GSDMD, an executor of pyroptosis, is involved in IL-1β secretion in Aspergillus fumigatus keratitis
Wenyi Zhao, Hua Yang, Leyu Lyu, Jie Zhang, Qiang Xu, Nan Jiang, Guibo Liu, Limei Wang, Haijing Yan, Chengye Che *
Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China

A R T I C L E I N F O

Keywords: Aspergillus fumigatus Keratitis
Pyroptosis GSDMD
Inflammation IL-1β

A B S T R A C T

The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1β was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1β secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1β and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.

1. Introduction

Fungal keratitis is an infectious disease caused by direct infection of the cornea by pathogenic fungi resulting in blindness. The disease is associated with agricultural trauma, especially during the harvest sea-son (Hu et al., 2014; Mascarenhas et al., 2014; Nielsen et al., 2015). In recent years, with the widespread use of antibiotics and glucocorticoids, its incidence has increased (Xie et al., 2006). There are many types of fungi that cause corneal infection, although most infections are mainly caused by filamentous fungi such as Aspergillus fumigatus and Fusarium (Niu et al., 2018; Yuan et al., 2017). Innate immunity, which is the first line of defense in the cornea against fungal invasion, is quickly activated early in the course of the disease to control infection (Liu and Lieberman, 2017; Miao et al., 2010; Shi et al., 2015). In the process of fungal infection of the cornea, this innate immune response recognizes pathogen-associated molecular patterns (PAMP) or damage-associated

molecular pattern (DAMP) through specific host receptors such as pattern recognition receptors (PRRs) (Niu et al., 2018). Subsequently, the intracellular signaling pathways are activated and a large number of cytokines are secreted outside the cell, triggering an inflammatory response against the pathogen.
Pyroptosis is a cell-programmed inflammatory death pattern newly discovered following apoptosis and cell necrosis (Fink and Cookson, 2005; Shi et al., 2017) and is mediated by inflammatory caspase (cas-pase-1/4/5/11) to punch pores in the cell membrane, resulting in cell lysis and releasing of pro-inflammatory factors and cellular contents (Aglietti et al., 2016; Chen et al., 2016; Ding et al., 2016; Liu et al., 2016). Recent research indicates that pyroptosis is closely related to the activation of NOD-like receptor (NLR), one of the anti-fungal immune response PRRs (He et al., 2015).
New research focused on understanding the key molecules involved in pyroptosis has suggested gasdermin-D (GSDMD), a common substrate

* Corresponding author. Department of Ophthalmology, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, 266003, China.
E-mail address: [email protected] (C. Che).

https://doi.org/10.1016/j.exer.2020.108375

Received 2 April 2020; Received in revised form 24 October 2020; Accepted 27 November 2020
Available online 3 December 2020
0014-4835/© 2020 Elsevier Ltd. All rights reserved.

W. Zhao et al. Experimental Eye Research xxx (xxxx) xxx

for caspase-l, caspase-4, caspase-5 and caspase-11, is a key performer of pyroptosis (Kayagaki et al., 2015; Shi et al., 2015). The GSDMD protein is 53 kDa in length and is cleaved to produce two major domains: 30 kDa GSDMD-NT and 20 kDa GSDMD-CT. In the normal resting state, the two domains combine with each other and are in an inactive self-inhibitory state (Kayagaki et al., 2015). GSDMD-NT is the main functional domain, which causes pyroptosis, but the full-length GSDMD protein has no such effect. Recently, GSDMD has been considered to be an essential factor downstream of caspase-1 or caspase-11, which mediates pyroptosis and releases interleukin (IL)-1β to the extracellular space (Quach et al., 2019). However, no research has been done in the role of GSDMD in mouse A. fumigatus keratitis.
The purpose of our research is to evaluate the expression of GSDMD and its function in A. fumigatus keratitis. Our study suggests that GSDMD is involved in the immune process of mice fungal keratitis and in IL-1β secretion induced by Aspergillus fumigatus.

