In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

Abstract

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe²⁺ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe²⁺ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

Keywords: infectious disease; labile iron; luciferin; metal homeostasis; molecular imaging.

Scheme 1.

Fe²⁺-dependent cleavage of iron-caged luciferin-1 (ICL-1), an endoperoxide-luciferin conjugate and in vivo probe of Fe²⁺.

Scheme 2.

Synthesis of ICL-1^a. ^aReagents and conditions: (i) triphosgene, 4-DMAP, toluene, 125 to 35 °C, 3 h; (ii) 1, NaH, toluene, 35 °C, 12 h; and (iii) d-cysteine, K₂CO₃, CH₂Cl₂, MeOH, H₂O, 0 °C, 12 h.

Fig. 1.

ICL-1 responds to Fe²⁺ over other metals and tightly bound biological iron species with metal and redox specificity. Bioluminescence response of ICL-1 incubated with (A) varying concentrations of Fe²⁺ [ferrous ammonium sulfate salt (FAS)] (gray bars) or 100 µM FAS with 100 µM of bipyridine (BPY) (gray patterned bars), (B) various biologically relevant s-block (1 mM), d-block (100 µM) metal ions, and (C) tightly bound iron species of biological relevance: transferrin (without iron, apoTf; with iron, holoTf), ferritin, hemin, and hemoglobin (Hb), and reductants at 3 mM, such as glutathione (GSH), N-acetyl cysteine (NAC), β-mercaptoethanol (BME), and ascorbic acid (as. acid). Signals are integrated over 30 min and expressed as photon fluxes normalized to ICL-1 bioluminescence with no treatment (buffer alone). Statistical analyses were performed with one-way ANOVA with multiple comparisons to the control with no metal treatment (*P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001). Error bars are ±SEM (n = 3).

Fig. 2.

Bioluminescent signals from PC3M-luc cells probed with ICL-1. Cells were supplemented with FAS for 90 min, BPY for 30 min, or a combination the two chemicals followed by addition of ICL-1 (20 μM). Total photon flux was integrated over 1 h and normalized to cells treated with buffer alone. Representative images of PC3M-luc cells with each treatment are shown below the corresponding data bar in the graph. Statistical analyses were performed with one-way ANOVA with multiple comparisons to the control with no metal treatment (*P ≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001). Error bars are ±SD (n = 3–5).

Fig. 3.

ICL-1 monitors labile iron dynamics in luciferase-expressing mice. FVB-luc⁺ mice were injected (i.p.) with ICL-1 (25 nmol) after i.p. injection of vehicle (DPBS), FAC (20 mg/kg), BPY (8 mg/kg), or both FAC and BPY. Mice were injected with vehicle or FAC 1 h before injection of ICL-1 and with BPY 20 min before injection of ICL-1. (A) Representative images of FVB-luc⁺ mice treated with vehicle, FAC, and/or BPY and imaged with ICL-1. (B) Ratios of the total photon fluxes from ICL-1 of treated animals to their basal signals. Bioluminescent photon fluxes were acquired 0–50 min postinjection of the ICL-1 (i.p. injection, 25 nmol). Statistical analyses were performed with a two-tailed Student’s t test (**P ≤ 0.01 and ***P ≤ 0.001). Error bars are ±SEM (n = 3–7).

Fig. 4.

ICL-1 imaging visualizes changes in tissue labile iron levels and distributions in systemic infection with A. baumannii. (A) Representative images of FVB-Luc⁺ mice mock-infected (PBS) or infected with A. baumannii through retroorbital injection (dorsal images at 30 min postinjection of ICL-1) and imaged with ICL-1 (25 nmol) at 24 h postinfection. (B) Bacterial burdens in the organs of infected mice represented as scatter plots; bars represent the medians, with each organ being significantly colonized (P ≤ 0.005, Wilcoxon signed rank test). Error bars are interquartile ranges (n = 9–15). (C) LA–ICP-MS analysis of iron in liver tissue slices from a mock-infected and an infected mouse (Top) and H&E stains of the corresponding slices (Bottom). (D) Liquid ICP-MS analysis of total iron in organs of mock-infected and infected mice (24 h postinfection). Data are represented as box-and-whiskers plots (n = 3–9). Statistical analyses were performed with a two-tailed Student’s t test (*P ≤ 0.05). (E) Gene expression analysis of iron proteins in homogenized liver tissues by real-time PCR; mRNA levels are normalized to GAPDH. Data are plotted as the log₂ fold change of the mean gene expression in the livers of infected mice from those of mock-infected mice. Ferroportin (FPN), transferrin receptor (TfR), divalent metal transporter-1 (DMT-1), hepcidin, lipocalin-2 (LCN2), ferritin heavy chain (FHC) and light chain (FLC), and regulatory proteins IRP1 and IRP2 (dark gray bars) were evaluated. Additional housekeeping genes (HMBS and RLPL0) are included as controls (light gray bars). Statistical analyses were performed on ΔΔC_t values with one-way ANOVA with multiple comparisons to the GAPDH control (*P ≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001). Error bars are ±SEM (n = 4–6).