| Schistosomiasis, the second major parasitic disease in the world after malaria,affects mainly developing countries. There are 200 million people worldwide infectedwith schistosomes, resulting in more than 250,000 deaths annually. In China,currently there are more than eight hundred thousand Schistosomiasis japonica(S.japonicum) patients and near one hundred million prople at risk according to thecountrywide investigation in 2005. Because of the increased mobidity in recent years,S.japonicum has been focused by Chinese government as one of the infectiousdiseases that should be controlled in stress.Chronic schistosome infection is known to skew the immune response towardsTh2, characterized by the production of interleukin-4 (IL-4), IL-5, IL-13, and highserum levels of immunoglobulin E (IgE). Individuals chronically infected withschistosomes usually develop lymphocyte hyporesponsiveness to parasite antigens,exhibited as a reduced ability of host cells to proliferate and induction of animmunomodulation or immunosuppression. However, recent reports show severalpathogen infection, such as S. mansoni, HIV and malrial, leads to the development ofa population of CD4+CD25+ T cells that make IL-10 and TGF-?or through celluarcontact, inhibit host protective immune response and develop the immunosuppressiveenvironment. However, it is still not clear how the CD4+CD25+ Tregs are inducedduring an infection.Our previous work have shown that S. japonicum egg antigens stimulated the generation of CD4+CD25+ Tregs, and then we have demonstrated that SjHSP60expressed in S.japonicum eggs and worms induces CD4+CD25+ Tregs. In this study,we further identified a peptide from SjHSP60, SJMHE1, which contained overlappedhuman and mouse CD4+ T cell epitopes, and was proved to induce CD4+CD25+ Tregswith immunosuppressive activity. The main results are as follows:1. Flow cytometry was used to analyze the CD4+CD25+Foxp3+ Tregs andshowed that only spleen and LN cells from SJMHE1-immunized mice or SJMHE1peptide-incubated pooled spleen and LN cells increased the proportion ofCD4+CD25+Foxp3+ T cells. To evaluate whether SJMHE1 treatment enhancedCD4+CD25+ Treg-mediated suppression, CD4+CD25+ T cells from SJMHE1-immunized mice, or CD4+CD25+ T cells from naive mice pretreated in vitro withSJMHE1 were co-cultured with responder naive murine CD4+CD25. T cells. Theproliferative responses were measured by methods of 3H-thymidine incorporation.Results showed that SJMHE1 significantly enhanced the inhibitory ability ofCD4+CD25+ T cells both in vivo and in vitro. Furthermore, CD4+CD25+ T cellsinduced by SJMHE1 significantly suppressed the DTH responses in mice, andsuppressed the OVA-stimulated proliferation of both pooled spleen and LN cells andCD4+CD25- T cells from DTH mice in vitro.2. To identify soluble mediators that might be involved in SJMHE1-triggeredimmunoregulation, CD4+CD25+ and CD4+CD25- T cells from SJMHE1-immunizedmice were co-cultured in separate Transwell chambers. Results suggested thatwithout SJMH1 stimulation in Transwell chambers, CD4+CD25+ T cells harvestedfrom SJMHE1-immunized mice were capable of suppressing responder cells only viadirect cellular contact. However, when SJMHE1 was added or SJMHE1 in vitropretreated CD4+CD25+ T cells were added, suppression of CD4+CD25- T cells wasrestored, indicating that some soluble factors also mediated SJMHE1-triggeredinhibitory effects. Adding either anti-TGF-?or anti-IL-10 neutralizing antibodiesonly slightly reversed the inhibition. However, adding both anti-IL-10 and anti-TGF-?blocked inhibition completely. These results suggested that both IL-10 and TGF-?contributed