miR-31, miR-155, and miR-221 expression profiles and their association with graft skin tolerance in a syngeneic vs. allogeneic murine skin transplantation model

Grafting is the gold standard for the treatment of severe skin burns. Frequently, allogeneic tissue is the only transient option for wound coverage, but their use risks damage to surrounding tissues. MicroRNAs have been associated with acute rejection of different tissues/organs. In this study, we analyzed the expression of miR-31, miR-155, and miR-221 and associate it with graft tolerance or rejection using a murine full-thickness skin transplantation model. Recipient animals for the syngeneic and allogeneic groups were BALB/c and C57BL/6 mice, respectively; donor tissues were obtained from BALB/c mice. After 7 days post-transplantation (DPT), the recipient skin and grafts in the syngeneic group maintained most of their structural characteristics and transforming growth factor (TGF)β1 and TGFβ3 expression. Allografts were rejected early (Banff grades II and IV at 3 and 7 DPT, respectively), showing damage to the skin architecture and alteration of TGFβ3 distribution. miRNAs skin expression changed in both mouse strains; miR-31 expression increased in the recipient skin of syngeneic grafts relative to that of allogeneic grafts at 3 and 7 DPT (p < 0.05 and p < 0.01, respectively); miR-221 expression increased in the same grafts at 7 DPT (p < 0.05). The only significant difference between donor tissues was observed for miR-155 expression at 7 DPT which was associated with necrotic tissue. Only miR-31 and miR-221 levels were increased in the blood of BALB/c mice that received syngeneic grafts after 7 DPT. Our data suggest that local and systemic miR-31 and miR-221 overexpression are associated with graft tolerance.

Bone marrow mesenchymal stem cell-derived exosomes attenuate the maturation of dendritic cells and reduce the rejection of allogeneic skin transplantation in mice model

Background Bone mesenchymal stem cells (BMSCs)-derived exosomes (B-exos) are attractive for applications in enabling alloantigen tolerance. An in-depth mechanistic understanding of the interaction between B-exos and dendritic cells (DCs) could lead to novel cell-based therapies for allogeneic transplantation. Herein, the potential of a B-exos-based application combined with DCs was explored for inducing allogeneic transplant tolerance. Methods After mixed culture of BMSCs and DCs for 48 hours, DCs from the upper layer were collected to analyze the expression levels of surface markers and mRNAs of inflammation-related cytokines. Then the DCs were co-cultured with B-exo before being collected to detect the mRNA and protein expression levels of indoleamine 2,3-dioxygenase (IDO). The treated DCs from different groups were co-cultured with naïve CD4 + T cells from the mouse spleen. The proliferation of CD4 + T cells and the proportion of CD4 + CD25 + Foxp3 + T cells were analyzed. Finally, the skins of BALB/c mice were transplanted to the back of C57 mice to establish a mouse allogeneic skin transplantation model. Results The coculture of DCs with BMSCs in a trans-well system downregulated the expression of the major histocompatibility complex class II (MHC-II) and CD80/86 costimulatory molecules on DCs, as well as the mRNA expression of interleukin-10 (IL-10), IL-12, and transforming growth factor-β (TGF-β). However, these findings were abolished when treated with GW4869. Moreover, B-exos (5 µg/mL) increased the expression of indoleamine 2,3-dioxygenase (IDO) in DCs treated with lipopolysaccharide (LPS) compared to the control cells. CD4 + CD25 + Foxp3 + T cells increased when cultured with B-exos-exposed DCs, which was attenuated by 1-methyl tryptophan (1MT). Mice recipients injected with B-exos-treated DCs significantly prolonged the skin allograft survival. The allografts showed slight cell infiltration and significantly preserved graft structure. Also, the level of CD4 + CD25 + Foxp3 + T cells was significantly higher in B-exos-exposed DCs recipient animals than that in other groups. Conclusions Taken together, these data suggested that the B-exos suppress the maturation of DCs and increase the expression of IDO, which might shed light on the role of B-exos in inducing alloantigen tolerance.

Cyclic Helix B Peptide Prolongs Skin Allograft Survival via Inhibition of B Cell Immune Responses in a Murine Model

Antibody-mediated rejection (AMR) represents a major cause of allograft dysfunction and results in allograft failure in solid organ transplantation. Cyclic helix B peptide (CHBP) is a novel erythropoietin-derived peptide that ameliorated renal allograft rejection in a renal transplantation model. However, its effect on AMR remains unknown. This study aimed to investigate the effect of CHBP on AMR using a secondary allogeneic skin transplantation model, which was created by transplanting skin from BALB/c mice to C57BL/6 mice with or without CHBP treatment. A secondary syngeneic skin transplantation model, involving transplantation from C57BL/6 mice to C57BL/6 mice, was also created to act as a control. Skin graft rejection, CD19+ B cell infiltration in the skin allograft, the percentages of splenic plasma cells, germinal center (GC) B cells, and Tfh cells, the serum levels of donor specific antibodies (DSAs), and NF-κB signaling in splenocytes were analyzed. Skin allograft survival was significantly prolonged in the CHBP group compared to the allogeneic group. CHBP treatment also significantly reduced the CD19+ B cell infiltration in the skin allograft, decreased the percentages of splenic plasma cells, GC B cells, and Tfh cells, and ameliorated the increase in the serum DSA level. At a molecular level, CHBP downregulated P100, RelB, and P52 in splenocytes. CHBP prolonged skin allograft survival by inhibiting AMR, which may be mediated by inhibition of NF-κB signaling to suppress B cell immune responses, thereby decreasing the DSA level.

Mouse Models for Human Skin Transplantation: A Systematic Review

Immunodeficient mouse models with human skin xenografts have been developed in the past decades to study different conditions of the skin. Features such as follow-up period and size of the graft are of different relevance depending on the purpose of an investigation. The aim of this study is to analyze the different mouse models grafted with human skin. A systematic review of the literature was performed in line with the PRISMA statement using MEDLINE/PubMed databases from January 1970 to June 2020. Articles describing human skin grafted onto mice were included. Animal models other than mice, skin substitutes, bioengineered skin, postmortem or fetal skin, and duplicated studies were excluded<b>.</b> The mouse strain, origin of human skin, graft dimensions, follow-up of the skin graft, and goals of the study were analyzed. Ninety-one models were included in the final review. Five different applications were found: physiology of the skin (25 models, mean human skin graft size 1.43 cm² and follow-up 72.92 days), immunology and graft rejection (17 models, mean human skin graft size 1.34 cm² and follow-up 86 days), carcinogenesis (9 models, mean human skin graft size 1.98 cm² and follow-up 253 days), skin diseases (25 models, mean human skin graft size 1.55 cm² and follow-up 86.48 days), and would healing/scars (15 models, mean human skin graft size 2.54 cm² and follow-up 129 days). The follow-up period was longer in carcinogenesis models (253 ± 233.73 days), and the skin graft size was bigger in wound healing applications (2.54 ± 3.08 cm²). Depending on the research application, different models are suggested. Careful consideration regarding graft size, follow-up, immunosuppression, and costs should be analyzed and compared before choosing any of these mouse models. To our knowledge, this is the first systematic review of mouse models with human skin transplantation.

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2-like sequences in rDNA promoter upon T cell activation in vitro. The elevated pre-rRNA level of T cells is also observed in both mouse heart or skin transplantation models, and in kidney transplanted patients. Importantly, T cell activation can be significantly suppressed by inhibiting NF45/NF90-dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off-target activity (i.e. toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90-mediated rDNA transcription as a novel signaling pathway essential for T cell activation and as a new target for the development of safe and effective immunosuppressants.