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 (Treg_IL-2) or IL-2 + IL-33 (Treg_IL-33) for 7d. BALB/c mice received 6 Gy of total body irradiation 24 h prior to transplantation. After irradiation, ex vivo cultured Treg_IL-2 or Treg_IL-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 Treg_IL-33 cells for 48 h. The M1/M2 macrophage population was detected at both mRNA and protein levels. For Treg_IL-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 Treg_IL-33 cells was more effective in preventing aGVHD than adoptive transfer of Treg_IL-2. In addition, recipients receiving Treg_IL-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 Treg_IL-33 cells. In in vitro study, Treg_IL-33 cells promoted M2 polarization and inhibited the M1 transition of BMDM compared with BMDM cultured with Treg_IL-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 Treg_IL-33 cells regulated M1/M2 macrophage polarization via the HPGD/PGE2 axis. Furthermore, blocking HPGD activity with SW033291 abolished the efficacy of Treg_IL-33 cells in protecting against aGVHD in vivo.

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

Graft-versus-host disease; HPGD; IL-33; Macrophage; PGE2; ST2.