Hypoxia Shapes Autophagy in LPS-Activated Dendritic Cells

During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.

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Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

Scientific Reports ◽

10.1038/s41598-020-77024-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

A. Casasola-LaMacchia ◽

M. S. Ritorto ◽

R. J. Seward ◽

N. Ahyi-Amendah ◽

A. Ciarla ◽

...

Keyword(s):

Mass Spectrometry ◽

Dendritic Cells ◽

Human Leukocyte ◽

Human Monocyte ◽

Fold Increase ◽

Protein Quantification ◽

Protein Levels ◽

Hla Dr ◽

Α Chain ◽

Hla Dqa1

AbstractThe major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3–7% and ≤ 1%, respectively.

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IL-1β Induces SOCS2 Expression in Human Dendritic Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20235931 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5931

Author(s):

Muamera Sarajlic ◽

Theresa Neuper ◽

Kim Tamara Föhrenbach Quiroz ◽

Sara Michelini ◽

Julia Vetter ◽

...

Keyword(s):

Dendritic Cells ◽

Myeloid Leukemia ◽

Inflammatory Responses ◽

Human Monocyte ◽

Cytokine Signaling ◽

Potent Inducer ◽

Protein Levels ◽

Surface Marker Expression ◽

Human Dendritic Cells

Dendritic cells (DCs) regulate immunity and inflammation and respond to various stimuli, including cytokines. IL-1β is a key cytokine in the course of both acute and chronic inflammatory responses, making it indispensable for protection of the host, but also linking it to several diseases. Thus, IL-1β signaling must be tightly regulated. As suppressor of cytokine signaling (SOCS) proteins effectively control immune responses, we investigated the role of SOCS2 in IL-1β-induced DC activation. Human monocyte-derived DCs were stimulated with IL-1β, and SOCS2 mRNA and protein levels were measured. DC activation was assessed by cytokine secretion and surface marker expression. For functional analysis, small interfering RNA (siRNA)-based SOCS2 silencing was performed. SOCS2 expression was also analyzed in a curated NCBI GEO dataset of myeloid leukemia patients. We found IL-1β to be a potent inducer of SOCS2 expression. By silencing SOCS2, we showed that SOCS2 specifically limits IL-1β-induced IL-8 secretion. Moreover, our analysis revealed that SOCS2 levels are significantly increased in patients with acute and chronic myeloid leukemia, two hematological malignancies where disease progression is closely linked to IL-1β. This study identifies SOCS2 as a novel IL-1β-inducible target gene and points toward a potential role of SOCS2 in IL-1β-mediated DC activation.

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Hypoxia Enhances the Expression of RNASET2 in Human Monocyte-Derived Dendritic Cells: Role of PI3K/AKT Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22147564 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7564

Author(s):

Sara Monaci ◽

Federica Coppola ◽

Gaia Giuntini ◽

Rossella Roncoroni ◽

Francesco Acquati ◽

...

Keyword(s):

Dendritic Cells ◽

Human Monocyte ◽

Cell Types ◽

Nuclease Activity ◽

Host Immunity ◽

Akt Pathway ◽

Tlr4 Ligand ◽

Tumor Growth And Metastasis ◽

Antigen Presenting

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O2) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs’ survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.

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A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell ResponsesIn Vivo

BioMed Research International ◽

10.1155/2013/864720 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 12

Author(s):

Laura Arribillaga ◽

Maika Durantez ◽

Teresa Lozano ◽

Francesc Rudilla ◽

Federico Rehberger ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Fusion Protein ◽

Fluorescent Protein ◽

Vaccine Development ◽

Human Monocyte ◽

Cell Immune Response ◽

Ns3 Protein ◽

Tlr4 Ligand ◽

Green Fluorescent

The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with aKd~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβby TLR4-expressing cells, as well as the production of TNF-αby the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

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Interleukin-1β and surface marker expression changes induced by tetrachloroplatinate in human monocyte-derived dendritic cells

Immunopharmacology and Immunotoxicology ◽

10.1080/08923970903095306 ◽

2009 ◽

Vol 00 (00) ◽

pp. 090824065221002-10

Author(s):

Joanna Arkusz ◽

Dobrosława Gradecka-Meesters ◽

Maciej Stępnik

Keyword(s):

Dendritic Cells ◽

Human Monocyte ◽

Surface Marker ◽

Interleukin 1Β ◽

Surface Marker Expression ◽

Marker Expression

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Faculty Opinions recommendation of Multiple signaling pathways contribute to synergistic TLR ligand-dependent cytokine gene expression in human monocyte-derived macrophages and dendritic cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1156938.617001 ◽

2009 ◽

Author(s):

Stephen Schwartz

Keyword(s):

Gene Expression ◽

Dendritic Cells ◽

Signaling Pathways ◽

Human Monocyte ◽

Cytokine Gene Expression ◽

Cytokine Gene ◽

Multiple Signaling

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Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells

Biology ◽

10.3390/biology10060490 ◽

2021 ◽

Vol 10 (6) ◽

pp. 490

Author(s):

Xueqi Qu ◽

Christiane Neuhoff ◽

Mehmet Ulas Cinar ◽

Maren Pröll ◽

Ernst Tholen ◽

...

Keyword(s):

Dna Methylation ◽

Dendritic Cells ◽

Epigenetic Modifications ◽

Dna Demethylation ◽

Dependent Manner ◽

Experimental Conditions ◽

Hdac Activity ◽

Protein Levels ◽

Anti Inflammatory ◽

Epigenetic Modulation

Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.

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Human Herpesvirus 6A Induces Dendritic Cell Death and HMGB1 Release without Virus Replication

Pathogens ◽

10.3390/pathogens10010057 ◽

2021 ◽

Vol 10 (1) ◽

pp. 57

Author(s):

Rasmus Gustafsson

Keyword(s):

Cell Death ◽

Dendritic Cells ◽

High Mobility ◽

Human Herpesvirus ◽

Human Monocyte ◽

Hmgb1 Protein ◽

Innate And Adaptive Immunity ◽

Th2 Polarization ◽

Dependent Cell ◽

Ifn Γ

Human herpesvirus 6A (HHV-6A) is a common virus that has important immunomodulatory effects. Dendritic cells (DC) are key players in innate and adaptive immunity and are implicated in the pathogenesis of many human diseases, including infections. (1) Background: Previous studies have demonstrated suppressive effects of HHV-6A on key DC functions. (2) Methods: human monocyte derived dendritic cells were inoculated with HHV-6A and viral replication, cell viability, and release of high mobility group box 1 (HMGB1) protein from DC and of the cytokines IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ after co-culture with allogenic CD4+ T cells were assessed. (3) Results: Nonproductive infection of HHV-6A in DC leads to titer-dependent cell death and the release of HMGB1 protein, and a Th2 polarization. (4) Conclusion: These immune responses aimed to clear the infection may also imply risks for inflammatory pathologies associated with HHV-6A such as multiple sclerosis.

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