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

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 ¹⁸ F-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?18 F-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.

SUBMITTER: Pannell M

PROVIDER: S-EPMC6916298 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Pannell Maria M Economopoulos Vasiliki V Wilson Thomas C TC Kersemans Veerle V Isenegger Patrick G PG Larkin James R JR Smart Sean S Gilchrist Stuart S Gouverneur Véronique V Sibson Nicola R NR

Glia 20190903 2

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 ...[more]

PMID: 31479168

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Project description:BACKGROUND:As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood. METHODS:Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes. RESULTS:Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1?, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-?B in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes. CONCLUSIONS:These results provide evidence that NF-?B signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.

Dataset Information

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

Publications

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

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