Aryl Hydrocarbon Receptor Controls Monocyte Differentiation into Dendritic Cells versus Macrophages.

After entering tissues, monocytes differentiate into cells that share functional features with either macrophages or dendritic cells (DCs). How monocyte fate is directed toward monocyte-derived macrophages (mo-Macs) or monocyte-derived DCs (mo-DCs) and which transcription factors control these differentiation pathways remains unknown. Using an in vitro culture model yielding human mo-DCs and mo-Macs closely resembling those found in vivo in ascites, we show that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DCs and mo-Macs, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Macs. AhR deficiency also impaired the in vivo differentiation of mouse mo-DCs. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DCs and mo-Macs are controlled by distinct transcription factors and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors.

TLR or NOD receptor signaling skews monocyte fate decision via distinct mechanisms driven by mTOR and miR-155.

Monocytes are rapidly recruited to inflamed tissues where they differentiate into monocyte-derived macrophages (mo-mac) or dendritic cells (mo-DC). At infection sites, monocytes encounter a broad range of microbial motifs. How pathogen recognition impacts monocyte fate decision is unclear. Here, we show, using an in vitro model allowing the simultaneous differentiation of human mo-mac and mo-DC, that viruses promote mo-mac while Mycobacteria favor mo-DC differentiation. Mechanistically, we found that pathogen sensing through toll-like receptor (TLR) ligands increases mo-mac differentiation via mTORC1. By contrast, nucleotide-binding oligomerization domain (NOD) ligands favor mo-DC through the induction of TNF-α secretion and miR-155 expression. We confirmed these results in vivo, in mouse skin and by analyzing transcriptomic data from human individuals. Overall, our findings allow a better understanding of the molecular control of monocyte differentiation and of monocyte plasticity upon pathogen sensing.

Culture System Allowing the Simultaneous Differentiation of Human Monocytes into Dendritic Cells and Macrophages Using M-CSF, IL-4, and TNF-α.

Monocytes circulate in the blood and infiltrate tissues where they differentiate into either macrophages or dendritic cells, in particular during inflammation. In vivo, monocytes are exposed to various signals that modulate their commitment toward macrophage or dendritic cell fate. Classical culture systems for human monocyte differentiation yield either macrophages or dendritic cells, but not both populations in the same culture. In addition, monocyte-derived dendritic cells obtained with such methods do not closely mimic dendritic cells that are present in clinical samples. Here, we describe a protocol to simultaneously differentiate human monocytes into macrophages and dendritic cells that resemble their in vivo counterparts from inflammatory fluids.

Assessing the Ability of Human Dendritic Cells to Stimulate Naive CD4+ and CD8+ T Cells.

Dendritic cells orient T cell responses via antigen presentation and provision of polarizing signals. The ability of human dendritic cells to polarize effector T cells can be assessed in mixed lymphocyte reactions. Here we describe a protocol that can be used with any human dendritic cell to assess their ability to polarize CD4+ T helper cells or CD8+ cytotoxic T cells.

Antigen presentation by mouse monocyte-derived cells: Re-evaluating the concept of monocyte-derived dendritic cells.

Antigen presentation is a key feature of classical dendritic cells (cDCs). Numerous studies have also reported in mouse that, upon inflammation, monocytes enter tissues and differentiate into monocyte-derived DCs (mo-DC) that have the ability to present antigens to T cells. However, a population of inflammatory cDCs sharing phenotypic features with mo-DC has been recently described, challenging the existence of in vivo-generated mo-DC. Here we review studies describing mouse mo-DC in the light of these findings, and evaluate the in vivo evidence for monocyte-derived antigen-presenting cells. We examine the strategies used to demonstrate the monocytic origin of these cells. Finally, we propose that mo-DC play a complementary role to cDCs, by presenting antigens to effector T cells locally in tissues.

In vivo Differentiation of Human Monocytes.

Circulating monocytes can infiltrate mucosal or inflamed tissues where they differentiate into either macrophages or dendritic cells. This paradigm is supported by numerous studies conducted in mice and in different in vitro settings for human cells. Determining whether it holds true in vivo in humans is essential for the successful design of monocyte-targeting therapies. Despite limitations inherent to working with human samples, there is accumulating evidence of the existence of in vivo-generated monocyte-derived cells in humans. Here, we review recent studies showing the recruitment of human monocytes into tissues and their differentiation into macrophages or dendritic cells, in normal or pathological settings. We examine the methods available in human studies to demonstrate the monocytic origin of infiltrating cells. Finally, we review the functions of human monocyte-derived cells and how they might contribute to pathogeny.

Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry.

The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used simultaneously. We used this protocol to specifically measure the expression of two transcription factor mRNAs, MAFB and IRF4, in human monocytes.