Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Purpose: Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Methods: Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72 hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Results: Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1ß were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Conclusions: Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

MiR-223-3p Regulates Autophagy and Inflammation by Targeting ATG16L1 in Fusarium solani-Induced Keratitis.

Purpose: Increasing evidence suggested that microRNAs (miRs) are implicated in the regulation of the inflammatory response and autophagy in multiple diseases. The present study aimed to explore the effect of miR-223-3p on inflammation and autophagy in fungal keratitis (FK). Methods: An FK mouse model was established, and primary corneal stromal cells were isolated by inoculation with Fusarium solani. The expression of miR-223-3p was determined by quantitative RT-PCR. Subsequently, the target gene of miR-223-3p was identified by a dual-luciferase reporter assay. The levels of miR-223-3p were altered by transfecting miR agomir/antagomir to evaluate its effects. Slit-lamp biomicroscopy and hematoxylin and eosin staining were employed to detect corneal damage. The levels of autophagy were assessed by immunofluorescence, Western blotting, mRFP-GFP-LC3 fluorescence microscopy, and electron microscopy. In addition, inflammation was demonstrated by determining the proinflammatory mediators IL-1ß and TNF-ɑ. Results: Our data suggested that miR-223-3p was increased and that autophagic flux was impaired in mouse FK. Then, we confirmed that autophagy-related gene 16L1 (ATG16L1) was a potential target of miR-223-3p and that this miR negatively regulated the expression of ATG16L1. The inhibition of miR-223-3p attenuated inflammation in FK, reduced P62 expression, and increased the ratio of LC3-II/LC3-I, whereas the overexpression of miR-223-3p displayed the opposite results. Conclusions: Taken together, miR-223-3p might regulate autophagy via targeting ATG16L1 in experimental F. solani keratitis and is associated with the inflammatory response. MiR-223-3p might be a potential therapeutic target for FK.

Impact of Sample Collection Order on the Diagnostic Performance of Metagenomic Deep Sequencing for Infectious Keratitis.

PURPOSE: The purpose of this article was to evaluate the impact of sample collection order on the diagnostic yield of metagenomic deep sequencing (MDS) for determining the causative pathogen in infectious keratitis. METHODS: We performed a cross-sectional diagnostic test evaluation among subjects with infectious keratitis at Aravind Eye Hospital in Madurai, India. All subjects underwent corneal scrapings of the affected eye to obtain potassium hydroxide smear, Gram stain, bacterial culture, and fungal culture, in this order. The order of MDS specimen collection relative to smear and culture samples was randomized and served as the primary predictor. Outcomes included the normalized copy number of pathogenic RNA detected by MDS, the proportion of MDS samples that were diagnostic, and the agreement of MDS results with cultures. RESULTS: MDS samples from 46 subjects with corneal ulcers were evaluated. MDS was positive in 33 subjects (76%) and had 74% overall agreement with culture results. There was no association between order of MDS sample collection and normalized copy number of genetic material detected (P = 0.62) or the likelihood of MDS positivity (P = 0.46). However, the likelihood of agreement between MDS and cultures decreased when MDS corneal swabs were collected after other diagnostic corneal scrapings (P = 0.05). CONCLUSIONS: The overall yield of MDS for detecting the cause of infectious keratitis was not affected by sample collection order. However, diagnostic agreement between MDS and cultures decreased when MDS samples were collected after other specimens. Additional investigation is warranted to determine whether this represents increased sensitivity of MDS compared with cultures or higher susceptibility to contaminants.

Inhibiting miR-129-5p alleviates inflammation and modulates autophagy by targeting ATG14 in fungal keratitis.

