TREM2 Promotes Immune Evasion by Mycobacterium tuberculosis in Human Macrophages

Abstract

Macrophage surface receptors are critical for pathogen defense, as they are the gatekeepers for pathogen entry and sensing, which trigger robust immune responses. TREM2 (triggering receptor expressed on myeloid cells 2) is a transmembrane surface receptor that mediates anti-inflammatory immune signaling. A recent study showed that TREM2 is a receptor for mycolic acids in the mycobacterial cell wall and inhibits macrophage activation. However, the underlying functional mechanism of how TREM2 regulates the macrophage antimycobacterial response remains unclear. Here, we show that Mycobacterium tuberculosis, the causative agent for tuberculosis, specifically binds to human TREM2 to disable the macrophage antibacterial response. Live but not killed mycobacteria specifically trigger the upregulation of TREM2 during macrophage infection through a mechanism dependent on STING (the stimulator of interferon genes). TREM2 facilitated uptake of M. tuberculosis into macrophages and is responsible for blocking the production of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and reactive oxygen species (ROS), while enhancing the production of interferon-β (IFN-β) and IL-10. TREM2-mediated blockade of ROS production promoted the survival of M. tuberculosis within infected macrophages. Consistent with this, genetic deletion or antibody-mediated neutralization of TREM2 reduced the intracellular survival of M. tuberculosis through enhanced production of ROS. Importantly, inhibition of type I IFN signaling in TREM2-overexpressing macrophages restored the ability of these cells to produce inflammatory cytokines and ROS, resulting in normal levels of intracellular bacteria killing. Collectively, our study identifies TREM2 as an attractive host receptor for host-directed antimycobacterial therapeutics. IMPORTANCE Mycobacterium tuberculosis is one of the most ancient bacterial pathogens and remains the leading cause of death from a single bacterial agent. The success of M. tuberculosis relies greatly on its ability to parasitize and disable its host macrophages. Previous studies have found that M. tuberculosis uses its unique cell wall lipids to manipulate the immune response by binding to specific surface receptors on macrophages. Our study reveals that M. tuberculosis binds to TREM2, an immunomodulatory receptor expressed on macrophages, to facilitate a "silent" mode of entry. Increased levels of TREM2 triggered by intracellular sensing of M. tuberculosis promoted the intracellular survival of M. tuberculosis through type I IFN-driven inhibition of reactive oxygen species (ROS) and proinflammatory cytokine production. Importantly, deletion of TREM2 reversed the effects of "silent" entry and resulted in increased production of inflammatory cytokines, generation of ROS, and cell death. As such, antibody-mediated or pharmacological targeting of TREM2 could be a promising strategy for novel treatments against M. tuberculosis infection.

Keywords: IFN; Mycobacterium tuberculosis; TREM2; macrophage cell death; phagocytosis; reactive oxygen species; triggering receptor expressed on myeloid cells; type I interferon.

FIG 1

Human TREM2 binds to M. tuberculosis and contributes to phagocytosis. (A) M. tuberculosis was incubated with recombinant human TREM2-Fc or control Fc proteins. APC-conjugated anti-human IgG Fc antibody was used to detect the TREM2-bound bacteria. Stained bacteria were analyzed by flow cytometry, with APC⁺ signal indicating TREM2-Fc-bound bacteria. (B and C) THP-NT, THP-ΔTREM2, THP-Vector, or THP-TREM2+ cells were analyzed (B) for TREM2 protein expression by Western blotting or (C) for surface expression of TREM2 by staining with APC-conjugated anti-TREM2 antibody followed by flow cytometric analysis. (D) THP-1 and its derivative cell lines were incubated for 4 h with a GFP-expressing M. tuberculosis strain (Mtb-GFP) that had been either mock treated (unopsonized condition) or pretreated with human serum for 20 min (opsonized condition) to allow for phagocytosis. The amount of phagocytosis was quantified by flow cytometry, as indicated by the percentage of GFP⁺ macrophages. (E and F) Anti-TREM2 antibody was used at various concentrations (1 to 10 μg/mL) to treat (E) THP-1 cells or (F) hMDMs, followed by infection with Mtb-GFP. At 4 h postinfection, macrophages were analyzed by flow cytometry to quantify levels of phagocytosis. The results in this figure are representative of three biological replicates, and error bars indicate the mean ± SD from three independent experiments.

