Effect of lipoarabinomannan (M. smegmatis) on RAW 264.7 murine macrophage nitric oxide production

Tuberculosis (TB) is a respiratory disease caused by the pathogenic bacteria Mycobacterium tuberculosis and is the leading cause of death by an infectious agent worldwide. Alveolar mammalian macrophages have a vigorous innate immune response to bacterial cell walls composed of Lipopolysaccharide (LPS). Unlike most bacteria, the Mycobacterium genus has a unique cell wall composition including Lipoarabinomannan (LAM). LAM is known to act through Toll-like receptor 2 (TLR-2), while LPS acts on TLR-4. In order to develop effective treatments for TB, we must better understand the disease process and how M. tuberculosis interacts with macrophages. RAW 264.7 mouse macrophages were maintained in culture by passage every 4 days and used for experiments at the time of passage. When stimulated with LPS, macrophages produce nitric oxide (NO) through the enzyme inducible nitric oxide synthase (iNOS). RAW 264.7 murine macrophages were stimulated in a dose-response fashion with LPS (0-100ng/mL) and M. smegatis-derived LAM (100-5000ng/mL). Nitrite levels in cell supernatant from treatment were measured using a Griess reaction and quantitated using a spectrophotometer. The current results indicate that LAM triggers a response (50ng/mL nitrite per 5000µL) but not as extreme as LPS treatment (90ng/mL nitrite per 100µL). LAM may act on TLR-4, producing NO through iNOS. The LAM reagent may be contaminated by endotoxin, which would stimulate TLR-4, or LAM may stimulate both TLR-2 and TLR-4 (to a lesser extent). Planned experiments using TLR-4 antagonist LPS-RS (Rhodobacter sphaeroides) should isolate the LAM response