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.

TGF-β-mediated epithelial–mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma

Epithelial–mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-β is a key regulator of EMT. Here, we demonstrate for the first time that TGF-β could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-β signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-β increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-β stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-β enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-β-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-β-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-β signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-β signaling and EMT.

Magnetism-Induced Cell-Targeting Transplantation Improves Early Hematopoietic Reconstitution in a Murine Bone Marrow Transplantation Model

Background Early hematopoietic reconstitution is essential for improving survival and reducing complications after hematopoietic stem/progenitor cell (HSC/HPC) transplantation (HSCT/HPCT). Increasing HSC/HPC homing to the bone marrow is a potential approach for promoting hematopoietic reconstitution. Methods We proposed the transplantation of HSCs/HPCs with a magnetism-induced cell-targeting transplantation (MagIC-TT) strategy. HSCs/HPCs were magnetized with CD45 microbeads. The biological characteristics (morphology, proliferation, viability, and ferroptosis) and target migration ability of these cells were studied in vitro. The hematopoietic reconstitution experiments were constructed in vivo in autologous and allogeneic bone marrow transplantation models with grouping showed as Table 1. The therapeutic effects were assessed by survival, donor chimerism, routine blood examination and histological analysis. We also performed transcriptomic sequencing for further mechanistic studies. Results The biological characteristics was found no significant difference between the MagIC-TT and non-MagIC-TT groups, while migration ability was greatly improved with MagIC-TT (Data not showed). The survival rate was higher in the MagIC-TT group and significantly different in the allogeneic model (P<0.05). Hematopoietic reconstitution of donor chimerism, WBCs, HGB, and PLTs was faster in the MagIC-TT groups (within 22 days) (Figure 1). Confocal observation showed that more donor cells (eGFP +) were retained in the injected femur of the MagIC-TT group than in the femur of the non-MagIC-TT group 7 days after transplantation (P<0.05) (Figure 2). The severity of aGVHD (Assessed by survival, body weight, back arching, fur losting, diarrhea etc.) was reduced in the MagIC-TT groups in the allogeneic model (P<0.05) (Data not showed). Transcriptome sequencing revealed differentially expressed genes (DEGs) involved in localization/locomotion and pathways, cytokine-cytokine receptor interactions and chemokine signaling pathways between the two groups (Figure 3). Conclusion The MagIC-TT strategy improves HSC/HPC homing, resulting in faster hematopoietic reconstitution in a murine bone marrow transplantation model. Key words Magnetism; Hematopoietic Stem Cell Transplantation (HSCT); Cell Homing; Hematopoietic Reconstitution; Ferroptosis Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Hormonally Induced Hepatocellular Carcinoma in Diabetic Wild Type and Carbohydrate Responsive Element Binding Protein Knockout Mice

Objective: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. Methods: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. Results: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. Conclusions: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma.