Dose response effect of Mycobacterium smegmatis-derived lipomannan in 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 the study was to determine if RAW 264.7 murine macrophage activity could be facilitated and intensified by stimulation with LAM from M. smegmatis. Stimulation with bacterial LAM, and lipopolysaccharide (LPS) as a positive control, yields functional endpoints: nitric oxide (NO) production measured by nitrites (NO2) in the culture supernatant and expression of proteins, such as tumor necrosis factor-α and inducible nitric oxide synthase (iNOS). RAW 264.7 cells were stimulated dose-responsively with LAM at concentrations of 1-1000 ng/mL. Use of 100 ng/mL LPS served as a positive control. Nitrite, an indirect product of the NO inflammatory response, was measured by the Greiss reaction and total protein was collected from the cells for western blotting to quantify iNOS. Preliminary results in experiment one showed that LAM NO production was minimally noticeable in the 1000 ng/mL dose and was still substantially lower than the LPS control when evaluated by the Greiss Assay. Experiments two and three were conducted with higher doses of Lam, 3000 ng/mL and 5000 ng/mL and showed an increased response. The nitrite concentration reached by both these LAM treatments still did not reach that of 100 ng/mL LPS. This result suggests 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