Fig 2: Interleukin-1 receptor signaling controls MyD88-dependent chemokine induction and neutrophil recruitment.Mean (+SEM) of (A, C) BALF, (B, D) lung neutrophil recruitment and (E, G, I) BALF and (F, H, J) lung, (E, F) CXCL1, (G, H) CXCL2 and (I, J) CXCL5 levels in WT (black bars), IL-1R(−/−) (bars with diagonal stripes), IL-18R(−/−) (bars with crosshatch), TLR2(−/−) (dark grey bars), and TLR4(−/−) (light grey bars) mice 10 h p.i. with 3 × 107 conidia. Data are from 2 (A-D) or 1 (E-J) experiment(s) with 5–7 mice per genotype in each experiment. Graphs from a single experiment (out of three independent experiments) are shown for E, G and I.

Fig 3: CARD9-dependent induction of ELR+ chemokines in vitro and in vivo.(A) The plots show mean (+SEM) CXCL1 and CXCL2 secretion by WT (black bars) or CARD9(−/−) (light grey bars) BMMs following stimulation with A. fumigatus germlings (MOI = 1) as measured by ELISA. Data are from 4–5 replicates per condition from a representative experiment. (B) Strategy to generate CXCL2 reporter mouse. The graph shows CXCL2 (black lines) and mean GFP fluorescence (green lines) in transgene-positive (circle) and transgene-negative (non-Tg, square) littermates that were administered indicated amounts of Pam3Cys4 i.p. (C) The plots show neutrophils (left panel), inflammatory monocytes (middle panel) and CD11b+ DCs (right panel) that were isolated from CXCL2-GFP transgenic mice (upper panel) and non-transgenic littermates (lower panel) and analyzed for GFP expression. Representative data from 2 experiments is shown. Mice were administered 3 × 107 conidia and lung cell suspensions were analyzed 36 h p.i. (D) The graphs show mean number (+SEM) of GFP+ lung neutrophils, inflammatory monocytes, or CD11b+ DCs from Tg+ CARD9(+/+) (black bars), Tg+ CARD9(−/−) (grey bars), Tg− CARD9(+/+) (black crosshatched bars) and Tg− CARD9(−/−) mice (grey crosshatched bars).

Fig 4: CXCL1 is controlled by MyD88 in lung epithelial cells and prolongs survival in MyD88(−/−) mice following A. fumigatus challenge.(A) BALF (B) lung neutrophil recruitment in WT → WT (black bars), MyD88(−/−) → WT (dark grey bars), WT → MyD88(−/−) (light grey bars), and MyD88(−/−) → MyD88(−/−) (white bars) BM chimeric mice 10 h p.i. with 3 × 107 conidia. Data are expressed as the fold change when compared to the WT → WT group and were pooled from 3 experiments with 12–15 mice per group. (C) BALF and (D) lung neutrophil recruitment in IL1R(−/−) → WT (black circles), and WT → IL1R(−/−) (white circles) BM chimeric mice 10 h p.i. with 3 × 107 conidia. Data are expressed as mean (±SEM) and are from an experiment with 9 mice per group. (E-H) Mean (+SEM) BALF (E) neutrophil recruitment, (F) CXCL1, (G) CXCL2 and (H) CXCL5 levels, in MyD88(−/−) CC10-MyD88 (CC10-MyD88+; black bars) and in MyD88(−/−) transgene-negative littermate controls (CC10-MyD88−; grey bars) 10 h p.i. with 3 × 107 conidia. Data were pooled from 2 experiments and include 7–9 mice per genotype. (I) Kaplan-Meier survival plot of MyD88(−/−) mice challenged with 6–7 × 107 conidia and treated 4 h p.i. with 50 ng rCXCL1 (white circles, n = 11), or PBS vehicle (grey circles, n = 12). Data were pooled from 2 experiments (p = 0.026, Gehan-Breslow-Wilcoxon test).

Fig 5: Genetic ablation of DPP1 or of all three major NSPs, or DPP1 inhibition, does not affect mouse neutrophil migration ex vivo. (A) Schematic representation of the transwell migration assay. (B) BM progenitor cell-derived neutrophils were loaded in the upper chamber of 3-µm pore size transwell plates, with 100 ng/mL CXCL2 in the lower chamber and 2 µg/cm2 laminin on the insert. Cells were further incubated at 37 °C for 30 or 60 min, and neutrophil migration rates were calculated as the # cells in the lower chamber/load control x100%. (C) Mouse BM progenitor cells from WT animals were treated with brensocatib on days 0, 3, and five at the indicated concentrations, or with vehicle control, while differentiating into neutrophils. On day 7, neutrophil migration was assessed as described in panel (B). Data is mean ± SEM from 4 independent experiments. Statistical analysis by two-way ANOVA (B,C).