To confirm the role of the TGF-ß signaling pathway in this study, two of the TGF-ß inhibitors, SIS3 and SB431542, were used to validate their effects on the induction of regulatory T cells and the proliferation inhibition of HpHsp60-treated PBMCs. Figure 10A showed the addition of SB431542 combined with HpHsp60 mildly reversed the proliferative inhibition of PBMCs without significance.
However, SIS3 had no effect on the proliferative inhibition (Fig. 10B).
Subsequently, since the treatment with HpHsp60 significantly increased Treg cell percentage on the second day (Fig. 11A), SB431542 and SIS3 were treated at the same time with HpHsp60 or on the second days after seeding respectively. Figure
11B showed the simultaneity addition of SB431542 and HpHsp60 significantly
decreased the percentage of HpHsp60-induced regulatory T cells. Furthermore, addition of SB431542 on the second day significantly reversed the proliferative inhibition and increased the proliferation index to 91% (Fig.12A). However, SIS3still did not influence the proliferative inhibition when treated on the second day after treatment with HpHsp60 (Fig.12B).
Discussion
Recently, microbial Hsp60s were demonstrated to serve as immunomodulators. Kinnune et al. found C. trachomatis Hsp60 responding T cells produce IL-10 to down-regulate hosts’ immune response, and the pathogenic Hsp60 has ability to shift the immune response from Th1 to Th2 [27]. In addition, mycobacterial Hsp60 can reduce the level of IFN-gamma and inhibit cell proliferation of PBMC isolated from periodontitis patients [41]. However, whether the Hsp60 of H. pylori play immunomodulater roles in their pathogenic mechanism is still unknown. In the study, HpHsp60 was found it could inhibit PBMCs proliferation. To exclude the possibility for the LPS contamination would cause the same inhibitory phenomenon, we boiled HpHsp60 to denatures proteins but not LPS. In the result, the effect of HpHsp60 on proliferation inhibition was completely inhibited by boiling, which indicated the inhibitory effect was not likely due to the LPS contamination. Furthermore, since rGFP could not induce the same inhibition of PBMCs proliferation, the inhibitory ability might be restricted to the HpHsp60.
Something interesting was, the inhibition effect could be abolished when additional IL-2 was administrate.
The effects of HpHsp60 on PBMCs were not only on cell growth but also on cytokine secretion. Figure 2 indicated HpHsp60 has different levels of influence on different cytokine. Cytokines and chemokines that involved in T cell activation and macrophage maturation were all down regulated with the treatment of HpHsp60.
On the scanning photograph of the forth day, the cytokine expression profile was quite similar with the first day, but the intensities of IL-8, IL-10, GRO-alpha, IL-6 and RANTES were much lower. This indicated activation of PBMCs continued to be blocked by HpHsp60.
HpHsp60 has effect on the proliferation of T cells but has no effect on non-T cells in PBMCs (Fig. 3). The results showed the proliferative inhibition is resulted from cell cycle arrest but not cell death. HpHsp60 induces cell cycle arrest at G0/G1 phase and lowers the cell population in S and G2/M phases. In addition, no significant change was observed in sub G1 phase, which indicated HpHsp60 could not induce apoptosis. And the results were coincided with the results from annexin-V staining (Fig. 4). Together these results, it was proposed that HpHsp60 could result in proliferative inhibition for T-cell by interfering the DNA replication but not apoptosis.
The effective molecules are on or in the HpHsp60-treated cell (Fig. 6-7).
(Fig. 8). Thus, Treg cells maybe play an important role in this inhibition. Figure 9 revealed percentages of regulatory T cells (CD4+ and FoxP3 + T cells) rise in the HpHsp60-treated PBMCs, which proved the proposition. Indeed, H. pylori have been found their infection will cause the increase of Treg cell at infectious sites [38-40]. Moreover, HpHsp60 are also revealed that they can be secreted out by H.
pylori [29]. Therefore, we proposed that H. pylori would cause increase in Treg cell
by Hsp60 secretion after the infection to impair host immunity.In this study, we first demonstrated that the pathogenic HpHsp60 acts on the increase of Treg cell. In 2006, Alexandra Z. Z. et al. also reported that human Hsp60 acts as a costimulator of human Tregs. Treatment of human Hsp60 enhanced the ability of the CD4+CD25+ T cells to inhibit the growth of untreated CD4+CD25- cells by cell-to-cell contact and the secretion of TGF-ß and IL-10 [42]. In addition, regulatory T cells have been indicated that they are associated with the suppression of inappropriate or excessive immune response in human immune system. There are two types of Treg cells: natural Treg (nTreg) cells and induced Treg (iTreg) cells in periphery bloods. Natural Treg cells are developed in the thymus whereas the iTreg cells can be generated through the TCR activation in the present of immunosuppressive cytokines like IL-10 and TGF-ß [43]. According the literatures, HpHsp60 can induce the TGF- and IL -10 expressions in monocytes [32].
preferentially enhancing the expressions of immunosuppressive cytokines in PBMCs. The suppressive mechanisms induced by Treg cells can be divided into three categories: cell–cell contact, secretion of inhibitory cytokines and competition for growth factors. Figure 4 showed the HpHs060-treated cells significantly contributed on the proliferative inhibition, which proposed the suppressive mechanism might be through the cell–cell contact.
Furthermore, TGF-ß signaling pathway was shown to be involved in the generation of HpHsp60-induced Treg cells and may have contribution to their suppressive functions (Fig. 10-12). SB431542 is an inhibitor specific to the ALK-4, 5, 7 (activin-like kinase, i.e. TGF-ß receptor I) [44-45] whereas SIS3 is an inhibitor selectively blocked the phosphorylation and functions of Smad3 [46]. In the study, we showed SB431542, but not SIS3, could efficiently block the forming of HpHsp60-induced regulatory T cell as well as their suppressive activity. There is one possibility for the phenomenon: HpHsp60 induced the generation of Treg cells engages the TGF-ß signaling different from classical Smad-dependent pathway.
According to literatures, tyrosine residues on both TGF-ß receptor I and II can be phosphorylated through TGF-ß stimulation thus resulted in activation of Erk, p38 and JNK MAP kinases [47-51]. On the other hand, phosphorylation of p38 and MAPK were both demonstrated up regulated in the induced regulatory T cells.
[52-Smad-independent TGF-ß signalling pathway. In the other hand, addition of SB431542 on the second day could reverse the proliferative inhibition about 57%, whereas SIS3 still had no effect on proliferative inhibition. These results indicated that the mechanism of how the HpHsp60-induced regulatory T cells cause the proliferative inhibition of PBMCs might be just partially relative to TGF-ß.
In conclusion, this study showed that Helicobacter pylori-derived Hsp60 has immune suppressive ability in a dose-dependent manner. The proliferative inhibition is on T cell populations mainly and caused by HpHsp60-induced Treg cells through stopping the cell cycles at G0/G1 phase. And the generation of regulatory T cell by HpHsp60 engages Smad-independent TGF-ß signaling pathway.