Figure 6.

FOXO1 regulates TLR expression in neutrophils. (A,B) Bacteria were incubated with neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L and littermate control LyzM.Cre⁻FOXO1^L/L mice. RNA was isolated and mRNA levels of TLR2 and TLR4 were measured by RT-PCR and normalized to ribosomal protein L32. (C–F) HL-60 neutrophils were transfected with FOXO1 or empty vector alone and stimulated with bacteria or vehicle alone. RNA was isolated and real-time PCR was carried out to assess mRNA levels of TLR2 and TLR4 or immunofluorescence was carried out with antibody specific for TLR2 or TLR4 and protein levels were assessed by mean fluorescence intensity (MFI). (G) Neutrophils were isolated from bone marrow of FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice and littermate control LyzM.Cre⁻FOXO1^L/L mice by break free centrifugation on Histopaque 1119 and 1077 and washed by phosphate-buffered saline (PBS) without calcium/magnesium. Alternatively HL-60 neutrophils were transfected with scrambled siRNA (SCR) or FOXO1 siRNA or were transfected with FOXO1 expression plasmid (FOXO1) or empty vector alone (pcDNA). Cells were incubated with P. gingivalis (Bact) (multiplicity of infection 1:10) for 12 h or vehicle alone (VEH) at 37°C with 5% CO₂ at normoxic moisture conditions. Apoptotic cells were assessed by flow cytometry with Annexin V labeling. The positive control was HL-60 neutrophils incubated with camptothecin. The data are representative of two or three independent experiments and expressed as the mean ± SEM from triplicate samples. *Significant difference between neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice and littermate control LyzM.Cre⁻FOXO1^L/L mice (P < 0.05). *Significant difference between HL-60 neutrophils from transfection of FOXO1 vs. empty vector alone or human neutrophils scrambled or FOXO1siRNA transfected (P < 0.05).