Mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress.

This study aims to investigate the beneficial effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on trabecular meshwork cells under oxidative stress and predict candidate genes associated with this process. Trabecular meshwork cells were pretreated with BMSC-derived exosomes for 24 h, and exposed to 0.1 mM H2O2 for 6 h. Survival rate of trabecular meshwork cells was measured with CCK-8 assay. Production of intracellular reactive oxygen species (iROS) was measured using a flow cytometer. RT-PCR and ELISA were used to detect mRNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs). Sequencing of RNA and miRNA for trabecular meshwork cells from Exo and control groups was performed on BGISEQ500 platform. Phenotypically, pretreatment of BMSC-derived exosomes improves survival rate of trabecular meshwork cells exposed to H2O2, reduces production of iROS, and inhibits expression of inflammatory cytokines, whereas increases expression of MMPs. There were 23 miRNAs, 307 lncRNAs, and 367 mRNAs differentially expressed between Exo and control groups. Exosomes derived from BMSCs may protect trabecular meshwork cells from oxidative stress. Candidate genes responsible for beneficial effects, such as DIO2 and HMOX1, were predicted.

Expression profiles and function of Toll-like receptors in human corneal epithelia.

BACKGROUND: Toll-like receptors play an important role in the human immune system. This study was conducted to investigate the expression profiles and function of Toll-like receptor (TLR) 1 - 9 in human corneal epithelium. METHODS: The expression of TLR1 - 9 mRNA in 20 human donor corneal epithelia samples abraded during photorefractive keratotomy (PRK) and cultivated telomerase-immortalized human corneal epithelial cells (THCEs) was examined by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Human peripheral blood mononuclear cells (PBMCs) were used as positive controls. The expression of the TLR2 and TLR4 proteins was detected by Western analysis. ELISA was used to detect IL-8 secretion from THCEs challenged with ligands for TLR3 and TLR4 with and without antibody blockade. RESULTS: The expression of TLR1 - 9 at the mRNA level was detected in the epithelia of 20 patients and in THCE. Significant differences among individuals were observed. One patient was found to lack of the expression of TLR3, 4, 6 and 8, whereas another did not express TLR5. The expression of TLR2 and TLR4 protein was detected in human corneal epithelial cells. As THCE cells express TLR1 - 9, cells were challenged with lipopolysaccharides (LPS) and poly I:C to determine whether TLR4 and TLR3 were functional. The results showed that secretion of IL-8 by cells stimulated with LPS and Poly I:C was 7 to 10 fold greater than secretion by unchallenged cells. Blocking TLR4 with an anti-TLR4 antibody significantly inhibited the LPS-induced IL-8 production by THCE (P < 0.05). CONCLUSION: Human corneal epithelial cells express multiple TLRs and are able to recognize LPS and poly I:C. Different expression profiles among individuals suggest that differences in the susceptibilities and sensitivities to bacterial and viral infection in human populations relate to different patterns of TLR expression.

[Aspergillus fumigatus activate human corneal epithelial cells via Toll-like receptor 2 and 4].

