Pathogenesis Of Fungal Keratitis Research Articles

Galectin-3 Is a Crucial Immunological Disease Marker in Patients with Fungal Keratitis.

Fungal keratitis, one of the most common infectious eye diseases in China, often results in a poor prognosis due to a delayed diagnosis and the insufficiency of effective therapy. There is an urgent need to identify specific biomarkers for the disease. In this study, we screened out tear proteins in patients with fungal keratitis by microsphere-based immunoassay analysis. Levels of cytokine expression were determined in both human corneal epithelial cell models in vitro and the corneas of patients by western blot, quantitative polymerase chain reaction (qPCR), and immunofluorescence analysis. Neutrophil activation was examined by flow cytometry analysis. The relationship between the cytokine expression and neutrophils was evaluated by immunofluorescence costaining and correlation analysis. These results demonstrated that the galectin-3 expression level was increased in both cell model and patient samples at the early and late stages of fungal keratitis. The neutrophils were significantly activated during the disease course of fungal keratitis. Meanwhile, colocalization and a positive correlation between galectin-3 and neutrophils were observed, suggesting that galectin-3 may play a crucial role in the recruitment of neutrophils and immune regulation of fungal keratitis. In conclusion, galectin-3 could be a key disease marker implying a beneficial immune response in the pathogenesis of fungal keratitis, which might be a target of therapeutic strategy in the future.

Immunologic mechanism of fungal keratitis.

Fungal keratitis (FK) is a refractory disease that poses a serious threat to vision, with common risk factors like eye trauma, contact lens wearing, topical corticosteroids and antibiotic abuse. Nowadays, topical and systemic anti-fungal drugs and ocular surgeries are still the main therapeutic modalities. However, the pathogenesis of FK, especially the immunologic mechanism within it, has not yet been deeply clarified. A better understanding of the pathogenesis of FK is imperative for more effective therapies and prognosis. Meanwhile, the immune protection strategies are also urgently required to manage FK. This review highlights recent advances in the immunologic mechanism in the pathogenesis of FK, in hope of providing valuable reference information for more effective anti-fungal treatment.

Therapeutic effects of an anti-IL-6 antibody in fungal keratitis: Macrophage inhibition and T cell subset regulation

Immune modulation plays a critical role in the pathogenesis of fungal keratitis (FK). However, the immune cell-mediated processes linking the innate immune response to the adaptive immune response are incompletely elucidated. IL-6 plays crucial roles in infectious and inflammatory processes in the cornea, regulating not only mononuclear macrophage differentiation but also lymphocyte activation, and IL-6 might be a useful target for immune intervention in FK. The frequencies of macrophages and T cells increased upon infection and were correlated with the severity of ocular pathogenesis. Additionally, protein profiling revealed that the expression of IL-6 and associated cytokines/chemokines was upregulated. Furthermore, anti-IL-6 intervention suppressed disease progression by reducing macrophage infiltration in the cornea and Th1, Th17, and Treg cell infiltration in draining lymph nodes (DLN) in an animal model of FK. Tocilizumab (TCZ), an antibody specific for IL-6, reduced the signal transducer and activator of transcription 3 (STAT3) activation in vivo and in vitro. In summary, fungal infection promoted macrophage and T cell activation via IL-6-mediated transcellular signaling to regulate immune cell migration and cytokine production, further demonstrating the role of IL-6 and providing a potential clinical therapeutic target in 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.

Histone deacetylase inhibitor attenuates experimental fungal keratitis in mice

Fungal keratitis is one of the leading causes of blindness of infected corneal diseases, but the pathogenesis of fungal keratitis is not fully understood and therefore the treatment of the disease by medication is still under investigation. In the current study, we sought to study the effect of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on experimental fungal keratitis in mice. SAHA (25 mg/kg) (n = 30) or vehicle (DMSO) (n = 30) was delivered through intraperitoneal injection (IP) 24 hours after the fungal inoculation, and the same amount of SAHA injection or DMSO was followed at day 2. The expression of histone H3 (H3), acetylated histone H3 (AC-H3), histone deacetylase 1 (HDAC)1, tumor necrosis factor-α (TNFα), and Toll-like receptor 4 (TLR4) in surgically excised specimens from the patients and mice with fungal keratitis were detected by immunohistochemistry. The expression of mRNAs for Interleukin-1β (IL-1β), TNFα, and TLR4 were evaluated in the corneas of the mice with fungal infection and the control corneas by real-time PCR. The quantification of IL-1β and TNFα in the corneas of the mice with fungal infection was determined by ELISA. The inhibitory effect of SAHA on mice fungal keratitis was revealed by GMS and H&E staining. We found that the downregulation of histone acetylation and upregulation of HDAC1 expression were associated with the increased inflammation response in fungal keratitis not only in humans but also in experimental animals. SAHA was able to inhibit experimental fungal keratitis in mouse by suppressing TLR4 and inflammatory cytokines such as TNFα and IL-1β; the inhibition of HDAC may be a potential therapeutic approach for the treatment of fungal keratitis.

