http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2015.1016578

RESEARCH ARTICLE

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Effect of the TLR2/MyD88/NF-kB axis on corneal allograft rejection after penetrating keratoplasty Wei Wu1y, Shengyou Yu2y, Songfu Feng1, Jize Yang1, and Xiaohe Lu1 1

Department of Ophthalmology, ZhuJiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China and Department of pediatrics, Guangzhou first people’s Hospital, Guangzhou 510282, Guangdong Province, China

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Abstract

Keywords

Purpose: To evaluate the effect of the TLR2 (Toll-like receptor 2)/MyD88/NF-kB axis on the allograft rejection after penetrating keratoplasty (PK). Methods: The PK rat models were randomly divided into four groups: allograft group, dexamethasone group, PDTC group and isograft group. The mean survival time (MST) and rejection index of corneal grafts were observed. The immunohistochemical staining of TGF-a was performed on day 15. The messenger RNA (mRNA) and protein expression of TLR2, MyD88 and NF-kB p65 in corneal grafts were detected by reverse transcription–polymerase chain reaction (RT–PCR) and western blotting. Results: On days 5, 7, 9, 11, 13 and 15, the rejection index in the allograft group was higher than in the other three groups (p50.05). The MST in the PDTC group (MST, 23.30 ± 0.42 days, n ¼ 10) and in the dexamethasone group (MST, 24.40 ± 0.50 days, n ¼ 10) were higher than in the allograft group (MST, 14.7 ± 0.70 days, n ¼ 10) (2 ¼ 18.02, p50.01; 2 ¼ 21.47, p50.01). The expression of TNF-a in the PDTC group and in the dexamethasone group decreased compared with the allograft group by immunohistochemistry. On day 15, the mRNA and protein expression of TLR2, MyD88 and NF-kB p65 in the PDTC group and the dexamethasone group were less than in the allograft group (p50.05). Conclusions: Expression of TLR2, MyD88 and NF-kB p65 in rat corneal graft increased significantly and concurred with the allograft rejection, but were effectively inhibited by the treatment with dexamethasone and PDTC after PK. Dexamethasone could improve corneal allograft survival by the TLR2/MyD88/NF-kB axis. PDTC could suppress corneal graft rejection by inhibiting the activity of NF-kB. The TLR2/MyD88/NF-kB axis maybe a potential therapeutic target for corneal allograft rejection.

Allograft rejection, cornea, dexamethasone, MyD88, NF-kB, Toll-like receptor 2

Introduction Recently, keratonosus is one major factor of corneal blindness, which affects the visual quality seriously. Penetrating keratoplasty (PK) is the important treatment of irreversible corneal blindness, which not only could improve visual quality, but also could control corneal infection and maintain the integrity of the eyeball (1–3). However, the immune privilege is abolished and immune rejection emerges as a major threat to the survival of corneal allograft, especially for high-risk keratoplasty (4–8). Although topical and systemic immunosuppressants like CsA could prolong the survival time of corneal grafts, therapeutic dosage is limited by drug toxicity and the potentially life-threatening complications associated with immune suppression (9,10). Although the mechanism of corneal graft rejection has been studied a yWei Wu and Shengyou Yu contributed equally to this study and share first authorship. Address for correspondence: Professor Xiao-he Lu, MD, PHD, Department of Ophthalmology, ZhuJiang Hospital of Southern Medical University, Guangzhou 515282, Guangdong Province, China. E-mail: [email protected]

History Received 12 January 2015 Revised 3 February 2015 Accepted 3 February 2015 Published online 24 March 2015

lot, the mechanism is still unclear that how to prevent and cure corneal allograft rejection better (4,5,7,8). Therefore, the mechanism of corneal graft rejection remains to be studied further for the survival of corneal allograft. Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune response, which could identify various kinds of pathogenic microorganisms. TLRs not only could trigger innate immunity by recognizing pathogen-associated molecular patterns (PAMPs), but also could induce and regulate adaptive immunity (11–13). TLRs and its downstream molecules were found to express widely in both corneal and conjunctival tissues and dendritic cells including myeloid differentiation protein 88 (MyD88) and nuclear transcription factor kB (NF-kB) (14,15). Our previous study indicated that the expression of TLR2 was descent during the preventive treatment of corneal allograft rejection by glucocorticoid (16), which indicated that the TLR2/MyD88/NF-kB axis may play an important role in the prevention of corneal graft rejection (17). It was consistent with the immune tolerance of kidney transplantation, which demonstrated that the immune tolerance of kidney transplantation may be induced by TLR2 inhibitor and MyD88 knockout (18–20). Therefore, if MyD88 and

