDH cytotoxicity assay 48 h after transient transfection of siRNAs as in (B) in BEAS-2B cells.(J) Expression of LYSMD3 protein in BEAS-2B cell with CRISPR-Cas9 knockout of LYSMD3 (sgD3). Hsp90 is the loading control.(K) ELISA of IL-8 in supernatants of BEAS-2B cell knockout of LYSMD3 as in (J), unstimulated or stimulated for 24 h with chitin DP7 (500 µg/mL) or FLA-ST (300 ng/mL). See also Figure S2F.(L) ELISA of IL-33 in supernatants of hBE33 cells transfected with siRNA as in (D) and then stimulated for 1 h with Alternaria extract (200 µg/mL).*p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test). Data are representative of three experiments (mean and SD of three samples per group in A, C, and E, or four samples per group in G, K, and I, or eight samples per group in H and L).

Fig 3: 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-?B 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).

Fig 4: LYSMD3 is located in the plasma membrane of human airway epithelial cells and in sites of fungal-epithelial interactions(A) Schematic illustration of the predicted domains of human LYSMD3.(B) Immunoblot analysis of membrane (M) and cytosolic (C) fractions from BEAS-2B and A549 cells probed with anti-LYSMD3.(C) Immunoblot analysis of biotinylated cell-surface proteins (eluate, 25 µL) and unbound lysate (25 µL) from BEAS-2B cells(D)Representative images of BEAS-2B cells infected with live C. albicans SC5314 and stained for LYSMD3 (green). Scale bars, 10 µm. Accompanying histogram (right) depicts enrichment in LYSMD3 at sites of contact with the fungus, compared to sites not in contact with the fungus. Results were calculated from seven pairs of fungal contact regions and non-contact regions. IgG control staining during C. albicans infection of lung epithelial cells revealed negligible staining (data not shown). **p < 0.01 (Student’s t test). Data are from one representative experimental of three performed with similar results (mean and SD of seven samples per group).