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Non‐Tuberculous Mycobacteria Infections Increase NOS2 While Decreasing NO Levels

Non‐tuberculous mycobacteria (NTM) are rod‐shaped bacilli that cause lung infections in patients with weakened immune system or underlying lung disease such as cystic fibrosis, COPD, or prior tuberculosis. While it is well‐established that NTM cause lung disease, the host response to infection with NTM has not been well‐characterized. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by innate immune cells is considered a major defense mechanism against mycobacteria. Nitric oxide (NO), a product of inducible nitric oxide synthase (NOS2) in macrophages, plays an essential role in M. tuberculosis (MTb) infection through its bactericidal and anti‐inflammatory characteristics. Therefore, we hypothesized that NTM infection may upregulate NOS2 expression, which in turn may contribute to the host response to NTM infection. RAW 264.7 and MH‐S cells, virus transformed murine macrophages and alveolar macrophages, respectively, were exposed to NTM (M. avium‐intracellulare, or MAI) at a MOI of 25 and NO levels in cell media were monitored 4, 8, 12, 24, and 48 hours after NTM infection using a colorimetric GSNO assay. mRNA expression of NOS isoforms (NOS1, NOS2, and NOS3) in MH‐S and RAW cells were analyzed 24‐ and 48‐hours post‐infection. MH‐S cells were also transfected with siRNA to NOS2 and infected with MAI for 0.5, 4, and 24 hours, and their intracellular NTM were quantified to assess macrophage function. NO levels decreased following 4 hours of NTM exposure and remained decreased for 48 hours (P<0.05). NTM infection increased the expression of NOS2 without affecting levels of other NOS isoforms (P<0.05). siNOS2 transfected cells infected with MAI for more than four hours had significantly greater levels of intracellular MAI compared to scrambled negative control (P<0.05). Our preliminary data are the first to demonstrate that NTM increase NOS2 expression while decreasing NO levels. Furthermore, decreasing NOS2 expression exacerbates NTM infections. Future studies will examine mechanisms whereby NTM decrease NO levels. Therefore, we propose that approaches that restore NO can enhance macrophage host defense against NTMs and improve infection clearance.