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

Abstract

Dendritic cells (DC) and monocytes are vital for the initiation of innate and adaptive immune responses. Recently, we identified bona fide DC subsets in blood of cattle, revealing subset‐ and species‐specific transcription of toll‐like receptors (TLR). In the present study, we analyzed phenotypic and transcriptional responses of bovine DC subsets and monocytes to in vitro stimulation with four to six different TLR ligands. Bovine DC subsets, especially plasmacytoid DC (pDC), showed a clear increase of CCR7, CD25, CD40, CD80, CD86, and MHC‐II expression both on mRNA and protein level. Flow cytometric detection of p38 MAPK phosphorylation 15 min after stimulation confirmed activation of DC subsets and monocytes in accordance with TLR gene expression. Whole‐transcriptome sequencing of sorted and TLR‐stimulated subsets revealed potential ligand‐ and subset‐specific regulation of genes associated with inflammation, T‐cell co‐stimulation, migration, metabolic reprogramming, and antiviral activity. Gardiquimod was found to evoke strong responses both in DC subsets and monocytes, while Poly(I:C) and CpG preferentially triggered responses in cDC1 and pDC, respectively. This in‐depth analysis of ligand responsiveness is essential for the rational design of vaccine adjuvants in cattle, and provides a solid basis for comparative studies on DC and monocyte biology across species.

Transcriptomic signature and metabolic programming of bovine classical and nonclassical monocytes indicate distinct functional specializations

“…Furthermore, transcripts for vimentin ( VIM ), reported to be a key positive regulator of the NLRP inflammasome (44), were clearly enriched in cM. While none of the monocyte subsets expressed nitric-oxide synthases at steady state (data not shown), we could recently show that bovine cM massively increase transcription of NOS2 upon in vitro stimulation with TLR ligands (16). Nonclassical monocytes expressed the highest levels of CASP4 and GBP5 , the latter being described as an activator of inflammasome assembly (45).…”

“…Furthermore, lymphmediated entry of antigen-presenting monocytes has been reported for mice (166). However, given the lack of CCR7 upregulation on stimulated bovine monocytes (16), the mechanisms that would enable this migration remain elusive. of the present study are highly valuable for ensuing functional investigations of monocyte subsets across species.…”

“…Stimulation with TLR ligands and staining for phosphorylated p38 MAPK was performed as reported previously (16). Prior to stimulation, cell surface staining was performed.…”

“…These lipid-activated nuclear receptors have evolved as key regulators linking lipid metabolism to inflammation, and in particular expression of PPARG is associated with anti-inflammatory functions (159). We recently showed that bovine cM downregulate PPARG transcription dramatically upon Gardiquimod (TLR7/8) stimulation in vitro (16). PPARA was recently proposed as an important mediator of antimicrobial responses to mycobacteria (160), inducing expression and translocation of TFEB, a key transcriptional activator of autophagy and lysosomal biogenesis.…”

“…Also bovine monocytes, particularly ncM, were reported to induce allogeneic T-cell responses (15). However, neither resting nor TLR-stimulated bovine monocytes express CCR7 (16), which is required for migration of antigen presenting cells from the site of inflammation to the draining lymph node. Classical monocytes are known for their pro-inflammatory role especially in bacterial infections (17), however the role of intM and ncM is less well described.…”