Dose Response Effect of Mycobacterium Smegmatis-Derived Lipoarabinomannan on RAW 264.7 Murine Macrophages

Tuberculosis is a debilitating respiratory disease caused by the bacterial species Mycobacterium tuberculosis, which acts by infecting the host’s macrophages and evading their immune responses. The purpose of this experiment is to characterize RAW 264.7 murine macrophage response to lipoarabinomannan (LAM) from Mycobacterium smegmatis. Stimulation with bacterial LAM, and LPS as a positive control, will yield two functional endpoints, nitric oxide (NO) production measured by nitrites (NO2) in the culture supernatant and expression of the protein inducible nitric oxide synthase (iNOS). RAW 264.7 cells are stimulated dose-responsively with LAM at concentrations of 1-1000 ng/mL as well as 100 ng/mL LPS. Nitrite, an indirect product of the NO inflammatory response, is measured by the Greiss reaction and the proteins are collected from the cells for western blotting to quantify iNOS. Preliminary results show that LAM NO production was minimally noticeable in the 1000 ng/mL dose, however it was significantly lower than the LPS control. This is suggestive that the pattern recognition difference between Toll-like receptor-2 (TLR-2) for LAM and Toll-like receptor-4 (TLR-4) for LPS has an impact on the output of NO