|Molecular Vision 2007;
Received 17 December 2006 | Accepted 16 July 2007 | Published 19 July 2007
Gene profiling in murine corneas challenged with Aspergillus fumigatus
State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China
Correspondence to: Dr. Lixin Xie, Shandong Eye Institute, #5 Yan'erdao Road, Qingdao, 266071, China; Phone: 86-532-8589-8703; FAX: 86-532-8589-1110; email: firstname.lastname@example.org
Purpose: Fungal keratitis (FK) is a blinding infection of the corneas and accounts for a significant portion of all keratitis in the world but little is known about the pathogenesis of FK, especially at the level of molecular biology. This study tried to determine the genes that are modulated in the process and thus deserve further investigation for understanding the pathogenesis of FK.
Methods: Agilent Mouse Oligo Microarray was used to compare the gene profiles in control corneas and in corneas challenged with heat-inactivated Aspergillus fumigatus spores. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to confirm the changes of interested genes.
Results: In the 18,335 genes detected by array, 61 showed an increase of more than one-fold in RNA expression and 48 showed a decrease of over 50%. Among the regulated genes some are known to be related to host defense such as IL3, mannose binding lectin A (MBL-A), and postaglandin D2 synthase (Psgd) while others like Dopachrome tautomerase (Dct) have never been correlated to ocular or host defense. The other 33 changed genes either have unknown functions or encode hypothetical proteins. The changes in the expression of early growth response 4 (Egr4), Dct, Psgd, MBL-A, and hemoglobin α adult chain 1 were confirmed by using RT-PCR. MBL-A expression regulation was further confirmed by using immunohistochemisty in both in vitro and in vivo challenged murine corneas.
Conclusions: MBL-A is among the primary responding genes during the onset of fungal keratitis. Also it was found that the microarray is a useful tool in elucidating the pathogenesis of FK.
Fungal keratitis (FK) is a blinding infection of the cornea, which can lead to the loss of the infected eye if not controlled properly. As a disease caused by infectious microbes, both the incidence and predisposing factors of FK vary greatly in different parts of the world. For example, FK was reported to account for only 2% of all the keratitis cases in New York but for 35% of all keratitis cases in south Florida. This percentage is 44%, 17%, 36%, and 37.6% for south India, Nepal, Bangladesh, and Ghana, respectively . When comparing these figures, we take into consideration that the spectra of causative pathogens of all infectious keratitis or just FK is continously changing due to the shift of dominant infectious agents along the time line [2,3]. For instance, with the treatment and control of viral and bacterial keratitis by using effective antibiotics and anti-viral therapeutics, the percentage of FK among all keratitis has been increasing during the last two decades in China. Other factors such as abuse or misuse of antibiotics, contact lens wearing, popularization of cataract surgeries, and laser in situ keratomileusis also contribute to the increase of FK cases in China. But compared to other infectious keratitis like herpetic simplex keratitis , much less is known about the pathogenesis of FK especially at the level of molecular progress . In the current study, we used microarray to profile the genes that might be involved in the response of murine corneal cells to fungal pathogens. The responding genes found in this model can guide which direction the study of the pathogenesis of FK should be taken.
Eight- to twelve-week old female Balb/c and C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China) and were housed at the Shandong Eye Institute Animal Facility. All animals were maintained and handled according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For induction of FK in Balb/c mice, the method described by Wu et al.  was used but with some modification. Briefly, about 30 superficial crosses were made with 30-gauge needles on the surface of the left corneas of the mice under a stereoscope. Five μl of Aspergillus fumigatus preparations, containing 1x106 viable spores, were applied to the left eye of a mouse in the FK group. Corneas in control mice were similarly sacrificed but received 5 μl saline buffer instead of fungal preparations (sham infected). Over 80% of the fungal-challenged eyes developed typical keratitis in three days and the right eyes of all mice as well as the left eyes in the control group remained unaffected.
