Role And Mechanism Of NOD1Mediated Corneal Innate Immune Response Against Aspergillus Fumigatus

BackgroundFungal keratitis is a potentially vision-threatening disease that requires timely accurate diagnosis and aggressive management to prevent vision loss, with a dramatic increase of incidence recently in both the US and China. Extended wear of contact lenses is a leading cause of fungal keratisis in US and other industrialized countries, while agricultural work is the primary risk factor when there is greater exposure to airborne soil and contaminated plant material. The extended use of immunosuppressive agents, long-term broad spectrum antibiotics, and AIDS has also considered as a risk factor for fungal keratisis. However, fungal keratisis is still one of the most challenging forms of infectious keratisis to treat, and the underlying cellular mechanisms that directly control inflammatory response are largely unresolved. Therefore, a better understanding of the molecular mechanisms by which corneal defense against fungal infection is required to develop therapeutic strategies.Cornea is the first barrier of defense against pathogenic infection. Fungal infection of the cornea is relatively infrequent when the cornea is intact. But after trauma, the corneal stroma is exposed and allows the fungi to invade. Corneal epithelial cells are an efficient physical barrier to infection and the first line of defense against microbial pathogens. Corneal innate immunity is necessary for host defense against invading pathogens. Therefore, the biological function of corneal epithelial cells to recognize and respond to microbial components is extremely important. The host corneal immune response triggered by fungi relies on innate immune recognition through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Activation of PRRs initiates a cascade of inflammatory signal transduction events, including infiltration of inflammatory cells (polymorphonuclear neutrophils and monocytes/macrophages), production of Th2-type cytokines (IL-4, IL-6and IL-10), chemokines (IL-8), pro-inflammatory cytokines (TNF-a and IL-1β), and antibacterial peptides (hBD2and LL37). The inflammatory response is necessary for corneal epithelial cells to mediate the initial hose defense, kill and clear the invading pathogens, such as fungi. Our recent work has demonstrated that both TLR2and TLR4play crucial roles in the recognition and response to A. fumigatus in HCECs by activation of multiple signaling pathways (NF-κB and ERK) and production of pro-inflammatory cytokines. However, whether there are intracellular receptors that respond to fungi has not been fully understood, while both TLR2and TLR4are mainly located in the membrane of HCECs.Recent studies suggest that another family of PRRs, the cytoplasmic-based NOD-like receptors (NLRs) also function as intracellular receptors to respond to microbes or microbial components. Among them, NOD1can recognize specific bacterial molecules and y-D-glutamyl-meso-diaminopilemic acids (iE-DAP), which induces secretion of pro-inflammatory cytokines through activation of the NF-κB and MAPK signaling pathways via the adaptor molecule RIP2. Recent studies revealed that NOD1also plays a crucial role in protozoa, virus and fungal infection in multiple organs, such as lung, intestine and oral cavity. However, little is known about the role of NOD1in the innate immunity and inflammatory response of HCECs to Aspergillus fumigatus (A. fumigatus) conidia.In the present study, we investigated the stimulatory effects of A. fumigatus on NOD1signaling pathways and the production of pro-inflammatory cytokines and antimicrobial molecules. Our results indicated that that A. fumigatus induces an innate immune and inflammatory response in HCECs characterized by secrestion of pro-inflammatory cytokine and chemokine via NOD1signaling pathway. Part I Role of nucleotide-binding oligomerization domain1in regulating the innate immune response in THCEs to Aspergillus fumigatus[Purpose]To study the expression and role of the nucleotide-binding oligomerization domain1(NOD1) in the innate immune response to Aspergillus fumigatus (A. fumigatus) in the immortalized human corneal epithelial cells (THCEs).[Methods]1. Human telomerase-immortalized corneal epithelial cells were kindly provided by Dr. Fu-Shin Yu of Wayne State University in the US and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12supplemented with50%defined keratinocyte serum free medium at37℃in a humidified5%CO2incubator.2. THCEs were seeded in six-well plates at a density of2×105cells per well. When the cells achieved80%confluence, they were challenged with different concentrations of the heat-inactivated A. fumigatus conidia and the NOD1ligand iE-DAP (10μg/ml) for0,2,4,8,12and24hours respectively. The cells and culture supernatants were then collected for further experiments.3. The control RNA and siRNA pool against NOD1were purchased from Invitrogen. The THCEs were grown to80%confluence, and siRNA transfection was carried out with Lipofectamine2000according to the manufacturer’s protocols. The transfection efficiency was detected by Western blot assay. THCEs were challenged with heat-inactivated A. fumigatus conidia at48h after transfection. Then the culture supernatants and cells were harvested for ELIS A and real time RT-PCR assay.4. Total RNA was isolated with TRIzol reagent. Briefly, the concentration and purity were determined by spectrophoatometry. Subsequently,1μg of total RNA was reversely transcribed first-strand cDNA synthesis kit according to the manufacturer’s directions according to the manufacturer’s instructions. PCR reactions were performed on ABI7000machine. The amplification process was as follows: pre-denaturation at95℃for5min, followed by40cycles of denaturation at95℃for30sec, annealing at58℃for30sec, and a final extension