Mice that express Cre recombinase under control of the lysozyme M promoter (LyzM⁺.Cre) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). FOXO1^L/L mice were generously provided by Dr. Ronald DePinho (University of Texas MD Anderson Cancer Center, Houston, TX, USA) (21). FOXO1^L/L mice were bred with LyzM.Cre mice to generate experimental mice (LyzM.Cre⁺FOXO1^L/L) and the control littermates (LyzM.Cre⁻FOXO1^L/L) as described (20). Genotypes were determined by PCR using primers specific for LyzM.Cre (5′-ATCCGAAAAGAAAACGTTGA-3′ and 5′-ATCCAGGTTACGGATATAGT-3′) and specific for FOXO1 (5′-GCTTAGAGCAGAGATGTTCTCACATT-3′, 5′-CCAGAGTCTTTGTATCAG GCAAATAA-3′, and 5′-CAAGTCCATTAATTCAGCACATTG A-3′). All procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Broth-grown Porphyromonas gingivalis (ATCC, #33277) in logarithmic growth phase was collected and washed three times with phosphate-buffered saline (PBS). Bacteria were then resuspended and counted with a standard CFU curve as previously described (22). Mice were challenged by injection of lightly fixed or live P. gingivalis (ATCC, #33277) or sham injection with vehicle alone (PBS) into the scalp connective tissue as described (23–25) and euthanized at indicated time points after the injection (26). Neutrophils were isolated from the vasculature, BM, and scalp connective tissues and assessed by flow cytometry after incubation with specific antibodies or control IgG as previously described (27). Neutrophil mobilization was calculated as described (28).

Primary mouse neutrophils were isolated from the BM (27). Briefly, BM cells from experimental mice (LyzM.Cre⁺FOXO1^L/L) and the control littermates (LyzM.Cre⁻FOXO1^L/L) were suspended in PBS without calcium and magnesium, placed over Histopaque^® 1119 and 1077 (Sigma Chemicals Ltd.) and centrifuged at 2,000 rpm, 25°C for 30 min. The neutrophil layer was collected and washed twice in PBS. Neutrophil purity was routinely >95%, as determined by flow cytometry after staining for Ly6G, F4/80, and CD3 (27). Primary human neutrophils were isolated from human peripheral blood (PBL) of healthy donors obtained from the Human Immunology Core at University of Pennsylvania following the procedure in Ref. (27). Neutrophil purity (routinely >95%) was determined by Wright–Giemsa staining (27).

The human promyelocytic leukemia HL-60 cells (ATCC CCL-240) were cultured at 37°C in 5% CO₂ in RPMI 1640 supplemented with 2 mM l-glutamine, 25 mM HEPES, and 10% heat-inactivated fetal bovine serum (MilliporeSigma, St. Louis, MO, USA). HL-60 cells were incubated with 1.3% DMSO (MilliporeSigma) for 4 days (29) and their differentiation into neutrophils (referred to as HL-60 neutrophils) was monitored by flow cytometry analysis by CD11b expression using antihuman CD11b mAb (BD Pharmingen) (27).

Chemotaxis was measured in primary mouse BM neutrophils with transwell chambers (polycarbonate filter, 5-µm pore size, Corning) with or without CXCL1 (Peprotech) for 2 h at 37°C in the bottom chamber. Neutrophils that migrated to the bottom side of the filter were counted by DAPI staining and fluorescence microscopy.