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NF-kB were blocked in the ocular surface, it would be very important for how to preventive treat corneal allograft rejection and to induce immune tolerance after KP. Therefore, the role of the TLR2/MyD88/NF-kB axis is studied further during the preventive treatment of corneal allograft rejection by glucocorticoid, which could help us to understand the mechanism of corneal allograft rejection better and to find a potential therapeutic target for corneal allograft rejection. It is also conducive to improve the postoperative vision and visual quality in patients with corneal blindness after KP.

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Materials and methods Animals Sixty eight Sprague–Dawley (SD) and 39 Wistar rats (weighing 200–220 g) were obtained from the Experimental Animal Center of Southern Medical University. Fifty two SD rats served as hosts, accepting corneal grafts from either other 13 SD rats (isograft) or Wistar donors (allograft). The residual three SD rats were normal control. The rats were quarantined and acclimatized 1 week before the experiments in the Animal Laboratory of Southern Medical University, Guangzhou, China. The experiments were performed on the rats under standard conditions throughout the study as follows: room temperature 25 ± 2  C, relative humidity 60 ± 10% and alternating 12-h light–dark cycles (8 AM–8 PM). All experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical Ethics Committee of Southern Medical University. Penetrating keratoplasty Penetrating orthotopic keratoplasty was performed on the unilateral right eye, as described previously (16). Before the surgical procedure, the animals were anesthetized by intraperitoneal injection of 3% barbital (1.5 ml/kg). Briefly, a 3.25-mm central area of the cornea was excised with Vannas scissors from the donor and secured in the host graft bed of 3.0-mm diameter with 8–10 interrupted 10-0 nylon sutures. Antibiotic ointment was applied to the corneal surface of the animals in all groups. Four experimental groups were included: allograft group, dexamethasone group, PDTC group (allograft treated with phosphate-buffered saline, 0.2% dexamethasone, 10 mg/ml pyrrolidine dithiocarbamic acid eyedrops, respectively) and isograft group (21). Phosphate-buffered saline was prepared with 150 mM NaCl, 6 mM Na2HPO4 and 4 mM KH2PO4 (pH 7.2). After the operation, all grafts were examined by slit-lamp microscopy once every other day to calculate the rejective index (RI) according to opacity, edema and neovascularization of grafts. When the RI grade was 5, rejection was acknowledged (16,22). Ten rats were evaluated by mean survival time (MST) followed up to 26 days in dexamethasone, PDTC, isograft and allograft groups, respectively. The animals were discarded when infection and graft failure occurred in the cornea, and follow up stopped when graft failure occurs. The RI of corneal grafts was always assessed by the two observers.

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Immunohistochemistry On day 15 after KP, the corneal grafts of the four groups were taken for immunohistological analysis. Immunohistochemical staining of whole-mount corneas was performed according to the manufacturer’s instructions for antibodies. The corneal grafts were enucleated, fixed with 10% formalin solution and imbedded in paraffin, then cut into 4 mm sections for immunohistochemical staining. Sections were baked at 55  C overnight, deparaffinized in xylene and rehydrated through a graded series of ethanol concentrations. A 5% goat serum solution was used for blocking non-specific antibody binding for 30 min. The primary antibody of rabbit anti TGF-a polyclonal antibody (Boster, Wuhan, China) was incubated overnight at 4  C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min before biotinylated anti-rabbit and anti-mouse were applied as secondary antibodies and avidin–biotin–peroxidase complex (ABC kit, Vector Laboratories, Burlingame, USA) served as the third reagent. Negative control sections were incubated in the absence of the primary antibody. The medial area of corneal section was obtained and evaluated by HMIAS-2000 high definition color medical image analysis software, on the expression of TGF-a in each group. Real-time RT-PCR The mRNA expression of TLR2, MyD88 and NF-kB p65 in corneal grafts was detected by reverse transcription–polymerase chain reaction (RT-PCR) on the 15th day after the operation. Methods of real-time RT-PCR to quantify the expression levels of TLR2 mRNA in corneas have been described by previous studies (16,23). Total RNA was isolated from frozen corneas using Trizol and digested with deoxyribonuclease I to remove genomic DNA contamination, according to the manufacturer’s instructions (Invitrogen, Waltham, MA). One microgram of total RNA was reverse transcribed to produce a complementary DNA template for PCR using a First Strand cDNA Synthesis Kit (GeneCopoeia, Rockville. MD). The primer pair sequences used for the PCRs were 50 -AGC GAAAATCTGA TGGTTGAA-30 and 50 -TGACTCAAAACC AAGCTTTGTAGA-30 for rat TLR2, 50 -GCTCATTGAGA AAAGGTGTCG-30 and 50 -TCAGTCGCTTCTGTTGGACA30 for rat MyD88, and 50 -CCAACCGTGAAAAGATGACC-30 and 50 -ACCAGAGGCATACAGGGACA-30 for rat b-actin. The primers were synthesized by Vipotion Biotechnology, Co, Ltd (Guangzhou, China). For the PCR amplification, 1 ml of complementary DNA template was used per 25 ml of PCR (23 AllinOne Q-PCR Mix; Gene Copoeia, Rockville. MD). Transcript quantification was performed in triplicate for each sample (IQ5 Real-Time PCR Detection System; Bio-Rad, Hercules, CA) and reported relative to normal rat corneas after normalized by b-actin using the DDCT method (16,23). Western-blot analysis Proteins (50 mg) extracted from corneal allograft were subjected to electrophoresis on a 12% SDS–PAGE and then transferred onto PVDF membranes. After blocking with 5%