at72℃for5min. All PCR products were normalized to the amount of GAPDH in the same sample. All PCR Primers were designed using Primer Express3.0software and synthesized by shanghai Boya biotechnology.5. The concentration of IL-6, IL-8and TNF-a in the culture supernatants was analyzed by ELISA. Briefly, THCEs were grown in6-well plates and treated with iE-DAP or A. fumigatus for different times. Then the culture media were harvested. For measurement of IL-6, IL-8and TNF-a, ELISA detection kits were used according to the manufacturer’s instructions. The amount of IL-6, IL-8and TNF-a were normalized with the total amount of cellular protein.6. THCEs were seeded onto coverslips in6-well plates and then challenged with A. fumigatus conidia (1×105/ml). After stimulation, the cells were fixed in4%buffered paraformaldehyde for20min at room temperature, and permeabilized for5min on ice in0.1%Triton X-100. Subsequently, the coverslips were blocked using5%BSA and incubated with primary antibody against NOD1(1:500) for1hour. Then they were washed with PBS and incubated with secondary antibody for1hour. The coverslips were mounted using DAPI. Fluorescent images were captured by using confocal immunofluorescence microscopy with an Imaris software.7. Total protein was extracted using1%RIPA lysis buffer and quantified with BCA protein assay kit. Various amount of protein lysates were electrophoresed on SDS-PAGE gel, transferred onto polyvinylidenedifluoride (PVDF) membrane, blocked with5%nonfat dried milk in Tris buffer with0.05%Tween20, and then incubated with the primary antibodies against NOD1overnight at4℃. After washing, the membrane was incubated with the secondary antibodies with HRP and detected by an enhanced chemiluminescence (ECL) reagent.8. The results were expressed as mean±SEM. SPSS for Windows v.18.0was used for statistical analysis. The T-test was used to determine the statistical significance of intra-group values. One-way ANOVA was also performed to demonstrate comparisons among groups. P<0.05was considered to be statistically significant.[Results]NOD1expressed in the normal THCEs. The stimulation of THCEs by DAP and A. fumigatus conidia respectively resulted in a significantly increased expression of NODI an, the secretion of TNF-α, IL-6, IL-8, hBD2and LL37was up-regulated simultanously. On knockdown of NOD1using small RNA interference, the production of cytokines and antibacterial peptides was reduced and the activation of NF-κB was blocked.[Conclusions] Our results suggested that NOD1existed and expressed in the THCEs. A. fumigatus conidia increased the expression of NOD1, RIP2and the production of cytokines and antibacterial peptides through the NF-κB pathway, which contributed to the innate immune response in fungal keratitis. Part II Mechanism of nucleotide-binding oligomerization domain1in regulating the innate immune response in THCEs to Aspergillus fumigatus[Purpose]To explore the key receptor and signal transduction pathways in the innate immune response to Aspergillus fumigatus (A. fumigatus) in the immortalized human corneal epithelial cells (THCEs).[Methods]1. Cultured human telomerase-immortalized corneal epithelial cells in vitro, THCEs were seeded in six-well plates at a density of2×105cells per well, and the application of anti-RIP2antibody treatment THCEs1h, then challeged THCEs with iE-DAP or heat-inactivated Aspergillus fumigatus conidia for12h, the cells and the cell supernatant were collected.2. The application of anti-NF-KB antibody treatment THCEs lh, then challenged THCEs with iE-DAP or heat-inactivated Aspergillus fumigatus conidia for12h, the cells and the cell supernatant were collected.3. The control RNA and siRNA pool against NOD1were purchased from Invitrogen. THCEs were treated with NOD1siRNA to knockdown NOD1for24h, then challenged with heat-inactivated Aspergillus fumigatus conidia for4h, the cells and the cell supernatant were collected. Then the culture supernatants and cells were harvested for ELIS A and real time RT-PCR assay.4. Real-time RT-PCR detected the expression of NOD1, NOD1-mediated downstream signaling molecular RIP2, NF-κB p65and the secretion of inflammatory cytokines TNF-α, IL-6, IL-8and antibacterial peptides hBD2, LL37. Western Blotting detected he expression of NOD1, NOD1-mediated downstream signaling molecular RIP2, NF-κB p65. ELIS A detected the secretion of inflammatory cytokines TNF-a, IL-6, IL-8.5. The results were expressed as mean±SEM. SPSS for Windows v.18.0was used for statistical analysis. The T-test was used to determine the statistical significance of intra-group values. One-way ANOVA was also performed to demonstrate comparisons among groups. P<0.05was considered to be statistically significant.[Results]NOD1siRNA transfected THCEs, the level of mRNA and protein of NOD1were inhibited significantly. After stimulated THCEs by iE-DAP or heat-inactivated Aspergillus fumigates conidia, the expressions of the key molecules of downstream signal transduction pathway RIP2, NF-κB p65, the secretion of inflammatory cytokines TNF-a, IL-6and IL-8and antimicrobial peptides LL37and hBD2was significantly increased, while in NOD1siRNA treated group, the expression of downstream signaling molecules, the secretion of inflammatory cytokines and antimicrobial peptide was significantly lower than the control groups With the application of anti-RIP2or anti-NF-xB neutralizing antibody treatment THCEs, the secretion of inflammatory cytokines and antimicrobial peptide was significantly lower than the control group.[Conclusions]NOD1, as the key receptor in the innate immune system of the corneal epithelial cells, can mediate the innate immune response to specific ligand or Aspergillus fumigates conidia. The expression of key molecules in RIP2-dependentment-NF-KB signaling pathway may play a key role in regulating the host immunity to Aspergillus fumigatus.