TREM2 recognizes mycobacteria
To screen novel ITAM-coupled receptors capable of recognizing mycobacteria, we analyzed the binding of 20 known ITAM-coupled CLR, TREM, and leukocyte mono-Ig- like receptor (LMIR; CD300) family receptors22,37–39 fused to Fc-antibody fragments to heat-killed mycobacterial strains, including the virulent strain Mtb H37Rv, the attenuated strain Mtb H37Ra, and the vaccine strain M. bovis BCG, using flowcytometry. In addition to several CLRs previously reported to recognize mycobacteria, including Mincle, specific ICAM-3 grabbing nonintegrin-related (SIGNR)1, SINGNR3, and DC-SIGN30,40, this screening identified TREM2 and LMIR5 as novel receptors capable of binding to mycobacteria (Fig. 1a). Especially, TREM2 demonstrated a strong binding capacity to all tested mycobacterial strains, which prompted us to further characterize this interaction.
To investigate whether recognition of mycobacteria by TREM2 activates intracellular ITAM signaling, we used nuclear factor of activated T cells (NFAT)-driven green fluorescent protein (GFP)-reporter cells (2B4)30 ectopically expressing TREM2 and its signaling subunit DAP1241,42. As reported previously30, Mtb H37Rv, Mtb H37Ra, and M. bovis BCG stimulated NFAT-GFP signaling in reporter cells expressing Mincle and FcRγ (Supplementary Fig. 1a). We found that all these mycobacterial strains clearly activated reporter cells expressing TREM2 and DAP12, but not in those expressing only DAP12, in a dose dependent manner (Fig. 1b). Whereas, none of these strains activate reporter cells expressing TREM1 plus DAP12 (Supplementary Fig. 1b). Importantly, the level of stimulation by TREM2 and Mincle differed between the strains (Fig. 1b and supplementary Fig. 1a), suggesting that TREM2 and Mincle recognize different ligands commonly expressed by these mycobacterial strains.
TREM2 recognizes MAs
TREM2 binding to mycobacteria implies that its ligand(s) exist on the cell-wall surface, where mycobacteria express a wealth of unique lipids that influence the host immune responses 43. Given that TREM2 binds to various endogenous mammalian lipids44,45, we first examined whether mycobacterial lipids contain TREM2 ligand(s). De-lipidation of Mtb H37Ra with chloroform and methanol (C:M) (Fig. 2a) markedly diminished the level of stimulation in TREM2-reporter cells (Fig. 2b), as well as in Mincle-reporter cells as reported30 (Fig. 2c). Accordingly, the lipid-containing C:M fraction (Fig. 2a) but not the hydrophilic M:W fraction showed strong stimulating activity in TREM2 reporter cells (Fig. 2b) as with Mincle reporter cells (Fig. 2c), implicating that TREM2 ligands were present in the cell wall lipid fraction as with so Mincle ligands 30. We then next tested the TREM2-stimulating activity using known major constituents of mycobacterial cell wall, including the glycans PGN and AbG, the immunostimulatory glycolipids LAM and TDM (Fig. 2d), and fMA (Fig. 2d). We observed that AbG, PGN, and LAM did not activate the TREM2-reporter cells, even at higher concentrations (Fig. 2e-g), whereas we observed substantial ligand activity with TDM and fMA (Fig. 2h). Importantly, loss of trehalose moiety from TDM (i.e. fMA) markedly increased the TREM2-stimulating activity (Fig. 2h), while it completely abolished the Mincle-stimulating activity as reported (Fig. 2i)30. These results suggest that TREM2 recognizes the MA moiety of TDM, and that the sugar moiety interferes with this recognition.
We then investigated the structure of MA necessary for TREM2 recognition. Extremely long (C60-C90) and branched alkyl chains are hallmarks of mycobacterial MAs. To assess the importance of the length and branching, we compared the stimulatory activity of mycobacterial MA with that of Rhodococcus equi (R. equi) MA, which has shorter alkyl chains (C35), and of behenic acid (BA), which is a linear fatty acid with an alkyl chain (C22) of similar length with the mero-chain of R. equi MA (Fig. 2j). R. equi MA exhibited a similar level of stimulatory activity in TREM2-reporter cells as mycobacterial MA, whereas the level of stimulation with BA was very low, even at higher concentrations of BA (Fig. 2j), suggesting that the branched structure rather than the alkyl-chain length was important for TREM2 recognition of MA. To further investigate this, we tested the ligand activity of palmitic acid (PA; C16), 2-tetradecylhexadecanoic acid (THA) (C14/C16), which bears a PA-based synthetic fatty-acid with similar branching structure as MA but lacking its 3-hydroxyl residue, and 3-hydroxybutyric acid (HBA), which has only the carboxyl and hydroxyl residue at the branching moiety of MA (Fig. 2k). We detected TREM2 stimulation with THA, albeit at a lower level than MA, but could not detect stimulation with PA and HBA (Fig. 2k), suggesting that TREM2 recognition requires a branched fatty acid structure with an alkyl chain.
