AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.METHODS: HCE cells cultured in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-κB and relative downstream cytokines such as TNF-α, IL-1β, IL-8 and IL-10 in supernatant fluid were measured by ELISA.RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-α and IL-1β reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner.CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-κB pathway. SP-D possibly mediates the recognition to AF mycelium.
AIM: To observe the expression of vitamin D receptor(VDR) in human specimen and immortalized human corneal epithelium cells(HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide(CAMP) in the downstream pathway of VDR.·METHODS: Immunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction(qPCR) was performed to observe the messenger ribonucleic acid(mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both m RNA and protein levels.·RESULTS: We found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The m RNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen(P <0.01) and it could be inhibited by toll like receptor 2(TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation(P <0.01).·CONCLUSION: The VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.
Lin CongYi-Ping XiaGui-Qiu ZhaoJing LinQiang XuLi-Ting HuJian-Qiu QuXu-Dong Peng
AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P <0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.
AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat's corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats'. There was no significantly difference of dectin-1 mRNA expression in group A and B (P >0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P <0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat's corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P<0.01). · CONCLUSION:Dectin-1 exists in rat's cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.
AIM: To explore the expression of S100 B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro.·METHODS: Immortalized human corneal epithelial cells(HCECs) were exposed to inactive Aspergillus fumigatus(A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24 h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48 h and the normal rat corneas were used for normal control. The m RNA level of S100 B was evaluated by real time quantitative reverse transcription-polymerase chain reaction(q RT-PCR).S100 B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining(IHC/ICC).· RESULTS: Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100 B at baseline. A. fumigatus significantly induced S100 B m RNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the m RNA began to rise significantly at8 h in vitro(P <0.05) and continue to rise as time prolonged(P <0.01). In vivo, S100 B m RNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12 h after infection(P <0.05) and reached to a peak at 24h(P <0.001). Immunochemistryrevealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100 B protein.·CONCLUSION: S100 B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation.S100 B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.
Jie ZhangGui-Qiu ZhaoJing QuCheng-Ye CheJing LinNan JiangHan ZhaoXue-Jun wang
AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1(TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection.METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis(FK) group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72 h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1through quantitative reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed.RESULTS: Corneal inflammation scores increased with time after fungal infection(F =49.74, P =0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole(F =137.78, P =0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h(P <0.001,compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24 h and 48 h after fungal infection.Immunofluorescence technique showed that TREM-1mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1expression in FK(r =0.942, P =0.000).CONCLUSION: TREM-1 may contribute to amplify theinflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.