2. Materials and methods

2.1. Clinical specimen

Overall, 6 samples of healthy corneas and 6 corneas of patients with Aspergillus keratitis were collected. All corneas were obtained from living patients. Healthy human corneal specimens were derived from patients with intraocular tumors such as malignant melanoma of the choroidal and with intact cornea. In addition, the collected corneas infected by Aspergillus fumigatus came from patients who had been clinically diagnosed to need “corneal transplantation”, and specimens needed to be retained for ophthalmic pathological examination after the operation. All the collected samples were used to examine the changes of GSDMD expression in cornea by immunofluorescence staining before and after Aspergillus fumigatus infection. The research aims and collection of specimens were completely in conformity with the participating patients right of informed consent. The corneas were treated in accordance with the ethics committee of the Affiliated Hospital of Qingdao University.

2.2. Murine model of corneal infection

Mice (female, 8 weeks of healthy clean grade C57BL/6) were ob-tained from Jinan Pengyue Experimental Animal Ltd. (Jinan, China). The standard A. fumigatus strain (strain 3.0772) was obtained from the department of clinical laboratory, Affiliated Hospital of Qingdao Uni-versity, Qingdao, China. Mice were anesthetized with 0.08 ml chloral hydrate (8%) via intraperitoneal injection. After this, they were placed
under a ×40 magnification stereoscopic microscope. A 30-gauge needle
was used to draw a scratch to the depth of the superficial stroma. Then
2.5 μL of 5.0 × 107/mL A. fumigatus conidial suspension were injected into the corneal stroma by the scratch. Mice corneas were observed and
collected at 1/2, 1, 2, 3, 5, 7, 10 and 14 days for real-time RT-PCR and Western blot. Mice eyeballs were removed for immunofluorescent staining at 1 day after infection. Laboratory animals were treated in ophthalmic and vision research to accordance with the guidance of the Association for Research in Vision and Ophthalmology (ARVO).

2.3. Clinical scoring

The mice were anesthetized with isoflurane. In the relative dark room, the corneal infection of mice was observed by slit lamp micro-scope, and the clinical scores were taken by camera. According to the three aspects in Table 1, a total score ranging from 0 to 5 as mild infection, a total score ranging from 6 to 9 as moderate infection, and a total score ranging from 10 to 12 as severe infection.

2.4. The human corneal epithelial cells (HCECs) culture and A. fumigatus stimulation

Immortalized HCEC cell lines were obtained from the Ocular Surface Laboratory of Xia Men Eye Center. The HCECs were seeded onto 6-well plates or 12-well plates of high DMEM/F12 (Hyclone) containing 12%
fatal bovine serun (FBS) and 1% penicillin and streptomycin and incubated in a humidified 5% CO2 incubator at 37 ◦C. When they grew to 80% confluence, HCECs were stimulated with A. fumigatus conidia (final concentration of 5.0 × 106 conidia/ml). Cells were harvested at 4 h, 8 h, 12 h, 16 h post-infection, then samples were detected for the expression
of GSDMD by PCR and Western blot.

2.5. GSDMD siRNA treatment of mice model

For siRNA treatment, the left eye of each mouse was injected sub-conjunctivally with 5 μL specific GSDMD siRNA (8 μm Santa Cruz) 1 d before infection and at the day just before infection. Similarly, 5 μL of non-targeting scrambled siRNA (8 μm Santa Cruz) were given to control eyes. One day after fungal infection, mice corneas were collected for RT-PCR and Western blot, and mice eyeballs were enucleated for immunofluorescent staining.

2.6. IFNR, JAK/STAT and caspase-1 inhibitor treatment of HCECs

HCECs were pretreated with inhibitors 2 h before A. fumigatus conidia infection. Inhibitors included: IFNR inhibitor hydrochloride (1 μm MedChemExpress), JAK/STAT inhibitor ruxolitinib (1 μm MedChe-mExpress), caspase-1 inhibitor belnacasan (10 μm MedChemExpress). Controls were also incubated with the same concentration of DMSO. Cells were harvested to evaluate the expression of GSDMD at 12 h for PCR and 16 h for Western blot. The HCECs were seeded onto 24-well plates and pretreated with the above inhibitors for 2 h and fungal stimulated for 12 h, and samples were collected for immunofluorescence staining.