to SJMHE1-mediated inhibition. We further investigated the expression of intracellular IL-10, TGF-?and CTLA4 in CD4+CD25+ T cells from SJMHE1immunized mice or in CD4+CD25+ T cells from co-cultures with SJMHE1 in vitro.Flow cytometric analysis showed that only SJMHE1, but not OVA323-339 stimulationshowed high levels of intracellular IL-10, TGF-?and CTLA4 in CD4+CD25+ T cells.These results further supported that production of IL-10, TGF-?and CTLA4 byCD4+CD25+ T cells contributed to SJMHE1-mediated inhibition.3. To investigate the role of antigen presenting cells (APCs) in SJMHE1-inducedCD4+CD25+ Tregs, BmDCs from BALB/c mice and the RAW264.7 M?cell linewere pulsed in vitro with SJMHE1, then incubated with allogenic CD4+ T cells fromnaive mice. Flow cytometric analysis showed that BmDCs or M?s pulsed withSJMHE1 stimulated a marked increase in CD4+CD25+Foxp3+ T cells. Parallel to theincrease in CD4+CD25+Foxp3+ T cells, CD4+ T cells co-cultured with SJMHE1-pulsed BmDCs or M?s proliferated poorly, compared to CD4+ T cell proliferationinduced by BmDCs or M?s co-cultured with either OVA323-339 or LPS. Furthermore,SJMHE1-treated BmDCs or M?s retained an immature morphology, and the levels ofsurface markers (e.g., CD40, CD80, CD86 and MHCII molecules) on BmDC/M?bySJMHE1 stimulated were consistently lower and showed an immature phenotype.4. Recent studies have shown that TLR2 and TLR4 signaling promotedCD4+CD25+ Treg proliferation. To further investigate the mechanism through whichSJMHE1 triggered CD4+CD25+ Treg induction, TLR2-/- and TLR4-/- mice were used.The results showed that spleen and LN cells harvested from SJMHE1-immunizedTLR4-/- mice or SJMHE1 in vitro pretreated TLR4-/- mice spleen and LN cellsresulted in a CD4+CD25+Foxp3+ T cell increase. Furthermore, CD4+CD25+ T cellsfrom SJMHE1-immunized TLR4-/- mice or SJMHE1-pretreated CD4+CD25+ T cellsfrom TLR4-/- mice were highly effective at suppressing CD4+CD25- T cellproliferation, which consistent with the results described in WT mice. However, anincrease in CD4+CD25+Foxp3+ T cell and an enhancement of suppression inCD4+CD25+ T cell were all not observed in TLR2-/- mice treated in a similar fashion.These results indicated that TLR2 but not TLR4 played an important role in specifically promoting the development of CD4+CD25+ Tregs induced by SJMHE1.Furthermore, TLR4-/- BmDCs (prepared from either SJMHE1-immunized mice orfrom an in vitro SJMHE1 pulse) increased the number of CD4+CD25+Foxp3+ T cells.In contrast, neither BmDCs nor BmM?s from TLR2-/- mice increased the CD4+CD25+T cell counts regardless of whether they were immunized in vivo or pulsed in vitrowith SJMHE1. These data further confirmed that the induction of CD4+CD25+ Tregsby SJMHE1 was dependent on TLR2.In summary, the results above suggested that SJMHE1 was able to increasethe number and suppressive potential of CD4+CD25+ T cells both in vivo and invitro. Release of IL-10 and TGF-?or expression of CTLA4 by CD4+CD25+ Tregsresulting from SJMHE1 stimulation contributed to inhibit the responder T cellsproliferation. SJMHE1-treated APCs were tolerogenic and induced CD4+ cells todifferentiate into suppressive CD4+CD25+ Tregs. Furthermore, data supported arole for TLR2 in SJMHE1-mediated CD4+CD25+ Tregs induction. Thesefindings suggested that CD4+CD25+ Tregs could be induced by schistosomeantigens via TLR2 signaling, a mechanism that likely contributes to controlinflammatory responses during infection, thereby protecting host tissues againstimmune-mediated damage while concomitantly providing the parasites with afavorable environment. |
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