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TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis.

Takahashi K, Prinz M, Stagi M, Chechneva O, Neumann H - PLoS Med. (2007)

Bottom Line: EAE was induced in mice by immunization with a myelin autoantigen.Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination.TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University of Bonn Life & Brain Center and Hertie-Foundation, Bonn, Germany.

ABSTRACT

Background: In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system (CNS), are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor. Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.

Methods and findings: EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination. TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

Conclusions: Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE. TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

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Myeloid Cells Transduced with TREM2 Showed Increased Phagocytic and Anti-Inflammatory Activity In Vitro(A) TREM2-transduced myeloid cells showed increased phagocytosis of beads and of apoptotic neuron–enriched cultures. Analysis of the phagocytosis of beads (left graph), TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars) after stimulation with TREM2-specific antibodies either untreated (−) or treated with ERK inhibitor (+). Phagocytosis assay (right graph) of apoptotic cells derived from neuron-enriched cultures by TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars), either treated with ERK inhibitor (+) or untreated (−). Transduction of myeloid cells (BM-MC) with TREM2 increased the phagocytosis of beads and apoptotic cells that were dependent on ERK phosphorylation. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with bead phagocytosis, and p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with phagocytosis of apoptotic neurons. Five independent experiments were performed, and each of them was done in quadruplicate.(B) Anti-inflammatory activity of TREM2 stimulation. Myeloid cells were transduced with TREM2 (open bars) or control GFP vector (filled bars). Myeloid cells were cultured on plates coated with TREM2 cross-linking antibodies (left graph). One hour later, cells were stimulated with LPS. Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. TREM2-transduced myeloid cells showed reduced gene transcript levels of IL-1β and NOS2 and an increased level of IL-10. In addition, apoptotic cells derived from neuron-enriched cultures were added to myeloid cells (right graph). Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) with TREM2 plus LPS for IL-1, p = 0.0205 (TREM2 versus GFP) with TREM2 plus LPS for IL-10, p = 0.0149 (TREM2 versus GFP) with coculture for IL-1, and p = 0.012 (TREM2 versus GFP) with coculture for NOS2. Four independent experiments were performed.(C) Phosphorylation of ERK after stimulation of TREM2. Myeloid cells were transduced with TREM2 and cultured for 1 h on plates coated with TREM2 cross-linking antibodies (+) or isotype control antibodies (−). Cross-linking of TREM2 increased the amount of phosphorylated ERK (phospho ERK) in relation to total ERK (ERK).(D) TREM2-transduced myeloid cells showed an increased release of IL-10 after stimulation of TREM2 plus LPS or coculture with apoptotic neuron–enriched cultures. Myeloid cells were transduced with TREM2 (open bars) or with control GFP vector (filled bars), cultured on plates coated with TREM2 cross-linking antibodies, and stimulated with LPS 1 h later or supplemented with apoptotic cells derived from neuron-enriched cultures. The amount of IL-10 released in the supernatant was determined by ELISA. Data are presented as mean ± SEM. Unpaired t-test: p = 0.044 (TREM2 versus GFP) for TREM2 plus LPS, and p = 0.0054 (TREM2 versus GFP) for coculture. Four independent experiments were performed.
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pmed-0040124-g003: Myeloid Cells Transduced with TREM2 Showed Increased Phagocytic and Anti-Inflammatory Activity In Vitro(A) TREM2-transduced myeloid cells showed increased phagocytosis of beads and of apoptotic neuron–enriched cultures. Analysis of the phagocytosis of beads (left graph), TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars) after stimulation with TREM2-specific antibodies either untreated (−) or treated with ERK inhibitor (+). Phagocytosis assay (right graph) of apoptotic cells derived from neuron-enriched cultures by TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars), either treated with ERK inhibitor (+) or untreated (−). Transduction of myeloid cells (BM-MC) with TREM2 increased the phagocytosis of beads and apoptotic cells that were dependent on ERK phosphorylation. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with bead phagocytosis, and p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with phagocytosis of apoptotic neurons. Five independent experiments were performed, and each of them was done in quadruplicate.(B) Anti-inflammatory activity of TREM2 stimulation. Myeloid cells were transduced with TREM2 (open bars) or control GFP vector (filled bars). Myeloid cells were cultured on plates coated with TREM2 cross-linking antibodies (left graph). One hour later, cells were stimulated with LPS. Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. TREM2-transduced myeloid cells showed reduced gene transcript levels of IL-1β and NOS2 and an increased level of IL-10. In addition, apoptotic cells derived from neuron-enriched cultures were added to myeloid cells (right graph). Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) with TREM2 plus LPS for IL-1, p = 0.0205 (TREM2 versus GFP) with TREM2 plus LPS for IL-10, p = 0.0149 (TREM2 versus GFP) with coculture for IL-1, and p = 0.012 (TREM2 versus GFP) with coculture for NOS2. Four independent experiments were performed.(C) Phosphorylation of ERK after stimulation of TREM2. Myeloid cells were transduced with TREM2 and cultured for 1 h on plates coated with TREM2 cross-linking antibodies (+) or isotype control antibodies (−). Cross-linking of TREM2 increased the amount of phosphorylated ERK (phospho ERK) in relation to total ERK (ERK).(D) TREM2-transduced myeloid cells showed an increased release of IL-10 after stimulation of TREM2 plus LPS or coculture with apoptotic neuron–enriched cultures. Myeloid cells were transduced with TREM2 (open bars) or with control GFP vector (filled bars), cultured on plates coated with TREM2 cross-linking antibodies, and stimulated with LPS 1 h later or supplemented with apoptotic cells derived from neuron-enriched cultures. The amount of IL-10 released in the supernatant was determined by ELISA. Data are presented as mean ± SEM. Unpaired t-test: p = 0.044 (TREM2 versus GFP) for TREM2 plus LPS, and p = 0.0054 (TREM2 versus GFP) for coculture. Four independent experiments were performed.