2.7. Real-time RT-PCR

Total corneal and cellular tissue were isolated with the RNAiso plus reagent (TaKaRa) and disrupted via ultrasonic crusher (Eppendorf). Then, the content and purity of retrieval RNA were rapidly measured by spectrophotometry (Eppendorf). The reverse transcription step was performed according to the recommended procedure of the PrimeScript RT Reagent Kit (TaKaRa). Quantitative RT-PCR was performed with 20 μL of total reaction volume (2 μL of cDNA diluted 1:12.5 with DEPC). The amplification conditions were fulfilled by the following cycling

Table 1
Clinical scoring criteria for murine fungal keratitis.
Infection grade I II III IV
Area of corneal 0%–25% 26%–50% 51%–75% 76%–100%

opacity
Density of corneal opacity

Slight cloudiness, outline of iris and pupil discernible

Cloudy, but outline of iris and pupil remain visible

Cloudy, opacity not uniform Uniform opacity

Surface regularity Slight surface irregularity Rough surface, some swelling Significant swelling, crater or serious
descemetocele formation

Perforation or descemetocele

Scoring 1 2 3 4

parameters: 95 ◦C for 30 s, then 95 cycles of 5 ◦C for 40 s and 60 ◦C for 30 s. β-actin (housekeeping gene) was used as the control primers. The oligonucleotide primers used in this study are shown in Table 2.

2.8. Western blot analysis

Mice corneas and HCECs were extracted and lysed in radio-immunoprecipitation assay (RIPA; Solarbio) lysis buffer containing 1% Phenylmethanesulfonyl fluoride (PMSF; Solarbio) and phosphatase in-hibitor on ice for 2 h, with shaking every 15 min. The cell lysate was
centrifuged at 12,000 rpm for 5 min at 4 ◦C, and the supernatant ob-
tained. The protein concentration was measured according to a bicin-choninic acid (BCA; Solarbio) Protein assay reagent. The protein samples were resolved by a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF; Millipore) membrane by electro-transfer, on ice for 2 h. The membrane was blocked with western blocking buffer (Beyotime) at room temperature for 2–3 h, and then
soaked in primary antibody at 4 ◦C overnight. After removed and
washing with PBST (1 mL Tween-20 in 1 L Phosphate buffer solution) three times, the membrane was incubated with the secondary antibody dilution for 1–2 h. The primary antibodies used in this study included GSDMD (Novus), IL-1β (R & D Systems) and anti-β-actin (Bioss). An anti–rabbit (or goat) antibody corresponding to the primary antibody were used as the secondary antibody. The protein bands was detected with ECL reagents (Thermo Fisher Scientific).

2.9. Immunofluorescence staining

The C57BL/6 mice eyeballs were removed at 1 d post-infection, together with the cornea of patients with fungal keratitis, placed at optimum cutting temperature (OCT) solution (Tissue-Tek), and imme-diately frozen with liquid nitrogen. Corneal frozen slices (10 μm) were cut by the freezing-microtome (Leica Germany) and stored at —20 ◦C
prior usage. Slices were successively fixed in acetone for 5 min and
rinsed 3 times with PBS buffer, blocked with 10% blocking buffer of the goat’s serum (Solarbio) for 30 min at 37 ◦C, and incubated with goat anti-rabbit GSDMD antibody (1:400; Abcam), Neutrophil Marker anti-
body (1:200; Santa Cruz Biotechnology) and the F4/80 antibody (1:100; Cell Signaling Technology) respectively, and labeled GSDMD, neutro-phils and macrophages successively, overnight at 4 ◦C. After rinsing
gently with PBS for three times, the slices were stained with goat anti-
rabbit secondary antibody (1:1000; Cwbio) and CY3 conjugated IgG (1:100; Elabscience) respectively in the dark for 1 h and then DAPI so-lution (1:10; Solarbio) for another 10 min at room temperature. Finally,
the slices were submerged in an antifade reagent, covered with the coverslip, and then observed and photographed under a × 40 magnifi-cation with a Zeiss Axiovert microscope.

