We investigated the immune suppression domain of HpHSP60 involved in the inhibitory activity to anti-CD3 antibody-induced T-cell proliferation. The mechanisms might be the same homological between HpHSP60 and human HSP60. Many literatures reptorted human HSP60 can act as a ligand bind both innate immune cells (macrophages and dendritic cells (Chen et al. 1999, Habich et al. 2002)) and be adaptive immune cells (T cells and B cells (Zanin-Zhorov et al. 2003, Cohen-Sfady et al. 2005)) TLR receptors. When human HSP60 binding with immune cells can secrete IL-10 and enhance regulator T cells function (Zanin-Zhorov et al. 2006a). The sequences alignment of H.
pylori, homo sapiens, S. japonicum HSP60 proteins showed that the
identity is 52% (Fig. 1), HpHSP60 can interact with immune cell by instead of human HSP60 to achieve inhibit PBMCs proliferation (Fig. 3).The effects of HpHSP60 on PBMCs were not only on cell growth but also on cytokine secretion. Since Treg cells inhibit proliferation of CD4+ T cells either via IL-10 (Quinn et al. 2000) and TGF-β (Yoshimura et al.
2010), we demonstrated which domains of HpHSP60 function for
25
immune suppression by monitoring the two cytokines. Fig 4 indicated different fragments of rHpHSP60 induced different cytokine expression, PBMCs treatment HpHSP60101-200 and HpHSP60300-547 could increase IL-10 cytokine expression. According to Song-Nan et al report, the region HpHSP60300-435 was associated with induction of IL-8 in monocytic cells, we truncated recombinant proteins HpHSP60300-547
confirmed the system workimg that PBMCs tremented with HpHSP60300-547 could expression IL-8 (Fig. 4).
Interestingly, the fragment of HpHSP60300-547 might have two functions including the inflammation and immune suppression. In other words, this fragment included immune suppression and inflammation domain. HpHSP60300-435 have been exploed that treated with this recombint proteins could induce dramatically IL-8 releases (Lin et al.
2005), immune suppression domains of HpHSP60300-435 was located in HpHSP60435-547. In 2008, Delaleu et al. reported human HSP60 a.a.
437-460 led to alter inflammatory chemotaxis and down-regulate Th1 and Th2 effector responses (Zanin-Zhorov et al. 2006a). The human HSP60437-460 (amino acid sequence: vlgggcallrcipaldsltpaned) compared with HpHSP60 a.a. 410-433 was similar, it was proposed that PBMC
26
after treated HpHSP60300-547 induced both IL-8 and IL-10 expression. On the other hand, HpHSP60101-200 also could inhibit T cells’ proliferation as HpHSP60 (Fig. 3) and induce dramatically IL-10 cytokine releases, but it would abolish this activity without (Fig. 4).
To exclude the possibility for the LPS contamination would cause the same inhibitory phenomenon, and investigate which amino secquences or structure of HpHSP60 fragments for lead to immune suppression, we boiled HpHSP60 to denature proteins but not LPS. In the result, the effect of HpHSP60101-200, HpHSP601-200, HpHSP601-250 on proliferation was completely inhibited by boiling, which indicated the inhibitory effect was not likely due to the LPS contamination (data not show). However, the proliferative inhibition of PBMCs didn’t completely revise by treatments of rHpHSP60 and HpHSP60300-547. Based on the above reaults, we knew HpHSP60101-200 and HpHSP60300-547 may be involved the immune suppression, and HpHSP60101-200 inhibited the proliferations of PBMCs by its structure, but HpHSP60300-547 inhibited the proliferations by amino acids sequence.
PBMCs were mainly composed of monocytes and lymphocytes that were involved in both adaptive and innate immunity, and many literatures
27
also reported HpHSP60 was correlation with monocytes (Gobert et al.
2004, Lin et al. 2005). Fig. 5 showed rHpHSP60 fragments could decrease THP-1 proliferation, and different fragments of rHpHSP60 induced different cytokine expression. According to our lab’s unpublished paper have shown that the generation of regulatory T cell by HpHSP60 engageing with Smad-independent TGF-β signaling pathway.
Thus, we presumed the HpHSP60 could effect to both monocytes and lymphocytes. In nature state, PBMCs were increase IL-10 and TGF-β cytokine expression after treated with HpHSP60. IL-10 is an anti-inflammatory cytokine (Quinn et al. 2000) and TGF-β can enhance the production of regulatory T cell (Yoshimura et al. 2010). The mechanism of proliferative inhibition was on T cell populations mainly and caused by HpHSP60-induced Treg.
