Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

presence or absence of BOS (0.52 g/mL).(B) Comparison of VRE CFU per infected wound of animals treated with five IP injections of vehicle or BOS (30 L of 5mg/kg).(C) Representative images of wounds (top panel) and summary of data from five mice (low panel) at the end of the multiple-treatment experiment.Scale bars, 2 mm.Wound area measured at 4 dpi after five treatments.(D) Comparison of VRE CFU per infected wound of animals treated with a single IP injection of vehicle or BOS (5mg/kg) 24h prior to infection.(E) Comparison of VRE CFU per infected wound treated topically a single dose of vehicle or BOS (5.20 g/mL).(F-I) A schematic diagram of experimental design (F).Mice were injected IP with clop-A (200 L, 6 mg/mL) 3 days prior to wounding and infection, and additional doses of clop-A on the day of wounding and infection and every 2 days afterwards.In addition, clop-A (10 L, 6 mg/mL) was applied to the wounds every 2 days.Following VRE infection, BOS (10L, 0.52 g/ml) was applied to the wounds daily for 5 days.Five days after wounding and infection, mice were sacrificed, and wounds were recovered for assaying macrophage depletion and VRE CFU.Representative flow cytometry of macrophages (CD45 + CD11b + F4/80 + ) from infected wounds (G).The number indicate percentages of cells within the gated areas.Comparison of the percentages of macrophages recovered from infected wounds with or without clop-A and/or BOS treatments (H).Comparison of the percentages of neutrophils recovered from infected wounds that were vehicle or BOS treated with five topical doses (I).Each symbol represents one mouse with the median indicated by the horizontal line (B, D-E, and H-I).Data were from at least two independent experiments with two to three mice per experiment.Statistical analysis was performed using unpaired t test with Welch's corrections (A), using the nonparametric Mann-Whitney test to compare ranks or using Kruskal-Wallis test with uncorrected Dunn's posttest (B-E, H-I)).NS, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.

Figure S2 -
Figure S2-Different doses of BOS enhance macrophage killing of intracellular bacteria in vitro.Comparison of VRE CFU in RAW264.7 cells treated with BOS (0.01 -10 M) for 15 h after initial infection of 3 h.Statistical analysis was performed using unpaired t test with Welch's corrections.NS denotes not significant; and *P ≤ 0.05.

Figure S4 -Figure S5 -
Figure S4-BOS stimulates macrophage phagocytosis across a range of doses in vitro.Comparison of the uptake of SYTO9-labelled VRE by RAW264.7 macrophages in the presence or absence of BOS with concentrations varying from 0.01 to 10 M.RAW264.7 macrophages with and without BOS pre-treatment were infected for 1h with SYTO9-labelled VRE, followed by quenching with trypan blue and measurement of fluorescence intensity by flow cytometry.Shown are the MFI from three independent experiments.Statistical analysis was performed using unpaired t test with Welch's corrections.NS denotes not significant; *P ≤ 0.05; and **P ≤ 0.01.

Figure S6 -
Figure S6-BOS-treated macrophages produce more ROS.(A) Western blot of whole-cell lysates for proteins of the canonical and non-canonical phagocytosis pathway.RAW264.7 cells with (+) and without (−) VRE infection were treated with BOS (+) or left untreated (−).Cell lysates were separated by SDS-PAGE and the

Figure S7 -
Figure S7-BOS promotes survival of infected macrophages.(A) Western blotting analysis of MLKL and pMLKL.RAW264.7 cells with (+) and without (−) VRE infection were treated with BOS (+) or left untreated (−).Whole-cell lysates were Western blotted with anti-MLKL, anti-pMLKL and anti-GAPDH antibodies.(B) Comparison of percentage of Annexin V + and PI + cells at the end of infection.RAW264.7 cells were either infected or not infected and were treated with BOS alone or in combination with the NF-B inhibitor QNZ (10 nM).Annexin V and PI reactivity was assayed by flow cytometry.Data (mean ± SEM) are a summary of at least three independent experiments.(C) RAW264.7 cells were infected with VRE in the presence of BOS (0.52 g/mL), and QNZ (1 nM), alone or in combination.Intracellular bacterial CFU was quantified after 18 h.Statistical analysis was performed using ordinary one-way ANOVA, followed by Tukey's multiple comparison test (B), or Brown-Forsythe and Welsh ANOVA test (C).NS, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.

Table S3 -
Cytotoxicity as measured by LDH assay of compounds used in this study.

Table S4 -
Comparison of transcript levels of cell surface markers associated with bacterial recognition, uptake, and presentation in RAW264.7 cells with and without BOS treatment.