Investigative Ophthalmology & Visual Science Cover Image for Volume 66, Issue 1
January 2025
Volume 66, Issue 1
Open Access
Immunology and Microbiology  |   January 2025
Piezo1 Enhances Macrophage Phagocytosis and Pyrin Activation to Ameliorate Fungal Keratitis
Author Affiliations & Notes
  • Jiahui Yang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Jing Zhong
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Zhenyuan Fu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Dalian He
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Jing Zhang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Jin Yuan
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
  • Correspondence: Jin Yuan, Zhongshan Ophthalmic Center, Sun Yat-Sen University, No. 7 Jinsui Rd., Clinical Building, 1827, Guangzhou, Guangdong 510623, China; [email protected]
  • Jing Zhang, Zhongshan Ophthalmic Center, Sun Yat-Sen University, No. 7 Jinsui Rd., Clinical Building, 1827, Guangzhou, Guangdong 510623, China; [email protected]
  • Footnotes
     Jiahui Yang and Jing Zhong contributed equally to this article.
Investigative Ophthalmology & Visual Science January 2025, Vol.66, 33. doi:https://doi.org/10.1167/iovs.66.1.33
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      Jiahui Yang, Jing Zhong, Zhenyuan Fu, Dalian He, Jing Zhang, Jin Yuan; Piezo1 Enhances Macrophage Phagocytosis and Pyrin Activation to Ameliorate Fungal Keratitis. Invest. Ophthalmol. Vis. Sci. 2025;66(1):33. https://doi.org/10.1167/iovs.66.1.33.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Fungal keratitis (FK) remains a treatment challenge, necessitating new therapeutic targets. Piezo1, a mechanosensitive ion channel, regulates calcium signaling and immune cell function. This study investigates its role in macrophage-mediated antifungal responses in FK.

Methods: Piezo1 and Pyrin expression in corneas and bone marrow-derived macrophages (BMDMs) were assessed by RNAseq, quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence. Intracellular calcium ion concentration was detected by Fluo-4 AM fluorescent probe staining. Heterozygous Piezo1 deficiency (Piezo1+/−) mice and Yoda1 were performed to regulate the expression of Piezo1.

Results: Our investigation demonstrates elevated expression of Piezo1 in the corneas of patients with FK and infected mice. This upregulation of Piezo1 corresponded with the swift recruitment of macrophages via the limbus. Additionally, Piezo1+/− mice exacerbate the progression of FK in the infection model. Furthermore, Piezo1 knockdown in macrophages exhibit a notable reduction phagocytic capacity, accompanied by an increase in viable colony-forming units in an in vitro model of fungal infection. Moreover, using a pharmacologic activator of Piezo1 (Yoda1), a calcium ion (Ca2+) chelator of BAPTA or Piezo1+/− mice, we demonstrate that Piezo1 activation triggers the Pyrin inflammasome via augmented calcium ion influx, which is required for protection against FK in murine hosts.

Conclusions: Piezo1 is crucial for innate immunity in FK, enhancing macrophage recruitment, activation, and Pyrin inflammasome-mediated antifungal activity via calcium signaling. Using Piezo1+/− mice and Yoda1, we confirm Piezo1’s role in fungal clearance. Targeting Piezo1 offers a novel strategy to improve FK outcomes by boosting macrophage function and immune response.