FIG 2

M. tuberculosis infection upregulates TREM2 expression. (A and B) TREM2 mRNA and protein levels of M. tuberculosis-infected THP-1 macrophages (MOI = 10) were assessed by (A) qRT-PCR and (B) Western blotting at the indicated time points. hMDMs were infected with M. tuberculosis (MOI = 10) and at indicated time points postinfection, TREM2 mRNA and protein levels were quantified by (C) qRT-PCR and (D) Western blot. Data shown in panels A and C were analyzed using the 2^−ΔΔCT method, normalized to ACTB as a reference gene, and are expressed as the relative fold change compared to uninfected cells. qPCR data represents the result of three technical replicates (mean ± SD). (E) M. bovis BCG and M. tuberculosis were mock treated or pretreated with gentamicin (150 μg/mL) for 1 h. Bacteria were then used to infect THP-1 macrophages, and TREM2 protein levels were analyzed at day 3. (F) Listeria monocytogenes-infected (MOI = 10) and M. tuberculosis-infected THP-1 macrophages at the indicated time points were analyzed by Western blotting, and vinculin was used as a loading control. (G) THP-1 macrophages were pretreated with the indicated inhibitors for 2 h and 6 h (in the case of SN-011) and subsequently infected with M. tuberculosis (MOI = 10). TREM2 expression was analyzed by Western blotting at 3 days postinfection using vinculin as a loading control. Blots in panels B, D, E, F, and G are representative of three independent biological replicates. The quantification of TREM2 expression is shown below each panel and is reported as normalized expression over vinculin. AU, arbitrary units.

FIG 3

TREM2 expression alters cytokine production in M. tuberculosis-infected macrophages. (A to D) THP-NT, THP-ΔTREM2, THP-Vector, and THP-TREM2+ macrophages were infected with M. tuberculosis (MOI = 10), and culture supernatants were collected at 24 h postinfection. Levels of (A) TNF-α, (B) IL-1β, (C) IL-10, and (D) IFN-β were measured using human ELISA kits. (E and F) THP-Vector and THP-TREM2+ macrophages were mock treated or pretreated with R406 (SYK inhibitor; 10 μM), anti-IFNAR1 (2.5 μg/mL), SN-011 (STING inhibitor; 1 μM), and RU.521 (cGAS inhibitor; 10 μg/mL) for 2 h and 6 h (in the case of SN-011), prior to M. tuberculosis infection (MOI = 10). Culture supernatants were collected at 24 h postinfection and levels of (E) TNF-α and (F) IL-1β were measured. Error bars represent the mean ± SD of three independent biological replicates.

FIG 4

TREM2 regulates cell death through a mechanism dependent on type I IFN-mediated inhibition of TNF-α production. (A) THP-NT, THP-ΔTREM2, THP-Vector, and THP-TREM2+ macrophages were infected with M. tuberculosis, and cell viability was assessed using CellTiter-Glo (measured as relative luminescence units [RLU]). (B) THP-NT, THP-ΔTREM2, THP-Vector, and THP-TREM2+ macrophages were infected with M. tuberculosis, stained with FVS780 at the indicated time points, and analyzed using flow cytometry to measure the levels of cell death. (C) hMDMs mock treated or pretreated with anti-TREM2 antibody were infected with M. tuberculosis and assessed for cell viability using CellTiter-Glo. (D) THP-NT and THP-ΔTREM2 macrophages were mock treated or pretreated with 100 ng/mL anti-TNF-α antibody for 2 h. Cells were then infected with M. tuberculosis, and cell viability was quantified at day 4 postinfection by flow cytometry using FVS780 stain (12). THP-Vector, and THP-TREM2+ macrophages were mock treated or pretreated with (E) anti-IFNAR1 (2.5 μg/mL), (F) SN-011 (STING inhibitor; 1 μM), or (G) RU.521 (cGAS inhibitor; 10 μg/mL) for 2 h and 6 h (in the case of SN-011). Cells were then mock treated or pretreated with anti-TNF-α (100 ng/mL) for 2 h, followed by M. tuberculosis infection. The percentage of macrophage death was assessed at day 4 postinfection by staining with FVS780. An MOI of 10 was used for all infections in this figure. Error bars in this figure represent the mean ± SD from three independent biological replicates.

