Fig. 6: Ornithine limits ammonia and activates AMPK.

AMs and KCs from C57BL/6 mice were isolated and infected with Mtb H37Rv at an MOI of 1:2.5. After 72 h. a NH₄⁺/NH₃ levels were measured in the cell lysates treated with ornithine or nor-NOHA (arginase inhibitor) by spectrophotometric analysis at 570 nm with extrapolation from the standard curve and expressed in nmol (mean ± SD). Data are representatives of five independent experiments. b A representative micrograph of LC3-B, ATG-7, ATG-5 and Beclin-1 in AMs treated with ornithine or NH₄Cl and KCs treated with NH₄Cl at respective concentrations. Blue: DAPI; green: LC-3B and ATG-7; red: ATG-5 and Beclin-1. Scale bar: 5 μm. Images of multiple fields at 63× magnification were taken with oil immersion. c The number of LC-3B puncta relative to the cell area (10 cells per condition) was quantified using ImageJ. p < 0.0012. d At 2 h post-infection, NH₄Cl was added at the indicated concentrations. Intracellular CFUs were demonstrated after 5 days of infection. p < 0.0195, p < 0.0042. n = 5 experiments. e Total protein was extracted from the above cells and western blotting was performed. A representative blot depicting the levels of p-AMPK, p-mTOR, p38 MAPK, LC-3 and p62/SQSTM1 in the control, ornithine or NH₄Cl treated and Mtb H37Rv-infected macrophages is shown. Normalized fold intensities of p-AMPK, p < 0.0013, p < 0.0032, p < 0.0051; p-mTOR, p < 0.0017, p < 0.0027, p < 0.0018; p38 MAPK, ns not significant; and p62 levels p < 0.0191, p < 0.0072 in Mtb-infected and ornithine or NH₄Cl treated macrophages compared with the levels in the uninfected control. n = 3 experiments. f At 2 h post-infection, ornithine and compound C were added at indicated concentrations. Intracellular CFUs were demonstrated after 5 days of infection. n = 5 experiments. Data are presented as the mean ± SD and analyzed using paired two-tailed t test. g Schematic representation of ornithine restricting Mtb growth in AMs. Red color indicates inhibitory line and green arrows indicates activation.