TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation

Abstract

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

Fig. 1. TREM2 recognizes mantihumanycobacteria.

a Heat-killed Mtb H37Ra or H37Rv, or M. bovis BCG were incubated with the indicated CLR, TREM, and LMIR family receptors fused to the Fc-antibody fragment or with the control Fc fragment, followed by staining with antihuman IgG-FITC secondary antibody. The binding of each receptor-Fc protein was analyzed by flow cytometry. Open histograms show binding data for the indicated receptor-Fc proteins. The gray-filled histograms show background fluorescence of the control staining. b NFAT-GFP reporter cells expressing TREM2 + DAP12 or DAP12 alone were stimulated with the indicated amounts of heat-killed Mtb H37Ra or H37Rv, or M. bovis BCG for 24 h, followed by analysis of GFP fluorescence by flow cytometry. The histograms on the left indicate the data from stimulation with 100 μg/ml of the indicated mycobacteria. Data in the right panels are presented as percent increase over control values of unstimulated cells. Data are presented as the mean ± SEM of duplicate assays and representative of three independent experiments. Source data are provided as a Source data file.

Fig. 2. TREM2 recognizes MAs.

a Schematic diagram of solvent-based de-lipidation and fractionation of Mtb H37Ra. Bacteria were de-lipidated by the treatment with chloroform/methanol. After centrifugation (cfg.), soluble extracts (sup) were mixed with water and separated into the lipid-soluble (C:M) and the water-soluble methanol:water (M:W) fractions. b, c NFAT-GFP reporter cells expressing TREM2 + DAP12 (b) or Mincle + FcRγ (c) were stimulated for 24 h with either untreated or de-lipidated bacteria (ppt) or plate-coated C:M or M:W fractions indicated in a, followed by analysis of GFP fluorescence by flow cytometry. d Chemical structure of TDM. The structure of α-mycolate-containing TDM is shown. e–g NFAT-GFP reporter cells expressing TREM2 + DAP12 or DAP12 alone were stimulated with the indicated amounts of ABG (e), PGN (f), and LAM (g) for 24 h, followed by analysis of GFP fluorescence by flow cytometry. Data are presented as percent increase over control values of unstimulated cells. h, i NFAT-GFP reporter cells expressing TREM2 + DAP12 or DAP12 only (h) or Mincle + FcRγ (i) were stimulated with the indicated amounts of TDM or fMA, and analyzed as described in e–g. j, k NFAT-GFP reporter cells expressing TREM2 + DAP12 were stimulated with the indicated amounts of BCG MA, R. equi MA, or BA (j) or THA, PA, or HBA (k) and analyzed as described in e–g. Data are presented as the mean ± SEM of duplicate (b, c, e–i, j) or triplicate (k) assays and representative of three independent experiments. Source data are provided as a Source data file.

Fig. 3. TREM2 and Mincle preferentially recognize distinct MA-containing lipids based on their glycosylation.

a Chemical structure of glycosylated (TDM and GMM) and non-glycosylated (GroMM and fMA) MA-containing lipids found in Mtb. The structures of α-mycolate-containing lipids are included. b The indicated amounts of TREM2 or Mincle-Fc fusion proteins were added to wells coated with 0.5 μg/well TDM, GMM, GroMM, or fMA, followed by incubation with anti-human IgG-horseradish peroxidase and detection of bound proteins using an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay. c NFAT-GFP reporter cells expressing TREM2 + DAP12 or Mincle + FcRγ were stimulated with the indicated amounts of TDM, GMM, GroMM, or fMA coated on the plates for 24 h, followed by analysis of GFP fluorescence by flow cytometry. Values are plotted as percent maximal response of the largest value. Data are presented as mean ± SEM of duplicate assays and are representative of at least three independent experiments. Source data are provided as a Source data file.

Fig. 4. Distinct lipid recognition and macrophage activation through Mincle and TREM2.

a–c Peritoneal macrophages from wild-type (WT), Trem2^−/−, or Clec4e^−/− mice (a), WT, Tyrobp^−/−, or Fcer1g^−/− mice (b), or WT or Card9^−/− mice (c) were stimulated with the indicated amounts of TDM, GMM, GroMM, or fMA coated on the plates or with LPS (100 ng/ml) for 24 h. WT cells were stimulated in the presence or absence of the Syk inhibitor BAY-613606 (BAY) (c). MCP-1 and TNF production in the culture supernatant was measured by ELISA. Data are presented as the mean ± SEM of triplicate assays and are representative of three independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.