Mentions: Stimulation of TREM2 has been shown to induce phagocytosis in primary microglia [14]. In order to determine the effects of TREM2 expression on phagocytic activity of BM-MC, we analyzed phagocytosis of microsphere beads and apoptotic cells by TREM2-transduced BM-MC. Phagocytosis of microsphere beads was increased in TREM2-stimulated TREM2-transduced BM-MC compared to control BM-MC and cultured monocytes (Figure 3A). Likewise, phagocytosis of apoptotic membrane fragments derived from mixed neuron-enriched cultures was increased after gene transduction of TREM2. In detail, 93.3% (SEM 6.6) of BM-MC phagocytosed apoptotic neurons after transduction with TREM2, while only 50.5% (SEM 5.6) of BM-MC phagocytosed apoptotic neurons after transduction with the control GFP vector (Figure 3A). Increased phagocytosis of beads and apoptotic cells after TREM2 stimulation was completely neutralized by the ERK inhibitor PD98059, suggesting that ERK signaling was involved in TREM2-mediated phagocytosis by BM-MC (Figure 3A). TREM2 cross-linking antibodies stimulated phosphorylation of ERK in TREM2-transduced BM-MC (Figure 3), but did not modify cell surface expression of MHC class II, CD80, CD86, CD36, CCR7, and CD11c (Figure S1).


TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis.

Takahashi K, Prinz M, Stagi M, Chechneva O, Neumann H - PLoS Med. (2007)