The underdeveloped countries face a significant challenge in the form of fungal keratitis, which ranks among the leading causes of blindness. Prompt and effective resolution has become imperative for addressing fungal keratitis (FK) due to its potential for rapid corneal tissue damage.1 Upon attachment of fungi to the cornea, both innate and adaptive immunity mechanisms are progressively activated by the body to combat this threat.24 As the primary defense against corneal fungi, innate immunity plays a vital role in FK by the phagocytosis of immune cells, such as macrophages and neutrophils. These immune cells maintain the ocular microenvironment by removing pathogens and debris resulting from cell death.5,6 Furthermore, they produce various cytokines that enhance their function and stimulate subsequent host adaptive immune responses.79 Therefore, the appropriate promotion of immune cell phagocytosis could potentially serve as an effective therapeutic strategy for the treatment of FK. 
Phagocytosis is a highly intricate process primarily regulated by membrane surface receptor proteins and cytoskeletal proteins.5,10 In the quiescent state, phagocytic cells possess anchored receptors and binding proteins that restrict protein movement and uphold cell membrane integrity. However, during pathogen invasion, disrupted actin structure induces membrane wrinkling, thereby enhancing receptor mobility and facilitating interaction with target particles.11,12 Consequently, this leads to receptor clustering and triggers activation of cytoskeletal proteins in diverse forms. The deformation of the cell membrane leads to the formation of synapses, gradually engulfing exogenous particles and eventually forming phagosomes as initial antimicrobial agents.1315 Moreover, under conditions of imbalanced cytoskeleton homeostasis, Pyrin, a cytoplasmic pattern recognition receptor, can be activated to form inflammasomes and exert antimicrobial effects in infectious diseases by the activation of caspase-1 and subsequent release of IL-1β and IL-18.16 Thus, in infectious diseases, cell membrane deformation plays a pivotal role in facilitating the phagocytosis and antimicrobial activity of immune cells. Additionally, it acts as a critical trigger for the activation of inflammatory factors, such as Pyrin, thereby amplifying the immune response. 
Piezo is a recently discovered family of mechanosensitive cation channels, comprising two subtypes: Piezo1 and Piezo2.17 Piezo1 is predominantly expressed in various mechanosensitive cells, including immune cells, whereas Piezo2 is not expressed in immune cells.1820 Piezo1 facilitates the influx of cations, particularly calcium ions (Ca²⁺), in response to diverse external forces, such as fluid flow, stretching, and cell membrane tension.2124 The downstream calcium ions play a crucial role as second messengers for various cellular activities.25 Several studies have demonstrated that calcium could activate Rac1, a calcium-dependent bridging protein, thereby inducing alterations in cell cytoskeletons and promoting the phagocytosis of pathogens.26,27 Furthermore, recent research has demonstrated that Piezo1 mediates cellular sensing in the innate immune system, enabling them to respond to fluctuations in local microenvironmental factors during inflammation, including temperature, pH value, oxygen levels, etc.28 Therefore, we propose that fungal infection in corneal tissue activates the Piezo1 channel, leading to calcium ion (Ca²⁺) influx. This calcium signaling likely influences the cytoskeletal homeostasis of immune cells, thereby enhancing their phagocytic function. Concurrently, this activation may trigger the expression of inflammatory factors, such as Pyrin, further amplifying the immune response to combat fungal pathogens. This study aims to investigate the effects and underlying mechanisms of Piezo1 on macrophage phagocytic activity and Pyrin activation in the context of FK using both animal models and cell models. 
Materials and Methods
Patient and Tissue Specimens
The tissue samples were obtained from patients with clinically diagnosed FK confirmed by corneal scraping culture, who underwent corneal transplantation at the Zhongshan Ophthalmic Center between May 2020 and May 2021. This study was approved by the Zhongshan Ophthalmic Center Medical Science Research Ethics Committee (protocol number: 2020KYPJ115), and all participants provided written consent. Infected corneal tissues were collected during the transplantation surgery, whereas normal donor corneas were sourced from the Guangdong Eye Bank. Samples were promptly stored in a cryogenic refrigerator at –80°C. Comprehensive medical records, clinical signs, anterior segment photography, and follow-up data were available for analysis. In this study, a patient presenting with clinical symptoms of suppurative keratitis was classified as a case of FK if the patient met one or more of the following criteria at the initial visit: (1) isolation of fungal organisms from corneal scrape or biopsy samples in one or more culture media, or visualization of fungal elements in light microscopy of corneal scrape samples, and (2) identification of fungal elements by in vivo confocal microscopy (IVCM). Subjects were excluded if they had (1) concurrent fungal infection confirmed by culture results, (2) corneal endophthalmitis and specific perforation on the day that cultures were obtained, (3) immune disorders, autoimmune diseases, allergic conditions, and (4) unwillingness to participate, pregnancy, or breastfeeding. 
Experimental Animals and Model Establishment
Six to 8-week-old pathogen-free male C57BL/6 mice were obtained from the Guangdong Yaokang Animal Center. Mice heterozygous for Piezo1 (strain number: T012739; common name: Piezo1 KO) were purchased from the Guangdong Yaokang Animal Center. These animals were bred and tested in the specific pathogen-free (SPF) animal room of the Zhongshan Ophthalmic Center and the Experimental Animal Center of South China Agricultural University. The protocols for the use of laboratory animals strictly adhered to the guidelines set by the American Academy of Ophthalmology and the Association for Research in Vision and Ophthalmology (ARVO), and were approved by the Institutional Review Board of the Zhongshan Ophthalmic Center (Ethics Batch No. 2020-011). Each mouse was injected with 5 µL of a fungal spore suspension, containing a concentration of 5 × 106 spores/mL. The injected corneal surface turned white, indicating the success of the procedure. The eyelids were gently rubbed together for a few seconds to ensure even distribution of the inoculum across the corneal surface. The untreated left eye of each mouse served as a control for the progression of FK. In addition, the mice in the experimental group were administered a daily subconjunctival injection of Yoda1 (S6678; Selleckchem, Shanghai, China) at a concentration of 2 µM following the modeling procedure (reagent concentration was determined by cell viability assay, 10-fold higher than the in vitro concentration; Supplementary Fig. S1C). 
At 0 days, 12 hours, 1 day, 3 days, and 5 days, intervals post-modeling, the mice were anesthetized and positioned under a stereomicroscope for anterior segment photography to monitor the evolution of corneal lesions at various time points. The severity of the ocular disease was assessed using a scoring system, as previously reported in the references, ranging from 0 to 12 points, and evaluating 3 aspects: corneal opacity area (0 to 4 points), corneal opacity degree (0 to 4 points), and corneal surface regularity (0 to 4 points).29 
Preparation of Fungal Spore Suspensions
The Aspergillus fumigatus strain used in this study was the standard AS 3.1320, obtained from the Guangdong Microbial Culture Collection Center. The potato dextrose agar (PDA) culture plate was utilized for the isolation of single colony through padding-plate passage, incubated at 28°C for 7 days. The culture plate was eluted with phosphate-buffered saline (PBST) and subjected to three rinsing cycles. The rinse solution containing spores is gathered and thoroughly mixed with a pipette. The spore suspension was centrifuged at 3500 revolutions per minute (rpm) for 4 minutes, and the supernatant was discarded. The spores were resuspended in sterile saline, centrifuged at 3500 rpm for 4 minutes, and the supernatant was discarded, this process was repeated twice for thorough washing. The concentration of the spore suspension (spores/mL) was determined using a cell counting plate. 
Bone Marrow-Derived Macrophages Culture and Cell Model Establishment
The mice were euthanized by cervical dislocation following anesthesia, and then placed in a beaker containing a sufficient amount of 75% ethanol for disinfection by immersion for 5 minutes. The femur and tibia were isolated and immersed in a PBS solution containing 1% penicillin-streptomycin solution. Bone marrow cell suspension was obtained by flushing the bone marrow cavity with a 1 mL syringe. After thoroughly mixing the medium containing bone marrow cells, the cells were screened using a 70 µm cell filter, and then centrifuged at 1000 rpm for 5 minutes to remove the supernatant. The bone marrow cells were continuously incubated in traditional 1640 medium containing 20 ng/mL induction factor M-CSF in a 37°C incubator. Semi-replacement of medium is carried out on days 3 and 5, and complete replacement of medium is carried out on day 7 to complete the induction of bone marrow-derived macrophages (BMDMs). After cell induction, the cells from various groups were pretreated with 50 μM BAPTA (GC1757; GlpBio, Montclair, CA, USA; reagent concentration was determined by cell viability assay; see Supplementary Fig. S1A), 0.2 μM Yoda1 (S6678; Selleckchem, Shanghai, China; reagent concentration was determined by cell viability assay; see Supplementary Fig. S1B), 0.1 mg/mL Cytochalasin D (GC13440-1; GlpBio, Montclair, CA, USA) or 0.1 mg/mL histamine (GC30775; GlpBio, Montclair, CA, USA) for 30 minutes. Subsequently, based on the cell: spore ratio of 1:4, the fungal spore suspension was added, and the mixture was incubated at 37°C for 0, 1, 2, 4, and 8 hours (0 hours represents the cell state without added fungal spores, serving as the control group for the experiment). The medium was then removed and the samples were washed three times with PBS to collect cell samples. 
Genotyping Analysis
Tail tissues from wild-type mice and Piezo1 het mice were collected, and the Quick Genotyping Assay Kit for Mouse Tail (D7283S; Beyotime, Shanghai, China) was used to conduct genetic identification analysis. Subsequently, the mouse tails were digested with a digestion solution and incubated to obtain genomic DNA. The PCR reaction system was then prepared according to the provided instructions and placed in a PCR machine for amplification. Upon completion, the samples were directly loaded for agarose gel electrophoresis. 
RNA- Sequencing Analysis
RNA was initially extracted from the corneas collected 5 days after infection in a mouse model of fungal keratitis, as well as normal mouse corneas, following the RNaesy Plus Mini Kit protocol (Qiagen, Valencia, CA, USA). The extracted RNA samples were subsequently sent to the company for sequencing. HaploX Biotechnology Co. Ltd. conducted the construction and sequencing of the RNA-sequencing (RNA-seq) library. Upon obtaining the raw data, Trimomatic software was used to eliminate aptamers, low-quality bases, and fragment information from the initial dataset. The effective RNA fragments were aligned with the mouse GRCm38 reference genome using Hisat2 software, and RSEM software was used to calculate the TPM value of each sample for successful construction of the RNA sequencing expression matrix in a mouse model of FK. The clinical public database was downloaded from Gene Expression Omnibus (GEO) datasets and the expression matrix can be accessed via the accession number GSE58291. Differential gene expression analysis was performed using the R package DESeq2, where a differentially expressed gene was defined as having an absolute log2 fold change (FC) greater than 1 and a false discovery rate (FDR) value less than 0.05. The selected differential genes underwent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using the R package Clusterprofiler, with differential screening criteria set at a P value less than 0.01 and Q-value less than 0.05. The relevant database is available in the Supplementary Materials
Immunohistochemical Staining
We placed the clinical corneal samples collected in EP tubes containing fixative and incubated them at room temperature overnight. Following dehydration, we proceeded with embedding the samples in paraffin and preparing the sections, followed by staining according to the instructions of Super PlusTM High Sensitive and Rapid Immunohistochemical Kit (pH9.0; Elabscience). After rapid dewaxing and antigen retrieval of the slices using Dewaxing/Antigen Retrieval Buffer, they were incubated overnight with an appropriate concentration of primary antibody (Piezo1, 15939-1-AP; Proteintech, Wuhan, China). Subsequently, SP Reagent C Polyperoxidase-anti-Rabbit/Mouse IgG was added and incubated for 30 minutes. Then, DAB chromogenic reagent and SP Reagent F Hematoxylin Staining Buffer (hematoxylin staining solution) were sequentially added to stain the target points and cell nuclei. Finally, alkaline water was used for bluing, followed by gradient alcohol dehydration, transparency agent treatment, addition of neutral gum resin, and sealing of the slides. 
Hematoxylin and Eosin Staining
The mice were euthanized by cervical dislocation after anesthesia and their eyeballs were removed. The eyeballs were then placed in EP tubes containing fixative solution and left overnight at room temperature. After dehydration and embedding in paraffin, the samples were subjected to dewaxing treatment using xylene and gradient alcohol. Subsequently, the slices were soaked in Harris hematoxylin solution for 3 to 8 minutes, rinsed with tap water, briefly immersed in 1% hydrochloric acid ethanol, rinsed again with tap water until turning blue after adding 0.6% ammonia water, and finally washed with tap water. Then, the slices were immersed in eosin staining solution for 1 to 3 minutes. Neutral resin mixed with xylene was dropped onto the slices and covered with a glass coverslip. 
Quantitative Real-Time PCR
Total RNA was extracted from human corneal epithelial cells (HCECs) or intact corneas using a RNeasy Mini Kit, according to the manufacturer’s instructions. The concentration of total RNA was measured using a spectrophotometer. cDNA synthesis was performed with PrimeScript RT Master Mix, and amplification was carried out using SYBR Green Supermix in a Light Cycler 480 Real-Time PCR System with specific primers. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal reference gene. The primer sequences can be found in the Table
Table.
 
Primer Sequences for Real-Time (RT)-PCR
Table.
 
