| Fungal keratitis is an important cause of blindness and visual impairment which accounts for about 65% of all corneal ulcers worldwide. Fungal keratitis usually occurs after trauma, which is the primary predisposing factor in the agricultural countries, such as China and India. The traumatizing factors can either directly implant fungal conidia into the corneal stroma or abrade the corneal epithelium, leading to the invasion by exogenous fungi. Other predisposing factors including immunological incompetence, previous use of corticosteroids and the use of contact lenses. Species of Fusarium and Aspergillus are the principal etiologic agents of fungal keratitis. In a study of 115 patientswith diagnosed filamentous fungal keratitis, 31.3% failure was reported using 5% natamycin monotherapy. Large ulcer size, hypopyon and Aspergillus forms should be direct indicator of treatment failure. Besides, previous studies have revealed that 42-60% of corneal infections due to Aspergillus species lead to keratoplasty, in comparison to 23-32% of corneal ulcers due to Fusarium species. In our study, we choose the Aspergillus fumigatus (A. fumigatus) to be our object.Mammals sense the microorganisms via multiple innate pattern recognition receptors (PRRs), which include toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) leucine-rich repeat-containing receptors (NLRs). PRRs recognize the conserved microbial structures called pathogen-associated molecular patterns (PAMPS) to initiate an intracellular signaling cascade. Several distinct members have been identified in both family with the two most studied being TLR2 and NOD2. TLR2 from the TLR family can recognize ligands with very diverse structures, including fungal polysaccharideslike zymosan. NOD2, a member of the NLR family, acts as an intracellular sensor for the peptidoglycan (PGN)-derived PAMP muramyl dipeptide (MDP), a component of both Gram-positive and Gram-negative bacteria. Upon ligands recognition, NOD2 and TLR2 can induce the transcriptional expression of inflammatory mediators via the intracellular activation of nuclear factor κB (NFκB) signalling pathway. In the process above, receptor interacting protein 2 (RIP2) is a critical mediator of NOD2 signaling pathway. The binding of NOD2 and RIP2 allow RIP2 to interacts with the inhibitory κB kinase (IKK) subunit IKKy, resulting in activation of the IKK complex. The IKK complex then phosphorylates the NFκB inhibitor IκB-α and lead to its ubiquitination. After IκB-α is degraded, free NFκB will translocate into the nucleus where it drives the transcription of KB-containing genes.The expression of NOD2 and TLR2 in corneal epithelium has already been reported. We found that HI A. fumigatus conidia can raise the expression of TLR2 and NOD2 in our preliminary experiments. Previous studies also confirmed that TLR2 plays an important role in A. fumigatus keratitis. Given that they share the common outcome of NFκB signalling pathway activation, we intent to reveal the relationship between TLR2 and NOD2 in HCECs against A. fumigatus in our study. Our results indicated that zymosan can increase the expression of NOD2 in a TLR2-dependent way while its pretreatment can decrease the secretion of inflammatory cytokines induced by MDP. Meanwhile, A. fumigatus conidia also promote the expression of NOD2 and RIP2 partly through the TLR2-dependent way. Besides, knockdown of NOD2 reduce the secretion of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia.[Purpose]To determine the basal expression of NOD2 in HCECs and its expressionwhen challenged with MDP or Aspergillus fumigates.[Methods]1. Cell culture:Human corneal epithelial cells (HCECs) were kindly provided by Professor Fu-Shin X. Yu (Wayne State University, Detroit, Michigan). HCECs were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 50% defined keratinocyte serum free medium (KSFM) in a humidified 5% CO2 incubator at 37℃. The cells were seeded into six-well plates at a density of 1×105 cells per well and cultured in normal growth medium.2. Aspergillus fumigatus strain and preparation of conidia:A. fumigatus strain CCTCC 93024, purchased from the China Center for Type Culture Collection, was cultured on Sabouraud dextrose agar 7 days at 37℃ after inoculation. Conidia was prepared and heat-inactivated (HI) as described previously. A concentration of 1 × 105ml was used in the experiments.3. Cell treatment:The HCECs were treated with HI conidia of A. fumigatus (1× 105/ml). Cells were harvested 12h or 24h later for real time-polymerase chain reaction (RT-PCR) and western blottingto measure the mRNA and protein expression of TLR2 and NOD2.Another group of HCECs were challenged with MDP (10 μg/ml), which is the specific ligand for NOD2. Cells were collected 12h after MDP treatment for RT-PCR to measure the mRNA level ofNOD2 and RIP2.Cells and culture supernatants were collected 24h after MDP treatment forwestern blotting and ELISA to measure the protein level of NOD2, RIP2, NFκB-p65, IκB-α, IL-6, IL-8 and TNF-α. Immunofluorescence staining was applied to determine the expression and location of NFκB-p65.