PET Imaging of Innate Immune Activation Using 11C Radiotracers Targeting GPR84

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11Svia carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies-11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology

The use of spatial intensity distribution analysis to examine G protein-coupled receptor oligomerization

Spatial Intensity Distribution Analysis (SpIDA) is a new approach for detecting protein oligomerization states that can be applied not only to live cells but also fixed cells and native tissue. This approach is based on the generation of pixel-integrated fluorescence intensity histograms from laser scanning fluorescence microscopy images. These histograms are then fit with super-Poissonian distribution functions to obtain density maps and quantal brightness values of the fluorophore that are used to determine the proportions of monomer and dimers/oligomers of the fluorophore-tagged protein. In this chapter we describe SpIDA and highlight its advantages compared to other biochemical or biophysical approaches. We provide guidelines that should be useful to readers who wish to perform SpIDA measurements and describe the application of SpIDA as a post-acquisition imaging histogram analysis software tool to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at the surface of mammalian cells in order to define the steady-state proportion of monomeric and dimeric/oligomeric forms and how this may be regulated by cellular challenges such as ligand treatment