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.