The R47H mutation in TREM2, which is associated with a higher risk of several neurodegenerative diseases46–49, impairs the recognition of brain lipids45,50. We found that the TREM2 R47H mutation also attenuated MA recognition (Supplementary Fig. 2), suggesting that this residue is important for MA recognition by TREM2.
TREM2 and Mincle preferentially recognize distinct MA-containing lipids based on their glycosylation
Mycobacteria express a variety of MA-containing lipids on their cell wall51. Since both Mincle and TREM2 have the capacity to recognize MA-containing lipids, we next compared their binding activity to the glycosylated (TDM and GMM) or the non-glycosylated (GroMM and fMA) MA-containing lipids (Fig. 3a) using the receptor-Fc fusion proteins. We observed that Mincle-Fc not only showed strong binding to TDM as expected, but also binding to GMM, whereas it showed no detectable binding to fMA and weak binding to GroMM only at higher concentrations (Fig. 3b). By contrast, TREM2- Fc showed strong binding to GroMM and fMA but showed relatively much weaker binding to TDM and GMM (Fig. 3b). We then examined whether the different binding of MA-containing lipids to TREM2 and Mincle reflect their receptor-stimulatory activities using NFAT-GFP-reporter cells. Consistent with the binding data, TREM2-reporter cells strongly responded to MA and GroMM but weakly to TDM and GMM, whereas Mincle- reporter cells responded strongly to TDM and GMM but weakly to GroMM only at higher concentrations and did not respond to fMA (Fig. 3c). Therefore, TREM2 and Mincle preferentially recognize non-glycosylated and glycosylated MA-containing lipids, respectively.
Glycosylated and non-glycosylated MAs elicit distinct macrophage activation through Mincle and TREM2
To investigate the relevance of TREM2 and Mincle recognition of MA-containing lipids in the activation of innate immune cells, we stimulated peritoneal macrophages from wild-type (WT), TREM2-deficient (Trem2–/–), or Mincle-deficient (Clec4e–/–) mice with either glycosylated or non-glycosylated MA-containing lipids and examined their production of MCP-1, a pivotal monocyte chemoattractant implicated in TB pathology52– 54, and TNF, an essential cytokine for granuloma formation and TB control55,56. Interestingly, we found that activation of WT macrophages by the glycosylated MA- containing lipids TDM or GMM or by lipopolysaccharide (LPS) induced production of both MCP-1 and TNF, whereas activation of macrophages with the non-glycosylated MA-containing lipids GroMM or fMA induced production of MCP-1 but not TNF (Fig. 4a), as well as other pro-inflammatory cytokines, including IL-6 and IL-12p40 (Supplementary Fig. 3a). Loss of Mincle almost completely abolished TNF, as previously30, as well as MCP-1 in response to TDM as expectedly30. We found that Mincle deficiency also abolished these productions induced by GMM (Fig. 4a). However, Mincle deficiency did not affect MCP-1 production in response to GroMM or fMA. By contrast, loss of TREM2 almost completely abolished the MCP-1 production in response to GroMM or MA, but not to TDM or GMM (Fig. 4a). These results demonstrated that glycosylated and non-glycosylated MA-containing lipids elicited distinct macrophage activation, which was dependent on Mincle and TREM2, respectively.
Triggering of ITAM-coupled receptors on myeloid cells activates the ITAM-Syk-CARD9 signaling pathway to induce cytokine production57. To investigate whether MCP-1 and TNF production induced by MA-containing lipids depends on this pathway, we first confirmed the requirement for DAP12 and FcRγ, which are the ITAM-containing signaling subunits of TREM2 and Mincle, respectively. As expected, we observed that FcRγ-deficient (Fcer1g–/–) and DAP12-deficinet (Tyrobp–/–) macrophages almost phenocopied Mincle-deficient and TREM2-deficient macrophages, respectively, with similar patterns of defects in TNF and MCP-1 productions in response to glycosylated or non-glycosylated MA-containing lipids (Fig. 4b). However, FcRγ deficiency slightly dampened MCP-1 induction by fMA or GroMM at higher concentrations, implicating that FcRγ might partly contribute to TREM2 signaling. We then examined the requirement for Syk and CARD9 for each of these responses. Treatment of WT macrophages with a Syk inhibitor (BAY-613606) abrogated both MCP-1 and TNF production in response to all MA-containing lipids (Fig. 4c), indicating essential role for Syk in the response to these lipids. Intriguingly, although TNF production induced by glycosylated MAs through Mincle was abolished in CARD9-deficient (Card9–/–) macrophages, as expected58, CARD9-deficiency did not affect MCP-1 production induced by both glycosylated and non-glycosylated MA-containing lipids (Fig. 4c). Collectively, these results demonstrated that mycobacterial MA-containing lipids induced TNF production through Mincle via the canonical FcRγ-Syk-CARD9 pathway, while they induced MCP-1 production via the ITAM-Syk pathway but independent of CARD9 signaling (Supplementary Fig. 3b).
