Transcriptional Classification and Functional Characterization of Human Airway Macrophage and Dendritic Cell Subsets

Abstract

The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Due to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole lung bronchoalveolar lavage. Six subsets of phagocytic antigen presenting (HLA-DR+) cells were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1− CD14+, BDCA1+ CD14+, BDCA1+ CD14−, and BDCA1− CD14− cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared to E. coli. Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared to the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole genome transcriptional profiling revealed a clade of “true dendritic cells” consisting of Langerin+, BDCA1+ CD14+, and BDCA1+ CD14− cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6. Each clade, and each member of both clades, were discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.

Human dendritic cell subsets: an update

“…Furthermore, blood cDC2 can be induced to express high langerin and Birbeck granules in response to transforming growth factor‐ β (TGF‐ β ) in vitro although it remains unknown whether cDC2 have any potential to form LC in vivo . Myeloid cDC2 have also been described in the lung, intestine and liver of humans . In the lymph node, most interdigitating cells of the T‐cell areas have a phenotype compatible with cDC2 lineage .…”

“…Populations of monocyte‐derived cells exist in human steady‐state tissues, including the skin, lung and intestine . It is not known if this is a distinct mechanism of recruitment or one that is accelerated and activated in inflammation.…”

“…Fluorescence flow cytometry is the most commonly used platform for identifying human DC. [2][3][4] This has been expanded from the analysis of blood, to single-cell suspensions of tissues including skin, [11][12][13] lung [14][15][16] intestine 17 and liver of humans 2,18,19 and to lymphoid tissue 3,4,[20][21][22] fetal tissues 23 and other body fluids. 24 The Tenth Human Leucocyte Differentiation Antigen workshop has recently reported a range of new antibodies characterized on human DC populations (Table 1).…”

“…Human cDC1 are found in blood and among resident DC of lymph node, tonsil, spleen and bone marrow 3,4,20,29,94,95 and non-lymphoid tissues, skin, lung, intestine and liver. [2][3][4]12,[14][15][16][17][18][19] There are suggestions that they are more enriched in tissues than in the blood, although this has not always been backed up by sufficiently accurate characterization.…”

Introductory Chapter: Dendritic Cells

“…Moreover, CD64 and MAR-1 are considered to be the most selective markers for the effective separation moDC from cDC. In addition to that, the GenChip technology application to DC research has recently demonstrated that DC subsets display different transcriptional factor's requirement in their development and function [7,8]. Several transcription factors including PU.1 (E26 transformation-specific family transcription factor), Bcl11a (C2H2 type zinc-finger protein), Irf4 and Irf8 (Interferon Regulatory Factors 4 and 8, correspondingly), E2-2 (basic helix-loop-helix transcription factor E protein), Id2 (Inhibitor of DNA…”