Adoptive transfer of TregIL-33 cells ameliorates acute graft-versus-host disease by regulating the HPGD/PGE2 pathway.

BACKGROUND

Adoptive transfer of regulatory T-cells (Tregs) has shown promising therapeutic efficacy in ameliorating graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. However, the underlying mechanisms have not been thoroughly studied.

OBJECTIVE

In this study, we investigated the potential application of adoptive transfer of IL-33-cultured Tregs to prevent acute graft-versus-host disease (aGVHD) and its underlying mechanism.

STUDY

DESIGN

Tregs were magnetically isolated from the spleen of C57BL/6 mice and subsequently cultured with IL-2 (TregIL-2) or IL-2 + IL-33 (TregIL-33) for 7d. BALB/c mice received 6 Gy of total body irradiation 24 h prior to transplantation. After irradiation, ex vivo cultured TregIL-2 or TregIL-33 cells were co-adoptively transferred with splenocytes and T cell-depleted bone marrow cells from C57BL/6 donors. On day 8 post transplantation, splenocytes and liver cells were collected and analyzed by flow cytometry for M1/M2 macrophage populations and T cell subsets. For in vitro experiments, bone marrow derived macrophages (BMDM) were cultured with TregIL-33 cells for 48 h. The M1/M2 macrophage population was detected at both mRNA and protein levels. For TregIL-33 cells, hydroxyprostaglandin dehydrogenase (HPGD) expression and prostaglandin E2 (PGE2) production were examined. To demonstrate that the regulation of macrophage polarization was mediated through the HPGD/PGE2 axis, the HPGD inhibitor SW033291 and an anti-PGE2 antibody were used in a co-culture setting.

RESULTS

Adoptive transfer of TregIL-33 cells was more effective in preventing aGVHD than adoptive transfer of TregIL-2. In addition, recipients receiving TregIL-33 cells showed increased Tregs, especially ST2+Tregs frequencies in the spleen and liver, while frequencies of IFN-γ and IL-17 producing conventional T cells were decreased. Meanwhile, the expression of CD86+ type I macrophages was downregulated and that of CD206+ type II macrophages was upregulated in mice co-transferred with TregIL-33 cells. In in vitro study, TregIL-33 cells promoted M2 polarization and inhibited the M1 transition of BMDM compared with BMDM cultured with TregIL-2. Mechanistically, IL-33 induced ST2 expression on Tregs, increased HPGD synthesis, and accelerated PGE2 degradation. Using the HPGD inhibitor SW033291, and an anti-PGE2 antibody, we demonstrated that TregIL-33 cells regulated M1/M2 macrophage polarization via the HPGD/PGE2 axis. Furthermore, blocking HPGD activity with SW033291 abolished the efficacy of TregIL-33 cells in protecting against aGVHD in vivo.

CONCLUSION

Our data demonstrate that adoptive transfer of TregIL-33 cells could ameliorate aGVHD by regulating the HPGD/PGE2 axis, thus regulating M1/M2 paradigm.