Gene profiling using microarray assay
Corneas were removed from Balb/c mice, cut into pieces of about 1 mm and put in RPMI 1640 culture medium with 10% fetal calf serum. Ten million heat-killed A. fumigatus spores were added to the culture of five corneas (the FC group), making the final total volume 1.3 ml. The medium control group (the MC group) contained similar amount of cornea tissue but was not treated with fungal preparation. After 24 h of incubation at 37 °C with 5% CO2, corneal pieces were picked out, rinsed with PBS, and used for RNA extraction by using EZ Spin Column Total RNA Isolation Kit (BioBasic Inc., Toronto, Canada). The integrity and high quality of RNA samples were confirmed by using agarose electrophoresis and the Lab-on-chip system. Four hundred nanograms of RNA were reverse-transcribed to cDNA during which process a T7 sequences was introduced into cDNA. T7 RNA polymerase-driven RNA synthesis was used for the preparation and labeling of cRNA with Cy3 (the FC sample) and Cy5 (the MC sample), respectively. A Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA) was used to purify fluorescent cRNA probes. An equal amount (1 μg) of Cy3 and Cy5 labeled probes were mixed and used for hybridization on one Agilent Mouse Oligo Microarray (catalog number G4121A, product serial number 5268; Agilent Techonologies Inc., Santa Clara, CA) following the protocol provided by the manufacturer. The hybridization signals were acquired by using Agilent 2100 Bioanalyzer (G2938B) and analyzed using Agilent G2567AA Feature Extraction Software (v7.5). Specifically, the Linear&Lowess method and the rank consistency filter were used for normalization of the features. The rank consistency filter selects features that fall within the central tendency of the data by observing consistent trends between the red and green channels. To be counted as a valid feature, the feature has to pass four criteria that they are positive and significant versus just the background, the signals are uniform in the spot, the signals are not saturated, and they are not population outliers in either channel. If the features failed any one of the criteria, they were flagged and excluded from further analysis.
Reverse transcription-polymerase chain reaction
The fungal challenge of the corneas and subsequent RNA extraction were carried out as described above. Twenty μl of RNA underwent reverse transcription using AMV First Strand cDNA Synthesis Kit (Biobasic Inc., Toronto, Canada). The RNA amount used for cDNA synthesis varied from 100 ng to 1 μg among different batches of experiments but was consistent for all samples in each single experiment. The RT reaction product was diluted ten-fold with water and 1 μl of the diluted mixture was used for the 20 μl PCR reaction. Semi-quantitative PCR was done with primers specific for each interested gene using similar denaturing and extension parameters (30 s at 93 °C, 1 min at 72 °C, respectively) but different annealing temperatures (Table 1). Each target was amplified for increasing the number of cycles of PCR with optimized annealing temperature. For targets expressed at an extremely low level, a second round of PCR with the dilution of the first round PCR product might be necessary. All PCR products were resolved in 1.5% agarose gel. The photograph was obtained with Electrophoresis Documentation and Analysis System 120 (Kodak Digital Science, New Haven, CT) and densitometry of the gel image was done with ImageJ 1.37v (NIH).
The eyeballs were removed from infected and control mice at different times after application of A. fumigatus, fixed with 4% paraformaldehyde in PBS for 2 h, then embedded in OCT compound for cryosecting. Corneas challenged in vitro with heat-killed spores were treated similarly. Conventional immunohistochemistry was done with rat anti-mouse mannose binding lectin-A (MBL-A; HyCult biotechnology, Uden, Netherlands) used as the primary antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (Golden Bridge Biotechnology, Beijing, China) as the secondary antibody. Samples were mounted with Ultra-Cruz mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA) and were observed using E800 Nikon microscope with Digital Sight DS-U1 CCD cassette (Nikon Ltd. Tokyo, Japan). For background staining monitoring, sections were processed similarly except that the primary antibody was omitted.
