In microglia, it has been previously demonstrated that TREM2 promotes phagocytosis of apoptotic neurons without upregulation of antigen presentation molecules or TNFα transcripts, but a role for target recognition by TREM2 had not been elucidated (Takahashi et al. 2005
). Thus, TREM2 might have bound to a stimulatory molecule on the microglia themselves, or may simply have facilitated the recruitment of DAP12 to a signaling complex during phagocytosis. In contrast, our current studies provide compelling evidence that microglial phagocytosis of apoptotic neurons involves direct recognition by TREM2 on microglia with ligands that are upregulated on apoptotic neurons. When neuronal cells undergo apoptosis, they increase the expression of TREM2-L with a corresponding increase in their phagocytosis by BV2 cells, which is blocked at least in part by our antibody to TREM2. The upregulation of TREM2-L on apoptotic neurons appears to reflect a phenomenon that is generalizable to multiple cell types; conditions that induce apoptosis as assessed by staining with Annexin V, increase binding by soluble TREM2 5-10 fold. TREM2 may thus be generally important in clearing apoptotic cells.
The engulfment of apoptotic cells is essential in the CNS to clear cell debris without eliciting an inflammatory response (Ravichandran & Lorenz 2007
, Napoli & Neumann 2009
). The upregulation of TREM2-L on apoptotic neuronal cells provides a means by which microglia can be directed to the phagocytic removal of these cells, and when this interaction is blocked with an anti-TREM2 mAb, phagocytosis is diminished. TREM2-L were also found at lower levels on non-apoptotic cultured neuronal cells, but we could not detect TREM2-L in vivo
on neurons in tissue sections from healthy adult mice (not shown) suggesting that the mere stress of being in culture may be enough to upregulate low levels of TREM2-L on neurons. Regardless, apoptotic cells expressed much more TREM2-L and they more effectively activated signaling through TREM2/DAP12.
Although TREM2 is important for phagocytosis of apoptotic neurons, TREM2 is not likely to be the only engulfment receptor on microglia that can recognize injured neurons. Other phagocyte receptors that may be involved include CD36, receptors for phosphatidylserine, and/or the vitronectin receptor (αv
integrin) (Savill et al. 2002
). Interestingly, a recent report indicates that another member of the TREM receptor family, TREM-like 4 (Treml4), also recognizes apoptotic cells (Hemmi et al. 2009
In our studies, transfection of TREM2 into CHO cells conferred phagocytic capacity for apoptotic cells while TREM1 did not, strongly supporting the evidence that recognition of apoptotic cells by TREM2 directly activates phagocytosis. Moreover, in BV2 microglia, while loss of TREM2 led to reduced phagocytosis of apoptotic cells, it did not lead to reduced phagocytosis of microbeads, indicating that TREM2 does not nonspecifically promote phagocytosis.
The ligands on apoptotic cells that are recognized by TREM2 are unknown. We have previously shown that TREM2 also binds broadly to bacteria, and that this binding is inhibited by anionic glycans (Daws et al. 2003
). We hypothesize that TREM2 binds to glycans on both bacteria and apoptotic cells. In this regard, it would be similar to other pattern recognition receptors, such as the mannose receptor, the Siglec receptors, and TLR4, all of which recognize ligands on pathogens as well as endogenous ligands (Allavena et al. 2004
, Crocker et al. 2007
, Akira & Hemmi 2003
Our studies also further clarify the expression of TREM2 on mouse microglia. TREM2 is selectively expressed by microglia in vivo
in the normal adult mouse brain in multiple regions of the CNS, including the cortex, hippocampus, spinal cord, and putamen, but TREM2 is not expressed by neurons in the adult mouse. Our data are consistent with findings from others that TREM2 is expressed on microglia, but we did not find evidence for its presence in neurons. It has previously been suggested that TREM2 marks a subset of microglia with variation in brain regions as determined at the RNA level by in situ
hybridization of TREM2, which costained with tomato lectin, a protein that binds both microglia and blood vessels (Schmid et al. 2002
). The highest previously reported percent of murine TREM2+
microglia in vivo
was 57±5.8% in the cortex (Schmid et al. 2002
). Our studies using a cocktail of specific TREM2 mAbs, which do not bind myeloid cells derived from TREM2 knockout mice, suggest that TREM2 is expressed in the majority (~90%) of CD11b+
microglia in vivo
at the protein level, though the expression levels may vary. In vitro
, nearly all cultured microglia from neonatal mice expressed low levels of TREM2 on the surface.
