For the first time we studied the effect of BDNF on the proliferation and differentiation of peripheral blood CD4+T cells into Th17 and Treg in SLE patients. The results showed that BDNF inhibited the development of Th17 and up-regulated the development of Treg. In addition, we also explored the signal transduction pathway, which indicated that BDNF affect the development of Th17 and Treg through inhibiting the activation of PI3K-Akt-mTORC1 axis and ERK1/2 pathway.
Recently the theory of neural-immune network has been proposed[25,26]. There were several reports on serum BDNF level in NPSLE patients, suggesting that BDNF was associated with the disease activity and brain parenchymal injury of NPSLE. We also reported BDNF level was significantly decreased in serum of SLE. However, the relationship between BDNF and T cell subpopulation is ambiguous.
Aberrant immune response of T lymphocytes plays a crucial role in the pathogenesis of SLE(Sup.3). Increasing quantity of T helper cells attributes to excessive secretion of inflammatory cytokines. It was reported that there existed an imbalance of cytokines in the serum of SLE[14,29], with elevated cytokines such as IL-4, IL-6, IL-17A. Such imbalance is not limited to SLE flares, but is the hallmark of the disease, since also patients with quiescent disease display a TH17/Treg ratio favoring Th17. Amount of evidences demonstrated that Th17 cells and IL-17A play important roles in the pathogenesis of SLE[28,30]. Differentiation of Th17 and Treg is interrelated and restricted. RORγt and Foxp3 are the key transcription factors for the differentiation of Th17 and Treg[4-6,31]. TGF-β can induce the expression of RORγt and Foxp3 simultaneously, but the interaction between Foxp3 and RORγt inhibits the action of RORγt. Only in the presence of IL-6 the inhibition of Foxp3 on RORγt can be removed and the development of Th17 be promoted[5,32]. Otherwise, Foxp3 promotes the development of Treg[33,34]. As we known, serum IL-6 level in SLE is increased, and IL-6 activates STAT3, thereby down-regulating the expression of Foxp3, resulting in the proportion of Th17 cells increases in SLE.
Our result showed that the proportion of CD4+IL-17A+(Th17) decreased significantly with the increasing BDNF concentration, while the proportion of CD4+CD25+CD127low(Treg) increased. In addition, we found that the expression of RORγtmRNA showed a decreasing intendancy, while the expression of Foxp3mRNA was increased. We proved that BDNF inhibited the differentiation of Th17 by down-regulating the expression of RORγtmRNA, and promoted the differentiation of Treg by up-regulating the expression of Foxp3mRNA. Previously, we have reported that serum BDNF level is lower in active stage of SLE. We speculated that decreased BDNF level in serum of SLE probably weakened its inhibition on the proliferation of Th17, and the deveolpment of Th17 increased, participating in the pathogenesis of SLE. However, BDNF level gradually increased during the convalescence of the disease, restored its inhibition on Th17 and finally up-regulated the deveolpment of Treg. From the results above we speculate that BDNF appears to have a vital role in maintaining the balance of Treg/Th17 ratio.
Previous studies confirmed that the activity of PI3K/Akt pathway was enhanced in murine lupus. The inhibitor of PI3K could improve the symptoms of glomerulonephritis in MRL/Faslpr SLE mice and reduced the mortality. MAPK/ERK was involved in the pathogenesis of SLE and MAPK inhibitor had been shown to reduce autoimmune responses. The differentiation and function of Th17 are controlled by a variety of intracellular signaling pathways and complex transcription factor networks. It was reported that the PI3K-Akt-mTORC1 axis had positive regulating effect on the differentiation of Th17[8,9,40]. In the CD4+T lineage, both PI3K and mTORC1 inhibitors can increase the differentiation of Treg. Kurebayashi Y also reported that the PI3K-Akt-mTORC1-S6K1 axis has positive regulating effect on the differentiation of Th17 by inhibiting the expression of Gfi1 and promoting nuclear translocation of RORγt. As the downstream of mTORC1, S6K1 induces the expression of transcription factors EGR1 and EGR2, and EGR1 and EGR2 directly bind to the Gfi1 promoter to inhibit the expression of Gfi1 and accelerate the differentiation of Th17. S6K2 is the nuclear counterpart of S6K1, which has the role of nuclear localization signal. S6K2 can transport RORγt to the nucleus after binding to RORγt by a back-loading manner. The expression of S6K2 is dependent partly on mTORC1 after TCR stimulation. Therefore, the PI3K-Akt-mTORC1-S6K2 pathway also up-regulates the differentiation of Th17 through the nuclear translocation of RORγt. Otherwise, Th17 differentiation is also positively regulated by HIF-1, a transcription factor induced by hypoxia. Recent studies have shown that STAT3-induced HIF-1 binding to Foxp3 leads to the degradation of Foxp3 proteome, which removes its inhibition on RORγt. Reports have shown that both hypoxia and HIF-1 have positive and negative regulatory effects on the differentiation of Th7 and Treg respectively[43,44].