[Results]1. TLR2 and NOD2 were basically expressed in HCECs. The expression of both TLR2 and NOD2 were upregulated after treated with HI conidia of A. fumigatus.2. After treated with MDP, the expression of NOD2 and its downstream molecules were upregulated, the translocation of NFκB-p65 into cell nuclei was significantly enhanced and the secretion of IL-6, IL-8 and TNF-α was increased.[Conclusion]1. HI conidia of A. fumigatus can upregulate the expression of TLR2 and NOD2.2. MDP can activate NOD2 pathway, therefore increase the secretion of inflammatory cytokines. TLR2 and NOD2 were upregulated after treated with HI conidia of A. fumigatus.2. After treated with MDP, the expression of NOD2 and its downstream molecules were upregulated, the translocation of NFκB-p65 into cell nuclei was significantly enhanced and the secretion of IL-6, IL-8 and TNF-α was increased.[Conclusion]1. HI conidia of A. fumigatus can upregulate the expression of TLR2 and NOD2.2. MDP can activate NOD2 pathway, therefore increase the secretion of inflammatory cytokines.Part Ⅱ TLR2 regulate the expression and downstream pathway of NOD2 in HCECs anti fungal immunity response[Purpose]To explore the regulating effect of zymosan towards NOD2 and its mechanism. To examine the effect of zymosan pretreatment on the expression of NOD2 and RDP2 and the production of inflammatory induced by MDPor A. fumigatus in hCECs. To study the secretion of inflammatory cytokines induced by HI conidia after NOD2 knockout.[Methods]1. Cell treatment:The HCECs were treated with diverse dosage of zymosan or depleted zymosan, which can only be detected by Dectin-1. Cells were harvested 12h or 24h later for RT-PCR and western blotting to measure the mRNA and protein expression of NOD2 and RIP2. Another group of HCECs were pretreated with diverse dosage of zymosan.12h after pretreatment, the medium was removed and HCECs were treated with MDP (10μg/ml). Cells were collected 12h after MDP treatment for RT-PCR to measure the mRNA level ofNOD2 and RIP2.Cells and culture supernatants were collected 24h after MDP treatment forwestern blotting and ELISA to measure the protein level of NOD2, RIP2,IL-6, IL-8 and TNF-α.2. TLR2 blocking:hCECs were seeded in 6-well plates at 1 ×105/well. At 50-60% confluency (24 h after seeded), cells were incubated with monoclonal anti-TLR2 blocking antibody (100 μg/ml). After 4 hours, the culture medium was replaced and the HI conidia were added. Cells were collected 12h after conidia treatment for RT-PCR to measure the mRNA level ofNOD2 and RIP2.Cells and culture supernatants were collected 24h after conidia treatment for Western blotting and ELISA to measure the protein level of NOD2, RIP2, IL-6, IL-8 and TNF-α. Immunofluorescence staining was applied to determine the expression and location of NFκB-p65.3. RNA interference:Three target sequences were chosen from the human Nod2 gene (GenBank, Gene ID:64127) todesign siRNA online (WI siRNA selection program, http://sirna.wi.mit.edu/), and a scramble siRNA that does not match any known mammalian GenBank sequence was designed using the Invivogen scramble siRNA online program. HCECs were seeded in 6-well plates at 1 × 105 cells/well. At 50-60% confluency, cells were transfected with 50 nM siRNA per well using Lipofectamine 2000 and Opti-MEM Ⅰ reduced serum medium according to the manufacturer’s protocols. The transfection efficiency was assessed by RT-PCR and Western blotting. Choose the most efficient NOD2 siRNA for further assays.4 hours after siRNA transfection, the HI conidia (1×105/ml) were added to the replacement medium. The plates were incubated for 24 h, and culture supernatants were collected for ELISA to measure the secretion of IL-6, IL-8 and TNF-α.[Results]1. The most efficient concentration of zymosan treatment is 10μg/ml. The expression of both NOD2 and RIP2 were upregulated after treated with zymosan but not depleted zymosan. This effect can be impeded by TLR2 antibody.2. The most efficient concentration of zymosan pretreatment is lOng/ml. Compared with those merely treated with MDP, zymosan pretreatment group reduced the expression of NOD2 and RIP2 induced by MDP and the secretion of IL-6, IL-8 and TNF-α was also decreased.3. After TLR2 blockade, the up-regulation of NOD2 and its downstream molecules induced by HI conidia was dampened, and the translocation of NFκB-p65 into cell nuclei was also significantly attenuated.4. NOD2 siRNA can significantly reduce the expression of NOD2 and RIP2. After NOD2 knockout, the increased secretion of IL-6, IL-8 and TNF-a induced by HI conidia was dampend.[Conclusion]1. Zymosan can upregulate the expression of NOD2 and RIP2 through a TLR2-dependent pathway.2. Zymosan pretreatment may induce a "cross tolerance" between different PRR families, therefore dampened the cell response towards MDP.3. HI conidia also activate the NOD2 pathway through a partly-TLR2-dependent pathway, which is similar to zymosan.4. NOD2 was successfully knocked out using siRNA. NOD2 plays a positive role in the inflammatory cytokines secretion initiated by HI conidia. |
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