Reactive Oxygen Species Level Defines Two Functionally Distinctive Stages of Inflammatory Dendritic Cell Development from Mouse Bone Marrow

Abstract

Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF–induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF–induced DCs is distinct in two developmental stages. Whereas division of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROSlo DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROShi DCs. ROShi DCs could not initiate and sustain a significant level of NF-κB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROSlo DCs. ROSlo DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROShi DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROShi DCs also demonstrated rapid cellular adhesion and H2O2 release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.

An active mitochondrial biogenesis occurs during dendritic cell differentiation

“…In a previous report, we showed that the differentiation process of peripheral blood monocytes to DC requires growth factor dependent production of intracellular Reactive Oxygen Species (ROS) (Del Prete et al, 2008; see also Sattler et al, 1999;Sheng et al, 2010). As revealed by ultrastructural analysis, DC exhibited an increased number of condensed mitochondria compared to monocyte precursors.…”

Alteration of early dendritic cell activation by cancer cell lines predispose immunosuppression, which cannot be reversed by TLR4 stimulation

“…They are comparable, if not better, to the GM-CSF + IL-4 equivalents. These early GM-CSFderived DCs express lower levels of activation markers than early GM-CSF + IL-4-derived DCs, however as we demonstrate herein, these early DCs derived from GM-CSF cultures rapidly increased the expression levels of CD80, CD86 and MHCII upon LPS and CpG stimulation [14,16], and are capable of secreting cytokines and chemokines after stimulation. In addition, these early GM-CSF-derived DCs, like the early GM-CSF + IL-4-derived DCs, can stimulate T-cell proliferation after exposure to LPS.…”

“…We have shown that the early GM-CSF derived DCs are fully functional in terms of endocytosis, up-regulating activation markers upon TLR stimulation [14,16]. They are comparable, if not better, to the GM-CSF + IL-4 equivalents.…”

“…In addition, we previously demonstrated that reactive oxygen species (ROS) induction in GM-CSF-derived DCs correlated with inflammatory DC functionality and expansion. Day 4 GM-CSF DC cultures expressed very high levels of ROS and day 3 ROS(lo) DCs were highly responsive to tolllike receptor stimuli (LPS) by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROS(hi) DCs [16]. Herein, we further explored the effects of the in vitro cancer cell line RMA on early GM-CSF bone marrow-derived DCs.…”

“…DCs express co-stimulatory markers such as CD80 and CD86 which are important in mediating T-cell functions [13][14][15][16]. Engagement of CD28 on T-cell by CD80 and CD86 induces T-cell activation, proliferation and secretion of immunostimulatory cytokines such as IL-1, IL-6 and IL-12 [17].…”