Umbilical mesenchymal stem cells attenuates corneal fibrosis induced by fungal keratitis through the TGFβ1/Smad2 signaling pathway

Background: Fungal keratitis (FK) is eye microbial infection disease which can leads to severe corneal blindness. Corneal scar formation is one of the major complications of fungal keratitis and is closely related to prognosis. The aim of the current investigation was to evaluate the anti-fibrosis effect of human umbilical cord mesenchymal stem cells (uMSCs) in FK model and further to explore underlying mechanisms. Methods: FK mice model was made by corneal epithelial scratching and F. solani inoculation. The C57BL/6J mice were randomly divided into four groups, including control group, FK group, vehicleinj FK group and uMSCsinj FK group. After injury, antifungal drug natamycin eye drops were used topically to FK mice eyes 6 times per day for 7 days to inhibit fungi growth. Mice received repeated subconjunctival injection of uMSCs or veichle for 3 times including the 1d, 4d and 7d after wounding. At 14d, 21d and 28d post-injury, clinical observation, histological examination, second harmonic generation, immunofluoresence staining and molecular assays were performed. Results: The uMSCs topical administration reduced corneal scar formation and corneal opacity, accompanying with decreased corneal thickness and inflammatory cell infiltration, following down-regulated fibrotic-related factors α-SMA, TGFβ1, CTGF, and COLⅠ and finally inhibited phosphorylation of TGFβ1/Smad2 signaling pathway, which indicating the potential anti-fibrotic and protective effect of human uMSCs against FK-induced corneal fibrosis. Conclusion: Human uMSCs can evidently inhibit corneal fibrosis after FK wounding through TGFβ1/Smad2 signaling pathway regulation. collagen Ⅰ(COLⅠ), forward sequencing: TCAGACCTGTGTGTTCCCTA, reverse sequencing: AGACGTGCTTCTTTTCCTTG, and housekeeping gene β-actin, forward sequencing: GGCACCACACCTTCTAC, reverse sequencing: CTGGGTCATCTTTTCAC. Gene expression was standardized to housekeeping gene expression in respective samples. Each individual experiment was done in triplicate. umbilical cord mesenchymal stem cell conditioned medium attenuates renal fibrosis by reducing inflammation and epithelial-to-mesenchymal transition via the TLR4/NF-kappaB signaling pathway in vivo and in vitro Stem Cell

4 [11]. Meanwhile, stromal thickness and collagen fibril defects in lumican null mice were restored by injection of human stromal stem cells [12]. Based on the anti-fibrosis effects of mesenchymal stem cells (MSCs) reported by numerous researchers, we conducted this research to explore potential mechanism and optimized utilization of MSCs therapy in the field of corneal fibrosis induced by infectious eye disease.
In the present study, we isolated and characterized human uMSCs, and investigated the role of uMSCs administration in the treatment of corneal fibrosis induced by F. solani keratitis through various technical means and further exploring the mechanism of TGFβ1/SMAD2 signaling pathway regulation during the healing period of FK, which provide a novel perspective and practice basis for cell therapy against corneal fibrosis induced by corneal microbial infections.

Methods
Male C57BL/6J mice were obtained from the Nanjing University Biomedical Research Institute (Nanjing, China). All mice were 6 8 weeks of age and 18 25g of weight. The mice were housed in an SPF-class animal laboratory at room temperature 20-25℃ with appropriate humidity, automatic feeding, and 12:12-day cycle natural light.
Intraperitoneal injection of 1 pentobarbital sodium (0.01ml/g; Sigma Aldrich, USA) was used in mice anesthesia and mice were sacrificed by cervical dislocation at different time points. 30 mice were involved in control group and 70 mice were involved in three experimental groups and 240 mice were involved in the study in total. All procedures used in this study were approved by the Henan Eye Hospital Ethics Committe in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Primary culture and identification of uMSCs
Human umbilical cords were acquired from the Henan Provincial People's Hospital. For the 5 use of umbilical cord, written informed consent was obtained from the donors. The

