CD34+ derived macrophage and dendritic cells display differential responses to paraquat

Abstract

Paraquat (PQ) is a redox cycling herbicide known for its acute toxicity in humans. Airway parenchymal cells have been identified as primary sites for PQ accumulation, tissue inflammation and cellular injury. However, the role of immune cells in PQ induced tissue injury is largely unknown. To explore this further, primary cultures of human CD34+ stem cell derived macrophages (MC cd34 ) and dendritic cells (DC cd34 ) were established and characterised using RNA-Seq profiling. The impact of PQ on DC cd34 and MC cd34 cytotoxicity revealed increased effect within DC cd34 cultures. PQ toxicity mechanisms were examined using sub-cytotoxic concentrations and TempO-seq transcriptomic assays. Comparable increases for several stress response pathway (NFE2L2, NF-kB and HSF) dependent genes were observed across both cell types. Interestingly, PQ induced unfolded protein response (UPR), p53, Irf and DC maturation genes in DC cd34 but not in MC cd34 . Further exploration of the immune modifying potential of PQ was performed using the common allergen house dust mite (HD). Co-treatment of PQ and HD resulted in enhanced inflammatory responses within MC cd34 but not DC cd34 . These results demonstrate immune cell type differential responses to PQ, that may underlie aspects of acute toxicity and susceptibility to inflammatory disease.

Mononuclear phagocyte sub-types in vitro display diverse transcriptional responses to dust mite exposure

“…Bone marrow derived CD34+ hemopoietic stem cells (StemCell Technologies, Cambridge, UK) were cultured as previously described 23 . Briefly, up to 5 different donors per experimental procedure were individually expanded for 1 week for both macrophage like cell (MLC) and dendritic like cell (DLC) protocols in Stemspan SFEM II media supplemented with human serum albumin (0.05%), pen/strep, FLT3L (50 ng/ml), TPO (50 ng/ml), SR-1 (1 µM), SCF (50 ng/ml), IL6 (20 ng/ml) and IL-3 (20 ng/ml) in ultra-low attachment plates.…”

“…In this study, we used human primary bone marrow derived CD34+ stem cells to generate different populations of MNPs, found within inflammatory airway disease, with the aim to test responses to pollutant particulate and allergen exposure 23,24 . These stem cells give rise to many if not all of the infiltrating MNP cells in allergic and asthmatic lung disease and have also been observed to replace alveolar macrophages 25 as they are depleted from the lung in disease.…”

“…We therefore considered this in vitro model an appropriate test system to establish a diverse array of MNPs from a primary cell source to best capture the inflammatory cell type responses to environmental adverse exposures as would occur during active lung inflammatory disease. Our previous work has shown, using a protocol specific for macrophages like cell (MLC) differentiation, overexpression of markers reflective of different macrophage cell types, including SPP1, MARCO and CD206, and similarly for dendritic cell (DC) types using a DC specific protocol 23 . Building from this work in our current study, we have used Scseq to further characterise the different cell types within these in vitro systems and tested how some of them respond to HDM exposure.…”

Induced pluripotent and CD34+ stem cell derived myeloid cells display differential responses to particle and dust mite exposure

“…Bone marrow derived CD34+ hemopoietic stem cells (StemCell Technologies, Grenoble, France) were cultured as previously described 19 . Briefly, CD34+ hemopoietic stem cells from 4 different donors were expanded for 1 week in Stemspan medium supplemented with human serum albumin (0.05%), pen/strep, FLT3L (50 ng/ml), TPO (50 ng/ml), SR-1 (1 µM), SCF (50 ng/ml), IL6 (20 ng/ml) and IL-3 (20 ng/ml) in low attachment plates.…”

“…CD34+ hematopoietic stem cells (HSC) isolated from human bone marrow or blood have the potential to self-renew and differentiate towards all lineages of immune cells. The ability of these cells to differentiate towards myeloid cell populations in vitro, including MC and DC using different combinations of growth and differentiation factors 16 – 18 has allowed for the development of mature cell populations useful for toxicological assessments 19 . However, CD34+ derived cells are difficult to obtain in sufficient amounts from healthy individuals and have a limited lifespan limiting their applicability to in vitro toxicity assays 20 .…”