Model Of Corneal Transplantation Research Articles

The ocular immunological alterations in the process of high-risk corneal transplantation rejection

PurposeThis study aims to reveal the immunopathogenesis of the high-risk corneal transplantation using a comparative proteomic approach. MethodsThe immunological properties of ocular tissues (including corneal grafts, aqueous humour, and iris-ciliary body) were analysed using a high-risk rabbit corneal transplantation model employing a comparative proteomic approach. ResultsThe corneal grafts revealed a dramatic increase in the immune response both at the early (postoperative day 7) and rejection stages, along with the appearance of transplantation stress-induced cellular senescence in the early stage. The aqueous humour (AH) displayed persistent pathological alterations, indicated by the significant enrichment of complement and coagulation cascades pathway in the early stage and interleukin (IL)-17 signalling pathway in the rejection stage. More surprisingly, the pronounced elevation of immune response was also observed in the iris-ciliary body (I-CB) tissues at the early and rejection stages. The enriched immune-related pathways were associated with antigen processing and presentation, complement and coagulation cascades, and IL-17 signalling pathway. Furthermore, proteomic analysis revealed that the implantation of Cyclosporine A drug delivery system (CsA-DDS) into the anterior chamber obviously mitigated corneal transplantation rejection by inhibiting immunoreaction both in the corneal grafts and I-CB tissues. ConclusionThe results highlighted the involvement of intraocular immunity both in the grafts and I-CB tissues during corneal transplantation rejection, further suggesting the anterior chamber as an optimal drug-delivery site for its treatment.

Cyclic guanosine monophosphate-adenosine monophosphate synthetase/stimulator of interferon genes signaling aggravated corneal allograft rejection through neutrophil extracellular traps

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected-corneal allografts compared with syngeneic (Syn) and normal (Nor) corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1and interferon regulatory factor 3. Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

Human mesenchymal stem cells derived from adipose tissue showed a more robust effect than those from the umbilical cord in promoting corneal graft survival by suppressing lymphangiogenesis

BackgroundMesenchymal stem cells (MSCs) have shown promising potential in allograft survival. However, few reports have focused on comparing the immunosuppressive capacity of MSCs from different sources and administered via different routes in inhibiting transplant rejection. Moreover, virtually nothing is known about the role of MSCs in the regulation of graft neovascularization and lymphangiogenesis. In this study, we compared the efficacy of human adipose MSCs (hAD-MSCs) and human umbilical cord MSCs (hUC-MSCs) in vitro and in corneal transplantation models to explore the underlying molecular mechanisms and provide a powerful strategy for future clinical applications.MethodshAD-MSCs and hUC-MSCs were generated, and their self-renewal and multi-differentiation abilities were evaluated. The inhibitory effect of human MSCs (hMSCs) was examined by T-cell proliferation assays with or without transwell in vitro. Two MSCs from different sources were separately adoptively transferred in mice corneal transplantation (5 × 105 or 1 × 106/mouse) via topical subconjunctival or intravenous (IV) routes. Allograft survival was evaluated every other day, and angiogenesis and lymphomagenesis were quantitatively analyzed by immunofluorescence staining. The RNA expression profiles of hMSCs were revealed by RNA sequencing (RNA-seq) and verified by quantitative real-time PCR (qRT‒PCR), western blotting or ELISA. The function of the differentially expressed gene FAS was verified by a T-cell apoptosis assay.ResultshAD-MSCs induced stronger immunosuppression in vitro than hUC-MSCs. The inhibitory effect of hUC-MSCs but not hAD-MSCs was mediated by cell–cell contact-dependent mechanisms. Systemic administration of a lower dose of hAD-MSCs showed better performance in prolonging corneal allograft survival than hUC-MSCs, while subconjunctival administration of hMSCs was safer and further prolonged corneal allograft survival. Both types of hMSCs could inhibit corneal neovascularization, while hAD-MSCs showed greater superiority in suppressing graft lymphangiogenesis. RNA-seq analysis and confirmation experiments revealed the superior performance of hAD-MSCs in allografts based on the lower expression of vascular endothelial growth factor C (VEGF-C) and higher expression of FAS.ConclusionsThe remarkable inhibitory effects on angiogenesis/lymphangiogenesis and immunological transplantation effects support the development of hAD-MSCs as a cell therapy against corneal transplant rejection. Topical administration of hMSCs was a safer and more effective route for application than systemic administration.