Primer Sequences for Real-Time (RT)-PCR
Western Blot Analysis
Total cellular and corneal protein was extracted using a Minute Total Protein Extraction Kit (Invent Biotechnologies). A bicinchoninic acid protein assay kit (Millipore) was used to measure the total protein concentration. Equal amounts of protein samples were then loaded onto sodium dodecyl sulfate-polyacrylamide gels and electrophoresed. The separated proteins in the gels were transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk in Tris-buffered saline with Tween 20 for 2 hours at room temperature, the membranes were incubated overnight with appropriate primary antibodies (Piezo1, 15939-1-AP; Proteintech, Wuhan, China; Pyrin [phospho S241], ab200420, Abcam, Cambridge, UK; Pyrin, ab195975, Abcam, Cambridge, UK; Phospho-RhoA [Ser188], AF8020-100, Affbiotech, Jiangsu, China; RhoA, 10749-1-AP, Proteintech, Wuhan, China; and β-actin, ab130935, Abcam, Cambridge, UK). After thorough rinsing, secondary antibodies (Abcam) were added and incubated for 1 hour at room temperature. Horseradish peroxidase signals were amplified using an ECL kit (Vazyme) and detected using a Bio-Rad Western blot detection system (Bio-Rad Laboratories, Inc.). Grayscale images of the Western blots were used for semiquantitative analysis with ImageJ software. 
ELISA Detection
Proteins were extracted from clinical corneas by using the Minute Total Protein Extraction Kit (Invent Biotechnologies). The total protein concentration was ascertained using the BSA protein assay kit (Millipore). In accordance with the manufacturer’s instructions, the protein samples were incubated with antibodies for Piezo1, washed, and stained with Human PIEZO-1 (Piezo-type mechanosensitive ion channel component 1) ELISA Kit (ELK Biotechnology). Subsequently, the color intensity was determined via spectrophotometry, enabling the quantification of the amount of antigen or antibody in the sample. 
Immunofluorescence Staining
The mouse eyeball slices and cell climbing tablets were utilized for immunofluorescence staining in both in vivo and in vitro infection models. Following fixation and permeabilization of the tissues and cells, they were incubated overnight at 4°C with a primary antibody dilution (F-actin, ab130935; Abcam, Cambridge, UK; LAMP-1, 121602, Biolegend, San Diego, CA, USA; Piezo1, 15939-1-AP, Proteintech, Wuhan, China; CD11b, ab52478, Abcam, Cambridge, UK; and F4/80, ab213200, Abcam, Cambridge, UK). Subsequently, they underwent washing with PBST for 20 minutes, followed by PBS for 10 minutes, and another wash with PBST for 20 minutes. An appropriate concentration of DAPI and fluorescent secondary antibody dilution (Abcam, Cambridge, UK) was then added and incubated at room temperature in the dark for 2 hours. Afterward, the slides were washed sequentially with PBST for 20 minutes, then twice with PBS for 10 minutes each time, followed by another wash with PBST for 20 minutes before sealing the slides using a mounting medium. Mouse IgG was used as the isotype control of the Piezo1 antibody to validate the specificity of its staining (Supplementary Fig. S2). Subjective analysis of the expression of Piezo1 and CD11b+F4/80 in the cornea was conducted after taking photographs with an inverted fluorescence microscope. 
Fluo-4 AM Calcium Concentration Measurement
An appropriate amount of Fluo-4 AM (FXP136-100, 4A; Biotech, Beijing, China) stock solution was diluted with PBS to obtain a working solution of 5 µmol/L, which was used immediately and not subjected to repeated freezing and thawing. The culture medium of the cells under study was removed, and the well plate was treated with an adequate volume of Fluo-4 AM working solution followed by incubation at 37°C for 30 minutes. After removing the excessive dye, the cells were washed thrice with Hanks’ balanced salt solution (HBSS) to eliminate any residual staining before being covered in HBSS. Subsequently, they were further incubated at 37°C for approximately 20 to 30 minutes until complete de-esterification of Fluo-4 AM stain within them occurred. Finally, positive fluorescence microscopy using a wavelength channel of 488 nm was used to observe these cells. The fluorescence results were semiquantitatively analyzed using ImageJ. 
Fungal Load Detection
Four hours after spore stimulation and on the fifth day of the in vivo mouse model, samples of cells and corneal tissue were collected. The corneal tissue was homogenized by adding 1 mL of sterile saline to obtain a homogeneous solution. Subsequently, it was sequentially diluted to achieve the desired number of colonies on each plate. Then, 100 uL of each dilution group was evenly spread onto the corresponding PDA plate using a coater. After incubating at 37°C for 18 to 24 hours, colony-forming unit (CFU) counts were calculated. The Aspergillus fumigatus load was expressed as CFU/mL. Petri dishes exposed only to a clean bench for 10 seconds served as negative controls. 
Cell Viability Assay
The HCECs were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle's medium Nutrient Mixture F-12 (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco, USA). The cells were grown to confluency in 25 cm2 polystyrene tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Confluent cells were subcultured every 2 to 3 days by trypsinization using trypsin/EDTA solution (Gibco). 
To assess the effects of Yoda1 on the morphology and proliferation of HCECs and BMDMs and the effect of BAPTA on BMDMs, the cells were cultured in 96-well tissue plates containing a gradient concentration of Yoda1 or BAPTA. Following a 6-hour or 24-hour incubation period, the proliferation of HCECs or BMDMs was quantitatively evaluated using the Cell Counting Kit-8 (CCK-8) assay at an optical density value of 450 nm with a microplate reader (BioTek Instruments). 
Statistical Analysis
All experiments were repeated three times, and the results were presented as the mean ± standard error of the mean (SEM). SPSS version 22.0 software was used for statistical analysis. Independent sample t-test was used to compare the differences between the two groups. One-way ANOVA with Bonferroni's multiple-comparison test or 2-way ANOVA with Bonferroni's multiple-comparison test were used for multiple comparisons to assess the significance of the experimental group versus the control group. Images were obtained from GraphPad Prism 8 for Windows (version 7.04; GraphPad software). A difference of 0.05 was considered statistically significant. 
Data Availability
The data and materials used to support the findings of this study are available from the corresponding author upon request. The clinical public database utilized by the research institute can be accessed via the NCBI accession number GSE58291. Raw data of the RNA-Seq of FK mouse model have been deposited in the SRA database as BioProject PRJNA930608. 
Results
Increased Expression of Piezo1 in the Corneas of Patients With Fungal Keratitis is Associated With the Regulation of the Innate Immune Response
To identify key targets in the pathogenesis of FK, we conducted a preliminary analysis using clinical samples. Patients with confirmed fungal infection exhibited toothpaste-like white infiltrates and extensive epithelial defects on the cornea (Figs. 1A1B). Confocal imaging of the cornea revealed the presence of corneal hyphae along with a significant accumulation of inflammatory cells (indicated by red arrows) clustering together (Figs. 1C1D). Upon further analysis of existing public databases, it was found that pathways mediated by inflammation factors, particularly those related to leukocytes, were significantly upregulated during fungal infection. The gene expression profile data (GSE58291) were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). GSE58291 included 12 healthy non-infected corneal tissues, 7 bacterial keratitis tissues, and 8 FK tissues. Twelve healthy non-infected corneal tissues and 8 FK tissues were selected for analysis. Additionally, phagocytosis was identified as playing a crucial role (Fig. 1E). Consequently, our focus shifted toward the analysis of the process of phagocytosis related targets (Fig. 1F). Subsequent quantitative PCR (qPCR) testing of several those genes (see Supplementary Figs. S1A–S1C) in clinical samples confirmed elevated PIEZO1 mRNA level in the disease (Fig. 1G). Furthermore, ELISA assay and immunohistochemical staining of sections from clinical corneal samples indicated increased levels of Piezo1 protein in infected samples (P < 0.05), particularly evident in inflammatory cells (as indicated by arrows; Fig. 1H, Supplementary Fig. S3D). Collectively, these results suggest that Piezo1 is an important target in FK, as it plays a role in regulating the innate immune response. 
Figure 1.
 
The upregulation of Piezo1 expression in the corneas of patients with FK was linked to the modulation of the innate immune response. (A–D) Anterior segment photography and corneal co-focusing are employed for the assessment of patients with fungal keratitis. (E) Enrichment analysis of pathways upregulated following infection. (F) Conduct RNA-seq analysis on targets associated with phagocytosis. (G) RNA analysis of PIEZO1 expression in the corneas of healthy individuals and patients with fungal keratitis. (H) Immunohistochemical analysis of Piezo1 protein levels in the corneas of healthy individuals and patients with fungal keratitis (the arrows point to inflammatory cells; n = 3 people/ group / time point). Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 1.
 
The upregulation of Piezo1 expression in the corneas of patients with FK was linked to the modulation of the innate immune response. (A–D) Anterior segment photography and corneal co-focusing are employed for the assessment of patients with fungal keratitis. (E) Enrichment analysis of pathways upregulated following infection. (F) Conduct RNA-seq analysis on targets associated with phagocytosis. (G) RNA analysis of PIEZO1 expression in the corneas of healthy individuals and patients with fungal keratitis. (H) Immunohistochemical analysis of Piezo1 protein levels in the corneas of healthy individuals and patients with fungal keratitis (the arrows point to inflammatory cells; n = 3 people/ group / time point). Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
The Upregulation of Piezo1 Corresponded With the Rapid Recruitment of Macrophages via the Limbus in Wild-Type Mice Model of Fungal Keratitis
To investigate the expression of Piezo1 in FK, a mouse model was established using matrix injection of Aspergillus fumigatus spore suspension. Clinical scoring through anterior segment photography demonstrated the sustained stability of the mouse model, with a gradual progression of the condition over time (Figs. 2A2B). The expression levels of Piezo1 and calcium-related factors (Camk2b2, Plcl2) were assessed at 0 days, 12 hours, 1 day, 3 days, and 5 days post-infection. We observed a significant increase in the expression levels of these factors following infection with peak levels observed at the first day (P < 0.01; Figs. 2C2D). These findings revealed accumulation of Piezo1 in infected corneas consistent with increased mRNA expression of calcium-related factors (see Fig. 2D). Furthermore, careful examination of the macrophage with its marker CD11b and F4/80 in FK mice demonstrated an increase in Piezo1 protein expression in the thickened central cornea after infection (P < 0.0001; Fig. 2E, Supplementary Fig. S4). This suggested a tightly regulated expression of Piezo1 protein in macrophage following infection. To directly examine the contribution of Piezo1 in recruiting the macrophage, we performed multiple immunofluorescences staining over time in the limbus of cornea revealed the onset of infiltration of macrophages (orange) corresponded with the rapid accumulation of Piezo1 protein (green; Fig. 2F). These findings suggest that Piezo1 may play a pivotal role in activation of macrophage. 
Figure 2.
 