Table 2
Nucleotide sequences of primers used in real-time RT-PCR.

Gene GenBank no. Primer sequence (5′-3′) Size (bp)
hβ-Actin NM_001101.3 F-GCT CCT CCT GAG CGC AAG 75
R-CAT CTG CTG GAA GGT GGA CA
hGSDMD NM_001166237.1 F-TGA ATG TGT ACT CGC TGA GTG 98
TGG
R-CAG CTG CTG CAG GAC TTT GTG
mβ-Actin NM_007393.5 F-GAT TAC TGC TCT GGC TCC TAG C 147
R-GAC TCA TCG TAC TCC TGC TTG
C
mGSDMD NM_026960.4 F-TCG CTT GGT GGA CCC AGA TAC 134
R-AGG CTG TCC ACC GGA ATG AG
mIL-1β NM_008361.4 F-CGC AGC AGC ACA TCA ACA AGA 111
GC
R-TGT CCT CAT CCT GGA AGG TCC
ACG

To pre-treat A. fumigatus keratitis HCECs, the aforementioned experimental steps were utilised, with the following differences: cell slices were fixed in paraformaldehyde for 15 min at 37 ◦C, 0.3% triton-
100 was used to break cell membrane for 30 min before blocking and
Confocal Laser Scanning Microscope (CLSM) was applied to collect images at a 40 × magnification.

2.10. Statistical analysis

The expression data were expressed as mean ± SD. The relative expression levels of each group were statistically analyzed using GraphPad 7.0 software. One-way analysis of variance was used to
compare the groups and LSD-t test was used for analysis between two groups. p < 0.05 was considered statistically significant.

3. Results

3.1. GSDMD increased in human A. fumigatus keratitis and A. fumigatus stimulated HCECs

Firstly, we confirmed that GSDMD is expressed in human corneal epithelium and HCEC infected by Aspergillus fumigatus. The following results were obtained by real time RT-PCR, Western blot and immunofluorescent staining. Immunofluorescence staining was used on control and A. fumigatus-infected human corneas to detect the localization and expression of activated GSDMD P30. As shown in Fig. 1A, the concentration of GSDMD P30 (green) in A. fumigatus keratitis samples was significantly higher than in the control group. And the positive expression was mainly located in the corneal stroma. In addition, we examined the changes of GSDMD in HCECs after A. fumigatus infection. Immunofluorescence images showed that the expression of GSDMD P30 (green) was significantly increased in HCECs after Aspergillus fumigatus stimulation, and mainly expressed in the cell membrane and cytoplasm (Fig. 1B). Moreover, results indicated that mRNA levels of GSDMD (Fig. 1C) were distinctly increased from 4 h, peaking at 12 h after infection (p < 0.05, p < 0.05, p < 0.001, respectively). Additionally, Western blot analysis also observed markedly elevated GSDMD P30 (Fig. 1D) in HCECs at 8 h, 12 h and 16 h (p < 0.001, p < 0.001, p < 0.001,
respectively).

3.2. GSDMD increased in cornea of A. fumigatus keratitis mouse

To determine whether or not GSDMD is related to mice A. fumigatus keratitis, we continued to study the mouse model of A. fumigatus keratitis 14 days after infection. We observed that the corneal opacity after infection gradually increased and even formed ulcers. With the extension of time, the inflammation gradually reduced and the scar healed (Fig. 2A). The result of the clinical score also showed that the infection began to increase on the first day, and gradually decreased after reaching the peak on the fifth day (Fig. 2B). Following this, we tested the expressions of GSDMD in corneas of normal uninfected and infected C57BL/6 mice. When compared with untreated corneas, GSDMD mRNA levels were elevated from 0.5 d and peaked at 5 d after A. fumigatus
infection (Fig. 2C; All = p < 0.001). Moreover, Western blot results also
revealed that protein levels of GSDMD P30 were gradually increased from 0.5 d to the highest at 5 d after infection (Fig. 2D; All = p < 0.001). To further confirm these data, we confirmed GSDMD protein (green)
expression noticeably enhanced in infected mouse corneas. The immunoreactivity of GSDMD P30 was rarely detected in corneal tissue of healthy mice (Fig. 2E).

