Imaging of translocator protein upregulation is selective for pro‐inflammatory polarized astrocytes and microglia

Maria Pannell,Véronique Gouverneur,James R Larkin,Vasiliki Economopoulos,Stuart Gilchrist,Nicola R Sibson,Sean Smart,Patrick G Isenegger,Veerle Kersemans,Thomas C Wilson

doi:10.1002/glia.23716

Abstract

Translocator protein (TSPO) expression is increased in activated glia, and has been used as a marker of neuroinflammation in PET imaging. However, the extent to which TSPO upregulation reflects a pro‐ or anti‐inflammatory phenotype remains unclear. Our aim was to determine whether TSPO upregulation in astrocytes and microglia/macrophages is limited to a specific inflammatory phenotype. TSPO upregulation was assessed by flow cytometry in cultured astrocytes, microglia, and macrophages stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin‐4 (Il‐4). Subsequently, mice were injected intracerebrally with either a TNF‐inducing adenovirus (AdTNF) or IL‐4. Glial expression of TSPO and pro‐/anti‐inflammatory markers was assessed by immunohistochemistry/fluorescence and flow cytometry. Finally, AdTNF or IL‐4 injected mice underwent PET imaging with injection of the TSPO radioligand 18F‐DPA‐713, followed by ex vivo autoradiography. TSPO expression was significantly increased in pro‐inflammatory microglia/macrophages and astrocytes both in vitro, and in vivo after AdTNF injection (p < .001 vs. control hemisphere), determined both histologically and by FACS. Both PET imaging and autoradiography revealed a significant (p < .001) increase in 18F‐DPA‐713 binding in the ipsilateral hemisphere of AdTNF‐injected mice. In contrast, no increase in either TSPO expression assessed histologically and by FACS, or ligand binding by PET/autoradiography was observed after IL‐4 injection. Taken together, these results suggest that TSPO imaging specifically reveals the pro‐inflammatory population of activated glial cells in the brain in response to inflammatory stimuli. Since the inflammatory phenotype of glial cells is critical to their role in neurological disease, these findings may enhance the utility and application of TSPO imaging.

Highlights

The translocator protein (TSPO), first described as the peripheral benzodiazepine receptor (PBR), is an 18 kDa protein found predominantly on the outer mitochondrial membrane (Liu et al, 2014)
The use of TSPO as a biomarker for activated microglia in neurological disease with positron emission tomography (PET) imaging has increased in popularity in recent years
Analysis of cultured microglia and macrophages stimulated with LPS and tumor necrosis factor (TNF) showed a significant upregulation of MHC II expression, in cultured astrocytes this was only seen after LPS stimulation

Summary

| INTRODUCTION

The translocator protein (TSPO), first described as the peripheral benzodiazepine receptor (PBR), is an 18 kDa protein found predominantly on the outer mitochondrial membrane (Liu et al, 2014). It has long been established that microglia/brain macrophages can adopt distinct inflammatory phenotypes: (a) the pro-inflammatory phenotype (historically termed M1), associated with neuroinflammation, inflammatory cytokine release (Laskin, Sunil, Gardner, & Laskin, 2011; Taylor, Jones, Kubota, & Pocock, 2005), increased phagocytosis (Fu, Shen, Xu, Luo, & Tang, 2014), and in many cases damage and destruction to healthy neurons (Fricker, Oliva-Martín, & Brown, 2012); and (b) the anti-inflammatory phenotype (historically termed M2), which is known as the neuroprotective phenotype and is characterized by anti-inflammatory cytokine release (Kigerl et al, 2009), immunoregulation and tissue repair (Cairo, Recalcati, Mantovani, & Locati, 2011) It is generally agreed, that the “M1/M2” distinction is far too simplistic and that microglia and macrophages undergo a spectrum of activation, which can be different depending on the type and length of stimulus, among other factors (Martinez & Gordon, 2014). The aim of this study, was to determine whether TSPO receptor expression in microglia, macrophages, and astrocytes is associated with a specific inflammatory response in the brain, and with a specific astrocytic or microglial phenotype

| METHODS

| RESULTS

Findings

| DISCUSSION