The expression of Piezo1 was increased in the cornea of wild-type mice model of fungal keratitis correlated with the rapid recruitment of macrophages through the limbus. (A, B) Anterior segment photography and clinical scores indicate the progression of fungal keratitis in mice. (C) Immunofluorescent expression of Piezo1 (green) in the cornea at different time points after infection, magnification = 100×. (D) RNA analysis of calcium-related factors (Camk2b and Plcl2) expression at different time points in vivo model. (E) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the central part of the cornea on the first day after infection (magnification = 200×). (F) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the peripheral cornea at different time points after infection, magnification = 200×, n = 3 to 6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 2.
 
The expression of Piezo1 was increased in the cornea of wild-type mice model of fungal keratitis correlated with the rapid recruitment of macrophages through the limbus. (A, B) Anterior segment photography and clinical scores indicate the progression of fungal keratitis in mice. (C) Immunofluorescent expression of Piezo1 (green) in the cornea at different time points after infection, magnification = 100×. (D) RNA analysis of calcium-related factors (Camk2b and Plcl2) expression at different time points in vivo model. (E) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the central part of the cornea on the first day after infection (magnification = 200×). (F) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the peripheral cornea at different time points after infection, magnification = 200×, n = 3 to 6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Heterozygous Piezo1 Deficient Mice Exacerbated the Pathological Damage of Fungal Keratitis in Mice
To investigate the function of the Piezo1 encoded protein in fungal keratitis, we generated mice that were heterozygous for a knockout allele at the Piezo1 locus (Piezo1+/− or Piezo1 het) and established infection mouse model in Piezo1+/− mice and their wild-type siblings (Piezo1WT). Prior to building models, it is imperative to validate the successful knockdown of Piezo1 in Piezo1+/− mice. Genotyping was used to confirm precise gene editing at the targeted site in the knockdown mice, whereas qPCR detection demonstrated effective inhibition of mouse Piezo1 expression at the RNA level (Figs. 3A3B; P < 0.05). We then observed the corneal pathological changes at 0 days, 12 hours, 1 day, 3 days, and 5 days post-infection. The results from anterior segment imaging and clinical scoring revealed that Piezo1+/− mice worsened corneal ulcer progression and increased perforation severity (Figs. 3C3D, P < 0.001) in this model. Moreover, there was a substantial increase in corneal fungal load following Piezo1 knockdown, indicating a potential mechanism for the accelerated progression of the disease (Fig. 3E, P < 0.01). Furthermore, hematoxylin and eosin (H&E) staining revealed significant inflammatory infiltration and tissue damage during late-stage corneal infection in the Piezo1+/− group compared with the wild-type group (Fig. 3F). Finally, we performed immunofluorescences in infected mice found that Piezo1 het mouse have decreased Piezo1 protein expression with significant inflammatory infiltration upon fungi invasion in cornea (Fig. 3G). These findings underscore that Piezo1 plays a protective role against fungal infection, influencing host antimicrobial defense mechanisms. 
Figure 3.
 
The knockdown of Piezo1 exhibited exacerbated pathological damage on the development of fungal keratitis. (A) Genotyping is employed for the identification of gene editing in Piezo1 het mice. (B) The levels of corneal Piezo1 RNA expression were compared between wild-type mice and Piezo1 het mice. (C, D). Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (E) Quantification of corneal colony-forming units (CFUs) on various groups of the in vivo model on the fifth day post-infection. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Immunofluorescent expression of Piezo1 (green) in the cornea on the first day post-infection in two groups, magnification: 100×. n = 3-6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8) or one-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 3.
 
The knockdown of Piezo1 exhibited exacerbated pathological damage on the development of fungal keratitis. (A) Genotyping is employed for the identification of gene editing in Piezo1 het mice. (B) The levels of corneal Piezo1 RNA expression were compared between wild-type mice and Piezo1 het mice. (C, D). Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (E) Quantification of corneal colony-forming units (CFUs) on various groups of the in vivo model on the fifth day post-infection. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Immunofluorescent expression of Piezo1 (green) in the cornea on the first day post-infection in two groups, magnification: 100×. n = 3-6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8) or one-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Inhibition of Piezo1 Attenuated the Skeletal Deformability of Macrophages
To further study the effect of Piezo1 on phagocytosis, we used the mouse model of fungal keratitis to analyze the activation of innate immune cells-related pathways. RNA-seq was performed using corneas from mouse model in early stage of infection (1 dpi and 0 dpi samples). The results of GO analysis demonstrated that macrophage-related pathways are highly upregulated, suggesting their crucial role in the early stage of innate immunity (Fig. 4A). Consequently, the experimental subjects in the in vitro model of this study were set as macrophages. The in vitro fungal infection model was established by introducing the spores of Aspergillus fumigatus to prompt primary macrophage reactions. The expression of Piezo1 was subsequently examined at 1 hour, 2 hours, 4 hours, and 8 hours post-infection. We observed a gradual increase of the mRNA and protein levels of Piezo1, with its transcript peaking at 4 hours post-infection (P < 0.001; Fig. 4B, 4C). This finding corroborates Piezo1 as a crucial target during fungal infection and identifies the observation time point in the in vitro model as 4 hours post-infection. 
Figure 4.
 
Inhibition of Piezo1 resulted in a reduction of the skeletal deformability of macrophages. (A) GO analysis of immune cell-related pathways during early infection. (B, C) The qPCR and Western Blot analysis of Piezo1 expression at different time points in an in vitro infection model. (D) Immunofluorescence staining of LAMP-1 (red) and F-actin (green) at different time points in an in vitro model of fungal infection, magnification = 600×. (E) Immunofluorescence staining comparing changes of F-actin (green) in the control group with the Piezo1 knockdown group in an in vitro model of fungal infection, magnification = 600×. (F) The qPCR analysis of Rac1 and Rac2 expression levels at different time points in an in vivo model. (G) Western Blot analysis of changes in RhoA-GTP levels among different groups using an in vitro model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 4.
 
Inhibition of Piezo1 resulted in a reduction of the skeletal deformability of macrophages. (A) GO analysis of immune cell-related pathways during early infection. (B, C) The qPCR and Western Blot analysis of Piezo1 expression at different time points in an in vitro infection model. (D) Immunofluorescence staining of LAMP-1 (red) and F-actin (green) at different time points in an in vitro model of fungal infection, magnification = 600×. (E) Immunofluorescence staining comparing changes of F-actin (green) in the control group with the Piezo1 knockdown group in an in vitro model of fungal infection, magnification = 600×. (F) The qPCR analysis of Rac1 and Rac2 expression levels at different time points in an in vivo model. (G) Western Blot analysis of changes in RhoA-GTP levels among different groups using an in vitro model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Simultaneously, the morphological alterations in macrophages following spore stimulation were observed. Immunofluorescence staining for the lysosomal marker protein LAMP-1 revealed that the fluorescent rings within the cells progressively increased with the prolongation of infection time, indicating an increase in the quantity of spores absorbed by the cells (Fig. 4D). Concurrently, the surface synapse formation of macrophages amplifies and the cytoskeletal homeostasis became destabilized. The qPCR analyses demonstrated a significant increase (P < 0.001) in the expression of Rac1 and Rac2 (Fig. 4F), members of the actin family typically upregulated in response to changes in the cell skeleton, further corroborating that the cytoskeleton of macrophages undergoes substantial changes following spore stimulation. We also observed the activation of RhoA, a cytoskeletal protein parallel to the Rac family, particularly its GTP phosphorylation levels. RhoA remained in a dephosphorylated state when the cell skeleton is stable without stimulation, and its level of GTP-bound form increased as the cell skeleton underwent more changes. Our research findings suggest that alterations in RhoA-GTP levels are in line with the mRNA expression of Rac1 and Rac2 (Fig. 4G). 
To investigate the influence of Piezo1 on the cytoskeleton, BMDMs were isolated from Piezo1 het mice and subsequently stimulated with spores to establish an in vitro Piezo1 knockdown model. Immunofluorescence staining of the cytoskeletal protein F-actin revealed a decrease in cell surface synapse formation after Piezo1 knockdown compared to the healthy control group (Fig. 4E). Furthermore, the expression levels of Rac1, Rac2, and RhoA-GTP were assessed in both groups. The findings demonstrated that mRNA expression levels of Rac1 and Rac2 and the protein expression levels of RhoA-GTP were diminished in the Piezo1 knockdown group relative to the simple infection group, consistent with the immunofluorescence results (see Figs. 4F4G). This suggests that Piezo1 plays a positive role in regulating cytoskeleton homeostasis. 
Piezo1 Boosts Fungal Spore Phagocytosis by Raising Intracellular Calcium Levels in the Cell Infection Model
Prior to spore stimulation, the cells were pretreated with Yoda1, a Piezo1 agonist. Immunofluorescence analysis confirmed that the application of Yoda1 markedly increases the cellular Piezo1 expression (Fig. 5A). Subsequently, the intracellular calcium concentration was dynamically monitored using the Fluo-4 AM fluorescent probe. We used Ca²⁺ ionophore histamine and calcium ion chelator BAPTA (to inhibit Ca2+ influx) as positive and negative control groups, respectively, to verify the detection effect of Fluo-4 AM in cell models (Supplementary Fig. S5). Results from immunofluorescence and grayscale analyses revealed a significant increase in the intracellular calcium concentration after Piezo1 activation, compared to the control group (P < 0.0001), which was reversed after Piezo1 knockdown (P < 0.001; Figs. 5B5C). This demonstrates that Piezo1 modulates calcium influx during fungal infection of macrophages. 
Figure 5.
 