To investigate the role of miR-129-5p in inflammation and autophagy in fungal keratitis, we established a keratitis mouse model infected with Fusarium solani (F. solani) and conducted experiments on corneal stromal cells infected with F. solani. The expression of miR-129-5p was detected via quantitative real-time polymerase chain reaction (PCR). The miR-129-5p antagomir was used to transfect cells and mice to study the regulatory role of miR-129-5p in autophagy and inflammation after fungal infection. The expression of Beclin1 and LC3B and colocalization of LC3B with lysosomes were detected via Western blotting and immunofluorescence. CCK-8 was used to determine the viability of corneal stromal cells. The expression of IL-1ß were detected by ELISA. Bioinformatics software was used to predict the potential targets of miR-129-5p, which were verified by a luciferase reporter gene assay. RT-PCR showed that miR-129-5p expression in mouse corneas was significantly increased after infection with F. solani. Subconjunctival injection of the miR-129-5p antagomir significantly enhanced the proteins Beclin-1 and LC3B. At the same time, inhibiting miR-129-5p expression could reduce the inflammatory response in FK and significantly increase the viability of corneal stromal cells infected with F. solan. Moreover, the dual luciferase reporter assay indicated that Atg14 was a direct target of miR-129-5p. Our study shows that miR-129-5p is a novel small molecule that regulates autophagy by targeting Atg14, indicating that it may be a proinflammatory and therapeutic target for fungal keratitis.

The Role of SREC-â in Innate Immunity to Aspergillus fumigatus Keratitis.

Purpose: To determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis. Methods: SREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1ß, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1ß, and TNF-α mRNA expression levels were tested before and after anti-SREC-Ⅰ treatment. Results: SREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1ß, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1ß, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ-neutralizing antibody treatment. Conclusions: SREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.

Antimicrobial peptides in human corneal tissue of patients with fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is the leading cause of unilateral blindness in the developing world. Antimicrobial peptides (AMPs) have been shown to play an important role on human ocular surface (OS) during bacterial, viral and protozoan infections. In this study, our aim was to profile a spectrum of AMPs in corneal tissue from patients with FK during the active pase of infection and after healing. METHODS: OS samples were collected from patients at presentation by impression cytology and scraping. Corneal button specimens were collected from patients undergoing therapeutic penetrating keratoplasty for management of severe FK or healed keratitis. Gene expression of human beta-defensin (HBD)-1, -2, -3 and -9, S100A7, and LL-37 was determined by quantitative real-time PCR. RESULTS: Messenger RNA expression (mRNA) for all AMPs was shown to be significantly upregulated in FK samples. The levels of HBD-1 and -2 mRNA were found to be elevated in 18/20 FK samples. Whereas mRNA for HBD-3 and S100A7 was upregulated in 11/20 and HBD9 was increased in 15/20 FK samples. LL-37 mRNA showed moderate upregulation in 7/20 FK samples compared with controls. In healed scar samples, mRNA of all AMPs was found to be low and matching the levels in controls. CONCLUSION: AMP expression is a consistent feature of FK, but not all AMPs are equally expressed. HBD-1 and -2 are most consistently expressed and LL-37 the least, suggesting some specificity of AMP expression related to FK. These results will help to identify HBD sequence templates for designing FK-specific peptides to test for therapeutic potential.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis.

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and IFN-ß. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

Fusarium infection alters the m6A-modified transcript landscape in the cornea.

N6-methyladenosine (m6A) is the most common post-transcriptional modification of RNA in eukaryotes that regulates the post-transcriptional expression level of genes without changing the base sequence. The role of m6A in fungal keratitis has not yet been elucidated. Here, we aimed to identify m6A modification changes and their potential roles in fungal keratitis. The murine model of fungal keratitis was established by inoculating mice with Fusarium solani (F. solani). The overall m6A level was detected via an m6A RNA methylation assay kit. The expression levels of key m6A modification-related genes were estimated by quantitative real-time polymerase chain reaction (PCR). The expression and localization of METTL (methyltransferase like)3, the key component of the m6A methyltransferase complex, was determined by immunostaining and Western blotting (WB). Immunoprecipitation methylation microarray was used to describe the changes in m6A modification in F. solani-infected corneal tissue. The overall m6A level in corneal tissue on the 5th day in the F. solani-treated group was upregulated compared with that in the control group. The demethylase levels were unaltered, but the level of the methylase METTL3 was increased significantly after fungal infection. Additionally, differences were found in m6A modifications in 1137 mRNAs, of which 780 were hypermethylated and 357 were hypomethylated. To the best of our knowledge, the present work is the first investigation on the m6A modification profiles in experimental fungal keratitis, and it may provide a potential therapeutic target.

Transcriptome Analysis of the Gene Expression Profiles Associated with Fungal Keratitis in Mice Based on RNA-Seq.

