Fig 1.
S. aureus biofilm infection promotes a shift towards OxPhos metabolism in monocytes.
Monocytes associated with tissues surrounding the knee joint of mice with sterile (S) or S. aureus-infected (I) orthopedic implants were stained with (A) the bi-potential dye JC-1 or (B) 2-NBDG at the indicated time points (days 1–7) as measures of OxPhos and glycolysis, respectively, and analyzed by flow cytometry. (A) OxPhos was calculated as the ratio of green:red monocytes (CD11bhighLy6G-Ly6C+F4/80-) following JC-1 staining and is reported as a percentage relative to animals receiving sterile implants. (B) Glycolytic activity is reported as the percentage of monocytes that were 2-NBDG+ relative to mice with sterile implants. Representative histograms of (C) JC-1 and (D) 2-NBDG at day 7 are shown. Data are presented as the mean ± SD (n = 3–10 animals/group) combined from two independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; Student’s t-test).
Fig 2.
Inhibition of OxPhos by oligomycin promotes MФ pro-inflammatory activity.
Mouse bone marrow-derived or human monocyte-derived MФs were plated overnight and the following day cells were pre-treated with IL-4 (10 ng/mL) for 1 h followed by various concentrations of oligomycin for 24 h, whereupon TNF-α (A and F) and IL-10 (C and H) production was determined by cytometric bead array or ELISA, CD86 (B and G) and CD204 (D and I) expression by flow cytometry, and arginase activity by an enzymatic assay (E and J). Data are presented as the mean ± SD combined from two independent experiments (n = 4–8 biological replicates) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; Student’s t-test).
Table 1.
Nanoparticle features.
Fig 3.
Preferential nanoparticle uptake by monocytes during PJI.
(A) C57BL/6NCrl mice received a single intra-articular injection of Cy5 (C) or Cy5/Tuftsin (CT) nanoparticles (10 μg) at day 7 post-infection and were analyzed on three consecutive days. (B) IVIS imaging of CT labeled nanoparticles in the infected joint at day 3 with (C) representative flow cytometry dot plots. Left panel shows all CD45+ cells and the right panel depicts Ly6G-Ly6C+ gated cells, where Cy5+ monocytes (CD11bhighLy6G-Ly6C+F4/80-) are highlighted in black. (D) Mice receiving C or CT nanoparticles were sacrificed at days 1–3 following nanoparticle injection, whereupon Cy5+ monocytes, MDSCs, and PMNs were quantified by flow cytometry. Data is presented as the mean ± SD (n = 4–5 mice/time point). (*, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t-test).
Fig 4.
Oligomycin-containing nanoparticles induce a metabolic shift in biofilm-associated monocytes that polarizes cells towards a pro-inflammatory phenotype in vivo.
(A-B) Nanoparticle-mediated delivery of oligomycin attenuates OxPhos metabolism in biofilm-associated monocytes. C57BL/6NCrl mice received a single intra-articular injection of free oligomycin only (Oligo; 100 ng), empty nanoparticles (CT), empty nanoparticles (CT) + free oligomycin (100 ng; not loaded), or oligomycin loaded nanoparticles (CTO) at day 7 post-infection and were analyzed at day 3 (A; 7–10 mice/group) or day 7 (B; 10 mice/group) after treatment. OxPhos was calculated as the ratio of green:red monocytes (CD11bhighLy6G-Ly6C+F4/80-) following JC-1 staining and glycolytic activity was calculated as the percentage of 2-NBDG+ monocytes. (C-G) C57BL/6NCrl mice received a single intra-articular injection of CT or CTO nanoparticles (10 μg) at day 7 post-infection. Mice were sacrificed 3 days following nanoparticle injection and monocytes (CD11bhighLy6G-Ly6C+F4/80-) were recovered from each individual animal by FACS (n = 5 mice/treatment group), whereupon intracellular metabolites were isolated for analysis by HPLC-MS/MS. (C) A principle component analysis (PCA) plot was generated using an algorithm in MetaboAnalyst with mean intensities and pareto scaling distribution. Ellipses represent a 95% confidence interval of the normal distribution for each cluster. (D) The heat map depicts the top 25 metabolite differences in monocytes recovered from CTO and CT treated mice. The color key indicates log2-fold changes of normalized mean peak intensities for metabolites in monocytes from CTO vs. CT nanoparticle treated animals. (E) Identification of the most significantly altered pathways in monocytes isolated from CTO treated mice as determined by pathway impact analysis. (F) Gene expression levels in monocytes pooled from 5 CTO-treated animals were calculated after normalizing signals to GAPDH and are presented as the fold-change relative to monocytes isolated from mice receiving CT (control) nanoparticles. Data is presented as the mean ± SD of monocytes combined from three independent experiments (n = 2–3, since some genes did not amplify in one experiment). (G) Reactive oxygen species production by monocytes was examined using CellROX Green (n = 8 mice).