Effect of aprotein kinaseB (Akt) inhibitor on the angiogenesis of HUVECs and corneal neovascularization.

Corneal neovascularization (CNV) is avision-threatening disease and an increasing public health concern. It was found that administering an Akt inhibitor in the second phase of retinopathy significantly decreased retinal neovascularization. This study investigated the effect of an Akt inhibitor on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and its impacts on the degree of CNV and corneal opacity in arat keratoplasty model. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, tube formation assays, cell scratch experiments, and afully allogeneic corneal transplant model were performed. It was found that an Akt inhibitor inhibited the proliferation, angiogenesis, and migration of HUVECs induced by vascular endothelial growth factor (VEGF). The results showed that both CNV and corneal opacity were decreased in rats after Akt inhibitor administration. The research illustrates the vital role of Akt inhibitors in mediating CNV. The analysis shows that the Akt inhibitor may provide anovel and feasible therapeutic approach to prevent CNV, but its mechanism needs further investigation.

Amplified Natural Killer Cell Activity and Attenuated Regulatory T-cell Function Are Determinants for Corneal Alloimmunity in Very Young Mice.

Corneal transplantation outcomes are generally less favorable in young children compared with adults. The purpose of this study was to determine the immunological mechanisms underlying this difference. A murine model of allogeneic corneal transplantation was used in the study, and graft survival was determined by evaluating opacity scores for 8 wk. Syngeneic transplantation in the very young host served as a surgical control. The frequencies of total and activated natural killer (NK) cells in cornea posttransplantation were kinetically evaluated using flow cytometry. The regulatory T cell (Treg) frequency and function in naive animals were assessed by flow cytometry and in vitro suppression assays, respectively. Finally, graft survival and immune responses were determined in NK cell-depleted, or adult naive Treg-transferred, young hosts. Corneal allograft survival in the very young recipients was significantly lower than in adult hosts. The frequencies of total NK cells and their interferon gamma-expressing subset in the cornea were significantly higher in the very young mice posttransplantation. In ungrafted mice, frequencies of Treg in draining lymph nodes as well as their capabilities to suppress NK-cell secretion of interferon gamma were lower in the very young compared with adults. In NK cell-depleted or adult Treg--transferred very young recipients, the allograft survival was significantly improved along with the suppressed NK-cell response. Our data demonstrate that amplified activity of NK cells, together with lower suppressive function of Treg, contributes to early rejection of corneal allografts in very young graft recipients.

Local and Systemic Injections of Human Cord Blood Myeloid-Derived Suppressor Cells to Prevent Graft Rejection in Corneal Transplantation.

Myeloid-derived suppressor cells (MDSCs) are therapeutic agents to prevent graft rejection in organ transplants by modulating inflammation. Herein, the immunosuppressive effect of human cord blood MDSCs on corneal allograft models was confirmed. CB-MDSCs were locally (subconjuctival, 5 × 105) or systemically (intravenous, 1 × 106) injected twice on days 0 and 7. A corneal transplantation model was established using C57BL/6 and BALB/c mice, and corneal graft opacity was measured to evaluate graft rejection up to 6 weeks. Results showed that graft survival in the MDSCs groups increased compared to vehicle groups after 42 days. Systemic and local MDSC administration inhibited the maturation (MHC-IIhi CD11c+) of dendritic cells (DCs) and the differentiation of interferon γ+ CD4+ Th1 in draining lymph nodes (LNs). However, vehicle groups increased the infiltration of CD3+ T cells and F4/80+ macrophages and produced prominent neovascular and lymphatic vessels into the graft site with increased mRNA expression of VEGF-A/C and VEGFR-1/R-3. Local MDSCs administration showed prominent anti-angiogenic/anti-lymphangiogenic effects even at lower MDSCs doses. Thus, CB-MDSCs could relatively suppress the infiltration of pathological T cells/macrophages into the corneas and the migration of mature DCs into draining LNs Therefore, ocular and systemic MDSCs administration showed therapeutic potential for preventing corneal allograft rejection.

