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Translocator Protein (18 kDa) (TSPO) Is Expressed in Reactive Retinal Microglia and Modulates Microglial Inflammation and Phagocytosis

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Translocator Protein (18 kDa) (TSPO) Is Expressed in Reactive Retinal Microglia and Modulates Microglial Inflammation and Phagocytosis

Marcus Karlstetter et al. J Neuroinflammation.

Abstract

Background: The translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions.

Methods: TSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173.

Results: TSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their neurotoxic activity on 661 W photoreceptor cells. Furthermore, XBD173 treatment of murine and human microglial cells promoted the formation of filopodia and increased their phagocytic capacity to ingest latex beads or photoreceptor debris. Finally, treatment with XBD173 reversed the amoeboid alerted phenotype of microglial cells in explanted organotypic mouse retinal cultures after challenge with LPS.

Conclusions: These findings suggest that TSPO is highly expressed in reactive retinal microglia and a promising target to control microglial reactivity during retinal degeneration.

Figures

Figure 1
Figure 1
TSPO expression as a marker for microgliosis in degenerating and aging retinas. In MacGreen mice, representative photomicrographs show low TSPO expression in retinal microglia (green GFP signal and red TSPO immunofluorescence) and constitutive expression in astrocytes (red TSPO immunofluorescence) (A-C) but strong up-regulation in retinal microglia from MacGreen/Rs1h-/Y mice (D-F). The overlap of TSPO and Iba1 immunostaining also indicates co-expression in human retinal microglia (G-I). TSPO immunostaining staining does not co-localize with the Müller cell marker GFAP (J, K) or the neuronal microtubule marker MAP2 (L, M). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer, GFP, green fluorescent protein; TSPO, translocator protein (18 kDa); GFAP, glial fibrillary acid protein; MAP2, microtubule-associated protein 2. Scale bar, 50 μm.
Figure 2
Figure 2
TSPO mRNA and protein expression in reactive microglia. (A) Strong induction of TSPO mRNA levels in isolated microglial cells from MacGreen/Rs1h-/Y mice compared to MacGreen mice. (B) TSPO protein induction in total retinas from MacGreen/Rs1h-/Y mice compared to MacGreen mice. (C) Temporal TSPO mRNA expression profiling shows a high early postnatal expression level and continuous decline to low levels in adult mouse retinas. (D,E) LPS activation of BV-2 microglial cells leads to the induction of TSPO transcripts (D) and protein (E). Data show mean ± SD (n = 3/group, measured in triplicates) **P <0.01 MacGreen/Rs1h-/Y versus MacGreen; ***P <0.001 BV-2 + 50 ng/ml LPS versus BV-2. LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).
Figure 3
Figure 3
The TSPO agonist XBD173 dampens gene transcription of pro-inflammatory markers and reduces microglial neurotoxicity. LPS-activated BV-2 microglial cells were cultured in the presence of various concentrations of XBD173 for 24 h and the pro-inflammatory transcript markers CCL2 (A), IL6 (B), iNOS (C) were determined by real time qRT-PCR. Data show mean ± SD (n = 3/group, measured in triplicates) *P <0.05, **P <0.01, ***P <0.001 XBD173 + LPS versus LPS-treated cells. (D-F), Knock-down of TSPO with two independent shRNAs abrogates the suppressing effects of XBD173 on CCL2 (D), IL6 (E) and iNOS (F) gene expression in BV-2 cells. (G), Production of NO as determined by detection of nitrite from BV-2 microglial cells treated with 50 μM XBD173 in the absence or presence of 50 ng/ml LPS. Data show mean ± SD (n = 9/group) ***P <0.001 XBD173 + LPS versus LPS-treated cells. (H), 661 W photoreceptor cell cultures were treated with conditioned media from BV-2 microglial cells for 48 hours. The supernatant from control-stimulated, 20 μM XBD173-treated, 50 ng/ml LPS-treated, or 20 μM XBD173 + 50 ng/ml LPS-treated cells was added to 661 W photoreceptor cells and apoptosis-related caspase 3/7 activation was determined. Data show mean ± SD (n = 6/group) **P <0.01 XBD173 + LPS versus LPS-treated cells. CCL2, (C-C motif) ligand 2; IL6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide.
