P038 Retinoic acid primed dendritic cells induce Interferon gamma (IFN ³ ) and reduce FOXP3 expression on human Th9 cells
R. Rampal*1, A. Awasthi2, V. Ahuja1
1All India Institute of Medical Sciences, Gastroenterology and Human Nutrition, New Delhi, India, 2Translational Health Science and Technology Institute , CHME, Gurgaon, India
Retinoic acid (RA) produced by intestinal dendritic cells (DCs) induces gut homing molecules, α 4 β 7 and CCR9 on T cells and in presence of TGF β drives the differentiation pathway towards FOXP3+ Treg cells while inhibiting the Th17 and Th1 differentiation. Th9 is a recently identified CD4 T helper cell subset, which secretes Interleukin (IL) -9 in the presence of TGF β , and IL-4.While TGF β initiates Treg differentiation, IL-4 inhibits FOXP3 and promotes Th9 differentiation. We investigated the effect of RA primed DCs on CD4+ T cell responses with and without TGF β and IL-4 in healthy human subjects.
Monocyte (Mo) derived DCs were generated from peripheral blood samples collected from healthy human subjects by incubating CD14+ cells with GM-CSF and IL-4 in the presence and absence of RA. LPS stimulated Mo-DCs and RA-DCs were co-cultured with sorted naïve CD4+ T cells in the presence and absence of TGF β and IL-4 (Th9 conditions)) and cytokine expression of T cells were checked by flowcytometry. The data was analyzed with Graph Pad Prism 5.0 software.
Characterization of RA-primed DCs showed an increased expression of CD103 while HLA-DR, CD86 and CD83 were decreased as compared to non-RA-primed DCs. These RA primed DCs, when co-cultured with naïve CD4+T cells induced elevated levels of IFN- γ and FOXP3 with reduction in IL-9 and IL-17. When naïve CD4 T cells and RA-DCs were cultured in presence of TGF- β and IL-4 , there was reduction in IL-9, however, it resulted in significantly reduced FOXP3 and elevated IFN- γ expression( as shown in figure below).
“Naïve CD4+ T cells were stimulated with Mo-DC and RA-DC in the presence of TGFb and IL-4. Percentages of (a) IL-9+ cells (b) IFNg+ cells (c) Foxp3+ cells were determined by flow cytometry. * P<0.05, *** P<0.0001”
We report a novel role of RA in immune regulation. RA inhibits DC maturation, thereby maintaining its anti-inflammatory role. On the other hand, RA not only reduces the IL-9 production by CD4+T cells but surprisingly also reduces FOXP3 while inducing IFN γ expression suggesting a pro-inflammatory role.