Table 2.
Intracellular metabolites from CT and CTO treated MDSCs and monocytes*.
Fig 5.
Oligomycin-containing nanoparticles targeted to monocytes induce metabolomic changes in MDSCs.
C57BL/6NCrl mice received a single intra-articular injection of Cy5/Tuftsin (CT) or Cy5/Tuftsin/Oligomycin (CTO) nanoparticles (10 μg) at day 7 post-infection (n = 5 mice/treatment group). Mice were sacrificed 3 days following nanoparticle injection and MDSCs (CD11bhighLy6G+Ly6C+F4/80-) were recovered from each individual animal by FACS, whereupon intracellular metabolites were isolated for analysis by HPLC-MS/MS. (A) A principle component analysis (PCA) plot was generated using an algorithm in MetaboAnalyst with mean intensities and pareto scaling distribution. Ellipses represent a 95% confidence interval of the normal distribution for each cluster. (B) The heat map depicts the top 25 metabolite differences in MDSCs recovered from CTO and CT treated mice. The color key indicates log2-fold changes of normalized mean peak intensities for metabolites in MDSCs from CTO vs. CT nanoparticle treated animals. (C) Identification of the most significantly altered pathways in MDSCs isolated from CTO treated mice as determined by pathway impact analysis.
Fig 6.
Nanoparticle-mediated delivery of oligomycin to monocytes reduces established biofilm infection.
(A) C57BL/6NCrl mice received a single intra-articular injection of Cy5 (C), Cy5/Tuftsin (CT), or Cy5/Tuftsin/Oligomycin (CTO) nanoparticles at day 7 post-infection and were analyzed out to 28 days following nanoparticle treatment. Bacterial burden was quantified in the (B) surrounding soft tissue, (C) knee, (D) femur, and (E) implant, where the dotted line represents the limit of detection (LOD). Infiltrating leukocytes were analyzed by flow cytometry and (F) MDSCs, (G) PMNs, (H) monocytes, and (I) macrophages are reported as the percentage of live CD45+ leukocytes (mean ± SD). Results are combined from three independent experiments (n = 15 mice/group/time point). (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; One-way ANOVA).
Fig 7.
Monocyte metabolic reprogramming with oligomycin-containing nanoparticles cooperates with antibiotics to clear established S. aureus biofilm infection.
C57BL/6NCrl mice received a single intra-articular injection of Cy5/Tuftsin (CT) or Cy5/Tuftsin/Oligomycin (CTO) nanoparticles (10 μg) at day 7 post-infection. Seven days later, animals received daily i.p. injections of antibiotics (Abx; 25 mg/kg/day rifampin and 5 mg/kg/day daptomycin) or vehicle (Veh) for one week, whereupon mice were sacrificed at day 21 post-infection. Bacterial burden was quantified from the (A) surrounding soft tissue, (B) knee, (C) femur, and (D) implant, where the dotted line represents the limit of detection (LOD). Infiltrating leukocytes were analyzed by flow cytometry and (E) MDSCs, (F) neutrophils, (G) monocytes, and (H) macrophages are reported as the percentage of live CD45+ leukocytes (mean ± SD). Results are combined from two independent experiments (n = 7–10 mice/group/time point). (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; One-way ANOVA).
Fig 8.
Metabolic reprogramming of monocytes promotes pro-inflammatory properties, MDSC crosstalk, and S. aureus biofilm clearance.
Nanoparticles containing the ATP synthase inhibitor oligomycin are targeted to Fc-receptor positive monocytes with tuftsin. Upon internalization, oligomycin inhibits ATP synthase of the mitochondrial electron transport chain and induces metabolic reprogramming to shift monocyte metabolism and promote pro-inflammatory gene expression. Metabolically reprogrammed monocytes also influence MDSC metabolism. Collectively, these changes promote increased antibiotic susceptibility and clearance of established biofilm infection. ADP, adenosine diphosphate; ATP, adenosine triphosphate; Fc-Receptor, fragment crystallizable region receptor; HIF-1α, hypoxia-inducible factor 1-alpha; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; MDSC, myeloid-derived suppressor cell; Pi, inorganic phosphate; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α.