Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Abstract

BackgroundDendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.MethodsOxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H2DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.Results Tert-BHP increased CM-H2DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.ConclusionsOur results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.

CD16+ monocytes give rise to CD103+RALDH2+TCF4+ dendritic cells with unique transcriptional and immunological features

“…CD16 2 compared with CD16 1 MDDCs, were also enriched in transcripts involved in the protection against oxidative stress including myeloperoxidase, DUOX1, and DUOAX1. 77 Interestingly, CD16 2 MDDCs also expressed the sensor for microbial DNA IFI16, 78 previously reported to sense HIV in CD4 1 T cells. 79 Pioneering studies by Sánchez-Torres et al demonstrated that CD16 1 vs CD16 2 MDDCs differ in their immunogenic potential, 43 with differences in CD86 and CD11A expression that are confirmed by our transcriptional studies.…”

Ultra-endurance exercise: unanswered questions in redox biology and immunology

“…For example, dendritic cells are important tissue sentinels that detect and ingest invading pathogens and parts of dying or infected body cells in order to initiate immune responses. PRDX2 allows dendritic cell differentiation by regulating hydrogen peroxide levels via its mild oxidation, providing protection from ROS-induced cell death [40]. However, if PRDX2 is depleted in dendritic cells due to over-oxidation and clearance, the ensuing nonreducing intracellular environment, associated with depletion of other antioxidants, such as thiols, could impair anti-viral immunity.…”

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

“…It should be considered that in the present study PRDX-2 was measured in PBMCs, a heterogenous population of cells consisting mostly of lymphocytes but also some monocytes. While the majority of PRDX-2 would have been derived from lymphocytes as monocytes contain little or no PRDX-2 [15] , the results could in principle, be influenced by the proportion of each cell type in the samples. For example, in the present study, samples collected immediately post-race contained fewer lymphocytes and more monocytes compared to pre-race samples.…”

“…Plasma and peripheral blood mononuclear cells (PBMCs) were processed within 60 minutes of collection and frozen at −80°C as previously described [4] . PBMCs are hereafter referred to as lymphocytes as the results reported below reflect changes in lymphocyte, and not monocyte, PRDX-2 levels: monocytes represent a small proportion of PBMCs and contain little or no detectable PRDX-2 [15] . Cell preparations were thawed and lysed using RIPA buffer (Thermo Scientific, UK) containing a protease inhibitor cocktail (Sigma Aldrich, Dorset UK).…”

“…Indeed, PRDX-2 is the most abundant antioxidant enzyme in the cytosol of lymphocytes, and its levels appear to be involved in the regulation of lymphocyte function [8,14] . Conversely, monocytes, a relatively minor proportion of mononuclear cells in blood, contain little or no PRDX-2 [15] .…”