Background/Purpose:  We found that Snapin, a SNARE complex protein required for synaptic vesicle docking and fusion, was significantly increased in rheumatoid arthritis (RA) synovial tissue compared to control synovial tissues. Snapin was highly expressed in CD68 positive macrophages (MΦs) in the sublining, which correlated with inflammation. Snapin expression in MΦs co-localized with Rab7, a marker of late endosomes. Therefore, studies were performed to determine the role of Snapin in the formation of autophagosomes and their fusion with lysosomes.

Methods:  The forced reduction of Snapin in primary human MΦs was performed using siRNA.  Snapin, Lamp1, 2 and LC3 protein levels were determined by Western blot analysis. MΦ phagocytosis of living bacteria was performed by infection of Staphylococcus aureus and intracellular bacteria number was determined by counting the bacterial colonies on LB-agar plates with MΦ lysates. Forced reduction of Snapin in Raw 264.7 macrophage cell line was performed by infection with a lentiviral vector expressing Snapin shRNA, followed by puromycin selection. Phagocytosis of fluorescent pHrodo-labeled dead S. aureus by Raw stable cell lines was performed and the amount of intracellular bacteria was determined either by mean fluorescence with flow cytometry or by cell fluorescence microscopy.

Results: The expression of Snapin progressively increased during human monocyte to MΦ differentiation. The forced reduction of Snapin in MΦs (documented by immunoblot analysis) resulted in elevated levels of LC3-II, a marker for autophagosomes, and Lamp-1, Lamp-2, two lysosomal markers. Snapin siRNA combined with cell starvation by FBS deprivation to promote autophagy, further increased LC3-II. However, the reduction of Snapin in MΦs co-treated with chloroquine, an autophagy efflux blocker, did not affect LC3-II levels, suggesting that the reduction of Snapin does not affect autophagy formation but leads to accumulation of autophagosomes by impaired autolysosome formation.  

Phagocytosis of pHrodo fluorescent labeled dead S. aureus by Raw 264.7 stably expressing Snapin shRNA demonstrated a 25% increase in mean fluorescence intensity (MFI) by flow cytometry compared to control shRNA infected Raw cells. Following the forced reduction of Snapin in primary human MΦs, phagocytized S.aureus numbers at 1 hour after infection did not show differences, compared to non-targeting siRNA transfected MΦs. However, after 5 hours of infection, there were significantly increased numbers of S aureus remaining intracellularly in Snapin siRNA transfected MΦs compared to control siRNA treated MΦs ( 2.6 verses 1.7 colony formation units per cell, p<0.05 ). These data suggested Snapin is important for phagosomal fusion with lysosomes and the clearance of bacteria in MΦs.  

Conclusion: Reduction of Snapin in MΦs resulted in the blockage of the fusion of autophagosmes and phagosomes with lysosomes. By promoting autophagy, the increased expression of Snapin in the RA joint may contribute to the long term survival of MΦs which contribute to the disease pathogenesis and joint destruction.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/snapin-is-critical-for-the-maturation-of-autophagosome-and-phagosome-in-macrophages/