Функціональний стан альвеолярних макрофагів та нейтрофілів кісткового мозку мишей різного віку

Abstract

Фагоцити відіграють ключову роль у тканинному гомеостазі і вродженому імунному захисті організму. Зміни фагоцитуючих клітин з віком досліджені недостатньо. В літературі наявні суперечливі дані стосовно змін фагоцитозу та продукції реактивних форм кисню при старінні. Метою нашої роботи було порівняти вікові зміни оксидативного метаболізму та фагоцитарної активності тканинних макрофагів ембріонального походження та нейтрофілів кісткового мозку. Виявлено достовірне зниження фагоцитарного числа та відсотку клітин, що продукували реактивні форми кисню, у альвеолярних макрофагів старих мишей. Статистично вірогідні відмінності між дослідженими метаболічними характеристиками нейтрофілів кісткового мозку старих і молодих тварин були відсутні. Це вказує на важливу роль тканинного мікрооточення старого організму в розвитку вікових порушень фагоцитуючих клітин.

Фагоциты играют ключевую роль в тканевом гомеостазе и врожденной иммунной защите организма. Изменения фагоцитирующих клеток с возрастом исследованы недостаточно. В литературе имеются противоречивые данные относительно изменений фагоцитоза и продукции реактивных форм кислорода при старении. Целью нашей работы было сравнить возрастные изменения оксидативного метаболизма и фагоцитарной активности тканевых макрофагов эмбрионального происхождения и нейтрофилов костного мозга. Обнаружено достоверное снижение фагоцитарного числа и процента клеток, продуцирующих реактивные формы кислорода, у альвеолярных макрофагов старых мышей. Статистически достоверные отличия между исследованными метаболическими характеристиками нейтрофилов костного мозга старых и молодых животных отсутствовали. Это указывает на важную роль тканевого микроокружения старого организма в развитии возрастных нарушений фагоцитирующих клеток.

Phagocytes play crucial role in tissue homeostasis and innate immune defence of the organism. Immune system functioning is deteriorated with aging. Phagocytosis and reactive oxygen species production are poorly studied processes with contradictory results being obtained in different experiments. Moreover, some studies suggest that resistance to certain infectious agents increase with age. It is known that distinct phagocytes differ in their ontogenetic origin. Furthermore, Linehan et al (2014) showed age-related decline in peritoneal macrophage phagocytosis, while such defect was absent in bone marrow-derived macrophages. Thus, the aim of the research was to compare age-related changes in oxidative metabolism and phagocytic activity of tissue macrophages of embryonic origin and bone marrow neutrophils. Young (2-3 mo) and aged (18-23 mo) male C57/B6 mice were used in the experiment. Neutrophils were obtained by percoll gradient centrifugation of bone marrow-derived cells. Alveolar macrophages were collected by bronchoalveolar lavage of the lungs. Cells were used to measure intracellular reactive oxygen species (ROS) production and phagocytic activity by flow cytometry. Percent of phagocytic cells, phagocytic index, percent of ROS producing cells and mean fluorescence intensity (MFI) per amount of ROS producing cells were analyzed. Statistical significance of the data was determined using Student’s t-test. There was statistically significant decrease in the percentage of alveolar macrophages obtained from aged mice, which phagocytized FITC-labeled Staphylococcus aureus (p=0,019). In neutrophils, however, there was no change in proportion of phagocytizing cells with aging. Percentage of alveolar macrophages producing ROS was significantly lowered in aged mice (p=0,025). Also, there was a tendency towards the decrease of ROS producing neutrophils with aging (p=0,056). There was slight trend toward increase of ROS production per cell in alveolar macrophages obtained from old mice, while in neutrophils there was no statistical difference in this parameter between cells from young and old animals Phagocytic index was unchanged in both alveolar macrophages and neutrophils obtained from old mice as compared to corresponding cells from young animals. Our data on age-related changes of alveolar macrophages is corroborated by previous findings of different authors. Percent of ROS producing alveolar macrophages was decreased in aged rats. Wong et al. (2017) found the significantly lower percent of alveolar macrophages which phagocytized apoptotic neutrophils and fluorophore labeled beads. Also, they found out that CD204 expression was down-regulated with aging, which can explain our results, due to the ability of CD204 to recognize bacterial ligands. As can be seen from our results, functional state of bone marrow neutrophils from aged mice was almost unaffected. Unimpaired phagocytosis in murine neutrophils from old animals was also shown in previous publications. There are controversial results regarding changes in ROS generation by neutrophils, with studies showing increased, unchanged and decreased production with aging. However, most of these studies were performed on neutrophils obtained from the blood of elderly. It was shown that phenotype and morphology of neutrophils change during their short life, with a shift toward more proinflammatory activation due to cell «aging». Bone marrow derived neutrophils must contain a larger proportion of «young» cells as compared to neutrophils from blood and tissues. Thus, different «age» of neutrophils used in experiments may be an important factor determining differences in the results of our experiment in comparison to aforementioned studies. However, this issue requires further study. In conclusion, there were more pronounced age-related changes in tissue macrophages as compared to neutrophils. It points out that tissue microenvironment of an old organism plays important role in the development of age-related impairment of phagocytes.