TLR2/MyD88/NF-B axis on corneal allograft

DOI: 10.3109/10799893.2015.1016578

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non-fat milk for 1 h, the membranes were washed 3 times with TBST for 5 min and then incubated overnight with polyclonal antibodies against TLR2, MyD88, NF-kB p65 and GAPDH (1:1000 dilution in 5% non-fat milk) in TBST, respectively. GAPDH was used as the control. After washing 3 times in TBST, membranes were incubated with secondary HRPconjugated anti-mouse IgG for 1 h. The membranes were again washed with TBST 3 times, and 1 time in TBS, for 5 min each. The blots were visualized using enhanced chemiluminescence reagent (Pierce, Waltham, MA). It was repeated 3 times. Results were quantified by capturing the exposed X-ray film image and using area measurements from image analysis software. Statistical analysis SPSS 13.0 statistical package (Chicago, IL) was used for statistical analysis. All data were expressed as mean ± standard deviation (mean ± SD). Corneal graft survival of recipients was compared using Kaplane–Meier survival curve. The clinical scoring data were compared among various groups by means of one-way ANOVA. Repeated-measures analysis of variance (factorial analysis) followed by Student– Newman–Keuls test were used for post hoc analysis among different groups. One-way analysis of variance for the rest statistical analysis, all tests were two-tailed and a

Figure 1. The status of corneal grafts in four groups by slit lamp. It was shown that on postoperative days 1, 7 and 15, the status of corneal grafts in isograft group, allograft group, dexamethasone group and PDTC group. Corneal graft appeared edema slightly and neovessels growth in allograft group on day 7. On the 15th postoperative days, corneal graft appeared seriously opacity and many neovessels in allograft group, whereas it was less opacity and neovessels in isograft group, dexamethasone group and PDTC group.

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probability value of 0.05 was considered as statistically significant.

Results The results of clinical observations and survival analysis During the first week after keratoplasty, slight epithelial and stromal edema was observed in all grafts in the allograft group, the dexamethasone group and the PDTC group. On day 7, the corneal opacity and edema could be obviously observed together with signs of neovasculature of the limbus in the allograft group. On day 9, the corneal neovessels in the allograft group began to migrate into the corneal bed and had begun to develop around the edge of the incision. Corneal neovessels of the limbus could be partly observed in the isograft group, the PDTC group and the dexamethasone group. On day 15, the allografts were gradually opaque, and the rejection line appeared in the corneal epithelium and endothelium in the allograft group. In contrast, corneal neovascularization and edema was significantly delayed in the isograft group, the dexamethasone group and the PDTC group. The edema, opacity and neovascularization of the corneal grafts occurred with the prolongation of the postoperative time in the allograft groups (Figure 1).