Purpose: Fungal keratitis (FK) is an eye disease that can lead to blindness and has a high incidence worldwide. At present, there is no effective treatment for this disease. There are innate immune response mechanisms that protect against fungal infections. One example is C-type lectin receptors (CLRs), which can identify fungal invaders and trigger signal transduction pathways and cellular responses to eliminate pathogens. However, previous studies have focused mostly on single-receptor factors, and a systematic analysis of the genetic factors underlying the pathogenesis of FK has not been conducted. This study aimed to investigate the molecular mechanisms of FK in terms of genomics and to further elucidate its pathogenesis. Methods: We performed a transcriptome analysis of a mouse model of FK using RNA sequencing to obtain the relevant gene expression profiles and to identify differentially expressed genes, signaling pathways, and regulatory networks of the key genetic factors in the pathogenesis of murine FK. Results: Several genes that are significantly associated with FK and serve as markers of FK, such as the inflammatory cytokine genes IL1B, IL6, IL10, IL23, and TNF, were identified. The mRNA and protein expression patterns of IL-1ß, IL-6, and TNF-α in the corneas of mice with FK were validated by quantitative RT-PCR and Luminex multiplex assay technology. The Wnt, cGMP-PKG, and Hippo signaling pathways were significantly enriched during fungal infection of mouse corneas. Conclusions: Our study may help to elucidate the mechanisms of FK pathogenesis and to identify additional candidate drug targets for the treatment of FK.

mRNA profiling reveals response regulators of decreased fungal keratitis symptoms in a tree shrew model.

Fungal keratitis is a corneal disease with a high blindness rate caused by pathogenic fungal infections. The pathogenesis of fungal keratitis and the immune response after fungal infection are still unclear. Notably, the pathological features of fungal keratitis in tree shrews are similar to those in humans. In the present study, mRNA profiling of tree shrew corneas with fungal keratitis was performed. GO and KEGG enrichment analyses were performed on the differentially expressed mRNAs, and the GO biological process ontology was used to analyze functional trends in the differentially expressed mRNAs. In total, 151 downregulated and 71 upregulated mRNAs were shared among the 7-day, 14-day and 30-day infection groups. These differentially expressed mRNAs were significantly enriched in the GO category immune response (GO: 0002376) and the KEGG pathways cytokine receptor binding (KEGG ID: tup04060) and cell adhesion (KEGG ID: tup04514). The downregulated mRNAs were significantly enriched in the corneal epithelial cell adhesion function. Fifty-eight initially upregulated mRNAs gradually decreased in expression, and these mRNAs were significantly enriched in the functions lipopolysaccharide (LPS) and antibacterial polypeptide recognition, cell differentiation, and cell rearrangement. Zeta chain of T-cell receptor associated protein kinase 70 (ZAP70), lymphocyte cytosolic protein 2 (LCP2), C-C motif chemokine and its receptor showed high degrees of connectivity in the protein-protein interaction (PPI) network. We speculate that the decrease in symptoms of tree shrew fungal keratitis may be related to the upregulation of genes involved in immune regulation and macrophage colony stimulation. This study showed that the C-C motif chemokine and its receptor may play a key role in regulating tree shrew fungal keratitis, providing a theoretical basis for studying the pathogenesis of human fungal keratitis.

Analysis of differentially expressed genes in bacterial and fungal keratitis.

Purpose: This study was aimed at identifying differentially expressed genes (DEGs) in bacterial and fungal keratitis. The candidate genes can be selected and quantified to distinguish between causative agents of infectious keratitis to improve therapeutic outcomes. Methods: The expression profile of bacterial or fungal infection, and normal corneal tissues were downloaded from the Gene Expression Omnibus. The limma package in R was used to screen DEGs in bacterial and fungal keratitis. The Co-Express tool was used to calculate correlation coefficients of co-expressed genes. The "Advanced network merge" function of Cytoscape tool was applied to obtain a fusional co-expression network based on bacterial and fungal keratitis DEGs. Finally, functional enrichment analysis by DAVID software and KEGG analysis by KOBAS of DEGs in fusion network were performed. Results: In total, 451 DEGs in bacterial keratitis and 353 DEGs in fungal keratitis were screened, among which 148 DEGs were found only in bacterial keratitis and 50 DEGs only in fungal keratitis. Besides, 117 co-expressed gene pairs were identified among bacterial keratitis DEGs and 87 pairs among fungal keratitis DEGs. In total, nine biological pathways and seven KEGG pathways were screened by analyzing DEGs in the fusional co-expression network. Conclusion: TLR4 is the representative DEG specific to bacterial keratitis, and SOD2 is the representative DEG specific to fungal keratitis, both of which are promising candidate genes to distinguish between bacterial and fungal keratitis.