FK model preparation
Mice were anesthetized, the beard was cut off, and the cornea was topically anesthetized with oxybuprocaine hydrpchloride eye drops (Santen Pharmaceutical Co., Ltd., Japan). The right eye of each mouse was selected as the experimental eye. The preparation of the model refers to the article published before by our research group [13]. Under the operating microscope (Topcon OMS-90, Japanese), the left hand was used to fully expose the cornea, and the right hand-held sterile blade (carbon steel, size 11, Shanghai Medical 6 Suture Needle Factory Co., Ltd., China) was used to make a cross scratch in the center of the cornea. The scratch depth was to exceed Bowman's membrane and stop at the corneal stroma superficial layers. Use a sterile bamboo stick tip (tip diameter 0.30mm, length 1.10mm) to pick up a small amount of hyphae, inoculate the corneal scratches, repeatedly apply on the wound surface, so that the hyphae adheres evenly to the scratches, simulate the natural infection of the cornea process. After injury, antifungal drug natamycin eye drops were used topically to FK mice eyes 6 times per day for 7 days to inhibit fungi Thirty mice were randomly selected as blank controls. The remaining mice with similar degree of corneal lesions were weighed and sorted according to body mass. According to random number table, mice were randomly divided into three groups, including FK group, vehicle inj FK group and uMSCs inj FK group. Control group referred to unwounded ones, whereas FK group referred to wounded ones without any injection but antifungal drug therapy, meanwhile vehicle inj FK group referred to wounded ones with vehicle subconjunctival injection and antifungal drug therapy, finally uMSCs inj FK group referred to 7 wounded ones with uMSCs subconjunctival injection and antifungal drug therapy.

Topical administration of uMSCs
Mice in the uMSCs inj FK group were anesthetized and the ocular surface was fully exposed with the help of left hand fixation. Topical anesthetic was applied onto the ocular surface.
Under the operating microscope, the Hamilton micro-syringe (Hamilton, Switzerland) with a 30G needle on it was used to carefully insert into the conjunctiva sac and slowly injected 5×10 4 uMSCs in 5μl PBS. The injection operation was performed for 3 times including the 1d, 4d and 7d after wounding. Others in vehicle inj FK group received a subconjunctive injection of 5μl PBS at the same time points using the same method. However, control group and FK group received no injection at all.

Observation and examination
The cornea was scored and photographed 14d, 21d and 28d after injury. A trained researcher blinded to the group was assigned to score and count corneal opacity and corneal area to prevent bias. The ocular surface pictures were taken by slit-lamp (SL-120; Zeiss, Jena, Germany) and the corneal opacity scoring criteria was as follows: grade 1 (mild corneal haze, pupil iris clearly visible), grade 2 (superficial corneal opacity, visible pupil and iris through the lesion), grade 3 (inhomogeneous full-thickness corneal opacity), and grade 4 (homogeneous and dense opacity [14]. The corneal scar formation areas (mm 2 ) in each group were analyzed by EyeStudio slit-lamp software according to the photographs taken by slit-lamp.

Pathlogical examination and hematoxylin-eosin (HE) staining
For the purpose of histological analysis, mice were sacrificed by cervical dislocation and eyeballs were dissected at 14d, 21d and 28d post-injury. The eyeballs were removed entirely along with some of the surrounding tissue and optic nerve to determine the location of the cornea. After a fixation in 4 paraformaldehyde for 24 hours, the tissues were dehydrated, dipped in wax, embedded, and sliced in sequence. The slices were under a routine operation of dewaxing and then HE staining was used for pathological examination. Photos were captured by Nikon 80i light microscope (Nikon, Sendai, Japan) and analyzed in terms of corneal thickness, histological structure, inflammatory cell infiltration and collagen destruction.

Second harmonic generation (SHG)
At 14d, 21d and 28d post-injury, mice were under deep anesthesia and fixed on a designated plate, with the ocular surface faced up. According to a previously described method [15], a modified plastic bowl containing sterile PBS was fixed on the ocular surface sealed by erythromycin eye ointment to ensure the use of water immersion objective. SHG imaging was performed using an inverted two-photon excitation fluorescence microscope (NLO780, Zeiss). The laser was tuned to 780 nm and a 20× water immersion objective (numerical aperture =1.0) was used to focus the excitation beam and to collect backward signals. The Z-stack (Z=5 μm) layer-by-layer scan was used, and the obtained images were three-dimensionally reconstructed using Imaris software (×64, version 7.4.2, Bitplane, Zurich, Switzerland) to calculate the average signal intensity of the image. The stronger the image signal intensity, the more regular the corneal matrix collagen fibers are arranged, the corneal matrix structure is intact, and the weaker the image signal, the corneal matrix collagen fiber structure is disordered or degraded. Collagen SHG signals were visually represented with grey-scale images.