Bacterial phagocytosis was performed as described (30) with modification. Briefly, bacteria were labeled with CFSE (#65-0850-84, carboxyfluorescein succinimidyl ester, Thermo Fisher Scientific, Waltham, MA, USA) (31) and incubated with neutrophils at multiplicity of infection (MOI) 1:10 (cell: bacteria) for 1 h. The neutrophils were fixed and stained with Alexa 647-labeled wheat germ agglutinin (WGA) (W32466, Thermo Fisher Scientific) to delineate the cell surface (red) and internalized bacteria (green) then visualized by fluorescent microscopy with deconvolution. Internalized bacteria were considered as those within WGA-decorated plasma membranes (27). Neutrophil-associated bacteria were determined by colocalizing CFSE-labeled P. gingivalis and Alexa 647-labeled WGA stained neutrophils. Internalized bacteria and neutrophils with associated bacteria were counted under fluorescent microcopy (27). Bacterial clearance in vivo was determined by inoculating P. gingivalis (1 × 10⁷ bacteria/injection) into the scalp connective tissue of experimental LyzM.Cre⁺FOXO1^L/L and LyzM.Cre⁻FOXO1^L/L control littermates, a well-characterized experimental model (23–26). The scalp soft tissue was harvested, mechanically processed by medimachine (BD Biosciences, San Jose, CA, USA), lysed, and divided into aliquots with multiple dilutions that were used for numeration of recovered CFU (colonies were counted after anaerobic culture on blood agar plates). Data are presented as total number of bacteria recovered per mouse (27). To assess bacterial killing in vitro mouse neutrophils were cocultured with P. gingivalis (MOI = 1:1) (cell: bacteria) at 37°C and 5% CO₂ for 2 h. The neutrophils were lysed and viable CFU were enumerated after anaerobic culture on blood agar plates. The neutrophil killing index was calculated according to the formula: [(CFU in the absence of neutrophils − CFU in the presence of neutrophils)/CFU in the absence of neutrophils] × 100 (27).

The inhibitors for reactive oxygen species (ROS, NAC, N-acetyl-l-cysteine, 5 mM) (32), nitric oxide synthase (NOS, l-NAME, NG-nitro-l-arginine methyl ester, 5 mM) (33), Sirt1 (Sirtinol, 10 µM), histone deacetylases (HDAC, Trichostatin A, TSA, 2 µM), TLR4 (TAK242, 1 µg/mL), and DMSO were obtained from MilliporeSigma (Billerica, MA, USA) or Cayman Chemical (Ann Arbor, MI, USA). TLR2 blocking antibody (10 μg/mL) and matched mouse IgG2a (10 µg/mL) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). HL-60 neutrophils were incubated with inhibitors or antibody compared to vehicle or matched control IgG for 1 h before P. gingivalis challenge and during incubation with bacteria. After 16 h, cells were fixed and examined by immunofluorescence with antibody to FOXO1 compared to matched control IgG. FOXO1 nuclear localization was assessed by FOXO1 colocalization with DAPI nuclear stain by image analysis with NIS-Elements software (Nikon, Melville, NY, USA).

HL-60 neutrophils were incubated with P. gingivalis in 96-well plates for 12 h at 37°C. Neutrophils were fixed in 3.7% formaldehyde for 10 min, permeabilized in 0.5% Triton X-100 for 5 min, blocked in 2% BSA, and stained with primary antibody and appropriate isotype-matched negative control antibody. This was followed by incubation with biotinylated secondary antibody and ABC reagent and visualized by incubation with Alexa Fluor 546-conjugated streptavidin (#S11225, Thermo Fisher Scientific) with DAPI counterstain (#62248, Thermo Fisher Scientific). Images were captured at a magnification of 200× with a fluorescence microscope (Nikon) with the same exposure time for experimental and negative control groups. The capture time was set so that control antibody images were negative. Image analysis was performed using NIS Elements AR image analysis software. The percentage of immunofluorescence positive cells or mean fluorescence intensity (MFI) was measured.

Neutrophils were transfected with a plasmid containing constitutively active FOXO1, FOXO1-AAA (referred to as FOXO1), or pcDNA empty vector as we have described (34) by electroporation with Amaxa Nucleofector Transfection Device (Lonza, Basel, Switzerland) or Neon Transfection System (Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were then stimulated with P. gingivalis as MOI 1:10 overnight. Total RNA was extracted from neutrophils and gene expression was then measured by quantitative real-time PCR with primers (IDT, Coralville, IA, USA) designed using the Universal Probe Library Assay Design Center (Roche Applied Science, Indianapolis, IN, USA) and labeled probes (Roche Applied Science). Each value was normalized to ribosomal protein L32 and represents the mean of three independent experiments.

