Targeting of Formyl Peptide Receptor 2 for in vivo imaging of acute vascular inflammation

Abstract

Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. Methods: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for in vivo applications. We tested internalization, toxicity and functional interactions with neutrophils in vitro for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, in vivo imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. Results: In vitro studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalization at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). In vivo studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive ("switchable") version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. Conclusions: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.

Keywords: Inflammation; formyl peptide receptors; intravital microscopy; neutrophils; small-molecule imaging probes.

Figure 1

Schematic of synthetic pathway for preparation of FPR2/ALX-targeted ligand (6) and the functionalized fluorescent probe (11, Rho-pip-C1). Reagents: i) 1-bromobutane, potassium carbonate, methanol, ii) hydrazine monohydrate, ethanol, iii) 2-nitro-benzoyl chloride, potassium carbonate, dichloromethane, iv) zinc dust, acetic acid, dichloromethane, v) 4-methoxy benzaldehyde, citric acid, ethanol, vi) tert-butyl piperazine-1-carboxylate, triethylamine, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate and dichloromethane, vii) trifluoroacetic acid, dichloromethane, viii) chloroacetyl chloride, triethylamine, dichloromethane ix) 6, N,N-diisopropylethylamine, acetonitrile.

Figure 2

Rho-pip-C1 binds to FPR2/ALX, without causing receptor internalization. (A) Flow-cytometric analysis of FPR2 internalization. HeLa cells stably expressing N-terminally FLAG-tagged human FPR2/ALX were used to determine whether Rho-pip-C1 and Rho-NH-C1 induce FPR2/ALX internalization at a concentration range of 10^-6 to 10^-9M. Results are expressed as a loss of FPR2/ALX upon agonist treatment relative to untreated cells. The lead compound Quin C1 was included for comparison. (B) Representative magnified images (100X) of fixed human neutrophils treated with Rho-pip-C1 and the nuclear stain DAPI (scale bar, 10 μM). (C) Representative immunofluorescence images from n = 5 independent donors in cells that had been treated with vehicle (PBS), the pan-FPR antagonist Boc2 (10 µM), or the FPR2 specific antagonist WRW4 prior to incubation with Rho-pip-C1 (scale bar, 100 μM). (D) Quantification of emission intensity ratios for vehicle (PBS), Boc2 (10 µM) and the FPR2/ALX specific antagonist WRW4 (10 µM) (n = 5 independent donors in each group). All imaging analysis was done in a double-blinded fashion. Statistical significance was determined using one-way ANOVA with Bonferroni post-hoc test and is presented as *p < 0.05 vs. PBS control.

Figure 3

Rho-Pip-C 1 does not elicit changes in neutrophil function. (A) Neutrophils isolated from peripheral blood of healthy volunteers were resuspended in DMEM with 3% FBS and added at 100,000 cells on top of each chemotaxis filter. Neutrophil chemotaxis towards PBS (control) or LTB₄ (10^-6 M) using a chemotaxis plate after 3 hours was quantified by counting the neutrophils with Neubauer hemocytometer under bright field microscope. Some neutrophils were pre-treated with Rho-Pip-C1 at the indicated concentrations before chemotaxis (n = 5 independent donors in each group, with samples run in duplicate). (B) Quantification of transmigrated neutrophils through fibronectin-coated HUVECs. After 72 hours, human neutrophils were allowed to transmigrate through HUVECs for 3 hours towards PBS (vehicle) or LTB₄ (10^-6 M) and were counted using Neubauer hemocytometer. Some neutrophils were pre-treated with Rho-pip-C1 at the indicated concentrations before transmigration (n = 5 independent donors in each group). (C) Neutrophils were treated with vehicle (PBS) or phorbol 12-myristate 13-acetate (PMA) for 3 hours, with and without Rho-pip-C1 (10^-6 M to 10^-9 M). MPO levels were then quantified (n = 5 independent donors in each group, with samples run in duplicate) and statistical significance was determined using a one-way ANOVA with Bonferroni post-hoc test (A, C) or Friedman test followed by Dunn's multiple comparison tests between groups (B) and is presented as ^*p < 0.05 vs. respective PBS control without chemoattractant. All experiments were done in a double-blinded fashion.

