Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to LC3-associated pathways involving single- or double-membrane vacuoles.

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3's association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis' intravesicular life cycle.

IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo.

Recent studies indicate that IL-1alpha functions intracellularly in pathways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-associated protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines, is an endothelium-derived, chromatin-associated nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-associated nuclear factor in HEV endothelial cells in vivo. Association of IL-33 with heterochromatin was also observed in human and mouse cells under living conditions. In addition, colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-association, and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, associated with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1alpha and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.