Background/Purpose: Our recent data indicate that Snapin, a SNAP associated protein, is significantly increased in macrophages (MΦs) in rheumatoid arthritis (RA) synovial tissue and its expression level was correlates with inflammation. Reduction of Snapin hindered the maturation of autophagosome and phagosome, resulted in autophagosome accumulation and delayed bacterial clearance in MΦs. In this study, we further explored the mechanisms by which Snapin is involved in autophagy in MΦs as well as its role on monocyte to MΦ differentiation.

Methods: The reduction of Snapin in primary human MΦs was performed using siRNA, while in J774A mouse MΦ cell line was implemented by infection with a lentiviral vector expressing Snapin shRNA. Snapin, Lamp1 and LC3B protein levels were determined by Western blot analysis. Cell fractions enriched in lysosomes were isolated using density gradient centrifugation. Autophagosomes were purified by anti-LC3B antibody and Dyna magnetic beads.  Autophagy in MΦs was detected by electron microscopy as well as by LC3B punctae determined by immunofluorescence microscopy. Human peripheral blood monocytes isolated by counter-flow elutriation were differentiated in 20% FBS plus CSF-1. Non-specific (NS), Snapin , or Beclin 1 siRNA was transfected into monocytes at Day0 and Day3 by lipofectamine. Monocyte to MΦ differentiation was measured by morphology and by the levels of differentiation markers CD163 and CD71 by flow cytometry.     

Results: The forced reduction of Snapin in primary human MΦs and the murine J774A MΦ cell line resulted in increased late autophagosome vacuoles that contain partially digested organelles and other cellular material. Large sized LC3 punctae were also dramatically increased in these MΦs detected by immunofluorescence.  These changes were similar to those observed by inhibition of lysosomal maturation with chloroquine but not the induction of autophagy by starvation or rapamycin. The results suggest that the reduction of Snapin blocked autophagy efflux. Snapin was abundant in lysosome-enriched cell organelles in MΦs. Snapin also co-isolated with LC3B and Lamp1 in autophagosomes which were isolated by anti-LC3B antibody from the lysosome-enriched fraction. By co-IP, Lamp1 was co-purified with Snapin employing MΦ cell lysates. These data suggest that Snapin directly interacted with Lamp1 and is necessary for the maturation of autophagosomes.     

The control monocytes transfected with NS siRNA differentiated normally after 5 days into MΦs determined phenotypically and by the strong expression of both CD163 and CD71. However, monocytes transfected with Snapin or Beclin1 siRNA appeared as small round cells that loosely attached to plastic and the expression of both CD163 and CD71 was greatly reduced.  These results indicate that arresting autophagy, by the reduction of Beclin1, or Snapin, suppressed monocyte to MΦ differentiation. 

Conclusion: Snapin is required for the fusion of lysosomes with autophagosomes and the maturation of autophagosomes which is critical for monocyte to MΦ differentiation.  These observations suggest that Snapin contributes to the pathogenesis of RA and may be a therapeutic target.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/snapin-is-required-for-functional-autophagy-and-is-critical-for-monocyte-to-macrophage-differentiation/