Novel molecular mediators in human dendritic cells adaptive responses to hypoxia.

Tissue anatomical structure and cell specific energy requirements impair O2 supply and increase O2 demand, participating in the establishment of hypoxia (defined as a reduction in O2 tensions) in several healthy tissues, including immunological niches. Moreover, O2 shortage is associated with inflammatory areas and with the tumor microenvironment, where immune cells are recruited to exert their functions. Dendritic cells (DCs) are the most efficient antigen presenting cells and they provide a link between innate and adaptive immune responses. Indeed, they exist in as immature cells (iDCs), patrolling peripheral tissues for pathogens detection, and as mature DCs (maDCs), mediating naïve T lymphocyte priming in primary and secondary lymphoid organs. Thus, during their lifespan DCs are frequently exposed to different and lower O2 tensions, such as in immunological niches, where pO2 ranges between 10 and 40 mmHg against 100 mmHg of arterial blood. Hypoxia represent a stressful condition for several cell types, including DCs, and evolution has led to the development of cellular and systemic strategies to cope with it. Most of them are mediated by the Hypoxia-inducible factors (HIFs), but in last decades other cellular processes and molecular mediators of cell adaptation to hypoxia have been identified, including PI3Ks, Erk1/2 signaling and autophagy. We here investigated whether p62, a multifunctional protein that acts as a selective autophagy receptor, and Vps34, the only member of Class III PI3K classically associated with the autophagic process, could be involved in hypoxia-induced adaptative responses, in either iDCs or maDCs, especially in cell survival. To this end, we used several chemical compounds and siRNAs and a combination of Western blot, RT-qPCR, autophagy and viability assays. We observed that p62 inhibition, by RNA interference approach, resulted in an impaired hypoxic iDC viability by affecting the Erk1/2 pro-survival program. In addition, SAR405, a potent and selective Vps34 inhibitor, was able to dysregulate autophagy in maDCs, not only for their survival but also for the autophagic-dependent dampening of inflammation. In addition, we evaluated whether RNASET2, a multifunctional ribonuclease, which also affects cell survival, could be involved in DCs responses to the hypoxic stress. We found out that prolonged hypoxic exposure significantly enhanced its expression along with the impairment of DC viability. Furthermore, RNASET2 upregulation was inhibited by LPS stimulation, which is known to exert not only DC final maturation but also a pro-survival function via activation of the PI3K/Akt pathway.