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The histopathological results of corneal grafts by hematoxylin and eosin staining Normal rat corneal epithelium was composed of five to six cellular layers, the collagen fibers were arranged regularly in normal corneal stromal layer without swelling, and without the infiltration of monocytes and lymphocytes and other inflammatory cells. In addition to the appearance of vacuole at the basal layers of epithelial cells, marked infiltration of inflammatory cells and neovascularization in corneal stroma were observed in the allograft group, which increased gradually along with the prolong of the postoperative time. On the 15th postoperative day, there was a certain degree edema and opacity with the infiltration of many inflammatory cells and neovascularization in the allograft group. However, the infiltrating inflammatory cells and stromal neovascularization reduced significantly in the PDTC group and the dexamethasone group (Figure 3). Expression of TNF-a by immunohistochemistry

Figure 2. The rejection index (RI) and survival analysis of corneal grafts in four groups. It was shown that the scores of RI were less in the isograft group, the dexamethasone group and the PDTC group compared with the allograft group (p50.05) (upside). It was shown that the MST was 19.20 ± 0.49, 14.70 ± 0.70, 24.40 ± 0.50 and 23.30 ± 0.42 days in the isograft group, allograft group, dexamethasone group and PDTC group, respectively (downside). The MST of PDTC group was longer than that of the allograft group (2 ¼ 18.02, p50.01). The MST of dexamethasone group was longer than that of the allograft group (2 ¼ 21.47, p50.01). The MST of dexamethasone group was longer than that of the PDTC group without statistical significance (2 ¼ 3.82, p ¼ 0.051). (*, # p50.05).

In following period, the rejection index of each group stepped up consistently, but for the isograft group, the PDTC group and the dexamethasone group that was much lower compared with the allograft group. According to the score of RI, on the postoperative 9th day, the rejection index of the allograft group, but not that of the isograft group, the dexamethasone group and the PDTC group, met the diagnostic criteria for graft rejection. However, at the postoperative 15th day, the RI in the PDTC group and the dexamethasone group partially reached the diagnostic criteria of rejection, the RI of each group was 7.20 ± 1.030, 6.60 ± 0.840, 6.40 ± 0.966 and 10.50 ± 0.707, respectively. On days 5, 7, 9, 11, 13 and 15, the score of RI in the allograft group was higher than in the other three groups (p50.05) (Figure 2). The MST was less in the allograft group (MST, 14.7 ± 0.70 days, n ¼ 10) than in the isograft group (MST, 19.20 ± 0.49 days, n ¼ 10), in the PDTC group (MST, 23.30 ± 0.42 days, n ¼ 10) and in the dexamethasone group (MST, 24.40 ± 0.50 days, n ¼ 10) with a significant statistical difference (2 ¼ 18.02, p50.01; 2 ¼ 21.47, p50.01). The MST in the dexamethasone group was higher than in the PDTC group with no statistical difference (2 ¼ 2.75, p ¼ 0.097) (Figure 2).

The corneal grafts were harvested on day 15 after the operation, the expression of TNF-a was not found in the normal cornea. The expression of TNF-a increased in the allograft group, which was mainly expressed in the corneal epithelial layer with the edema of corneal stromal, whereas it decreased in the isograft group, the dexamethasone group and the PDTC group. Therefore, the results of immunohistochemistry revealed that TNFa was mainly expressed in the corneal allografts (Figure 3). The mRNA expression of TLR2, MyD88 and p65 in corneal grafts Some mRNA expression of TLR2, MyD88 and NF-kB P65 were found in the normal cornea. At the postoperative 15 days, the mRNA expression of TLR2 was significant difference among the allograft group, the isograft group, the dexamethasone group and the PDTC group (F ¼ 73.07, p50.001). The mRNA expression of TLR2 was higher in the allograft group than in the dexamethasone group (p50.01) and the PDTC group (p50.01). The mRNA expression of TLR2 was higher in the PDTC group than in the isograft group with significantly statistical difference (p ¼ 0.030). The mRNA expression of TLR2 in the dexamethasone group was not higher than in the PDTC group (p ¼ 0.112) and in the isograft group (p ¼ 0.419) without significant difference. On day 15 after the operation, the mRNA expression of MyD88 was significant difference among the four groups (F ¼ 75.07, p50.001). The mRNA expression of MyD88 was higher in the allograft group than in the dexamethasone group (p50.01) and the PDTC group (p50.01). The mRNA expression of MyD88 was higher in the PDTC group than in the dexamethasone group with significantly statistical difference (p ¼ 0.037) which was not significant difference with the isograft group (p ¼ 0.116). On day 15 after the operation, the mRNA expression of NF-kB P65 was significant difference among the four groups (F ¼ 162.68, p50.001). The mRNA expression of NF-kB P65 was higher in the allograft group than in the