FIG 5

TREM2 facilitates intracellular survival of M. tuberculosis. (A) THP-NT, THP-ΔTREM2, THP-Vector, and THP-TREM2+ macrophages were infected with the autoluminescent Mtb-lux strain at an MOI of 10. Luminescence signal (RLU) representing viable M. tuberculosis was analyzed at the indicated time points. (B) THP-1 macrophages and (C) hMDMs were mock treated or pretreated with anti-TREM2 antibody for 20 min, followed by infection with Mtb-lux (MOI = 10), and RLU were analyzed at the indicated time points postinfection. (D) THP-Vector and THP-TREM2+ macrophages were mock treated or pretreated with R406 (SYK inhibitor; 10 μM), anti-IFNAR1 (2.5 μg/mL), SN-011 (STING inhibitor; 1 μM), or RU.521 (cGAS inhibitor; 10 μg/mL) for 2 h and 6 h (in the case of SN-011) and infected with Mtb-lux (MOI = 10). RLU were analyzed at day 4 postinfection. Error bars in this figure represent the mean ± SD from three independent biological replicates.

FIG 6

TREM2-mediated induction of type I IFNs disables ROS production (A) THP-NT, THP-ΔTREM2, THP-Vector, and THP-TREM2+ macrophages were infected with M. tuberculosis (MOI = 10) and stained with 5 μM DCFH-DA for 30 min at 37°C. ROS was quantified at the indicated time points postinfection by relative fluorescence unit (RFU) measurements. (B) THP-1 macrophages (THP-NT, THP-ΔTREM2, and THP-TREM2+) and (C) Mock or anti-TREM2 pretreated hMDMs were pretreated with 10 mM N-acetyl cysteine (NAC) or 25 μM GSK2795039 for 24 h, followed by infection with Mtb-lux (MOI = 10). RLU were analyzed at day 4 postinfection. (D) THP-Vector and THP-TREM2+ macrophages were mock treated or pretreated with R406 (SYK inhibitor; 10 μM), anti-IFNAR1 (2.5 μg/mL), SN-011 (STING inhibitor; 1 μM), or RU.521 (cGAS inhibitor; 10 μg/mL) for 2 h and 6 h (in the case of SN-011), prior to M. tuberculosis infection (MOI = 10). At day 4 postinfection, cells were stained with 5 μM DCFH-DA for 30 min at 37°C and ROS was quantified by fluorescence measurements. Error bars in this figure represent the mean ± SD from three independent biological replicates.

FIG 7

Role of TREM2 in macrophage antibacterial defense against M. tuberculosis. (A) Entry of M. tuberculosis via TREM2 induces a STING-dependent upregulation of TREM2 expression, which in turn increases IL-10 and IFN-β production. Increased levels of IFN-β is responsible for type I IFN-driven inhibition of ROS production and proinflammatory cytokine production, which results in the increased intracellular survival of M. tuberculosis. (B) Targeting the TREM2 signaling pathway by genetic deletion or antibody-mediated neutralization of TREM2, antibody-mediated neutralization of IFNAR1, or pharmacological inhibition of STING, restores proper proinflammatory cytokine and ROS production to promote bacterial clearance.