Fungal keratitis is one of the major eye diseases in the world, especially in tropical and subtropical regions (Srinivasan, 2004). It is also one of the strongest blinding diseases (Thomas et al., 2005; Burton et al., 2011). Wearing contact lenses and plant trauma were major factors (Iyer et al., 2006; Ahearn et al., 2008; Gower et al., 2010). In fungal keratitis, Fusarium and Aspergillus are the most common fungi (Khurana et al., 2022). Fusarium is everywhere in our living environment. It is highly infectious and causes more serious consequences. It has a series of symptoms, including corneal tissue feathery margins, satellite lesions, immune ring, ulcers, perforations and even progression to endophthalmitis (Xie et al., 2006; Edelstein et al., 2012). Fusarium infection causes many immune responses in the body, such as macrophage activation, leukocyte infiltration and secretion of various inflammatory factors (Mitchell et al., 2007; Li et al., 2016). These biological processes will produce large amounts of reactive oxygen species (ROS), which will kill the Fusarium. Large amounts of ROS can cause damage to surrounding normal cells, promote inflammatory processes, and cause REDOX imbalance. Similar to other fungal keratitis, Fungal smear and culture is the gold standards (Sharma, 2012; Chidambaram et al., 2016). There are several approaches to treating Fusarium keratitis, such as topical and systemic antifungal drugs, laser therapy and surgery (Thomas and Kaliamurthy, 2013; Maharana et al., 2016; Prajna et al., 2016; Sharma et al., 2017). Although there are many treatments for Fusarium keratitis, the results are still poor and we still see many people who have lost their vision about it (Danielescu et al., 2022). Now, it is well established that multiple immune factors and enzymes were involved in the development of Fusarium keratitis, and they caused many different types of cell death. Ferroptosis, as an important mode of death, has not been reported in Fusarium keratitis (Mitchell et al., 2007; Karthikeyan et al., 2011; Hu et al., 2014; Li et al., 2016).

Ferroptosis is a new form of cell death. It is a non-apoptotic, iron-dependent form of regulated cell death (RCD) occurring. Its main feature is that lipid oxides can’t be metabolized by glutathione reductase reaction, and then Fe²⁺ oxidizes lipids to generate reactive oxygen species, thereby causing cellular damage. It is mainly due to the disruption of redox balance, the production of reactive oxygen species greater than antioxidants, which increases oxidation in the body and disrupts iron balance, causing an imbalance in cellular homeostasis and leading to cell death (Jiang et al., 2021). Ferroptosis is widespread in cellular metabolisms, such as substance metabolism (lipid metabolism, amino acid metabolism), cellular respiration, etc (Gao and Jiang, 2018; Lang et al., 2019). Ferroptosis is involved in the development of many diseases in current studies. These include infections, cancer, immune diseases, cardiovascular diseases, etc (Stockwell et al., 2017). Ferroptosis has become a new hot spot, and it provides a new pathway for the diagnosis and treatment of diseases (Weiland et al., 2019). Given the important role of oxidative stress in ferroptosis, we hypothesized that ferroptosis was involved in the development of Fusarium keratitis.

Currently, ferroptosis in Fusarium keratitis has not been studied. Therefore, we used data mining and data analysis approach to identify differentially expressed genes (DEGs) in corneal tissue between Fusarium keratitis and healthy people. Then, the DEGs intersected with the ferroptosis associated genes. We acquired ferroptosis related to DEGs (Ferr-DEGs). Meanwhile, we obtained hub genes and verified their association with immune infiltration. Finally, we verified the existence of this process by qPCR. Our study explained the potential role of ferroptosis in Fusarium keratitis and provides a new direction for treatment.

Our analyses suggested that ferroptosis was activated during Fusarium keratitis. At the same time, we obtained the relevant hub genes and analyzed them. It provided a new direction for the treatment of Fusarium keratitis. We got 1368 DEGs, including 828 upregulated and 540 downregulated genes from the datasets GSE58291. By KEGG enrichment analysis, we found that ferroptosis plays an important role in Fusarium keratitis. Then we got 26 intersecting genes from the intersection of DEGs and FerrDb. By GO and KEGG enrichment analysis, they were mainly associated with oxidative stress and ferroptosis, then hub genes were obtained. The top 5 genes with the highest scores were selected, which are HMOX1, CYBB, GPX2, ALOX5 and SRC. Analysis of hub genes using GSEA software further suggested the role of ferroptosis in Fusarium keratitis. Then the samples were analyzed for immune heterogeneity and their correlation with hub genes. Finally, the expression of the mRNA of hub genes were demonstrated by the experiment. SRC expression showed no statistically significant difference. So SRC might not serve as the hub gene.