Mesenchymal stem cells augment regulatory T cell function via CD80-mediated interactions and promote allograft survival

Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) both have been shown to modulate the alloimmune response and promote transplant survival. Mounting evidence suggests that MSCs augment Treg function, but the mechanisms underlying this phenomenon have not been fully deciphered. Here, we identified that MSCs express substantial levels of CD80 and evaluated its immunoregulatory function using in vivo and in vitro experiments. Our in vitro culture assays demonstrated that MSCs induce expression of FoxP3 in Tregs in a contact-dependent manner, and the blockade of CD80 abrogates this FoxP3 induction and Treg-mediated suppression of T cell proliferation. Moreover, supplementation of soluble CD80significantly upregulated FoxP3 expression. Using a well-characterized murine model of corneal transplantation, we show that silencing CD80 in MSCs diminishes the capacity of MSCs to promote selective graft infiltration of Tregs, promote FoxP3 expression and upregulate suppressive function of Tregs. Consequently, MSCs, following CD80knockdown, failed to promote corneal allograft survival.

Pre-incubation of corneal donor tissue with sCD83 improves graft survival via the induction of alternatively activated macrophages and tolerogenic dendritic cells

Immune responses reflect a complex interplay of cellular and extracellular components which define the microenvironment of a tissue. Therefore, factors that locally influence the microenvironment and re-establish tolerance might be beneficial to mitigate immune-mediated reactions, including the rejection of a transplant. In this study, we demonstrate that pre-incubation of donor tissue with the immune modulator soluble CD83 (sCD83) significantly improves graft survival using a high-risk corneal transplantation model. The induction of tolerogenic mechanisms in graft recipients was achieved by a significant upregulation of Tgfb, Foxp3, Il27, and Il10 in the transplant and an increase of regulatory dendritic cells (DCs), macrophages (Mφ), and T cells (Tregs) in eye-draining lymph nodes. The presence of sCD83 during in vitro DC and Mφ generation directed these cells toward a tolerogenic phenotype leading to reduced proliferation-stimulating activity in MLRs. Mechanistically, sCD83 induced a tolerogenic Mφ and DC phenotype, which favors Treg induction and significantly increased transplant survival after adoptive cell transfer. Conclusively, pre-incubation of corneal grafts with sCD83significantly prolongs graft survival by modulating recipient Mφ and DCs toward tolerance and thereby establishing a tolerogenic microenvironment. This functional strategy of donor graft pre-treatment paves the way for new therapeutic options in the field of transplantation.

Novel adaptation of a running suture technique in a mouse model of corneal transplantation.

Several murine models of corneal transplantation have been developed over the years to study the immunopathological processes that lead to the failure of grafted corneas. In all of them, the classic eight interrupted sutures technique is utilized for transplanting the donor cornea on the host bed. However, in clinical practice, a single continuous suture with a single knot is generally performed for corneal transplantation. Here, we describe the adaptation of the single continuous suture technique in a mouse model of corneal transplantation.

Subconjunctival administration of low-dose murine allogeneic mesenchymal stromal cells promotes corneal allograft survival in mice

BackgroundSystemic administration of mesenchymal stromal cells (MSCs) has been efficacious in many inflammatory disease settings; however, little data are available on the potential immunomodulatory effects following local MSC administration in the context of corneal transplantation. The purpose of this study was to assess the potential of subconjunctival injection of MSCs to promote corneal allograft survival.MethodsMSCs were isolated from female C57BL/6 (H-2k) or Balb/c (H-2d) mice and extensively characterized. An allogeneic mouse corneal transplant model was used with Balb/c mice as recipients of C57BL/6 grafts. A dose-finding study starting with 5 × 105 MSCs injected subconjunctivally at day − 7 was tested first followed by a more clinically translatable low-dose single or dual injection strategy on day − 1 and day + 1 before/after transplantation. Graft transparency served as the primary indicator of transplant rejection while neovascularization was also recorded. Lymphocytes (from draining lymph nodes) and splenocytes were isolated from treatment groups on day 2 post-transplantation and characterized by flow cytometry and qRT-PCR.ResultsBoth high- and low-dose injection of allogeneic MSCs on day − 7 led to 100% graft survival over the observation period. Moreover, low-dose dual subconjunctival injection of 5 × 104 allogeneic MSCs on day − 1 or day + 1 led to 100% allograft survival in transplant recipients (n = 7). We also demonstrate that single administration of allogeneic MSCs on either day − 1 or day + 1 promotes rejection-free graft survival in 100% (n = 8) and 86% (n = 7) of transplanted mice, respectively. Early time point ex vivo analysis suggests modulation of innate immune responses towards anti-inflammatory, pro-repair responses by local MSC administration.ConclusionThis work demonstrates that low-dose subconjunctival injection of allogeneic MSCs successfully promotes corneal allograft survival and may contribute to refining future MSC immunotherapies for prevention of corneal allograft rejection.

The role of soluble programmed death protein-1 (sPD-1) and soluble programmed death ligand-1 (sPD-L1) in rat corneal transplantation rejection.

Immunological rejection is one of the problems in corneal transplantation. Recently, some research found out that soluble programmed death protein-1 (sPD-1) and soluble programmed death ligand protein-1 (sPD-L1) play a significant role in immunologic suppression. To explore expression of sPD-1 and sPD-L1 in a penetrative corneal transplantation model and its relationship with transplant rejection. Autologous corneal transplantation rat models and allogeneic corneal transplantation rat models were used as the control group and the experimental group, respectively. Changes of the transplanted grafts were observed under a slit-lamp microscope. Hematoxylin-eosin (H&E) staining was applied to examine the histopathological features of the corneal grafts. Flow cytometry was used to analyze CD4+CD25+Treg in the serum and spleen. The sPD-1, sPD-L1, interleukin 10 (IL-10) and interleukin 4 (IL-4) levels in serum and the aqueous humor of the rats were detected using enzyme-linked immunosorbent assay (ELISA). After the operation, no transplant rejection occurred in the control group. Flow cytometry results showed that expressions of CD4+CD25+Treg in serum in the experimental group were lower than those in the control group (p < 0.05). The ELISA results showed that after the operation, sPD-1 and sPD-L1 expression levels in serum in the experimental group were higher than in the control group (all p < 0.05). After the operation, lL-10 and IL-4 content in serum in the experimental group was lower than in the control group (all p < 0.05). The sPD-1/sPD-L1 ratio in the experimental group was higher than in the control group. Increases of sPD-1 content and decreases of CD4+CD25+Treg, IL-10 and IL-4 levels may be involved in corneal allograft rejection. Dynamic detection of the content of sPD-1 and sPD-L1 in serum and aqueous humor after the operation would help in understanding the local immune response in a clinical setting and predicting the occurrence of corneal graft rejection.

Different Innate Immune Responses in BALB/c and C57BL/6 Strains following Corneal Transplantation

Purpose: To investigate immunological differences and the role of CD38+/F4/80 + M1 macrophages in C57BL/6J- and BALB/c-recipient mouse corneal transplantation models. Methods: Allogeneic transplantation was performed crosswise in BALB/c mice and C57BL/6J mice; syngeneic transplantation was performed in both strains. Anterior chamber depth (ACD) was measured before and central corneal thickness (CCT) was measured both before and after transplantation. In vivo graft rejection was monitored using anterior eye segment optical coherence tomography (ASOCT) evaluating the CCT and grading of corneal oedema using a well-established clinical score (CS). Histology of corneal grafts was performed 18 or 21 days after surgery. Immunohistochemistry with anti-F4/80 antibody and anti-CD38 antibody was used for detecting M1 macrophages within the grafts. Results: High CS and CCT values after allogeneic transplantation persisted in both BALB/c (n = 18) and C57BL/6J recipients (n = 20). After syngeneic transplantation, CS and CCT values increased in both models in the early phase after surgery due to the surgical trauma. Surprisingly, in the syngeneic C57BL/6J model, high CCT values persisted. Furthermore, anterior synechiae developed in C57BL/6 recipients after both syngeneic and allogeneic transplantation, whereas BALB/c recipients showed almost no synechiae – even though C57/BL6J animals tended to have a deeper anterior chamber (281 ± 11 pixels [mean ± SD]) compared with BALB/c animals of the same age (270 ± 9 pixels [mean ± SD]). Immunohistochemistry revealed numerous CD38+/F4/80 + M1 macrophages in grafts of C57BL/6J recipients following both syngeneic and allogeneic transplantation. However, in BALB/c-recipient mice only sparse M1 macrophages were detectable (CD38 + M1 macrophages relative to all F4/80 + cells: 75 vs. 17% [after allogeneic transplantation] and 66 vs. 17% [after syngeneic transplantation]; p < 0.05). Conclusions: Allogeneic corneal transplants are rejected in BALB/c as well as C57BL/6J mice, but tissue alterations with anterior synechiae are more pronounced in C57BL/6J recipients. Following syngeneic transplantation, C57BL/6J-recipient animals show a persistent graft swelling with increased numbers of CD38+/F4/80 + M1 macrophages in grafted tissue, in contrast to the common model using BALB/c-recipient mice. Our data strongly suggest that strain-dependent differences convey different innate immune responses in BALB/c and C57BL/6J strains. Accordingly, in murine keratoplasty experiments, the mouse line of both donor and recipient animals must be carefully considered. C57BL/6J-recipient mice might be particularly suited to study corneal graft rejection in a clinical setting considered “high risk.”

Topical VEGF-C/D Inhibition Prevents Lymphatic Vessel Ingrowth into Cornea but Does Not Improve Corneal Graft Survival.

Vascular endothelial growth factor-C/D (VEGF-C/D) regulates lymphangiogenesis. Ingrowth of lymphatic vessels is negatively associated with corneal transplantation success. In this study, we therefore analyzed the effect local blockade of VEGF-C/D has on inflamed corneas. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation. Mice were treated with a VEGF-C/D trap prior to transplantation. Topical inhibition of VEGF-C/D significantly reduced lymphatic vessel ingrowth, but increased Macrophage numbers in the cornea. Furthermore, corneal transplantation success was not improved by the topical application of the compound. This study demonstrates that local VEGF-C/D inhibition is insufficient to increases corneal transplantation success, likely due to interaction with immune cells.

Effect of RMT1-10 on the immunological characteristics of dendritic cells cultured in vitro and corneal transplantation in vivo.

Corneal allograft rejection is an immunological hypersensitive reaction caused by the antigenicity of the donor cornea. This study aimed to explore the effects of RMT1-10 on the prevention of corneal graft rejection by modifying immunological characteristics of dendritic cells (DCs). DCs and CD4+T cells were sorted using flow cytometry and used for in vitro mixed lymphocyte culture. The cultured cells were prepared for the characterization of the DC cell phenotypes using the markers CD11c, CD80, MHC II, CD54, and TIM-4. Cytokine concentrations of IL-4, IL-12, and IL-10 of supernatants were measured by the enzyme-linked immunosorbent assay. CD4+T cells were examined by flow cytometry for apoptosis and proliferation. We also investigated the effect of RMT1-10 in the prevention and treatment of high-risk corneal graft rejection using a mouse model of corneal transplantation. DCs were identified as the CD11c+MHC-II-expressing subset. RMT1-10 suppressed the expression of CD11c, CD80, MHC II, CD54, and TIM-4 of DCs using the blockade of TIM-1 signaling. Moreover, TIM-1 blockade inhibited the production of IL-12 and IL-10 in a mixed lymphocyte culture system. However, a TIM-1 blockade had no effect on the apoptosis of CD4+T cells. RMT1-10 suppressed DC maturation, inhibiting the proliferation of CD4+T cells. RMT1-10 significantly improved the survival rate of the corneal allografts in mice compared with saline-injected controls. This clinical improvement from RMT1-10 occurred through the inhibition of CD4+T cell proliferation. Moreover, RMT1-10 induced antigen-specific detection of receptor immune tolerance. The cross-linking of TIM-1 on CD4+T cells with the agonist mAb provided a costimulatory inhibition signal for T cell activation or proliferation.

The purinergic receptor antagonist oxidized adenosine triphosphate suppresses immune-mediated corneal allograft rejection

Adenosine triphosphate (ATP) is released into the extracellular environment during transplantation, and acts via purinergic receptors to amplify the alloimmune response. Here, using a well-established murine model of allogeneic corneal transplantation, we investigated the immunomodulatory mechanisms of the purinergic receptor antagonist oxidized ATP (oATP). Corneal transplantation was performed using C57BL/6 donors and BALB/c hosts. oATP or sterile saline was administered via intraperitoneal injection for 2 weeks postoperatively. Frequencies of CD45+ leukocytes, CD11b+MHCII+ antigen presenting cells (APCs), CD4+IFN-γ+ effector Th1 cells and CD4+Foxp3+ regulatory T cells (Tregs) were evaluated by flow cytometry. Slit-lamp microscopy was performed weekly for 8 weeks to evaluate graft opacity and determine transplant rejection. Treatment with oATP was shown to significantly reduce graft infiltration of CD45+ leukocytes, decrease APC maturation and suppress effector Th1 cell generation relative to saline-treated control. No difference in Treg frequencies or Foxp3 expression was observed between the oATP-treated and control groups. Finally, oATP treatment was shown to reduce graft opacity and increase graft survival. This report demonstrates that oATP limits the alloimmune response by regulating APC maturation and suppressing the generation of alloreactive Th1 immunity.