Human primary neutrophils were transfected with FOXO1 siRNA as described (27). Briefly, ON-TARGET plus SMART pool siRNAs specific for FOXO1 and control scrambled non-targeting control pool siRNA were obtained from GE Healthcare Lifesciences (Pittsburgh, PA, USA) and transfection was performed using lipofectamine 3000 Transfection Reagent (L3000008, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Neutrophils were lysed with lysis buffer (sc-24948, Santa Cruz Biotechnology) containing protease inhibitor cocktail and phosphatase inhibitor cocktail. Protein concentration was measured using a protein assay with BSA as a standard (#26149, Thermo Fisher Scientific). The 30–60 µg cell lysate was resolved in 4–20% SDS-PAGE (#4561084, Bio-Rad Laboratories) and transferred onto PVDF membrane (#88518, Thermo Fisher Scientific). The membranes were incubated with primary antibodies against FOXO1 (#2880S, 1:500, rabbit mAb, Cell Signaling Technology) and β-actin (A5316, 1:1,000, MilliporeSigma) after blocking with 5% milk. The samples were then incubated with horseradish peroxidase-labeled donkey antirabbit IgG (NA934, 1:5,000, GE Healthcare) or antimouse IgG (HAF018, 1:5,000, R&D), and immunoreactive bands were detected with ECL Western blotting reagents (#32209, Thermo Fisher Scientific).

Chromatin immunoprecipitation assays were performed using a ChIP-IT Kit (#53035, Active Motif, Carlsbad, CA, USA). Cells were fixed in 1% formaldehyde, DNA sheared enzymatically and immunoprecipitated with anti-FOXO1 or matched control antibody and captured with magnetic protein G beads. The precipitated DNA was then amplified by real-time SYBR green real-time PCR using primers for CD11b and CXCR2.

Apoptosis were measured by flow cytometry with Annexin V FITC apoptosis detection kit (#88-8005, Thermo Fisher Scientific) according to the manufacturer’s instructions. In brief, neutrophils were coculture with P. gingivalis for overnight and assessed by flow cytometry after stained with Annexin V. Data were analyzed by Flow Jo software.

Experiments were carried out a minimum of two to three times with similar results. Statistical significance was determined by t-test or ANOVA with Turkey’s post hoc test at P < 0.05.

To examine host bacteria interactions in vivo, we utilized a well-defined animal model in which bacteria are inoculated into the scalp connective tissue (23–25). The number of mature neutrophils, T cells, B cells, and macrophages recruited to the site of inoculation following injection of bacteria was measured by immunofluorescent flow cytometry using specific antibodies or matched control antibody. As expected, the major leukocyte recruited were neutrophils, which were far greater than T cells, B cells, and macrophages (P < 0.05) (Figure 1A). To determine whether FOXO1 plays a role in bacteria-induced neutrophil recruitment, experimental mice were examined in which floxed FOXO1 was deleted in myeloid cells by Cre recombinase under the control of a LyzM promoter element (LyzM.Cre⁺FOXO1^L/L) and compared with littermate controls (Figures 1B–F). The expression of CD11c.Cre⁺ recombinase has no apparent affect as demonstrated by comparison with wild-type mice as reported (35). Bacterial inoculation stimulated a 16-fold increase in the number of neutrophils recruited in WT control mice at 24 h, which was reduced 80% in LyzM.Cre⁺FOXO1^L/L experimental mice (P < 0.05) (Figure 1C). The number of mature neutrophils in the blood (Figures 1B,D) and BM (Figure 1E) was measured by immunofluorescent flow cytometry to examine the role of FOXO1 in neutrophil redistribution following inoculation of bacteria. After 12 h, there was a 4.6-fold increase in neutrophil numbers in the PBL of control mice, which was 68% lower in experimental LyzM.Cre⁺FOXO1^L/L mice (P < 0.05) (Figure 1D). This coincided with a 52% decrease in the number of neutrophils in the BM of control mice 12 h following bacterial inoculation. Thus, deletion of FOXO1 in LyzM.Cre⁺FOXO1^L/L experimental mice caused a significant reduction in the movement of neutrophils from the BM to PBL after bacterial inoculation (P < 0.05) (Figure 1E). Quantitatively, bacteria induced a fivefold increase in neutrophil mobilization from the BM compartment to the peripheral vasculature in WT control mice at 12 h, which was reduced by more than 50% in FOXO1-deleted experimental mice in vivo (Figure 1F). To ensure that FOXO1 was deleted, neutrophils from LyzM.Cre⁺FOXO1^L/L and matched control mice were examined (P < 0.05) (Figures 1G,H). At both the mRNA and protein level, the results demonstrate efficient FOXO1 deletion in neutrophils in the experimental but not control groups.