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DOI: 10.3109/10799893.2015.1016578

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Figure 3. The histopathological result and the expression of TNF-a in corneal grafts on the 15th postoperative day (100). It was shown that the histopathological results in each group by hematoxylin and eosin staining on day 15 (upside). The cornea was observed without swelling and the infiltration of inflammatory cells in normal control and isograft group. There were various degrees of congregation of inflammatory cells and neovascularization in the allograft group. There was less inflammatory cells and neovascularization in the PDTC group and dexamethasone group. It was shown that the expression of TNF-a on corneal graft on day 15 after PK (DAB  100) (downside). On the 15th postoperative day, the high expression of TGF-a was observed with many inflammatory cells infiltration in the allograft group. No expression of TGF-a on corneal graft was observed with moderate swelling in the isograft group, the PDTC group and dexamethasone group.

dexamethasone group (p50.01) and the PDTC group (p50.01). The mRNA expression of NF-kB P65 was less in the dexamethasone group than in the isograft group (p ¼ 0.001) and in the PDTC group (p ¼ 0.027) with significantly statistical difference. It indicated that the mRNA expression of TLR2, MyD88 and NF-kB p65 in the dexamethasone group and the PDTC group decreased significantly in comparison with that of the allograft group (Figure 4). The proteinic expression of TLR2, MyD88 and p65 in corneal grafts On the 15th postoperative day, the proteinic expression of TLR2, MyD88, NF-kB p65 in the dexamethasone group was less than in the isograft group and in the allograft group with significant difference (p50.05). The proteinic expression of TLR2 in the dexamethasone group was less than in the PDTC group with significant difference (p50.01) (Figure 5).

Discussion Toll-like receptors are widely expressed in corneal and conjunctival tissues and dendritic cells, which could identify ocular pathogen associated molecules antigen in the early phase, causing innate immune response in ocular surface (11–13). TLRs play an important role in the regulation of ocular infectious and non-infectious inflammation. TLR2 could recognize the PAMPs from bacteria, fungi, parasites and viruses, which could form the polymerization with TLR1 and TLR6 to increase binding target site to the ligand (14,15). TLR2 could upregulate the expression of TNF-a and costimulatory molecules by the TLR2/MyD88/NF-kB axis, which could stimulate ocular obtained immune response (24,25). Therefore, TLR2 is not only an important receptor to recognize the pathogenic microorganisms in the natural

immune system, but also the bridge between innate immunity and acquired immunity in ocular surface. Recent studies demonstrated that TLR2 not only played an important role in the process of autoimmune and allergic ophthalmopathy, but also in the process of corneal allograft rejection (16). As a type I transmembrane receptor, the expression of TLR2 increased in the process of acute rejection after PK, and what about the variation and the role of MyD88 and NF-kB in the TLR2/MyD88/NF-kB axis on the preventive cure of corneal allograft rejection by dexamethasone, which remained to be studied further. The study indicated that the scores of RI in the dexamethasone group and the PDTC group were less than in the allograft group with significantly statistical difference on days 5, 7, 9, 11, 13 and 15 after the operation. On the 15th day after KP, the serious degree of corneal opacity, edema and neovascularization in the dexamethasone group and the PDTC group were significantly lighter compared with the allograft group by histopathological section. The survival time of corneal graft in the dexamethasone group and the PDTC group was longer than the allograft group. It indicated that dexamethasone and PDTC could significantly suppress corneal graft rejection and prolong the survival time of corneal graft. We further found that the expression of TLR2, MyD88 and NF-kB p65 decreased in the preventive cure of rat corneal graft rejection by dexamethasone, while the expression of TLR2, MyD88 and NF-kB p65 increased in the process of corneal allograft rejection. It indicated that the TLR2/MyD88/ NF-kB axis played an important regulatory role in the process of corneal graft rejection, whereas dexamethasone could prevent and cure corneal graft rejection to some extent by regulating the TLR2/MyD88/NF-kB axis. NF-kB is a heterodimer including five members of Rel, P65 (RelA), RelB, P50 and P52 in mammalian. After NF-kB was activated by cytokine, the translocation signal of the P50 homopolymer and the binding sites of DNA on the P65

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Figure 4. The mRNA expression of TLR2, MyD88 and p65 in corneal grafts on the 15th operative day. Some mRNA expression of TLR2, MyD88 and p65 was found in the normal cornea. At the postoperative 15 days, the mRNA expression of TLR2, MyD88 and NF-kB p65 were less in the dexamethasone group, isograft group and PDTC group than that of the allograft group (**p50.05).

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Figure 5. The proteinic expression of TLR2, MyD88 and p65 in corneal grafts on the 15th operative day. It was shown that the proteinic expression of TLR2, MyD88, NF-kB p65 in the dexamethasone group was less than that of the isograft group and the allograft group with significant difference on the 15th postoperative day (**p50.05). The proteinic expression of TLR2 in the dexamethasone group was less than in the PDTC group with significant difference (*p50.01).

homopolymer revealed after IKB phosphorylation. P65/P50 heterodimer translocated from the cytoplasm to the nucleus by the role of P50 homopolymer in which P65 (RelA) plays the regulative role of nuclear transcription by binding with the KB sequence (24,26). Therefore, the mRNA and proteinic expression of P65 (RelA) could reflect the transcriptional activity of NF-kB to some extent. The study revealed that PDTC was a specific inhibitor of NF-kB, which could inhibit the activity of NF-kB. PDTC eyedrops has been applied in the study of herpetic stromal keratitis and corneal neovascularization (21). Our study revealed that the activity of NF-kB decreased in the dexamethasone group and the PDTC group. The survival time of corneal graft was significantly longer in the dexamethasone group and the PDTC group than in the allograft group. It indicated that PDTC could prevent corneal graft rejection by inhibiting the activity of NF-kB. The activity of NF-kB in ocular surface could be suppressed to prevent and cure corneal allograft rejection and prolong the survival time of corneal graft. It demonstrated that Th1/Th2 immune deviation played an important regulative role in the process of corneal graft rejection. Helper T cells (Th cells) were the starting point of the immune response. According to the different cytokine by the secretion of Th cells, Th cells could be divided into two subgroups: Th1 and Th2. Th1 cells could secrete TNF-a, IFN-g, IL-2 and IL-12, which could mediate cellular immunity, involved in the proliferation and the functional differentiation of T cells and in the process of delayed hypersensitivity. Th2 cells could secrete IL-4, IL-6 and IL-10, which could mediate humoral immunity, involved in the proliferation and differentiation of B cells (27–29). It revealed that Th1/Th2 homeostasis played an important role to induce and maintain immune tolerance, Th1 to Th2 immune deviation could induce immune tolerance of organ transplantation (30,31). TLR2 signaling pathway could induce the secretion of many cytokines, including TNF-a, IL-6, IL-2 and IL-12, etc, which could involve in the process of Th1/Th2 immune response. Our study revealed that the expression of TNF-a increased in the allograft group, whereas it decreased in the dexamethasone and PDTC group. It indicated that Th1 to Th2 immune deviation may be existent during the prevention and cure of corneal graft rejection by dexamethasone and PDTC, which could prevent corneal graft rejection. It remains to be studied that whether the role of the TLR2/MyD88/NF-kB axis

in corneal graft rejection was related with Th1/Th2 immune deviation. It was found that TNF-a played an important role during corneal allograft rejection. TNF-a was one important downstream cytokine of the activated NF-kB, which could reflect the activity of NF-kB and be the hint of Th1/Th2 immune deviation to a certain extent during corneal allograft rejection (25). In conclusion, the expression of TLR2, MyD88 and NF-kB decreased in the process of prevention and cure of corneal graft rejection after KP by topical application of dexamethasone. Dexamethasone could prevent corneal allograft rejection by the TLR2/MyD88/NF-kB axis in ocular surface. The activity of NF-kB in ocular surface could be suppressed to prevent corneal allograft rejection by topical application of PDTC. The TLR2/MyD88/NF-kB axis may play an important role in the prevention of corneal graft rejection, which may be a potential therapeutic target for corneal allograft rejection after KP.

Declaration of interest The authors contributed equally to this work. The authors declare no conflict interests. This research was supported by grant No. 81371067 (L.X.H.) from the National Science Foundation of China.

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NF-κB axis on corneal allograft rejection after penetrating keratoplasty.

To evaluate the effect of the TLR2 (Toll-like receptor 2)/MyD88/NF-κB axis on the allograft rejection after penetrating keratoplasty (PK)...
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