A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia

Abstract

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor-ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.

Figure 1. Neuronal cells express ligands for TREM2

Neuro2A cells (top), primary cortical neurons (middle), and primary ventral midbrain neurons (bottom) were cultured and stained for TREM2-L expression by cytochemistry, using a soluble TREM2-Fc fusion protein. All neuronal cells examined bound soluble TREM2 (brown), but not TREM1 (right column). Data are representative of at least 3 experiments for each cell type.

Figure 2. Apoptosis increases the expression of functional TREM2-L on neuronal and non-neuronal cells

(A) TREM2-L expression was quantified by flow cytometry before (left) and after (right) induction of apoptosis in Neuro2A cells by staurosporine (sts) or MPP⁺. The median fluorescence intensity (MFI) of TREM2-Fc or TREM1-Fc binding of each gate is shown. Apoptotic Neuro2A cells (Annexin V^hi) express 3-10 fold greater levels of TREM2-L compared to Annexin V^lo Neuro2A cells, but do not express TREM1-L (n=7). (B) TREM2-L expression is increased on non-neuronal cells during apoptosis. BWZ, WEHI-231, and P388D1 cells bind to TREM2-Fc, but not TREM1-Fc. Treatment with staurosporine boosts TREM2-Fc binding to Annexin V^hi cells by 7-10 fold (n=3). (C) Neuronal cells activate TREM2/DAP12 signal transduction as assessed by BWZ.TREM2/DAP12 reporter cells. Healthy Neuro2A cells induce a modest level of cellular activation in the BWZ.TREM2/DAP12 cell line, and this is increased to near-maximal levels when the Neuro2A are treated with MPP⁺ to induce apoptosis (top panel). Pretreating the BV2 cells with a blocking TREM2 mAb (black bars), but not with a control rat IgG1 mAb (gray bars), significantly reduces cellular activation in response to both untreated and apoptotic Neuro2A. Ventral midbrain neurons (VMN) elicit similar responses (third panel), while cortical neurons (CN) demonstrate little stimulation unless they are apoptotic (second panel). BWZ.TREM2/DAP12 reporter cell activation in response to primary neurons was effectively blocked with the TREM2 mAb. None of the neuronal cells activate the parental BWZ reporter cell line (representative results for VMN are shown in the bottom panel). *p-value <0.05, **p-value < 0.005, ***p-value < 0.0005.

Figure 3. TREM2/TREM2-L interactions are important for phagocytosis, and TREM2 is required for efficient phagocytosis of apoptotic neuronal cells but not of beads

(A) Phagocytosis of apoptotic Neuro2A cells (>40% Annexin V^hi) by BV2 microglial cells cocultured at an E:T of 1:10 as assessed by flow cytometry (left column) or fluorescence confocal microscopy (right panels). Histograms indicate the percent of BV2 cells that have internalized CM-DiI-labeled staurosporine-treated Neuro2A cells during 1h assays. Images of single optical sections (<1 μm) were obtained by confocal microscopy with a 60x magnification lens of BV2 cells labeled with an APC-conjugated anti-CD11b mAb (blue) and cocultured with CM-DiI⁺ apoptotic Neuro2A cells (red). Images indicate uptake of neuronal debris by BV2 cells. Phagocytosis is inhibited by cytochalasin D in both assays. Scale bars represent 10 μm. (B) Phagocytosis of apoptotic Neuro2A cells is reduced in BV2 cells following lentiviral-mediated RNAi against TREM2. RNAi reduced the surface expression of TREM2 up to 84% as detected by flow cytometry (left). Quantification of 6 experiments shows that phagocytosis by BV2 cells deficient in TREM2 is reduced to 15±5% (mean ± SEM) from 36±5% for untreated BV2 cells and from 34±5% for BV2 cells transduced with empty virus (*p-value < 0.05) (right). (C) A mAb to TREM2 partially but significantly inhibits phagocytosis of apoptotic Neuro2A cells by BV2 microglia. Representative flow cytometric histograms assessing phagocytosis in the presence of a blocking TREM2 mAb (Clone 78.18) or an isotype control mAb (rat IgG1) is shown (left). Summary of 6 experiments, showing a reduction to 23.7 ± 0.9% of effector cells engulfing targets compared to untreated (34.8 ± 2.9%) and control mAb (36.7 ± 2.1%) treated BV2 cells (right). The TREM2 mAb partially decreases phagocytosis by 32-35%. (**p-value ≤ 0.005, ***p-value ≤ 0.0005). (D) Reduction of microglial TREM2 by RNAi does not reduce BV2 phagocytosis of microspheres (here at 1:50 E:T ratio, top row), although the same cells again show a loss of phagocytosis of apoptotic Neuro2A cells at 1:10 E:T (sts N2A, bottom row) during 1h assays. BV2 cells were left untreated or subjected to cytochalasin D or infected with TREM2 shRNA or empty virus. Representative flow cytometric histograms are shown (n=2).

Figure 4. TREM2 is sufficient to confer phagocytosis of apoptotic Neuro2A

CHO cells stably transfected to express TREM2/DAP12 or TREM1/DAP12 chimeras were assessed for their phagocytosis of apoptotic Neuro2A cells. Flow cytometry confirmed that the transfected CHO cells express either TREM2 (filled histogram, left) or TREM1 (filled histogram, middle) on cells gated for GFP, while isotype controls (open histograms) did not bind to the CHO cells. The expression of TREM2/DAP12 (black bar) was sufficient to increase phagocytosis of apoptotic Neuro2A cells by 1.7 fold over untransfected cells CHO cells (white bar), while expression of TREM1/DAP12 did not (gray bar) (n=8, * p-value < 0.05).

Figure 5. TREM2 is not expressed by normal adult neurons but is expressed by adult and neonatal microglia

(A) Semi-quantitative RT-PCR analysis for TREM2 using cDNA from Neuro2A neuroblastoma cells, BV2 microglial cells, and sorted adult microglia (CD45^lo CD11b⁺). TREM2 transcript levels are normalized to GAPDH RNA levels. Transcripts for TREM2 are not found in Neuro2A cells, but are strongly expressed in BV2 cells and microglia. (B) Immunofluorescence confocal microscopy of histologic sections from brains of normal adult C57BL/6 mice to determine TREM2 expression in the brain. Single optical sections of <1 μm were imaged at 60x magnification from the cortex, hippocampus (CA1 and dentate gyrus regions), putamen, and spinal cord tissues. A TREM2 mAb cocktail (red) colocalized with CD11b (green) on microglia, but not with neuronal soma as detected by an antibody to NeuN (blue) in neurons. Scale bars indicate 10 μm. (C) Flow cytometric analysis of TREM2 on cultured neonatal microglia. Mixed glial cells were cultured from CX3CR1^GFP/+ mice. TREM2 is expressed on microglia (GFP⁺CD11b⁺), but not on non-microglial (GFP^-CD11b^-) cells, which include astrocytes. (D) Immunofluorescence confocal microscopy images of neonatal microglia isolated from CX3CR1^GFP/+ mice and sorted for GFP. TREM2 is detected on nearly all microglia, although the expression level varies from one small patch of TREM2 on the cell membrane to expression around the entire cell membrane. Single optical sections (<1 μm) at 60x magnification were imaged. Scale bars indicate 10 μm.