To determine whether FOXO1 facilitates neutrophil migration, in vitro studies were carried out. Neutrophils were stimulated with the chemokine CXCL1 and examined in a transwell assay. CXCL1 induced a dose-dependent increase in migration that was stimulated up to sevenfold in control mice and reduced by 45% when FOXO1 was deleted (P < 0.05) (Figure 2A). To examine how FOXO1 may affect neutrophil migration, CXCR2, a receptor for CXCL1, was assessed. Bacteria induced a 2.3-fold increase in CXCR2 mRNA levels (P < 0.05) that was 44% lower in similarly stimulated neutrophils from experimental littermates (P < 0.05) (Figure 2B). Regulation of CXCR2 by FOXO1 was further examined by transfection of HL-60 neutrophils with a FOXO1 expression plasmid. FOXO1 plasmid increased CXCR2 mRNA levels 2.2-fold and protein levels by 1.6-fold compared to empty vector (Figures 2C,D). Moreover, the increase was further enhanced in cells stimulated with bacteria, suggesting that FOXO1 enhances CXCR2 in cooperation with other factors that are induced in neutrophils stimulated with bacteria. Direct interaction between FOXO1 and the CXCR2 promoter was demonstrated by ChIP assay, which was also significantly enhanced in bacteria stimulated neutrophils (P < 0.05) (Figure 2E). Furthermore, FOXO1 protein levels were increased in neutrophils transfected with a FOXO1 expression plasmid (P < 0.05) (Figure 2F). Thus, FOXO1 mediates CXCR2 transcription stimulated by bacteria and deletion of FOXO1 in neutrophils reduces chemotaxis induced by its cognate ligand.

The impact of FOXO1 deletion on neutrophil phagocytosis was examined. Phagocytosis was assessed by internalization of labeled bacteria. Neutrophils phagocytosis of bacteria was reduced ~60% (Figures 3A–C) (P < 0.05) when FOXO1 was knocked down. The number of neutrophils with associated bacteria was reduced in half (P < 0.05) (Figure 3D). Since a primary function of neutrophils is bacterial killing, we assessed this parameter in vivo 12 h after injection of bacteria and in vitro 2 h after coincubation, time points at which neutrophils are the primary antibacterial defense (36). In vivo, FOXO1 deficient mice were 70% less efficient in clearing bacteria than matched littermate control mice (P < 0.05) (Figure 3E). Similar results were obtained in vitro. Neutrophils from experimental mice with lineage specific FOXO1 deletion were 50% less efficient than control littermates in bacterial killing (P < 0.05) (Figure 3F). Experiments were carried out to ensure that FOXO1 was efficiently knocked down by RNAi. At both the mRNA (Figure 3G) and protein levels (Figure 3H), FOXO1 siRNA substantially reduced FOXO1 compared to scramble siRNA.

