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. 2022 Nov 18;11(22):3659.
doi: 10.3390/cells11223659.

Oxidized Proteins Differentially Affect Maturation and Activation of Human Monocyte-Derived Cells

Ramona Clemen  1 , Kevin Arlt  1 , Lea Miebach  1   2 , Thomas von Woedtke  1   3 , Sander Bekeschus  1
Affiliations

Oxidized Proteins Differentially Affect Maturation and Activation of Human Monocyte-Derived Cells

Ramona Clemen et al. Cells. .

Abstract

In cancer, antigen-presenting cells (APC), including dendritic cells (DCs), take up and process proteins to mount adaptive antitumor immune responses. This often happens in the context of inflamed cancer, where reactive oxygen species (ROS) are ubiquitous to modify proteins. However, the inflammatory consequences of oxidized protein uptake in DCs are understudied. To this end, we investigated human monocyte-derived cell surface marker expression and cytokine release profiles when exposed to oxidized and native proteins. Seventeen proteins were analyzed, including viral proteins (e.g., CMV and HBV), inflammation-related proteins (e.g., HO1 and HMGB1), matrix proteins (e.g., Vim and Coll), and vastly in the laboratory used proteins (e.g., BSA and Ova). The multifaceted nature of inflammation-associated ROS was mimicked using gas plasma technology, generating reactive species cocktails for protein oxidation. Fourteen oxidized proteins led to elevated surface marker expression levels of CD25, CD40, CD80, CD86, and MHC-II as well as strongly modified release of IL6, IL8, IL10, IL12, IL23, MCP-1, and TNFα compared to their native counterparts. Especially IL8, heme oxygenase 2, and vimentin oxidation gave pronounced effects. Furthermore, protein kinase phospho-array studies in monocyte-derived cells pulsed with native vs. oxidized IL8 and insulin showed enhanced AKT and RSK2 phosphorylation. In summary, our data provide for the first time an overview of the functional consequences of oxidized protein uptake by human monocyte-derived cells and could therefore be a starting point for exploiting such principle in anticancer therapy in the future.

Keywords: ROS; antigen uptake; calreticulin; heat shock protein 27; heme oxygenase; high-mobility group box 1; inflammation; interleukin-8; kINPen; reactive oxygen species; tumor microenvironment.

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Conflict of interest statement

The authors declare no conflict of interest regarding this paper publication.

Figures

Figure A1
Figure A1
Correlation analysis between protein molecular weight and activation of myeloid-derived cells. (a) characteristics of proteins used in this study; (b) Pearson’s correlation between protein molecular weight (native; in kDa) and fold change expression of CD25, CD40, MHC-II, and CD80/86 on myeloid-derived cells after stimulation with oxidized proteins; (c,d) Pearson’s correlation between protein molecular weight (native; c) and cysteine and tyrosine numbers (d) and fold change cytokine release after stimulation of myeloid-derived cells with oxidized proteins.
Figure 1
Figure 1
Monocyte-derived cells surface marker expression after uptake of different proteins. (a) scheme of the workflow to investigate the effect of proteins on monocyte-derived cells; (b) representative histogram of surface marker expression of CD25, CD40, HLA-DR, and CD80 investigated using flow cytometry 24 h after stimulation with the proteins; (c–f) quantification of activation marker CD25 (c), co-stimulatory receptor CD40 (d), maturation marker HLA-DR (e), and co-stimulatory and activation markers CD80/CD86 (f). Data are mean of six independent experiments, normalized to each corresponding native protein. Statistical analysis was performed using Mann–Whitney Test comparing each protein to PBS (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Figure 2
Figure 2
Secretion profiles of monocyte-derived cells incubated with different proteins. (a–h) supernatants of stimulated monocyte-derived cells were analyzed for pro-inflammatory and anti-inflammatory cytokines (in pg/mL) using a multiplex assay: IL1β (a), IL6 (b), IL8 (c), IL10 (d), IL12 (e), IL23 (f), MCP-1 (g), and TNFα (h); (i) principal component analysis of surface marker and secretion profiles. Data are mean of technical replicates of an assay with six independent experiments. Gas plasma-treated proteins were normalized to each corresponding native protein; LPS was normalized to PBS. Statistical analysis was performed using Mann–Whitney Test by comparing the proteins to PBS (p < 0.05, p < 0.01, p < 0.001 are grouped and given as *).
Figure 3
Figure 3
Gas plasma-oxidized proteins stimulate activation and maturation in phagocytes. (a) scheme of the experimental workflow; (b) gas plasma treatment of protein solutions; (c–f) representative histogram of surface marker expression of CD25 (c), CD40 (d), HLA-DR (e), and CD80 (f) investigated using flow cytometry 24 h after stimulation with proteins; (g–j) quantification of activation marker CD25 (g), co-stimulatory receptor CD40 (h), maturation marker HLA-DR (i), and activation markers CD80/CD86 (j). Data are mean of six independent experiments, normalized to each corresponding native protein. Statistical analysis was performed using Mann–Whitney Test comparing each protein to its native (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Figure 4
Figure 4
Secretion profiles of monocyte-derived cells incubated with oxProteins. (a–h) stimulated monocyte-derived cells’ supernatants were analyzed for cytokines using a multiplex assay: IL1β (a), IL-6 (b), IL8 (c), IL10 (d), IL12 (e), IL23 (f), MCP-1 (g), and TNFα (h); (i) principal component analysis of surface marker and secretion profiles. Data are mean of technical replicates of an assay with six independent experiments. Gas plasma-treated proteins were normalized to each corresponding native protein. Statistical analysis was performed using Mann–Whitney Test by comparing the oxidized proteins to the native (significances p < 0.05, p < 0.01, p < 0.001 are grouped and given as *).
Figure 5
Figure 5
Gas plasma-treated insulin and IL8 reveal different modes of action in signaling cascades. (a,b) IL8 and insulin were chosen for phosphorylation assay (upper panel: antibody loading (a); lower panel: representative membranes (b)); (c) phosphorylation state of signaling proteins after stimulating monocyte-derived cells with oxIL8, oxIns, oxOva, or LPS; (d) simplified scheme of assignment of the proteins to different signaling pathways leading to phosphorylation of transcription factors CREB (e), MSK (f), and mTOR (g) using phospho-arrays. ctr = native protein/ PBS in case of normalized LPS. Data are mean of six to nine independent experiments.
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Grants and funding

This work was funded by the German Federal Ministry of Education and Research (BMBF), grant numbers 03Z22DN11 and 03Z22Di1 (both to SB). The funding source had no role in the design of this study or its execution, analyses, interpretation of the data, or decision to publish results.