†These authors contributed equally.
Academic Editors: Shikun He and Graham Pawelec
Background: Mesenchymal stem cells (MSCs) are promising candidates for
immunomodulatory therapy that are currently being tested in corneal allograft
rejection. In this study, we tested the effects of Mesenchymal stem cells derived
exosomes in the corneal allograft rejection model. Methods: Mesenchymal
stem cells derived exosomes (MSC-exo) were collected and characterized.
Wistar-Lewis rat corneal allograft rejection models were established. PKH26
labeled exosomes were used for track experiment. Models were randomly separated
into four groups and treated with graded doses of exosomes or same volumn of PBS.
Corneal grafts were assessed for rejection degree using slit-lamp biomicroscopy.
Grafts were examined histologically using hematoxylin-eosin (H-E) staining and
immunohistochemically using antibodies against CD4, CD8 and CD25. A comprehensive
graft mRNA gene expression array analysis was conducted and checked by real-time
polymerase chain reaction (PCR). Results: The nanovesicles obtained were
expressing exosome specific protein markers CD9, CD63, CD81. The labeled exosomes
could be detected in both cornea and anterior chamber two hours after
injection.The 10
Corneal transplantation is widely accepted in corneal blindness for visual rehabilitation. However, it is reported that graft rejection occurred in almost 50% of the cases, and the rejection rate of high-risk cornea could reach to 70% to 90% [1]. The mechanism of graft rejection is complicated and multifactor involved, among which immune responses is the predominant reason for graft failure. Current gold treatments for anti-rejection are systemic corticosteroids and immunosuppressants [2]. Yet its effect in high-risk corneal allograft rejection is limited due to its complications such as drug toxicity and life-threatening potential. New therapies to ensure the viability of corneal transplantation are in need.
Mesenchymal stem cells (MSCs) offer great hope and promise in a series of allograft rejection models due to its immunomodulatory effect [3]. Our and other studies have demonstrated that MSCs possess strong immunosuppressive ability, which can inhibit corneal allograft rejection in animal model in vivo [4, 5, 6]. However, stem cell based therapy is associated with certain disadvantages like poor targeted migration, engraftment and the survival of the transplanted cells. MSCs may play their role via a paracrine action [7]. Recent studies identifying exosomes as the secreted agents, mediating MSCs therapeutic efficacy, which could potentially replace a cell-based drug by a safer biologic based alternative [8]. Several studies have compared the therapeutic effects of MSCs and their extracellular vesicles (EVs) and did not discover significant differences [9, 10].
Based on MSCs’ effects on immunity on immunity and our previous work, the current effort presented here sought to improve upon these therapeutic results by extending MSCs efficacy and reducing cell based therapeutic risk. In this study, we investigated the immunosuppressive capacity of MSCs-derived exosomes in corneal allograft rejection model and tried to clarify its immunomodulatory mechanism and functional way.
Male Lewis rats (6–8 weeks old) and male Wistar rats (4 weeks old and 6–8 weeks old) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were housed under pathogen-free conditions. All procedures of experiments involving rats were approved by the Laboratory Animal Care and Use Committee of Tianjin Medical University, and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Approximately 90–110 g weighted male Wistar Rats at 4 weeks were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).
After being sacrificed and instantly soaked in 75% ethanol for 10 minutes, the
femurs
and tibias
of the rats were collected under sterile conditions and placed in phosphate
buffered saline (PBS, Gibco). Needles were used to drill numerous holes on both
ends of the bone and the bone marrow was washed out into the PBS by another
needle. Samples were centrifuged and suspended in complete culture medium
containing Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 (DF-12,
Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL
penicillin and 100
The supernatant of MSCs from passages 3 to 5 were collected for exosomes
isolation. Supernatant fractions collected from 48 h cell cultures were
centrifuged at 300
For identification of isolated exosomes, the pellets were fixed with 200
Corneal allograft rejection model was induced in Lewis recipient rats by transplanting the cornea grafts from Wistar donor rats. Lewis rats were anesthetized sequentially by intraperitoneal injection of chloral hydrate (10%), 0.3 mL/100 g bodyweight. The pupil of the recipient eye was fully dilated by 0.5% tropicamide. The corneal grafts were 3.5 mm in diameter excised of sacrificed Wistar and instantly kept in wet chamber before transplantation. A 3.0 mm-diameter corneal bed was prepared in the right eye of Lewis. The graft was then placed onto the bed and secured with 8 interrupted 10-0 nylon sutures. The anterior chamber was reformed by injection of air bubble.