3.3. GSDMD production in A. fumigatus keratitis was dependent on IFNR, JAK/STAT and Caspase-1

The inhibitors (IFNR inhibitor hydrochloride, JAK/STAT inhibitor ruxolitinib and caspase-1 inhibitor belnacasan) were respectively used

Fig. 1. GSDMD increased in human A. fumigatus keratitis and A. fumigatus stimulated HCECs. Immunofluorescent staining demonstrated expression of GSDMD in human A. fumigatus keratitis (A). In HCECs, images taken by fluorescence microscope showed that GSDMD expression markedly increased after infection compared with normal control (B). Blue: nuclear staining (DAPI); Green: GSDMD staining. (C–D) The mRNA and protein expression of GSDMD significantly increased in HCECs following fungal infection. mRNA levels of GSDMD (C) peaked at 12 h and protein levels of GSDMD (D) peaked at 16 h after A. fumigatus infection. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

to pre-treat A. fumigatus keratitis HCECs, in vitro. When compared with the DMSO control, results indicated that relative GSDMD mRNA levels (Fig. 3A–C) decreased after treatment with inhibitors (p < 0.01, p < 0.001, p < 0.001, respectively). Moreover, Western blot analysis of GSDMD P30 (Fig. 3D–F) showed a similar descending trend to the PCR outcome (p < 0.01, p < 0.001, p < 0.01, respectively). And the protein levels of p-STAT1 and Caspase P20 decreased significantly after in-
hibitors treatment (All = p < 0.001). By using immunofluorescence staining, GSDMD P30 positive staining (green) was also detected in
HCECs of pre-treated cells with inhibitors versus the DMSO treatment group (Fig. 3G).

3.4. Disease response after siRNA GSDMD treatment

Aiming to study the function of GSDMD on A. fumigatus infection, the corneas of C57BL/6 mice were pretreated with GSDMD siRNA, photographs were taken by slit lamp and clinical scores (Fig. 4A–B; p < 0.001) at 1 d after infection. GSDMD siRNA treatment significantly reduced the disease severity compared with the A. fumigatus infected control. After pre-treatment with GSDMD siRNA, we found that the trend of increased GSDMD mRNA (Fig. 4C; p < 0.05) and protein (Fig. 4D; p < 0.001) expression levels were significantly suppressed at 1 d post-infection. In

addition, the immunofluorescence staining showed GSDMD expression in mouse corneal stroma was significantly reduced after GSDMD siRNA treatment corneas (Fig. 4E). All these results demonstrate that GSDMD siRNA successfully knocked down the gene in mice corneas of
A. fumigatus keratitis models. Immunostaining was used to measure neutrophils (Fig. 4F) and macrophages (Fig, 4G) after GSDMD siRNA treatment at 1 d after infection. The apparent positive staining figures for GSDMD siRNA-treated corneas after infection were distinctly decreased compared with the control. (a, corneal epithelium; b, stroma; c, anterior chamber).

3.5. Knockdown of GSDMD decreased IL-1β production in response to A. fumigatus

The role of GSDMD in IL-1β secretion induced by A. fumigatus infection was demonstrated by real-time RT-PCR and Western blot. Pretreatment of mice corneas was performed with siRNA of specifically targeted GSDMD before A. fumigatus infection. When compared with control infection, the knockdown of GSDMD infection group significantly reduced mRNA levels of IL-1β in the corneal epithelium (Fig. 5A; p < 0.01). Similarly, protein expression of IL-1β (Fig. 5B; p < 0.01) was also noticeably reduced when compared with control treatment mice at

Fig. 2. GSDMD increased in cornea of A. fumigatus keratitis mouse. The photograph of A. fumigatus keratitis mouse model by a slit lamp (A) and the result of clinical score during the period of infection (B). The mRNA levels of GSDMD (C) increased in mice corneas from 0.5 d and peaked at 5 d after infection. GSDMD (D) protein were significantly increased from 0.5 d. Immunofluorescent staining demonstrated that GSDMD expression (green) increased in mice infected cornea compared with the normal (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

1 d post-infection.

