Original Article
Dissecting the target leukocyte subpopulations of clinically relevant inflammation radiopharmaceuticals

https://doi.org/10.1007/s12350-019-01929-zGet rights and content

Abstract

Background

Leukocyte subtypes bear distinct pro-inflammatory, reparative, and regulatory functions. Imaging inflammation provides information on disease prognosis and may guide therapy, but the cellular basis of the signal remains equivocal. We evaluated leukocyte subtype specificity of characterized clinically relevant inflammation-targeted radiotracers.

Methods and Results

Leukocyte populations were purified from blood- and THP-1-derived macrophages were polarized into M1-, reparative M2a-, or M2c-macrophages. In vitro uptake assays were conducted using tracers of enhanced glucose or amino acid metabolism and molecular markers of inflammatory cells. Both 18F-deoxyglucose (18F-FDG) and the labeled amino acid 11C-methionine (11C-MET) displayed higher uptake in neutrophils and monocytes compared to other leukocytes (P = 0.005), and markedly higher accumulation in pro-inflammatory M1-macrophages compared to reparative M2a-macrophages (P < 0.001). Molecular tracers 68Ga-DOTATATE targeting the somatostatin receptor type 2 and 68Ga-pentixafor targeting the chemokine receptor type 4 (CXCR4) exhibited broad uptake by leukocyte subpopulations and polarized macrophages with highest uptake in T-cells/natural killer cells and B-cells compared to neutrophils. Mitochondrial translocator protein (TSPO)-targeted 18F-flutriciclamide selectively accumulated in monocytes and pro-inflammatory M1 macrophages (P < 0.001). Uptake by myocytes and fibroblasts tended to be higher for metabolic radiotracers.

Conclusions

The different in vitro cellular uptake profiles may allow isolation of distinct phases of the inflammatory pathway with specific inflammation-targeted radiotracers. The pathogenetic cell population in specific inflammatory diseases should be considered in the selection of an appropriate imaging agent.

Introduction

Inflammation plays a major role in cardiovascular pathology and is a promising target for novel gene- and small peptide-based therapies.1,2 Leukocyte infiltration is associated with atherosclerotic plaque vulnerability,3 myocardial infarction,4 cardiac sarcoidosis,5 and progressive heart failure.6 Several specific leukocyte populations participate in the propagation and resolution of tissue inflammation and healing.7 Severity of inflammation can predict functional outcome,8,9 as evidenced by leukocyte count after hospitalization.10,11 But conventional blood-based measurements do not accurately reflect local tissue inflammation.8 Accordingly, non-invasive imaging techniques such as positron emission tomography (PET) are an attractive method to assess tissue inflammation.8

Clinically, inflammation imaging has relied on 18F-FDG, which accumulates in activated macrophages due to high glucose metabolism.12 Uptake of 18F-FDG correlates well with macrophage content in atherosclerotic plaque and infarct tissue.13,14 However, 18F-FDG is not specific for leukocytes, such that high cardiomyocyte signal and requisite myocyte suppression protocols complicate image interpretation.15

In recent years, a range of alternative tracers have been characterized for preclinical and clinical delineation of inflammatory cells, including amino acid metabolism, leukocyte-expressed chemokine receptors, and macrophage-expressed TSPO and SSTR2.13,16, 17, 18 Preclinical and clinical studies using imaging agents targeting these biomarkers have demonstrated specificity of for inflammation.18, 19, 20, 21, 22, 23 However, the range of leukocyte subpopulations and their diverse adverse and protective roles necessitates a closer examination of the precise cellular basis of the imaging signal.

Accordingly, we undertook a side-by-side in vitro comparison of the uptake profile for metabolism-targeted and molecular-targeted inflammation radiotracers in isolated leukocyte subpopulations and polarized macrophages. For comparison, uptake by cardiomyocytes and cardiac fibroblasts was assessed to determine potential contribution to in vivo non-inflammatory signal.

Section snippets

Methods

Additional details are provided in the online supplement.

Validation of Purified Cell Populations

To ensure the maintained health of all cell populations evaluated, metabolic viability of isolated leukocytes and polarized macrophages was tested. Viability between all cell subtypes was comparable. Likewise, exposure to radioactivity dose used in uptake assays did not adversely impact metabolic viability (Online Figure 1A and B). Trypan blue exclusion assay confirmed identical viability at time of assay between M0, M1, M2a, and M2c macrophages (Online Figure 1C). To verify the distinct

Discussion

The range of inflammation-targeted radiotracers has expanded, but specificity for leukocyte subtypes is poorly defined. We evaluated the in vitro binding characteristics of inflammation imaging agents to demonstrate three major findings. First, elevated metabolic rate drives selective uptake of 18F-FDG and labeled amino acids by monocytes and pro-inflammatory M1-like macrophages over other leukocyte subtypes including reparative M2-like macrophages, though metabolic tracers also accumulate in

Limitations

Some limitations of the current study should be considered. First, because uptake assays were conducted using common incubation periods, absolute tracer uptake may be underestimated compared to 18F-FDG. The lower absolute uptake of 68Ga-labeled tracers may be influenced by the shorter radioactive half-life, saturable receptor-limited binding, and rapid turnover of receptors at the cell surface, as we did not account for internalization or efflux of the tracer. Nonetheless, uptake assays for

New Knowledge Gained

Direct comparison of in vitro uptake identified distinct cell populations targeting for clinically validated inflammation PET radiotracers. These cellular substrates provide valuable insights into the suitability of each metabolic or molecular imaging tracer for infiltrative cardiovascular diseases characterized by different dominant leukocyte subtypes, guiding the selection of appropriate imaging markers of inflammatory and reparative macrophages, granulocytes, and lymphocytes.

Conclusion

The present data indicate the variability of the cellular substrate for imaging agents targeting inflammation. Importantly, the optimal radiotracer for post-infarct or atherosclerotic plaque inflammation may be different from post-transplant rejection or sarcoidosis imaging. The temporal pattern of tracer retention and specific targeting of leukocyte subtypes can provide critical insights into disease progression and response to therapy. Different inflammation radiotracers target divergent

Acknowledgments

This study was supported by the German Research Foundation (DFG, Clinical Research Group KFO311, Excellence Cluster REBIRTH-2, and research Grant TH-2161/1-1 (JT)). The authors thank the Preclinical Molecular Imaging Core Facility, Radiochemistry Laboratory, and the Molecular Cardiology Laboratory for skilled assistance.

Disclosure

All authors declare that they have no conflict of interest.

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