Lung epithelial cells are necessary and sufficient to generate protective antimicrobial responses, even in the presence of leukemia cells and chemotherapy. (A) Wild-type C57BL6/J mice challenged with P. aeruginosa 24 hours after inhaled treatment with Pam2-ODN (4 µM Pam2 and 1µM ODN M362) or PBS (sham). (B) Neutrophil-depleted mice challenged with P. aeruginosa 24 hours after inhaled treatment with Pam2-ODN or PBS. (C) Lung epithelial MyD88 deletant mice challenged with P. aeruginosa 24 hours after inhaled treatment with Pam2-ODN or PBS. (D) Bacterial burden of lungs removed immediately after P. aeruginosa challenge in C. (E) Bacterial burden of MLE15 (top) and HBEC3kt (bottom) monolayer cultures 8 hours after infection with P. aeruginosa. Cell were exposed to the indicated treatment of 4 hours prior to infection. The escalating doses of Pam2 (µM):ODN (µM) were 0.31:0.072, 0.93:0.22; 3.1:0.72; 9.3:2.2; and 52:12. (F) Schematic of epithelial-leukemia coculture model. (G) Apical bacterial burden of MLE15 cultures grown in coculture with FBL3 cells (or not), in the presence or absence of the indicated chemotherapy. Cells were treated for 4 hours with Pam2-ODN (9.3 and 2.2 µM, respectively) or PBS, infected for 4 hours, and then samples were collected. (H) Apical bacterial burden of primary mouse tracheal epithelial cells from the indicated genotypes grown in coculture with FBL3 cells. Cells were treated for 4 hours with Pam2-ODN or PBS, infected for 4 hours, and then samples were collected. (I) Quantitative real-time PCR of HBEC3kt cells for inflammatory cytokine or antimicrobial peptides genes 30 minutes after treatment with Pam2-ODN or PBS. Shown are RQ values relative to 18s expression. All data are representative of ≥3 experiments. N = 8 to 10 mice for all groups in survival experiments, N = 4 mice or culture wells per group for bacterial burden experiments. *P < .05 vs PBS treated; **P < .005 vs PBS treated; †P < .005 vs Pam2-ODN–treated Myd88 fl/fl mice.