Quantitative real-time PCR
The mice were sacrificed by cervical dislocation at 14d, 21d and 28d post-injury and the corneas were trimmed as described above. Corneas were cut into small pieces and grinded for RNA extraction. RNeasy Mini Kit (Qiagen, US) was used to extract total RNA according to the manual and cDNA was generated by reverse transcription (TIANScript RT Kit,

Enzyme-linked immunisorbent assay (ELISA)
The mice were sacrificed by cervical dislocation at 14d post-injury and the corneas were

Western blot assay
The mice were sacrificed by cervical dislocation at 14d post-injury and the corneas were

Statistical analysis
Each part of the experiment was repeated at least 3 times, and the statistical analysis is based on the summary of the results. Data were expressed as the mean ± S.D. and analyzed using GraphPad Prism software (GraphPad Prism7, GraphPad software company, San Diego, California, US). Corneal opacity score, corneal scar area, corneal thickness and mRNA level fibrosis-related factors were 3 or 4 grouped data at 3 time points, which were analyzed with regular two-way ANOVA (not repeated measures) along with multiply comparison corrected by Turkey test. As for SHG, ELISA and westernblot analysis, nonparametric one-way ANOVA was performed along with multiply comparison corrected by Turkey test as well. P < 0.05 was considered to be statistically significant.

Primary cell culture and identification of uMSCs
The morphology of the cultured cells is characterized according to their adherence to plastic surfaces with a fibroblast-like morphology (Fig. 1C). Cultured cells are positive for mensenchymal stem cell surface antigen CD29 and CD44 (Fig. 1B), whereas negative for hematopoietic stem cell marker CD45 and CD34 (Fig. 1A). According to the results of flow cytometry, cell number of CD45-CD34-CD29+CD44+ counts for 96.30 among total number of cells, which demonstrate the cultured cells belong to a member of mesenchymal stem cells.

uMSCs prevented corneal scar formation and enhanced slit-lamp observation and clinical score
Corneal scar formation is one of the major complications of FK and is closely related to prognosis. 12 mice of each group were involved in ocular surface examination. At 14d, 21d and 28d post-injury, uMSCs inj FK group showed decreased corneal scar formation, and enhanced corneal transparency, compared with vehicle inj FK group and FK group (Fig. 2a).
The comparison of corneal opacity scores and scar formation area (mm 2 ) between uMSCs inj FK group and the other two groups were statistically significant differences both at the 14d, 21d, and 28d post-injury ( Fig. 2b and c).

Corneas from uMSCs inj FK group exhibited reduced corneal thickness and inflammatory cell infiltration after FK in histological examination.
Histological examination was performed on 14d, 21d and 28d after injury. 6 mice of each group were involved. The vehicle inj FK group and FK group exhibited corneal thickening, multiply layers of irregularly aligned, excessive collagen deposition and extensive inflammatory infiltrates. In contrast, corneas from uMSCs inj FK group showed less corneal thickness, relative normal structural arrangements, fewer inflammatory cell infiltrations ( Fig. 3a-i). The comparison of average corneal thickness (μm) in uMSCs inj FK group and the 13 other two groups had statistically significant difference at 14d, 21d and 28d post-injury (Fig. 3m). Thus, histological examination provided further evidence of the therapeutic effects of uMSCs application on FK.

Collagen destruction was restored by uMSCs treatment in SHG
SHG pictures were taken by two-photon confocal microscopy in vivo at 14d, 21d and 28d post-injury. 6 mice of each group were involved. The mice corneas of control group exhibited high intensity transient signals due to regular arrangement of corneal collagen fibers (Fig. 4a, e, and i). However collagen degradation and abnormal collagen deposition caused by infection and inflammation in FK group and vehicle inj FK group exhibited weak signals excited by confocal microscopy (Fig. 4b, f, and j). Of note, collagen destruction was restored by uMSCs treatment with an average optical intensity (AOD) 69.97±7.09 at 14d post-injury, which had statistically significant differences compared with FK group (26.09±1.27) and vehicle inj FK group (28.98±3.32) (Fig. 4m). Light scattering were also markedly reduced in uMSCs treatment group compared with the other two groups.