Myeloid Cells Transduced with TREM2 Showed Increased Phagocytic and Anti-Inflammatory Activity In Vitro(A) TREM2-transduced myeloid cells showed increased phagocytosis of beads and of apoptotic neuron–enriched cultures. Analysis of the phagocytosis of beads (left graph), TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars) after stimulation with TREM2-specific antibodies either untreated (−) or treated with ERK inhibitor (+). Phagocytosis assay (right graph) of apoptotic cells derived from neuron-enriched cultures by TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars), either treated with ERK inhibitor (+) or untreated (−). Transduction of myeloid cells (BM-MC) with TREM2 increased the phagocytosis of beads and apoptotic cells that were dependent on ERK phosphorylation. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with bead phagocytosis, and p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with phagocytosis of apoptotic neurons. Five independent experiments were performed, and each of them was done in quadruplicate.(B) Anti-inflammatory activity of TREM2 stimulation. Myeloid cells were transduced with TREM2 (open bars) or control GFP vector (filled bars). Myeloid cells were cultured on plates coated with TREM2 cross-linking antibodies (left graph). One hour later, cells were stimulated with LPS. Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. TREM2-transduced myeloid cells showed reduced gene transcript levels of IL-1β and NOS2 and an increased level of IL-10. In addition, apoptotic cells derived from neuron-enriched cultures were added to myeloid cells (right graph). Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) with TREM2 plus LPS for IL-1, p = 0.0205 (TREM2 versus GFP) with TREM2 plus LPS for IL-10, p = 0.0149 (TREM2 versus GFP) with coculture for IL-1, and p = 0.012 (TREM2 versus GFP) with coculture for NOS2. Four independent experiments were performed.(C) Phosphorylation of ERK after stimulation of TREM2. Myeloid cells were transduced with TREM2 and cultured for 1 h on plates coated with TREM2 cross-linking antibodies (+) or isotype control antibodies (−). Cross-linking of TREM2 increased the amount of phosphorylated ERK (phospho ERK) in relation to total ERK (ERK).(D) TREM2-transduced myeloid cells showed an increased release of IL-10 after stimulation of TREM2 plus LPS or coculture with apoptotic neuron–enriched cultures. Myeloid cells were transduced with TREM2 (open bars) or with control GFP vector (filled bars), cultured on plates coated with TREM2 cross-linking antibodies, and stimulated with LPS 1 h later or supplemented with apoptotic cells derived from neuron-enriched cultures. The amount of IL-10 released in the supernatant was determined by ELISA. Data are presented as mean ± SEM. Unpaired t-test: p = 0.044 (TREM2 versus GFP) for TREM2 plus LPS, and p = 0.0054 (TREM2 versus GFP) for coculture. Four independent experiments were performed.
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pmed-0040124-g003: Myeloid Cells Transduced with TREM2 Showed Increased Phagocytic and Anti-Inflammatory Activity In Vitro(A) TREM2-transduced myeloid cells showed increased phagocytosis of beads and of apoptotic neuron–enriched cultures. Analysis of the phagocytosis of beads (left graph), TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars) after stimulation with TREM2-specific antibodies either untreated (−) or treated with ERK inhibitor (+). Phagocytosis assay (right graph) of apoptotic cells derived from neuron-enriched cultures by TREM2-transduced myeloid cells (open bars), control GFP vector–transduced myeloid cells (filled bars), or cultured monocytes (grey bars), either treated with ERK inhibitor (+) or untreated (−). Transduction of myeloid cells (BM-MC) with TREM2 increased the phagocytosis of beads and apoptotic cells that were dependent on ERK phosphorylation. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with bead phagocytosis, and p < 0.001 (TREM2 versus GFP) for BM-MC without ERK inhibitor and with phagocytosis of apoptotic neurons. Five independent experiments were performed, and each of them was done in quadruplicate.(B) Anti-inflammatory activity of TREM2 stimulation. Myeloid cells were transduced with TREM2 (open bars) or control GFP vector (filled bars). Myeloid cells were cultured on plates coated with TREM2 cross-linking antibodies (left graph). One hour later, cells were stimulated with LPS. Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. TREM2-transduced myeloid cells showed reduced gene transcript levels of IL-1β and NOS2 and an increased level of IL-10. In addition, apoptotic cells derived from neuron-enriched cultures were added to myeloid cells (right graph). Gene transcript levels for TNFα, IL-1β, NOS2, and IL-10 were quantified by real-time PCR after 48 h. Data are presented as mean ± SEM. Unpaired t-test: p < 0.001 (TREM2 versus GFP) with TREM2 plus LPS for IL-1, p = 0.0205 (TREM2 versus GFP) with TREM2 plus LPS for IL-10, p = 0.0149 (TREM2 versus GFP) with coculture for IL-1, and p = 0.012 (TREM2 versus GFP) with coculture for NOS2. Four independent experiments were performed.(C) Phosphorylation of ERK after stimulation of TREM2. Myeloid cells were transduced with TREM2 and cultured for 1 h on plates coated with TREM2 cross-linking antibodies (+) or isotype control antibodies (−). Cross-linking of TREM2 increased the amount of phosphorylated ERK (phospho ERK) in relation to total ERK (ERK).(D) TREM2-transduced myeloid cells showed an increased release of IL-10 after stimulation of TREM2 plus LPS or coculture with apoptotic neuron–enriched cultures. Myeloid cells were transduced with TREM2 (open bars) or with control GFP vector (filled bars), cultured on plates coated with TREM2 cross-linking antibodies, and stimulated with LPS 1 h later or supplemented with apoptotic cells derived from neuron-enriched cultures. The amount of IL-10 released in the supernatant was determined by ELISA. Data are presented as mean ± SEM. Unpaired t-test: p = 0.044 (TREM2 versus GFP) for TREM2 plus LPS, and p = 0.0054 (TREM2 versus GFP) for coculture. Four independent experiments were performed.
Mentions: Stimulation of TREM2 has been shown to induce phagocytosis in primary microglia [14]. In order to determine the effects of TREM2 expression on phagocytic activity of BM-MC, we analyzed phagocytosis of microsphere beads and apoptotic cells by TREM2-transduced BM-MC. Phagocytosis of microsphere beads was increased in TREM2-stimulated TREM2-transduced BM-MC compared to control BM-MC and cultured monocytes (Figure 3A). Likewise, phagocytosis of apoptotic membrane fragments derived from mixed neuron-enriched cultures was increased after gene transduction of TREM2. In detail, 93.3% (SEM 6.6) of BM-MC phagocytosed apoptotic neurons after transduction with TREM2, while only 50.5% (SEM 5.6) of BM-MC phagocytosed apoptotic neurons after transduction with the control GFP vector (Figure 3A). Increased phagocytosis of beads and apoptotic cells after TREM2 stimulation was completely neutralized by the ERK inhibitor PD98059, suggesting that ERK signaling was involved in TREM2-mediated phagocytosis by BM-MC (Figure 3A). TREM2 cross-linking antibodies stimulated phosphorylation of ERK in TREM2-transduced BM-MC (Figure 3), but did not modify cell surface expression of MHC class II, CD80, CD86, CD36, CCR7, and CD11c (Figure S1).

Bottom Line: EAE was induced in mice by immunization with a myelin autoantigen.Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination.TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University of Bonn Life & Brain Center and Hertie-Foundation, Bonn, Germany.

ABSTRACT

Background: In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system (CNS), are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor. Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.

Methods and findings: EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination. TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

Conclusions: Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE. TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Show MeSH
Related in: MedlinePlus