The models were randomly divided into 4 groups with 6 rats in each group. For
treatment, operated eyes of the Lewis rats were administrated with different
doses of MSCs derived exosomes (1
Rat corneal allograft rejection model. Rejection model was made in a Lewis recipient rat by transplanting the cornea graft from a Wistar donor rat. Injections of exosomes were performed immediately after transplantation and two days post-operation. The slit-lamp observation was performed from day 3 post-operation.
The rats were observed post operation by slit-lamp bio-microscope from day 3.
The Larkin method [12] based on opacity, edema, and vascularization scoring is
used in graft rejection degree assessment. The graft reached rejection level as
indicated by an opacity score
On day 10, four corneas of each group (PBS-treated and exosome-treated) were collected for gene expression array, which was performed by Genechem Co., Ltd., Shanghai, China. Total RNA was extracted and the quality was monitored using the Agilent 2100 Bioanalyzer. GeneChip 3’ IVT Express Kit was used to prepare the amplified RNA (aRNA). After the aRNA was purified and fragmented, it was then hybridized using Genechip Hybridization Oven 645 (Affymetrix, Inc., CA, USA), washed by GeneChip Fluidics Station450 and scanned by GeneChip Scanner 3000. Raw data were analyzed by GeneSpring GX software version 11.5 (Agilent Technologies, CA, USA).
On day 10, both the PBS-treated group and the exosome-treated group were
sacrificed with double-dose of chloral hydrate. The eyeballs (3 eyes per group)
were collected and quickly fixed in 10% neutral buffered formalin
(Sigma-Aldrich, St. Louis, MO, USA), dehydrated in gradient ethanol
(Sigma-Aldrich, St. Louis, MO, USA), embedded in paraffin block, and sagittally
sectioned. Continuous sections (4
The animals were killed on day 10, eyes were enucleated. Fixed and dehydrated
tissues were embedded in paraffin wax and cut into 4
Grafts of PBS-treated Lewis rats, and exosome-treated Lewis rats (3 rats per
group) were collected and frozen in liquid nitrogen on day 10. Total RNA was
extracted using Trizol (Thermo Fisher Scientific, CA, USA) under the instruction
of the manual. The concentration and purity of total RNA were examined by a
Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). 1
On day 10, the spleens and lymph nodes were collected from PBS-treated
and exo-treated rats. The monocytes were isolated by Ficoll (GE Healthcare Life
Sciences, Beijing, China), 1
For exosomes’ tracking, purified exosomes and PBS were both labeled using PKH26
(red) membrane dye (Sigma-Aldrich) according to the manufacture’s protocol.
Labeled exosomes and PBS with dye were washed in 40 mL of PBS, collected by
ultracentrifugation and re-suspended in 200
All statistics were processed by GraphPad Prism version 6.00 for Mac (GraphPad
Software, La Jolla, CA, USA). Results of survival time were assessed by
Kaplan-Meier analysis. The relative mRNA level was analyzed by one-way ANOVA
followed by Bonferroni multiple comparisons test. Data are expressed as mean
MSCs were characterized by their multilineage differentiation potentials, including chondrocytes, osteoblasts, and adipocytes. The vesicles of MSCs in our experiments were identified by electron microscopy and the vesicles were cup-shaped and measured 30–150 nm in diameter (Fig. 2A). Western blot confirmed that the vesicles from MSCs expressed markers of exosomes, including CD63, CD9 and CD81 (Fig. 2B).
Identification and characteristics of exosomes. (A) Electron microscopy image of exosome isolated from BMSCs-medium. (B) Western-blot results. CD63, CD9, CD68 were as the markers of exosomes. Three independent samples from vesicles of MSCs (marked as S1, S2 and S3) were performed here.
