IL-37 overexpression promotes endometrial regenerative cell-mediated inhibition of cardiac allograft rejection

Abstract

Background: Endometrial regenerative cells (ERCs) play an important role in attenuation of acute allograft rejection, while their effects are limited. IL-37, a newly discovered immunoregulatory cytokine of the IL-1 family, can regulate both innate and adaptive immunity. Whether IL-37 overexpression can enhance the therapeutic effects of ERCs in inhibition of acute cardiac allograft rejection remains unknown and will be explored in this study.

Methods: C57BL/6 mice recipients receiving BALB/c mouse heterotopic heart allografts were randomly divided into the phosphate-buffered saline (untreated), ERC treated, negative lentiviral control ERC (NC-ERC) treated, and IL-37 overexpressing ERC (IL-37-ERC) treated groups. Graft pathological changes were assessed by H&E staining. The intra-graft cell infiltration and splenic immune cell populations were analyzed by immunohistochemistry and flow cytometry, respectively. The stimulatory property of recipient DCs was tested by an MLR assay. Furthermore, serum cytokine profiles of recipients were measured by ELISA assay.

Results: Mice treated with IL-37-ERCs achieved significantly prolonged allograft survival compared with the ERC-treated group. Compared with all the other control groups, IL-37-ERC-treated group showed mitigated inflammatory response, a significant increase in tolerogenic dendritic cells (Tol-DCs), regulatory T cells (Tregs) in the grafts and spleens, while a reduction of Th1 and Th17 cell population. Additionally, there was a significant upregulation of immunoregulatory IL-10, while a reduction of IFN-γ, IL-17A, IL-12 was detected in the sera of IL-37-ERC-treated recipients.

Conclusion: IL-37 overexpression can promote the therapeutic effects of ERCs to inhibit acute allograft rejection and further prolong graft survival. This study suggests that gene-modified ERCs overexpressing IL-37 may pave the way for novel therapeutic options in the field of transplantation.

Keywords: Acute allograft rejection; Endometrial regenerative cells; Interleukin-37; Mice.

Fig. 1

Identification of ERCs and IL-37-ERCs. A Flow cytometry analysis of surface markers of ERCs. B Morphology of ERCs, NC-ERCs, and IL-37 ERCs at passage 2 (magnification 40×). The successful transfection of ERCs with vector or IL-37 was shown by GFP. C Concentration of IL-37 in the culture supernatant of ERCs, NC-ERCs, and IL-37-ERCs. Statistical analysis was done by one-way ANOVA, ***P < 0.001

Fig. 2

IL-37 overexpression promoted ERCs to prolong graft survival and alleviate acute allograft rejection. A Kaplan–Meier curves of allograft survival of fully MHC-mismatched cardiac transplants, in which BALB/c hearts were transplanted into C57BL/6 recipients. The allograft survival was significantly prolonged in the ERC group when compared with the untreated group, and further improved in the IL-37-ERC group (n = 6 per group). Statistics by Log-rank test. B Representative histology of cardiac allografts retrieved at the time of rejection. ERC-treated recipients revealed a milder lymphocytes infiltration and milder myocyte damage, while IL-37-ERCs further alleviated these changes (n = 6 per group). C Grading of graft rejection in different groups. Score criteria: 0, no change; 1, minimum change; 2, mild change; 3, moderate change; and 4, marked change compared with normal tissues (n = 6 per group). Statistics by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD

Fig. 3

IL-37-ERCs decreased the infiltration of CD4⁺ cells while promoting Treg generation in the graft. A Representative graft sections for immunohistological staining of intra-graft CD4⁺ cell infiltration. The positive staining area was shown by an arrow. B The mononuclear cells were isolated from the grafts and analyzed by flow cytometry. Treg (CD4⁺Foxp3⁺ cell) populations were significantly raised in the ERC-treated group compared to the untreated and further increased in the IL-37-ERC-treated group (n = 6 per group). The percentage of intra-graft CD4⁺ cells (C) and Tregs (CD4⁺Foxp3⁺ cells) (D) from heart allografts of different groups. Data were analyzed with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD

Fig. 4

IL-37-ERCs inhibited the antigen-presenting capacity and stimulatory function of recipients' dendritic cells. Single-cell suspensions of splenocytes obtained from the untreated, ERC-treated, NC-ERC-treated, and IL-37-ERC-treated groups were analyzed for the frequency of CD11c⁺MHC-II⁺ cells by flow cytometry, gated on live cells. The representative pseudocolor plots (A) and statistical graphs (B) were depicted (n = 6 per group). In addition, the expression of the costimulatory molecules CD86 was analyzed via flow cytometry. The representative histogram (C) and mean fluorescence intensity (MFI) (D) are depicted (gated on CD11c⁺ population; n = 6 per group). E The proliferative response of allogeneic T cells was represented by OD₄₅₀ value, which was measured via the CCK-8 test in one-way MLR analyses (n = 6 per group). Statistical analysis was performed using one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD

Fig. 5

IL-37-ERCs reduced recipients' splenic Th1 and Th17 populations while increasing the generation of Tregs. Splenocytes obtained from the C57BL/6 recipients of each group were stained with CD4⁺IFN-γ⁺IL-17A⁺ and CD4⁺CD25⁺Foxp3⁺, respectively. The representative pseudocolor plots of Th1 cells (A), Th17 cells (B), and Tregs (C) were exhibited. The percentage of D Th1 cells, E Th17 cells, and F Tregs (CD4⁺CD25⁺Foxp3⁺) were analyzed by one-way ANOVA, n = 6 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD

Fig. 6

IL-37-ERCs further reduced the levels of pro-inflammatory cytokines but enhanced the level of IL-10 in recipient sera. To evaluate the overall function of the recipients' immune system, the production of pro-inflammatory cytokines and the regulatory cytokine IL-10 in the sera were detected via ELISA assay. The sera were collected from recipients of each group at day 8 post-transplantation. Here, the levels of A IFN-γ, B IL-17A, C IL-12, and D IL-10 were shown (n = 6 per group). Data were analyzed using one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD