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

ABSTRACT:

Background

Systemic 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.

Methods

MSCs were isolated from female C57BL/6 (H-2^k) or Balb/c (H-2^d) 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.

Results

Both 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.

Conclusion

This 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.

SUBMITTER: Treacy O

PROVIDER: S-EPMC8025388 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-? expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8?±?1.2 days). Injection twice of MSCs (MST 12.6?±?1.4 days) was more effective than a single injection (MST 10.8?±?1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4?+?T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-? expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Project description:Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro. Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68+ macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68+ macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft.

Project description: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.

Project description:BackgroundMesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration.MethodsBone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and during all assays. Autologous and allogeneic MSCs were then directly co-cultured with responder leukocytes from the Connemara horse in varying concentrations of MSCs to leukocytes (1:1, 1:10, and 1:100). MSCs were also cultured with complement present and heat-inactivated complement to determine whether complement alone would decrease MSC viability. MSCs underwent haplotyping of their equine leukocyte antigen (ELA) to determine whether the MHC factors were matched or mismatched between the donor MSCs and the responder leukocytes.ResultsAll allogeneic MSCs were found to be ELA mismatched with the responder leukocytes. MHC II-low and universal blood donor MSCs caused no peripheral blood mononuclear cell (PBMC) proliferation, no increase in B cells, and no activation of CD8 lymphocytes. Universal blood donor MSCs stimulated a significant increase in the number of T regulatory cells. Neutrophil interaction with MSCs showed that universal blood donor and MHC II-high allogeneic MSCs at the 6 h time point in co-culture caused greater neutrophil activation than the other co-culture groups. Complement-mediated cytotoxicity did not consistently cause MSC death in cultures with active complement as compared to those with inactivated complement. Gene expression assays revealed that the universal blood donor group and the MHC II-low MSCs were more metabolically active both in the anabolic and catabolic gene categories when cultured with allogeneic lymphocytes as compared to the other co-cultures. These upregulated genes included CD59, FGF-2, HGF, IDO, IL-10, IL-RA, IL-2, SOX2, TGF-β1, ADAMSTS-4, ADAMSTS-5, CCL2, CXCLB/IL-8, IFNγ, IL-1β, and TNFα.ConclusionsMHC II-low MSCs are the most appropriate type of allogeneic MSC to prevent activation of the innate and cell-mediated component of the adaptive immune systems and have increased gene expression as compared to other allogeneic MSCs.

Project description:Mesenchymal stem cells (MSCs) have unique and beneficial properties and are currently used to treat a broad variety of diseases. These properties include the potential for differentiation into other cell types, secretion of different trophic factors that promote a regenerative microenvironment, anti-inflammatory actions, selective migration to damaged tissues, and non-immunogenicity. MSCs are effective for the treatment of ocular surface diseases such as dry eye, corneal burns, and limbal stem cell deficiency (LSCD), both in experimental models and in humans. LSCD is a pathological condition in which damage occurs to the limbal epithelial stem cells, or their niche, that are responsible for the continuous regeneration of the corneal epithelium. If LSCD is extensive and/or severe, it usually causes corneal epithelial defects, ulceration, and conjunctival overgrowth of the cornea. These changes can result in neovascularization and corneal opacity, severe inflammation, pain, and visual loss. The effectiveness of MSCs to reduce corneal opacity, neovascularization, and inflammation has been widely studied in different experimental models of LSCD and in some clinical trials; however, the methodological disparity used in the different studies makes it hard to compare outcomes among them. In this regard, the MSC route of administration used to treat LSCD and other ocular surface diseases is an important factor. It should be efficient, minimally invasive, and safe. So far, intravenous and intraperitoneal injections, topical administration, and MSC transplantation using carrier substrata like amniotic membrane (AM), fibrin, or synthetic biopolymers have been the most commonly used administration routes in experimental models. However, systemic administration carries the risk of potential side effects and transplantation requires surgical procedures that could complicate the process. Alternatively, subconjunctival injection is a minimally invasive and straightforward technique frequently used in ophthalmology. It enables performance of local treatments using high cell doses. In this review, we provide an overview of the current status of MSC administration by subconjunctival injection, analyzing the convenience, safety, and efficacy for treatment of corneal failure due to LSCD in different experimental models. We also provide a summary of the clinical trials that have been completed, are in progress, or being planned.