Dataset Information

Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells.

ABSTRACT: Inflammatory monocyte-derived dendritic cells (Mo-DCs) have been described in several chronic inflammatory disorders, such as rheumatoid arthritis (RA), and are suspected to play a detrimental role by fueling inflammation and skewing adaptive immune responses. However, the characterization of their phenotype is still limited, as well as the comprehension of the factors that govern their differentiation. Here, we show that inflammatory Mo-DCs generated in vitro expressed a large and atypical panel of C-type lectin receptors, including isoforms of CD209 and CD206, CD303 and CD207, as well as intracellular proteins at their surfaces such as the lysosomal protein CD208. Combination of these markers allowed us to identify cells in the synovial fluid of RA patients with a close phenotype of inflammatory Mo-DCs generated in vitro. Finally, we found in coculture experiments that RA synoviocytes critically affected the phenotypic differentiation of monocytes into Mo-DCs, suggesting that the crosstalk between infiltrating monocytes and local mesenchymal cells is decisive for Mo-DCs generation.

SUBMITTER: Coutant F

PROVIDER: S-EPMC8307578 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells.

Coutant Frédéric F Pin Jean-Jacques JJ Miossec Pierre P

Cells 20210702 7

Inflammatory monocyte-derived dendritic cells (Mo-DCs) have been described in several chronic inflammatory disorders, such as rheumatoid arthritis (RA), and are suspected to play a detrimental role by fueling inflammation and skewing adaptive immune responses. However, the characterization of their phenotype is still limited, as well as the comprehension of the factors that govern their differentiation. Here, we show that inflammatory Mo-DCs generated in vitro expressed a large and atypical pane ...[more]

PMID: 34359833

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Project description:West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Recent studies in humans have found that dysfunctional T cell responses strongly correlate with development of severe WNV neuroinvasive disease. However, the contributions of human dendritic cells (DCs) in priming WNV-specific T cell immunity remains poorly understood. Here, we demonstrate that human monocyte derived DCs (moDCs) support productive viral replication following infection with a pathogenic strain of WNV. Antiviral effector gene transcription was strongly induced during the log phase of viral growth, while secretion of type I interferons (IFN) occurred with delayed kinetics. Activation of RIG-I like receptor (RLR) or type I IFN signaling prior to log phase viral growth significantly diminished viral replication, suggesting that early activation of antiviral programs can block WNV infection. In contrast to the induction of antiviral responses, WNV infection did not promote transcription or secretion of proinflammatory (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], CCL3, CCL5, and CXCL9) or T cell modulatory (IL-4, IL-12, and IL-15) cytokines. There was also minimal induction of molecules associated with antigen presentation and T cell priming, including the costimulatory molecules CD80, CD86, and CD40. Functionally, WNV-infected moDCs dampened allogenic CD4 and CD8 T cell activation and proliferation. Combining these observations, we propose a model whereby WNV subverts human DC activation to compromise priming of WNV-specific T cell immunity.IMPORTANCE West Nile virus (WNV) is an encephalitic flavivirus that remains endemic in the United States. Previous studies have found dysfunctional T cell responses correlate to severe disease outcomes during human WNV infection. Here, we sought to better understand the ability of WNV to program human dendritic cells (DCs) to prime WNV-specific T cell responses. While productive infection of monocyte-derived DCs activated antiviral and type I interferon responses, molecules associated with inflammation and programming of T cells were minimally induced. Functionally, WNV-infected DCs dampened T cell activation and proliferation during an allogeneic response. Combined, our data support a model whereby WNV infection of human DCs compromises WNV-specific T cell immunity.

Project description:The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.

Dataset Information

Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells.

Publications

Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells.

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