As depicted in Fig. 2a, VIP displayed a slight increase of FoxP3 expression in maternal PBMCs from both groups of women under study. Interestingly, we observed
a significant decrease in FoxP3 expression levels in RSA PBMCs after interaction with trophoblast cells in comparison with that observed in fertile PBMCs. FoxP3 modulation was also accompanied by a significant increase in TGF-β expression CH5424802 mouse and IL-10 secretion, the main anti-inflammatory cytokines, in co-cultures performed with PBMCS from both groups of women (see Fig. 2b,c). In particular, even though the co-cultures performed with RSA PBMCs displayed significantly lower IL-10 secretion after trophoblast interaction, VIP was able to normalize this level to that observed in the cultures with fertile PBMCs. To confirm these results using a more specific technique, we performed co-cultures in the absence or presence Selleck Acalabrutinib of VIP along with its specific antagonist (10−6 M), and Treg frequency was evaluated by FACS. As shown in Fig. 3a, VIP significantly increased the frequency of CD4+CD25+FoxP3+ cells in maternal PBMCs and the specific VIP antagonist prevented this modulation in both
groups of women. The present results suggest that after the interaction with trophoblast cells, VIP specifically shifted the maternal Th1/Treg balance towards a tolerogenic response, increasing the frequency of CD4+CD25+FoxP3+ cells, TGF-β and IL-10 production. In addition, RSA PBMCs displayed an exacerbated Th1 effector response and decreased
frequency of Treg cells after interaction with trophoblast cells. Because VIP added exogenously promoted a tolerogenic profile towards trophoblast antigens, and RSA PBMCs displayed alterations in the Th1/Treg effector responses, SPTBN5 we investigated the VIP/VPAC system under physiological and pathological conditions. First, we evaluated VIP receptor expression, VPAC1 and VPAC2, in maternal PBMCs from RSA and fertile women after the interaction with trophoblast cells. Real-time RT–PCR showed that VPAC1 is expressed constitutively in both groups of women; however, RSA PBMCs displayed significantly lower expression compared with fertile PBMCs (Fig. 4a). Conversely, VPAC2 was not detected by RT–PCR in maternal PBMCs after culture with trophoblast cells (data not shown). We then quantified VIP production by real-time RT–PCR and, as shown in Fig. 4b, RSA PBMCs presented a significant reduction of VIP expression after culture with trophoblast cells in comparison with fertile PBMCs. To confirm this result, we quantified VIP production by maternal CD4+ cells after trophoblast stimulation by means of intracellular staining and FACS analysis. We observed a significant decrease in the frequency of CD4+VIP+ cells in co-cultures performed with RSA PBMCs in comparison with those performed with fertile PBMCs (Fig. 4c).