LC3-Associated Phagocytosis Is Impaired in Bone Marrow Macrophages during Ageing Leading to Defective Processing of Apoptotic Cells and Suppression of Sting Activation

Abstract

Introduction Macrophage phagocytic function declines with ageing, with potential deleterious consequences on inflammation, immune response, maintenance of tissue health, and cancer. Paradoxically, we and others have found that with increasing age, macrophage number increases within the bone marrow (BM) microenvironment, supporting the hypothesis that the ageing macrophages are functionally impaired. Phagocytosis by mononuclear cells occurs through either an LC3-dependent or -independent mechanism. LC3-associated phagocytosis (LAP) is physiologically triggered by pathogens and cellular debris via interaction with phagocyte surface receptors, including T cell membrane protein 4 (TIM-4) and FcR. Here, we investigate whether age-related macrophage functional impairment is associated with LC3. Results BM was isolated from young (8-12 weeks) and aged (18-24 months) female C57Bl/6 mice and macrophages were analysed using flow cytometry and isolated via FACS. We show increased BM macrophage numbers in old BM (2 fold), but no difference in total phagocytic potential as measured by uptake of E.coli bioparticles or apoptotic debris, when compared to young. Furthermore, using immunofluorescent microscopy to visualise macrophage phagocytosis, we observed that the phagosomes generated in BM macrophages from aged animals did not recruit LC3, whereas in young animals LC3 was recruited. Consequently, macrophages from older animals accumulated large amounts of E.coli bioparticles and apoptotic bodies when compared to young macrophages. This suggests defective processing of the E.coli bioparticles by the phagosome once the phagosome has formed. To understand if this is due to lack of LC3 protein, we next compared gene expression and protein levels of LC3 in young and old animals. There was no loss in LC3 RNA or protein expression in macrophages from old and young. As we have recently reported that BM macrophage LAP mediates the stimulator of interferon genes (STING) pathway, we examined STING activation in old and young BM macrophages by measuring Gbp2, Irf7, and Ifit3 gene expression. Results showed a significant reduction in STING mediated gene expression in BM macrophages from aged mice compared to young. Summary These data suggest the decline in macrophage phagocytic function associated with age occurs because of a failure of LC3-mediated phagosome clearance. Furthermore, we hypothesise that the observed increase in BM macrophage numbers in older animals is, at least in part, a compensatory mechanism for the defective individual macrophage phagocytic processing seen, helping preserve overall BM phagocytic function.