Effect of CFP-10/ESAT-6 secretory proteins on long-term non-specific immunological memory in mouse macrophages

Abstract

Innate immune cells (monocytes/macrophages, NK) can also develop immune memory, which means that these cells are trained after their first encounter with pathogens so that they exhibit a nonspecific immunological response to the same or another pathogen. Bacilli Calmette–Gu rin (BCG) induces nonspecific innate memory (trained immunity) in innate immune cells. We examined nonspecific innate memory in macrophages of BALB/c mice in response to mycobacteria with or without the RD1 region in the genome. Mice were immunized with BCG vaccine, and peritoneal macrophages were isolated on day 7, and then stimulated with bacterial lipopolysaccharide, CFP-10, or ESAT-6. In addition, mice were immunized with Mycobacterium tuberculosis uro-BCG vaccine (RD1-) and Mycobacterium tuberculosis strain H37Rv (RD1+) subcutaneously or intravenously; peritoneal macrophages were isolated and stimulated with lipopolysaccharide on day 4. Alveolar macrophages were obtained from lung explants of mice infected with Mycobacterium tuberculosis strain H37Rv mice, were expanded to confluence 70-80% and further stimulated with lipopolysaccharide. Lactate, cytokines, and glucose levels were examined in conditioned macrophage medium. Peritoneal macrophages from mice primed with BCG vaccine were shown to increase IL-1b, TNFa, and lactate production in response to CFP-6 and ESAT-10 (p < 0.05). Of note is the fact that lipopolysaccharide also increased production of IL-1b, TNFa, and also increased glucose uptake by peritoneal macrophages primed with BCG vaccine (p < 0.05). Peritoneal macrophages primed with Uro-BCG were shown to increase spontaneous production of IL-1b and decrease spontaneous production of TNFa (p < 0.05). When macrophages were primed by subcutaneous or intravenous administration of Mycobacterium tuberculosis strain H37Rv differentially affected cytokine production, by decreasing IL-1b production and increasing TNFa and IL-10, was observed. In response to lipopolysaccharide, peritoneal macrophages increased IL-1b, TNFa, IL-10 production and glucose consumption (p < 0.05). The mode of priming of macrophages with Mycobacterium tuberculosis strain H37Rv also led to multidirectional levels of cytokine production. Alveolar macrophages were shown to retain trained immunity, as they produced elevated levels of IL-1b, TNFa, and IL-10 (p < 0.05). Thus, mouse macrophages formed a trained immunity phenotype in response to different types of mycobacteria, which persists for a long time after primary contact with the pathogen, particularly in alveolar macrophages.