Experiments were undertaken to better understand mechanisms through which bacteria stimulate FOXO1 nuclear localization, a key step in induction of FOXO1 activity. Bacteria stimulated FOXO1 nuclear localization by approximately fourfold. Bacteria-stimulated FOXO1 nuclear localization was dependent upon TLRs since inhibitors of TLR2 and TLR4 blocked most of this increase (P < 0.05) (Figures 4A,B). Inhibition of ROS and NOS, intermediates in TLR signaling also substantially reduced bacteria-induced FOXO1 nuclear localization. Furthermore, deacetylation of FOXO1 played an important role as inhibitors that blocked FOXO1 deacetylation, Sirt1 and HDAC largely blocked the ability of bacteria to stimulate translocation of FOXO1 to the nucleus (P < 0.05) (Figures 4A,B).

To investigate a potential mechanism by which FOXO1 affects several aspects of neutrophil function including migration and phagocytosis (37), we examined FOXO1 regulation of CD11b. Under basal conditions neutrophils from mice with FOXO1 deletion had 50% less CD11b mRNA than control littermates (P < 0.05) (Figure 5A). Bacterial stimulation increased neutrophil CD11b mRNA levels almost fivefold in vitro (P < 0.05). More than 50% of this increase was blocked in neutrophils from experimental FOXO1-deleted mice (P < 0.05) (Figure 5A). To further investigate regulation of CD11b, HL-60 neutrophils were transfected with FOXO1 or empty vector alone and stimulated with bacteria. Overexpression of FOXO1 induced a significant 3.9-fold increase in CD11b mRNA levels and 1.6-fold increase at the protein level compared to empty vector (Figures 5B–D). To determine whether FOXO1 regulates neutrophil activity directly, interaction between FOXO1 and the CD11b promoter was examined by ChIP assay. Under basal conditions FOXO1 was shown to interact directly with the CD11b promoter, which was increased almost threefold in neutrophils stimulated by bacteria (P < 0.05) (Figure 5E).

Bacterial stimulation increased TLR2 and TLR4 mRNA levels twofold to threefold and most of this increase was blocked in neutrophils with deleted FOXO1 (P < 0.05) (Figures 6A,B). Similarly, overexpression of FOXO1 increased TLR2 and TLR4 mRNA and protein levels particularly in neutrophils coincubated with bacteria (Figures 6C–F). Thus, FOXO1 can potentially sensitize neutrophils to bacterial stimulation through upregulation of TLRs to enhance inflammatory responses. To determine whether changes in neutrophils modulated by FOXO1 could be due to apoptosis, experiments were carried out assessing apoptosis by Annexin V. Bacteria stimulated a small increase in neutrophil apoptosis that was much less than the positive control, camptothecin (Figure 6G). However, deletion of FOXO1 by Cre recombinase or knockdown of FOXO1 by siRNA had no effect on neutrophil apoptosis.

FOXO1 may contribute to inflammatory responses of neutrophils by upregulating cytokine expression. FOXO1 deletion in neutrophils reduced the capacity of bacteria to induce TNFα and IL-1β mRNA levels by 50–70% (P < 0.05) (Figures 7A,B). Although IL-1β mRNA levels are not directly related to mature IL-1β protein levels, the results do show FOXO1 regulation IL-1β mRNA. The effect of transfection of a FOXO1 expression plasmid on TNFα and IL-1β was also assessed. Transfection with FOXO1 stimulated approximately fivefold increase in TNFα and IL-1β mRNA (Figures 7C,D). When neutrophils were stimulated with bacteria plus FOXO1 overexpression the levels were further increased to 25-fold for TNFα and >100-fold for IL-1β. A similar synergy between FOXO1 overexpression and bacterial stimulation was observed when the protein levels were measured by MFI (Figures 7E,F).