4. Discussion

Fungal keratitis, an important disease especially in many developing countries, can easily induce corneal ulcers and even blindness. With the development of modern medicine, the cure rate for this disease has been improved significantly (Maharana et al., 2016; Papaioannou et al., 2016). However, the treatment of fungal keratitis is still a major problem due to the lack of effective antifungal drugs (Peng et al., 2018; Yuan et al., 2017). Pyroptosis plays an important role in the anti-microbial innate immune defense (Bergsbaken et al., 2009; Kayagaki et al., 2015). The protein GSDMD is an important mediator of pyroptosis, and a generic substrate for inflammatory caspases (Shi et al., 2014, 2015). Previous studies proved that GSDMD is highly expressed in the gastro-intestinal (epithelial) and cutaneous systems and is a key participant in inflammation and pathogen defense caused by overactivation of pyroptosis(Liu and Lieberman, 2017). However, reports of GSDMD related to fungal pathogenesis are still unclear.
This study reports, for the first time, the GSDMD expression in human A. fumigatus keratitis and HCECs after A. fumigatus infection. Moreover, the expression of GSDMD was also significantly increased in murine keratitis model infected by A. fumigatus. The present findings indicated that GSDMD is involved in the immune response process of

A. fumigatus keratitis.
Previous published studies (Bergsbaken et al., 2009; Liu and Lie-berman, 2017) demonstrated that when the body is subjected to various external stimuli, the pattern recognition receptors (PRRs) initiate a downstream signaling by activating nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) and interferon (IFN) regulatory factors (IRF) dependent pathways to cause transcription of inflammatory genes, including inflammatory caspases and pro-inflammatory cytokines, IFNs and IFN-regulated genes (IRG). This study showed that inhibitors IFN, JAK/STAT and caspase-1 suppressed the production of GSDMD caused by the fungal infection. We concluded that the fungus stimulates the cornea to induce the production of IFN. The binding of IFN to homologous IFNR causes dimerization of receptor molecules, which activates the phosphorylation of intracellular receptor-coupled JAKs, and then phosphorylates STATs, transferred to the nucleus, combined with the corresponding target gene promoters and activates the gene transcription and expression. This result is consistent with Sun et al. who found that Caspase-11 is regulated by IFNR through JAK-STAT signal transduction in fungal keratitis (Sun et al., 2018). Caspase-11 is necessary for Caspase-1 activation and IL-1 β processing during infection. In this process, pro-IL-1β and pro-IL-18 were processed into IL-1β, IL-18 mature bodies, which are released into the extracellular matrix. Based on the above findings, we confirmed that IFN, JAK/STAT and caspase-1 signaling pathways participate in

Fig. 3. GSDMD production in A. fumigatus keratitis was dependent on IFNR, JAK/STAT and caspase-1. After these inhibitors were used to pre-treat A. fumigatus keratitis HCECs, mRNA (A-C) of GSDMD was obviously reduced at 12 h post-infection compared with infected DMSO control. And the protein (D–F) of GSDMD was also decreased at 16 h after treatment with these inhibitors. Immunofluorescence images (G) showed that the expression of GSDMD significantly was reduced in the inhibitors pre-treated group versus the DMSO pre-treated group.