TREM2/DAP12 signal inhibits macrophage activation through Mincle/FcRγ
Interestingly, we observed that loss of TREM2 markedly enhanced MCP-1 and TNF production by peritoneal macrophages in response to TDM or GMM (Fig. 4a). This was also true in Tyrobp–/– macrophages (Fig. 4b), suggesting an inhibitory role of TREM2/DAP12 signaling in Mincle/FcRγ-induced macrophage activation. We did not observe an increase in cytokine response when the same preparation of Trem2–/– macrophages were stimulated with TLR ligands, such as Pam3CSK4 (for TLR2), LPS (for TLR4), Poly (I:C) (for TLR3), CpG-ODN (for TLR9), or with the Dectin-1 ligand zymozan (Fig. 4a and supplementary Fig. 4a, b). This selective enhancement of Mincle activation in the absence of TREM2 was more prominent in BMDMs, where TDM- induced TNF was detected at very low levels in WT BMDMs. Nevertheless, Trem2–/– or Tyrobp–/– BMDMs showed substantial TNF production in response to TDM (Supplementary Fig. 4c), whereas this enhancement was not observed following Pam3CSK4 or LPS stimulation. Additionally, we observed an increased cytokine response following stimulation of Trem2–/– BMDMs with mycobacterial total lipids (C:M fraction in Fig. 2a) prepared from Mtb R37Ra or M. bovis BCG (Supplementary Fig. 4d). This response was largely dependent on Mincle, as Clec4e–/– BMDMs produced markedly lower TNF in response to the total lipids compared to WT BMDMs (Supplementary Fig. 4d). These data clearly indicated that TREM2/DAP12 signaling selectively inhibited macrophage activation induced by Mincle-FcRγ signaling.
Triggering of TREM2 induces permissive macrophages
Given that NO plays a pivotal role in controlling mycobacterial infections59,60, we investigated the action of TREM2 and Mincle on NO production by macrophages. We observed that TDM stimulation strongly induced NO production by bone marrow-derived macrophages (BMDMs), as reported previously30, while fMA did not substantially induce NO production (Fig. 5a). This was consistent with the observed increase in Nos2 expression, which encodes iNOS, after stimulation with TDM but not fMA (Supplementary Fig. 5a). Additionally, the TDM-induced NO production by macrophages was inhibited following the addition of a TNF blocking antibody (Fig. 5b). Inversely, addition of recombinant TNF to the fMA-stimulated culture induced NO production by macrophages (Fig. 5b), indicating that NO production was dependent on TNF, which is consistent with previous findings61–63.
We then characterize the inflammation triggered via Mincle and TREM2 in vivo. Because TDM induces lung granulomas in mice via the Mincle/FcRγ pathway30, we examined whether fMA had a similar effect. Intravenous injection of a TDM (oil-in- water) emulsion into mice induced massive granulomatous lesions in the lungs, which was completely abolished in Clec4e–/– mice (Fig. 5c), as reported previously30. Additionally, this effect was also absent in Card9–/– mice, indicating that TDM-induced granuloma formation was dependent on Mincle/CARD9 signaling. By contrast, we did not observe lung granulomatous lesions in mice injected with an fMA emulsion (Fig. 5c), suggesting that fMA cannot activate CARD9 signaling. To further explore the innate immune response induced by TDM or MA in vivo, we intraperitoneally injected the TDM or fMA emulsion into mice and measured the level of MCP-1 and TNF, and Nos2 mRNA levels, as well as the number of recruited inflammatory cells, induced in the peritoneal cavities. We detected MCP-1 in peritoneal lavages following respective fMA and TDM administrations at similar levels, with peak MCP-1 levels observed at 4- and 24-h post- injection, respectively (Fig. 5d). However, TNF production and Nos2 mRNA expression were induced only by TDM but not fMA (Fig. 5d), which was consistent with results from the in vitro-stimulated macrophages (Fig. 4). Moreover, we observed that TDM recruited substantial numbers of macrophages [CD11b+Ly6G–F4/80low small peritoneal macrophages (SPMs)], Supplementary Fig. 5b) and neutrophils (CD11b+Ly6G+ F4/80–, Supplementary Fig. 4b) to the cavity, whereas fMA recruited comparable numbers of macrophages as TDM but fewer neutrophils only immediately after administration (Fig. 5e). Importantly, MCP-1 level (Fig. 5f) and the number of recruited cells (Fig. 5g) induced by fMA in Trem2–/– mice were almost the same as those induced by the control vehicle administration, indicating that the fMA-induced inflammation was dependent on TREM2.