The expression of TREM2 on microglia is in contrast to circulating monocytes on which TREM2 is not detected. It is consistent, however, with evidence that TREM2 is expressed by tissue macrophages as well as by other cells of the monocyte/macrophage lineage, including immature dendritic cells and osteoclasts (Bouchon et al. 2001
, Turnbull et al. 2006
, Colonna 2003
Macrophages have been classified into at least 2 main subtypes. Classical (M1) macrophages develop in response to cytokines that promote Th1 immune responses while the development of alternatively activated (M2) macrophages is promoted by the Th2 cytokines IL-4 and IL-13 (Gordon 2003
). M1 macrophages are proinflammatory while M2 macrophages inhibit inflammation and instead promote tissue repair in part through the phagocytosis of apoptotic cells. It is tempting to suggest that TREM2+
microglia in their resting state are similar to alternatively activated macrophages and are already poised to respond to TREM2-L. The failure of this recognition may impair the clearance of neuronal debris by microglia, resulting in the degenerative brain disease, Nasu-Hakola disease.
The recognition of TREM2-L on neuronal cells may also regulate microglial functions in addition to phagocytosis. In this regard, blockade of TREM2 has been shown to exacerbate EAE, while infusion of TREM2+
cells improves it. Inflammation contributes not only to multiple sclerosis, but also to Alzheimer’s disease and Parkinson’s disease. With regard to Alzheimer’s disease, TREM2 was found to be specifically upregulated in amyloid plaque-associated microglia in APP23 transgenic mice (Frank et al. 2008
). Further studies are warranted to examine the function of microglia, TREM2, and TREM2-L in Alzheimer’s disease
TREM2 and TREM-2L join other receptor/ligand pairs that mediate crosstalk between microglia and neurons. Neurons express CD200, which tonically inhibits microglia through interaction with its receptor, CD200R. Mice deficient in CD200 have augmented microglial responses following transection of the facial nerve, and they have accelerated onset of EAE (Hoek et al. 2000
). Similarly, microglia are inhibited by the fractalkine receptor, CX3CR1, through the expression and release of fractalkine by neurons, and mice lacking CX3CR1 have increased neuronal loss in Parkinson’s disease and amyotrophic lateral sclerosis (Cardona et al. 2006
). While neurons are capable of inhibiting microglia through CD200R and CX3CR1, apoptotic neurons may engage TREM2 to influence microglial differentiation towards an “alternative” phenotype that facilitates phagocytosis of neurons. Interestingly, DAP12, the adapter protein associated with TREM2, has been implicated in microglia-mediated neuronal cell death in the developing hippocampus exclusively in postnatal day 1-2 mice (Wakselman et al. 2008
). TREM2 and DAP12 may thus provide important functions at different developmental stages as well as during disease states.
In sum, our data suggest that TREM2 is a phagocyte receptor that is stimulated by an unknown “eat-me” signal on apoptotic neurons. This unknown signal may be commonly expressed on all apoptotic cells. The nature of the ligands for TREM2, however, has not yet been identified. Characterization of TREM2-L will greatly facilitate studies about TREM2 and its role in the CNS. In the meantime, understanding the mechanisms by which TREM2-L regulate microglial activity may prove important to ameliorate neurodegenerative diseases and brain injury.