Figure 4

Preferential uptake of Rho-Pip-C 1 on neutrophils in TNFα treated mice. Mice were treated with saline (control), TNFα (250 ng in 200 µL saline, intrascrotal injection) or TNFα+BMS-470539 (20 mg/kg via intraperitoneal injection). Confocal intravital microscopy was performed and mice were injected with Gr-1 antibody conjugated with eFluor 488 fluorochrome (green) and Rho-pip-C1 (red). Representative confocal intravital microscopy pictures of cremasteric vessels of (A) saline treated mice and (B)TNFα treated mice. Scale bar, 10 μM. Dotted line represents the edges of the vessel. The number of adherent (within) or extravasated (outside) neutrophils in the cremaster of (C) saline treated (control) (n = 5 mice per group) or (D) TNFα treated (n = 6 mice per group) mice was quantified. Mice were treated with saline (control), TNFα or TNFα+BMS-470539 and the number of (E) adherent (n = 5-6 mice per group) and (F) extravasated neutrophils (n = 5 mice per group) were quantified. Neutrophils were identified by their Gr-1 label and classified as either cells that were positive for Gr-1 (Gr-1⁺. Shown in green) or as cells that had taken up both Gr-1 and Rho-pip-C1 (Gr-1⁺/Rho⁺, dually labelled, shown in red), i.e. neutrophils that had taken up the probe. Statistical significance was determined using unpaired t-test, Mann-Whitney U test (C+D) or ANOVA with Bonferroni post-hoc test (E+F). ^$p < 0.05 vs. Gr-1; ^*p < 0.05 vs. saline control and ^#p < 0.05 vs. vehicle (saline) + TNFα control. All imaging analysis was done in a double-blinded fashion.

Figure 5

“Switchable” Rho-NH-C1 is an effective tool for imaging inflammation. (A) Emission spectra of 13 (Rho-NH-C1. 1 μM, 1:1 v/v methanol and water solutions) at varying pH values (λ_exc = 350 nm or λ_exc = 500 nm as indicated). (B) Absorption spectra of 13 (33 μM, 1:1 v/v methanol and water solutions) at varying pH values. (C) Determination of pK_cycl value using the normalized absorption intensity at 560 nm and a sigmoidal fit (in-built OriginPro 2017 function). (D) Representative images of human neutrophils (n = 5 independent donors) that had been treated with vehicle (saline, tops panels) and phorbol 12-myristate 13-acetate (PMA) (100 nM, bottom panels) prior to incubation with Rho-NH-C1 (scale bar, 100 μM). (E) Quantification of emission intensity ratios demonstrating a statistically significant difference between saline and PMA-treated human neutrophils (n = 5 independent donors in each group). Statistical significance was determined using unpaired t-test and is presented as ^*p < 0.05 vs. PBS control. All imaging analysis was done in a double-blinded fashion.

Figure 6

Design and application of Rho-pip-C1 as a novel small molecule in vivo imaging agent for acute inflammation. (A) Schematic of design elements in FPR2/ALX-targeted fluorescent probe, termed Rho-pip-C1. (B) Under non-inflammatory conditions (i.e. treatment with phosphate buffered saline (vehicle for tumor necrosis factor alpha [TNFα])), Rho-pip-c1 does not bind to FPR2/ALX on neutrophils, therefore no fluorescence is observed in the murine microcirculation using confocal intravital microscopy. (C) Acute inflammation is induced via injection of the pro-inflammatory cytokine TNFα. Rho-pip-C1 binds to FPR2/ALX on activated neutrophils and fluorescence can been observed and quantified using confocal intravital microscopy (recorded at λ_exc = 595 nm/ λ_em = 645 nm).