Interactions of thymic stromal lymphopoietin with TLR2 and TLR4 regulate anti-fungal innate immunity in Aspergillus fumigatus-induced corneal infection.

Thymic stromal lymphopoietin (TSLP) is an interleukin 7 (IL-7)-like four helix bundle cytokine that plays diverse roles in the regulation of immune responses. In fungal infection, pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors, recognize pathogen-associated molecular patterns to initiate downstream signal cascades to active immune responses. Our previous studies reported that, in vitro human cornea epithelium cells represented a novel target of TSLP and that TSLP/TSLPR/STAT5 signaling played an important role in the response to Aspergillus fumigatus challenge. TSLP downstream signaling molecules upregulated TLR2 and MyD88/NF kappa B-p65 signaling. This phenomenon suggested that TSLP had an impact on PRRs in antifungal immunity. In mouse fungal keratitis induced by A. fumigatus, TSLP was mainly expressed in the epithelium as well as in some infiltrated immune cells in a time-dependent manner. Exogenous TSLP with Aspergillus led to severe keratitis and worse corneal recovery with higher levels of TLR2, TLR4, IL-6, and IL-8 as well as increased neutrophil infiltration. By contrast, when TSLP was suppressed by siRNA, fungal keratitis was mild with higher levels of antimicrobial peptides such as human beta-defensin (hBD9). Taken together, our data revealed an unreported function of TSLP in mediating an anti-fungal inflammatory response and serving as a target to control tissue injury and infection in A. fumigatus keratitis.

Fenretinide Inhibits Neutrophil Recruitment and IL-1ß Production in Aspergillus fumigatus Keratitis.

PURPOSE: Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. Fenretinide, a derivative of vitamin A, has been shown to suppress inflammation in a multitude of diseases. In this study, we aimed to characterize the effect of fenretinide in Aspergillus fumigatus keratitis of the eye in a mouse model. METHODS: In vivo and in vitro experiments were performed in mouse models and THP-1 macrophage cell cultures infected with A. fumigatus, respectively. Experimental subjects were first pretreated with fenretinide, and then the effect of the compound was assessed with clinical evaluation, neutrophil staining, myeloperoxidase assay, quantitative polymerase chain reaction (qRT-PCR), and western blot. RESULTS: We confirmed that fenretinide contributed to protection of corneal transparency during early mouse A. fumigatus keratitis by reducing neutrophil recruitment, decreasing myeloperoxidase (MPO) levels and increasing apoptosis. Compared with controls, fenretinide impaired proinflammatory cytokine interleukin 1 beta (IL-1ß) production in response to A. fumigatus exposure with contributions by lectin-type oxidized LDL receptor 1 (LOX-1) and c-Jun N-terminal kinase (JNK). CONCLUSIONS: Together, these findings demonstrate that fenretinide may suppress inflammation through reduced neutrophil recruitment and inflammatory cytokine production in A. fumigatus keratitis.

Phospholipase Cγ2 is critical for Ca2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells.

