Trehalose 6,6'-dimycolate on the surface of Mycobacterium tuberculosis modulates surface marker expression for antigen presentation and costimulation in murine macrophages

Abstract

Trehalose 6,6'-dimycolate (TDM) is the most abundant lipid extracted from Mycobacterium tuberculosis (MTB). TDM promotes MTB survival by decreasing phagosomal acidification and phagolysosomal fusion in macrophages. Delipidation of MTB using petroleum ether removes TDM and decreases MTB survival within host cells. TDM reconstituted onto MTB restores its virulent wild-type characteristics. We investigated the role of TDM in regulating surface marker expression in MTB-infected macrophages. Macrophages were infected with wild-type, delipidated, and TDM-reconstituted MTB for 24h and measured for changes in surface marker expression. TDM on MTB was found to specifically target MHCII, CD1d, CD40, CD80 and CD86. Both wild-type and TDM-reconstituted MTB suppressed or induced no change in expression of these surface markers, whereas delipidated MTB increased expression of the same markers. MTB-infected macrophages were also overlaid with MHCII-restricted T cell hybridomas which recognize Antigen 85B. Macrophages infected by wild-type and TDM-reconstituted MTB did not present antigen as well as delipidated MTB-infected macrophages. The evidence shown furthers supports the notion that TDM present on MTB promotes its survival and persistence in host macrophages.

Fig. 1

Thin layer chromatography of petroleum ether extracts from MTB strain H37Rv (#2) against commercial purified TDM standard (#1).

Fig. 2. Effects of TDM on MTB on the expression of MHCII, CD1d, CD40, CD80 and CD86 by murine macrophages

Macrophages were infected with wild-type, delipidated (dMTB), or TDM-reconstituted (rMTB) MTB at a MOI of 2:1 for 24 hours, and examined for surface expression of MHCII, CD1d, and CD40 using fluorescent antibodies and flow cytometry. a) Data shows percent cells positive for MHCII, CD1d, and CD40 in alveolar-like, peritoneal-like, and bone marrow macrophages. b) Percent positive cells for CD80 and CD86 in BMMs. Data collected from triplicate experiments (SEM; * p < 0.05 vs. uninfected macrophage controls). Horizontal dotted line represents fold change of percent positive cells compared to uninfected controls. c) Histograms of BMM experiments show mean fluorescence intensity (MFI) against number of cells for MHCII, CD1d, CD40, CD80 and CD86. Grey solid line is isotype control, black solid line is uninfected control, and dotted line is infection with MTB, dMTB, or rMTB.

Fig. 2. Effects of TDM on MTB on the expression of MHCII, CD1d, CD40, CD80 and CD86 by murine macrophages

Macrophages were infected with wild-type, delipidated (dMTB), or TDM-reconstituted (rMTB) MTB at a MOI of 2:1 for 24 hours, and examined for surface expression of MHCII, CD1d, and CD40 using fluorescent antibodies and flow cytometry. a) Data shows percent cells positive for MHCII, CD1d, and CD40 in alveolar-like, peritoneal-like, and bone marrow macrophages. b) Percent positive cells for CD80 and CD86 in BMMs. Data collected from triplicate experiments (SEM; * p < 0.05 vs. uninfected macrophage controls). Horizontal dotted line represents fold change of percent positive cells compared to uninfected controls. c) Histograms of BMM experiments show mean fluorescence intensity (MFI) against number of cells for MHCII, CD1d, CD40, CD80 and CD86. Grey solid line is isotype control, black solid line is uninfected control, and dotted line is infection with MTB, dMTB, or rMTB.

Fig. 2. Effects of TDM on MTB on the expression of MHCII, CD1d, CD40, CD80 and CD86 by murine macrophages

Macrophages were infected with wild-type, delipidated (dMTB), or TDM-reconstituted (rMTB) MTB at a MOI of 2:1 for 24 hours, and examined for surface expression of MHCII, CD1d, and CD40 using fluorescent antibodies and flow cytometry. a) Data shows percent cells positive for MHCII, CD1d, and CD40 in alveolar-like, peritoneal-like, and bone marrow macrophages. b) Percent positive cells for CD80 and CD86 in BMMs. Data collected from triplicate experiments (SEM; * p < 0.05 vs. uninfected macrophage controls). Horizontal dotted line represents fold change of percent positive cells compared to uninfected controls. c) Histograms of BMM experiments show mean fluorescence intensity (MFI) against number of cells for MHCII, CD1d, CD40, CD80 and CD86. Grey solid line is isotype control, black solid line is uninfected control, and dotted line is infection with MTB, dMTB, or rMTB.

Fig. 3. Effects of TDM-coated beads on expression of surface markers in bone marrow macrophages

BMMs were treated for 24 hours with TDM-coated or plain biodegradable beads and assayed for surface marker expression. Histograms show mean fluorescence intensity (MFI) against number of cells for MHCII, CD1d, CD40, CD80 and CD86. Solid black line is untreated control BMMs, grey solid line is plain bead treatment, and black dotted line is TDM-coated bead treatment.

Fig. 4. Removal of TDM from MTB increases Ag85B presentation to BB7 T cells

a) BMMs with or without IFN-γ stimulation were infected with MTB, dMTB, or rMTB for 4 hours, then washed and overlaid with BB7 T cells. Supernatants were collected at 24 hours and measured for IL-2. Graph displays pg ml⁻¹ of IL-2 concentration of triplicate experiments (SEM; * p < 0.05 vs wild-type MTB infection). b) BMMs infected with fbpA mutant or TDM-reconstituted fbpA mutant, then overlaid with BB7 T cells. Data collected from triplicate experiments (SEM; * p < 0.05 vs. uninfected macrophage controls).

Fig 5. TDM promotes SOCS production in bone marrow macrophages

BMMs were infected with LPS, BCG, MTB, dMTB, or rMTB for 4 hours. Whole cell lysates were immunoblotted for SOCS-1, SOCS-3, and actin.