uMSCs inhibited α-SMA production in mice corneal after FK
The α-SMA is a biochemical marker for myofibroblast. The corneal fibroblasts grown in presence of TGFβ1 expressed α-SMA and acquired fibroblastic phenotype [16]. Since α-SMA has been identified as a key mediator in corneal fibrogenesis, we determined the effect of uMSCs on α-SMA production during corneal fibrosis by immunofluorescence at 14d post-injury. 4 mice of each group were involved. Mice corneas of control group could only be found of α-SMA expression at the pericorneal vascular region due to vascular wall smooth muscle cells staining, but was not observed in the central area of cornea (Fig. 5a).
14 The α-SMA production was up-regulated in the lesion area of corneas 14d post-injury in FK group and vehicle inj FK group (Fig.5c and d), whereas was found down-regulated in uMSCs inj FK group (Fig. 5b), which was consistent with the results of clinical manifestations. The results demonstrated that uMSCs application could inhibit keratocyteto-myofibroblast transition during the healing period of FK. Discussion FK leads to corneal fibrosis. The keratocyte-to-myofibroblast transition plays crucial role during the process of corneal scar formation and TGFβ is the key regulatory factor during this period. At the early stage of injury, damaged epithelium and basement membrane 16 release inflammatory cytokines, mainly IL-1 (α and β), which leads to the necrosis and activation of keratocytes [17]. Quiescent keratocytes become activated fibroblasts, and keratocytes proteins such as cystallins and keratin sulfate proteoglycans are downregulated [18].α-SMA was up-regulated due to myofibroblast transdifferentiation, which leads to excessive extracellcular matrix deposition and lamella disorganization. Compared with other available sources of MSCs, the uMSCs have the advantages of lower immunogenicity, higher capacity in proliferation, conveniently available, and less ethical controversy [11,33]. The mechanisms involved in the therapeutic effects exerted by uMSCs, mainly focused on paracrine effects to suppress inflammation and myofibroblast differentiation. Researchers demonstrated that mRNA quantification of TSG-6 in MSCs predicted their efficacy in sterile inflammation models for corneal injury [34]. Exosomes are closed nanoscale membrane vesicles (30-150 nm) that transport active substances between different cells and identified as a new kind of major paracrine factor released by uMSCs [35]. Yu [36] reported that intravitreally injection of uMSCs-derived exosomes could reduce damage, inhibit apoptosis and suppress inflammation responses in laser-induced retinal injury. uMSCs-exosomal microRNAs has been found to play key roles in suppressing myofibroblast differentiation by inhibiting excess α-SMA and collagen deposition [11].

Fibrosis-related factors were down-regulated by uMSCs administration
Allogeneic transplantation often results in graft versus host response or host rejection.
Since uMSCs do not express MHC class II molecules on the surface and can inhibit lymphocyte proliferation by producing some soluble cytokines, uMSCs can induce host immune tolerance and reduce transplant rejection [37]. In this experiment, the transplanted animals did not see significant immunological rejection, the eye showed normal corneal transparency, no corneal neovascularization, clear pupil and iris texture, no ocular reaction, such as uveal reaction. Studies suggest that uMSCs transplantation did not result in host anti-graft rejection.
Of note, the anti-fibrosis effect of uMSCs mainly occurs in cases with mild symptoms, and it is ineffective for those cases with severe infection, strong inflammatory response and perforation. This suggests that for the FK, a kind of infectious keratopathy, the use of antifungal drugs is essential at early stage, and further in combination with antiinflammatory agent to reduce the inflammatory response to tissue damage, which is also our next research direction.

Conclusion
The current study suggests that human uMSCs can evidently inhibit corneal inflammation and corneal fibrosis after FK wounding, the corneal opacity, scar formation area and corneal thickness can be reduced accompanying with down-expression of α-SMA, TGFβ1, CTGF, and COLⅠ through TGFβ1/Smad2 signaling pathway regulation. Our study has an implication for further exploration of MSCs as a novel therapy for patients with infectious eye disease and other inflammatory and fibrosis ocular diseases. However, further studies should be established to explore the in-depth mechanism underlying mesenchymal stem cells regulation in corneal fibrosis for new therapies.

Ethics approval and consent to participate
Mice were maintained in a SPF-class animal laboratory in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For the use of umbilical cord, written informed consent was obtained from the donors. All procedures used in this study were approved by Henan Provincial Eye Hospital Ethics Committe in compliance with the National Institutes of Health guidelines.

Availability of data and material
All the data supporting the findings was contained within the manuscript.   Values are presented as means ± SD, n=6, Mean values with asterisks are significantly different (P < 0.05). Scale bar, 50μm.