Hypoxia and physiological adaptations: autophagy as a cell survival pathway in dendritic cells

Hypoxia consists of a reduction in oxygen availability that may occur because of an increased oxygen demand of an impaired oxygen supply. Hypoxia-inducible factors (HIFs) are transcriptional activators that are expressed in response to cellular hypoxia and mediates multiple cellular and systemic homeostatic responses to hypoxic conditions, as well as the regulation of immune system. HIF expression and stabilization in immune cells can be induced not only by hypoxia, but also by other factors such as inflammation and infectious microorganism. Dendritic cells (DCs) are the most potent antigen presenting cells and they have a broad range of functions including pathogens detection, phagocytosis, antigen processing and activation of adaptive immune system. During their lifespan DCs are exposed to different oxygen tensions, since they patrol several tissue microenvironments and interact with T cells in primary and secondary lymphoid organs, including thymus and lymph nodes, which are characterized by hypoxia (e.g., 2–10% oxygen tension against a 21% atmospheric oxygen tension). Oxygen deprivation can induce autophagy which is a catabolic process involving an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, in order to maintain cellular homeostasis and facilitate adaptation to adverse conditions. Autophagy has a great variety of physiological and physio-pathological roles such as intracellular protein and organelle clearance, elimination of microorganisms, anti-aging activity, tumour suppression, antigen presentation, development and cell death. While the autophagy in DCs was shown to affect Tool-like receptors, its regulation by the lipopolysaccharides (LPS) is still unclear. For this reason, we investigated whether hypoxia can affect autophagy in unstimulated and LPS-stimulated DCs. Along HIF-1α expression, we observed a modulation in the expression of autophagic markers as well as enhanced lysosomal activity after exposure of DCs to hypoxia. To this purpose, using immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon oxygen levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxic conditions, LPS induced a significant increase of functional lysosomes, LC3B-II accumulation, Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This was associated with the activation of signaling pathways and expression of cytokines typically associated with DC survival and autophagy. Finally, by using Bafilomycin A1 and chloroquine, which are well known autophagic inhibitors, we confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. Our data underline the importance of hypoxia in the physiology of DCs and may contribute to further understand DC function. Moreover, understanding the autophagy role in DC adaptation to hypoxia may have a major impact on vaccine development since it may help to improve DC survival and antigen presentation. In conclusion, our results show that autophagy plays a pivotal role in the regulation of DC survival under hypoxia upon LPS stimulation.