Figure 4
Figure 4
The TSPO agonist XBD173 reduces microglial migration and proliferation. (A) Scratch assay to mimic wound-healing in cultured BV-2 microglia treated with vehicle, 50 ng/ml LPS, 50 μM XBD173 or both. Microphotographs from scratched areas were quantified 8 h after treatment (B). Data show mean ± SEM (n = 5/group) *P <0.05 XBD173 versus control, *P <0.001 XBD173 + LPS versus LPS-treated cells. (C), CFSE-based proliferation assay of BV-2 microglial cells treated with vehicle, 100 ng/ml LPS, 50 μM XBD173 or 100 ng/ml LPS + 50 μM XBD173. The proliferation rate of BV-2 microglia was analyzed 24 hours after treatment using flow cytometry and a representative graph out of four repetitions is shown. CFSE, carboxyfluorescein diacetate succinimidyl ester; LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).
Figure 5
Figure 5
The TSPO ligand XBD173 promotes microglial filopodia formation. (A-D) Representative images of phalloidin-stained murine BV-2 microglial cells treated with 50 μM XBD173 in the absence or presence of 50 ng/ml LPS. (E-H) Representative images of phalloidin-stained human iPS-derived microglia (iPSdM) treated with 30 μM XBD173 in the absence or presence of 250 ng/ml LPS. LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).
Figure 6
Figure 6
The TSPO ligand XBD173 increases microglial phagocytosis. Phagocytosis was monitored in BV-2 cells incubated with latex beads (A, B) or CM-DiI-stained apoptotic 661 W photoreceptor material (C, D). Data show mean ± SEM (n = 9/group) **P <0.01 XBD173 + LPS versus LPS-treated cells, *P <0.05 XBD173 versus vehicle-treated cells. Phagocytosis was monitored in human iPSdM cells incubated with latex beads (E, F) or CM-DiI-stained apoptotic 661 W photoreceptor material (G, H). Data show mean ± SEM (n = 9/group) ***P <0.01 XBD173 + LPS versus LPS-treated cell and XBD173 versus vehicle-treated cells. LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).
Figure 7
Figure 7
The TSPO ligand XBD173 increases pregnenolone levels and CYP11A1 inhibition prevents XBD173-induced phagocytosis. (A) Pregnenolone levels in cell culture supernatants of BV-2 cells treated with 20 μM XBD173, 50 ng/ml LPS, or 20 μM XBD173 + 50 ng/ml LPS for 24 hours. Data show mean ± SD (n = 3/group) ***P <0.001 XBD173 versus vehicle-treated cells, **P <0.01 XBD173 + LPS versus LPS-treated cells. (B) Quantification of latex bead phagocytosis of BV-2 cells treated with 20 μM XBD173 or 20 μM XBD173 + 100 μM aminoglutethimide for 24 hours. Data show mean ± SEM (n = 30/group) ***P <0.0001 XBD173 versus control cells. LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).
Figure 8
Figure 8
The TSPO ligand XBD173 reduces the number of alerted amoeboid retinal microglia ex vivo. (A-D) Representative GFP images of the retinal microglia network in explanted mouse retinas treated for 24 hours with vehicle (A), 20 μM XBD173 (B), 1 μg/ml LPS + vehicle (C) and 1 μg/ml LPS + 20 μM XBD173 (D). (E) Quantification of ramified and amoeboid microglial cells in 10 independent image areas of two individual flat mounts (mean ± SEM). **P <0.01 for amoeboid cells in LPS versus control explants; ***P <0.0001, for amoeboid cells in XBD173 + LPS versus LPS-treated explants. GFP, green fluorescent protein; LPS, lipopolysaccharide; TSPO, translocator protein (18 kDa).

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