SBI coordinated with MSCs to modulate IL-6/pSTAT3 signaling.
In the current and previous studies[6], we revealed that Tfh cells were also an important cell type under MSC regulation. To explore the mechanism of SBI in collaboration with MSCs to inhibit Tfh cells, we examined the proliferation and apoptotic status of Tfh cells in splenocytes of R848-induced mice after different treatments in vitro , and found that both treatments had no effect on the Ki67+ Tfh cell proportion, while the effect on the Annexin V+Tfh cell proportion was quite opposite (Fig. 4A).
Next the expression levels of genes closely related to Tfh cell differentiation were examined by qRT-PCR. Compared with acetone-treated mice, mRNA expression of inducible T cell costimulator (Icos ), Stat3 and IL-6 was significantly increased in the splenocytes of R848-induced mice, while SBI treatment reduced mRNA expression of Stat3 and IL-6(Fig. 4B). SBI enhanced the ability of MSCs to suppress mRNA expression of Icos , Stat3 , and IL-6 in the splenocytes of R848-induced mice. (Fig. 4C). Consistently, SBI inhibited the phosphorylation of STAT3 protein in the splenocytes of R848-induced lupus mice (Fig. 4D), implying that SBI may coordinate with MSCs to modulate IL-6/pSTAT3 signaling, thereby inhibiting Tfh cell differentiation.
IL-6 and its downstream pathways counteracted the effect of SBI on Tfh cell production.
To confirm that SBI regulated Tfh cell production through the inhibition of IL-6, we added IL-6 to R848-induced mice splenocyte cultures with the presence of SBI. As expected, IL-6 blocked the effect of SBI on Tfh cell expansion (Fig. 5A). Since IL-6 may initiate downstream signaling through the mTOR and STAT3 pathways, we subsequently measured the mean fluorescence intensity of the mTOR downstream targets phosphorylated serine/threonine kinase Akt (pAKT) and phosphorylated ribosomal protein S6 (pS6), as well as pSTAT3, by flow cytometry and found that their expression in Tfh cells was all inhibited by SBI and rebounded upon addition of IL-6 (Fig. 5B-C). Correspondingly, both the mTOR inhibitor rapamycin and the STAT3 inhibitor Stattic partially dampened IL-6-mediated Tfh cell expansion (Fig. 5D-E), whereas both the STAT3 agonist colivelin and the mTOR agonist MHY1485 partially attenuated the inhibitory effect of SBI on Tfh cells, with colivelin showing a more pronounced effect compared to MHY1485 (Fig. 5F). These findings suggest that that SBI inhibits Tfh cell production by regulating IL-6 and its downstream pathways.