BACKGROUND: Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. This study aimed to identify the role of PLCγ2 in Dectin-1-mediated Ca2+ Flux and its effect on the expression of proinflammatory mediators at the exposure to Aspergillus fumigatus (A. fumigatus) hyphae antigens in human corneal epithelial cells (HCECs). METHODS: The HCECs were preincubated with or without different inhibitors respectively before A. fumigatus hyphae stimulation. Intracellular calcium flux in HCECs and levels of PLCγ2 and spleen-tyrosine kinase (Syk) were detected by fluorescence imaging and Western Blotting. The expression of proinflammatory mediators was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We demonstrated that an intracellular Ca2+ flux in HCECs was triggered by A. fumigatus hyphae and could be reduced by pre-treatment with PLCγ2-inhibitor U73122. A. fumigatus hyphae induced PLCγ2 phosphorylation was regulated by Dectin-1 via Syk. Furthermore, PLCγ2-deficient HCECs showed a drastic impairment in the Ca2+ signaling and the secretion of IL-6, CXCL1 and TNF-α. CONCLUSIONS: PLCγ2 plays a critical role for Ca2+ Flux in HCECs stimulated by A. fumigatus hyphae. Syk acts upstream of PLCγ2 in the Dectin-1 signaling pathway. The expressions of proinflammatory mediators induced by A. fumigatus are regulated by the activation of Dectin-1-mediated PLCγ2 signaling pathway in HCECs.

Multiplex Polymerase Chain Reaction Assay for Screening of Mycotoxin Genes From Ocular Isolates of Fusarium species.

PURPOSE: To identify mycotoxin genes among clinical ocular isolates of Fusarium species and to correlate these with clinical outcomes of Fusarium keratitis. METHODS: Fifty-four clinical isolates of Fusarium were retrieved from the Bascom Palmer Eye Institute Ocular Microbiology Laboratory data bank. Multiplex polymerase chain reactions were run to confirm the identification of Fusarium species [internal transcribed spacer sequence, translation elongation factor 1-α (TEF) and ß-tubulin] and to detect the presence of genes encoding production of fumonisin B mycotoxins (FUM1 and FUM8) and trichothecene mycotoxins (deoxynivalenol and nivalenol). The presence or absence of mycotoxins was compared with patient outcomes. RESULTS: Forty-three (79%) of the 54 isolates were confirmed as Fusarium species, by an internal transcribed spacer sequence in 3 (5.6%) and by TEF in 43 (79.6%) of the 54 isolates. Fumonisin biosynthetic gene 1 (FUM1) was detected in 57.4% (n = 31/54) of the Fusarium isolates. No FUM8, deoxynivalenol genes, and nivalenol genes were detected among these in the clinical isolates group. Initial best-corrected visual acuity ranged from 20/25 to 20/80 in the FUM1 gene-negative group and from 20/20 to light perception in the FUM1 gene-positive group. There was no difference in the time to cure between both groups. The presence of FUM1 genes in 5 fungal isolates seemed to be associated with progression to penetrating keratoplasty in the 5 patients from whom the fungi were isolated. Fusarium solani was recovered from all patients requiring penetrating keratoplasty. CONCLUSIONS: Fumonisin B biosynthetic gene 1 may be common among clinical Fusarium isolates and contribute to worse initial visual acuity and high-risk progression to penetrating keratoplasty.

Aspergillus fumigatus Increased PAR-2 Expression and Elevated Proinflammatory Cytokines Expression Through the Pathway of PAR-2/ERK1/2 in Cornea.

Purpose: To determine the role of protease-activated receptor-2 (PAR-2) in cornea infected by Aspergillus fumigatus. Methods: PAR-2 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of PAR-2 antagonist (FSLLRY-NH2). Polymorphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of FSLLRY-NH2. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of PAR-2, IL-1ß, TNF-α, IFN-γ, MIP-2, and p-ERK1/2. PMN infiltration was assessed by myeloperoxidase assay and immunofluorescent staining. Results: PAR-2 expression was significantly elevated by A. fumigatus, whereas the upregulation was significantly inhibited by FSLLRY-NH2 in mice corneas. FSLLRY-NH2 decreased disease response, PMN infiltration, and proinflammatory cytokine expression compared with infected control. In PMNs, PAR-2 expression was also significantly increased by A. fumigatus, which was significantly inhibited by FSLLRY-NH2. FSLLRY-NH2 significantly inhibited proinflammatory cytokine protein expression, as compared with that in infected control cells, which may be modified by p-ERK1/2. Conclusions: These data provide evidence that A. fumigatus increased PAR-2 expression and elevated disease, PMN infiltration, and proinflammatory cytokine expression through PAR-2, which may be modified by p-ERK1/2.

ISG15 in Host Defense Against Candida albicans Infection in a Mouse Model of Fungal Keratitis.

