Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells

Abstract

The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

“…When analysing B‐cells expressing activation/plasma cell markers CD38+ and CD27+, both RA alone and RA complexed in holo ‐Alt a 1 significantly enhanced its expression (Figure 2H). This is in line with the observation that RA mediates differentiation of human peripheral B lymphocytes into plasma cells with enhanced CD38+ and CD27+ expression 38,39 . These studies also proposed that the cytokine TGF‐β may synergize with RA in the IgA class switching of B‐cells, 38,39 with a role in immune homeostasis and peripheral tolerance induction 40 .…”

“…This is in line with the observation that RA mediates differentiation of human peripheral B lymphocytes into plasma cells with enhanced CD38+ and CD27+ expression. 38,39 These studies also proposed that the cytokine TGFβ may synergize with RA in the IgA class switching of B-cells, 38,39 with a role in immune homeostasis and peripheral tolerance induction. 40 Indeed, in our samples (Figure 2I) we detected enhanced total IgA levels rather when PBMCs were treated with RA complexed to holo-Alt a 1, than with RA or Alt a 1 alone.…”

QUAS-R: Glutamine (Q) Uptake Assay with Single cell Resolution reveals metabolic heterogeneity with immune populations

“…One barrier to applying this approach for use in complex multiparametric flow cytometry analysis is the fact that copper-click chemistry has the potential to significantly quench the fluorescence of protein fluorophores, such as phycoerythrin (PE), thereby significantly reducing the multiplexing power of this assay 38,39 .…”