This study investigates the interaction between Mycobacterium tuberculosis (Mtb) and macrophages, focusing on the role of TLR-2 in phagocytosis and immune evasion. The findings highlight how Mtb manipulates host cell signaling to persist within macrophages.
Obtain a culture of fully differentiated macrophages in a multi-well plate. These macrophages have surface markers, such as toll-like receptors or TLR-2 — a pattern recognition receptor that identifies various microbial components.
Add an appropriate amount of fluorescently labeled Mycobacterium tuberculosis or Mtb cell suspension into each well and incubate.
TLR-2 recognizes lipoarabinomannan, or LAM, a complex glycolipid component of the Mtb cell wall, initiating phagocytosis. The macrophage internalizes Mtb, sequestering the bacterium within a phagosome — a membrane-bound compartment.
The phagosome matures, by fusing with lysosomes, forming a phagolysosome — an acidic and enzymatic-rich compartment.
Within the phagolysosome, the Mtb inhibits the vacuolar-type proton pump responsible for generating an acidic pH. Consequently, the pH within the phagolysosome becomes near-neutral, deactivating enzymes that degrade engulfed pathogens.
The interaction between LAM and TLR2, also affects host cell signaling pathways, leading to the modulation of surface markers involved in antigen presentation.
The inactivation of phagolysosome and inhibition of host immune response enable Mtb to persist and replicate within the macrophage.
To infect the monocyte-derived cells, dilute the bacteria to a 5 times 10 to the sixth colony-forming units per milliliter concentration in serum-free medium, and replace the supernatant in each well of the six-well cell culture plate, with 1 milliliter of fresh serum-free medium and 1 milliliter of bacteria suspension per well to obtain a multiplicity of infection of 5.
After a 4-hour incubation in the cell-culture incubator, wash each well three times with 1 milliliter of sterile wash buffer per wash, tilting the plate carefully to remove the entire volume of buffer from the corners of the wells after each wash. Then, resuspend the MTb-infected monocyte-derived cells in 2 milliliters of complete medium without antibiotics.
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