Results & Discussion
Validation of the technique
The technique of microarray provides a valuable tool for screening out genes involved in any biological process like FK. As with the G4121A array used in this study, 1,075 internal control probes are loaded to monitor the quality of the whole experimental process. Besides these control probes, 50 biological probes are assigned 10 duplicates that are evenly distributed over the array. The Cy3/Cy5 ratio for each probe spot was obtained for all 10 duplicates and the average and standard deviation (Stdev) of each probe were used for calculating the coefficient variant (CV) of that specific probe according to formula Stdev/mean x100. Among all of the 50 duplicated probes, only five gave CV over 15% and none of the probes that produced Cy3 and Cy5 signals over 300 gave CV over 10% (Figure 1A). The average CV for the 50 biological control probes was 7.33%, indicating the high quality and reliability of the data obtained with this array, especially for those probes that produced high intensity of Cy3 and Cy5 signals (such as over 300).
Changes of gene expression patterns in fungal challenged corneas
Both our own work and the work of others have shown that the corneas are heavily infiltrated with inflammatory cells during fungal keratitis development (Figure 2F inset, and data not shown). Since, in this study, we were more interested in the primary response of the residential corneal cells per se than the response of immigrant inflammatory cells, we used in vitro fungal-challenged corneas for microarray profiling instead of corneas from mice with preset FK, minimizing the possible interference of infiltrating inflammatory or immune cells (Figure 2C,F, upper insets). Among the 20,868 genes or biological probes on the chip, 18,335 were detectable, giving an overall detection rate of 87.86%. Among the detectable genes, most of them (99.41%) remained unchanged in the abundance of mRNA or the change was less than two-fold (Figure 1B). However, 61 genes manifested an expression increase of at least one-fold in the infected group compared to the control (Table 2) while 48 showed a decrease of over 50% of the control after treatment (Table 3).
We divided these genes into different categories according to their main biological properties. Interestingly, about one-third (23/61) of the upregulated genes and one-fifth (10/48) of the downregulated genes are not cloned yet or had only been suggested to encode hypothetical proteins. Involvement of these unknown genes in FK as shown in this study implies that some of them might be related in local or systemic antifungal response of the host. For example, ectopic expression of the olfactory receptor genes/pseudogenes family has been reported in many tissues . The findings that two olfactory receptors (NM_147090, NM_146320) were downregulated while two olfactory receptor pseudogenes (NG_001719, NG_001878) were upregulated in fungi-challenged corneas not only demonstrated their expression in corneas for the first time but also proved that individual members of this family respond differently to the same challenge.
Among the upregulated genes, over one-third of them are functionally involved in biologically fundamental processes like metabolism, signal transduction, mass transportation, and cytoskeleton organization. Only three of the upregulated genes, namely mannose binding lectin (MBL)-A [8,9], CD37 , and chemokine ligand 10 (CXCL10) , are known to be closely related to immune response to pathogens. MBL is a member of the collectins family. Binding of pathogens to MBL activates a complement pathway or opsonizes the engulfment of pathogens by macrophages [8,12] thus enhancing the antimicrobial reactions of host immune system. MBL-deficient mice were found to be more susceptible to pathogens-induced lethality [13,14], further demonstrating the importance of MBL in controlling infections in affected hosts. Though mainly produced in the liver, MBL-A is also expressed in other tissues [15,16] and the expression of MBL-A increases in response to acute phase stimuli in the liver . For the first time, our array result showed that MBL-A is expressed in corneas and it might be one of the local responders to challenging pathogens. Overexpression of MBL-A was confirmed using immunohistochemisty in both corneas challenged with heat-killed spores in vitro (Figure 2A-C) and corneas from experimental FK mice (Figure 2D-F). It was noteworthy to point out that in corneas cocultured with heat-killed spores, MBL-A expression was restricted to epithelial layers (Figure 2C). In eyes from FK mice, however, MBL-A expression was detected in all layers of the cornea as well as in the infiltrating inflammatory cells located in the anterior chamber of the infected eye (Figure 2F). DAPI staining showed that corneas from FK mice were heavily infiltrated with inflammatory cells (Figure 2F, inset).
