S pneumoniae-induced CXCL1 production requires activation of TLR and NF-kB signaling. (A) C57BL6/J, A/J and BALB/c mice were inoculated intratracheally with 5 × 10⁴ CFU S pneumoniae 6303. (B) C57BL6/J mice were infected intratracheally with S pneumoniae strain 6303 (5 × 10⁴ CFU), A66.1 (2 × 10⁵ CFU), WU2 (5 × 10⁷ CFU), and D39 (5 × 10⁴ CFU) strains or PBS. (A-B) Mice were euthanized at 48 hours postinfection, and CXCL1 was measured in BALF. (C) BMDMs from WT mice were generated as described in supplemental Methods. BMDMs were pretreated with TLR agonists Pam2CSK4 (500 ng/mL), Pam3CSK4 (500 ng/mL), HKSA (10⁸/mL), HKSP (10⁸/mL), LTA (5 μg/mL), and LPS (500 ng/mL) for 4 hours and infected with S pneumoniae 6303 (MOI 10) for 8 hours. CXCL1 level was measured in the supernatant. (D) BMDMs from WT, Nlrp3^−/−, Nlrc4^−/−, and Nlrp6^−/− were infected with S pneumoniae 6303 (MOI 10) for 8 hours. CXCL1 was measured in supernatant. (E) BMDMs from WT, MyD88/Trif^−/−, and Asc^−/− were infected with S pneumoniae 6303 (MOI 10) for 8 and 18 hours. CXCL1 was measured in supernatant. (F) WT BMDMs were pretreated with 10 μM of inhibitors of NF-κB, p38 (SB203580), JNK (SP600125), ERK (PD098059), or dimethyl sulfoxide and then infected with S pneumoniae 6303 (MOI 10) for 8 hours. CXCL1 was measured in the supernatant. All experiments were performed 3 times. (n = 5-6 mice/infection group; n = 3 mice/control group). In vitro experiments have at least 4 biological replicates. Statistical significance was determined by 1-way ANOVA (followed by Bonferroni’s post hoc comparisons). *P < .05; **P < .01; ***P < .001. HKSA, heat-killed S aureus; LTA, lipoteichoic acid; MOI, multiplicity of infection.