Purpose: ISG15, a di-ubiquitin-like protein, is critical for controlling certain viral and bacterial infections. We sought to determine if ISG15 plays a role in corneal innate immunity against Candida albicans (C. albicans) using a C57BL/6 (B6) mouse model of human fungal keratitis. Methods: Scarified corneas of adult B6 mice were pretreated with TLR5 ligand flagellin and then inoculated with C. albicans. The expression of ISG15 and other genes involved in ISG15 conjugation (ISGylation) was determined by real-time PCR. ISG15 expression and distribution in infected corneas were assessed by immunohistochemistry. ISGylation was examined by Western blotting. siRNA knockdown and recombinant ISG15 were used to elucidate the effects of ISG15 on controlling fungal keratitis by clinical scoring, fungal number plate counting, ELISA cytokine determination, and polymorphonuclear leukocytes (PMN) infiltration measurement. Results: Heat-killed C. albicans induced expression of ISG15, and hBD2 was markedly enhanced by flagellin-pretreatment in cultured human primary corneal epithelial cells (CECs). In vivo, C. albicans infection induced the expression of ISG15, ISGylation-associated genes (UBE1L, UBCH8, and HERC5), and ISGylation in mouse CECs, all of which were enhanced by flagellin-pretreatment. siRNA knockdown of ISG15 increased keratitis severity, dampened flagellin-induced protection, and greatly suppressed the expressions of ISGylation enzymes, IFN-γ, but not CXCL2 in B6 mouse CECs. Recombinant ISG15, on the other hand, enhanced corneal innate immunity against C. albicans and suppressed infection-induced IL-1ß, but not IL-Ra expression. ISG15 alone induced the expression of IL-1Ra, CXCL10, and CRAMP in mouse CECs. ISG15 was upregulated and secreted in cultured human CECs in response to challenge in a type 1 IFN-dependent manner. Conclusions: Our data, for the first time, demonstrate that ISG15 acts as an immunomodulator in the cornea and plays a critical role in controlling fungal keratitis.

Regulatory effect of caspase-11 on interleukin-1ß in the fungal keratitis.

Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1ß in the fungal keratitis. neutrophils were the main cell lineage of IL-1ß to take part in the innate anti-fungi immunity in the cornea; IL-1ß generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1ß generation; while Dectin-1/syk pathway was involved in IL-1ß generation in the fungal keratitis; Caspase-l participated in the modification of IL-1ß to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1ß generation; Caspase-11 was involved in IL-1ß generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1ß generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1ß generation.

Human Corneal MicroRNA Expression Profile in Fungal Keratitis.

PURPOSE: MicroRNAs (miRNAs) are small, stable, noncoding RNA molecules with regulatory function and marked tissue specificity that posttranscriptionally regulate gene expression. However, their role in fungal keratitis remains unknown. The purpose of this study was to identify the miRNA profile and its regulatory role in fungal keratitis. METHODS: Normal donor (n = 3) and fungal keratitis (n = 5) corneas were pooled separately, and small RNA deep sequencing was performed using a sequencing platform. A bioinformatics approach was applied to identify differentially-expressed miRNAs and their targets, and select miRNAs were validated by real-time quantitative PCR (qPCR). The regulatory functions of miRNAs were predicted by combining miRNA target genes and pathway analysis. The mRNA expression levels of select target genes were further analyzed by qPCR. RESULTS: By deep sequencing, 75 miRNAs were identified as differentially expressed with fold change greater than 2 and probability score greater than 0.9 in fungal keratitis corneas. The highly dysregulated miRNAs (miR-511-5p, miR-142-3p, miR-155-5p, and miR-451a) may regulate wound healing as they were predicted to specifically target wound inflammatory genes. Moreover, the increased expression of miR-451a in keratitis correlated with reduced expression of its target, macrophage migration inhibitory factor, suggesting possible regulatory functions. CONCLUSIONS: This is, to our knowledge, the first report on comprehensive human corneal miRNA expression profile in fungal keratitis. Several miRNAs with high expression in fungal keratitis point toward their potential role in regulation of pathogenesis. Further insights in understanding their role in corneal wound inflammation may help design new therapeutic strategies.