We have previously reported that conjunctiva injection of MSCs could prevent the
rejection of allograft in rats. To explore whether exosomes released from MSCs
could inhibit the allograft-rejection, we applied three different doses of
MSC-exo (1
The role of MSC-exo in corneal allograft rejection. (A) 10
MSC-exo was labeled by PKH26 to investigate the trace of exosomes after subconjunctival injection. The labeled MSC-exo were detected via confocal laser scanning microscopy (Fig. 4). Two hours after MSC-exo injection, there were numerous exosomes could be detected both in the cornea and anterior chamber. 24 hours after, the fluorescent intensity was weaker and the quantity was markedly decreased. However, there was no positive finding in spleen tissue either 2 hours or 24 hours after exosomes injection.
Tracking of BMSC-exo in sub-conjunctiva. The exosome in the recipient rat’s subconjunctiva after 2h/24h administrating PKH26 labeled-exo as the images of left row showed. The controlled group were injected PKH26-incubated PBS as the right column showed. Labeled-exo still can be observed after 24h with a weaker fluorescent intensity (n = 2).
CD4+, CD8+ and CD25+ cells infiltration were assessed by immunohistochemistry. As shown in Fig. 5, the numbers of CD4+, CD25+ cells in the allografts were decreased in MSC-exo treatment group compared to PBS group (p = 0.007, p = 0.001, respectively). No statistical significance was found in the number of CD8+ cells infiltrating corneal grafts compared to control groups (p = 0.937).
Representative immunohistochemistry staining of CD4, CD8, and
CD25 in corneal allografts. The numbers of the cells positive for CD4, CD8, and
CD25 staining were quantified and compared with PBS groups (n = 3). *p
MSC-exo also up-regulated the proportions of CD4+CD25+Foxp3+cells in the
splenocytes and lymph nodes by flow cytometry (Fig. 6). The ratio of
Foxp3+expressing Treg cells/CD4+ T cells increased in the MSC-exo group (8.56
Flow cytometry was used for analysis of proportion of
CD4+CD25+Foxp3+Tregs. Splenocytes from recipient rats were harvested at day 10
after allograft transplantation. Representative splenocytes sample of the
percentages of CD4+, CD4+CD25+Foxp3+ T cells were represented. Mean
To examine the possible mechanisms behind the effectiveness of local MSC-exo
therapy, we further performed the mRNA array. Three independent samples were
studied for each group. And three duplications were made for an independent
sample. Quantitative real-time polymerase chain reaction was performed to confirm
the results from mRNA array. Five obviously altered genes, including
IFN-
Th1 pathway map. In this experiment, Th1 pathway was significantly inhibited, compared with Supplementary Fig. 1. The green color indicated the gene was significantly down regulated and red color indicated the gene was markedly up regulated.
Verification of relative expression of Th1
cytokine-IFN-
Penetrating keratoplasty is the last resort for patients who suffer the irreversible blindness caused by corneal diseases. However, the demand of recipients for transplantable corneas outnumbers the supply of donor corneas. Meanwhile, transplanted patients are facing the risk of immune response and graft rejection on a lifelong basis. Despite the advancement of corneal transplantation in recent years, the penetrating keratoplasty has an irreplaceable role in some corneal diseases, especially disorders affecting both the stromal layers and endothelial layers. With regard to the penetrating keratoplasty, the allograft survival is limited though under the administration of immunosuppressants. Although it has been demonstrated that MSCs could promote graft survival by inhibit immune response, cell based treatment has potential uncertainty about safety and efficacy [13]. Exosomes are considered a form of extracellular vesicles with a size ranging from 30 to 150 nm. Exosomes contain cell-derived specific functional components, including proteins, lipids, mRNAs and miRNAs, which enable them to play a role similar as their original cells [14]. The potential advantages of exosomes in cargo delivery including easily sterilized, low to none-immunogenicity for allogenic use, easy manipulation for loading of therapeutic agents, better crossing through the biological barriers, which make them ideal for cell-free therapeutic strategies. In the light of this statement, MSC-exo has been used in a plethora of degeneration and allograft rejection models and has inspiring results [15, 16, 17]. Effects of exosomes from different MSCs types are not equivalent, and the quality of exosomes obviously depend on the quality of the secreting stem cells [18, 19]. Bone marrow-derived mesenchymal stem cells were used in our research. In this study, the grafts survival time were accessed to be prolonged after MSC-exo treatment. It indicated that the use of exosomes may represent a cell-free alternative approach in corneal allograft rejection.