Mesenchymal Stromal Cells Modulate Corneal Alloimmunity via Secretion of Hepatocyte Growth Factor.

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and possess considerable immunomodulatory potential. MSCs have been shown to promote allograft survival, yet the mechanisms behind this phenomenon have not been fully defined. Here, we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation, we report that MSCs promote graft survival in an HGF‐dependent manner. Moreover, our data indicate that topically administered recombinant HGF (a) suppresses antigen‐presenting cell maturation in draining lymphoid tissue, (b) limits T‐helper type‐1 cell generation, (c) decreases inflammatory cell infiltration into grafted tissue, and (d) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF‐based therapeutics. stem cells translational medicine 2019;8:1030–1040

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.

AICAR prolongs corneal allograft survival via the AMPK-mTOR signaling pathway in mice

Immune rejection is a critical complication that results in the graft failure after corneal transplantation. Thus, there remains a need for new therapies for allograft rejection. AICAR (aminoimidazole-4-carboxamide ribonucleoside) is an, as adenosine monophosphate–activated protein kinase (AMPK) activator and a purine nucleoside with a wide range of metabolic effects, including activation of AMPK. More recently, it was reported that it is possible to inhibiting organs rejection and prolong the graft survival time in various models of organ transplantation. In this study, we systematically evaluated the efficacy of AICAR as a treatment modality for inhibiting allograft rejection in a mouse model of corneal transplantation. We found that AICAR significantly suppressed the opacity, edema, and vascularization of the graft, resulting in prolonged corneal allograft survival. AICAR treatment also significantly decreased central corneal thickness. Moreover, the AICAR-treated group showed decreased expression of IB4 and VEGF as compared to the control group. In addition, the mRNA expression of T helper 1 cytokines (IL-2, INF-γ, and TNF-α) was suppressed, and the expression of T helper 2 cytokines (IL-4, IL-5, and IL-13) was elevated by AICAR. Furthermore, the western blotting results revealed that AICAR stimulated AMPK activation and inhibited angiogenesis and inflammation possibly by subsequently suppressing mTOR phosphorylation. By contrast, the AMPK inhibitor Compound C (also called dorsomorphin) had the opposite effect. Our results showed that Compound C blocked AMPK-mTOR signaling and promoted the angiogenesis and inflammation, thus compromising the graft survival. These results suggest that AICAR may be a potential option for inhibiting the corneal graft rejection and for prolonging the graft survival.

Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation

High-risk cornea transplant recipients represent a patient population with significant un-met medical need for more effective therapies to prevent immunological graft rejection due to heightened anti-donor immune response. In this study, a rat model of pre-existing anti-donor immunity was developed in which corneal allografts were rejected earlier than in non-pre-sensitized recipients. In this model, third-party (non-donor, non-recipient strain) allogeneic mesenchymal stromal cells (allo-MSC) were administered intravenously 7 and 1 days prior to transplantation. Rejection-free graft survival to 30 days post-transplant improved from 0 to 63.6% in MSC-treated compared to vehicle-treated control animals (p = < 0.0001). Pre-sensitized animals that received third-party allo-MSC prior to transplantation had significantly higher proportions of CD45+CD11b+ B220+ monocytes in the lungs 24 h after the second MSC injection and significantly higher proportions of CD4+ FoxP3+ regulatory T cells in the graft-draining lymph nodes at the average day of rejection of control animals. In in vitro experiments, third-party allo-MSC polarized primary lung-derived CD11b/c+ myeloid cells to a more anti-inflammatory phenotype, as determined by cytokine profile and conferred them with the capacity to suppress T cell activation via prostaglandin E2 and TGFβ1. In experiments designed to further validate the clinical potential of the protocol, thawed cryopreserved, third-party allo-MSC were shown to be similarly potent at prolonging rejection-free corneal allograft survival as their freshly-cultured counterparts in the pre-sensitized high-risk model. Furthermore, thawed cryopreserved third-party allo-MSC could be co-administered with mycophenolate mofetil without adversely affecting their immunomodulatory function. In conclusion, a clinically-relevant protocol consisting of two intravenous infusions of third-party allo-MSC during the week prior to transplantation, exerts a potent anti-rejection effect in a pre-sensitized rat model of high-risk corneal allo-transplantation. This immune regulatory effect is likely to be mediated in the immediate post-transplant period through the promotion, by allo-MSC, of alternatively-activated macrophages in the lung and, later, by enhanced regulatory T-cell numbers.