This was confirmed using one mAb prepared by immunizing mice with HpHSP60101-200
fragments. The monoclonal Ab can specific reacted
with HpHSP60101-200fragments; even human HSP60 has high homology;
yet, it still not reacted. HpHSP60101-200 mAb reacted with rHpHSP60, and datas showed anti-HSP60101-200 antibodies inhibit the proliferation decreasing of PBMCs in the present of anti-CD3 antibody (Fig 7c and 7d).
28
This result confirms that HpHSP60101-200 maybe is immune suppression domain.
In conclusion, this study showed that Helicobacter pylori-derived HSP60 might have two functions, included the inflammation and immune suppression. The proliferative inhibition is on T cell populations mainly through IL-10 expression or TGF-β could enhance the production of regulatory T cell. Furthermore, the monoclonal Abs against rHpHSP60101-200 could block the immune suppression ability of HpHSP60.
These results propose that the sequence from 101 to 200 of H. pylori HSP60 may be closely associated with immune suppression. However, it was still not clear about rHpHSP60300-547 for immune suppression. In the future, we will continued to prepare the monoclonal Abs against rHSP60 (sequence: a. a. 300-547) to prove that rHSP60 (sequence: a. a. 300-547) could down-regulate the immune responses. On the other hand, we will synthetized peptide of rHpHSP60101-200 and rHpHSP60300-547 for demonstrating the immune suppression domain located on above fragments. This finding may be useful to further study for the chronic infection of H. pylori.
29
Figure and Legend
Figure 1: The blast on the sequences of HSP60 derived from different
specisis. The amino acid sequences of H.pylori HSP60, human HSP60
and Schistosoma japonicum HSP60 were retrieved from Pumed database and analyzed by BioEdit.30
Figure 2: The expressionss of different HpHSP60 fragments. a) The figure simply showed that the different sequence of HpHSP60. b) SDS-PAGE and c) Western blotting analyses of recombinant HpHSP60. Commassie blue staining of different fragments run on a 10%
SDS-PAGE. Lane M means the molecular marker and different fragments (lanes 1: whole protein; lanes 2: 101-200; lanes 3: 1-200; lanes 4: 1-250;
lanes 5: 300-547).
(a)
(b) (c)
31
Figure 3: Treatment with whole and different fragment HpHSP60 proteins to PBMCs decrease the proliferation in the present of anti-CD3 antibody. PBMCs were isolated from healthy donors and
seeded in 96-well, which was coated with anti-CD3 antibody (OKT3, 1ug/ml). Different fragments of recombinant proteins were co-culture with PBMCs. MTT assay was used to detect the proliferation of PBMCs.*, P<0.05 compared to only coated anti-CD3. (n=4)
* *
*
32
Figure 4: Production of cytokines in PBMCs. a) IL-10, b) TGF-β, c)
IL-8, d) TNF-α secretion in PBMCs in response to whole and different fragment HpHSP60 proteins. Recombinant heat shock protein 60 was added to medium containing 2 ×105 cells to a final concentration of 1 μg/ml incubated in 96-well plate for 96 h. Cytokines were measured by ELISA . *, P<0.05 compared to only coated anti-CD3. (n=4)(a)
(b) (a)
(c) (d)
33
Figure 5: Treatment with whole and different fragment HpHSP60 proteins to THP-1 cells decrease the proliferation. Different dosages of
whole and different fragment HpHSP60 proteins were co-culture with THP-1 cells. MTT assay was used to detect the proliferation of THP-1 cells. *, P<0.05 compared to without HpHSP60 treatment. (n=3)*
*
*
*
*
34
Figure 6: Production of cytokines in THP-1 cells. a) IL-10, b) TGF-β, c)
IL-8, d) TNF-α secretion in THP-1 cells in response to whole and different fragment HpHSP60 proteins. Recombinant heat shock protein 60 was added to medium containing 2 ×105 cells to a final concentration of 1 μg/ml incubated in 96-well plate for 96 h. Cytokines were measured by ELISA. *, P<0.05 compared to only coated anti-CD3. (n=3)(a) (b)
(c)
(d) (c)
35
Figure 7: The characterizations and application of monoclonal Ab. a)
Dot blotting for investigating the HpHsp60 monoclonal to recognize which fragments. 50 ng of different fragments HpHsp60 were spotted on the nitrocellulose membrane and probed with HpHsp60 mAb (1:10000) followed by goat anti-mouse IgG Abs conjugated with HRP (1:10000). b) Determine of the isotype of HpHSP60 mAb by ELISA. c) Anti-HSP60101-200 antibodies inhibit the proliferation decreasing of PBMCs in the present of anti-CD3 antibody. d) Anti-HSP60101-200antibodies inhibit the IL-10 cytokines expression of PBMCs in the present of anti-CD3 antibody.
*, P<0.05 compared to with HpHsp60 treatment and with coated
36
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