Piezo1 can enhance the phagocytic ability of macrophages to fungal spores by promoting calcium influx in the cellular infection model. (A) Immunofluorescence detection to assess the expression of Piezo1 (green) in BMDM macrophages (CD11b [yellow] + F4/80 [red]) following treatment with Yoda1, magnification = 400×. (B, C) Different groups and time points of the in vitro model of fungal infection underwent calcium ion Fluo-4 AM fluorescence staining (green) and relative quantitative analysis, magnification = 100×. (D) The qPCR analysis of the expression changes of cell cytoskeleton-related factors (Rac1 and Rac2) in different groups of the in vivo model. (E) Western Blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (F) CFU fungal load analysis on cells from different groups after 4 hours post-spore infection, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 5.
 
Piezo1 can enhance the phagocytic ability of macrophages to fungal spores by promoting calcium influx in the cellular infection model. (A) Immunofluorescence detection to assess the expression of Piezo1 (green) in BMDM macrophages (CD11b [yellow] + F4/80 [red]) following treatment with Yoda1, magnification = 400×. (B, C) Different groups and time points of the in vitro model of fungal infection underwent calcium ion Fluo-4 AM fluorescence staining (green) and relative quantitative analysis, magnification = 100×. (D) The qPCR analysis of the expression changes of cell cytoskeleton-related factors (Rac1 and Rac2) in different groups of the in vivo model. (E) Western Blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (F) CFU fungal load analysis on cells from different groups after 4 hours post-spore infection, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
To examine the correlation between alterations in intracellular Ca2+ and the cytoskeleton, we treated cells with BAPTA and Yoda1. The detection outcomes of intracellular calcium ion concentration have demonstrated that BAPTA can inhibit calcium ion influx and Yoda1 can counteract this effect (P < 0.001; see Figs. 5B5C). Under diverse intervention conditions of intracellular calcium ion concentration, the mRNA expression of Rac1 and Rac2 in various groups was assessed by qPCR. The findings revealed that the mRNA expression of Rac1 and Rac2 in the group where cells were pretreated with BAPTA was significantly reduced compared to the simple infection group (P < 0.0001), but it increased when combined with Yoda1 (Fig. 5D; P < 0.0001). We also observed that alterations in RhoA-GTP levels are in line with the mRNA expression of Rac1 and Rac2 (Fig. 5E). These results imply that Piezo1 is capable of mediating cytoskeletal deformation through the regulation of calcium ion influx. The activation of cytoskeletal proteins is intricately linked to phagocytosis. Therefore, we subsequently assessed the fungal load in the cell supernatant following spore stimulation to discern the phagocytosis capacity of macrophages toward spores. As illustrated in Figure 5F, the quantity of intracellular spores following Piezo1 knockdown demonstrates a significant reduction compared to the simple infection group (P < 0.05), whereas a significant increase (P < 0.001) was observed, indicating a decline in the cell’s ability to phagocytose spores. 
Expression of Pyrin by Macrophages With Fungi Stimulation Is Increased
To investigate the key target of Piezo1-mediated inflammation, we extracted 0-, 1- and 5-day post-infection samples, 3 animals for each group, from the FK mouse model for RNA-seq analysis, focusing on cytoskeleton-related inflammatory factors for screening. Our findings revealed significant expression of the gene Mefv, which encodes for a protein called Pyrin (Fig. 6A). The gene has been identified as being regulated by the cell cytoskeleton, particularly the Rho protein family. It is readily activated by dephosphorylation and form inflammasomes through a combination of toxin stimulation and the cell cytoskeleton, recruiting inflammatory factors and facilitating pathogen clearance. The results from qPCR and Western Blot of 1-day post-infection corneal samples (Fig. 6C) and cell samples (Fig. 6B) at 4 hours post-infection also demonstrated upregulation of Pyrin and downregulation its phosphorylation in mRNA Protein levels in both in vitro and in vivo infection models (Figs. 6D6E, P < 0.05). This indicates that Pyrin is a crucial inflammatory target associated with the cytoskeleton during fungal infection. 
Figure 6.
 
The expression of Pyrin was significantly increased in both animal and cell models of fungal infection. (A) RNA-seq analysis of inflammation-related gene expression on the first day and the fifth day post-infection of a mouse model infection compared to the control group. (B) The qPCR analysis of the Mefv gene expression levels in the in vitro model before and after infection. (C) The qPCR analysis of the Mefv gene expression levels in the in vivo model before and after infection. (D) Western Blot analysis of changes in Pyrin protein expression levels in the cell model. (E) Western Blot analysis of changes in Pyrin protein expression levels in the mouse model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 6.
 
The expression of Pyrin was significantly increased in both animal and cell models of fungal infection. (A) RNA-seq analysis of inflammation-related gene expression on the first day and the fifth day post-infection of a mouse model infection compared to the control group. (B) The qPCR analysis of the Mefv gene expression levels in the in vitro model before and after infection. (C) The qPCR analysis of the Mefv gene expression levels in the in vivo model before and after infection. (D) Western Blot analysis of changes in Pyrin protein expression levels in the cell model. (E) Western Blot analysis of changes in Pyrin protein expression levels in the mouse model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Pyrin Activation Is Associated With the Cytoskeleton and Regulated by Piezo1-Ca²⁺
To elucidate the regulatory relationship between Piezo1 and Pyrin, the expression of Pyrin in the control group and Piezo1 knockdown group was examined. The results of qPCR and Western blot showed a decrease in Pyrin activation level following Piezo1 inhibition (Figs. 7A7C, P < 0.05), indicating that Piezo1 can positively regulate Pyrin expression during fungal infection. Next, to further investigate whether Piezo1 regulates Pyrin expression through Ca2+, we inhibited Ca2+ influx using BAPTA, followed by Western blot analysis in the cell model. The results demonstrated a significant reduction in expression of Pyrin and its related factors after calcium influx inhibition compared to the control group, whereas an increase in expression was observed after co-treatment with Piezo1 agonist Yoda1 (Figs. 7D7F, P < 0.05). These findings suggest that calcium can influence Pyrin activation and is regulated by Piezo1 in the upstream. 
Figure 7.
 
Pyrin was associated with cytoskeleton deformation and positively regulated by Piezo1. (A–C). The qPCR and Western blot analysis to compare the expression of Pyrin-related factors between the control group and Piezo1 knockdown group in an in vitro model. (D–F) The qPCR and Western blot analysis to determine the activation status of Pyrin-related factors among different groups within an in vitro model. (G) The qPCR analysis to examine alterations in the expression of cytoskeletal factors Rac1 and Rac2 in an in vitro model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control and CytoD groups of an in vitro model, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 7.
 