OBJECTIVE: To determine Toll-like receptors (TLR) 2 and TLR4 expression in human corneal epithelial tissue and cell line (THCE), and its activation by aspergillus fumigatus (AF) in inflammatory response. METHODS: The expression of TLR2 and TLR4 protein in human corneal epithelial tissue and THCE was detected by Western blot and immunocytochemistry. THCE was challenged with AF mycelium fragment (5 x 10(6)/ml) and supernatant extract agent (equivalent to bovine serum albumin 10 microg/ml). IL-8 and TNF-alpha in THCE supernatant were detected by ELISA at 1, 2, 4 and 8 h post stimulation. The protein of IkappaBalpha in THCE cells was assayed by Western blot at 30 min, 1 h and 2 h after treatment. Antibody blocking test was utilized to evaluate the effect on IL-8 and TNF-alpha expression of THCE by blocking TLR2 and (or) TLR4 before challenge with AF agent. RESULTS: TLR2 and TLR4 protein were expressed in human corneal epithelial tissue and THCE. The IL-8 and TNF-alpha level in THCE supernatant was elevated at 1 h, increased to (64.71 +/- 5.15) pg/ml and (32.46 +/- 3.28) pg/ml (AF mycelium challenge group), (94.94 +/- 11.92) pg/ml and (48.70 +/- 3.32) pg/ml (AF supernatant challenge group) 8 h post-challenged, which was 3.0 times and 2.5 times, 4.5 times and 3.5 times to that of control group respectively (P < 0.01). The activity of IkappaBalpha in THCE cells was decreased to 10.31 +/- 1.30 (gray scale value) and 8.15 +/- 2.37 at 30 min after challenged with AF mycelium or supernatant extract agent compared to 51.57 +/- 5.58 and 49.23 +/- 3.49 of control group (P < 0.01), and was reverted at 2 h. The secretion of IL-8 and TNF-alpha was partly inhibited by blocking TLR2 or TLR4 (P < 0.05), obviously inhibited by blocking TLR2 and TLR4 (50% and 40% compared to that of control group) (P < 0.01) when challenged with AF mycelium. And that was markedly inhibited by blocking TLR4 or blocking TLR2 and TLR4 when challenged with AF supernatant (P < 0.01). The secretion of IL-8 and TNF-alpha was not inhibited by blocking TLR2 when challenged with AF supernatant (P > 0.05). CONCLUSIONS: AF agent may induce human corneal epithelial cells express inflammatory cytokines via TLR-NF-kappaB pathway. TLR2 and TLR4 possibly mediate the recognition to AF mycelium, and TLR4 may dominate the recognition to AF supernatant agent.

[The expression and functionality of Toll-like receptors in human corneal epithelium and tolerated human corneal epithelial cell line].

OBJECTIVE: To investigate the expression and functionality of Toll-like receptors (TLRs) 1 - 9 in human corneal epithelium and cell line THCE (tolerated human corneal epithelial). METHODS: Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was used to determine the expression of TLR1 - 9 mRNA in the specimens of human corneal epithelial cells from 20 persons, 5 males and 10 females, aged 18 - 25, undergoing photorefractive keratectomy (PRK), and in cultured THCE cells. Human peripheral blood mononuclear cells (PBMCs) were used as positive control. The expression of TLR2 protein and TLR4 protein were detected with Western blotting. KEM-2 fluid containing anti-TLR4 antibody was used to block the THEC cells for 30 minutes, then KEM-2 fluid containing PolyI: C or LPS, ligands for TLR 3 and 4, was used to stimulate the THEC cells, 1, 4, and 8 hours after the concentrations of IL-8 in the supernatant were determined by ELISA. RESULTS: The human corneal epithelial cells strongly expressed TLR1, 2, 3, 5, 6, and 9 mRNA, weakly expressed TLR7 and 8 mRNA, and very weakly expressed TLR4 mRNA. Negative expression of TLR3, 4, 6, and 8 mRNA was seen in 1 case, a female aged 22. Weak expression of TLR5 mRNA was seen in 1 case, a female aged 20. The THCE cells showed the same expression pattern as the healthy human corneal epithelial cells, except for the strong expression of TLR9. The human PBMCs, as positive controls, strongly expressed TLR1 approximately 4 and 8 mRNA, weakly expressed TLR5, 6, and 9 mRNA, and very weakly expressed TLR7 mRNA. The human corneal epithelial cells, cultured THEC cells, and PBMCs all expressed TLR2 and 4 proteins. The concentrations of IL-8 in the supernatant of the culture fluid of THEC cells increased along with the time of stimulation of LPS and Poly: C, ligands for TLR3 and 4, and reached to 297.33 pg/ml and 229.67 pg/ml respectively 8 hours after, 10 times and 7 times those of the control group (both P < 0.05). 30 minutes after the blocking of TLR4 with anti-TLR4 antibody LPS was used to stimulate the THEC, the concentration of IL-8 was 88.54 pg/ml, being 30% that of the un-blocked group (P < 0.05). CONCLUSION: Human corneal epithelium expresses TLR1 approximately 9 at different levels. THCE cell line is an excellent line to study TLRs function.