Ferroptosis is a new type of cell death that is different from other cell death processes. It is mainly depending on Fe²⁺. It catalyzes the high expression of unsaturated fatty acids on cell membranes through Fenton reaction and peroxidation reaction, and lipid peroxidation occurs and induces cell death. At the same time, the lack of reducing substances is also an important factor. Lipid peroxidation and ROS production are important features of ferroptosis. It is regulated by a variety of cellular activities including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids, lipids and sugars, in addition to various signaling pathways relevant to disease (Gao and Jiang, 2018; Jiang et al., 2021). Ferroptosis is associated with many diseases, including cancer, blood diseases, brain and neurodegenerative diseases, heart diseases and infectious diseases (Qiu et al., 2020). It is well known that Fusarium keratitis activated a variety of immune cells and enhances oxidative stress in corneal tissue. It could stimulate neutrophils to produce pro-inflammatory factors, which then promote the production of reactive oxygen species by epithelial cells (Ruban et al., 2018). Some studies have also shown that the Candida albicans or Aspergillus fumigatus can infect alveolar macrophages and may trigger feroptosis in cells. (Bagayoko and Meunier, 2022). In our findings, we enriched many ferroptosis-associated functions and pathways including response to oxidative stress, response to metal ion, oxido reductase activity acting on NADPH, regulation of lipid metabolic process, and so on. Fusarium keratitis is an infectious disease that causes cell death by activating multiple immune responses, producing ROS, increasing ROS, and disrupting the metabolism of multiple substances. As a result, we could hypothesize that disturbance of iron and ROS metabolism were a major important cause of corneal tissue damage in Fusarium keratitis patients.

The HIF-1 signaling pathway associated with ferroptosis in Fusarium keratitis was discovered using GO, KEGG, and GSEA enrichment analysis. HIF-1 (hypoxia-inducible factor 1), the transcriptional activator that functions as a master regulator of oxygen homeostasis. We proposed the following model based on our data. When Fusarium infected the cornea, it caused local hypoxia and mitochondrial generation of superoxide in the corneal epithelial tissue. Then, HIF-1 was activated (Semenza, 2001). Next, HIF-1 would induce the expression of HO-1. Then HO-1 increased heme degradation, releasing iron ions from the heme, and the increased iron promotes ferroptosis (Lei et al., 2019). So, in the GSEA enrichment analysis, the pathway to hematopoietic cells was enriched. On the other hand, we found that the NF-κB signaling pathway might also be associated with ferroptosis. Nuclear factor-κB (NF-κB) consists of multiple transcription factors. It is associated with a variety of biological processes, including inflammation, immunity, cell proliferation, differentiation, and survival (Oeckinghaus and Ghosh, 2009). Fusarium infection of corneal tissues might induce the release of inflammatory factors and activate the NF-κB signaling pathway. The current study finds that inhibition of RSL3 activates the NF-κB signaling pathway and can induce ferroptosis (Li et al., 2021). Beyond the initial, in the results of GSEA enrichment analysis, lysosomes were shown to be involved in Fusarium keratitis ferroptosis. Some studies have proven that lysosome inhibitors could inhibit ROS generation and local iron accumulation can be involved in ROS generation within lysosomes (Dixon et al., 2012). Erastin induce enhanced lysosomal membrane permeabilization and lysosomal disruption, leading to ferroptosis (Zhou et al., 2020).