Pyrin was associated with cytoskeleton deformation and positively regulated by Piezo1. (A–C). The qPCR and Western blot analysis to compare the expression of Pyrin-related factors between the control group and Piezo1 knockdown group in an in vitro model. (D–F) The qPCR and Western blot analysis to determine the activation status of Pyrin-related factors among different groups within an in vitro model. (G) The qPCR analysis to examine alterations in the expression of cytoskeletal factors Rac1 and Rac2 in an in vitro model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control and CytoD groups of an in vitro model, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Furthermore, given the intimate association between Pyrin and the cellular cytoskeleton, as well as our findings on the regulatory impact of Piezo1 on the cytoskeleton, we sought to delve deeper into the regulatory interplay between Pyrin and the cellular cytoskeleton in the context of fungal infection. To achieve this, we used pretreatment with a cell relaxant Cytochalasin D (CytoD) to impede alterations in the cellular cytoskeleton. The qPCR results demonstrated a significant downregulation of Rac1 and Rac2 by CytoD (Fig. 7G, P < 0.001), indicating its potential to impede cell deformation. Furthermore, the expression of Pyrin and its associated factors in the CytoD group was markedly lower than that in the control group (Figs. 7H7I, P < 0.05), suggesting a positive correlation between Pyrin and cytoskeleton homeostasis. Based on our research findings, we posit that following fungal infection, Piezo1 triggers cellular cytoskeletal deformation, subsequently resulting in Pyrin activation and thereby mediating disease progression. 
Upregulation of Piezo1 Activates Macrophage Phagocytosis and Pyrin Inflammasome to Alleviate Fungal Keratitis in Mice
To validate the therapeutic potential of Piezo1 as a crucial target, we utilized the agonist Yoda1 for the treatment of FK. The qPCR and immunofluorescence analysis confirmed that Piezo1 expression was upregulated at both RNA and protein levels in the cornea of the Yoda1 group compared to the control group on the first day after Yoda1 injection, thus confirming the efficacy of the agonist (Figs. 8A8B, P < 0.001). Concurrently, immunofluorescence detection of Piezo1 expression levels in the Yoda1 injection group within the infection model revealed a significant increase compared to the simple infection group, further demonstrating the impact of the agonist (Fig. 8C). Moreover, changes in disease progression were observed at various time points. The clinical scores of model mice significantly decreased on the first day following Yoda1 intervention, and by the fifth day, marked improvement in corneal inflammation infiltration was evident (Figs. 8D8E, P < 0.05). Additionally, H&E staining revealed a reduction in inflammatory cell numbers and alleviation from corneal edema (Fig. 8F). Furthermore, using a pharmacologic activator of Piezo1 (Yoda1), or Piezo1+/− mice, we observed changes in the expression of Pyrin and related factors at the RNA level as well as alterations in the activation status of cytoskeletal protein RhoA (Figs. 8GI, P < 0.001). These findings were consistent with those obtained from cell experiments and demonstrated that Piezo1 positively regulates Pyrin and cytoskeleton. Meanwhile, we also performed immunofluorescence detection of the infiltration of corneal macrophages (F4/80+CD11b) in diverse groups on the fifth day after infection. The outcomes demonstrated that the quantity of macrophages in the posterior cornea of the Yoda1 treatment group was conspicuously decreased in contrast to the control group, whereas that in the Piezo1+/− group was the converse, suggesting that Piezo1 possesses a certain anti-inflammatory effect (Supplementary Fig. S6). Collectively, our results suggest that therapies targeting the activation of Piezo1 could become an important and effective tool in the treatment of FK in patients (Fig. 9). 
Figure 8.
 
The enhancement of Piezo1 expression activated macrophage phagocytosis and the Pyrin inflammasome leading to amelioration in the pathology of fungal keratitis. (A, B) The levels of corneal Piezo1 RNA and protein expression were compared between wild-type mice and mice treatment with Yoda1. (C) Immunofluorescence detection to assess the expression of Piezo1 (green) in cornea between the control group and Yoda1 group in an in vivo model. (D, E) Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Western blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control, Yoda1, and knockdown groups of an in vivo model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test, 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 8.
 
The enhancement of Piezo1 expression activated macrophage phagocytosis and the Pyrin inflammasome leading to amelioration in the pathology of fungal keratitis. (A, B) The levels of corneal Piezo1 RNA and protein expression were compared between wild-type mice and mice treatment with Yoda1. (C) Immunofluorescence detection to assess the expression of Piezo1 (green) in cornea between the control group and Yoda1 group in an in vivo model. (D, E) Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Western blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control, Yoda1, and knockdown groups of an in vivo model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test, 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 9.
 
A schematic illustration of the role of Piezo1 in fungal keratitis (FK). We demonstrate targeting Piezo1 with Yoda1 effectively treats FK using a murine model of Aspergillus fumigatus (AS3.1320) infection-induced FK. Subconjunctival administration of Yoda1 activates Piezo1 mechanosensitive channels (Ca2+) on macrophages within the corneal stroma. This activation triggers increased intracellular calcium influx, subsequently activating RhoA-GTP. This cascade promotes heightened cellular cytoskeletal deformation, enhancing phagocytic clearance capacity against pathogens. Simultaneously, RhoA activation can initiate phosphorylation-dependent activation of Pyrin protein, leading to inflammasome formation. All processes are interconnected to mediate Fungi killing in FK. ASC, apoptosis-associated speck-like protein; P, phosphorylation; RhoA-GTP; IL18, interleukin-18; RhoA, RhoA GTPase.
Figure 9.
 