To investigate the phenotype of TDM- or fMA-induced macrophages, we examined the expression of the inflammatory M1-macrophage markers iNOS and CD38 64 in the recruited macrophages (Fig. 5h). Intraperitoneal administration of TDM and fMA recruited comparable numbers of macrophages to the cavity (Fig. 5i); however, although TDM-induced macrophages exhibited a CD38highiNOS+ phenotype (Fig. 5j), those induced by fMA had reduced CD38 expression and were negative for iNOS (Fig. 5k). This phenotype resembles a previously reported phenotype associated with permissive macrophages that are recruited by a subset of pathogenic mycobacteria expressing the virulence lipids PDIM and PGL in an MCP-1 and CCR2-dependent manner and provide a niche for mycobacterial propagation21,65.
Collectively, these results suggested that triggering of the Mincle-CARD9 pathway elicited lung granuloma formation and recruited M1-type mycobactericidal macrophages producing TNF and NO, whereas TREM2 activation recruited mycobacterium-permissive macrophages lacking TNF and NO production.
TREM2 deficiency exacerbates Mincle-induced inflammation
To investigate the relevance of TREM2 inhibition of Mincle-induced macrophage activation in vivo, we assessed the impact of TREM2 deficiency on tissue inflammation induced by TDM administration. Intraperitoneal injection of TDM emulsion into Trem2–/– mice resulted in significantly higher levels of TNF and MCP-1 production and increased Nos2 mRNA expression (Fig. 6a), as well as higher numbers of macrophages and neutrophils to the peritoneal cavities (Fig. 6b), as compared to those observed in WT mice. Intravenous injection of the TDM emulsion into mice induces lung swelling (increased lung weight index: LWI) and thymic atrophy (decreased thymic weight index: TWI) dependent on the Mincle-FcRγ pathway30. We observed that Trem2–/– mice displayed significantly higher LWI and lower TWI than WT mice followingr TDM injection (Fig. 6c). Histopathological analysis of the lungs revealed that the granulomatous lesions were markedly more prominent in Trem2–/– mice (Fig. 6d). In addition, specific pathological features in the form of prominent vasculitis and edema, indicating accelerated inflammation, were observed in the lungs of Trem2–/–, but not WT mice (Supplementary Fig. 6). Consistent with these results, the levels of TNF and MCP-1 production and Nos2 mRNA expression in the inflamed lungs were significantly higher in Trem2–/– mice than in WT mice (Fig. 6e). These results suggested that TREM2 suppressed Mincle-induced inflammation in vivo.
TREM2 deficiency accelerates the clearance of mycobacterial infection
Since our data suggested a suppressive role for TREM2 in the microbicidal innate immune response via Mincle, we investigated the impact of TREM2 deficiency on the clearance of mycobacterial infection. First, we infected WT and Trem2–/– BMDMs with M. bovis BCG in vitro and assessed NO production and mycobacterial killing in the macrophages. NO production in Trem2–/– BMDMs was significantly higher than in WT BMDMs (Fig. 7a). Accordingly, the number of intracellular BCG colony-forming units (CFUs) was significantly lower in Trem2–/– BMDMs than in WT BMDMs (Fig. 7b). Next, to investigate the relevance of this observation in vivo, we intraperitoneally infected WT and Trem2–/– mice with M. bovis BCG and measured bacterial clearance. The bacterial burden (CFUs) in the peritoneal cavities at day 1 and 3 after infection were significantly lower in Trem2–/– mice than in WT mice (Fig. 7c). Consistently, early MCP-1 production (Fig. 7d) and the number of infiltrated macrophages (Fig. 7e) after infection were significantly higher in Trem2–/– mice than in WT mice. Moreover, Nos2 expression in infiltrated cells tended to be higher (Fig. 7f, left), whereas expression of the M2 marker arginase 1 (Arg1) was significantly lower in Trem2–/– mice (Fig. 7f, right) relative to that in WT mice, suggesting that lack of TREM2 signaling favored M1 polarization. These results suggested that TREM2 contributed to immune evasion by mycobacteria by inhibiting the activation of microbicidal M1 macrophages.