GSDMD-induced pyroptosis in mice A. fumigatus keratitis.
Pyroptosis can remove the habitat for invading pathogens by directly killing infected host cells. In addition, inflammatory factors are released through GSDMD pores, which results in immune cells migration to the site of infection. This way, GSDMD can be identified as new target for anti-inflammatory treatment (Lamkanfi and Dixit, 2014; Liu and Lie-berman, 2017). Additionally, neutrophils and macrophages were considered to be essential immune cells involved in the cornea to counter with A. fumigatus infection (Taylor et al., 2014). Despite the importance of immune cell defense in acute inflammation in fungal pathogenesis, inappropriate migration can cause excessive side re-actions and destructive inflammation that contributes to impair normal tissues (Hamidzadeh et al., 2017; Oishi and Manabe, 2018; Wang, 2018). Their contradictory role in inflammation suggests that excessive infiltration of immune cells is not beneficial in A. fumigatus keratitis. Therefore, the inflammatory response must be tightly regulated to bal-ance fungal clearance and corneal transparency (Che et al., 2018; Tang et al., 2019). We demonstrated that treatment with GSDMD siRNA reduced the expression level of GSDMD after A. fumigatus infection and this knockdown further reduced the migration of neutrophils and macrophages to the corneal stroma during infection, which is consistent with the previous reported findings (Karmakar et al., 2020).
Previous studies have reported that IL-1β, an important inflamma-
tory cytokine, is involved in the inflammatory immune responses

induced by A. fumigatus keratitis (Gao et al., 2016; Gringhuis et al., 2012; Underhill and Pearlman, 2015). The expression level of IL-1β can change accordingly with the severity of disease. GSDMD is essential for IL-1β secretion (Kanneganti et al., 2018; Kayagaki et al., 2015; Shi et al., 2015). Shi et al. found that the absence of GSDMD protein does not affect the activation of caspase-1 and cleavage of downstream IL-1β, but inhibited the release of active IL-1 β to the extracellular environment (Shi et al., 2015). In other words, GSDMD controls the release of IL-1 β, but not its maturation. In this study, after knockdown of GSDMD, we found that the trend of increased IL-1β expression was significantly inhibited in the mice Aspergillus fumigatus. This can indicate that GSDMD, which activates IL-1β to recruit immune cells into the corneal stroma, plays an important role in A. fumigatus keratitis.
In conclusion, the research data presented here demonstrated that the expression of GSDMD was significantly elevated in human
A. fumigatus keratitis, mouse model and HCECs experiments. Moreover, the inhibition of GSDMD can decrease the disease response by reducing inflammatory cytokine IL-1β secretion and the recruitment of neutrophils and macrophages in mice A. fumigatus keratitis. Also, we provided evidence that GSDMD-mediated pyroptosis was closely related to IFN, JAK/STAT, Caspase-1 in HCECs stimulated by A. fumigatus. Taken together, GSDMD plays an essential role in mediating the immune response during A. fumigatus keratitis. A novel therapeutic target for fungal keratitis is provided by regulating the production of GSDMD.

Fig. 4. Disease response after siRNA GSDMD treatment. Compared with the pre-treatment of control, the GSDMD siRNA reduced the degree of corneal inflammation, as shown by slit-lamp photographs(A). There was a statistical difference in clinical disease scores between the two groups(B). PCR(C), Western bolt (D) and immunostaining(E) were used to validate the interference effect of GSDMD siRNA treatment. Immunostaining images showed that treatment with GSDMD siRNA markedly reduced neutrophils(F) and macrophages(G) number compared with the scrambled siRNA group. (a, corneal epithelium; b, stroma; c, anterior chamber).

Fig. 5. Knockdown of GSDMD decreased IL-1β secretion in response to A. fumigatus. The mRNA levels (A) of IL-1β in corneal tissue after knockdown of GSDMD were significantly decreased compared with infected scrambled control. Similarly, GSDMD siRNA also decreased relative protein levels of IL-1β (B) compared with infected control.

Disclosure

W. Zhao, None; H. Yang, None; L. Lyu None; J. Zhang, None; N. Jiang, None; L. Wang, None; H. Yan, None; C. Che, None
Wenyi Zhao, Hua Yang and Leyu Lyu contributed equally to this

work.

Acknowledgments

Supported by the National Natural Science Foundation of China

(81300730), China Postdoctoral Science Foundation (2018M630482), Key Research Project of Shandong (2018GSF118193), Science and Technology Project of Qingdao (19-6-1-39-nsh) and Qingdao Outstanding Health Professional Development Fund.

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