ROS-induced Oxidative Injury involved in Pathogenesis of Fungal Keratitis via p38 MAPK Activation

This study was to explore the mechanism by which reactive oxygen species (ROS)-induced oxidative stress involved in the pathogenesis of fungal keratitis using an in vivo experimental keratitis mouse model and an in vitro culture model of human corneal epithelial cells (HCECs). Compared to normal control mice and HCECs, ROS production was markedly increased in fungal corneas and HCECs exposed to Candida albicans, accompanied by p38 mitogen-activated protein kinases (MAPK) activation. Increased products of oxidative markers, malondialdehyde (MDA), 4–hydroxynonenal (HNE), mitochondria DNA 8-OHdG and aconitase-2 were observed in fungal infected corneas and HCECs. Fungal infection also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), with suppressed levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPx1) and peroxiredoxin-4 (PRDX4). Interestingly, the levels of ROS, oxidative markers and oxygenases were significantly reduced by co-cultured p38 inhibitor SB203580. Furthermore, SB203580 restored the levels of antioxidant enzymes suppressed by fungus. Our findings demonstrated for the first time that ROS-induced oxidative injury is involved in pathogenesis of fungal keratitis via p38 MAPK pathway, suggesting the novel therapeutic targets for the potential treatment of fungal keratitis.

The crosstalk between TLR2 and NOD2 in Aspergillus fumigatus keratitis

Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection.

Toll‐like receptor 2 siRNA suppresses corneal inflammation and attenuates Aspergillus fumigatus keratitis in rats

Toll-like receptors (TLRs) are key components of innate immunity that detect microbial infection and trigger host defense responses. However, they are capable of initiating both protective and damaging immune responses, as exaggerated expression of inflammatory components can have devastating effects on the host. We previously reported that TLR2 in corneal epithelium has an important role in the pathogenesis of fungal keratitis, however, how the corneal inflammation is modulated remains to be elucidated. This study aims to investigate the effect of targeting TLR2 on Aspergillus fumigatus keratitis in rats. The control or TLR2 small interfering RNA (siRNA) was applied sub-conjunctively and topically to the cornea. TLR2 immunostaining was performed to determine the feasibility of TLR2 siRNA delivery. Production of inflammatory cytokines and chemokines were determined by real-time quantitative PCR. Polymorphonuclear leukocyte (PMN) infiltration was assessed by myeloperoxidase activity. It was found that rat corneas treated with TLR2 siRNA showed a significant reduction of TLR2 expression in corneal epithelium. TLR2 siRNA treatment improved the outcome of keratitis, which was characterized by decreased corneal opacity, less corneal perforation, suppressed PMN infiltration, reduced production of inflammatory cytokines and chemokines, and less fungal burden. In conclusion, TLR2 siRNA treatment attenuated A. fumigatus keratitis by suppressing corneal inflammation and preventing fungal invasion, suggesting a novel avenue to control fungal infection and avert damage caused by excessive inflammation.

Advance in pathogenesis of fungal keratitis

Fungal keratitis is the most common and severe infectious corneal diseases in many developing countries. The disease has become the leading cause of corneal blindness in China. It is imperative to study deeply on pathogenesis of fungal keratitis for lowering its incidence, increasing therapeutic option and enhancing therapeutic efficacy. This article reviewed the advances of related factors in pathogenesis of fungal keratitis including fungal virulence factors and host defense mechanisms.

Extracellular proteases of Aspergillus flavus: Fungal keratitis, proteases, and pathogenesis

To get a better understanding of the possible role of proteases in the pathogenesis of fungal keratitis, the extracellular proteases of a clinical isolate of Aspergillus flavus, from a severe case of kerititis, were identified and partially characterized. This strain, designated CU226/88, was grown with a variety of substrates as nitrogen sources, under conditions that would be expected to derepress the production of extracellular proteases. When grown on minimal medium with milk protein as a nitrogen source, the fungus appeared to produce primarily a metalloprotease, which has a zinc cofactor. When grown with insoluble collagen or elastin as a nitrogen source, a serine protease and cysteine protease, as well as the metalloprotease, are produced. Strain CU226/88 can grow with collagen, but not elastin, as the sole source of carbon as well as nitrogen. It is possible that the collagenase activity is a mediator of the severe corneal destruction caused by this isolate.