Ascorbic acid supports ex vivo generation of plasmacytoid dendritic cells from circulating hematopoietic stem cells
- Aarhus University, Denmark
- Aarhus University Hospital, Denmark
- niels uldbjerg, Denmark
Abstract
Plasmacytoid dendritic cells (pDCs) constitute a rare type of immune cell with multifaceted functions, but their potential use as a cell-based immunotherapy is challenged by the scarce cell numbers that can be extracted from blood. Here, we systematically investigate culture parameters for generating pDCs from hematopoietic stem and progenitor cells (HSPCs). Using optimized conditions combined with implementation of HSPC pre-expansion, we generate an average of 465 million HSPC-derived pDCs (HSPC-pDCs) starting from 100,000 cord blood-derived HSPCs. Furthermore, we demonstrate that such protocol allows HSPC-pDC generation from whole blood HSPCs, and these cells display a pDC phenotype and function. Using GMP compliant medium, we observe a remarkable loss of TLR7/9 responses, which is rescued by ascorbic acid supplementation. Ascorbic acid induces transcriptional signatures associated with pDC-specific innate immune pathways suggesting an undescribed role of ascorbic acid for pDC functionality. This constitutes the first protocol for generating pDCs from whole blood, and lay the foundation for investigating HSPC-pDCs for cell-based immunotherapy.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Sequencing data have been deposited in Dryad (doi:10.5061/dryad.69p8cz92z).
Article and author information
Author details
Albert Gris-Oliver
Funding
Lundbeckfonden (R238-2016-3349)
- Rasmus O Bak
Aarhus Institute of Advanced Studies, Aarhus Universitet
- Renée M van der Sluis
- Rasmus O Bak
European Union (609033)
- Rasmus O Bak
Lundbeckfonden (R238-2016-2708)
- Martin R Jakobsen
Independent Research Fund Denmark (8020-00201B)
- Martin R Jakobsen
Novo Nordisk Fonden (NNF18OC0053146)
- Martin R Jakobsen
European Union (754513)
- Renée M van der Sluis
Carlsbergfondet (CF17-0129)
- Rasmus O Bak
Carlsbergfondet (CF20-0424)
- Rasmus O Bak
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Carlos Isales, Medical College of Georgia at Augusta University, United States
Ethics
Human subjects: De-identified umbilical cord blood (UCB) samples were obtained following scheduled caesarean section deliveries of healthy infants at Department of Gynecology and Obstetrics, Skejby University Hospital. Consent was obtained from the mothers, but studies on anonymized samples, such as those used in the present study, are exempt from ethical permissions in Denmark (Kommiteeloven {section sign} {section sign}14. 3).Buffy coat samples were obtained from normal healthy donors from Aarhus University Hospital Blood Bank. These were de-identified samples and studies on anonymized samples are exempt from ethical permissions in Denmark (Kommiteeloven {section sign} {section sign}14. 3).
Version history
- Received: December 7, 2020
- Accepted: September 1, 2021
- Accepted Manuscript published: September 2, 2021 (version 1)
- Version of Record published: September 16, 2021 (version 2)
Copyright
© 2021, Laustsen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Ascorbic acid supports ex vivo generation of plasmacytoid dendritic cells from circulating hematopoietic stem cells
eLife 10:e65528.
https://doi.org/10.7554/eLife.65528
Further reading
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- Immunology and Inflammation
- Medicine
Background:
Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin.
Methods:
Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors.
Results:
We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01–2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 μg/mL, p=0.004).
Conclusions:
Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin.
Funding:
LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust).
Clinical trial number:
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- Immunology and Inflammation
Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.