A schematic illustration of the role of Piezo1 in fungal keratitis (FK). We demonstrate targeting Piezo1 with Yoda1 effectively treats FK using a murine model of Aspergillus fumigatus (AS3.1320) infection-induced FK. Subconjunctival administration of Yoda1 activates Piezo1 mechanosensitive channels (Ca2+) on macrophages within the corneal stroma. This activation triggers increased intracellular calcium influx, subsequently activating RhoA-GTP. This cascade promotes heightened cellular cytoskeletal deformation, enhancing phagocytic clearance capacity against pathogens. Simultaneously, RhoA activation can initiate phosphorylation-dependent activation of Pyrin protein, leading to inflammasome formation. All processes are interconnected to mediate Fungi killing in FK. ASC, apoptosis-associated speck-like protein; P, phosphorylation; RhoA-GTP; IL18, interleukin-18; RhoA, RhoA GTPase.
Discussion
Poorly controlled FK is a devastating and opportunistic infection of the cornea, characterized by a significant risk of blindness and a refractory condition that threatens vision.1 Compared with other forms of infectious keratitis, FK has a relatively poor prognosis due to the limited efficacy of antifungal agents and the broad range of drug sensitivity with current medications.7,9,30,31 Piezo1, a mechanically activated calcium-permeable nonselective cation channel protein, has been recently discovered and is widely expressed in various tissues and cells.32,33 The recent studies have revealed that Piezo1 regulates the inflammatory response and might play a crucial role in the immune system.24,34 In this study, the significant upregulation of Piezo1 were observed in both the in vitro and in vivo models suggesting it serves as an important target during fungal infection. In vivo downregulation of Piezo1 leads to accelerated corneal perforation and increased fungal load, indicating compromised host clearance ability against pathogens. Conversely, treatment with the Piezo1 activator Yoda1 remarkably ameliorates the condition of mice. Moreover, previous studies have demonstrated that Piezo1 plays a critical role in inflammation and immune regulation. It contributes to immune homeostasis and the inflammatory response by modulating immune cell polarization, activation, metabolic reprogramming, and interactions with other molecular pathways.22,3537 These findings highlight the potential therapeutic value of targeting Piezo1 for the effective management of FK. 
The clinical prognosis of FK primarily depends on the disease’s aggressiveness and the host’s immune defense mechanisms.2,38,39 The key phagocytes involved in fungal killing include macrophages and neutrophils, functioning as gatekeepers to manage and resolve infections.4,5,8,9,40,41 Studies have demonstrated that a decrease in mononuclear phagocytes can facilitate fungal proliferation in tissues, suggesting that macrophages play a crucial role in fungal phagocytosis and elimination.5,1214,41,42 In the disease context of fungal infection, macrophages depend on various mechanisms, such as phosphorylation or cytoskeletal protein deformation, to capture pathogens for phagocytosis.12,43,44 In this study, LAMP-1 and F-actin immunofluorescence staining were used to demonstrate that with the prolongation of spore stimulation, the number of spores absorbed by macrophages incrementally increased, accompanied by an increase in the number of synapses on the cell surface. In addition, Piezo1 knockdown decreased surface synapse formation in macrophages after spore stimulation. The observation of an increased number of extracellular fungal spores following Piezo1 downregulation suggests that Piezo1 enhances the phagocytic capacity of macrophages. This effect is mediated through cytoskeletal deformation, which amplifies their fungal clearance. 
High molecular-weight transmembrane proteins are typically stabilized through direct interaction with the cytoskeleton or by bridle proteins, which function like the pickets of a fence to maintain the cytoskeleton’s homeostatic balance.45,46 However, upon pathogen invasion, inflammatory receptors are activated and aggregate on the cell membrane surface, triggering the activation of skeletal-related proteins and leading to the gradual encapsulation of fungal-related components within the cytoskeleton.7,43,47 The Rho family GTPases, including RhoA, RhoB, RhoC, Rac1, and Rac2, participate in regulation of cytoskeleton through specific targets.16,45,48 Previous studies have shown that in bacterial infections, Piezo1 collaborates with TLR4 to regulate calcium ion-mediated signaling pathways, particularly the cytoskeletal factor Rac1, thereby modulating the bactericidal response.49 In this study, we observed a significant increase in the levels of Rac1, Rac2, and RhoA-GTP in macrophages following spore stimulation. In addition, Piezo1 knockdown reduced the expression of Rac1, Rac2, and RhoA-GTP. These data suggest that Piezo1 enhances cytoskeletal deformation and the phagocytic capacity of macrophages by upregulating the expression of Rho family GTPases. This also supports the previously reported regulatory connection between Piezo1 and Rac during infection. Furthermore, by measuring intracellular calcium concentration changes upon upregulating and downregulating Piezo1 expression during fungal infection, we confirmed that Piezo1 facilitates calcium ion influx. Based on the established relationship between calcium ions and the cytoskeleton,2527 as well as the observed reduction in Piezo1-induced RhoA activation upon treatment with the calcium ion chelator BAPTA, we conclude that Piezo1 mediates macrophage phagocytosis through calcium influx, which induces RhoA phosphorylation. This mechanism contributes to the regulation of the actin cytoskeleton by Piezo1. 
Pyrin is recognized as an intrinsic sensor that undergoes negative regulation by the cellular cytoskeletal protein Rho.50 Its activation occurs subsequent to phosphorylation modifications induced by bacterial toxins on Rho proteins, and it plays a crucial role in assembling inflammasome complexes involving caspase recruitment domain-containing proteins (CARDs) along with ASC proteins.16,51 In addition, the autocleavage of pro-caspase-1 results in the formation of the active caspase-1 p10/p20 tetramer, which subsequently processes pro-IL-1β and pro-IL-18 into their mature forms. These cytokines then act as powerful triggers and amplifiers of the innate immune response, leading to the activation of diverse defensive mechanisms.52 Therefore, the Pyrin activation is intricately linked to cytoskeletal deformation and could potentially play a role in infectious diseases. In our study, we have elucidated how macrophages undergo cytoskeletal protein reconstruction post-fungal infection while observing heightened levels regarding phosphor-RhoA activity simultaneously. Moreover, the results from examining Pyrin indicated significantly increased dephosphorylated states during early-stage infections both intracellularly and extracellularly, which suggests obvious activations taking place then. Furthermore, either knockdown of Piezo1 or treatment of CytoD (cytoskeleton relaxant) inhibited the expression of Pyrin, cytoskeletal factors (Rac1 and Rac2) and Pyrin-related factors (Mefv, Pycard, Caspase1, and IL-18) in BMDMs after fungi infection. These data suggest that Piezo1 may enhance the host’s antimicrobial capacity by increasing the expression of Pyrin and Pyrin-related factors. Furthermore, Yoda1 effectively counteracted the effect of BAPTA (a Ca2+ chelator) on inhibiting the expression of Pyrin, cytoskeletal factors (Rac1 and Rac2) and Pyrin-related factors (Mefv, Pycard, Caspase1, and IL-18). These data indicate that Piezo1 triggers alterations in the cellular cytoskeleton and activates Pyrin inflammasome pathway through an elevation in calcium ion influx to participate in the subsequent regulation of immune defense. 
Furthermore, upon activation of Piezo1 using the activator Yoda1, we observed a significant reduction in macrophage infiltration in the cornea during the later stages of infection, which could be linked to a decrease in fungal load. This finding suggests that Piezo1 not only plays a role in enhancing fungal clearance but may also exert anti-inflammatory effects. For clinical management of FK, both antifungal and anti-inflammatory treatments are essential.1,53 Previous studies have shown that neither antifungal nor anti-inflammatory therapies alone lead to satisfactory outcomes.39,54 Given the limited effectiveness of antifungal drugs and the challenges in developing new ones, exploring immune-based therapies with dual antifungal and anti-inflammatory effects is crucial.38,55,56 In this study, the upregulation of Piezo1 significantly improved fungal sterilization and reduced inflammatory cell infiltration, highlighting Piezo1 as a promising therapeutic target with dual potential. 
In this study, despite technical limitations, we made efforts to present our data effectively. However, the insufficient detection of cytoskeletal proteins hindered the identification of key targets. As a result, our investigation of Piezo1 and cellular homeostasis was largely phenomenological, and the exploration of specific immune mechanisms was limited. Moving forward, we aim to conduct further research to better understand the role of Piezo1 in corneal pathophysiology and its interactions with other immune cells. Nonetheless, our study marks the first discovery of Piezo1 and Pyrin targets in fungal infection. Collectively, Piezo1 triggers alterations in the cellular cytoskeleton and activates Pyrin inflammasome pathway through an elevation in calcium ion influx. This activation ameliorated fungal keratitis by enhancing the phagocytic capacity of macrophages and the host's antimicrobial capacity, suggesting its therapeutic potential for FK and other infectious keratitis. 
Acknowledgments
Supported by the National Natural Science Foundation of China (Nos. 81870633, 82101086, and 82401217) and Guangdong MS Institute of Scientific Instrument Innovation (No. MSXZ2023025). 
Author Contributions: Jin Yuan and Jing Zhang guided the project. Jiahui Yang and Jing Zhong conceived the project and designed the experiments. Jiahui Yang and Dalian He carried out the experiments. Jiahui Yang and Zhenyuan Fu analyzed the data. Jing Zhang and Jiahui Yang wrote the manuscript draft. Jin Yuan and Jing Zhang revised the manuscript. All the authors have read and approved the submission for publication. 
Disclosure: J. Yang, None; J. Zhong, None; Z. Fu, None; D. He, None; J. Zhang, None; J. Yuan, None 
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Figure 1.
 
The upregulation of Piezo1 expression in the corneas of patients with FK was linked to the modulation of the innate immune response. (A–D) Anterior segment photography and corneal co-focusing are employed for the assessment of patients with fungal keratitis. (E) Enrichment analysis of pathways upregulated following infection. (F) Conduct RNA-seq analysis on targets associated with phagocytosis. (G) RNA analysis of PIEZO1 expression in the corneas of healthy individuals and patients with fungal keratitis. (H) Immunohistochemical analysis of Piezo1 protein levels in the corneas of healthy individuals and patients with fungal keratitis (the arrows point to inflammatory cells; n = 3 people/ group / time point). Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 1.
 
The upregulation of Piezo1 expression in the corneas of patients with FK was linked to the modulation of the innate immune response. (A–D) Anterior segment photography and corneal co-focusing are employed for the assessment of patients with fungal keratitis. (E) Enrichment analysis of pathways upregulated following infection. (F) Conduct RNA-seq analysis on targets associated with phagocytosis. (G) RNA analysis of PIEZO1 expression in the corneas of healthy individuals and patients with fungal keratitis. (H) Immunohistochemical analysis of Piezo1 protein levels in the corneas of healthy individuals and patients with fungal keratitis (the arrows point to inflammatory cells; n = 3 people/ group / time point). Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 2.
 
The expression of Piezo1 was increased in the cornea of wild-type mice model of fungal keratitis correlated with the rapid recruitment of macrophages through the limbus. (A, B) Anterior segment photography and clinical scores indicate the progression of fungal keratitis in mice. (C) Immunofluorescent expression of Piezo1 (green) in the cornea at different time points after infection, magnification = 100×. (D) RNA analysis of calcium-related factors (Camk2b and Plcl2) expression at different time points in vivo model. (E) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the central part of the cornea on the first day after infection (magnification = 200×). (F) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the peripheral cornea at different time points after infection, magnification = 200×, n = 3 to 6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 2.
 
The expression of Piezo1 was increased in the cornea of wild-type mice model of fungal keratitis correlated with the rapid recruitment of macrophages through the limbus. (A, B) Anterior segment photography and clinical scores indicate the progression of fungal keratitis in mice. (C) Immunofluorescent expression of Piezo1 (green) in the cornea at different time points after infection, magnification = 100×. (D) RNA analysis of calcium-related factors (Camk2b and Plcl2) expression at different time points in vivo model. (E) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the central part of the cornea on the first day after infection (magnification = 200×). (F) Immunofluorescent expression of Piezo1 (green) and macrophages (CD11b (yellow) + F4/80 (red) in the peripheral cornea at different time points after infection, magnification = 200×, n = 3 to 6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 3.
 
The knockdown of Piezo1 exhibited exacerbated pathological damage on the development of fungal keratitis. (A) Genotyping is employed for the identification of gene editing in Piezo1 het mice. (B) The levels of corneal Piezo1 RNA expression were compared between wild-type mice and Piezo1 het mice. (C, D). Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (E) Quantification of corneal colony-forming units (CFUs) on various groups of the in vivo model on the fifth day post-infection. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Immunofluorescent expression of Piezo1 (green) in the cornea on the first day post-infection in two groups, magnification: 100×. n = 3-6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8) or one-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 3.
 
The knockdown of Piezo1 exhibited exacerbated pathological damage on the development of fungal keratitis. (A) Genotyping is employed for the identification of gene editing in Piezo1 het mice. (B) The levels of corneal Piezo1 RNA expression were compared between wild-type mice and Piezo1 het mice. (C, D). Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (E) Quantification of corneal colony-forming units (CFUs) on various groups of the in vivo model on the fifth day post-infection. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Immunofluorescent expression of Piezo1 (green) in the cornea on the first day post-infection in two groups, magnification: 100×. n = 3-6 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by two-tailed unpaired Student's t-test (GraphPad Prism 8) or one-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
Figure 4.
 