Fig. 5. Triggering of TREM2 by MA induces the permissive macrophages.

a, b BMDMs were stimulated for 24 h with the indicated amount of TDM or fMA in the presence of 10 ng/ml IFN-γ (a), 1 μg of TDM with or without the indicated amounts of a TNF-blocking antibody (αTNF Ab) or an isotype-matched control antibody (Control Ab) (b), or 1 μg of fMA with or without recombinant TNF (rTNF) (b). NO production was determined by Griess assay. Data represent as the mean ± SEM of triplicate assays from three independent experiments. The statistical significance was calculated by one-way ANOVA followed by Bonferroni’s test. **p < 0.01, ***p < 0.001, n.s. not significant. c Representative images of the hematoxylin and eosin-stained lungs from WT, Clec4e^−/−, or Card9^−/− mice at day 7 after intravenous injection of 50 μg TDM or 250 μg fMA emulsion. The images are representative of two independent experiments. Scale bars: 0.1 mm. d–f Peritoneal lavages were collected at 4, 24, and 72 h after intraperitoneally injection of 500 μg fMA or 100 μg TDM emulsion to WT mice (n = 4). MCP-1 and TNF concentrations in the lavages were measured by ELISA, and Nos2 mRNA levels were measured by quantitative RT-PCR. e Flow cytometric analysis of peritoneal neutrophils (CD11b⁺Ly6G⁺F4/80⁻) and monocyte-derived macrophages (CD11b⁺Ly6G⁻F4/80^low SPMs) from WT mice (n = 4) at 48 and 72 h post injection of MA or TDM emulsions as in d. f, g WT or Trem2^−/− mice were injected intraperitoneally with fMA or control (vehicle) emulsion. The concentrations of MCP-1 (control, n = 4; fMA, n = 6; at 4 h) and TNF (n = 4; at 72 h) in the peritoneal lavages was measured by ELISA (f). Peritoneal exudate cells (n = 4; at 72 h) was analyzed as in e and g. h–k WT mice were injected intraperitoneally with TDM, fMA, or control (vehicle) emulsion, and infiltrated macrophages at days 1, 2, and 3 were analyzed by flow cytometry for the expression of CD38 and iNOS (h). The numbers of total recruited monocyte-derived macrophages (CD11b⁺Ly6C⁺F4/80⁺) (i), the M1 macrophages (CD11b⁺Ly6C⁺F4/80⁺CD38^highNOS⁺) (j), and permissive macrophages (CD11b⁺Ly6C⁺F4/80⁺CD38^dullNOS⁻) (k) at day 3 are shown. Data in d–k represent as the mean ± SEM from at least three independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test (f–g) or by two-tailed unpaired t test (i–k). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant. Source data are provided as a Source data file.

Fig. 6. TREM2 deficiency exacerbates Mincle-induced inflammation.

a, b WT or Trem2^−/− mice (n = 4) were intraperitoneally injected with 100 μg TDM or control (vehicle) emulsion. TNF and MCP-1 in the peritoneal lavages were measured by ELISA and Nos2 mRNA levels in the peritoneal cells were measured by qRT-PCR at 24 h post injection (a). The numbers of macrophages and neutrophils in the peritoneal cavities at 72 h post injection were analyzed by flow cytometry (b). c–e WT or Trem2^−/− mice (n = 5) were intravenously injected with 50 μg TDM or control emulsion, and the lungs and thymuses were collected at day 7 after the injection. LWI and TWI are shown (c). Representative hematoxylin and eosin-stained sections of lung lobes from WT and Trem2^−/− mice (scale bars: upper panels, 1 mm; lower panels, 0.1 mm) are shown (d). Cytokine concentration in lung homogenates was measured by ELISA, and Nos2 mRNA levels in the lungs were measured by qRT-PCR (e). Data in a–c and e are presented as mean ± SEM and are representative of at least two independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test (a, b) or by two-tailed unpaired t test (c, e). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.

Fig. 7. TREM2 deficiency accelerates the clearance of mycobacterial infection.

a, b WT or Trem2^−/− BMDMs were infected in vitro with M. bovis BCG (MOI 10) for 4 h. After washing, the cells were further cultured for 24 h, and nitrite concentration in the culture supernatant was measured by Griess assay (a). The cells were collected, lysed, and CFUs of M. bovis BCG were counted (b). The statistical significance was calculated by two-tailed unpaired t test. c–f WT or Trem2 ^−/− mice (day 3, n = 5; day 14, n = 7) were infected intratracheally with 7.5 × 10⁶ CFU of M. bovis BCG. The lung tissues were collected at days 3 and 14 after infection, homogenized, and CFU were determined (c). The expressions of Ccl2, Adgre1, and Nos2 in the lungs from uninfected control (n = 5) and infected mice were analyzed by qRT-PCR (d–f). Data are presented as the mean ± SEM and are representative of two independent experiments. The statistical significance was calculated by two-tailed unpaired t test. *p < 0.05, **p < 0.01. Source data are provided as a Source data file.