Modulation of regulatory B cells through differential activation ofiNKT cells with glycolipids contained in liposomes

Abstract

Invariant NKT (iNKT) cells, a subpopulation of NKT cells, have attracted attention because of their ability to be activated by glycolipid antigens. Once activated, iNKT cells can secrete various cytokines to modulate immune responses. The activation of iNKT cells (mainly NKT10 cells, an iNKT cell population capable of secreting IL-10) with the α-galactosylceramide (α-GalCer) can contribute to the recruitment and suppressive effect of regulatory B and T cells (Breg and Treg). Nevertheless, the strong activation of iNKT cells elicited by α-GalCer exhibit limited therapeutic efficacy in asthma and allergies, mainly due to the induction of a mixed pro- and anti-inflammatory cytokine response. However, there have been developed multiple structural analogs of α-GalCer that elicit more selectively cytokine responses by iNKT cells. Thus, it would be highly relevant to determine whether α-GalCer analogs will increase interaction between iNKT cells and B reg cell types during allergic inflammation. We propose the following hypothesis: "The differential activation of iNKT cells with α-GalCer analogs, which expand NKT10 cells and elicit the regulatory- skewed response, increases the expansion of Breg cells during allergic inflammation". Firstly, we identified NKT10 cells in hCD1d-KI mice (a partially humanized murine model for NKT cell responses). Hence, we evaluated different experimental conditions, such as immunization schemes, glycolipid activators of iNKT cells, and the uses of glycolipid delivery systems. We observed a significant expansion of NKT10 cells only in hCD1d-KI mice treated with α-GalCer at seven days, like the proliferation of NKT10 cells reported during the immunization scheme of 30 days. In addition, we obtained glycolipid-contained liposomes. It was observed that incorporating the glycolipid ligands into liposomes remarkably increased the expansion of NKT10 cells. We evaluated the anti-allergic effect of liposomes containing the glycolipids (α-GalCer and AH10-7) in a prophylactic and therapeutic scenery. Our results demonstrated that liposomes containing AH10-7 and OVA (Lp/OVA/AH10-7) induced an anti-allergic effect in BALB/c mice with OVA-induced allergy in a prophylactic scenery. Particularly, we observed a significant decrease in the inflammatory score and the number of mucus-producing cells in the lungs of mice with allergic induction treated with Lp/OVA/AH10-7. Besides, we could not appreciate an expansion of IL-10-producing regulatory B cells (B10 cells) in mediastinal lymph nodes (MLN) from mice with OVA-induced allergy and treated with Lp/OVA/AH10-7 in a therapeutic scenery. Since this expansion was not statistically significant, future experiments must verify the proliferation of B10 cells induced by Lp/OVA/AH10-7. On the other hand, we observed, a complete decrease in lung inflammation and goblet cell hyperplasia in mice with OVA-induced allergy treated with two regimens of doses of liposomes containing OVA the α-GalCer. Finally, we could appreciate that it is tricky to induce an OVA model in hCD1d-KI mice (C57BL/6J background). Additionally, we didn’t obtain a severe allergic response in the lung and the BAL of mice challenged with HDM. Consequently, it is necessary to improve the obtention of the HDM induced-allergic model in hCD1D-KI mice.