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Contains fulltext : 206940.pdf (Publisher’s version ) (Open Access) Solid tumors grow at a high speed leading to insufficient blood supply to tumor cells. This makes the tumor hypoxic, resulting in the Warburg effect and an increased generation of reactive oxygen species (ROS). Hypoxia and ROS affect immune cells in the tumor micro-environment, thereby affecting their immune function. Here, we review the known effects of hypoxia and ROS on the function and physiology of dendritic cells (DCs). DCs can (cross-)present tumor antigen to activate naive T cells, which play a pivotal role in anti-tumor immunity. ROS might enter DCs via aquaporins in the plasma membrane, diffusion across the plasma membrane or via extracellular vesicles (EVs) released by tumor cells. Hypoxia and ROS exert complex effects on DCs, and can both inhibit and activate maturation of immature DCs. Furthermore, ROS transferred by EVs and/or produced by the DC can both promote antigen (cross-)presentation through phagosomal alkalinization, which preserves antigens by inhibiting proteases, and by direct oxidative modification of proteases. Hypoxia leads to a more migratory and inflammatory DC phenotype. Lastly, hypoxia alters DCs to shift the T- cell response towards a tumor suppressive Th17 phenotype. From numerous studies, the concept is emerging that hypoxia and ROS are mutually dependent effectors on DC function in the tumor micro-environment. Understanding their precise roles and interplay is important given that an adaptive immune response is required to clear tumor cells.

Cells are exposed to reactive oxygen species (ROS) as a by-product of mitochondrial metabolism, especially under hypoxia. ROS are also enzymatically generated at the plasma membrane during inflammation. Radicals cause cellular damage leading to cell death, as they react indiscriminately with surrounding lipids, proteins, and nucleotides. However, ROS are also important for many physiological processes, including signaling, pathogen killing and chemotaxis. The sensitivity of cells to ROS therefore likely depends on the subcellular location of ROS production, but how this affects cell viability is poorly understood. As ROS generation consumes oxygen, and hypoxia-mediated signaling upregulates expression of antioxidant transcription factor Nrf2, it is difficult to discern hypoxic from radical stress. In this study, we developed an optogenetic toolbox for organelle-specific generation of ROS using the photosensitizer protein SuperNova which produces superoxide anion upon excitation with 590 nm light. We fused SuperNova to organelle specific localization signals to induce ROS with high precision. Selective ROS production did not affect cell viability in most organelles except for the nucleus. SuperNova is a promising tool to induce locally targeted ROS production, opening up new possibilities to investigate processes and organelles that are affected by localized ROS production.

Contains fulltext : 200162.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-alpha secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Ethylene is a major plant hormone mediating developmental processes and stress responses to stimuli such as infection. We show here that ethylene is also produced during systemic inflammation in humans and is released in exhaled breath. Traces of ethylene were detected by laser spectroscopy both in vitro in isolated blood leukocytes exposed to bacterial lipopolysaccharide (LPS) as well as in vivo following LPS administration in healthy volunteers. Exposure to LPS triggers formation of ethylene as a product of lipid peroxidation induced by the respiratory burst. In humans, ethylene was detected prior to the increase of blood levels of inflammatory cytokines and stress-related hormones. Our results highlight that ethylene release is an early and integral component of in vivo lipid peroxidation with important clinical implications as a breath biomarker of bacterial infection.

Contains fulltext : 170881.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer.