Precise Delineation and Transcriptional Characterization of Bovine Blood Dendritic-Cell and Monocyte Subsets

Abstract

A clear-cut delineation of bovine bona fide dendritic cells (DC) from monocytes has proved challenging, given the high phenotypic and functional plasticity of these innate immune cells and the marked phenotypic differences between species. Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 (cDC1, cDC2), plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4−, cDC2 being Flt3+CD172a+CD13−CD4−, pDC being Flt3+CD172adimCD13−CD4+, and monocytes being Flt3−CD172ahighCD13−CD4−. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16−) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for “marker molecules” that—when targeted by flow cytometry—will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another.

Transcriptomic profiling of bovine blood dendritic cells and monocytes following TLR stimulation

“…Plasmacytoid DC stimulated with Gardiquimod and Resiquimod also showed the most pronounced upregulation of CD40, CD80, CD86, and MHC‐II, with individual animals showing exceedingly high increases in expression of these molecules. As bovine cDC1, cDC2, and monocytes already express high levels of co‐stimulatory molecules and MHC‐II ex vivo [14] and partly following incubation in culture medium, as shown in Supporting Information S1, stimulation with TLR ligands did not always result in a clear further upregulation of these molecules (Fig. 1 and Supporting Information S2).…”

“…Overall, these phenotypic analyses highlight that bovine DC subsets upregulate certain activation markers upon TLR stimulation, CCR7 representing the most consistent activation marker. However, our data also reveal possible bystander effects in bulk cultures of PBMC, acting, for example, on cDC1, which were found responsive to LPS despite the lack of TLR4 transcription in these cells [14].…”

“…In order to identify activation markers for bovine DC subsets and monocytes, bovine PBMCs were incubated with different TLR agonists: Pam2CSK4 (TLR2/6), Poly(I:C) (TLR3, RIG‐I and MDA5), LPS (TLR4), Gardiquimod (TLR7), Resiquimod (TLR7/8), and CpG‐ODN (TLR9) for 4 h. Subsequently, the expression of potential activation markers CCR7, CD80, CD86, CD40, and MHC‐II was analyzed by flow cytometry. Phenotypic definitions of DC subsets and monocytes were based on our previous work [14], which identified cDC1 as Flt3 + CD4 − CD13 + , pDC as Flt3 + CD4 + CD13 − , cDC2 as Flt3 + CD4 − CD13 − , and total monocytes as Flt3 − CD172a high (Fig. 1A).…”

“…Monocytes, on the other hand, are divided into subsets based on their chemokine receptor expression and the presence of specific surface molecules [1,2,12,13]. According to the studies characterizing bovine monocytes [14‐17], bovine peripheral blood monocytes can be split into at least two distinct populations transcriptionally resembling human and murine counterparts [12,18]. Classical pro‐inflammatory CD14 + CD16 − monocytes (cM) constitute the main monocyte subset in blood of human and cattle.…”

“…Recently, our group identified bona fide DC subsets in blood of cattle and delineated them transcriptionally from bovine monocyte subsets [14]. Apart from the confirmation of subset identity, these ex vivo transcriptomic analyses provided important insights into possible functional specialization of bovine DC and monocyte subsets, including subset‐and species‐specific PRR expression.…”