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Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype

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Abstract

Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1β and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the ischemic penumbra. While TREM-2+ macrophages were abundant at 3 days post-lesion (DPL) in the ischemic core, TREM-2+ astrocytes persisted in the penumbra until 14DPL. This study demonstrates that TLR4 expression increases astroglial sensitivity to ligands facilitating astrocyte conversion towards a proinflammatory phenotype, and that astroglial TREM-2 modulates this response reducing the downstream NF-κB activation. Therefore, the availability of TLR4 and TREM-2 ligands in the ischemic environment may control proinflammatory astroglial conversion to the neurodegenerative phenotype.

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Acknowledgements

GR and VC are doctoral fellows from CONICET. AJR and VM are researchers from CONICET (Argentina). We thank Dr. Marina Snitcofsky, Biot. Andrea Pecile, and Manuel Ponce for the animal care and Dr. Carla Bonavita for the correction of the manuscript.

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Author notes

  1. Alejandro Villarreal

    Present address: Department of Molecular Embryology, Institute for Anatomy and Cell Biology, University of Freiburg, 79104, Freiburg, Germany

Authors and Affiliations

  1. Universidad de Buenos Aires, Facultad de Medicina, Departamento de Histología, Embriología, Biología Celular y Genética, Buenos Aires, Argentina

    Gerardo Rosciszewski, Vanesa Cadena, Veronica Murta, Jeronimo Lukin, Alejandro Villarreal & Alberto Javier Ramos

  2. Laboratorio de Neuropatología Molecular, Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis” Facultad de Medicina, Universidad de Buenos Aires, Calle Paraguay 2155 3er piso (1121),, Ciudad de Buenos Aires, Argentina

    Gerardo Rosciszewski, Vanesa Cadena, Veronica Murta, Jeronimo Lukin & Alberto Javier Ramos

  3. Infectious Diseases Service, Department of Medicine, Lausanne University Hospital, Chemin des Boveresses 155, 1066, Epalinges, Switzerland

    Thierry Roger

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  1. Gerardo Rosciszewski
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Contributions

GR, AV, and AJR designed the experiments; GR, VC, VM, JL, and AV did the experimental work and analysis; GR, VC, VM, and AJR analyzed and interpreted the data; TR provided the proprietary essential reagents developed by his group; AJR wrote the article; and AJR, AV, VM, VC, JL, and TR revised the manuscript.

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Correspondence to Alberto Javier Ramos.

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Availability of Data and Materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing Interests

The authors declare that they have no competing interests.

Ethics Approval and Consent to Participate

The animal care for this experimental protocol was in accordance with the NIH guidelines for the Care and Use of Laboratory Animals, the principles presented in the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience and the ARRIVE guidelines, and it was approved by the CICUAL committee of the School of Medicine, University of Buenos Aires.

Funding

Supported by grants CONICET PIP, ANPCYT PICT2012–1424, and UBACYT assigned to AJR and by grants from the Swiss National Science Foundation (SNF 145014 and 149511) to TR. Funding agencies provided the research grants that supported all materials, reagents, and services required to perform the experiments included in this manuscript.

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Supplementary Figure 1

Specificity of anti-TLR4 antibodies. Images of anti-TLR4 staining in the ischemic core infiltrated with blood-borne leukocytes in wild type and TLR4 −/− mice and nuclear counterstaining with DAPI. Note the absence of TLR4 immunostaining in TLR4 −/− mice. Bar = 60 μm. (GIF 232 kb)

High resolution image (TIFF 3095 kb)

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Rosciszewski, G., Cadena, V., Murta, V. et al. Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype. Mol Neurobiol 55, 3875–3888 (2018). https://doi.org/10.1007/s12035-017-0618-z

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