Inhibition of Piezo1 resulted in a reduction of the skeletal deformability of macrophages. (A) GO analysis of immune cell-related pathways during early infection. (B, C) The qPCR and Western Blot analysis of Piezo1 expression at different time points in an in vitro infection model. (D) Immunofluorescence staining of LAMP-1 (red) and F-actin (green) at different time points in an in vitro model of fungal infection, magnification = 600×. (E) Immunofluorescence staining comparing changes of F-actin (green) in the control group with the Piezo1 knockdown group in an in vitro model of fungal infection, magnification = 600×. (F) The qPCR analysis of Rac1 and Rac2 expression levels at different time points in an in vivo model. (G) Western Blot analysis of changes in RhoA-GTP levels among different groups using an in vitro model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 4.
 
Inhibition of Piezo1 resulted in a reduction of the skeletal deformability of macrophages. (A) GO analysis of immune cell-related pathways during early infection. (B, C) The qPCR and Western Blot analysis of Piezo1 expression at different time points in an in vitro infection model. (D) Immunofluorescence staining of LAMP-1 (red) and F-actin (green) at different time points in an in vitro model of fungal infection, magnification = 600×. (E) Immunofluorescence staining comparing changes of F-actin (green) in the control group with the Piezo1 knockdown group in an in vitro model of fungal infection, magnification = 600×. (F) The qPCR analysis of Rac1 and Rac2 expression levels at different time points in an in vivo model. (G) Western Blot analysis of changes in RhoA-GTP levels among different groups using an in vitro model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 5.
 
Piezo1 can enhance the phagocytic ability of macrophages to fungal spores by promoting calcium influx in the cellular infection model. (A) Immunofluorescence detection to assess the expression of Piezo1 (green) in BMDM macrophages (CD11b [yellow] + F4/80 [red]) following treatment with Yoda1, magnification = 400×. (B, C) Different groups and time points of the in vitro model of fungal infection underwent calcium ion Fluo-4 AM fluorescence staining (green) and relative quantitative analysis, magnification = 100×. (D) The qPCR analysis of the expression changes of cell cytoskeleton-related factors (Rac1 and Rac2) in different groups of the in vivo model. (E) Western Blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (F) CFU fungal load analysis on cells from different groups after 4 hours post-spore infection, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 5.
 
Piezo1 can enhance the phagocytic ability of macrophages to fungal spores by promoting calcium influx in the cellular infection model. (A) Immunofluorescence detection to assess the expression of Piezo1 (green) in BMDM macrophages (CD11b [yellow] + F4/80 [red]) following treatment with Yoda1, magnification = 400×. (B, C) Different groups and time points of the in vitro model of fungal infection underwent calcium ion Fluo-4 AM fluorescence staining (green) and relative quantitative analysis, magnification = 100×. (D) The qPCR analysis of the expression changes of cell cytoskeleton-related factors (Rac1 and Rac2) in different groups of the in vivo model. (E) Western Blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (F) CFU fungal load analysis on cells from different groups after 4 hours post-spore infection, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 6.
 
The expression of Pyrin was significantly increased in both animal and cell models of fungal infection. (A) RNA-seq analysis of inflammation-related gene expression on the first day and the fifth day post-infection of a mouse model infection compared to the control group. (B) The qPCR analysis of the Mefv gene expression levels in the in vitro model before and after infection. (C) The qPCR analysis of the Mefv gene expression levels in the in vivo model before and after infection. (D) Western Blot analysis of changes in Pyrin protein expression levels in the cell model. (E) Western Blot analysis of changes in Pyrin protein expression levels in the mouse model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 6.
 
The expression of Pyrin was significantly increased in both animal and cell models of fungal infection. (A) RNA-seq analysis of inflammation-related gene expression on the first day and the fifth day post-infection of a mouse model infection compared to the control group. (B) The qPCR analysis of the Mefv gene expression levels in the in vitro model before and after infection. (C) The qPCR analysis of the Mefv gene expression levels in the in vivo model before and after infection. (D) Western Blot analysis of changes in Pyrin protein expression levels in the cell model. (E) Western Blot analysis of changes in Pyrin protein expression levels in the mouse model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 7.
 
Pyrin was associated with cytoskeleton deformation and positively regulated by Piezo1. (A–C). The qPCR and Western blot analysis to compare the expression of Pyrin-related factors between the control group and Piezo1 knockdown group in an in vitro model. (D–F) The qPCR and Western blot analysis to determine the activation status of Pyrin-related factors among different groups within an in vitro model. (G) The qPCR analysis to examine alterations in the expression of cytoskeletal factors Rac1 and Rac2 in an in vitro model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control and CytoD groups of an in vitro model, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 7.
 
Pyrin was associated with cytoskeleton deformation and positively regulated by Piezo1. (A–C). The qPCR and Western blot analysis to compare the expression of Pyrin-related factors between the control group and Piezo1 knockdown group in an in vitro model. (D–F) The qPCR and Western blot analysis to determine the activation status of Pyrin-related factors among different groups within an in vitro model. (G) The qPCR analysis to examine alterations in the expression of cytoskeletal factors Rac1 and Rac2 in an in vitro model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control and CytoD groups of an in vitro model, n = 3 cell samples / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 8.
 
The enhancement of Piezo1 expression activated macrophage phagocytosis and the Pyrin inflammasome leading to amelioration in the pathology of fungal keratitis. (A, B) The levels of corneal Piezo1 RNA and protein expression were compared between wild-type mice and mice treatment with Yoda1. (C) Immunofluorescence detection to assess the expression of Piezo1 (green) in cornea between the control group and Yoda1 group in an in vivo model. (D, E) Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Western blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control, Yoda1, and knockdown groups of an in vivo model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test, 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 8.
 
The enhancement of Piezo1 expression activated macrophage phagocytosis and the Pyrin inflammasome leading to amelioration in the pathology of fungal keratitis. (A, B) The levels of corneal Piezo1 RNA and protein expression were compared between wild-type mice and mice treatment with Yoda1. (C) Immunofluorescence detection to assess the expression of Piezo1 (green) in cornea between the control group and Yoda1 group in an in vivo model. (D, E) Anterior segment photography and clinical scoring were conducted on distinct groups of an in vivo model of fungal keratitis. (F) H&E staining was utilized to observe the corneal pathological changes in two groups, magnification = 200×. (G) Western blot analysis of the expression changes of RhoA-GTP in different groups of the in vivo model. (H, I) The qPCR analysis to investigate changes in the expression of Pyrin-related factors in the control, Yoda1, and knockdown groups of an in vivo model, n = 3 animals / group / time point. Data are shown as mean ± SD. Statistical analyses were performed by 2-tailed unpaired Student's t-test, 1-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8) or 2-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism 8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.
Figure 9.
 
A schematic illustration of the role of Piezo1 in fungal keratitis (FK). We demonstrate targeting Piezo1 with Yoda1 effectively treats FK using a murine model of Aspergillus fumigatus (AS3.1320) infection-induced FK. Subconjunctival administration of Yoda1 activates Piezo1 mechanosensitive channels (Ca2+) on macrophages within the corneal stroma. This activation triggers increased intracellular calcium influx, subsequently activating RhoA-GTP. This cascade promotes heightened cellular cytoskeletal deformation, enhancing phagocytic clearance capacity against pathogens. Simultaneously, RhoA activation can initiate phosphorylation-dependent activation of Pyrin protein, leading to inflammasome formation. All processes are interconnected to mediate Fungi killing in FK. ASC, apoptosis-associated speck-like protein; P, phosphorylation; RhoA-GTP; IL18, interleukin-18; RhoA, RhoA GTPase.
Figure 9.
 
A schematic illustration of the role of Piezo1 in fungal keratitis (FK). We demonstrate targeting Piezo1 with Yoda1 effectively treats FK using a murine model of Aspergillus fumigatus (AS3.1320) infection-induced FK. Subconjunctival administration of Yoda1 activates Piezo1 mechanosensitive channels (Ca2+) on macrophages within the corneal stroma. This activation triggers increased intracellular calcium influx, subsequently activating RhoA-GTP. This cascade promotes heightened cellular cytoskeletal deformation, enhancing phagocytic clearance capacity against pathogens. Simultaneously, RhoA activation can initiate phosphorylation-dependent activation of Pyrin protein, leading to inflammasome formation. All processes are interconnected to mediate Fungi killing in FK. ASC, apoptosis-associated speck-like protein; P, phosphorylation; RhoA-GTP; IL18, interleukin-18; RhoA, RhoA GTPase.
Table.
 
Primer Sequences for Real-Time (RT)-PCR
Table.
 
Primer Sequences for Real-Time (RT)-PCR
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