Ovalbumin-Derived Peptides Activate Retinoic Acid Signalling Pathways and Induce Regulatory Responses Through Toll-Like Receptor Interactions

Abstract

This study investigates the potential of a hydrolysate of ovalbumin with pepsin (OP) to preclude Th2-type immunity by the enhancement of tolerogenic dendritic cells (DCs) and regulatory T (Treg) cells. Through Toll-like receptor (TLR) stimulation, OP enhances the retinoic acid pathway on DCs by means of the induction of aldehyde dehydrogenase enzymes and transforming growth factor beta (TGF-β), and it confers upon DC the ability to upregulate interleukin 10 (IL-10) as well as other tolerance-promoting mediators downstream of TRL signalling, such as IL-27, IL-33, Notch ligands, OX40L, and the transcription factors IRF4 and IRF8. OP-conditioned DCs induce the expansion of Foxp3+ and Tr1 cells in co-culture with CD4+ T cells. Furthermore, OP directly conditions CD4+ T cells from naïve mice, without the mediation of DCs, to express aldehyde dehydrogenase (ALDH) enzymes and, in the presence of the Th2 cytokine IL-4 and exogenous TGF-β, it enhances Foxp3 expression. It is noteworthy that, on CD4+ T cells isolated from egg-allergic mice, OP significantly enriches the levels of Foxp3+ and Foxp3+ RORγt+ CD4+ T cells. In conclusion, we show that food peptides may work, analogously to microbial-driven signals, through TLRs, to promote a tolerogenic phenotype on cells of the innate and adaptive immune system, a property that is further enhanced in the context of a Th2 cytokine-rich environment.

Intestinal factors promoting the development of RORγt+ cells and oral tolerance

“…Ex vivo studies confirmed that the hydrolysate upregulated Aldh1a1 in IECs, which, in turn, conditioned DCs to overexpress Aldh1a2 and Tgfb1 , to release IL-6 and IL-10, and to promote the generation of TGF-β-secreting Foxp3 + RORγt + cells from CD4 + T cells ( 163 ). TLR stimulation is a possible mechanism through which food peptides enhance RALDH activity on murine DCs ( 164 ). Food protein hydrolysates can activate TLRs depending on peptide size and sequence ( 165 ), which may explain why intact proteins are less suited than hydrolysed proteins to generate Foxp3 + RORγt + Tregs, even after having undergone an in vivo digestion process ( 34 ).…”

“…Food protein hydrolysates can activate TLRs depending on peptide size and sequence ( 165 ), which may explain why intact proteins are less suited than hydrolysed proteins to generate Foxp3 + RORγt + Tregs, even after having undergone an in vivo digestion process ( 34 ). Thus, hydrolysates confer specific properties on DCs by means of the upregulation of tolerance-promoting mediators downstream of TRL signalling, such as Aldh1a2 , Tgfb1, Il10, Il27 (IL-27), Il33 (IL-33) , Jag2 and Dll4 (coding for the Notch ligands Jagged2 and Delta4 respectively) and Tnfsf4 (OX40L) ( 164 ). IL-27 supports the differentiation of Foxp3 - Tregs and their IL-10 production ( 166 ).…”

“…However, unlike RA ( 79 ), the hydrolysates significantly upregulate Il10 , possibly because TLR signalling on DCs enhances IL-10 independently of the induction of RALDH activity ( 154 , 171 ). Accordingly, DCs conditioned with the hydrolysate enhance Foxp3 expression in cocultured CD4 + T cells and, in line with their high expression of Il27 and Il10 , they also increase the level of Foxp3 - IL-10 + cells, depending on the concentration of exogenous TGF-β ( 164 ). Interestingly, a singular phenotype of Foxp3 - IL-10 - CD4 + T cells effective against Th2 responses in food allergy was claimed to be induced by DC differentiated in the presence of RA and exposed to TLR stimulation ( 67 ), illustrating that the modulation of RA, IL-10, and IL-27 levels by TLR ligands may modify the balance of Treg subsets with different properties ( 68 , 69 ).…”

“…As indicated, signalling by commensal bacteria on TLRs expressed on Tregs may directly control immune responses in the absence of APCs. Consequently, incubation of CD4 + T cells from naïve mice in the presence of hydrolysate, without DCs, upregulated Aldh1a1 , Aldh1a2 , Tgfb1 , and Il6 expression and induced RALDH activity through TCR stimulation ( 164 ). Although information regarding RALDH expression and subsequent production of RA by CD4 + T cells is very scarce, it has been reported that allogenic, and likely other types of stimulation, increase RALDH activity in conventional CD4 + Tregs, and in particular in Tregs, delivering resistance to cytotoxic agents and immunological tolerance ( 173 ).…”

“…Noteworthy, unlike the case of sensitized mice, the administration of hydrolysates to naïve mice does not modify the expression of enzymes involved in the generation of RA, which implies the absence of effects on vitamin A metabolism under homeostatic conditions ( 34 ). Accordingly, it was found that a Th2-skewed environment is favourable to the induction of a Foxp3 + RORγt + phenotype ( 163 , 164 ). These observations are consistent with the positive effect that certain Th2 mediators, such as IL-4 or IL-13, exert, in synergy with RA, GM-CSF, and TLR ligands, on the activation of Aldh1a2 expression and on the suppression of the production of pro-inflammatory cytokines by DCs, contributing to promote Tregs ( 75 , 175 ).…”