In this study, PKH26-labeled MSC-exo was administrated in the subconjuctiva of corneal allograft rejection model. The results showed that there was a small quantity of MSC-exo which could be observed in subconjunctiva and some of the exosomes could be tracked in allografts as well as anterior chamber. Our previous results showed that MSCs were accumulated in the subconjuctiva after local injection [6], this indicated that MSCs acted via a paracrine way, rather than direct cell contact. This is in accordance with the work of Lin et al. [20]. They found that after topical administration of MSCs to the murine corneal epithelial wounding model, the corneal stroma rather than epithelium is the retention place of most MSCs. So the attenuating effect to the inflammation and promotion effect to the regeneration of epithelium are explained by soluble factors released from MSCs. Extracellular vesicles, including exosomes can mediate the paracrine effect of MSCs [21, 22, 23]. Our results verified that exosomes could cross biological barrier and play better role directly towards target tissue.
Cornea is located at the surface of the eye, which makes it easy to access and
manipulate. A few studies have reported the therapeutic effects of MSC-exo on
corneal wound models [24, 25]. It is reported that human corneal mesenchymal
stromal cells derived exosomes promoted murine corneal epithelial wound healing
[24]. Topical MSC-exo treatment was also reported could suppress corneal
inflammation and corneal scarring [25]. These data showed the potential use of
MSC-exo in ocular surface diseases therapy. Local delivery routine is a regular
treatment way in ocular disease. Two different delivery routes to treat corneal
allograft rejection were compared in this study. The grafts survival observation
results showed that subconjunctival injection, other than topical application,
was effective. In this study, the failure of eye drops therapy may be due to
frequently blink, rapid tear turnover as well as low drug bioavailability [5].
Previous studies showed that exosomes might play their roles in a dose-dependent
way. Graded doses of exosomes derived from MSCs were applicated in the study. The
results showed 10
Corneal allograft rejection is a form of delayed-type hypersensitivity (DTH)
response, which predominantly mediated by CD4+ T cells. CD4+ T cells have the
potential to differentiate into different subgroups. Among the numerous subsets,
the helper T-cell (Th) and regulatory T-cell (Treg) subtsets are the best
characterized. Treg plays a key role in immune tolerance maintaining in corneal
allograft rejection [27]. It’s interesting that the proportion of Foxp3
expressing CD4+CD25+ regulatory T cells were found increased in the MSC-exo
treated group. This result suggest that MSC-exo therapy can induce immune
tolerance by upgrading the expressing of Tregs. The homeostasis of CD4+Th cells
subtypes plays key roles in the pathogenesis of allograft rejection. In corneal
allograft rejection T-cell differentiation is primarily polarized toward the Th1
response, driven by pro-inflammatory cytokines, particularly IFN-
To sum up, our results indicated that subconjunctival injection of MSC-exo can prolong corneal allograft survival by inhibiting Th1 response. These findings indicate that local MSC-exo therapy is a promising alternative method for the prevention and treatment of immune rejection after corneal transplantation.
ZJ and SZ designed the research study. ZJ, YL and WZ performed the research. SZ performed the transplant operations and revised the manuscript. XZ revised the manuscript. XZ, FL and XL provided help and advice on research performed. ZJ and YL analyzed the data. ZJ and YL wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
All procedures of experiments involving rats were approved by the Laboratory Animal Care and Use Committee of Tianjin Medical University (permission number: SYXK2009-0001), and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Not applicable.
This work was funded by Tianjin Binhai New Area Health Youth Project,grants number 2019BWKQ034, Tianjin Clinical Key Discipline Project, grants number TJLCZDXKT002, and Tianjin Science and Technology Youth Project, grants number 20JCQNJC00230.
The authors declare no conflict of interest.