In addition to RORγt, STAT3 is also an important transcription factor for the differentiation of Th17, and IL-6 is the necessary factor for the activation of STAT3. Ren reviewed the mechanism that mTORC1 up-regulated the expression of IL-17 by STAT3, HIF-1, S6K1 and S6K2. In addition, as the downstream pathway of IL-6 and TGF-β, MAPK/ERK is also involved in differentiation of Th17 and the development of autoimmune diseases. Studies have shown that blocking the activation of ERK pathway can alleviate autoimmune response mediated by Th17 in EAE mouse model. Liu reported the role of ERK in the development of Th17 and Treg. They demonstrated that blocking the activation of the IL-6-induced ERK pathway under the condition of Th17 polarization could down-regulate the expression of RORγt, inhibiting the differentiation of Th17, and up-regulate the differentiation of Treg. In vitro, they also demonstrated that T cells treated with ERK inhibitor produced more TGF-β, reduced differentiation of Th17, and reduced intestinal inflammatory response in colitis.
We explored the signal transduction pathway that BDNF affected differentiation of CD4+T. Our data showed that the phosphorylation of Akt, mTORC1 and ERK1/2 were increased after the addition of TGF-β+IL-6, and the proportion of CD4+IL-17A+ were increased, indicating that we successfully induced the differentiation of Th17 by activating the PI3K-Akt-mTORC1 axis and the ERK1/2 pathway. While the phosphorylation of Akt, mTORC1, ERK1/2 decreased in the BDNF group, the corresponding CD4+IL-17A+ ratio also declined. In the group pre-treated with TrkBIgG, there was no significant change in the phosphorylation of Akt and mTORC1. From the results above we hypothesized that combination of BDNF and TrkB may directly or indirectly down-regulate the development of Th17. We also proved that the PI3K-Akt-mTORC1 axis and ERK1/2 pathway were indeed affected by BDNF/TrkB complex. Similarly, Under the condition of Treg polarization induced by TGF-β, the phosphorylation of Akt and mTORC1 was also reduced in the BDNF group, nevertheless, the percentages of CD4+CD25+CD127low were increased. In the TrkBIgG group, there was no significant change in the phosphorylation of Akt and mTORC1. The phosphorylation of ERK1/2 was also decreased. Our data demonstrated that BDNF inhibited the development of Th17 regardless of Th17 or Treg polarization, and in turn promoted the development of Treg. Compared with initial CD4+T, under the condition of Th17 polarization, the activation of PI3K-Akt-mTORC1 axis was inhibited more obviously(Fig.3A). The amount of Th17 may be one of the reasons, or there may exist a certain correlation between BDNF and TGF-β or IL-6, or the downstream pathway of TGF-β and IL-6. Whether BDNF has a synergistic effect with TGF-β is indecisive. Although expressions of both Foxp3 and RORγt require TGF-β, the signaling cascade of downstream of TGF-β is different. For example, Smad4 seems to be necessary to induce both Foxp3 and RORγt, nevertheless, however, TGF-β induces Foxp3 expression through Smad2-/Smad3. Smad pathway is necessary for TGF-β-induced Foxp3 expression. It was reported that the ERK pathway negatively regulated the expression of Foxp3 and inhibited the differentiation of Treg, which is also dependent on the cytokine TGF-β. In conclusion, the deep understanding about the molecular mechanism of BDNF affecting the PI3K- Akt - mTORC1 axis and ERK1/2 pathway remains to be further explored.