Human Monocyte-Derived Dendritic Cells

NOX2 Assembles in the Phagocytic Cup of Monocyte-Derived Dendritic Cells and Rapidly Produces ROS

Reactive oxygen species (ROS) are crucial in both pathogen elimination and antigen presentation. Neutrophils exhibit short, intense bursts of ROS mainly for pathogen killing. Whereas dendritic cells sustain lower ROS production rates, essential for antigen cross-presentation and adaptive immunity activation. However, the exact ROS production rates in dendritic cells, unlike neutrophils, have not been quantified. Therefore, Paardekooper's team aimed to quantify ROS production in human monocyte-derived dendritic cells by measuring their oxygen consumption rate during phagocytosis.

When neutrophils, macrophages, or dendritic cells engulf a pathogen, they quickly activate the NOX2 protein complex to generate superoxide anions within the phagosome. Hence, Paardekooper et al. examined the timing of NOX2 assembly post-phagocytosis. Immunofluorescence labeling showed overlap of NOX2 components, gp91phox and p67phox, at the phagosomal membrane just 5 minutes after zymosan addition, indicating rapid NOX2 assembly (Fig. 1A). Further microscopy using FITC-conjugated zymosan revealed NOX2 formation before phagocytic cup closure (Fig.1B-E), suggesting NOX2 activation occurs swiftly upon pathogen contact. Supporting this, about 75% of early endosomal marker EEA1-positive phagosomes exhibited gp91phox (Fig. 2). For insights on ROS production timing after zymosan uptake, we co-incubated dendritic cells with fluorescent zymosan and NBT, observing formazan crystal formation within 2 minutes of contact (Fig. 1F), continuing for at least 25 minutes (Fig. 1G). Together, these results show that NOX2 is assembled already at the nascent cup of emerging phagosomes and ROS production occurs rapidly (i.e., within minutes) after zymosan attachment.

Fig. 1. NOX2 assembly at the phagocytic cup in monocyte-derived dendritic cells (Paardekooper LM, Dingjan I, et al., 2019).

Fig. 2. gp91^phox is present on EEA1-positive phagosomes (Paardekooper LM, Dingjan I, et al., 2019).

Toxicity towards Argon Plasma Treatment and ROS/RNS in Human Monocyte-Derived Dendritic Cells

Recent research suggests that cold physical plasma, which generates reactive oxygen and nitrogen species (ROS/RNS), can be used to treat cancer by its immunological effects. Antigen presentation and inflammation regulation are crucial in triggering antitumor immunity, with dendritic cells (DCs) being central to this process. However, the impact of plasma on DC inflammatory responses remains unclear.

Bekeschus's team aimed to explore the impact of plasma on human monocyte-derived DCs, assessing metabolic activity. They first tested the toxicity towards argon plasma treatment and ROS/RNS. The kINPen argon plasma jet was used for treating monocyte-derived dendritic cells (moDCs) 24 h (Fig. 3a). To assess sensitivity, moDCs and lymphocytes were treated together; dead cells were separately counted (Fig. 3c), revealing lymphocytes as more sensitive to argon plasma-induced cytotoxicity than moDCs (Fig. 3d). They then examined moDCs' metabolic activity and viability post-exposure to argon plasma or various ROS/RNS. Significant differences in metabolic activity were found when assessed kinetically after exposure to pilot ROS/RNS or plasma treatment (Fig. 4a), indicating detectable stress-induced activity changes. Endpoint experiments were performed at 24 hours after exposure to different durations of argon plasma (Fig. 4b) and concentrations of hydrogen peroxide (H₂O₂, Fig. 4c), hypochlorous acid (HOCl, Fig. 4d), peroxynitrite (ONOO, Fig. 4e), and lipopolysaccharide (LPS, Fig. 4f). With the exception of low-toxic LPS, all agents decreased their metabolic activity over time, consistent with flow cytometry viability measurements (Fig. 4g–k) and heatmap display (Fig. 4l).

Fig. 3. Study protocol and toxicity comparison (Bekeschus S, Dorothee M, et al., 2021).

Fig. 4. Argon plasma treatment and reactive oxygen and nitrogen species (ROS/RNS) have dose-dependent toxicity profiles (Bekeschus S, Dorothee M, et al., 2021).