In the present study, we obtained the top5 hub genes. We verified the differential expression of hub genes in HCECs by qPCR. HMOX1 (heme oxygenase 1) is an enzyme in the catabolism of heme and catalyzes the degradation of heme to biliverdin (Hassannia et al., 2019). In fungal keratitis, HMOX1 can be increased and suppressed levels of antioxidant enzymes, inducing ferroptosis (Hua et al., 2017). CYBB (Cytochrome B-245 Beta Chain) can transport electrons through the plasma membrane to generate ROS and activate ferroptosis (Yang et al., 2020). However, the exact mechanism by which it activates ferroptosis in Fusarium keratitis was currently unclear and needed to continue to explore. GPX2 (glutathione peroxidases2) is a member of the peroxidase family and similar to GPX4 in that it is an antioxidant and anti-inflammatory enzyme. In addition to serving as an antioxidant function, it also has anti-apoptotic, anti-inflammatory, and functions that are closely related to tumor cell growth, invasion, and metastasis (Brigelius-Flohé and Flohé, 2020). Current studies have shown that fungal keratitis can cause an experimental inflammatory response in the corneal tissue, resulting in a decrease in the levels of cyclooxygenase-2 (COX2), superoxide dismutase (SOD-1), and glutathione peroxidase (GPX2) in human corneal epithelial cells (HCECs), as well as activation of ferroptosis (Qin et al., 2022). ALOX5 (arachidonate 5-lipoxygenase) is a protein that is required for the activation of the lipid peroxidation reaction. It is an iron-containing enzyme that is associated with several modes of cell death. It currently activates ferroptosis primarily by promoting inflammatory responses as well as lipid peroxidation pathways (Shi et al., 2013; Sun et al., 2019).

In Fusarium keratitis, the immune response is an important biological process. In the enrichment analysis, we enriched many immune-related pathways, and we also analyzed the abundance of immune cells. T cells follicular helper, monocytes, macrophages and mast cells played an important role. This was essentially the same as the previous study. The main role of macrophages is to engulf useless cells and dead cells. And in fungal keratitis, it can inhibit spore germination and kill spores or mycelium of fungi (Zhang et al., 2009). Xie et al. (Xie et al., 2018). demonstrate the role of mast cells in fungal keratitis by mast cell inhibitors. The role of T cells follicular helper and monocytes in fungal keratitis was poorly documented. There have been numerous studies showing a correlation between ferroptosis and immune response in infectious diseases. For example, the macrophages are less effective in phagocytosing ferroptotic Jurkat T cells as compare with their apoptotic counterparts (Klöditz and Fadeel, 2019); T cells and helper T cells are regulated by lipid peroxidation and ferroptosis (Funes et al., 2018). Therefore, we further examined the correlation between hub genes and infiltrating immune cells. Macrophages can regulate the dynamic balance of iron by removing aging red blood cells. This might induce ferroptosis in macrophages, thereby limiting their immune activity. For example, long-term storage of red blood cells in the refrigerator leads to increased phagocytosis, which is inhibited by the iron sag inhibitor ferrostatin-1 in splenic red marrow macrophages (Youssef et al., 2018). To my surprise, the neutrophil difference was not statistically significant in the CIBERSORT algorithm. Many studies have demonstrated that neutrophils are the main immune cells in early Fusarious keratitis (Karthikeyan et al., 2011; Leal et al., 2012). Bridget Ratitong et al. (Ratitong and Pearlman, 2021). have summarized these studies. The neutrophil system generates O²⁻, hydrogen peroxide (H₂O₂) and hydroxyl radical. They can generate ROS (Urban and Backman, 2020). The ROS might activate ferroptosis. The main reason for calculation of neutrophils might be a problem with the CIBERSORT algorithm calculation formula. CIBERSORT is a tool for deconvolution of expression matrices of immune cell subtypes based on the principle of linear support vector regression. It depends primarily on fidelity of reference profiles (Newman et al., 2015). Perhaps because there were too few reference documents, resulting in inaccurate results. However, the molecular mechanism between ferroptosis and immunity remains unclear.

Although we used many bioinformatics analysis methods and statistical methods to analyze fungal keratitis. However, we should also be aware of the shortcomings. Firstly, the sample size of this study was not sufficiently large. Second, this study was retrospective, so clinical data were lacking. Thirdly, the data in this study relied on the GEO database, and we would integrate data from other sources in future studies. Finally, the study used DON to simulate Fusarium infection, which had some limitations, and in future studies, we would improve the experimental protocol to make the analysis more convincing.

In this study, we suggested several hub genes and pathways that are closely associated with ferroptosis in Fusarium keratitis. These results contribute to a better understanding of ferroptosis in Fusarium keratitis and provide us with new directions for the treatment of Fusarium keratitis. Unfortunately, our samples are not large enough and the specific mechanism is not clear enough, further research is needed.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/gds/?term=gse58291 http://www.zhounan.org/ferrdb/.

XT provided idea. XT, XX, XS and YT analyzed the data and performed the experiments. YL, JL, and YD wrote the manuscript. JZ and LL assisted in revising the manuscript. LL gave important guidance and analysis in the review process. All authors contributed to the article and approved the submitted version.