Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Abstract

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-γ (IFN-γ) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20–60%) copiously expresses iNOS (iNOS hi) upon IFN-γ/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOS lo). Monocyte-derived macrophages (mMϕ) are more homogeneous with regard to iNOS expression. The number of cells within the iNOS lo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-γ/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-γ/HKLM. A contamination of mDC with iNOS-expressing mMϕ was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOS hi mDC than in mMϕ. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOS hi mDC upon triggering with IFN-γ/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-γ/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.