Human Monocyte-Derived Dendritic Cells Produce Millimolar Concentrations of ROS in Phagosomes Per Second

Abstract

Neutrophils kill ingested pathogens by the so-called oxidative burst, where reactive oxygen species (ROS) are produced in the lumen of phagosomes at very high rates (mM/s), although these rates can only be maintained for a short period (minutes). In contrast, dendritic cells produce ROS at much lower rates, but they can sustain production for much longer after pathogen uptake (hours). It is becoming increasingly clear that this slow but prolonged ROS production is essential for antigen cross-presentation to activate cytolytic T cells, and for shaping the repertoire of antigen fragments for presentation to helper T cells. However, despite this importance of ROS production by dendritic cells for activation of the adaptive immune system, their actual ROS production rates have never been quantified. Here, we quantified ROS production in human monocyte-derived dendritic cells by measuring the oxygen consumption rate during phagocytosis. Although a large variation in oxygen consumption and phagocytic capacity was present among individuals and cells, we estimate a ROS production rate of on average ~0.5 mM/s per phagosome. Quantitative microscopy approaches showed that ROS is produced within minutes after pathogen encounter at the nascent phagocytic cup. H 2 DCFDA measurements revealed that ROS production is sustained for at least ~10 h after uptake. While ROS are produced by dendritic cells at an about 10-fold lower rate than by neutrophils, the net total ROS production is approximately similar. These are the first quantitative estimates of ROS production by a cell capable of antigen cross-presentation. Our findings provide a quantitative insight in how ROS affect dendritic cell function.

A guide to measuring phagosomal dynamics

“…This Potential for hydrogen (pH) decrease is in principle necessary for complete degradation of phagosomal cargo, since several hydrolases acquired by phagolysosomes have acidic pH optima. Additionally, reactive oxygen species (ROS) are generated in phagosomes to various degrees and with temporal variations depending on the nature of the phagocyte [32,34,35].…”

Low-dose immune challenges result in detectable levels of oxidative damage

“…Some of these costs may be linked to pro-inflammatory mechanisms, which are valuable for destroying pathogens, but can also cause damage to the host. For example, immune system activation increases production of reactive oxygen species (Paardekooper et al, 2019), which induce oxidative damage in pathogens, resulting in their destruction. However, reactive oxygen species also interact with the host's own tissues (Hoffmann and Griffiths, 2018), resulting in increased levels of oxidative damage in the host as well (Hasselquist and Nilsson, 2012;Arulselvan et al, 2016;Baylor and Butler, 2019).…”

Endoplasmic Reticulum-Associated Degradation-Dependent Processing in Cross-Presentation and Its Potential for Dendritic Cell Vaccinations: A Review

“…This occurs at very high rates (mM/s) and typically eliminates pathogens [105]. However, in DCs, ROS reacts with the protons in the luminal space, which causes an active alkalization [99,106,107]. NOX2 is made up of six subunits: Rac1 or Rac2, gp91phox (containing heme), p22phox, p40phox, p47phox, and p67phox [107].…”

“…However, in DCs, ROS reacts with the protons in the luminal space, which causes an active alkalization [99,106,107]. NOX2 is made up of six subunits: Rac1 or Rac2, gp91phox (containing heme), p22phox, p40phox, p47phox, and p67phox [107]. Active alkalization by NOX2 was shown to be regulated by Rab27a [106], a plasma membrane SNARE protein called VAMP-8 (in both BMDCs and human moDCs) [108], phagosomal SNARE syntaxin-4, and SNAP-23 (in BMDCs) [109].…”

“…While activation of the non-canonical UPR is essential for CP in cDC1s [164], overactivation of the UPR in DCs sometimes has the opposite effect [164,165]. Although ROS play an essential role in antimicrobial defense [105] and take part in CP by actively alkalizing the luminal space [99,106,107] or by the membrane rupture pathway [92], ROS oxidize the cellular membrane and produce peroxidized lipids. In cDCs, dysregulated activation of UPR is induced by peroxidized lipids, leading to aberrant triglyceride synthesis and blunt anti-cancer immunity [165].…”