Dct and Egr4 are among the genes that responded most strongly to the fungal challenge in our study (Figure 3). Dct has been known as a melanogenic enzyme and its main producers are pigmented cells like melanocytes or retinal pigment epithelial cells both in the human and mice system [18,19]. Very recently, Dct was found to regulate neural progenitor proliferation . Interestingly enough, six other genes that are closely related to neural cell functions were also upregulated in fungal spore-challenged corneas (Table 2). Not yet knowing the biological significance of an increased expression of these genes in FK corneas, we propose that these changes might be the reflection of changes occuring in numerous neural cells in corneas. Egr4 is a member of the Egr family that acts as a nuclear effector of various extracellular signals like tissue injury or apoptotic stimuli. Though no immunomodulating activity has been ascribed to Egr4 by itself, Egr4 may regulate immune response by interacting with proinflammatory cytokine genes [21,22]. Proline rich proteins are a group of molecules that work as lubricants in certain body parts such as saliva  and some of these proteins have been detected in corneal cells [24,25]. As a member of this family, the B-type proline-rich protein, MP4 gene (NM_053251) was first cloned in 1991  but its function remains unknown.
Overexpression of MP4 in fungi-challenged corneas implied that it might be involved in biological processes that are related to a host's defense against infections. More studies are needed to learn the exact roles of Dct, Egr4, and MP4 in pathogenesis of FK.
Down regulated genes
Of the 48 genes that were downregulated in the experimental fungal keratitic corneas, roughly one-third of them (15/48) are primarily involved in the cellular metabolism process. The many other gene products are proteins with high molecular weights such as IgM chain, polydomain protein, perital skeletal myosin, procollagen, etc. (Table 3). We think this might be a reflection of corneal structure destruction caused by fungi challenge. Only three of the downregulated molecules were related to immunology or host defense but we did not go into details of any other downregulated genes in this study except for confirming the decrease of Hba-a1 (NM_008218) mRNA by RT-PCR.
Though some promising results were obtained, this study bears several limitations that require further studies to clarify. For example, we used heat-killed fungal spores instead of viable ones in the in vitro study to eliminate the potential risk of nutrition deficiency in the culture that would be caused by an overgrowth of live fungi. But the potential difference of the response to viable and heat-killed fungi was not monitored in this study. It has been well-documented that live and killed microbes of the same strain may provoke greatly different responses. Specifically, with A. fumigatus, this phenomenon has been observed in both the cells [27,28] and the whole body . Rivera et al.  showed that when both are administrated intratracheally, only live A. fumigatus spores recruit fungus-specific and IFNγ-producing CD4+ T cells in airways while heat-inactivated spores prime CD4+ cells more efficiently in draining lymph nodes than live ones. Similarly, the possible bias caused by using an in vitro fungal challenge model instead of a real in vivo FK model in gene profiling has to be considered in further study. Furthermore, Huang et al.  reported that about 10% of all detected genes are upregulated or downregulated in mouse corneas one day after infection with viable Pseudomonas aeruginosa and a significant part of those regulated genes belong to cytokines, chemokines, or inflammatory factor groups. They also showed that many genes showed significantly different or even opposite changes during their response to the same infection in Balb/c and C57BL/6 mice . In our study, however, comparison of gene expression changes in heat-killed spore-challenged corneas from Balb/c or C57BL/6 strain mice was not attempted beyond Egr4 and Dct (Figure 3B). But these limitations do not weaken the significance of this study. On the contrary, it shows the strength of using microarray technology in elucidating the mechanism of fungal keratitis. Further intensive research will be following to solve the present limitations.
In summary, this is the first time the microarray technique was applied in the study of the pathogenesis of fungal keratitis. Our preliminary results revealed some genes that are possibly related to the innate response of corneas during FK initiation such as MBL-A. Besides serving as a good start in a more detailed search of the pathogenesis of FK, this study also gives clues on how to understand the biological functions of those uncloned hypothetical genes that were shown to respond to the fungal challenge in the corneas.
This work was supported by the National Natural Science Foundation of China (30630063, 30271394) and by the Department of Science and Technology of Shandong Province (2004GG2202154). Dr. Yiqiang Wang is partially supported by the Taishan Scholar Program QDU-EYE, Qingdao University, China.
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